Vol 3 issue 1 2013 Part-2

ISSN: 2321-3272 (Print) ISSN: 2230-7605 (Online) Int. J. Pharm. Biol. Sci. CODEN: IJPBK3

INTERNATIONAL JOURNAL OF PHARMACY AND BIOLOGICAL SCIENCES A Peer Reviewed (Refereed/ Juried) International Journal

www.ijpbs.com or www.ijpbsonline.com

I

Volume 3 Issue 1 Jan-March, 2013

Associate Editors Mr.E.Venkateshwarlu Department Of Pharmacology, India Mr.K.Venu Department Of Pharmacology, India Mr.K.Naresh Department Of Pharmaceutical Chemistry, India Mr.M.Satish Department Of Pharmaceutical Chemistry, India

Editorial Advisory Board Members Prof.Dr.J.Venkateshwar Rao Department Of Pharmaceutical Chemistry, India Dr.J.Raju, Department Of Pharmaceutics, India

Dr.A.Srinivas Department Of Pharmaceutical Chemistry, India

Dr.K.S.Nataraj Department Of Pharmaceutical Analysis, India

Dr.G.K.Mallaiah Department Of Pharmacognosy, India

Dr.G.Shyam Prasad Department Of Microbiology, India

Dr.B.Srinivas Department Of Pharmaceutical Chemistry, India

Dr.B.K.Prusty Department Of Pharmacology, India

Dr.T.Sreekanth Department Of Pharmaceutical Chemistry, India

Dr.A.Shyam Sunder Department Of Pharmacology, India

Dr. M.R.Jayapal Department Of Organic Chemistry, India

Dr.Deepak Prashar Department Of Pharmacy, Kullu (H.P.),India

Prof.Dr.Prakash.MMS.Kinthada Department Of Chemistry, India

Dr.Seshikala Durisetti Kakatiya University College Of Engineering

Dr.Anshu Srivastava Department of Applied science & Humanities, India

Dr.Arvind R.Umarkar Department of Pharmaceutical Chemistry, India

Dr.M.Jagadishnaik Department Of Zoology, India

Dr.Rajsekhar Paul Principal Scientist, Novartis Pharma, SWITZERLAND

Dr.Shidlingappa Shirol Department of Plastic Surgery, K.L.E.S ,India

Dr. DSVGK Kaladhar Department of Bioinformatics, GITAM University, India

Prof. Dr.G.Vidyasagar Dean, Faculty Of Pharmaceutical Sciences, India

Dr.Surapaneni Krishna Mohan Department of Biochemistry, India

Dr.N.N.Rajendran Director of PG Studies and Research, India

Dr.Mohammed Rageeb Md Usman Department of Pharmacognosy, India

Dr.Thirumalai.T Post graduate and Research Department of Zoology, India

Dr.Pulak Majumder Department of Pharmacognosy, India

Dr. Mirza Rafiullah Baig Department of Clinical Pharmacy, Malaysia

Dr. Kammuluri Ratna Kumar Department of Pharmaceutics, USA

Mr. Sai Krishna Department of Pharmaceutics, UK

Mr.Vamshi Krishna Department of Pharmaceutics, USA

Editor-In-Chief

JAYAPAL REDDY GANGADI M.Pharm, M.Phil.,FICCP, Ph.D




II

GENERAL INFORMATION

International Journal of Pharmacy and Biological

Sciences is a peer-reviewed international journal

scheduled to appear quarterly, which publishes

innovative research works in the fields of pharmacy

and biological sciences. IJPBS publishes papers of

Original Research work related all aspects of

pharmacy and biological sciences. The journal is

published quarterly in the Months of January, April,

July and October.

ANNUAL SUBSCRIPTION INFORMATION

Subscription is payable to: Jayapal Reddy Gangadi

bank account (Refer: Subscription application form)

Claims for missing issues will be received within 45

days of the publication date for Indian subscribers,

75 days in case of Overseas Subscribers.

** For single issue: 2000 INR

ETHICAL /LEGAL CONSIDERATIONS

Authors involving in the usage of experimental

animals and human subjects in their research article

should seek approval from the appropriate

Institutional Animal Ethics committee in accordance

with "Principles of Laboratory Animal Care". The

Method section of the manuscript should include a

statement to prove that the investigation was

approved and that informed consent was obtained.

While submitting the manuscript the corresponding

author ( on behalf of all the authors) should declare

that the manuscript is original and is not published

or communicated for publication elsewhere either

in part or full. Without the declaration the

manuscripts are not considered for their

publication. “No responsibility is assumed by the

publisher” for any injury and/or damage to persons

or property as a matter of products liability,

negligence or otherwise, or from any use or

operation of any methods, products, instructions or

ideas contained in the material herein. The

respective individual author(s) are responsible for

the facts and views expressed in their articles.

“Authors are solely responsible for their

manuscripts and data published in the journals”

and any mistakes in the published articles. The

publisher does not take any such responsibilities.

Copying and reproducing any parts of the published

articles should be duly acknowledged, otherwise, it

will be considered as an offense as per international

Publications law.

COPYRIGHT

All authors must Sign a copy of the Journal’s “Cover

Letter” and “Copy Right form” and submit it at the

time of Manuscript submission (Without this signed

undertaking your paper would not get displayed).

Copyright Clearance will be the sole responsibility of

the individual author for his/her contribution to

IJPBS.

PUBLICATION POLICY

Papers will normally be published in order of

acceptance by the editors, quickly as soon as the

corrected electronic manuscripts are received. The

details of the published paper (s) will be

communicated to the corresponding author.

ADVERTISEMENT AND MANUSCRIPT SUBMISSION

Manuscript submission, request for permission to

use published material and advertisement inquires

should be addressed to [email protected];

[email protected]

You can submit your manuscript through online

also: http://ijpbs.com/submitmanuscript.php

CHANGE OF ADDRESS

The request should be sent to the Editor-In-Chief.

The request should contain both old and new

addresses. Subscription copies returned due to

incomplete; incorrect or changed address for two

consecutive occasions, would force us to delete the

names from the mailing list of the Journal.

CORRESPONDING ADDRESS

JAYAPAL REDDY GANGADI Editor-In-Chief, IJPBS #2-2-83/13, Rajaji Nagar, Naim Nagar, Hanamkonda, Warangal-506 001; Phone: 09912185510;

PUBLISHED BY

Jp Research Publications (JPRP)

HYDERABAD (INDIA)

© 2013, IJPBS, HYDERABAD

Annual

Subscription

Fees

India

(INR)

Foreign

(USD)

Individual Rs. 4000/- $ 90/ €30

Institutional Rs. 6000/- $ 150/ €60

mailto:[email protected]


http://ijpbs.com/submitmanuscript.php




III

Volume 3 Issue 1 Jan-March, 2013

CONTENTS

MANUSCRIPTS

INSTRUCTIONS TO AUTHORS

MODEL COVER LETTER

COPY RIGHT FORM

SUBSCRIPTION/MEMBERSHIP FORM

NOTE: Submit your manuscript along with your COVER LETTER as an attachment file to: [email protected], [email protected].

@@@

Editorial Office:

INTERNATIONAL JOURNAL OF PHARMACY AND BIOLOGICAL SCIENCES [ISSN: 2321-3272 (Print), 2230-7605 (Online)]

#2-2-83/13, Rajaji Nagar, Naim Nagar, Hanamkonda, Warangal-506 001; Phone: 09912185510; Website: www.ijpbs.com; www.ijpbsonline.com ; Email: [email protected], [email protected]



http://www.ijpbs.com/

http://www.ijpbsonline.com/



Available Online through

www.ijpbs.com (or) www.ijpbsonline.com IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|474-482

Research Article

Biological Sciences

International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

B.K.Mohanty*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e47

4

EFFECT OF MERCURY AND CADIMIUM ON THE ENZYME ACTIVITY OF HYDRILLA PLANT

Goutam C. Das**, K. Bijay Kumar and B.K.Mohanty*

* P.G.Department of Botany &Biotechnology, Khallikote Autonomous College, BERHAMPUR-760001, ODISHA

**Lecturer in Botany, A.S. Collge, Balia-755005, Jajpur, ODISHA *Corresponding Author Email: [email protected]

ABSTRACT The study of toxicity of metals on the enzyme activity in the Hydrilla plant indicated that in general there is induction

or inhibition in enzyme activity. The enzymes studied here in relation Hg and Cd toxicity like catalase, peroxidase

and polyphenol oxidase found in the plant responded differently to the metal treatment. Catalase and polyphenol

oxidase showed a decrease in the activity whereas peroxidase enzymes were stimulated by the metals. When the

metals were treated along with growth hormone Kinetin, there was substantial recovery of the enzyme activity in

the test plant. Other enzymes like acid phosphatase, acid/alkaline protease, acid /alkaline prophophatase and

RNase were also studied to correlate the decrease in the molecular content in the test plant responded a decline in

its activity with the metal treatment. However, the addition of Kinetin to the metals (HgCl2 and CdSo4) showed a

recovery in the enzyme activity in the hydrilla plant .All these observations indicates that the aquatic plant hydrilla

can be used as an indicator plant to assess the metal toxicity in the aquatic system.

KEY WORDS Mercury, Cadimium, Enzymes, Hydrilla

INTRODUCTION

Life depends on a complex network of chemical

reactions brought about by a specific enzyme. The

enzymes are the primary instruments for the

expression of gene action. Enzymes are of

biological importance in metabolic function,

which have the most highly specialized class of

proteins. The change in metabolism can well be

correlated with the disturbance in behavior and

action of the enzymes. The reduction in active

metabolism has been correlated with the toxic

effective different types of toxicants. The

disturbance in enzymatic action can be related to

the specific action of certain toxicants by

inhibiting the enzymes to act, bind the active sites

of the enzyme and enzymes become

nonfunctional due to disintegration .Inhibition of

enzymes by chemical agents may be reversible or

irreversible, competitive or noncompetitive.

There are many different mechanisms through

which thiol groups of enzymes can be acted upon

by heavy metals. The importance of enzymatic

studies in the system lies in the functional

interpretation of the pollutant causing different

types of disorders.

Induction of catalase activity is brought about

when toxic does of various metals are present

(Van Assche and Clijsters, 1990). Taking into

consideration the effect of the metal application,

some workers have reported an induction of

catalase in response to various heavy metals

(Mukharji and Das Gupta, 1972; Subhadra et al.,

1991). There are reports that cadmium decreased

the activity of catalase, an antioxidant enzyme in

germinating seedlings of mung bean phaseolus

vilgaris. (Somasekharaiah et al., 1992). A decline










Pag

e47

5

in catalase activity was also observed in rhy leaves

when exposed to cadmium (Streh et al., 1993).

Catalase activity increased significantly in

Posidonia oceanica following 48h exposure to low

concentrations of HgCl2 (0.01 μg L-1 and 0.1 μg L-1)

as compared to controls (Ferrat et al., 2002b).

Increase in Peroxidase has been observed in

Glycine max (Lee et al., 1976) and Phaseolus

Vulgaris (Van Assche et al., 1988) in response to

cadmium. Reddy and Prasad (1992) observed an

increased peroxidase Oryza sativa treated with

different concentrations of cadmium. Karataglis

et al., (1991) reported that increasing

concentrations of copper, zinc, lead, nickel,

chromium caused complex changes in the forms

of isoperxidases in the seedlings of wheat. In

unicellular green alga Scenedesmus subspicatus

Reinhold et al., (1994) observed observed a

significant rise in peroxidase activity in response

to cadmium. Patro et al.,(2001), reported that all

concentrations of the effluents found to have

strong effect on the activity of peroxidase in the

leaves of Oryza sativa L. In Ceratophyllum

demersum mercury induced oxidative stress

increased anti-oxidant enzymes like peroxidase.

Rama Devi and Prasad, 1998, Sandalio et al., 2001

Metwally, 2003, 2004; Gallego et al., 1996;

Balestrasse et al., 2003). Mukherji and Maitra

(1976) have demonstrated the stimulated

activities of peroxidase under conditions of lead

toxicity of growth of rice seedlings. Very low

cadmium levels in vitro have shown to stimulate

the activities of certain enzymes like peroxidase,

acid phosphatase etc. (Ernst 1980, Shah and

Dubey, 1997).

MATERIALS AND METHODS

TEST MATERIAL:

The submerged rooted macrophyte Hydrilla

Verticillata casp is an aquatic weed belonging to

family Hydrocharitaceae.It grows abundantly in

lakes, ponds and ditches of tropical and

temperature climates. The coarsely serrated

leaves of this plant occur in whorls of three to

eight and have characteristic spines on the

underside of the midrib. Hydrilla produces

reproductive propagates called turions and

tubers. Turions are compact dormant buds that

are produced in leaf axils and fall from the plant,

when they are mature. Tubers are formed

terminally on rhizomes and can be found up to a

foot deep in the sediment. A pond near

A.S.College, Balia Dist: Jajpur (Orissa) was

selected for the collection of Hydrilla verticillata

plants. Samples of the plant were obtained from

the site and grown in experimental ponds near

the college premises.

TEST CHEMICALS:

The chemicals used in the present study were of

the purest grade available and were obtained

from renowned laboratories. Mercuric chloride

[Hg Cl2] (M.W-272)] and cadmium sulphate [3

CdSO4, 8H2O (M.W.769.51)] were used at the test

chemicals. Different concentration of the test

chemicals were prepared by using double distilled

water as the solvent. The selected concentration

of the test chemicals used in the present study for

both Mercury and cadmium were 1, 5, 10, 25mg-1

for experimentations.

EXPERIMENTAL SETUP:

Hydrilla growth in experimental tanks were

collected in the form of sample and after

acclimatization that would be subjected to tested

in four different concentrations.(1,5,10.25mgL-1)

of cd and Hg for 5 days and 10 days separately.

Various physiological and biochemical

parameters of the plant were measured in

response to the metal treatment.

After assessing the toxic effects of CD and Hg at

various selected concentration on the test plant,

an attempt would be made to study the

amelioratine effects of growth regulators, Kintein

(5mg-1) on toxic effects were assessed by adding

2ml in each concentrations of Cd and Hg.









Pag

e47

6

The enzymes like Catalase, Peroxydase, Poly

Phenyl oxidase (Kar and Mishra, 1976), Acid

Phosphatase (Sacher and De Leo, 1977)

Acid/Alkaline Protease (Moore and Stein, 1948)

were estimated following standard procedures.

RESULTS

The data pertaining to the effect of different

concentrations of mercury and cadmium on the

various enzyme activities and their percent

increase/decrease was depicted in Table No. 1

and 2.The catalase enzyme activity was least

effected and the highest percent decrease was

only up to 5.28% in case of Hg and 7.69% in case

of Cd.

The peroxydase enzyme was elevated with the

increase in Hg and Cd treatment and highest

%increase was seen in case of 25 mg Hg treatment

for 10 days. The polyphenol oxidase enzyme

showed a decrease in activity with the increase in

metal concentration and the maximum decrease

was seen in case of 25 mg Cd treatment for 10

days. The results obtained with enzymes like

Peroxydase and Poly phenyl oxidase was

statistically significant. The Acid Phosphatease

enzyme showed an increase in activity with the

increase in metal concentration but the trend was

not statistically significant.The Acid protease

activity showed a decreasing trend with the

increase in metal concentration and the highest

decrease (71.25%) was seen in case of 25 mg Hg

treatment for 5 days.

DISCUSSION

The catalase activity decreased following

exposure to elevated cadmium concentrations

(Shim et al., 2003; Dalurzo et al., 1997; 2001;

Fornazier et al., 2002) Romero – Puertas et al.,

(2004) studied the involvement of H2O2 and O2 in

the signaling events that lead to the variation of

the transcript levels of catalase in Pea plants

under cadmium stress. The activity of several

enzymes like RNase, catalase etc., also decrease

due to toxic action of chromium compounds

(Panda et al., 1997).

In the present investigation catalase showed a

decreased trend in response to increasing

concentrations of mercury and cadmium. There

was a –ve correlation between various

concentrations of both metals and the enzyme

activity in Hydrilla plant after 5 and 10 days of

interval. Similar decreasing trend also been

observed when treated with metals and kinetin.

Compared to metals the activity of catalase is

more expressed in presence of kinetin.

Peroxidase is present in grana and stroma

lamellae (Henry, 1974, 1975.a.b. Henry et al.,

1981.b) and thus may be closely associated with

the changes in chloroplast including variation in

chlorophyllII Pigment. Peroxidase is considered as

a reliable indicator of various physiological

processes. Some workers observed a marked

change in peroxidase activity during root

initiation and considered as an indicator of the

process (Gasper et al., 1982, 1985; Moncousin

1986; Moncousin et al., (1987). There are also

reports of other metals inducing peroxidase

activity viz. mercury in Oryza sativa, mercury in

Lemna minor (Subhadra et al.1991), choromium

in Eichhornia crassipes (Satyakala and Jamil,

1993).

In the present investigation there was induction

of peroxidase activity in Hydrilla plant in response

to both mercury and cadmium after 5 to 10 days

of treatment, though there were some

differences in degree of induction. There was a

+ve correlation between induction of peroxidase

and metal concentration both for mercury and

cadmium.









Pag

e47

7

TABLE NO: 1: EFFECT OF DIFFERENT CONCENTRATIONS OF HgCl2 AND CdSO4 ON VARIOUS ENZYME ACTIVITIES OF HYDRILLA Sp. (EACH DATUM IS THE MEAN OF SIX

REPLICATES) (Values in parentheses indicate the percent increase / decrease in content from control values)

Treatment Metal

Conc.

CATALASE

In µ moles of H2O2

utilized / min-1 g-1

fresh weight / min-1

PEROXIDASE

In absorbance (A) units

POLYPHENOL OXIDASE


ACID PHOSPHATASE


ACID PROTEASE


5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days

Hg Cl2

Control 119.25 119.18 1.88 1.89 1.24 1.25 1.18 1.18 1.6 1.6

1mg 118.23

(-0.855)

117.89

(-2.591)

2.02

(7.446)

2.01

(6.349)

1.09

(-12.096)

1.08

(-13.6)

1.03

(12.711)

1.01

(-14.406)

1.04

(-35)

1.03

(-35.625)

5mg 116.16

(-2.591)

115.07

(-3.448)

3.04

(61.702)

3.01

(59.259)

1.02

(-17.741)

0.96

(-23.2)

2.09

(77.118)

2.06

(74.576)

1.01

(-36.875)

0.98

(-38.75)

10mg 114.04

(-4.368)

113.98

(-4.363)

3.8

(106.914)

3.71

(96.296)

0.96

(-22.580)

0.90

(-28)

1.94

(64.406)

1.84

(55.932)

0.82

(-48.750)

0.99

(-51.25)

25mg 113.05

(-5.199)

112.88

(-5.286)

4.12

(119.148)

4.08

(115.873)

0.80

(-35.483)

0.76

(-39.2)

2.48

(110.169)

2.46

(108.474)

0.46

(-71.250)

0.68

(-57.5)

r Value -0.893* -0.858* 0.868* 0.889* -0.924** -0.888* 0.849* 0.853* -0.868* -0.767 Ns

Cd SO4

Control 119.24 119.19 1.89 1.88 1.24 1.25 1.18 1.18 1.6 1.6

1mg 117.18

(-1.727)

117.18

(-1.686)

1.92

(1.587)

1.89

(0.531)

1.11

(-10.483)

1.09

(-12.8)

1.06

(-10.169)

1.02

(-13.559)

1.63

(1.875)

1.60

(0.00)

5mg 115.14

(-3.438)

115.01

(-3.507)

2.11

(11.640)

1.90

(1.063)

0.96

(-22.580)

0.84

(-32.8)

2.06

(74.576)

2.03

(72.033)

1.48

(-7.5)

1.46

(-8.75)

10mg 111.98

(-6.088)

111.08

(-6.804)

3.29

(74.074)

3.08

(63.829)

0.78

(-37.096)

0.72

(-42.4)

1.05

(-11.016)

1.03

(-12.711)

0.98

(-38.75)

0.96

(-46)

25mg 110.46

(-7.363)

110.02

(-7.693)

4.06

(114.814)

4.01

(113.297)

0.45

(-63.709)

0.36

(-71.2)

2.20

(86.440)

2.11

(78.813)

0.88

(-45)

0.85

(-46.875)

r Value -0.900* -0.883* 0.958** 0.962** -0.977*** -0.957** 0.643 Ns 0.617 Ns -0.897* -0.902*

( *- Significant at P ≤ 0.05, **- Significant at P ≤ 0.01, ***- Significant at P ≤ 0.001, NS- Non-Significant.)









Pag

e47

8

TABLE NO: 2: EFFECT OF DIFFERENT CONCENTRATIONS OF HgCl2 WITH KINETIN AND CdSO4 WITH KINETIN ON VARIOUS ENZYME ACTIVITIES OF HYDRILLA Sp. (EACH

DATUM IS THE MEAN OF SIX REPLICATES)(Values in parentheses indicate the percent increase / decrease in content from control values)

Treatment Metal

Conc.

CATALASE

In µ moles of H2O2

utilized / min-1 g-1

fresh weight / min-1

PEROXIDASE


POLYPHENOL OXIDASE


ACID PHOSPHATASE


ACID PROTEASE


5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days

Hg Cl2

Kinetin

Control 119.43 119.25 1.9 1.9 1.25 1.26 1.19 1.19 1.7 1.7

1mg 118.63

(-0.669)

117.95

(-1.090)

2.12

(11.578)

2.05

(7.894)

1.11

(-11.2)

1.10

(-12.698)

1.06

(-10.169)

1.04

(12.605)

1.05

(-38.235)

1.04

(-380823)

5mg 116.56

(-2.403)

115.87

(-2.834)

3.14

(65.263)

3.11

(63.684)

1.06

(-15.2)

1.02

(-19.047)

2.11

(78.813)

2.09

(75.630)

1.02

(-40)

1.01

(-40.588)

10mg 117.34

(-1.749)

114.02

(-4.385)

3.96

(108.421)

3.92

(106.315)

0.98

(-21.6)

0.87

(-30.952)

1.98

(67.796)

1.88

(57.983)

0.89

(-47.647)

0.86

(-49.411)

25mg 113.35

(-5.090)

113.12

(-5.140)

4.22

(122.105)

4.18

(120)

0.82

(-34.4)

0.79

(-37.301)

2.58

(118.644)

2.56

(115.126)

0.56

(-67.058)

0.54

(-68.235)

r Value -0.948** -0.878* 0.870* 0.869* -0.940** -0.880* 0.898* 0.870* -0.813* -0.813*

Cd SO4 with

Kinetin

Control 119.25 119.28 1.90 1.90 1.25 1.26 1.19 1.19 1.7 1.7

1mg 117.58

(-1.400)

117.43

(-1.550)

1.95

(2.631)

1.93

(1.578)

1.13

(-9.6)

1.12

(-11.111)

1.08

(-9.243)

1.03

(-13.445)

1.65

(-2.941)

1.62

(-4.7.5)

5mg 115.34

(-3.278)

115.11

(-3.495)

2.21

(16.315)

2.19

(15.263)

0.98

(-21.6)

0.88

(-30.158)

2.09

(75.630)

2.06

(73.109)

1.58

(-7.058)

1.56

(-8.235)

10mg 112.63

(-5.551)

111.98

(-6.120)

2.12

(11.578)

3.28

(72.631)

0.82

(-934.4)

0.76

(-39.682)

1.11

(-6.722)

1.09

(-8.403)

1.08

(-36.470)

1.06

(-37.647)

25mg 111.56

(-6.448)

110.76

(-7.142)

4.26

(124.210)

4.11

(116.315)

0.47

(-62.4)

0.38

(-69.841)

2.24

(88.235)

2.15

(80.672)

0.98

(-42.352)

0.95

(-44.117)

r Value -0.884* -0.888* 0.951** 0.966** -0.982*** -0.967** 0.660 Ns 0.636 Ns -0.899* -0.902*

(*- Significant at P ≤ 0.05, **- Significant at P ≤ 0.01, ***- Significant at P ≤ 0.001, NS- Non-Standard)









Pag

e47

9

In the present study it is observed that polyphenol

oxidase activity was reduced in Hydrilla plant in

response to mercury and cadmium after 5 to 10

days of interval. There was a close-ve correlation

between metal concentration applied and the

decrease in the activity of the enzyme in response

to both the metals. Our investigation is

contradictory to the views of Garg et al., (1994)

that an increase in polyphenol oxidase activity in

Marsilea minuta under cadmium induced stress.

Activity of polyphenol oxidase increased into a lot

by the addition of kinetin with heavy metals and

was observed in Hydrilla plant after 5 to 10 days

of interval. As far as Hydrilla plant is concerned

polyphenol oxidase can safely be used as an

indicator of mercury and cadmium toxicity.

A rise in acid phosphate activity was observed in

Glycine max in response tyo cadmium (Lee et al.,

1976). Medicago polymorpha in response to

drought stress. (Ehsanpour et al., 2003). Trticum

aestivum in response to chromium (Sharma and

Sharma, 1996). On the other hand an inhibition of

acid phosphatase activity was observed by Kong

and Chen, (1995) in response to Al and Zn, and

Rajni et al., (1991) in response to Cu, Zn, Hg in

potato tubers are available.

In Hydrilla at low concentrations of mercury and

cadmium levels (5 mg L -1) stimulate the activity of

acid phosphatase. Whereas at higher

concentrations of both metals (10mg L-1) have

inhibitory to thjis enzyme. Further increasing the

concentrations of both metals (25mg L -1) the

activity of acid phosphatase again increases after

5 to 10 days of interval. This observations was

supported by Ernst, (1980); Shah and

Dubey,(1997); Kumar and Banerjii, (1992).

Similar trend was also observed when the test

plant was both metals and kinetin. There was a

+ve correlation between mercury and enzyme

activity and mercury and kinetin and enzyme

activity both after 5 to 10 days of treatment but a

non-significant correlation was observed

between the enzyme activity with cadmium and

enzyme activity with cadmium and kinetin. The

trends in acid phosphatase activity in response to

metals shows that the enzyme cannot be used as

a general indicator of metal toxicity as its

responses are dependent on species and organ.

Sneh Lata(1989) on Phaseolus aureus had shown

that cadmium influences the activity of protease

which seem to inhibit the seedling growth.

Cadmium inhibits protease activity in germinating

pea seeds was reported by Bansal et al.,(2001).

The protease enzyme inhibition with HgCl 2 was

also observed in germinating mungbean seeds.

(Yamaoka et al., 1990). Oats (Drivdhal and

Thimann, 1978). Common bean (Rascusen and

foote, 1970), Soybean (Ragster and Chrispeels,

1979) and Agave (Du Toit, 1976). In Hydrilla we

report the presence of two sulfhydryl protease,

one alkaline with pH optimum of 8.0 and the

other a highly acidic one, with optimum pH of 3.0.

Activity of acidic and alkaline protease were

inhibited by the response of heavy metals in

Kalachoe leaves. (Jasrai et al., 1992). In the

present investigation an attempt is made to

analyse the activity of both acidic protease and

alkaline protease in Hydrilla plant. There was a

significant decrease in the activity of both acidic

and alkaline protease Hydrilla in response to both

mercury (after 5 and 10 days) of interval. But a

non-significant correlation was observed after 10

days of interval with mercury treatment.

In the present investigation the acid

pyrophsphatase showed decreasing trend in

response to both the metal and enzyme activity

after 5 and 10 days interval. Similar trend exists

when treated with metals and kinetin and showed

non-significant correlation between the enzyme

and metal with kinetin.

The alkaline pyrophosphatase enzyme also

showed a decreased trend in response to both the









Pag

e48

0

metals and there was a –ve correlation and

enzyme activity after 5 days of treatment but

after 10 days non-significant correlation exists

between them. In response to metals and kinetin

similar decreasing trend also observed in Hydrilla

and showed a –ve correlation between enzyme

activity and metal with kinetin after 5 days of

treatment but the non-significant correlation was

existing after 10 days of interval. Thus inorganic

pyrophosphatase can be used as an indicator of

metal toxicity response.

Change in synthesis or alterations in levels of

RNase may be involved in the regulation of RNA

content. To explore the second possibility RNase

was included in the present investigation. There

was a decline in the RNase activity in Hydrilla in

response to both mercury and cadmium after 5

and 10 days of interval. A non-significant

correlation exists between mercury and enzyme

activity after subsequent days but a close –ve

significant correlation exists between cadmium

and enzyme acivity.

Induction of kinetin to the metals showed similar

trend in Hydrilla plant and a non-significant

correlation exists between enzyme activity and

mercury with kinetin, but with cadmium and

kinetin a significant close –ve correlation exist

after 5 and 10 days of interval. Compared to only

metal treatment RNase activity is more

pronounced when treated with metal and kinetin

Hydrilla after 5 and 10 days of interval. It may be

concluded that RNase may be involved in the

regulation of RNA content in Hydrilla in response

to metals under study.

ACKNOWLEDGEMENT

Authors are thankful to Principal, Khallikote

autonomous College, Berhampur for providing

necessary laboratory facilities.

REFERENCE

o Balestrasse, K.B.; Benavides, M.P.; Gallego, S, M. and

Tomaro, M.L; Effect on cadmium stress on nitrogen

metabolism in nodules and roots of soybean plants.

Func. Plant Biol. 30: 57-64, 2003

o Bansal, P.; Sharma, P. and Dhindsa, K.S. Impact of Pb2+

and Cd2+ on activities of hydrolytic enzymes in

germinating pea seeds. Ann. Agri. Bio. Res.6: 113-122,

2001

o Chen, C. and Chen, S.C.; Effects of some Physico-

chemical treatments on the cadmium uptake by the

alga chlollera sp. Cs-10. J. Chin. Agri. Chem. Soc. 32(2):

132-140, 1994,

o Dalurzo, H.C.; Sandalio, L.M.; Gomez, M. and Del Rio,

L.A, Cadmium infiltration of detached pea leaves: effect

on its activated oxygen metabolism. Iphyton-Ann. Rei.

Bot. 37: 59-64, 1997

o Drivdhal, R.H. and Thimann, K.V.; proteases of senscing

oat leaves II: reaction to substrates and inhibitors. Plant

physiol 61: 501-505, 1978

o Du Toit, P.J.; Isolation and partial characterization of

protease from Agave Americana variegate, Biochim.

Biophys. Acta 429: 85-911, 1976

o Ehsanpour, A.A. and Amini F.; Effect of salt and drought

stress on acid phosphates activity in medicago sativa L.

explants under in vitro culture. Indian J. plant physiol:

273-275, 2003

o Ernst WHO; Biochemical aspects of cadmium in plants.

In JD Nragu, ed. Cadmium in the Environment. Part-I.

Ecological cycling, Wiley Interscience, New York, 639-

653, 1980

o Ferrat, L.; Romeo, M.; Gnassia Barelli, M. and Pergent

Martini, C.; Effect of mercury on antioxidant

mechanisms in the in the marine phanerogam posidonia

oceanic. Dis. Aquat. Org. 50, 157-160, 2002b

o Fornazier, R.F.; Ferreira, R.R.; Vitoria, A.P.; Molina,

S.M.G.; Lea, P.J. and Azevedo, R.A. ;Effects of cadmium

on antioxidant enzyme activities in sugarcane. Biol.

Plant. 45: 91-97, 2002

o Gallego, S.M.; Benavides, M.P. and tomaro, M.L.; Effect

of heavy metal ion excess sunflower leaves: evidence

for involvement of oxidative stress. Plant Sci. 121: 151-

159, 1996

o Garg P.; Vishwanathan, V. and Singh, J.; Cadmium

induced variations in phenolic, polyphenol oxidase and

peroxidases in Marsilea minuta L. J. Environ. Sci. Engin.,

29(7): 1323-1333, 1994

o Gasper, J.; Pene, C.; Raudet, C.; Monocousin, C. and

Greppin, H.; A two-step control of basic and acidic

preoxidases and its significance for growth and

development. Physiol. Plant. 64: 418-423, 1985

o Gasper, T.; Penel, C.; Thorpe, T. and Greppin, H.;

Preoxidases, 1970-1980. A survey of their biochemical









Pag

e48

1

and physiological roles in higher plants. Univ. of Geneva,

Switzerland. 1982

o Henry, E.W.; Ultrastructural localization- polyphenol

oxidase activity in chloroplasts byNicotina tobacum

chloroplast. Plant Physiol. I50 (Suppl.): 357.1974

o Henry, E.W; Ployphenoloxidase activity in thylakooids

and membrance bound granular components of

Nicotina tobacum- Chloroplasts. J. Microsco. 22: 10-

116, 1975a

o Henry, E.W.; preoxidase in tobacco abscission zone. J.

Ultrastruct. Res. 289-299, 1975b

o Henry, E.W.; De Poore, M.J.; O’ Conner, M.N. and De

Morrow, J.M. Photobiochem. Photobiophys., Sorbitol

disrupted spinach (Spinacea oleracea L.) chloroplasts. II.

ATPsae, per oxidase and chlorophyll.3:145-151,1981b

o Jasrai, Y.T., Bhatt, P.N. and Mehta, A.R.; Highly acidic

and alkaline proteases in Kalanchoe leaves and their

activity during dormant epiphyllous bud reactivation.

Plant physiol and Biochem. Vol. 19; 88-92, 1992

o Karataglis, S.; Moustakes, M. and Symeonidis, L.; Effect

of heavy metals on isoperoxidates of wheat. Biol. Plant.

(Prague). 33(I): 3-9, 1991

o Kumar, S., Banerjee, D.; effect of some heavy metals on

in vitro activities of certain enzymes, Plant physiol.

Biochem. 19: 33-35, 1992

o Lee, K.C.; Cunningham, B.A.; Paulsen, G.M.; Liang, G.H.

and Moore, R.B.; effects of cadmium on respiration rate

and activities of several enzymes in soybean seedlings.

Physiol. Plant. 36: 4-6, 1976a

o Lee, K.C.; Cunningham, B.A.; Paulsen, G.M.; Chung, K.H.

and Liang, G.H.; Lead effects on several enzymes and

nitrogenous compounds in soybean leaf. Journal of

Environmental Quality. 5: 357-35, 1976b

o Maier, R.; Aktivitat und multiple Foremen der

Peroxidase in unverbleiten und verbleiten Pflanzen von

Zea mays und Medicago sativa. Phyton. 19: 83-96,

1978a

o Metwally, A.; Finkemeier, I.; Georgi, M. and Dietz, K.J.;

salicylic acid alleviates the cadmium toxicity in barley

seedlings. Plant physiol. 132: 272-281, 2003

o Monocousin, C.; Preoxidase as a marker for rooting

improvement of clones of Vitis cultured in vitro. In:

Molecular and physiological aspects of plant

peroxidases. Univ. of Geneva, Switzerland, 1986

o Monocousin, C.; Fevre, J.M.; Gasper, T.; In; Physiology

and Biochemistry of auxines in plants. (M.Kuttaceak,

R.S. Bandurski and J.Krekuleeds) Academia, Praha 331-

337, 1987

o Mukherji, S.; maitra, P.; Toxic efftecs of lead on growth

and metabolism of germinating rice seeds and on

mitosis of cells. Ind. J.Exp. boil. 14: 519-571, 1976

o Mukherji, S. and Dasgupta, B.; characterization of

copper toxicity in lettuce seedlings. Physiologia

Plantarum. 27: 126-129, 1972

o Panda, S.K. and Patra, H.K.; Physiology of chromium

toxicity in plants. A review plant physiol and Biochem,

vol. 24(I): 10-17,1997

o Patro, B.; Panigrahi, S.; Das, M. and tripathy, S.; the

effectiveness of industrial effluents on chlorophyll

degradation and hydrogen peroxide utilization in rice

leaves during senescence. Asian J. of Microbiol. Biotech.

And Env. Sc. Vol.3: 183-190,2001

o Racusen, D.; Foote, M., An endopeptidase of bean

leaves. Canadian J. Bot. 48:1017-1021,1970

o Ragster, L. and Chrispeels, M.J.; Azocoll-digesting

Proteinase in Soybean leaves. Plant physiol. 64: 857-

862, 1979

o Rajni, K.; Malhotra, N.; and Dua, S.; Characteristics of

acid phosphateses from Ambari (Hibiscus Cannabinus

L.) seedlings. Plant physiol and Biochem. Vol. 18(I): 47-

53,1991

o Rama Devi, S., Prasad, M.N.V. (1998) - Copper toxicity in

Ceratophyllum demersum L. (Coontail), a free floating

macrophyte; response of antioxidant enzymes and

antioxidants. Plant Sci. 138, 157-165,1998

o Reinheld, D.; Hoefner, W. and Koehler, W.; Influence of

Cu, Cd and altrazine on the metabolism of the

unicellular green alga scenedesmus subspicatus.

Zeitschrift fuer Pflanzenernaehrung und Bodenkunde.

157 (2): 145-150 ,1994

o Romero-Puertas, M.C.; Rodriguez-Serrano, M.; Corpas,

F.J.; Gomez, M.; del Rio, L.A. and Sandalio, L.M.

Cadmium induced subcellular accumulation of O2 and

H2O2 in pea leaves. Plant cell Environ. 27:1122-1134,

2004

o Sandalio, L.M.; Dalurzo, H.C.; Gomez, M.; Romero-

Puertas, M.c. and del Ri; Cadmium induced changes in

the growth and oxidative metabolism of pea plants. J.

Exp. Bot. 52: 2115-2126, 2001

o Satyakala, G. and Jamil, K.; Effect of trace metal (Cu and

Cr) on the aquaticweed Eichornia crassipes. Int.J.Toxic

Occup Envir. Health. (2):41 (abstr), 1993

o Sharma, D.C. and Sharma, C.P.; Chromium uptake and

toxicity effects on growth and metabolic activities in

wheat, ev 2003. Indian J. Expt. Biol. 34: 689-691, 1996

o Shim, I.S.; Monose, Y.; Yamamoto, A.; Kim, D.W. and

Usui, K. ; Inhibition of catalase activity by Oxidative

Stress and its relationship to salicyclic acid accumulation

in plants. Plant Growth Regul. 39: 285-292, 2003

o Sneh Lata ; Pretreatment effects of cadmium on

seedlings growth, mobilization and activity of hydrolytic

enzymes in phaselous aureus Cv.T-44. Mendel. 6, 697-

72, 1989









Pag

e48

2

o Somashokaraiah, B.K.; Padmaja, K. and Prasad, A.R.K.;

Phytotoxicity of cadmium ions on germinating seedlings

of mung bean (Phaselous vulgaris): involvement of lipid

peroxides in chlorophyll degradation. Physiol. Plant.

85(I); 85-89, 1992

o Streh, P.; Michael-knauf, A. and Feirabend, J.;

Preferential photoinactivation of catalase and

photoinhibition of photosystem II are common early

symptoms under various osmotic and chemical stress

conditions. Physiol. Plant. 88(II): 590-598, 1993

o Subhadra, A.V.; Nanda, A.K.; Behera, P.K. and Panda,

B.B. ;Acceleration of catalase and preoxidase activities

in Lemna minor L. and Allium cepa L. in response to low

levels of aquatic mercury. Environ. Pollut. 69; 169-197,

1991

o Van Assche, F. and Clijsters, H.; Effects of metals on

enzyme activity in plants. Plant, Cell and Environ. 13:

195-20, 1990.

o Van Assche, F.; Cardinales, C. and Clijsters, H.; Induction

of enzyme capacity in plants as a result of heavy metal

toxicity: dose-response relations in Phaseolus vulgaris

L., treated with zinc and cadmium. Environmental

Pollution. 52: 103-115, 1998

o Yamadka, Y.; Takimura, O.; Fuse, H. and Kamimura, K.;

Effect of magnetism on growth of Dunaliela salina. 51:

87-90, 1993

*Corresponding Author: Dr. B.K.Mohanty, Reader in Botany P.G.Department of Botany &Biotechnology, Khallikote Autonomous College, BERHAMPUR-760001, ODISHA, [email protected], Mob: 09437216341








Research Article

Pharmaceutical Sciences


KRISHNA H. FATTEPUR*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e48

3

DIELECTRIC STUDY OF DIMETHYL FORMAMIDE WITH ETHOXY ETHANOL USING TIME

DOMAIN REFLECTOMETRY TECHNIQUE

KRISHNA H. FATTEPUR*1, JAGADISH G. BARAGI 2 1Department of Physics, National College, Basavanagudi, Bangalore-560 004, Karnataka, India

2Department of Chemistry, J.S.S. College, Vidyagiri, Dharwad-580004, Karnataka, India *Corresponding Author Email: [email protected]

ABSTRACT Dielectric relaxation studies of Dimethyl Formamide (DMF) with Ethoxy Ethanol (EE) mixture for different

concentrations over the frequency range 10 MHz to 20 GHz in the temperature range 278K to 308K have been

investigated using time domain reflectometry (TDR) technique. Static permittivity (s), dielectric constant at high

frequency () and relaxation time () were found through dielectric measurements. The excess dielectric

parameters, Bruggeman factor, Kirk wood correlation factor and thermodynamic parameters like change in

enthalpy (H) and change in entropy (S) have been determined and discussed to yield information on the molecular

interactions and microdynamics of the mixture of the system.

KEY WORDS Dielectric Relaxation, Dimethyl Formamide, Ethoxy Ethanol, Bruggeman Factor, Cole Davidson model, Kirkwood

Correlation factor.

INTRODUCTION

Studies on frequency dependent dielectric

relaxation of binary mixture DMF and aqueous

solutions have been reported 1-2 to understand

the microdynamics and hydrogen bond structure

in these systems. The hydrogen bonding causes a

significant effect upon the structure,

thermodynamic properties like change in

enthalpy (H) and change in entropy (S).

Referring work of Gestblom and J. Sjoblom 3-4

small - chain alcohols like methanol have faster

relaxation time and low activation energy where

as long chain molecules have slower relaxation

and high activation energy.

Several researchers have studied the

temperature dependent dielectric relaxation

parameters, activation energy and Kirkwood

correlation factor of various binary mixtures 5-7. In

this paper an attempt has been made to report a

systematic investigation of dielectric properties

and dielectric mechanism, thermodynamic

properties, Bruggeman factor and Kirkwood

correlation factor and the results are discussed

over the seven different mole fractions at four

different temperatures from 278K to 308K.


(i) Chemicals and Sample Preparation:

Analytical reagent grade samples of Dimethyl

Formamide (DMF) and Ethoxy Ethanol (EE)) were

procured from S.D. fine-Chem. Pvt. Ltd., Mumbai,

India. In the present study they are used without

further purification. The solutions were prepared

at seven different mole fractions of DMF and EE

at room temperature. The mole fraction of

components DMF (1) and EE (2) is calculated

using the following Equation 1 7-8.










Pag

e48

4

B

BB

A

AA

A

AA

M

V

M

V

M

V

X

1

And B

BB

A

AA

B

BB

M

V

M

V

M

V

X

2

---------- (1)

Where MA, MB are the molecular weights VA, VB

are the volumes and A, B are the density of

liquid A and B respectively. The limiting high

frequency of pure ideal solvents and m of the

binary mixtures were taken as the square of

refractive index nD at the wavelength of sodium D-

line.

(ii) Apparatus:

The Hewlett Packard HP54750A sampling

oscilloscope with HP54754A TDR plug-in module

has been used. After observing TDR response for

the sample under study, the time window was

kept to 5 ns. Also by observing TDR response for

the sample under study, the SMA sample cell with

1.35 mm effective pin length has been used. To

reduce noise, time-dependent response curve

was averaged for 64 times and then stored in the

memory of the oscilloscope with 1024 points per

wave-form. First, the reflected pulse from the

empty cell is acquired and stored in the memory

and then, the reflected pulse from the cell with

sample is acquired and stored in the memory. The

empty cell wave-form is used as the reference

wave-form. Both response wave-forms are the

reflected wave-forms from the sample cell with

open termination of transmission line.

The data acquisition is carried out for 7

concentrations in the temperature range 278K to

308K with an accuracy of ±1 0C. At each time the

response wave-forms without sample and with

sample were recorded. The time-dependent

response wave-form without sample is referred

as R1 (t) and with sample is referred as Rx (t).

In this process, the time-dependent data were

processed to obtain complex reflection

coefficient spectra *() using Fourier

transformation 9-10 in the frequency range of 10

MHz to 10 GHz.

(iii) Data Analysis:

As explained earlier the reflection coefficient is

related to dielectric response of a sample under

study for the frequency range 10 MHz to 10 GHz

in terms of complex permittivity spectra. The

complex permittivity * can be written as ’ – i”,

where ’ is real part, is called dielectric dispersion

and it is proportional to stored energy. Where ”

is imaginary part and is called dielectric loss. The

value of is not so sensitive to ε* and it is taken

as a fixed value 3.0. A sample of complex

permittivity dispersion spectra with ’ and ” is

shown in Graph 1. This corresponds to 0.5438

mole fraction of EE at 278K.









Pag

e48

5

Graph 1: Complex permittivity dispersion spectra with’ and ” corresponds to 0.5438 mole fraction of

EE at 278K

The frequency dependent complex permittivity

ε* = ε'-iε'' data was obtained by using

dielectric parameters of raw data. The static

dielectric constant (s) and relaxation time () for

the mixture obtained by using Havriliak-Negami

equation 2 11.

1)(1

)(*

ti

s

------ (2)

Where s is the static dielectric constant, is the

high frequency dielectric constant, is the

angular frequency and is the relaxation time,

and are the experimental parameters for the

distribution of relaxation time. While fitting the

data, equation 2 includes Cole-Cole (=1) 12,

Davidson–Cole (=0) 13 and Debye ((=0, =1) 14

relaxation models.

RESULTS

The Physical properties of pure liquids DMF and EE used for calculation are listed in Table 1.

Table 1: Literature values of Physical properties of DMF and EE

Name Mol. Formula Mol. Wt. Density

gcm-3

R I (nD) Dipole

moment µ

DMF C3H7NO 73.094 0.944255 1.430520 3.86 D

EE C4H10O2 90.121 0.925235 1.405425 2.19 D

Source: Hand book of Chemistry and Physics David R Lide 84th Edition CRC Press

(i) Permittivity and Relaxation Time:

The evaluated values of s, as a function of mole fraction of EE at four different temperatures are given

in Table 2. A graphical representation of s, and is shown in Graphs 2 and 3 respectively.









Pag

e48

6

Table 2: Temperature dependent dielectric parameters for binary mixture of DMF+EE

Mole Fraction of EE 278K 288K 298K 308K

s (ps) s (ps) s

(ps)

s

(ps)

0.0000 45.0 25.2 41.1 19.3 39.4 11.1 34.7 8.4

0.1658 38.8 30.2 35.3 25.3 34.3 22.1 32.1 16.8

0.3463 33.2 40.4 32.2 34.5 30.7 30.2 27.1 27.3

0.4428 25.2 64.7 21.8 60.3 19.2 57.0 17.8 50.1

0.5438 29.4 58.6 4.4 54.4 22.7 48.6 19.0 46.6

0.7607 38.6 56.4 34.5 48.9 27.7 41.9 23.5 36.2

1.0000 17.5 55.8 16.5 46.5 15.7 38.6 14.2 34.5

Graph 2: Variation of static dielectric constant (s) as a function of mole fraction of EE at different

temperatures.

Graph 3: Variation of static relaxation time (s) as a function of mole fraction of EE at different

temperatures.









Pag

e48

7

(ii) Excess Permittivity and Excess Inverse

Relaxation Time:

The excess permittivity, E

s at various

concentrations is calculated using the equation 3.

E

s = (s)m – [(s)A XA + (s)B XB] -------(3)

Where X is the mole fraction and subscripts m, A

and B represent the binary mixtures and

components DMF and EE of the binary mixture

respectively.

The excess permittivity provides qualitative

information about structural aspects of the

mixture.

If E

s = 0 indicates the liquids A and B do not

interact at all.

If E

s < 0 indicates the liquids A and B interact in

such a way that the total effective dipole gets

reduced. This shows that liquids A and B may form

multimers leading less effective dipoles or there

is a tendency of dipoles to align in anti-parallel

direction.

If E

s > 0 indicates the liquids A and B interact in

such a way that the total effective dipole moment

increases. There is a tendency of dipole aligned in

parallel direction.

The excess parameter

E

1

at various

concentrations is calculated using the Equation 4.

(1/)E = (1/)m – [(1/)A XA + (1/)B XB] --------(4)

Where X is the mole fraction and subscripts m, A

and B represent the binary mixtures and

components DMF and EE of the binary mixture

respectively.

It provides the qualitative information about the

formation of multimers and monomers in the

following way –

(1/E= 0: Indicates there was no change in the

dynamics of liquids A and B.

(1/0: Indicates liquids A and B interaction

produced a field such that the effective dipole

rotated slowly.

(1/0: Indicates liquids A and B interaction

produced a field such that the effective dipoles

rotated rapidly.

The evaluated E

s and

E

1

values of the DMF-

EE binary mixture are given in Table 3.

Table 3: Excess permittivity and excess inverse relaxation time (1/)E of DMF+EE

Mole Fraction

of EE

278K 288K 298K 308K

(1/)E

(1/)E

(1/)E

(1/)E

0 0 0.0000 0 0.0000 0 0.0000 0 0.0000

0.1658 -1.6419 -0.0030 -1.7225 -0.0073 -1.172 -0.0342 0.7979 -0.0446

0.3463 -2.2760 -0.0074 -0.3803 -0.0123 -0.492 -0.0347 -0.5 -0.0512

0.4428 -7.6227 -0.0136 -8.4068 -0.0218 -9.705 -0.0441 -7.822 -0.0592

0.5438 -0.6451 -0.0108 -23.322 -0.0169 -3.812 -0.0346 -4.552 -0.0486

0.7607 14.5190 -0.0054 12.1133 -0.0083 6.3287 -0.0174 4.3944 -0.0229

1 0 0.0000 0 0.0000 0 0.0000 0 0.0000

A graphical representation of excess permittivity and excess inverse relaxation time against mole fraction

of EE for four different temperatures are shown in Graphs 4 and 5 respectively.

E

s

E

sE

sE

sE

s









Pag

e48

8

Graph 4: Variation of Excess permittivity SE as a function of mole fraction of EE at different

temperatures

Graph 5: Variation of excess inverse relaxation time (1/)E as a function of mole fraction of

Ethoxy Ethanol at different temperatures.

(iii) Bruggeman Factor (fb):

The Bruggeman mixture formula can be used as

first evidence of molecular interactions in binary

mixture A and B respectively 15. The equation for

Bruggeman factor (fB) is given by

Vfm

A

BA

Bm

B

1

31

0

0

00

00

---------- (5)

According to equation (5) a linear relation is

expected between Bruggeman factor and mole

fraction of EE. Any deviation from this linear

relation indicates existence of intermolecular









Pag

e48

9

interactions. Many similar observations have

been reported in 16-17. The evaluated values of

Bruggeman factor fB for binary mixture DMF+EE

at four different temperatures are given in Table

4.

Table 4: Bruggeman Factor, fB of DMF+EE

Mole Fraction

of EE

fB

278K 288K 298K 308K Ideal

0.0000 1 1 1 1 1

0.1658 0.81378 0.804 0.82192 0.8961 0.8342

0.3463 0.63182 0.6923 0.6878 0.6833 0.6537

0.4428 0.3397 0.2662 0.18767 0.2194 0.5572

0.7607 0.80753 0.7757 0.56943 0.5166 0.2393

1.0000 0 0 0 0 0

A graphical representation of fB against mole fraction is shown in Graph 6.

Graph 6: Variation of Bruggeman Factor fB as a function of mole fraction of Ethoxy Ethanol at different

temperatures.

(iv) Effective Kirkwood Correlation Factor (geff):

The structural information about the liquids from

the dielectric relaxation parameter may be

obtained using the effective Kirkwood correlation

factor, geff 17. This factor is also a parameter for

obtaining information regarding orientation of

electric dipoles in polar liquids.

The Kirkwood equation for the binary mixture can

be expressed as 18-19

2

22

2

24

msm

msmmsmeff

B

B

BBA

A

AA gXM

XMKT

N

-- (6)

Where XA and XB are mole fractions liquids A and

B respectively, s is static permittivity, is

permittivity at high frequency, is dipole

moment, is density at temperature T, M is

molecular weight, K is Boltzmann’s constant, N is

Avogadro’s number. The values of geff are

evaluated for different concentrations at









Pag

e49

0

different temperatures using equation 6. The

evaluated values of geff as a function of mole

fraction of EE at four different temperatures are

given in Table 5.

Table 5: Kirkwood Correlation Factor geff of DMF + EE

Mole

fraction

Of EE

geff

278K 288K 298K 308K

0.0000 1.27 1.20 1.19 1.08

0.1658 1.25 1.18 1.18 1.14

0.3463 1.27 1.27 1.25 1.14

0.4428 1.06 0.94 0.85 0.81

0.5438 1.40 0.15 1.15 0.98

0.7607 2.57 2.37 1.95 1.70

1.0000 1.92 1.87 1.83 1.70

The graphical representation of variation of geff with mole fraction of EE are given in Graph 7.

Graph 7: Variation of Kirkwood correlation factor geff as a function of mole fraction of Ethoxy Ethanol

at different temperatures.

(v) Arrhenius Plot:

The thermodynamic parameters like molar

enthalpy of activation H* and molar entropy of

activation S* are calculated by Eyring’s rate

equation 7 using least square fit method. 20-21

RTG

KT

h *

exp

--------- (7)

Where G* is molar free activation energy,

*** STHG ------ (8)

Equation 7 can also be written as

RT

STH

K

hT

**

lnln

RT

H

R

S

K

hT

**

ln)ln(

------- (9)

The slope of the linear plot between ln(T) and

(1/T) gives

R

H

.









Pag

e49

1

Using values of molar enthalpy H* and molar

entropy S*, molar free activation energy, G*

can be calculated using equation 8. The evaluated

values of ln(T) and (1/T) are tabulated in table 6

and the graphical representation of Arrhenius

plot is shown in Graph 8.

Table 6: Arrhenius Plot for DMF + EE

T in K

1000/T

K-1

ln(T)

Mole Fraction of EE

0 0.1658 0.3463 0.4428 0.5438 0.7607 1.000

278 3.597 -18.777 -18.596 -18.305 -17.897 -17.933 -17.971 -17.980

288 3.472 -19.130 -18.737 -18.427 -17.869 -17.972 -18.078 -18.130

298 3.356 -19.420 -18.838 -18.526 -17.891 -18.050 -18.199 -18.280

308 3.247 -19.770 -19.080 -18.590 -17.99 -18.060 -18.310 -18.360

Graph 8: Arrhenius plot to calculate thermodynamic parameters for different concentrations.

From Graph 8 linear nature of Arrhenius plot

shows that equivalent changes in values of molar

enthalpy of activation H in the observed

temperature range from 278 K to 308 K. The slope

of Arrhenius plot changes with concentration,

which shows the change in activation energy of

the system.

6. Molar Enthalpy and Molar Entropy:

The evaluated values of molar enthalpy and molar

entropy from Arrhenius plot for different molar

concentrations are tabulated in Table 7.









Pag

e49

2

Table 7: Change in Enthalpy and Entropy of DMF + EE

Mole

Fraction of

EE

ΔH in KJ ΔS in KJ

0.0000 24.941395 0.047927

0.1658 11.000624 -0.003533

0.3463 6.908211 -0.020428

0.4428 2.0059208 -0.041773

0.5438 3.2762430 -0.036662

0.7607 8.1292074 -0.018940

1.0000 9.1884923 -0.014922

The graphical representation of molar enthalpy and molar entropy are shown in Graph 9.

Graph 9: Variation of molar enthalpy and molar entropy as a function of mole fractions (EE).

DISCUSSION

From Graph 2, the nonlinear behavior of these

parameters confirms the H-Bond complex

function over the entire concentration range of

DMF-EE mixture.

The maximum decrease in s value is observed

when mole fraction of EE is 0.4428 reveals that

dipole interaction is minimum for all observed

temperatures 278, 288, 298 and 308K.This

suggests that weak intermolecular interaction

due to shielded charge distribution in DMF. The

maximum value of s observed for temperatures

278, 298 and 308K when mole fraction of EE is

0.7607 reveals the dipole interaction between

DMF-EE binary mixtures is maximum.

From graph 3, Relaxation time is found to be

maximum for all temperatures when mole

fraction of EE is 0.4428.

The Excess parameters related to s and provide

valuable information regarding interaction

between polar-polar liquid mixtures. It also gives









Pag

e49

3

the information of formation of multimers in the

mixture due to intermolecular interaction.

From Graph 4 it is observed that the maximum

negative value of ES when mole fraction of EE is

0.4428 for all observed temperatures. It

represents that effective value of dipole moment

is get reduced due to anti-parallel alignments of

dipoles in the mixture. Maximum negative value

of ES at 288K confirms the formation of strong

H-bond hetero molecular interactions in the

mixture and these interactions are relatively

stronger compare to other observed

temperatures. Excess permittivity SE is maximum

positive value is observed when mole fraction of

EE is 0.7607. It indicates the molecular interaction

of DMF & EE increases the effective dipole

moment. There is a tendency of dipole aligned in

parallel direction also it is maximum at

temperature 278K. As temperature increases

alignment of dipoles in parallel direction

decreases.

From Graph 5 it is observed that

E

1

is negative

for entire range of concentration and for all

observed temperatures. The negative value of

inverse relaxation time indicates molecular

interaction produced hindering field making

effective dipole rotation slowly. The maximum

negative value is observed when mole fraction of

EE is 0.4428 for all observed temperatures.

It is observed in Graph 6, that there is a non-linear

relationship between fB and mole fraction. The

Bruggeman factor shows a positive deviation

throughout from the ideal value. Maximum

deviation from ideal value is observed when mole

fraction of EE is 0.7607. This indicates the

existence of inter molecular interaction in the

mixture.

According to Graph 7 the values of geff are greater

than unity for all temperatures shows parallel

orientation of electric dipoles. The maximum geff

value is observed for all temperatures when mole

fraction of EE is 0.7607 shows maximum inter

molecular interaction within the system.

From graph 8 linear nature of Arrhenius plot

shows that equivalent changes in values of molar

enthalpy of activation H in the observed

temperature range from 278 K to 308 K. The slope

of Arrhenius plot changes with concentration,

which shows the change in activation energy of

the system.

It is observed that molar enthalpy of activation H

decreases till mole fraction of EE is 0.4428 and

then it increases. It means less energy is needed

for group of dipole reorientation up to mole

fraction of EE 0.4428. Negative value of entropy

S for all mole fractions of EE except zero value of

EE confirms relatively high ordered arrangement

of molecules in the system.

CONCLUSION

The dielectric parameters, Kirkwood correlation

factor, Bruggeman factor, thermo dynamic

parameters have been reported for DMF-EE

binary mixture for different concentrations at

various temperatures. The relaxation time of the

mixture is found to be higher for mole fraction of

EE is 0.4428. The maximum negative value of

excess relaxation time

E

1

indicates the slower

rotation of effective dipoles of the system. The

more deviation of Bruggeman factor from its ideal

value for mole fraction of EE 0.7607 shows more

energy is needed for group dipole reorientation.

REFERENCES 1. G.M. Dharne, A.P. Maharolkar, S.S.Patil, P.W.Khirade

and S.C. Mehrotra. IJPBS V1 (2), (2010).

2. R.J. Sengwa, Sonu Sankhela, Vinita Khatri. J. Mol. Liq.,

151, 17-22, (2010).









Pag

e49

4

3. R.H. Fattepur, M.T. Hosmani, D.K. Deshpande, R.L. Patil

and S.C. Mehrotra. Pramana, Vol. 44 No.1, 33-44,

(1995).

4. B.Gestblom and J. Sjoblom, J. Acta. Chem. Scan. A38,

575, (1984).

5. Prabhakar Undre, S.N.Helambe, S.B.Jagadale,

P.W.Khirade & S.C.Mehrotra, Pramana J.Phys. Vol. 68,

No 5, 851-861, (2007).

6. P.W. Khirade, A.S. Chaudhari, J.B. Shinde, S.N. Helambe

and S.C. Mehrotra, J. Soln, Chem. 28 (8), 1031-1043,

(1999).

7. V.P. Pawar and S.C. Mehrotra, J. Mol. Liq., 108/1-3, 95-

105, (2003).

8. K. Dharmalingam, K. Ramachandran, P. Shivgurunathan,

B. Prabhakar Undre, P.W. Khirade, S.C. Mehrotra., J.

Appl. Polym. Sci. Vol. 107 No.4, 2312-2316, (2008).

9. C.E. Shanon, Proc. IRE, 37, 10, (1949).

10. H.A. Samulan, Proc. IRE, 39, 175, (1951).

11. S. Havriliak and S.Negami, J. Polym, Sci. C14, 99, (1966).

12. K.S. Cole, R.H. Cole, J. Chem Phys. 9, 341, (1941).

13. D.W. Davidson, R.H.Cole, J. Chem. Phys. 18, 1484,

(1950).

14. P. Debye, Polar molecules, Chem. Catalog Co., New

York, (1929).

15. D.A.G. Bruggeman, Ann. Phys. (Leipzig) 5, 636, (1935).

16. S.M. Puranik, A.C. Khumbharakhane and S.C. Mehrotra

J. Mol. Liq. 59, 173, (1993).

17. M.I. Aralaguppi, T.M. Aminbhavi, R.H. Balundugi and

S.S. Joshi, J. Phys. Chem. 95, 5299, (1991).

18. A.C. Kumbharakhane, S.M. Puranik and S.C. Mehrotra,

J. Mol. Liq. 51, 261, (1992).

19. S.S. Dubal, S.B. Sayyad, S.S. Patil, P.W. Khirade, E-I. Sci.

J. V (3) Issue-3, 2094-1749, (2012).

20. S. Glasstone, K.J. Laidler and H. Eyring, Theory of rate

Process, Mc Graw-Hill, New York, 541, (1941).

21. R.H. Fattepur, S.B. Sayyad, N.H. Ayachit. P.W. Khirade

and S.C. Mehrotra. Int.M.S. 9, (2010).

*Corresponding Author: Email:[email protected] Telephone:+91 9480702543








Research Article

Biological Sciences


Anirban Mullick*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e50

4

IN-VITRO ASSAY OF ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY OF LEAF EXTRACT AND

LEAF DERIVED CALLUS EXTRACT OF ACALYPHA INDICA L.

ANIRBAN MULLICK1, SUNANDA MANDAL1, RITUPARNA BHATTACHARJEE1

AND ANINDITA BANERJEE*

Department of Microbiology

St. Xavier’s College, 30 Mother Teresa Sarani, Kolkata-700016. West Bengal, India. 1 The authors contributed equally to the work

*Corresponding Author Email: [email protected]

ABSTRACT Acalypha indica (Indian Acalypha), commonly known as Mukhtajhuri in West Bengal is an important medicinal plant

of India. The leaves are known to possess antimicrobial and antifungal properties. Leaf decoction is said to have

anti-inflammatory properties. The present study shows the presence of antioxidant and antibacterial properties of

Acalypha indica leaf extract and leaf-derived callus extract. Antioxidant activity was studied through DPPH assay.

Whereas, in vitro antibacterial study of the extracts were carried out by adopting agar well diffusion technique using

the pathogens Escherichia coli (E. coli), Klebsiella sp, Salmonella sp, Bacillus sp. After 24 hours of incubation

maximum zone of inhibition was found against Bacillus sp. for leaf extract and Salmonella sp. for callus extract.

These activities may be due to presence of flavonoids, phenolic compounds and other secondary metabolites present

in these extracts.

KEY WORDS Acalypha indica, Antibacterial, Callus extract, Leaf extract, Radical Scavenging Activity.

INTRODUCTION

Acalypha indica, a common weed in many parts

of Asia including of India, Bangladesh, Pakistan,

Sri Lanka as well as tropical Africa and South

America [1] belongs to the family Euphorbiaceae.

In Ayurveda, it is known as “Kuppi”,

“Muktavarchaa” or “Haritamanjari”. Previous

reports have also shown that Acalypha indica (A.

indica) contains acalyphine which is used in the

treatment of sore gums and to have a post-coital

antifertility effect [2], anti-venom properties [3],

and wound healing effects [4], antioxidant activity

[5], anti-inflammatory effects [6], acaricidal

effects [7], diuretic effects [8] and antimicrobial

activity [9]. The roots of Acalypha indica is used as

laxative and leaves for scabies and others

cutaneous diseases [10]. The leaf sap is used to

treat wounds [11] as well as eye and skin

infections. Historically these plants have provided

a good source of anti-infective agents in

treatment of asthma [12] and pneumonia and so

have increasingly gained importance during

recent years. Moreover, antioxidant compounds

[13] like phenolic acids, polyphenols and

flavonoids [14] present in this plant extract,

scavenge free radicals such as peroxide,

hydroperoxide or lipid peroxyl and inhibit the

oxidative mechanisms that lead to degenerative

diseases [15]. This weed also has antimicrobial

property. Previous reports have shown flavonoids

[16] (kaempferol glycosides mauritianin, clitorin,

nicotiflorin and biorobin) and secondary










Pag

e50

5

metabolites present in leaves and flowers of this

medicinal plant can be efficiently used in

treatment of diseases caused by antibiotics

resistant strains of bacteria. For these reasons,

various parts of this plant are utilized in

production of valuable drugs to cure nosocomial

infections of Staphylococcus sp [17]. Moreover, in

vitro production of callus from somatic plant

tissues can be helpful in future drugs production

for infectious diseases. So, this has prompted us

to investigate antioxidant and antibacterial

activity of leaf and callus derived from the leaves

of this plant.

MATERIALS AND METHOD

Acalypha indica used for sample preparation

Figure 1: Collection of whole plant of Acalypha

indica from Barisha in South Kolkata, West

Bengal, India.

(i) Plant material:

The whole plant of A.indica (Figure 1) was

collected from the adjoining areas of Barisha

(22.470N, 88.310E), South Kolkata locality and was

used in preparation of leaf extract, callus

induction and preparation callus extract.

(ii) Callus Induction:

The leaf explants were washed with sterile water.

Then the leaves were dipped in 1% sodium

hypochlorite solution for 30 minutes, followed by

rinsing twice with sterile water. The leaves were

washed with 0.1% mercuric chloride solution for

30 seconds followed by rinsing twice with sterile

water. Finally, the leaves were washed with

fungicide Bavistin for 45 seconds followed by

rinsing with sterile water.

(iii) Callus Production:

The surface sterilized leaves were trimmed and

placed on Murashige and Skoog’s (MS) medium

(HiMedia, Mumbai) supplemented with 5µM 2,4-

dichlorophenoxyaceticacid (2,4-D) and kept in

culture rack at 22-250C temperature and

alternative light for 16 hours and dark for 8 hours

at Relative Humidity maintained at 60-70%. The

complete formation of calli was obtained in 14

days and was allowed to propagate for 3 months

(Figure 2) before taken for extraction.

Completely formed Callus

Figure 2: Induction of leaf-derived callus in MS

medium supplemented with 5µM 2,4-D.

(iv) Preparation of leaf and callus extracts:

The air dried leaves were crushed with mortar

and pestle and were extracted with methanol for

three consecutive days at room temperature. The

methanolic extract was evaporated to dryness

and the crude residue left behind was weighed

down. The residue was named as methanolic

extract (LME).

The callus obtained was also air dried, crushed,

extracted using methanol and filtered. The filtrate

was evaporated and dissolved in methanol to

obtain callus methanolic extract (CME).









Pag

e50

6

(v) Determination of total phenolic content:

The extracts were dissolved in methanol to obtain

a concentration of 1mg/ml. 100µl of these

solutions were taken in a test tube and to it 100µl

of 50% Folin Ciocalteau (Merck Specialist Pvt. Ltd)

reagent was added. The mixture was then

incubated for 3 minutes at room temperature and

2ml of 2% sodium carbonate solution was added.

The volume was made up to 3ml with double

distilled water [18]. The mixture was kept for 1

minute in water bath at 1000C and allowed to cool

in dark. The absorbance of the samples was noted

at 720 nm using a UV-Vis spectrophotometer. The

total phenolic content of LME and CME was

calculated from standard curve of Gallic acid

1mg/ml.

(iv) Determination of total flavonoid:

To determine total flavonoid content, 150 μl of

5% NaNO2 was added to 20 μl of LME and CME (1

mg/ml) and was incubated at room temperature

for 5 minutes, followed by the addition of 2.5ml

of 10% AlCl3 solution and incubated further for 6

minutes at room temperature [19]. The

absorbance of the samples were noted at 510 nm.

The total flavonoids contents of LME and CME

was calculated from standard curve of quercetin

1mg/ml.

(vi) Determination of Radical Scavenging

Activity:

Antiradical activity was measured by a decrease

in absorbance at 517 nm of DPPH (2,2-Diphenyl-

1-Picrylhydrazyl) solution [20] brought about by

plant extracts. In this assay DPPH acts as an

indicator for “Radical Scavenging Activity” and

changes its deep violet colour to colourless or

pale yellow in presence of antioxidant and help us

to determine Radical Scavenging Activity (RSC) of

the substances. Therefore, to determine RSC of

the extracts a stock solution of DPPH (0.12 mg/ml)

was prepared in methanol and the samples

mixture taken in the test tubes was of 2ml out of

which LME and CME were present in varied

amount (5µl, 10µl, 20µl) as per the concentration

and rest was methanol (1995µl, 1990µl, 1980µl)

.Then to these test tubes, 1ml of DPPH solution

was added to achieve the final volume of 3ml and

kept for 20 minutes incubation in dark. After 20

minutes of incubation in dark the absorbance was

measured at 517 nm. Decrease in the absorbance

of the DPPH solution indicates an increase of the

DPPH antioxidant activity and percentage of

Radical Scavenging Activity (% RSC) was

calculated from the following equation:

% RSC = (Ao−As)

Ao x 100

(Ao = DPPH solution without the sample, As=

DPPH solution with the sample.)

(vii) Determination of Antibacterial Activity:

Antibacterial property of A. indica was

determined using Kirby Bauer method. The

antibacterial activity was studied by spreading

100 µl of 24 hours old culture of (Bacillus sp., E.

coli, Salmonella sp. and Klebsiella sp.) on Mueller

Hinton Agar (HiMedia,Mumbai) and 100 µl of LME

and CME were loaded in the wells of these plates

and their zone of inhibitions were calculated in

millimeter (mm) after 24 hours of incubation at

370C and the zone of inhibitions were compared

with methanol as a negative control [21].

RESULTS

1. Calculation of total phenolic and flavonoid

content of extracts:

Total phenolic and flavonoid content of LME and

CME are summarized in Table1. Total phenolic

concentration was found out using Gallic acid

(GAE) as standard. Total phenolic content for LME

was found out to be 26.6 ± 2.5mg GAE/g of

methanol extract. In the same way, total flavonid

concentration was found out using Quercetin

(QE) as standard. The total flavonoid content LME









Pag

e50

7

was found to be 21.5 mg Quercetin/g of extract.

Whereas, the total flavonoid content of CME was

found to be 19.0 ± 3 mg/g of extract.

Table 1: Total phenolic and flavonoid content of extracts:

Sample Total phenolic content (mg

GAE± 2.5/g of extract)

Total flavonoid content

(mg QE± 3/g of extract)

LME 26.60 ± 2.5 21.50 ± 3

CME 26.00 ± 2.5 19.0± 3

2. Calculation of Radical scavenging activity:

Percentage of RSC (Table2) was determined by

DPPH assay at different concentrations for the

extracts of which 250µg of LME showed

maximum 43.57 % of RSC. Whereas, same

concentration of CME had only 8.65% RSC.

Table 2: Percentage of Radical scavenging activity

Sample Concentration (µg) % RSC

LME 250 43.57

CME 250 8.65

3. Antibiotic sensitivity test:

Antibacterial test was done by measuring the

diameter of the zone of inhibition (Table 3) by all

the extracts on the test organisms and the

antibacterial activity was classified [22] into the

following types of test organism(s):

>12 mm zone of inhibition is high sensitivity

9-12 mm zone of inhibition is moderate

sensitivity

6-9 mm zone of inhibition is less sensitivity and

< 6 mm zone of inhibition is resistant.

Thus, from the classification it was seen that

Bacillus sp. was maximum sensitive to LME on

with zone of inhibition 17±0.5 mm followed by

Salmonella sp with diameter of zone of inhibition

15±0.5 mm. Whereas, E. coli has shown moderate

sensitivity with diameter of zone of inhibition

10.06 ± 0.4mm in LME. The assay has also shown

Klebsiella sp. was resistant to LME with diameter

of zone of inhibition 3±0.2mm. This assay has also

shown Salmonella sp. was less sensitive to CME

with zone of inhibition of 8±0.2 mm. Whereas,

Klebsiella sp., E. coli and Bacillus sp. was found to

be resistant to CME with zone of inhibitions 2±0.3

mm,2±0.2 mm and 2.3±0.2 mm respectively.

Table 3: Antibiotic sensitivity of test organisms:

DISCUSSIONS

Test Organism Zone of inhibition(mm)

LME CME

Klebsiella sp. 3.3± 0.2 2.0± 0.3

E. coli 10.06± 0.4 2.0± 0.2

Bacillus sp. 17.00± 0.5 2.3± 0.2

Salmonella sp. 15.00± 0.5 8.0± 0.2









Pag

e50

8

In recent times due to beneficial effect of

Acalypha indica researchers have gained interest

in studying the antioxidant activity of this type of

medicinal plant extracts to treat degenerative

diseases [23] because antioxidant compounds of

these medicinal plants may acts as radical

scavengers when added to the food products and

prevent the radical chain reaction of oxidation

and increase shelf life by retarding the processes

of lipid peroxidation [24]. This antioxidant activity

may be due to presence of phytochemicals like

phenolics and flavonoid compounds. In this

investigation we have also found that all the

extracts have significant phenolic and flavonoid

compounds in them. The result has also shown

LME has maximum phenolic and flavonoid

contents, compared to CME. So, due to presence

of maximum phytochemical compounds, 250 µg

of LME has shown 43.57% Radical scavenging

activity and may be due to presence of less

phytochemical compounds, 250 µg of CME has

shown 8.65% of Radical scavenging activity.

Previous reports have also stated that

Euphorbiaceae [25] showed antimicrobial activity

due to presence of high concentration of different

compounds like flavonoids, phenols and alkaloids

in it. This may be the reason in our investigation

we have found out both extracts have

antibacterial property against many common

pathogens. Maximum antibacterial property is

shown by LME against Bacillus sp. (zone of

inhibition 17±0.5 mm). This extract has also

shown highly sensitivity to Salmonella sp. (zone of

inhibition 15±0.5mm), moderate sensitive to E.

coli (zone of inhibition 10.06±0.4mm) and

resistant to Klebsiella sp. (zone of inhibition

3.3±0.2 mm).Whereas on the other hand, CME

has shown only less sensitivity to Gram negative

Salmonella sp. (zone of inhibition 8±0.2 mm) and

resistant to all other test microorganisms. From

the different zone of inhibitions, we can conclude

that the maximum antibacterial activity of LME

may be due to the presence of maximum phenolic

and flavonoid content in it. Our maximum

antibacterial activity of LME towards Gram

positive Bacillus sp. may be due to difference in

cell wall compositions compared to Gram

negative bacteria. This fact can be checked by

employing more bacterial strains to find out the

actual reason behind it. Moreover, various

reports [26, 27] have already shown entrances of

antibiotics through cell envelope (the outer and

cytoplasmic membrane) are highly efficient for

Gram positive bacteria depending on reaction

with the protein layer (mucopolysaccharides or

peptidoglycans). Moreover, the factors

responsible for more sensitivity of the leaf extract

towards bacteria are not exactly known but may

be the presence of soluble secondary plant

metabolites in LME. Hence, the present study

showed both LME and CME have antioxidant and

antibacterial property but LME has a potential to

be a better antioxidant and antimicrobial agent in

future.

CONCLUSION

This is first time a comparison is made on

antioxidant and antibacterial activity of leaf

extract (LME) and callus extract (CME). However,

CME did not show any significant antioxidant and

antibacterial activity even at high doses. So,

further studies are required to increase the

antioxidant and antibacterial activity of CME and

identify the exact composition of the phenols,

flavonoids and other secondary metabolites of

both LME and CME.

ACKNOWLEDGEMENT

We express our sincere thanks to Rev. Fr. Dr. J.

Felix.Raj S.J, Principal of St. Xavier’s College

(Autonomous), Kolkata, India for his support to

carry out these experiments in the college. We

are also grateful Dr. Arup Kumar Mitra, Head

Department of Microbiology of St. Xavier’s









Pag

e50

9

College (Autonomous), Kolkata, India and other

faculty members for their help and cooperation in

this work. Lastly, we would like to extend our

sincere thanks to our laboratory attendants for

their time to time help in this work.

REFERENCES 1. Ramachandran J. Herbs of Siddha Medicine.The First

3D Book on Herbs. Murugan P Patthipagam, Chenna,

India, 156. (2008).

2. Bedon, E., G.M. Hatfield. An investigation of the

antiviral activites of Phodophyllum peltun. Lloydia,

45(6):725. (1982).

3. Shirwaikar A, Rajendran K, Bodla R, Kumar CD.

Neutralization potential of Viper russelli russelli

(Russell's viper) venom by ethanol leaf extract of

Acalypha indica. J Ethnopharmacol, 94(2-3):267-73.

(2004).

4. Suresh Reddy, J., P. Rajeswara Rao S.R.Mada. Wound

healing effects of Heliotropium indicum, Plumbago

zeylanicum and Acalypha indica in rats. J

Ethnophharmacol, 79: 249-51. (2002).

5. Ruchi, G.M., Majekodunmi of Ramia, B.V. Gouri, A.

Hussain, S.B. Suad Khamis. Anti-oxidant capacity of

some edible and wound healing plants in Oman. Food

chem, 101:465-70. (2007).

6. Mohana Vamsi, N., M. Venkata Sunil Kumar, N.

Kodandaram, Y. Padmanbha Reddy. Evalution of Anti-

inflamatory activity of Acalypha indica. Ind pharm,

7:89-91. (2008).

7. Singh, D.A.P., M. Raman, V. Saradha, P. Jayabharathi,

R.S. Kumar. Acarcidal Property of Kuppaimemeni

(Acalypha indica) against natural Psoroptes cuniculi

infestion in broiler Rabbits. Indian J Anim Sci,

74(10):1003-6. (2004).

8. Das, A.K., F.Ahmed, N.N. Biswas, S. Dev, M.M. Masud.

Diuretic Activity of Acalypha indica. Dhaka Univ J

Pharm Sci, 4:1-2. (2005).

9. M. N. Somchit, R. Abdul Rashid, A. Abdullah, A.

Zuraini, Z. A. Zakaria, M. R. Sulaiman, A. K. Arifah and

A. R. Mutalib. In vitro antimicrobial activity of leaves

of Acalypha indica Linn. (Euphorbiaceae).African

Journal of Microbiology Research Vol. 4(20). 2133-

2136 (2010).

10. Perry, L. M. Medicinal plants of East and Southeast

Asia: attributed properties and uses. MIT Press,

Cambridge. Mass. U.S.A. 109. (1980).

11. Suresh Reddy, J., P. Rajeswara Rao S.R.Mada. Wound

healing effects of Heliotropium indicum, Plumbago

zeylanicum and Acalypha indica in rats. J

Ethnophharmacol, 79: 249-51. (2002).

12. H. Panda Herbal Soaps & Detergents Hand Book

National Institute of Industrial Research Delhi. 48 –

49. (2003).

13. Isolation of potential antibacterial and antioxidant

compounds from Acalypha indica and Ocimum

basilicum. K. Ramya Durga, S. Karthikumar and K.

Jegatheesan. Journal of Medicinal Plants Research

3(10).703-706. (2009).

14. V.T.Narwade, A. A.Waghmare, A. L. Vaidya. Detection

of Flavonoids from Acalypha indica L. Journal of

Ecobiotechnology.3 (11): 05-07(2011).

15. Halliwell, B. Free radicals, antioxidants and human

disease: curiosity, cause or consequence. Lancer,

344: 721-724.(1994).

16. Nahrstedt A, Hungeling M, Petereit F. Flavonoids

from Acalypha indica. Fitoterapia. 77(6):484-

6.(2006).

17. Rubin, R.J., Harrignton, C.A., Poon, A., Dietrich, K.,

Grene, J.A. and Moiduddin, A.The economic impact

of Staphylococcus infection in New York City

hospitals. Emerging infectious diseases. 5: 9-17.

(1999).

18. Waterman, PG and Mole, S. Analysis of Phenolic Plant

Metabolites, Blackwell Scientific Publication.83-85.

(1994).

19. Jia ZS, Tang M C, Zhu XR. Study on effect of

scavenging superoxide free radical on mulberry

flavonoids. J. Zhejiang Agric. Univ., 22(5): 519-523.

(1996).

20. R.Shanmugapriya,T.Ramanathan, .Thirunavukkarasu.

Evaluation of Antioxidant Potential and Antibacterial

Activity of Acalypha indica Linn. Using in vitro model.

Asian Journal of Biomedical and Pharmaceutical

Sciences 1 (1), 18-22. (2011).

21. P. Saranraj, D. Stella, K. Sathiyaseelan and Sajani

Samuel. Antibacterial Potentiality of Ethanol and

Ethyl Acetate Extract of Acalypha indica against

Human Pathogenic Bacteria. Journal of

Ecobiotechnology 2(7): 23-27. (2010).

22. Uma Devi, P., S. Murugan, S. Suja, S. Selvi, P.

Chinnasamy, E. Vijayaanad.Antibacterial invitro lipid

per-oxidation and phytochemical observation on

Achyranthes bidentata blume. Pak. J. Nutrition, 6(5):

447-451. (2007).

23. Willcox, J.K., S.L. Ash, G.L. Catignani. Antioxidant and

prevention of chronic disease. Crit. Rev. Food Sci.

Nutrition, 44: 275-295. (2004).

24. Young, I.S. and J.V. Woodside. Antioxidants in health

and disease. J. Clinical Pathol, 54: 176-186. (2001).









Pag

e51

0

25. Falodun A, Ali S, Mohammed Quadir I, Iqbal MI,

Choudhary IMI. Phytochemical and biological

investigation of chloroform and ethylacetate

fractions of Euphorbia heterophylla leaf

(Euphorbiaceae). J. Med. Plants Res., 2(12): 365-369.

(2008).

26. Baron EJ, Peterson LR and Finegold SM. Baily & Scott's

Diagnostic Microbiology.9th ed. Mosby-Year book,

Inc., St. Louis. (1994).

27. Lennette, EH, Balows A, Hausler WJ and Shadomy HJ.

Manual of Clinical Microbiology. 4 th ed. Amer. Soc.

Microbiol., Washington. (1985).

*Corresponding Author: “Dr. Anindita Banerjee” Assistant Professor Department of Microbiology St. Xavier’s College 30 Mother Teresa Sarani Kolkata-700016 E-mail: [email protected] Telephone: +91-(033)-22551276 Fax: 2280-1927








Research Article

Biological Sciences


Suresh Sukumar*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e51

1

CT SCAN – AUTOMATIC TUBE CURRENT MODULATION

Nimma purna chander reddy1, Suresh sukumar2, Sushil Yadav3,Albin Babu M Wilson4

1Clinical SME, Apollo Health street,. 2Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal

College of Allied Health Sciences, Manipal University, Manipal 3Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal

College of Allied Health Sciences, Manipal University, Manipal. 4Lecturer. MSc. Medical Imaging Technology, Department of Medical Imaging Technology, KSHEMA,Nitte

university, mangalore. *Corresponding Author Email: [email protected]

ABSTRACT AIM OF THE STUDY: To compare the dose difference and state the significance of Automatic Tube Current

Modulation (ATCM) in abdominal MDCT examination while maintaining optimum image quality for diagnosis.

Results: The varying in mAs values according to ACS for different body counter of the patients was observed. In

group A resulted in estimations of dose saving in range of 10-27 %.In group B 8.1-36.6 %, in group C 8.3-33.4%,

group D 5.7-19 % with application of ATCM technique. The overall radiation output results shown for forty patients

as follows the least is 5.7% and highest is 36.6%.According my study statistics shows there is no correlation between

BMI and the estimated dose savings. CONCLUSSION: The use of Automatic Tube Current Modulation (angular dose

modulation and z-axis dose modulation) helps in

Radiation Dose reduction Up to 36.6% was achieved with acceptable diagnostic image quality.

Reduces over all irradiation time.

Helps in reduce stochastic and genetic effects.

Helps in Not only reduce the patient dose as well as occupational dose.

Scan duration is less compare to standard protocol so we can save the time at clinical side.

Effective in breath hold scans (Coronary Angio, Pulmonary Angio, Thorax and Abdomen).

Required dose is applied according to patient body counter.

Helps in decrease scan time in uncooperative and trauma patients.

I conclude that according to the observed results there is no requirement to

INTRODUCTION

The invention of computed tomography (CT) has

revolutionized the practice of radiology and is so

remarkable that in many cases it generates a

dramatic increase in diagnostic information in a

short duration comparing to other imaging

modalities such as MRI and ultrasonography. CT

imaging continues to be on the increase due to its

varied advantage, despite the large radiation

dose imparted to patients. Due to wide spread

use variety of geometries has been developed to

acquire transmission data in a shortest time

period &according to anatomical region such as

heart. The advancement of the MDCT makes

possible rapid volume acquisition and has opened

new diagnostic fields such as CT cardiac

angiography, virtual colonoscopy, and

bronchoscopy. Fulfilling the demands for










Pag

e51

2

effective diagnosis has led to a steady increase in

the use of CT. With this trend of increasing use of

CT department strive to scan with

ALARAprinciple. According to literature the risk of

radiation induced cancer in patients from CT

examination is not negligible¹.

Generally the radiation doses to patients are

about 30-50% greater with the use of MDCT as

due primarily to scanoverlap, positioning of the x-

ray tube closer to the patient, over beaming,

increased significance of over scanning and

possibly increased scattered radiation with wider

x-raybeams. A reduction in the radiation dose

delivered from CT has become an important issue

and various dose reduction and optimization

techniques have been formulated.Modulation of

the x-ray tube current during scanning is one

effective method of reducing the dose and the

adaption of Automatic Tube Current Modulation

(ATCM) technique should permit overall

reduction in radiation exposure. Automatic tube

current modulation in CT is analogous to the

automatic exposure control (AEC) or photo timing

technique used for automatically terminating

radiographic exposure. The techniques used are

angular (x and y-axis) and z-axis tube current

modulations. The x and y-axis modulation

involves variation in tube current as the x-ray tube

rotates about the patient, while the z-axis

modulation involves variation in tube current

along the z-axis of the patient².

The current study aims to compare radiation dose

and image quality achieved with weight-based

protocol, along with the dose modulation

software available in the machine i.e. dynamic

dose modulation (D-DOM) and z-axis dose

modulation (Z-DOM) dose modulation techniques

using a sixty-four slice, Philips Brilliance CT

scanner. The D-DOM and Z-DOM are based on the

angular and z-axis tube current modulation

respectively.

AIM OF THE STUDY

To compare the dose difference and state the

significance of Automatic Tube Current

Modulation (ATCM) in abdominal MDCT

examination while maintaining optimum image

quality for diagnosis.

REVIEW OF LITERATURE

Livingstone RS, et al in year 2009 studied426

patients on a six-slice CT scanner for comparison

of radiation dose and image quality using dose

modulation techniques and weight- based

protocol exposure parameters for biphasic

abdominal CT. The use of dose modulation

technique resulted in a reduction of 16 to 28% in

radiation dose with acceptable diagnostic

accuracy.

A reduction of current-time product of

approximately three to five percent using D-DOM

and 37 to 55% using Z-DOM was achieved for

arterial and portal venous phases compared to

the weight based protocol settings. A reduction of

approximately 30 to 50% of tube current-time

product was noted within D-DOM and Z-DOM

respectively for arterial and portal venous phases.

Yoshinori Funama, et al in year 2007

investigated64 patients with known or suspected

lung or abdominal disease about the possibility of

obtaining adequate images at uniform image

noise levels and reduced radiation exposure with

automatic tube current modulation (ATCM)

technique for 64-detector CT.

The mean image noise ranged from 8.40 at the

center of the left ventricle to 11.31 at the porta

hepatis; the mean tube current ranged from

105.9 mAs at the center of the left ventricle to

169.6 mAs at the center of the spleen. The mean

dose reduction rate per constant tube current at

175 mAs ranged from 3.1 to 39.5%. By use of the

ATCM technique, it is possible to maintain a

constant image noise level with a 64-detector CT.









Pag

e51

3

Campbell J, et al in year 2005 studied one

hundred and forty-eight consecutive chest CT

examinations (70 men, 78 women; age range, 15-

90 years) with theobjective to determine

additional radiation dose associated with

scanning beyond the anatomic limits of the thorax

in chest CT protocol and to assess the effect of z-

axis modulation on the additional radiation dose

associated with the scanning protocol.

With z-axis modulation, the mean DLP for

supraapical and infrapulmonary extra images was

39.98 mGy x cm and 132.59 mGy x cm,

respectively. With fixed tube current, the mean

DLP for supraapical and infrapulmonary extra

images was 30.31 mGy x cm and 95.91 mGy x cm,

respectively


MACHINERY FEATURES – SPECIFICATIONS

Philips (Brilliance) 64 row MDCT

Output Capacity-60KW

KV-80, 120,140

MA-A.800mA

-B. Dose modulated mA

Anode storage capacity-8MHU

Dose right ACS (Automatic current selection)

Optimizes the dose for each patient based on the

planned scan by suggesting the lowest possible

setting mAs to maintain constant image quality at

low dose throughout the exam.

Dose Right D-DOM (Dynamic Dose Modulation)

Automatically controls the tube current x and y

axis rotationally increasing the signal over areas

of higher attenuation (lateral) and decreasing

signal over area of less attenuation (AP).

Dose Right Z- DOM (Longitudinal Dose

Modulation)

Automatically controls the tube current along z

axis adjusting the signal along the length of the

scan, increasing the signal over regions of higher

attenuation (shoulder, pelvis) and decreasing the

signal over regions less attenuation(neck, legs).

Study designis a randomized prospective, blinded

study involving40 patients undergoing abdominal

CT scan, performed using a sixty-four row CT

scanner (Brilliance, Philips medical systems at

Kasturba Hospital Manipal. The tube potential,

tube current-time product, volumetric CT dose

index (CTDI vol) and dose length product (DLP)

values will be recorded. The tube potentials

available in the machine are 80kV, 120 kV and 140

kV. Various other parameters such as the total

time duration of the scan, field of view and pitch

selection will be recorded. This protocol involves

a complete examination of the region of interest

along with a topogram, spiral or sequential ranges

and reconstruction modes. The preprogrammed

scan protocols used are based on recommended

exposure factors specified by the manufacturers

as a starting point for clinicalwork. During the

course of the study, exposure parameters will be

selected according to the patient's body weights

and will be lower than the preset protocols.

An appropriate tube current time product will be

used for patients based on their body weight for

arterial and venous phases.

INCLUSION CRITERIA

Age20 to 80 years, both genders

Advised CT scan abdomenby respective

clinician for clinical condition.

EXCLUSION CRITERIA

Larger body matrix, children,

Critical patients.









Pag

e51

4

Study of forty patients will be performed on sixty-

four slice CT scanner (Brilliance, Philips medical

systems at Kasturba Medical College, Manipal) as

per the method described above.

Following procedure to enable the ACS

1. In the processing window clicked the

preference button.

2. Clicked scanner.

3. Selected dose right.

4. Selected desired dose right ACS setting:

Yes or Auto.

5. Clicked ok to accept the settings and close

the dialog box.

Creating specific protocol

We can also enable Dose Right ACS for

specific protocol

1. Clicked home on the workflow bat.

2. Clicked generate protocols.

3. Selected the abdomen protocol.

4. Selected the helix scan step advanced

tab.

5. Selected the Dose Right ACS.

6. Clicked ok.

7. Selected from generate main form age

group as adult and weight as all.

8. Selected save option :

Clicked save as to rename the protocol

before saving given name as ACS

Abdomen Protocol.

Set dose right standards

When dose right is set to yes, we have several

options for setting and altering the protocol

standards.

Selected the reference image

The patient details were entered in the console

including height and weight and ACS Abdomen

Protocol was selected, entered the length of the

scan and acquired the scano by using surview test

later axial sections are planned on scano from

diaphragm level to the Symphysis pubis level.

No of images, scan time, CTDI & DLP entered in

the data chart as DLP from standard technique

and after the scan values are taken as DLP after

applying ATCM technique and noted down

estimation of dose savings according displayed on

the console.

PROTOCOL FOR CT ABDOMEN

Patient preparation: 4 hrs fasting.

Blood parameters: Urea: 8-38,

Creatinine level: 0.6 - 1.6 mg/dl

Oral Contrast –neutral or positive

depending on clinical indications









Pag

e51

5

PROTOCOL

Scan Protocol Abdomen Helical

Patient Position Supine- Feet First

Scano PA-1800

Area Coverage Domes of diaphragm- Symphysis pubis

Scan Direction Cranio caudal

Gantry Angle NO

Breathing instructions Yes

Routine Plain + Contrast Scan

Start Location Domes of diaphragm

End Location Symphysis pubis

Slice thickness 5 mm

Increment 5 mm

kV, mAs/slice 120,250

Resolution Standard

Filter Standard (C)

Collimation 64X0.625

Rotation Time 0.75sec

FOV Varies

Matrix 512X512

Image Enhancement 0.0

Contrast(Bolus Tracking) volume and flow rate depending on clinical index

Image Quality Acceptable-

Not acceptable-

Radiation dose Plain scan with ATCM-

OBSERVATION

There were forty adult patients in our study

including 27 males and 13female.Divided into

four groups depends upon body mass index. That

is Group-A underweight with BMI less then18.5,

Group-B normal weight with BMI 18.5-24.9,

Group-Overweight with BMI 25-29.9, Group-D

obesity with BMI of 30 or greater.

In each group we studied male female ratio,

height and weight, differences between the dose

from the standard techniques and dose from the

ATCM technique and estimations of dose saving

percentage varies individual to individual due to

applied ATCM technique.

In group A

There were total seven patients 4 males, 3

females with patient’s height in range of 153-183

centimeters and weight in range of 31-61

kilograms. A difference between the dose from

the standard technique is in range of 196.8-645.1

mg y*cm and dose from the ATCM technique is in

range of 155-492mgy* was observed which

resulted in estimations of dose saving in range of

10-27 % with application of ATCM technique.

In group-B, There were total twenty two patients

16 males, 6 females with patient’s height in range

of 152-175 centimeters and weight in range of

42.5-71 kilograms. A difference between the dose









Pag

e51

6

from the standard technique is in range of 259.6-

1279.5 mg y*cm and dose from the ATCM

technique is in range of 202-1067mgy* was

observed which resulted in estimations of dose

saving in range of 8.1-36.6 % with application of

ATCM technique.

In group-C, there were total eight patients 4

males, 4 females with patient’s height in range of

131-171 centimeters and weight in range of 51-86


the standard technique is in range of 361.7-

1196.8 mg y*cm and dose from the ATCM

technique is in range of 331.6-1098mgy* was


saving in range of 8.3-33.4% with application of

ATCM technique.

In group-D, There were total three patients 3

males, no females. With patient’s height in range

of 151-165 centimeters and weight in range of 71-

82 kilograms. A difference between the dose from

the standard technique is in range of 623.5-992.5

mg y*cm and dose from the ATCM technique is in

range of 524-935.5 mg y* was observed which

resulted in estimations of dose saving in range of

5.7-19 % with application of ATCM technique.

Total

There were total forty patients 27 males, 13

females. With patient’s height in range of 131-183

centimeters and weight in range of 31-86


the standard technique is in range of 199.6-

1279.5mgy*cm and dose from the ATCM

technique is in range of 155-1098 mg y* was


saving in range of 5.7-36.6 % with application of

ATCM technique.

Results

The varying in m As values according to ACS for

different body counter of the patients was

observed.

In group A resulted in estimations of dose saving

in range of 10-27 %.

In group B 8.1-36.6 %, in group C 8.3-33.4%, group

D 5.7-19 % with application of ATCM technique.

The overall radiation output results shown for

forty patients as follows the least is 5.7% and

highest is 36.6%.

According my study statistics shows there is no

correlation between BMI and the estimated dose

savings.

DISSCUSSION

The standard protocol setting is giving same

radiation dose output for every patient who is

going for CT scan examination. There are various

factors being used for CT image acquisition like

mA, KVP, m As(1dr roshan Livingstone) and they

are fixed in standard protocol. These fixed factors

should not be used in every patient as they have

different body habitus and body contours(synthia

teaching manual).Fixed factors not only degrade

image quality in the case of obese patients but are

also giving excess radiation dose in the case of

thin patients(chap bell).

Image quality is mainly depends upon number of

transmitted photons which are detected by the

detector (teaching manual). Decrease in photon

number lead to statistical noise that is due to

quantum mottle. This can be overcome in two

ways that is by increasing the mA or increasing the

mAs. But according to justification of laws

increase in exposure time results in increasing

patient dose twice as well as increases scanning

time for the particular region(synthia teaching

manual). So the other option left is to change the

mA to get the optimum image quality and

decrease the radiation dose at the same time

(denis tack).

In modern modalitiesmanufacturers’

implemented Automatic Exposure Control system

(AEC), which permit empirical adjustment of

radiological technique factors according to size of

the patient (from dr roshan 13). According to

them we should enter weight and height of the









Pag

e51

7

patient in control console but my study results

shown there is no correlation when we compare

BMI with the estimation of dose saving

percentage. According to my study the output

results shown dose reduction not depending

upon weight and height of the patient its upon

the tissue characteristics of the patient tissues

attenuation. As I noticed without entering height

and weight of the patient details in console can

lead to dose reduction.

According to previous referred study ATCM

technique may fail to control the tube current if a

pitch value larger than 1.0 is adopted.They found

that the image noise in overweight and obese

patients was significantly higher than that in

normal weight and underweight patients,

although with the ATCM technique it was possible

to maintain an almost constant image noise level

in these individuals.

The use of Automatic Tube Current Modulation

(angular dose modulation and z-axis dose

modulation) helps in


Helps in reduce stochastic and genetic

effects.

Helps in Not only reduce the patient dose

as well as occupational dose.

Scan duration is less compare to

standard protocol so we can save the

time at clinical side.

Effective in breath hold scans (Coronary

Angio, Pulmonary Angio, Thorax and

Abdomen).

Required dose is applied according to

patient body counter.

Helps in decrease scan time in

uncooperative and trauma patients.

I conclude that according to the

observed results there is no requirement

to enter weight and height of the patient

in the console so that we can save the

time especially in the case of bedside

patients very useful.

CONCLUSSION

The use of Automatic Tube Current Modulation

(angular dose modulation and z-axis dose

modulation) helps in

Radiation Dose reduction Up to 36.6%

was achieved with acceptable diagnostic

image quality.


Helps in reduce stochastic and genetic

effects.

Helps in Not only reduce the patient dose

as well as occupational dose.

Scan duration is less compare to

standard protocol so we can save the

time at clinical side.

Effective in breath hold scans (Coronary

Angio, Pulmonary Angio, Thorax and

Abdomen).

Required dose is applied according to

patient body counter.

Helps in decrease scan time in

uncooperative and trauma patients.

I conclude that according to the

observed results there is no requirement

to enter weight and height of the patient

in the console so that we can save the

time especially in the case of bedside

patients very useful.

BIBILOGRAPHY 1. A Comparison of radiation doses using weight-based

protocol and dose modulation techniques for patients

undergoing biphasic abdominal computed tomography

examinations. Livingstone RS, Dinakaran PM, Cherian

RS, Eapen A. Journal, The official journal of Association

of Medical Physicists of India,Year-2009, Volume-

34,issue- 4,page- 217-222.

2. Automatic tube current modulation technique for

multidetector CT, it is effective with a 64-detector CT?

Yoshinori Funama , Kazuo Awai, Masahiro Hatemura,

Masamitchi Shimamura, Yumi Yanaga, Seitaro Oda and









Pag

e51

8

Yasuyuki Yamashita. Journal, Radiological physics and

technology, publisher springer Japan.Year-january2008,

volume-1, number-1, pages- 33-37.

3. Scanning beyond anatomic limits of the thorax in chest

CT: findings, radiation dose, and automatic tube current

modulation. AJR Am J Roentgenoel Campbell J, Kalra

Mk, Rizzo S, Maher MM, Sheppard JA.Year-Dec2005,

volume-185, number-6, pages- 1525-30.

4. Mulkens TH, Bellinck P, Baeyaert M, Ghysen D, Van Dijck

X, Mussen E, et al. Use of an automatic exposure control

mechanism for dose optimisation in multidetectorrow

CT examinations: Clinical evaluation. Radiology

2005;237:21323.

5. Nakayama Y, Awai K, Funama Y, Hatemura M, Imuta M,

Nakaura T, et al. Abdominal CT with low tube voltage:

Preliminary observations about radiation dose,contrast

enhancement, image quality, and noise. Radiology

2005;237:945-51.

6. Brenner DJ, Hall EJ. Computed tomography: An

increasing source of radiation exposure. N Engl J Med

2007;357:2277-784.

7. Rehani MM, Berry M. Radiation doses in computed

tomography: The increasing doses of radiation need to

be controlled. BMJ 2000;320:593-4.

8. McCollough CH, Zink FE. Performance evaluation of a

multi-slice CT system. Med Phys 1999; 26:2223-30.

9. Hidajat N, Maurer J, Schroder RJ, Wolf M, Vogl T, Felix

R. Radiation exposure in spiral computed tomography:

Dose distribution and dose reduction. Invest Radiol

1999; 34:51-7.

10. Brenner D, Elliston C, Hall E, Berdon W. Estimated risks

of radiation-induced fatal cancer from pediatric CT. AJR

Am J Roentgenol 2001;176:289-96.

11. Brenner DJ, Elliston CD. Estimated radiation risks

potentially associated with full body CT screening.

Radiology 2004; 232:735-8.

12. Brenner DJ. Radiation risks potentially associated with

low-dose CT screening of adult smokers for lung cancer.

Radiology 2004; 231:440-5.

13. Yoshinaga S, Mabuchi K, Sigurdson AJ, Doody MM, Ron

E. Cancer risks among radiologists and radiologic

technologists: Review of epidemiologic studies.

Radiology 2004;233:313-21.

14. Wagner LK. Overconfidence, overexposure, and

overprotection. Radiology 2004;233:307-8.

15. Hart D, Hillier MC, Wall BF. Doses to patients from

medical x-ray examinations in the UK - 2000 review.

Chilton, NRPBW14 2002.

16. McCollough CH, Bruesewitz MR, James M, Kofler T Jr.

Dose reduction and dose management tools: Overview

of available options1. RadioGraphics 2006;26:503-12.

17. Tack D, Maertelaer VD and Gevenois PA. Dose reduction

in multidetector CT using attenuation-based online tube

current modulation. AJR Am J Roentgenol

2003;181:331-4.

18. Rizzo S, Kalra M, Schmidt B, Dalal T, Suess C, Flohr T, et

al. Comparison of angular and combined automatic

tube current modulation techniques with constant tube

current CT of the abdomen and pelvis. AJR Am J

Roentgenol 2006;186:673-9.

19. Huda W, Nickoloff EL, Boone JM. Overview of patient

dosimetry in diagnostic radiology in the USA for the past

50 years. Med Phys 2008;35:5713-28.

20. European Commission. European guidelines on quality

criteria for computed tomography. Brussels, Belgium:

Report EUR 16262 EN 1999.

21. Kalra MK, Prasad S, Saini S, Blake MA, Varghese J,

Halpern EF, et al. Clinical comparison of standard- dose

and 50% reduced-dose abdominal CT : Effect on image

quality. AJR Am J Roentgenol 2002;179:1101-6.

22. ACR. Computed Tomography Accreditation program:

Phantom testing criteria. 1891, Preston White Drive,

Reston VA20191.

23. Wade JP, Weyman JC, Goldstone KE. CT standard

protocols are of limited value in assessing actual patient

dose. Br J Radiol 1997;70:1146-51.

24. Tsapaki V, Aldrich JE, Sharma R, Staniszewska MA,

Krisanachinda A, Rehani M, et al. Dose reduction in CT

while maintaining diagnostic confidence: Diagnostic

eference levels at routine head, chest, and abdominal

CT-IAEA-coordinated research project. Radiology

2006;240:828-34.

25. Goddard CC, Al-Farsi A. Radiation doses from CT in the

Sultanate of Oman. BJR 1999;72:1073-7.

26. Van der Molen AJ, Veldkamp WJ, Geleijns J. 16-slice CT:

Achievable effective doses of common protocols in

comparison with recent CT dose surveys. Br J Radiol

2007;80:248-55.

27. Brix G, Nagel HD, Stamm G, Veit R, Lechel U, Griebel J,

et al. Radiation exposure in multi-slice versus single-

slice spiral CT: Results of a nationwide survey. Eur Radiol

2003;13:1979-91.

28. Shrimpton PC, Hillier MC, Lewis MA, Dunn M. Doses

from computed tomography examinations in the UK -

2003 review. Report NRPB-W67. [Published on 2005

March].









Pag

e51

9

Appendix A

Communication of the decision of the institutional ethics committee

Protocol title : Automatic Tube Current Modulation

Date of review (DD/M/YYYY) :

Please note*

Informed IEC immediately in case of any adverse events and serious adverse events

Informed IEC any amendments to the protocol, change of study procedure, site and investigator

and premature termination of study with reasons along with summery.

Final & six months reports to be submitted to IEC.

Members of IEC have right to monitor the trial with prior information.

A copy of the consent document to be given study participant giving the concern.

Appendix B

Informed consent form

Serial Number :

Hospital Number :

Name :

Age :

Sex :

I ……………………. hereby voluntarily give consent to Mr. PURNA CHANDER REDDY for taking Computed

Tomographic Scan (CT Scan) of my Abdomen for studying the Automatic Tube Current Modulation, which

will be used for the dissertational study. I have been explained about the procedure in the language that

I understand and I am aware that I can refuse unconditionally.

Signature of the Individual

Place:

Date:









Pag

e52

0

Appendix C

Proforma

DATA SHEET

Name: Date:

Serial. No Hosp No Age Sex Weight Height-

Male Female

Clinical

Information

Dose Measurements

Pre

Measurements

No of images CTDI

Scan time DLP

mA kVp 120 mAs 250 FOV

Post

Measurements No of images CTDI-

Scan time DLP

Est. Dose saving

mA varies kVp 120 mAs FOV

*Corresponding Author: Suresh Sukumar Assistant professor, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal.udupi – 576104. Email [email protected]. 9886118811








ResearchArticle

BiologicalSciences


Razia Muthusamy*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e52

1

PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF

METHANOL EXTRACT OF TRIDAX PROCUMBENS

Razia Muthusamy, Karthiga Vasu, Lavanya Kanagaraj, Deboral Ponnampallam, Bernala Wilson

Department of Biotechnology,Mother Teresa Women’s University,Kodaikanal-624101, Tamilnadu

*Corresponding AuthorEmail:[email protected]

ABSTRACT The leaves of Tridax procumbens were collected, grounded and subjected to methanol for extraction with soxhlet

apparatus. The extract was screened phytochemically for its chemical components. The presence of alkaloids,

reducing compounds (carbohydrates), cardiac glycosides, flavonoids, saponins, terpenes and steroids was

recorded. Antibacterial activity bydisc diffusion method revealed that the methanol extract have a broad spectrum

activity on gram positive, negative organisms respectively. The highest activity was shown in S. typhiS. flexneri

and least activity on E.coli.

KEY WORDS Tridax procumbens,Escherichia coli, Salmonella typhi, Shigella flexneri

INTRODUCTION

Tridax procumbens Linn. (Asteraceae) is one of

the medicinally important plants commonly

found in subtropical countries growing primarily

during raining season. It is a common weed in

Tamilnadu present along with economically

important crops (Suseela etal., 2002). It habitats

waste places, road sides and hedges throughout

India. The leaves of the plant are known to be

used for the treatment of wound in traditional

medicine (Collier 2001). The extracts of T.

procumbens have been reported to have various

pharmacological effects, antimicrobial activity,

wound healing property and immunomodulatory

activity on the experimental animals (Taddel and

Rosas, 2000). Phytochemical constituents are the

basic source for the establishment of several

pharmaceutical industries (Savithramma et al.,

2011). Pathogenic microorganisms are always

trying to develop resistance to the various

commercial antibiotic drugs for their control

(Beegum and Devi, 2003). High cost and adverse

side effects are commonly associated with

popular synthetic antibiotics (such as

hypersensitivity, allergic reactions,

immunosupression etc.) and are major burning

global issues in treating infectious diseases

(Schinor et al., 2007). Hence, recent attention

has been paid to biologically active extracts and

compounds from plant species used in herbal

medicines (Essawi and Srour, 2000).

The present study was designed to study the

preliminary phytochemical analysis and

antibacterial activity of Tridax procumbens.


Plant collection

The Leaves of Tridax procumbens were collected

from kodaikanal region of dindigul district of

Tamilnadu.






Pag

e52

2

Preparation and Extraction of Plant material

The plant materials (leaves of Tridax

procumbens) were air-dried at room

temperature for 2 weeks and grinded to a

uniform powder. The Methanol extract was

prepared by soaking 10 g of powdered plant

materials in 100 ml of methanol at room

temperature for 48 h. Extract was filtered after

48 h, with Whatmann filter paper No. 42

(125mm) and then through cotton wool. The

extract was concentrated using a rotary

evaporator with the water bath set at 40°C.

Phytochemical screening

The methanol extract was screened for

phytochemical constituents for the presence of

saponins, tannins, alkaloids, flavonoids,

anthraquinones, glycosides and reducing sugars

(Sharma and Sharma, 2010).

Antibacterial assay

The agar disc diffusion method as described by

(Parekh and Chanda, 2006) was used in the

antibacterial screening procedure. Mueller-

Hinton (MH) agar plates were prepared using

petridishes. For the agar disc diffusion method,

the disc was saturated with 100 μl of the test

compound, allowed to dry and then placed on

the upper layer of the seeded agar plate.

Antibacterial activity was determined by

measuring the diameter of the zone of inhibition

(mm) surrounding bacterial growth.

RESULTS AND DISCUSSION

The phytochemical screening of methanol

extract of T. procumbens was presented in Table

1.T. procumbens leaves contains alkaloids,

tannin, flavonoids, terpenoids, phenols, saponin,

carbohydrate, glycosides, and cardiac glycosides.

Flavonoids are known to be synthesized by

plants in response to microbial infection. Tannins

(commonly referred to as tannic acid) are also

known as antimicrobial agents (Sharma and

Sharma 2010). Tannins have been reported to

prevent the development of microorganisms

(Taddei, and Rosas-Romero 2000). The result of

antibacterial activity of Tridax procumbens by

disc diffusion method showed in the (Table 2).

The methanol extract showed significant zone of

inhibition against selected bacterial species. S.

flexneri, S. typhi, P. aeruginosa, and P. mirabilis

showed greater zone of inhibition than Klebsiella

pneumoniae and Escherichia coli which showed

lesser inhibition zone. Similar antibacterial

activity has been reported (Janovska et al.,

2003).

Table1: Phytochemical analysis of T. procumbens

S.No Test T. procumbens(Leaf)

1. Alkaloids +

2. Flavonoids +

3. Terpenoids +

4. Phenols +

5. Tannins +

6. Anthraquinone _

7. Free anthroquinine _

8. Saponin +

9. Carbohydrate +

10. Carotenoid _

11. Glycoside +

12. Cardiac glycoside +





Pag

e52

3

Table 2. Antibacterial activity of methanolic extract of Tridaxprocumbens

S.No Organisms (Zone of Inhibition mm)

Tridaxprocumbens

25µg 50µg 75µg 100µg

1. E. coli 4 6 12 14

2. K. pneumoniae 5 7 13 16

3. P. aeruginosa 6 9 18 19

4. P. mirabilis 5 10 15 18

5. S. flexneri 6 12 19 22

6. S. typhi 8 14 20 24

CONCLUSION

The study revealed that Tridax procumbens was

rich in secondary metabolites particularly tannins

and flavonoids which are responsible for

antibacterial activity. Further, detailed

investigation of the active compounds of the

plant for the exact mechanism of action will

contribute greatly to the development new

pharmaceuticals.

REFERENCES o Beegum B.N.R., and Devi T.G. Antibacterial activity of

selected sea weeds from Kovalam South West coast of

India.Asian Journal of Microbiol Biotech Env Sci, 5(3):

319-322,(2003)

o Collier M., An assessment plan for wound

management. J comm. Nurs, 16 (6): 22-26, (2001)

o Essawi T., Srour M. Screening of some Palestinian

medicinal plants for antibacterial activity. J

Ethanopharmacol, 46: 343-349,( 2000)

o Janovska, D., Kubikova K., and Kokoska L. Screening for

antimicrobial activity of some Medicinal plant species

of traditional Chinese medicine. Czech J. Food Sci., 21:

107-111,(2003)

o Parekh J, Chanda S. In vitro antimicrobial activities of

extracts of Launaeaprocumbens Roxb. (Labiateae),

VitisviniferaL. (Vitaceae) and CyperusrotundusL.

(Cyperaceae). Afr. J. Biomed. Res., 9: 89-93, (2006).

o Savithramma N., Rao M. L., and Bhumi G.

Phytochemical screening of Thespesiapopulnea (L.)

Soland and Tridax procumbens L. J. Chem. Pharm. Res.,

3(5):28-34, (2011)

o Sharma.M.K. and Sharma.S. Phytochemical and

Pharmacological Screening of Combined Mimosa

pudica Linnand Tridax procumbens for In vitro

Antimicrobial Activity. Intl. J. Microbiol. Res., 1 (3): 171-

174, (2010)

o Schinor EC, Salvador MJ, Ito IY, Dias DA. Evaluation of

the antimicrobial activity of crude extracts and isolated

constituents from Chrestascapigera. Brazilian J.

MIcrobiol,38: 145-149, (2007)

o Suseela L, Sarsvathy. A, Brindha. P. Pharmacognostic

studies on Tridax procumbensL.(Asteraceae). Journal of

Phytological Research. 15 (2): 141-147, (2002).

o Taddel A., Romero AJ.R. Bioactivity studies of extracts

from Tridax procumbens. Phytomedicine. 7(3): 235-

238. (2000).





Pag

e52

4

*Corresponding Author: M. Razia Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal– 624 102 TamilNadu, India. Email*: [email protected] Mobile: +91 8012137535




Case Report

Biological Sciences


Nagakumar J S* et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e52

5

ANEURYSMAL BONE CYST OF THE LATERAL END OF CLAVICLE IN A FOURTEEN YEAR OLD BOY

– CASE REPORT

Nagakumar J S1, Nazeer BS2, Samarth Arya3 1Assistant Professor, Department of Orthopaedics, SDUMC, Kolar, India

2Professor, Department of Orthopaedics, SDUMC, Kolar, India 3Jr.Resident, Department of Orthopaedics, SDUMC, Kolar, India

ABSTRACT Aneurysmal bone cysts are enigmatic lesions of unknown cause and presentation and are difficult to distinguish

from other lesions. We present a case of aneurysmal bone cyst involving clavicle. It presented difficulties in diagnosis

because of the uncommon Location. The boy was treated surgically with curettage and autologous bone grafting as

discussed.

KEY WORDS Aneurysmal bone cyst (ABC), Clavicle.

INTRODUCTION

Primary bone tumours of flat bones like clavicle

are rare. True benign tumours are much

uncommon than metastatic or malignant lesions.

Aneurysmal bone cyst (ABC) is a benign but locally

aggressive lesion of the bone which accounts for

3% of all bone tumours. Its histology is

characterized by multiloculated cystic tissue filled

with blood. Etiology and pathogenesis of this

lesion remains unclear and it affects 0.14 per lakh

every year 1. It is a disease mainly of the young

with a peak incidence in the second decade.

However it may on occasion occur in the elderly

and the very young 2. ABC may involve almost any

bone but the most frequent sites are long tubular

bones and vertebrae. Among flat bones, the

pelvis and scapula are well known locations.

Despite very characteristic radiological features,

the unusual age coupled with the uncommon site

led to diagnostic difficulties in present case. The

clavicle is a rare site for these lesion and not many

have been reported in literature. Smith in 1965

could find only 25 cases in the medical literature,

textbooks and atlases 3. Because of these factors,

this report is felt to be of interest.

CASE REPORT

A 14 year old boy presented with swelling in his

left clavicular region that had been increasing in

size progressively since last six months to reach

the size of a lemon. The swelling at the acromial

end had distinct edges, was smooth surfaced (Fig.

1) and overlying skin temperature was normal.

The mass was bony hard, non-tender and the skin

over the swelling was pinchable. Swelling was

immobile and Egg shell crackling sensation was

noted. No functional impairment, skin changes or

dilated veins or signs of neurological deficits and

lymphadenopathy was noted. There was no

history of trauma or history of recurrent fever and

loss of weight. No other lumps or swellings were

present anywhere else.

Radiograph showed a cystic expansile lesion of

the lateral end of the left clavicle bounded by a








Nagakumar J S*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e52

6

thin layer of bone (Fig. 2). Based on the

appearance various possibilities including simple

bone cyst, ABC, eosinophilic granuloma, and

enchondroma were considered.

Basic hematological work up including complete

blood count, ESR, CRP and alkaline phosphatase

were within normal limits. The lesion was further

studied with MRI left shoulder (Fig.3) and fine

needle aspiration cytology. MRI pointed to the

possibilities of ABC, Giant cell tumour and

Chondroblastoma. FNAC report came out to be

inconclusive.

Therapeutic options which were considered at

that point were resection of lesion and curettage

with autologous bone grafting. The conservative

approach was preferred as resection could have

resulted in weakening of the shoulder.

Intraoperatively, initially a 10cc disposable

syringe was used to aspirate the contents of the

cavity. The content was found to be a blood-fluid

(Fig. 4).

The lesion was approached after incising the

periosteum longitudinally. It was a multiloculated

cyst containing streaks of thrombi (Fig. 5). The

inner wall was curetted and electrocautery done

to seal the bleeding walls of the cavity. Cavity was

further irrigated with iodine containing alcohol

solution and cavity was filled with cortico-

cancellous strip of autologous iliac crest bone

graft. Periosteal tube was repaired and limb was

immobilized in cuff and collar sling. Postoperative

period was uneventful. The preoperative

diagnosis was confirmed with the

histopathological examination of the curetted

specimen.

Figure 1: Photograph depicting swelling at the left clavicular region.









Pag

e52

7

Figure 2: Radiograph showing cystic expansile lesion bounded by a thin layer of

bone at the lateral end of the left clavicle.

Figure 3: MRI Left shoulder (Plain and contrast) Scan showing multilobulated T1

hypointense, T2 hyperintense arising from end of left clavicle with destruction of

lateral end and peripheral enhancement, central non-enhancing areas.









Pag

e52

8

DISCUSSION

Despite the long experience of orthopedicians,

radiologists and pathologists, there is limited

knowledge regarding the cause of the lesion, its

natural history, and the results of treatment4-8.An

interesting theory about the aetiology of primary

ABCs is that the lesions occur because of

haemorrhage in the bone as a result of increased

venous pressure. The haemorrhage is thought to

lead to osteolysis. The osteolysis, in turn, causes

further haemorrhage, leading to exponential

growth of the tumour. This theory would perhaps

Figure 4: Intraoperative photograph showing blood-fluid in

the cyst.

Figure 5: Photograph depicting multiloculated cyst

containing streaks of thrombi.









Pag

e52

9

explain why ABCs are uncommon in the clavicle

and bones of the facial skeleton, where

thevenous pressure is low. On the other hand,

ABCs are common in long bones, where the

venous pressure is high and the marrow content

is greater9. Conventional thinking; however,

ascribes these lesions either to a reaction to

physical injury, which may sometimes be remote,

or to a vascular disturbance. The concept that the

lesion represents a vascular degenerative process

for some benign bone lesions is an attractive one,

but the pathologic findings, with rare exception,

do not really support this proposal. Few

pathologic specimens contain tissues that are

highly characteristic or diagnostic of giant cell

tumor, chondroblastoma, hemangioma,

osteoblastoma, non ossifying fibroma,

chondromyxoid fibroma & others6, 7, 8, 10.Thus, it is

often thought that ABC is more of a

pathophysiological change in a pre-existing

primary bone lesion rather than a single, unique

entity11.

Difficulty can occur in diagnosing these lesions.

The imaging studies, even CTs and MRIs,

sometimes do not provide clearly diagnostic

criteria for the diagnosis of ABC, and ABC is

sometimes added on to a list of diagnoses

including eosinophilic granuloma, giant cell

tumor, non ossifying fibroma, unicameral bone

cyst, fibrous dysplasia, chondroblastoma,

chondrosarcoma, chondromyxoid fibroma,

Ewing's tumour etc. 6, 7, 12.

In the past, curettage alone was employed in the

treatment later methods like saucerisation,

resection, radiotherapy, cryotherapy and vascular

occlusion are being employed. Nevertheless,

there is no consensus among treating physicians

regarding how these methods should be used. As

a result, there are quiet contradictory reports

regarding results and complications13. Resection

of lesion offers low recurrence rate but this

option cannot be exercised everywhere. A

combination of cryosurgery and curettage has

been reported by few authors that reported local

control after the first treatment in 82% patients14.

Radiotherapy can result in radiation induced

sarcomas and can cause radiation induced injury

to physis13. Thus radiotherapy is reserved in cases

that cannot be operated because of their location

and to prevent damage to the function of

important structure7. In some cases embolisation

of a feeding vessel may help to decrease

vascularity, making the surgical procedure less

bloody, especially in difficult locations such as

spine and pelvis but it is a highly demanding

technique and may not be available at all centres.

Recurrence rate in young children with ABC may

be as high as 100%1. Autograft implantations or

utilization of intercalary allografts are quite

successful and for the most part, are used for

patients with lesions that are large or seem to

threaten the integrity of the bone. Our patient

responded nicely to this form of treatment and

we feel that this case enriches existing data

regarding treatment option of an ABC in an

unusually young patient and in unusual location.

REFERENCES 1. Leithner A, Windhager R, Lang S.,Aneurysmal bone cyst:

A population-based epidemiologic study and literature

review. Clin Orthop Relat Res. 1999; 363:176-79.

2. Dominok G. W., Knoch H. G., Manza B., SchulzeK. J., Die

aneurysmatischeKnochencyste.LangenbecksArchievefu

rChirurgie. 1971; 328: 153-168.

3. Smith J, Aneurysmal bone cyst of clavicle.Br J Radiol.

1977; 50:706-9.

4. Aho HJ, Aho AJ, Einola S: Aneurysmal bone cyst: A study

of ultrastructure and malignant transformation.

Virchows Arch A PatholAnatHistol. 1982; 395:169-79.

5. Aho HJ, Aho AJ, Peliniemi LJ, Ekfors TO, Foidart JM:

Endothelium in aneurysmal bone cyst.Histopathology.

1985; 9:381-87.

6. Campanacci M: Aneurysmal bone cyst:In Campanacci M

- Bone and Soft Tissue Tumors 2ndedn. New York, NY,

Springer Verlag, 1999, 812-840.

7. Dorfman HD, Czerniak B: Cystic lesions, In Dorfman HD,

Czerniak B: Bone Tumors. St Louis, MO, Mosby, 1998,

855-912.









Pag

e53

0

8. Szendroi M, Aratgto G, Ezzati A, Huttl K, Szavcsur P.,

Aneurysmal bone cyst: Its pathogenesis based on

angiographic, immunohisto chemical and electron

microscopic studies. PatholOncol Res. 1998; 4:277-81.

9. Boyd RC. Aneurysmal bone cysts of the jaws. Br J Oral

Surg. 1979; 16: 248-53.

10. Bollini G, Jouve JL, Cottalorda J, Petit P, Panuel M et al.,

Aneurysmal bone cyst in children: Analysis of twenty-

seven patients. J PediatrOrthop B.1998; 7:274-285.

11. Kransdorf MJ, Sweet DE. Aneurysmal bone cyst:

concept, controversy, clinical presentation, and

imaging. Am J Roentgenol. 1995; 164: 573-80.

12. Mahnken AH, Nolte Ernsting CC, Wildberger JE, et al:

Aneurysmal bone cyst: Value of MR imaging and

conventional radiography. EurRadiol. 2003; 13: 118-24.

13. Tillman B., Dahlin DC, Lipscomb PR, Stewart JR.,

Aneurysmal bone cyst: an analysis of ninety-five cases.

Mayo Clin Proc. 1968; 42:478-95.

14. Marcove R,Sheth DS, Takemoto S, Healey H., The

treatment of aneurysmal bone cyst. ClinOrthop. 1995;

311: 157-63.

*Corresponding Author: Dr. Nagakumar J S Assistant Professor, Dept of Orthopaedics, SDUMC, Kolar, India, Ph: 9448543804







Research Article

Biological Sciences


NITHYAMALA I*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e53

1

DIURETIC ACTIVITY OF JALAMANJARI CHENDOORAM IN RATS

NITHYAMALA I *, KUMAR A, BANUMATHI V, VELPANDIAN V, PITCHIAHKUMAR M, AYYASAMY S

Post Graduate Department of Gunapadam (Pharmacology), Government Siddha Medical College, Chennai,

Tamilnadu, India. Pin Code: 600 106. *Corresponding Author Email: [email protected]

ABSTRACT Aim: In the Siddha System of medicine, innumerable drugs are available, one such valuable diuretic drug Jalamanjari

Chendooram (JC) from Siddha literature, has been identified, which till now not scientifically evaluated has been

chosen and a detailed study has been done to evaluate diuretic activity of the drug. Therefore the present study was

planned to evaluate the diuretic potential and effect on urinary electrolytes of JC in male Wistar rats. Methods: For

the evaluation of diuretic activity the methods of Lipchitz et al., 1943 and Murugesan et al., 2000 were followed.

Different concentrations of JC (25 mg and 50 mg/kg of body weight) and the standard drug Furosemide (20 mg/kg)

were administrated orally to hydrated male Wistar rats and their urine output was measured at several intervals of

time after a single dose administration. The parameters measured for diuretic activity were urine volume at different

time intervals, sodium, potassium and chloride content. Results: JC 50mg/kg showed remarkable increase in volume

of urine, sodium, potassium and chloride content. Conclusion: Conclusively, JC is an effective diuretic confirming the

traditional use of the drug.

KEY WORDS Jalamanjari Chendooram, diuretic activity, electrolytes, urine volume, furosemide.

INTRODUCTION

The most common condition which causes a

major financial and emotional burden on the

community is renal diseases. It is also a medical

condition with limited treatment options in the

modern medicine. But since ancient times

innumerable complementary and alternative

medicines especially Siddha system of medicines

possess with it a treasure of renal protective and

effective treatments and are followed here and

there successfully.

One among the important renal protective drugs

is the diuretic group of drugs. Diuretics increase

the urine formation either by increasing the

glomerular filtration rate (or) by decreasing the

rate of reabsorption of fluid from the tubules.

Such drugs which induce diuresis are known as

diuretics. Diuretic compounds are those which

stimulate water excretion potentially from our

body. So diuretics play a vital role in many

oedematous diseased conditions such as

congestive heart diseases, nephritis, and toxemia

of pregnancy also in hypertensive conditions and

pulmonary congestion. The mode of action of

diuretics is that they decreases cardiac work load,

oxygen demand, plasma volume and thereby

decreases blood pressure [1].

Diuretics cure clinical conditions like acute and

chronic renal failure, hypercalciuria, and cirrhosis

of liver. Though these are the good effects of

synthetic diuretics, many adverse effects have

also been reported. These are hyperuricaemia,

acidosis, gastric irritation & high blood sugar level [2].










Pag

e53

2

So there arises an emergent need to search for a

safe renal protective diuretic drug and

innumerable such drugs are available in Siddha

literatures but yet to be standardized since it

is deficit in scientific valuation. One such valuable

diuretic drug Jalamanjari Chendooram from

Siddha literature, has been identified, which till

now not scientifically evaluated has been chosen

and a detailed study has been done to evaluate

diuretic activity in rats.


Preparation of the drug Jalamanjari

Chendooram [3]

The raw drugs required for the preparation of

Jalamanjari Chendooram as mentioned in “Yoogi

Karisal - 151” are Salt Petre, Borax, Conch shells,

Alum, Ammonium Chloride, crystallized foliated

Gypsum, Asbestos, Red ochre, Magnetic oxide of

iron, Asphaltum, Sulphur , Iron Filings. The raw

drugs were subjected to ‘Suddhi’ (purification

process) as per Classical Siddha text [4, 5].

The purified drugs were powdered separately.

The powders were mixed and grinded again into

a very fine powder.

A shallow container was heated and some

amount of the powder was sprinkled in to it. The

mixture first melted and then solidified. The solid

was taken and allowed to cool. Similarly all the

powders were used and the solidified products

obtained were grinded into a very fine powder.

PHARMACOLOGICAL ACTIVITY

Drugs and Chemicals

Furosemide was procured from Himedia

Laboratories, Mumbai, Other chemicals and

reagents used in this study were analytical grade

was purchased from SRL labs.

Preparation of stock solution

The Jalamanjari Chendooram was further diluted

with distilled water so as to prepare 100mg/ml

concentration at room temperature for oral

administration by gastric intubation method.

Animal selection:

For the diuretic study, male Wistar rats weighing

between 180-220 g were used. The animals were

acclimatized to standard laboratory conditions

(temperature: 25±2°C) and maintained on 12-h

light: 12-h dark cycle. They were provided with

regular rat chow and drinking water ad libitum

(Approval number: XIII /VELS /PCOL /17 /2000

/CPCSEA /IAEC / 08.08.2012).

Evaluation of Diuretic activity:

The methods of Lipchitz et al., 1943 and

Murugesan et al., 2000 were followed [6-8]. The

screening was performed on healthy rats.

Furosemide (20 mg/kg) was used as reference

standard and Jalamanjari Chendooram were

dissolved in saline solution for administration

while normal saline (25 ml/kg) was used as

vehicle. The rats were divided in 4 groups each

containing 6 rats (n = 6). Rats were kept for fasting

for 18 hrs before the study.

The control group received normal saline and test

groups received 25 and 50mg/kg of Jalamanjari

Chendooram dissolved in normal saline. The

doses of Jalamanjari Chendooram were decided

on the basis of acute toxicity study. The doses

were given by oral route and rats were kept in

specially designed metabolic cages for the

collection of urine for 6 hrs. The urine volume

during 6 hrs is measured and urine electrolyte

estimation was carried out for Na+, K+ using flame

photometer and Cl – was estimated by titration.

Na+, K+ estimation was carried out using flame

photometry [9, 10]. The Cl – ion concentration was

estimated by titration with 0.02 N AgNO3 using 5%

potassium chromate solutions as indicator [11].









Pag

e53

3

STATISTICAL ANALYSIS

All results were expressed as mean ± standard

error. The data was analyzed statistically using

ANOVA followed by Dunnet’s Multiple

Comparison Test.


Jalamanjari Chendooram has traditional use as a

diuretic, the effect of this drug, standard drug

Furosemide and control group on urination and

other parameters related to diuretic assay were

investigated in Wistar rats and the results of the

evaluation carried out were tabulated in Table 1

& 2.

Table 1 shows the urine volumes collected at

different time intervals control group,

Furosemide and trial drug Jalamanjari

Chendooram treated orally at dose levels of 25

and 50mg/kg. Table 2 shows the parameters

related to electrolyte excretion (Na+, K+ and Cl–

concentrations in mMol/L).

Table-1: Showing the urine volume at different intervals in rats

Values are mean ± SEM, * p< 0.01, ** p< 0.05 when compared to normal saline (control)

Table 2: Effect of Jalamanjari Chendooram on electrolyte levels in urine

Group Treatment Sodium

(mMol/l)

Potassium

(mMol/l)

Chloride

(mMol/l)

Control Normal saline (25 ml/ kg) 62.380.08 92.101.24 10.201.21

Standard Furosemide (20 mg/ kg) 104.110.51** 118.494.15** 14.341.74*

Test 1 JC 25mg/kg 87.211.02** 92.400.18 14.010.04*

Test 2 JC 50mg/kg 96.100.66** 92.562.88 13.650.80

Values are mean ± SEM, * p< 0.01, ** p< 0.05 when compared to normal saline (control)

Effect on urine volume

Two dose levels of Jalamanjari Chendooram (25

mg/kg and 50mg/kg) were selected for study and

the urine volume after 15 minutes of

administration of drug were found to be

0.250.03 and 0.290.04 respectively.

Furosemide (20mg/kg) treated group was found

to be 0.340.05. Urine volume after 120 minutes

of administration of two dose levels of drug were

found to be 2.150.30 and 3.960.42

respectively. Furosemide (20mg/kg) treated

group was found to be 4.870.24. Furosemide

treated rats showed a significant increase in

volume of urine as compared to control while

Jalamanjari Chendooram 25mg treated rats did

not show any significant increase in urine volume

but Jalamanjari Chendooram 50mg/kg showed

remarkable increase in volume of urine.

Group Treatment Urine volume at different time intervals (in ml)

15 min 30 min 45 min 60 min 120 min

Control Normal 0.270.04 0.510.02 1.060.05 1.020.08 1.540.22

Standard Furosemide

(20 mg/ kg)

0.340.05 1.460.1** 2.280.12** 3.380.18 4.870.24**

Test 1 JC 25mg/kg 0.250.03 0.740.01* 1.240.05 1.880.11 2.150.30

Test 2 JC 50mg/kg 0.290.04 0.620.01 1.360.07* 2.690.10 3.960.42**









Pag

e53

4

Fig No.1. Effect of Jalamanjari Chendooram on electrolyte levels in urine

Fig No.2. Effect of urinary volume of Jalamanjari Chendooram treated rat

Effect on urinary electrolyte excretion

The effect of standard drug Furosemide and

different doses of Jalamanjari Chendooram on

electrolyte (Na+, K+ and Cl-) excretion in urine is

tabulated in Table 2.

The dose of 25mg/kg Jalamanjari Chendooram

produced a moderate increase in Na+, K+ and Cl-

excretion, compared with the control group (Na+

=87.211.02, K+=92.400.18 and Cl-=

14.010.04). The dose of 50 mg/kg Jalamanjari

Chendooram produced a significant increase in

excretion of sodium, potassium and chloride ions

in the urine to an extent similar to that of

0

20

40

60

80

100

120

140

Normal JMC 25mg/kg JMC50mg/kg Frusemide (20mg/ kg)

Co

nce

ntr

atio

n (m

Mo

l/l)

Effect of Jalamanjari Chendooram on electrolyte levels in urine

Sodium Potassium Chloride

-1

0

1

2

3

4

5

6

15 min 30 min 45 min 60 min 120 min

Urine volume at different time intervals (in ml)

Diuretic activity of Jalamanjari Chendooram in rats

Normal JMC 25mg/kg JMC50mg/kg Frusemide (20 mg/ kg)









Pag

e53

5

Furosemide (Na+ = 96.100.66, K+ = 92.562.88

and Cl- =13.650.80).

CONCLUSION

From this study it can be suggested that the drug

Jalamanjari Chendooram is an effective and

significant hyponatraemic, hypochloraemic and

hypokalaemic diuretic with values close to the

standard drug Furosemide, which supports the

claim about the Jalamanjari Chendooram being

used as a diuretic in Siddha system of medicine.

On the basis of the results of present

investigation, we can conclude that Jalamanjari

Chendooram might be a good diuretic.

ACKNOWLEDGEMENT

The authors are very much grateful to the

Principal and HOD, Government Siddha Medical

College, Chennai for their constant support to

carry out this work successfully.

REFERENCES 1. Finkel R, Clark MA, Cubeddu LX.Lippincott’s Illustrated

Reviews: Pharmacology 4th Edn, Lippincott Williams

&Wilkins, Florida, 2009, 190.

2. Deniels, T.C. and E.C. Jorgensen, Text Book of Organic

Medicinal and Pharmaceutical Chemistry, 7th ed.,

Lippincott Co. Ltd., Toronto, 1977,575.

3. Ramachandran, Yoogi Karisal 151, Edn 2, Tamarai Press,

Chennai, 2004,46

4. Thiyagarajan R. Gunapadam Thathu – Jeeva Vaguppu

Part (2 & 3) Edn 2, Indian Medicine and Homeopathy

Dept. Chennai. 2006, 305, 397, 408, 437, 442, 530,534,

546.

5. Aanaivaari Anandan, Sarakku Suddhi Sei Muraigal, Edn

1, Indian Medicine and Homeopathy Dept. Chennai-

106. 2008, 25, 28 & 100.

6. R. A. Turner, The Organization of Screening. In:

Screening Methods in Pharmacology, Vol. I, New York

and London, Academic Press; pp. 21(1965).

7. W.L. Lipchitz, Z.Haddian and A.Kerpscar. Bioassay of

diuretics. J. Pharmacol. Exp.Ther. 79: 97-110 (1943).

8. T. Murugesan, L. Manikandan, K.B. Suresh, M. Pal and

B.P. Saha. Evaluation of diuretic potential of Jussaea

suffruticosa Linn extract in rats. Indian J.Pharm.Sci.

62(2): 150-151(2000).

9. Jeffery GH, Basset J, Mundan J, Denny R. 5th ed.

England: Addison Westerly Longmann Ltd; 1989. Vogel's

Textbook of Quantitative Chemical Analysis; p. 801.

10. Bhakuni DS, Dhar ML, Dhar MM, Dhawan BN, Mehrotra

BN. Screening of Indian plants for biological activity, II.

Indian J Exp Biol. 1969; 7:250–62.

11. Beckett AH, Stanlake JB. 1st Edn. New Delhi: CBS

Publishers and Distributor; 1997. Practical

pharmaceutical chemistry; p. 197.

*Corresponding Author: Dr.I.Nithyamala Email: [email protected] Phone No: 044-26222682 / 9444183646 Fax: 044 - 26222683








Research Article

Biological Sciences


Suresh sukumar*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e53

6

COMPARISON BETWEEN CT SCAN AND MANUAL METHOD OF AREA MEASUREMENT OF

MASTOID PROCESS IN SEX DETERMINATION OF SOUTH INDIAN POPULATION

Albin Babu M Wilson1, Nimma purna chander reddy2, Suresh sukumar3,

Sushil Yadav4, Karuna paliwal5

1Lecturer. MSc. Medical Imaging Technology, Department of Medical Imaging Technology,

KSHEMA, Nitte University, mangalore. 2Clinical SME, Apollo Health Street.

3Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal

College of Allied Health Sciences, Manipal University, Manipal. 4Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal

College of Allied Health Sciences, Manipal University, Manipal. 5MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied

Health Sciences, Manipal University, Manipal. *Corresponding Author Email: [email protected]

ABSTRACT Aim: The purpose of this study was to evaluate the significance for sex determination of the measurement of the

area formed by projection of 3 craniometric points related to the mastoid process (the porion, asterion, and

mastoidale points) of 3D reconstructed computed tomography (CT) of skull and the result is compare the result of

manual measurement of the area formed by projection of 3 craniometric points related to the mastoid process.

Method: 3D reconstructed CT of 40 males and 40 females were analysed. The three craniometric points were located

and marked on both side of the 3D skull and the measurement was done by advanced post processing techniques. In

manual measurement 40 males and 40 female skull were analyzed by digital caliper (0.01mm). The area of mastoid

triangle was calculated by means of the Heron’s formula. The result of CT reading and Manual reading is analyzed

for the sex determination and the both result is compared. Result: From this study the areas of the male CT

measurement of Mastoid is 828.53±118.54 mm2which is greater than female Mastoid which is 578.24±71.53 mm2 (T

test value 9.901and P Value 001). Area of the male Mastoid which is measured by manual 805.87±90.99 mm2 which

is greater than female Mastoid which is 620.56±90.63 mm2 (T test value 10.202and P Value 001). Conculsion:The

area of the mastoid triangle measured by manual and 3D reconstructed CT skull are used to determine the sex of

the skull and there is no significant deference between CT scan and Manual method of area measurement of

mastoid process in sex determination of south Indian population .

KEY WORDS 3D reconstructed computer tomography, Sex determination, Mastoid process

INTRODUCTION

In the skull, the temporal bone is highly resistant

to physical damage; thus it is commonly found as

remainder in skeletons that are very old; of this,

the petrous portion has been described as

important for sex determination (Kalmey &










Pag

e53

7

Rathbun, 1996). Paiva & Segre (2003) introduced

an easy technique for sex determination starting

from the temporal bone, with a small

observational error and with a high predictability

degree. The technique is based on the triangular

area calculation obtained between the point’s

porion, mastoidale, and asterion, measured

from xerographic copy of skulls. They found

significant differences in the area between the

right and left mastoid triangle when comparing

male and female skulls, but owing to the

asymmetries present in the skulls, it is

recommended to observe the value of the total

area (adding right and left sides), which was also

significant, so that when it is higher than or equal

to 1447.40 mm2, the skull is diagnosed as male

skull, and a value near to 1260.36 mm2 or less is

indicative of female skull (De Paiva & Segre). The

present study is aimed at describing radiological

methods identification sex of skull by 3D

computer tomography image.

OBJECTIVE

The purpose of this study was to evaluate the

significance for sex determination of the

measurement of the area formed by projection

of 3 craniometric points related to the mastoid

process (the porion, asterion, and mastoidale

points) of 3D reconstructed computed

tomography (CT) of skull and the result is

compare the result of manual measurement of

the area formed by projection of 3 craniometric

points related to the mastoid process.

METHOD

3D reconstructed CT of 40 males and 40 females

were analysed. The three craniometric points

were located and marked on both side of the 3D

skull and the measurement was done by

advanced post processing techniques. In manual

measurement 40 males and 40 females skull

were analyzed by digital caliper (0.01mm). The

area of mastoid triangle was calculated by means

of the Heron’s formula. The result of CT reading

and Manual reading is analyzed for the sex

determination and the both result is compared.

RESULT

From this study the areas of the male CT

measurement of Mastoid is 828.53±118.54 mm2

which is greater than female Mastoid which is

578.24±71.53 mm2 (T test value 9.901and P

Value 001). Areas of the male Mastoid which is

measured by manual 805.87±90.99 mm2 which is

greater than female Mastoid which is

620.56±90.63 mm2 (T test value 10.202and P

Value 001) and there is no significant deference

between CT scan and Manual method of area

measurement of mastoid process in sex

determination of south Indian population Table

– 1:- The areas of male and female mastoid

measured using manual and 3D computer

Tomography imaging. (About here)

DISCUSSION

The analysis of the mastoid process

characteristics is important in the determination

of sex for forensic purposes. The mastoid region

used in this study, being a part of the temporal

bone, is recognized as being the most protected

and resistant to damage, due to its anatomical

position at the base of the skull. This has been

demonstrated by Kloiber (1953), Wells (1960),

Schäefer (1961), Gejval (1963), and Spence

(1967), as cited by Wahl and Henke10 (1980)

According to Paiva & Segre (2003), When it is

higher than or equal to 1447.40 mm2 singel side,

the skull is recognized as male skull and When









Pag

e53

8

the total area was lower than or equal to

1260.36 mm2 single side, the skull is recognized

as female skull. In our present study The areas of

the male right and left side From our study the

areas of the male CT measurement of Mastoid is

828.53±118.54 mm2 which is greater than female

Mastoid which is 578.24±71.53 mm2 for single

side (T test value 9.901 and P Value .001). Areas

of the male Mastoid which is measured by

manual is 805.87±90.99 mm2 for single side

which is greater than female Mastoid which is

620.56±90.63 mm2 for single side (T test value

10.202and P Value .001).

CONCULSION

The area of the mastoid triangle measured by

manual and 3D reconstructed CT skull are used

to determine the sex of the skull and there is no

significant deference between CT scan and

Manual method of area measurement of

mastoid process in sex determination of south

Indian population .

ACKNOWLEDGEMENT

Author acknowledges the immense help

received from the scholars whose articles are

cited and included in references of this

manuscript. The authors are also grateful to

authors / editors /publishers of all those articles,

journals and books from where the literature for

this article has been reviewed and discussed. The

author is highly thankful to the referees for their

very constructive, valuable suggestions and

useful technical comments, which led to a

significant improvement of the paper.

REFERENCES 1. Kalmey, J. K. & Rathbun, T. A. Sex determination by

discriminant function analysis of the petrous portion of

the temporal bone. J. Forensic Sci., 41:865-7, 1996

2. De Paiva, L. A. & Segre, M. sexing the human skull

through the mastoid process. Rev. Hosp. Clin. Fac.

Med. São Paulo, 58:15-20, 2003.

3. Standring S, ed. Gray’s Anatomy. 40th Ed.,

Philadelphia, Elsevier, Churchill Livingstone. 2005.

4. Kemkes, A. & Gobel, T. Metric assessment of the

"mastoid triangle" for sex determination: a validation

study. J.Forensic Sci., 51:985-9, 2006

5. Walsh M, Reeves P, Scott S. When disaster strikes; the

role of the forensic radiographer. Radiography 2004;

10:33-43.

6. Al Ekrish AA, Ekram M. A comparative study of the

accuracy and reliability of multidetector computed

tomography and cone beam computed tomography in

the assessment of dental implant site dimensions.

Dental Maxillofacial Radiol 2011; 40:67-75.

7. SUAZO, G. I. C.; ZAVANDO, M. D. A. & SMITH, R. L. Sex

determination using mastoid process measurements

in Brazilian skulls. Int. J. Morphol., 26(4):941-944, 2008









Pag

e53

9

Table – 1:- The areas of male and female mastoid measured using manual and 3D computer

Tomography imaging.

CT scan Vs.

Manual

Measurement

gender N Mean Std. Deviation T test P Value

CT scan

Measurement

male 40 828.53 118.54 9.901 <0.001

female 40 578.24 71.53

Manual

Measurement

male 40 805.87 90.99 10.220 <0.001

female 40 620.56 90.63

*Corresponding Author: Suresh Sukumar Assistant professor, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. udupi – 576104. Email: [email protected].








Research Article

Biological Sciences


NAGAKUMAR J S*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e54

0

MANAGEMENT OF COMPOUND FRACTURES OF TIBIA BY EXTERNAL FIXATION:

A PROSPECTIVE STUDY FROM A RURAL HOSPITAL OF SOUTH INDIA

NAGAKUMAR J S1, S S GUBBI2, S B KAMAREDDY3. 1Assistant professor in department of orthopaedics, Sri Devaraj Urs medical college, Kolar, Karnataka

2Professor in department of orthopaedics, M R medical college, Gulbarga, Karnataka 3Associate Professor in department of orthopaedics, M R medical college, Gulbarga, Karnataka

ABSTRACT Background: Introduction of external fixator is a revolution in the management of compound fractures tibia for it

has saved many limbs from amputation. Objectives: to study the usage of external fixator in the treatment of

compound tibial fracture and to assess the functional outcome of patient. Methods: During October 2005 to

September 2007, 20 cases of open fracture tibia were selected based on Gustilo Anderson’s classification with

exclusion of Type 1 and type 3C wounds. Fractures were managed by using bilateral frame with transfixing pins and

biplanar fixators. Patients were followed at 4 weeks interval with clinical and radiological assessment. The results

were classified as good, moderate and poor depending upon the degree of deformity, degree of shortening, range

of motion at neighbouring joint. Results: All patients were male belonging to age group 20 - 40 years with road

traffic accidents. Eighty percent of the fractures were of Type III with middle 1/3 of leg common site. Good outcome

was noted in 14 cases (70%) while 15% each of moderate and poor outcome. Conclusions: External fixators could

be the choice of fixation in open fracture tibia and was found to be simple, economical and effective.

KEY WORDS Open tibial fracture, external fixation, rural hospital

INTRODUCTION

The management of compound fractures of tibia

is a challenge to orthopedic surgeon. Number of

methods have described and tested with varying

results on the management of compound

fractures1-3.Introduction of external fixator is a

revolution in the management of compound

fractures tibia, for it has saved many limbs from

amputation4-7.

OBJECTIVES

1. To study the usage of external fixator in the

treatment of compound tibial fracture,

2. To assess the functional outcome of patient

with reference to rate of fracture union and

range of movement at ankle joint and knee

joint and to study the restoration of function

of the limb.

METHODOLOGY

The study was conducted in Basaveshwar

Teaching & General Hospital attached to

Mahadevappa Rampure Medical College,

Gulbarga. During October 2005 to September

2007, 20 cases of open fracture tibia were

selected based on Gustilo Anderson’s

classification 8(table 1) as Type 1, 2, 3A, 3B and #

based on the size of wound, degree of soft tissue

injury, level of contamination, degree of bony

injury and presence of neurovascular injury. Type









Pag

e54

1

1 and type 3C wounds were excluded as type I

were treated with primary intra-medullary

interlocking nailing and type 3C were referred to

vascular surgeon due to associated vascular

injury. Patients belonging to age groups 20 to 40

years were included. Patients were initially

examined in casualty regarding head injury,

respiratory, cardiovascular and abdomen status.

Intravenous fluids, antibiotics and intramuscular

tetanus toxin and tetanus immunoglobulin were

given. After haemo-dynamically stabilized were

shifted to major OT for wound debridement and

external fixator application within 24 hours.

OPERATIVE TECHNIQUES (Fig 1 & 2)

The basic frame components are the adjustable

clamps used for articulation of the schanz pin to

the steel tube. The clamps allow screw insertion

in any desired plane, Hollow tubes with outside

diameter of 11mm and length from 100-600mm,

Schanz pins of diameter 4.5mm and Steinmann

pins are used. The triple trocar is a universal

instrument for guiding insertion of the schanz pin

with conus. It consists of 5mm and 3.5mm drill

sleeve with a 3.5mm trocar. The pin is first

predrilled with a 3.5mm drill bit, then over drilled

in the near cortex with a long 4.5mm drill bit. The

universal chuck with a T-handle holds the schanz

pins during insertion, while the wrenches are

used to tighten the clamp nuts. Other

instruments include the open compressor and

distractor9-10.

The fixator components are generally assembled

into one of two basic frame types of

configurations namely unilateral uniplanar,

unilateral biplanar.

The one plane configurations are less obstructive

and generally suffice for most injury situations.

Two plane frames are more effective in

neutralizing multi directional bending and

torsional movements. However, they are only

needed when dealing with severe comminuted

fractures or with bone loss. The safe corridor for

schanz pin insertion in the tibia is at level proximal

to the tibial tubercle, schanz pins can be safely

inserted within the arc of 220 degree. At level B,

just below the tibial tubercle, the safe arc

decreases to 1400. At level C, in the distal third of

the leg, the safe arc remains 1400 but anterior

tibial vessels and deep peroneal nerve become

vulnerable as they cross the lateral tibial cortex.

At level D, above the ankle joint, the safe arc is

120 degree. At levels E and F, steinmann pins in

the tarsal or metatarsal bones may be used to

splint the ankle joint if neurological or soft tissue

injuries prevent the application of an external

support. External fixators are usually applied

under general or regional anaesthesia with the

limb draped free so as to leave all pertinent

skeletal land marks visible11. The insertion of

schanz pin should be done in the following

manner.

a. Assemble the triple trocar and penetrate soft

tissue (through a stab incision) down to the

bone surface.

b. Remove the trocar and drill through both

cortices using a long 3.5mm drill bit.

c. Remove the drill sleeve, through the

remaining 6 or 5mm sleeve over drill the near

cortex using a long 4.5mm drill bit. The use of

oscillating attachment combined with the

three fluted drill bit is recommended.

d. Insert the depth gauge probe through the drill

sleeve hooking the far cortex.

e. Loosen the locking pin, advance the knurled

disk to the top of the drill sleeve and tighten

the locking pin.

f. Remove the probe, place the threaded tip of

the schanz pin in to the schanz pin recess of

the knurled disk

g. Advance the universal chuck over the non-

threaded end of the schanz pin until the tip of

the probe touches the end of the universal









Pag

e54

2

chuck. Tighten the universal chuck on to the

schanz pin in this position.

h. Insert the schanz pin until the universal chuck

nearly touches the top of the drill sleeve, the

schanz pin is now fully inserted into far

cortex.

i. Remove the drill sleeve and attach the

adjustable clamp.

Unilateral uniplanar frame: This is applied as

follows

Step I: Application of schanz pin close to the

distal joint. The tube with the planned number of

adjustable clamps is fixed to the schanz pin.

Step II: Application of second schanz pin across

the most proximal adjustable clamp. At this time,

three dimensional reduction of fracture is easy,

observing axis and rotation of the foot by

comparing it with uninjured leg.

Step III: The inner schanz pin is inserted about

2cm from the fracture area. The tubular fixator

allows individualization of schanz pin placement

according to fracture configuration. Prevention

of drop foot by connecting metatarsal I and II to

tube by means of schanz pin.

Unilateral Biplanar Frame: This consists of 2

interconnects simple unilateral frames to allow

optimal wound access. The plane for the second

frame should lie at an angle of 60-100 degree with

the plane of the first frame. First the ventral

fixator is applied in a nearly sagittal plane aiming

towards the medial posterior tibial cortex. Next

the medial fixator is applied at an angle between

60-100 degree and fixed with either 2 or 4 schanz

pins. The tubes are interconnected by smooth

pins.

Maneuvers for reduction of Fracture: In simple

transverse fractures, stabilization at the fracture

site is achieved by compressing main fragments

against each other, taking care to avoid the

tendency to angulate the fragments. The fixators

are then used as a neutralization frame. In

diaphyseal fractures, however this maneuver

should only be considered in treating simple two

fragment fracture with relatively long contact

areas. In comminuted fracture neutralization

frame is applied.

To diminish motion at fracture site and increase

the stiffness of frame, the following was

considered:

a. Principal frame should be applied in saggital

plane.

b. Preloading of schanz pins

c. Increasing the number of pin in each bony

fragment

d. Increasing the pins spread with in each

fragment

e. Reducing the distance between bone and the

longitudinal tube.

Frame application: It depends on site of wound

that is opposite to the site of wound. If a soft

tissue coverage procedure is required lateral on,

then the side of frame application should be such,

as to leave enough free area for the plastic

surgery. The fixator was placed in neutralization

mode in case of comminuted and butterfly

fragment fractures, compression mode incase of

transverse, oblique and segmental fractures so as

to narrow fracture gap and improve stability.

Relaxing skin incisions were placed around the pin

tracts to avoid skin compression, bones were

covered with overlying muscles, skin

approximated with stay sutures. The foot and

ankle were manipulated at the end of procedure

to ensure absence of musculotendinous tethering

by transverse pins. All these patients were

followed at 4 weeks interval. Clinical and

radiological assessment of the patients at follow

up comprised of wound healing, tenderness at

fracture site, degree of weight bearing, presence

of callus, gap at fracture site, sclerosis at fracture

ends and obliteration of medullary canal. Once

the wound is clean and covered with healthy

granulation tissue, plastic surgeon opinion sought









Pag

e54

3

and treated accordingly. Static Quadriceps

exercise was started immediate post-operatively.

Knee and ankle motion was allowed 4 week from

the operative day. Partial weight bearing was

allowed in non-comminuted fractures 4-6 weeks

later. In case of comminuted fractures 10-12

weeks from the day of surgery. Full weight

bearing was allowed when there was radiological

evidence of union. The results were classified as

good, moderate or poor depending upon the

degree of deformity, degree of shortening, range

of motion at neighbouring joint. The degree of

deformity and limb length discrepancy was

assessed using the modified Anderson and

Huntchins Criteria (table 2). The ankle and knee

movements were graded as Full range-Normal;

Significant loss of movement -In the knee - loss of

extension up to 100, and flexion up to 400, ankle

- 250, but < 500 of flexion / extension; Insignificant

loss - Anything less than but above the normal.

Severe loss - both in knee and ankle, with loss of

> 500 of flexion and extension. The results were

classified as good, moderate and poor (Table 2).

Table 1: Gustilo – Anderson’s classification of type of fracture

Types No. of Patients

Type-I 0

Type -II 4

Type – III A 8

Type III B 8

Type – III C 0

Table 2: Modified Anderson &Huntchins Criteria to assess degree of deformity&limb length

discrepancy.

Results Shortening Grade of deformity in Angulation (Malunion)

Good <1 cm Up to 50Varus / Valgus up to 100 Anterior / Posterior

Moderate 1-2cm 5-100 Varus / Valgus

10-200 Anterior / Posterior

Poor > 2cm > 100 Varus / Valgus

> 200 anterior / Posterior









Pag

e54

4

Table 3: Management of Associated Injuries

Associated Injury Gustilo’s Type Side/Type Management

Closed fracture both bone

Right Forearm

Type –II Right Closed reduction and internal

fixation with square nail

Closed Fracture right

Femur M/3rd

Type III-B Right Intra-medullary interlocking nail

for the fracture femur

Crush injury Right foot

with fracture 1st and 2nd

metatarsal bone

Type III A Right Wound debridement and fixed

with k-wire

Fig 1: Wound debridement









Pag

e54

5

Fig 2: Open wound









Pag

e54

6

Fig 3: Operative procedure in progress

Fig 4: Operative procedure completed with fixators









Pag

e54

7

Fig 5a: Results showing good outcome

Fig 5b: Results showing good outcome

OBSERVATIONS

All patients were male belonging to age group 20

- 40 years with road traffic accidents. Eighty

percent of the fractures were of Type III with

middle 1/3 of leg common site. Good outcome

was noted in 14 cases (70%) while 15% each of

moderate and poor outcome (Fig 3a & 3b). Eight

patients needed split skin grafting. One patient









Pag

e54

8

had contra lateral closed fracture of both bone for

which close reduction and internal fixation with

square nailing was done. In two patients, one with

associated ipsilateral closed fracture shaft middle

third of right femur was treated by IM interlocking

nail and another patient associated with

ipsilateral crush injury of right foot with

metatarsal I and II fractures was managed by K-

wire fixation and split skin grafting (Table 3). In

the 4 Gustilo – Anderson type 2 fractures which

were 1 oblique, 1 segment, transverse and 1

butterfly external fixator was removed after 6

weeks and intramedullary interlocking nailing was

done. In 8 Gustilo –Anderson Type 3 A fractures

which were 2 butterfly and 6 comminuted, the

butterfly fractures were treated with

intramedulary nailing 6 weeks after being in

external fixator. The remaining 6 which were

comminuted, 5 were treated with external fixator

for 4 months followed by POP cast (PTB cast) for

another 6 weeks. One of them showed signs of

sclerosis of 1 to 2 fragments which were excised

after 4 months. In the 8 cases of Gustilo –

Anderson type – 3B all were comminuted

fractures, 6 were treated with external with

external fixator for 5-6 months followed POP cast

for another months 2 cases showed. Signs of non-

union and had cancellous bone grafting, after 6

months of external fixator application followed by

POP cast above Knee next 2 months.

DISCUSSION

Seventy percent of patients in the present study

had good results. All were male with mean age of

28years whereas in Thakur and Patanakar12 study,

females and males represented 16.5% and 83.5%

respectively with mean age of 38 years suggesting

higher level of activities and mobility in the these

age groups. The present study documents road

traffic accident as the cause of injury in all cases

whereas on an average 85.9% and 87.3% road

traffic accidents were recorded by Pedro

AntichAdrover et al13 and Thakur and Patankar12

series respectively. An equal number of cases of

Type III A and Type IIIB (40% each) and type II

(20%) noted in the present study in comparison

with Thakur and Patankar12 series where 12 spiral

(or) long oblique, 27 Transverse (or) short

oblique, 40 comminuted fractures were

documented. The present study also records 50%

of the fracture middle third, 30% fracture distal

third and 20% fracture proximal third while it was

78% middle third, 10% distal third and 4%

proximal third from a study by Henley MB, et al14.

In our series, 8 patients underwent split skin

grafting (40%), 1 patient muscle pedicle flap (10%)

and one case of type III A and 2 cases of type IIIB

bone grafting where as in the Thakur and

Patankar12 series, skin grafting was required in 43

patients, 5 flap coverage and 44 cases (60.3%) of

bone grafting. Superficial wound infection (20%)

and pin tract infection (10%) were the common

complications in the present study whereas

superficial and deep wound infections of 42.2%

and 16.1% respectively were noted in the series

by Bhandariet al15suggesting infection rate was

lower in the present study and was successfully

managed with parenteral antibiotics.

CONCLUSION

Open fractures of tibia are quite common,

because of its subcutaneous location, high energy

trauma, which is quite often encountered during

high speed moving vehicles, especially on national

highway. The study shows that reasonable

outcome may be attained in open tibial fractures

with the external fixation technique allowing

early definitive treatment. Complications are

minimal with good range of movements in knee

and ankle.

Competing interest: NIL









Pag

e54

9

Author’s contributions: NAGAKUMAR J S

participated in acquisition of data, literature

search and carried out analysis, interpretation,

and drafting the manuscript. S S GUBBI, S B

KAMAREDDY did acquisition of data and

participated in drafting and revising the

manuscript. All authors read and approved the

article.

REFERENCES 1. Dillin L, Slabaugh P: Delayed wound healing, infection,

and non-union following open reduction and internal

fixation of tibial plafond fractures. J Trauma 1986;26

(12):1116–1119.

2. McFerran MA, Smith SW, Boulas HJ, Schwartz HS:

Complications encountered in the treatment of pilon

fractures. J Orthop Trauma 1992;6(2):195–200.

3. Teeny SM, Wiss DA: Open reduction and internal

fixation of tibial plafond fractures. Variables

contributing to poor results and complications.

ClinOrthopRelat Res 1993, 292:108–117.

4. Sisk TD: General principles and technique of external

fixation. ClinOrthopRelat Res 1983; 180: 96-100.

5. Sisk TD. External fixation: historic review, advantages,

disadvantages, complications and indications. Clin

Orthop Relat Res1983; 180: 15-22.

6. Edwards CC. Staged reconstruction of complex open

tibial factures using Hoffman external fixation: Clinical

decisions and dilemmas.ClinOthop 1983; 178: 180.

7. Caudle, R.J., and Stern, P.J. Sever open fractures of the

tibia. J Bone Joint SurgAm 1987; 69(6): 801-807.

8. Gustilo RB, Mendoza RM, Williams DN. Problems in the

management of type III (Severe) open fractures: New

classification of type III open fractures.JTrauma 1984;

24: 742.

9. Weber, B.S. and Mageri F. The external fixator. New

York, spring – Verlag. 1985.

10. Weber B.G and Magerl F. “A.O. Manual of external

fixation”, Berlin, Hedelberg: Springer – Verlag. 1985, pp-

459.

11. Behrens F, and Searls K. External fixation of the Tibia

Basic concepts and prospective evaluation.J Bone Joint

Surg 1986, 68B (2):246-254.

12. Thakur AJ andPatnakar J. Open tibial fractures –

Treatment by uniplanar external fixation and early bone

grafting. J Bone Joint Surg (Br) 1991; 73-B: 448-51.

13. Pedro Antich – Adrover, David Martin – Garin, Juan

Murias-Alvarez, Carlos Puente-Alonso. External fixation

and secondary intramedullary nailing of open tibial

fractures. J Bone Joint Surg (Br) 1997; 79-B: 433-7.

14. Henley MB, Chapman JR, Agel J, Harvey EJ, Whorton

AM, Swiontkowski MF. Treatment of type II, III A, and

III B open fractures of the tibial shaft: a prospective

comparison of unreamed interlocking intramedullary

nails and half-pin external fixators: JOrthop Trauma

1998; 12(1): 1-7.

15. Bhandari M, Guyatt GH, Swionkowski MF, Schemitsch

EH. Treatment of open fractures of the shaft of tibia. J

bone Joint Surg Br2001; 83(1): 62-8.

*Corresponding Author: NAGAKUMAR J S Assistant professor in department of orthopaedics, Sri Devaraj Urs medical college,Kolar, Karnataka







Research Article

Biological Sciences


Andrew Nwaka C*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e55

0

COMPARATIVE EFFECTS OF CITRULLUS LANATUS AND CAJANUS CAJAN DIETS

ON THE LIPID PROFILE AND BODY WEIGHT OF ALBINO RATS

*Andrew Nwaka C.1, Christian Odunze S.1, John Ihedioha I.2 Ugwu Okechukwu P.C.3

Ossai Emmanuel C3., Ada Ikeyi4 and Bayim P.R.5 1Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria.

2 Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine, University of Nigeria,

Nsukka, Enugu State, Nigeria. 3Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria.

4Department of Science Laboratory Technnology, Institute of Management and Technology, Enugu, Nigeria. 5Department of Sciences, Cross-River State University of Technology, Calabar.


ABSTRACT This study evaluated the comparative effects of diets containing 50 and 70% Citrullus lanatus or Cajanus cajan on

the serum lipid profile and body weight of albino rats. Thirty male albino rats randomly assigned into five groups (A

– E) of six each were used for the study. The groupings and their diets were as follows: A – Standard rat feed (control),

B – diet containing 50% C. lanatus, C – diet containing 70 % C. lanatus, D - diet containing 50% C. cajan, and E - diet

containing 70% C. cajan. The rats were fed their group specific diets for 28 days during which they were weighed at

weekly intervals. At the end of the 28 days of feeding, blood was collected from the rats and the serum lipid profile

was assayed following standard procedures. Results showed that the rat groups fed diets containing 50 and 70 % C.

lanatus had a significantly higher (p < 0.05) serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-

C) and low density lipoprotein cholesterol (LDL-C) but significantly lower (p < 0.05) serum triglyceride when

compared to the control group fed on standard rat feed. The rat group diet fed containing 70% C. cajan however

had a significantly lower serum TC, HDL-C, VLDL-C and triglyceride when compared to the control group fed standard

rat feed. The rat group fed diet 70% C. cajan also had a significantly higher (p < 0.05) weight gain all through the

study period when compared to all other groups. It was concluded that findings in this study suggest that diets

containing 70% C. cajan can be recommended for its ability to lower serum cholesterol and triglycerides, and thus

can possibly be used to prevent and manage atherosclerosis.

KEY WORDS Serum lipid profile, body weight, rats, Citrullus lanatus, Cajanus cajan

INTRODUCTION

Citrullus lanatus and Cajanus cajan are legume

staples widely consumed in Nigeria and other

parts of the world. Legumes are edible seeds of

leguminous plants, belonging to the Leguminosae

family. Legumes can be divided into two main

broad classes, pulses and oil seeds. Pulses are the

dried edible seeds of cultivated legumes and they

include peas, beans, pigeon pea (Cajanus cajan)

etc. (Olusanya, 2008). The oil seeds are legume

seeds that contain appreciable amount of oil and

they include soya beans, melon (Citrullus lanatus)

and groundnut. Legumes constitute the second

largest family of seed plants and contain about

600 genera with 13 species. Apart from their

nutritive value to human beings, they are also










Pag

e55

1

important in fixing atmospheric nitrogen, thereby

making the element available to other plants in

the soil (Olusanya, 2008). Legumes are rich in

protein, but their protein has a well-recognized

deficiency of the sulphur amino acids, methionine

and cystein but is comparatively rich in lysine

(Ihekoronye and Ngoddy, 1987). Cereals on the

other hand contain these amino acids which

legumes lack. Hence a combination of legumes

and cereals such as beans and maize would

provide an ideal source of dietary protein for

human beings.

Cajanus cajan (pigeon pea) is a tropical legume

grown mainly in India. The true origin of pigeon

pea is still disputable. However, immigrants who

moved to Africa to become railway workers and

storekeepers (Hillocks et al., 2000) most likely

introduced the crop into East Africa from India in

the 19th century. The legume is increasingly

becoming an important sustenance crop in the

whole of Africa (Johansen et al., 1993). Some

potential uses of pigeon pea in Africa include the

production of noodles (Singh et al., 1989) and

other fermented products (Onofiok et al., 1996).

Pigeon pea leaves have been used to treat malaria

(Aiyeoloja and Bello, 2006) in Nigeria, while in the

Southern African; pigeon pea is currently one of

the indigenous crops being promoted for

potential medicinal uses (Mander et al., 1996).

Also, clinical studies have reported the seed

extracts to inhibit red blood cell sickling and as

potential benefit for people with sickle cell

anaemia (Akinsole and Solanke, 2011).

Citrullus lanatus (melon) is a creeping annual

legume which belongs to the Eucurbitaceae

family (Olusanya, 2008). It is a water-loving crop

and therefore is cultivated predominantly in the

southern part of Nigeria (Olusanya, 2008). The flat

seeds are embedded in white fleshy material in a

circular fruit. Melon seed is rich in protein and oil

(Fuller and Harvey, 2006; Olusanya, 2008) and low

in carbohydrate but contains good amounts of

minerals especially phosphorus, magnesium and

potassium and a fair amount of carotene and

vitamin D (Olusanya, 2008). Melon is a high

calorie source because of its high oil content.

Melon seeds are an excellent source of

tryptophan and arginine (Olusanya, 2008; Ojieh et

al., 2008).

Cholesterol and triglycerides are the major

blood/serum lipids of clinical significance in

humans and animals (Ononogbu, 1988; Oslon,

1998; Nelson and Cox, 2000). Cholesterol is an

essential component of mammalian cell

membranes which play major roles in membrane

permeability and fluidity and also as precursor of

bile acids, steroid hormones and fat soluble

vitamins (Oslon, 1998; NCEP, 2002). Triglycerides

play important role in metabolism as energy

sources and transporters of dietary fat

(Ononogbu, 1988; Nelson and Cox, 2000). Though

cholesterol and triglycerides are physiologically

important in the body, high levels of them in the

blood have been found to be a major risk factor

for the development of atherosclerosis (Brown

and Goldstein, 1992; Oslon, 1998; Schoen, 2004;

Brunzell et al., 2008). The possible pathological

consequences of atherosclerosis include

myocardial infarction (heart attack), cerebral

infarction (stroke), aortic aneurysms, peripherial

vascular disease, sudden cardiac death, chronic

ischaemic heart disease etc (NCEP, 2002; Schoen,

2004; Brunzell et al., 2008).

The consumption of food items that will

significantly reduce the overall blood levels of

cholesterol and triglyceride and / or those

components of cholesterol that have been

associated with increased risk of atherosclerosis

is one of the major strategies at prevention and

management of atherosclerosis (Law, 1999;

NCEP, 2002; Brunzell et al., 2008). Hence the

present study, which evaluated the comparative

effects of feeding diets containing 50 or 70 %









Pag

e55

2

Citrullus lanatus or Cajanus cajan on the serum

lipid profile and body weight gain of albino rats.


The rats used for the study were mature male

Sprague-Dawley albino rats weighing 150 – 250

grams, procured from the Laboratory Animal

House of Faculty of Veterinary Medicine

University of Nigeria, Nsukka. The rats were

randomly assigned to five groups (groups A – E) of

six each and acclimatized for seven days before

the commencement of the study. The rats were

kept in clean cages in a fly-proof animal house and

provided with feed and water ad libitum all

through the study. Guidelines for the humane use

and handling of laboratory animals for research

(NAS, 2011) were followed all through the study.

The standard rat feed used in this study was

sourced from Grand Cereals and Oil Mills Ltd., Jos

Nigeria. The C. lanatus and C. cajan seeds were

procured and processed into a mash.

The proximate composition of the standard rat

feed, C. cajan and C. lanatus used for the study

were determined following standard procedures.

The protein content was determined by micro

Khjelldahl method, while the ash, moisture and

fat contents were determined by gravimetric

method (AOAC, 2000; Changsam, 2003). The total

carbohydrate was then estimated using the

standard formula (Bemiller, 2003).

After acclimatization, the rats were weighed and

the different groups were fed their group-specific

diets for 28 days. The group specific diets were as

follows: Group A – Standard rat feed (control),

Group B – diet containing 50% C. lanatus, Group

C – diet containing 70 % C. lanatus, Group D - diet

containing 50% C. cajan, and Group E - diet

containing 70% C. cajan. The rats were further

weighed at weekly intervals and weight gain was

computed by comparing with the body weight

before commencement of feeding of the

experimental diets. After the 28 days of feeding

the experimental diets, 3 ml of blood was

collected from the orbital sinus of the retrobulbar

plexus of the rats following the orbital technique

(Bolliger and Everds, 2010). The blood dispensed

into clean plain glass test tubes and allowed to

stand for 30 minutes to clot. After clotting, it was

centrifuged at 3,000 revolutions per minute to

separate the serum from clot. The clear serum

was aspirated into clean labeled sample bottles

and used immediately for the lipid profile assay

following standard procedures.

The serum lipid profile was assayed using Quimica

Clinica Aplicada (QCA) test kits (QCA, Spain). The

serum total cholesterol (TC) was determined by

the enzymatic colorimetric method (Allain et al.,

1974). The serum high density lipoprotein

cholesterol (HDL-C) was determined by the

dextran sulphate-magnesium (II) precipitation

method (Albers et al., 1978). The glycerol

phosphate oxidase enzymatic method was used

to determine the serum triglyceride (Bucolo and

David, 1973). The very low density lipoprotein

cholesterol (VLDL-C) was calculated by dividing

the serum triglyceride by 5, while the serum low

density lipoprotein cholesterol (LDL-C) was

calculated using the Friedewald formular

(Friedewald et al., 1972; Warnick et al., 1990).

Data generated from the study were subjected to

one way analysis of variance and variant means

were compared with the control post hoc using

the least significant difference (LSD) method.

Significance was accepted at p < 0.05.

RESULTS

Results of the proximate analysis for the standard

feed, C. cajan and C. lanatus showed that C. cajan

had the highest percentage composition of crude

protein (26.52%) when compared to that of the

standard feed (19.53%) and that of the C. lanatus

(23.4%) (Table 1). The proximate analysis also

showed that C. lanatus had the highest

percentage composition of fat (45.7%) when









Pag

e55

3

compared to that of the standard feed (7.86%)

and C. cajan (3.14%) (Table 1). The standard rat

feed had the highest percentage composition of

carbohydrate (56.91%) while C. lanatus has the

least (10.6%) (Table 1).

Table 1. The proximate composition of the rat feed, Cajanus cajan and Citrullus lanatus used for the

study.

Composition Means ± standard deviation.

Standard rat feed Cajanus cajan Citrullus lanatus

Crude protein

19.53 ± 0.44

26.52 ± 0.43

23.40 ± 0.20

Moisture

6.11 ± 0.26

7.46 ± 0.25

5.80 ± 0.18

Ash

5.76 ± 0.44

4.25 ± 0.31

4.60 ± 0.30

Crude fibre

3.83 ± 0.11

6.99 ± 0.34

12.00 ± 0.11

Fat

7.86 ± 0.27

3.14 ± 0.28

45.70 ± 0.17

Carbohydrate

56.91 ± 0.62

51.64 ± 0.54

10.60 ± 0.20

Table 2. The serum lipid profile of albino rats fed for 28 days with diets containing varied percentages

of Cajanus cajan or Citrullus lanatus.

Groups

(Diets fed)

Means ± standard deviation.

Total

cholesterol

(mg/dl)

HDL-C (mg/dl) LDL-C

(mg/dl)

VLDL-C

(mg/dl)

Triglyceride

(mg/dl)

Group A

(Standard rat

feed)

56.10 ± 11.63 27.15 ± 1.92 16.85 ± 7.83 12.10 ± 3.37

60.49 ± 18.66

Group B

(50% C. lanatus)

85.61 ± 8.64* 49.41 ± 14.60* 28.95 ± 7.78* 7.24 ± 3.94 36.20 ± 19.68*

Group C

(70% C. lanatus)

75.96 ± 11.04* 39.61 ± 9.20* 30.38 ± 6.31* 5.96 ± 1.84* 29.80 ± 9.20*

Group D

(50% C. cajan)

50.82 ± 5.55 21.64 ± 0.63* 20.79 ± 8.05 8.30 ± 3.36 41.94 ± 16.80*

Group E

(70% C. cajan)

43.71± 7.52 20.83 ± 0.95* 16.62 ± 5.18 6.27 ± 1.48* 31.34 ± 7.41*

* Asterisk superscript on any mean indicates that it is significantly different from the control group (A) fed standard rat feed

(p < 0.05).









Pag

e55

4

Table 3. The body weight gain of albino rats fed for 28 days with diets containing varied percentages

of Cajanus cajan or Citrullus lanatus.

Groups

(Diets fed)

Means ± standard deviation.

Day 7

Day 14

Day 21

Day 28

Group A

(Standard rat

feed)

10.06 ± 3.29 24.12 ± 9.27 35.05 ± 5.64 38.69 ± 3.89

Group B

(50% C.

lanatus)

7.34 ± 2.86 18.68 ± 7.87 35.87 ± 6.30 39.94 ± 5.94

Group C

(70% C.

lanatus)

7.77 ± 3.62 19.54 ± 8.46 32.17 ± 4.18 36.77 ± 4.80

Group D

(50% C. cajan)

14.67 ± 5.91 33.34 ± 8.14 36.17 ± 4.18 36.77 ± 4.80

Group E

(70% C. cajan)

28.03 ± 8.87* 60.06 ± 7.94* 65.53 ± 9.83* 69.16 ± 6.41*

* Asterisk superscript on any mean indicates that it is significantly different from the control group (A) fed standard rat feed

(p < 0.05).

The rat groups fed diets containing 50% and 70%

C. lanatus (Groups B and C) had a significantly

higher (p < 0.05) serum TC, HDL-C and LDL-C when

compared to the control group fed standard rat

feed, while the group fed 70% C. cajan (Group E)

had a significantly lower (p < 0.05) serum TC, and

HDL-C when compared to the control group fed

standard rat feed (Table 2). The serum VLDL-C of

the rat groups fed 70% C. lanatus (Group C) and

70% C. cajan (Group E) were significantly lower (p

< 0.05) than that of the rat group fed standard rat

feed (Table 2). However, the serum triglyceride of

groups B, C, D and E rats were significantly lower

(p < 0.05) than that of the rat group fed the

standard rat feed (Table 2).

The body weight gain computations showed that

the rat group fed with 70% C. cajan diet (Group E)

had significantly higher (p < 0.05) body weight

gain when compared to all other rat groups all

through the study (Table 3).

DISCUSSION

The significantly higher total serum TC, HDL-C and

LDL-C recorded for the rat groups fed diets

containing 50% and 70% C. lanatus can be

attributed to the high fat composition of C.

lanatus as indicated by its proximate

composition. This suggests that consumption of

diets with up to 50 and 70 % C. lanatus could

predispose the consumer to development of

atherosclerosis and its associated pathological

consequences (Brown and Goldstein, 1992;

Oslon, 1998; Schoen, 2004; Brunzell et al., 2008).

In contrast, the significantly lower serum TC,

VLDL-C and triglyceride recorded for the rat group

fed 70% C. cajan could be as a result of its low fat

composition, and by implication, consumption of

diets with up to 70% C. cajan could be used

therapeutically to reduce blood TC, VLD-L and

triglyceride and thus prevent/manage

atherosclerosis. The findings in this study of the

ability of diet containing 70% C. cajan to reduce

blood cholesterol is in agreement with the reports









Pag

e55

5

of Luo et al. (2008) that showed that

administration of extracts of C. cajan was able to

significantly reduce serum total cholesterol of

hyperlipidemic mice.

The findings in this study of significantly higher

serum HDL-C in the rat groups fed 50 and 70 % C.

lanatus is a positive finding as HDL-C, often

referred to as “good cholesterol”, is known to

facilitate the removal of “bad cholesterol” (LDL-C

and VLDL-C) from the blood vessels and

transferring them to the liver where they are

metabolized and excreted (Libby et al., 1998;

NCEP, 2002; Barter et al., 2007). The significantly

lower serum triglyceride recorded for the rat

groups fed diets containing 50 and 70% C. lanatus

and C. cajan and also the significantly lower

serum VLDL-C recorded for the rat groups fed 50

and 70% C. cajan are considered positive

developments when viewed against the

background of the role that LDL-C and VLDL-C play

in the development of atherosclerosis and its

pathological consequences (Libby et al., 1998;

Barter et al., 2007). These findings in the C. cajan

fed rats is in agreement with the reports of Luo et

al. (2008)

Results of the body weight gain computation

which showed that the rats fed with 70% Cajanus

cajan diet (Group E) had significantly higher body

weight gain all through the study when compared

to other groups is believed to be as a result of the

high carbohydrate and protein content of C. cajan

when compared to the standard feed and C.

lanatu. It was worthy of note that the higher

weight gain was not associated with a

corresponding higher serum TC, rather the C.

cajan fed rats had a lower serum TC.

Based on the results of this study, it was

concluded that rats fed diets containing 50 and 70

% C. lanatus had significantly higher serum TC,

HDL-C and LDL-C but significantly lower serum

triglyceride when compared to the control fed

standard rat feed, while the rat group fed 70 % C.

cajan had significantly lower serum TC, HDL-C,

VLDL-C and triglyceride. In addition, rats fed diets

containing 70% C. cajan had a significantly higher

body weight gain when compared to all other

groups.

REFERENCES o Aiyeoloja, A.A., Bello, O.A. (2006). Ethnobotanical

potentials of common herbs in Nigeria: A case study of

Enugu State. Educational Research and Review, I: 16 –

22.

o Akinsole, O.A., Salonke, O.O. (2011). Clinical evaluation

of extract of Cajanus cajan (ciklavit) in sickle cell

anaemia. Nigerian Journal of Animal Production, 28: 243

– 250.

o Albers, J.J., Warnick, G.R. and Cheung, M.C. (1978)

Quantification of high density lipoproteins. Lipids, 13:

926 – 932.

o Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W. and Fu,

P.C. (1974), Enzymatic determination of total

cholesterol. Clinical Chemistry, 20: 470 – 475.

o AOAC (2000). Official Method of Analysis of Association

of Official Analytical Chemist. Washington D.C., 16th

Edition, Pp. 12 – 18.

o Barter, P., Gotto, A.M., LaRosa, J.C., Maroni, J., Szarek,

M., Grundy, S.M., Kastelein, J.J.P., Bittner, V., and

Fruchart, J. (2007) HDL Cholesterol, Very Low Levels of

LDL Cholesterol, and Cardiovascular Events. New

England Journal of Medicine, 357: 1301 - 1310.

o Bemiller, N.J. (2003). Carbohydrate Analysis. Food

Analysis Third Edition. Kluwer Academic publisher, New

York. Pp. 143 – 146.

o Bolliger, A. P. and Everds, N. E. (2010) Hematology of

laboratory rodents: mouse (Mus musculus) and rat

(Rattus norvegicus). In: Weiss D. J. and Wardrop K. J.

Eds., Schalm’s Veterinary Hematology, 6th edition,

Wiley-Blackwell, Iowa, pp 852 – 862.

o Brown, M.S. and Goldstein, J.S. (1992) Koch’s postulates

for cholesterol. Cell, 71: 187 – 188.

o Brunzell ,J.D., Davidson, M., Furberg, C.D., Goldberg,

R.B., Howard, B.V., Stein, J.H. and Witztum ,J.L. (2008)

Lipoprotein management in patients with

cardiometabolic risk – Consensus statement from the

American Diabetes Association and the Amarican

College of Cardiology Foundation. Diabetes Care, 31:

811 – 822.

o Bucolo, G. and David, H. (1973) Quantitative

determination of serum triglycerides by use of enzymes.

Clinical Chemistry, 19: 476 – 482.









Pag

e55

6

o Changsam, K.C (2003). Protein Analysis. Food Analysis.

Third Edition. Kluwer Academic / Plenum Publisher,

New York, pp. 134 – 135.

o Friedewald ,W.T., Levy R.I., Fredrickson ,D.S. (1972)

Estimation of the concentration of low density

lipoprotein cholesterol in plasma, without use of the

preparative ultracentrifuge. Clinical Chemistry, 18: 499

– 502

o Hillocks, R.J., Minja, E., Nahdy, M.S,Subrahmanyam, P.

(2000). Diseases and pests of pigeon pea in Eastern

Africa. International Journal of Pest Management, 46: 7

– 18.

o Ihekoronye, A.I. and Ngoddy, P.O. (1987). Integrated

food science technology for the trpics. Macmillian

Publication, London, pp. 330 – 332.

o Johansen, C., Silim, S.N., Laxman, S. (1993) Towards a

database for pigeon pea in Africa. International

Pigeon Pea Newsletter, 18: 2 -5.

o Law, M.R. (1999) Lowering heart disease risk with

cholesterol reduction: evidence from observational

studies and clinical trials. European Heart Journal

Supplement, 1:S3-S8.

o Libby, P., Schoenbeck ,U., Mach, F., Selwyn, A.P., Ganz,

P. (1998) Current concepts in cardiovascular pathology:

the role of LDL cholesterol in plaque rupture and

stabilization. American Journal of Medicine, 104: 14S -

18S.

o Luo ,Q.F., Sun, L.,Si. J.,Y., Chen ,D.H., DU, G.H. (2008).

Hypocholesterolemic effect of stilbene extract from

Cajanus cajan on serum and hepatic lipid in diet induced

hyperlipidemic mice. Phytomedicine, 43: 145-149

o Mander, M., Mander, J., Brean, C. (1996). Promoting the

cultivation of indigenous plants for markets in

domestication and commercialization of non-timber

forest products in Agronomy system. Non-wood Forest

Products, 9: 298– 104.

o NAS (National Academy of Sciences) (2011) Guide for

the Care and Use of Laboratory Animals, 8th ed. The

National Academies Press, Washington DC.

o NCEP (National Cholesterol Education Program) (2002)

Third Report of the National Cholesterol Education

Program (NCEP) Expert Panel on Detection, Evaluation

and Treatment of High Blood Cholesterol in Adults –

Adult Treatment Panel III, Final Report, NCEP, National

Heart, Lung, and Blood Institute, National Institutes of

Health, USA.

o Nelson, D.L. and Cox, M.M. (2000) Lehninger, Principles

of Biochemistry, 3rd Ed. Worth Publishing, New York.

o Olusanya, J.O. (2008). Legume nuts. Essential of Food

and Nutrition.1st ed. Apex Books Limited, Lagos, pp. 95

– 96.

o Onofiok, N., Nnanyelugo, D.O., Ukwondi, B.E.

(1996).Usage patterns and contribution of fermented

foods to the nutrient intakes of low income house holds

in Emene, Nigeria. Plant Foods for Human Nutrition, 49:

199 – 211.

o Ononogbu, I.C. (1988) Lipid and Lipoproteins:

chemistry, methodology, metabolism, biochemical and

physiological importance. New Africa Publishing Co. Ltd.

Owerri, Nigeria.

o Oslon, R.E. (1998) Discovery of the lipoproteins, their

role in fat transport and their significance as risk factors.

Journal of Nutrition, 128: 439S – 443S.

o Schoen, F.J. (2004) Atherosclerosis. In: Kumar V., Abbas

A.K., Fausto N. (Eds.), Robbins and Cotran Pathologic

Basis of Disease, 7th ed. Saunders, Philadelphia, pp. 515

– 525.

o Singh, U., Voraputhaporn, W., Roa, P.V., Jambunathan,

R. (1989). Physicochemical characteristic of pigeon

pea and mung bean starches and their noodle quality.

Journal of Food Science, 54: 1293 – 1207.

o Warnick, G.R., Knopp R.H., Fitzpatrick V. and Branson L.

(1990) Estimating low-density lipoprotein cholesterol

by the Friedewald equation is adequate for classifying

patients on the basis of nationally recommended cut

points. Clinical Chemistry, 36: 15 – 19.

*Corresponding Author: Andrew C. Nwaka, Ph.D, Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria. E-mail: [email protected] Phone: +234 (0) 8037834171.








Research Article

Biological Sciences


Thulasi G.Pillai*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e55

7

IN VIVO RADIO PROTECTIVE PROPERTIES OF FUNGAL POLYSACCHARIDES

Thulasi G.Pillaia and K.K.Janardhananb aDivision of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi,

Thrissur- 680 653, Kerala, India. bAmala Cancer Research Centre, Amalanagar, Thrissur.


ABSTRACT In vivo radio protective properties of polysaccharides isolated from Ganoderma lucidum, a macro fungi was

examined. Swiss albino mice were exposed to 4 Gy gamma irradiation. Serum lipid peroxidation and tissue GSH

were taken end points on 1st, 3rd, 5th, 7th and 9th day after irradiation. Polysaccharides were administered just after

irradiation at 10 and 20 mg/kg body wt. Administration of polysaccharides reduced the Serum MDA levels compared

to the irradiated group. Tissue GSH was maintained at normal levels after administration of polysaccharides. The

polysaccharides possess radio protective property.

KEY WORDS Ganoderma lucidum, polysaccharides, radioprotection, radiotherapy.

INTRODUCTION

Ganoderma lucidum, commonly known as Reishi

in Japan and Ling Zhi in China, is well known for its

medicinal properties. G.lucidum contains a

number of compounds among which the

polysaccharides and triterpenoids have been

identified as the major active components. Crude

or partially purified polysaccharides of G.lucidum

have been reported to inhibit tumor metastasis in

mice1. The immunomodulating property of this

mushroom provides a promising approach for

cancer prevention and its administration is found

useful alone or in combination with

chemotherapy and radiotherapy [1]. Our earlier

studies suggest that the aqueous extract of this

mushroom has significant radioprotective activity

ex vivo [2]. Polysaccharides are among the major

source of pharmacologically active constituents

of the aqueous extract. Polysaccharides from

G.lucidum was reported to markedly restore the

mitotic activity of bone marrow cells that has

been suppressed by anti-neoplastic drugs [3] The

present study was undertaken to examine the

protection offered by the polysaccharides from

the macro fungi G.lucidum against radiation

induced damage.


Chemicals

All chemicals used in the study were of analytical

grade obtained from reputed local manufactures.

Animals

Swiss albino mice, 6-8 weeks of age and weighing

28 ± 2 g, were selected for the study. They were

maintained in air-conditioned animal house and

fed on standard mouse food and water ad libitum.

Animal handling and experiments were done

according to the guidelines of the Committee for

the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Government

of India, and were approved by Institutional

Animal Ethics Committee.










Pag

e55

8

Isolation of polysaccharides

The fruiting bodies of G.lucidum were collected

from the outskirts of Thrissur district, Kerala,

South India. The type specimen was deposited in

the herbarium of Centre for Advanced Studies in

Botany, University of Madras, Chennai, India

(HERB. MUBL. 3175). Polysacchrides were

isolated by the method of Mizuno [4] with slight

modification [5] The confirmation of

polysacchrides were done by Anthrone [6] and

phenol sulphuric acid test [7] Structural

confirmation was done by IR and NMR spectrum

which were recorded at Sophisticated Analytical

Instrument Facility, Indian Institute of

Technology, Mumbai, India. From the Gel

filtration analysis the molecular weight of

Polysaccharides were found to be 1.5 x 106

Daltons. The powder was dissolved in double

distilled water and administered orally in the

experiments.

Irradiation

The cobalt therapy unit with Gamma Cell 220

(AECL, Canada) facility of Amala Cancer Hospital,

Thrissur was used for irradiation. Anaesthetized

animals, were exposed to 4 Gy -irradiation at a

dose rate of 1 Gy/min.

Experimental Design

Five groups with 15 animals each were used for

the study.

Group I – Normal Control (Double distilled water)

Group II – Radiation alone (4 Gy)

Group III – Amifostine (300 mg/Kg body wt) +

Radiation 4 Gy

Group IV - Radiation 4 Gy + Polysaccharides (10

mg/ Kg body wt)

Group V - Radiation 4 Gy + Polysaccharides (20

mg/ Kg body wt)

Animals were sacrificed in alternate days from 1

to 9 (1, 3, 5, 7, and 9)

Polysaccharides were administered orally just

after irradiation.

Tissue protein and serum protein

Tissue and serum protein were determined by

Bradford’s [8] method. The protein determination

was done according to the procedure given in the

kit purchased from Banglore Genei. BSA was used

as standard and diluted to get 1mg/ml.Different

concentrations of BSA and unknown samples

were pipetted into different test tubes. 2 ml of

Bradford’s reagent was added, mixed and kept at

room temperature for 10 mins. OD was recorded

at 595 nm using Varian DMS 200 UV-visible

spectrophotometer. The value of unknown

sample was recorded from the standard graph.

Serum lipid peroxidation

Serum lipid peroxidation was determined by

Ohkawa et al [9] after precipitating the protein

according to the method of Satoh [10] to 0.5 ml

serum, 2.5 ml of 0.02% TCA was added and the

tube is left to stand for 10 min at room

temperature. After centrifugation at 3500 rpm for

min, the precipitate was washed. A 4ml reaction

mixture containing 0.4 ml of serum, 1.5 ml of 0.8%

TBA, 1.5 ml of acetic acid (20% pH 3.5) and

distilled water was kept for 1 hour in a boiling

water bath at 950 C. After 1hour, the reaction

mixture was removed from water bath, cooled

and added 1 ml of distilled water. 5 ml of butanol:

pyridine mixture (15:1) was added to the reaction

tube, mixed thoroughly and centrifuged at 3000

rpm for 10 min. Absorbance of the clear

supernatant was measured at 532 nm against

butanol: pyridine mixture. The MDA was

calculated with the help of a standard graph made

by using different concentrations (1-10

nanomoles) of 1’1’3’3 – tetramethoxy propane in

1 ml distilled water and is expressed as nmol of

MDA/mg protein.

Determination of tissue GSH

Reduced glutathione (GSH) in tissue was

determined by the method of Moron et al,[9]

0.5ml of tissue homogenate was mixed with 0.1

ml of 25% TCA and kept on ice for few minutes









Pag

e55

9

and then subjected to centrifugation at 3000g for

few minutes to settle the precipitate. 0.3ml of the

supernatant was mixed with 0.7ml of 0.2M

sodium phosphate buffer (pH8). The yellow colour

obtained was measured after 10 min at 412 nm

against a blank which contained 0.1 ml of 5 % TCA

in place of the supernatant. A standard graph was

prepared using different concentrations of GSH in

0.3 ml of 5% TCA. The GSH content was calculated

with the help of this standard graph and

expressed as n mole/mg protein.


Tissue GSH was found to be reduced from 3rd to

7th day in a radiation alone group. Administration

of polysaccharides at 20 mg/kg body wt restored

GSH on the 7th and 9th day (Figure1). Serum MDA

was increased at 4 Gy gamma radiation (Figure.2).

Administration of polysaccharides at 20 mg/kg

body wt reduced significantly on 5th, 7th and 9th

day. The presence of amifostine at 300mg/kg

body wt also reduced MDA significantly on 5th

day after radiation exposure.

Antioxidant enzymes are among the endogenous

system that are available for the removal or

detoxification of these free radicals and their

products formed by ionizing radiation. The

GSH/GST detoxification system is an important

part of cellular defense against a large array of

endogenously or exogenously formed injurious

agents. GSH offers protection against oxygen-

derived free radicals and cellular lethality

following exposure to ionizing radiation. GST

enzymes also possess peroxidase activity and can

directly attack the peroxides that may be

generated via oxidative reduction recycling,

resulting in decreased cytotoxicity. The present

study demonstrates that a significant reduction in

GSH in radiation treated group. This could be due

to the enhanced utilization of antioxidant defense

system in an attempt to detoxify the radicals

generated by radiation. In the intact and healthy

cells the enzymes are restored immediately after

each interaction and GSH is also restored by

synthesis [11]. But in the irradiated animals, the

normal synthesis/repair will be disrupted due to

damage to DNA and membranes. As a result,

restoration will be delayed till the cells are

recovered. This could explain the slow recovery

in the levels of GSH and antioxidant enzymes after

radiation treatment.The antioxidant property of

the polysaccharides scavenge free radicals and

neutralize it, thus reducing its capacity to

damage. The balance between the production of

free radicals and the antioxidant defences in the

body has important health implications. This

antioxidant property may be a contributing factor

for the radioprotective properties offered by the

polysaccharides.

The interaction of ionizing radiation with

biological system results in generations of free

radicals, H and OH radicals, H2 and H2O2.

Radiations induced free radicals in turn impair the

antioxidant defense mechanism leading to

increased membrane lipid peroxidation, which

results in the damage of membrane bound

enzymes [12-13] .The increased lipid peroxidation

is due to the low concentration of GSH. The

membrane damage due to lipid peroxidation is

confirmed by the activity of hepatic enzyme GPT.

Antioxidant enzymes are among the endogenous

system that are available for the removal or

detoxification of these free radicals and their

products formed by ionizing radiation.

Polysaccharides reduced chromosomal

aberrations in mice exposed to 4 Gy gamma

radiation [14] As a result, restoration will be

delayed till the cells are recovered. This could

explain the slow recovery in the levels of GSH and

antioxidant enzymes after radiation treatment.

The polysaccharides from Ganoderma

administered to mice (5g/kg p.o for 30 days)

produced no changes in body wt, organ wt or

hematological parameters and produced no









Pag

e56

0

adverse effect [13]. Amifostine is an FDA

approved radioprotector used clinically.

Amifostine was used as a standard drug to

compare the activity of Ganoderma

polysaccharide. The protection offered by

amifostine at 300mg/kg body wt, a dose which

provided maximum protection with minimum

toxicity and by the polysaccharides at 20mg/kg

body wt was comparable. Thus the dose at which

the polysaccharide renders protection is much

lower than that of amifostine. Moreover, the

polysaccharide is effective by oral administration,

which is the most convenient mode of

administration in treatment of human diseases. In

conventional radiotherapy, the use of a

radioprotector, which can be administered orally

is of significant advantage.

CONCLUSION

The present finding that polysaccharides gives

significant radioprotection when given after

irradiation points to its advantage over the other

pre-administered radioprotectors and potential

for use both in medical non-medical exposures.

Figure .1. Effect of polysaccharides on serum MDA after gamma Irradiation (4 Gy).

*p < 0.01 compared to radiation alone.

** p< 0.05 compared to normal control.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

1 3 5 7 9

No of days

Seru

m M

DA

nm

ole

s/m

l

Normal Radiation alone(RT) 4 Gy

RT 4 Gy + Amifostine (300mg/kg body wt) Polysaccharides PS 10mg/kg body wt + RT 4Gy

Polysaccharides PS 20mg/kg body wt + RT 4Gy

****

*

*









Pag

e56

1

Figure .2. Effect of polysaccharides on tissue GSH after 4 Gy gamma irradiation.

*p < 0.01 compared to radiation alone.

REFERENCES [1] Gao Y., Zhou S., Cancer prevention and treatment by

Ganoderma, a mushroom with medicinal properties, Fd.

Rev. Intl.19: 275-325. (2003)

[2] Pillai T.G., Salvi V.P., Maurya D.K., Nair C.K.K.,

Janardhanan K.K. Prevention of radiation induced

damages by aqueous extract of Ganoderma lucidum

occurring in Southern parts of India, Curr. Sci. 91:341-

344. (2006)

[3] Wasser SP., Reishi or Ling zhi (G. lucidum). Encyclopedia

of Dietary supplements. Marcel Dekker. 5:

603-622.2005.

[4]Mizuno T., Development of an antitumor biological

response modifier from Phellinus linteus Teng.

(Review). Int.J. of. Med. Mushrooms. 2: 21-23. (2000)

[5] Pillai T.G., Nair C.K.K., Janardhanan K.K. Enhancement of

repair of radiation induced DNA strand breaks in human

cells by Ganoderma mushroom polysaccharides, Fd.

Chem. 119: 1040-1043. (2009)

[6] Yemn E.W., Wills A.J. The estimation of carbohydrate in

plant extract by anthrone. Biochem. J 57: 508-514.

(1954)

[7] Duboi S.M., Gilles G.A., Hamilton J.K., Colourimetric

estimation of Carbohydrates by Phenol Sulphuric acid

method, Anal.Chem. 28:350-356. (1956)

[8] Bradford, M M., A rapid and sensitive method for the

quantification of microgram quantities of protein

utilizing the principle of protein-dye binding. Anal.

Biochemistry. 72: 248-254. (1976)

[9] Ohkawa H., Ohishi N., Yagi K., Assay for lipid peroxide in

animal tissues by thiobarbituric acid reaction. Ann.

Biochem. 95: 351-358. (1979)

[10] Moron MA., Depierre JW., Mannervik B., Levels of

glutathione reductase and glutathione S transferase

activities in rat lung and liver. Biochem Biophys Acta.

582: 67-78, (1979)

[11] Satoh K., Serum Lipid Peroxide in cerebrovascular

disorders determined by a new colorimetric method.

Clin. Chem.Acta, 90: 37-43, (1987)

[12] Meister A., Anderson M.E., Transport and Direct

Utilization of -glutamylcysteine for Glutathione

Synthesis. PNAS. 3: 707-711 (1983)

[13] Halliwell B., John H., Gutteridge NC., Production of

hydroxyl radicals in living systems, In- Free Radicals

Biology and Medicine, Clarendon Press, Oxford, 1989,

31.

[14] Thulasi G Pillai and Uma Devi., Mushroom beta glucan:

Potential candidate for post irradiation

protection.Mutation Research.751 (2013) 109-115.

0

5

10

15

20

25

1 3 5 7 9

No of days

Tis

su

e (

GS

H U

/mg

pro

tein

)

Normal Radiation alone (RT) 4 Gy RT 4 Gy + Amifostine (300 mg/kg body wt)

Rt 4 GY + Polysaccharides (10 mg/kg body wt) RT 4 Gy + 20 mg/kg body wt)

*

*









Pag

e56

2

*Corresponding Author: Thulasi G.Pillai Division of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi, Thrissur- 680 653, Kerala, India.







Research Article

Biological Sciences


Andrew C. Nwaka*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e56

3

EFFECT OF SOYABEAN DIET ON THE SERUM IMMUNOGLOBULIN PROFILE AND CD4+ COUNT

OF MALE DIABETIC RATS CHALLENGED WITH K.PNEUMONIAE

*ANDREW C. NWAKA1, PETER N. UZOEGWU2, ONYECHI OBIDOA3, UGWU OKECHUKWU P.C.4, OSSAI

EMMANUEL C.4 and CHIGOR VINCENT N.5 1Department Of Biochemistry, Anambra State University, Uli, Nigeria.

2Tropical Diseases Research Unit, Department of Biochemistry, University of Nigeria, Nsukka, Nigeria. 3 Department of Biochemistry, Kogi State University, Anyingba, Kogi State.

4Department of Biochemistry, University of Nigeria, Nsukka. 5Department of Microbiology, University of Nigeria, Nsukka.


ABSTRACT In this study, the effect of soyabean supplemented diet on the immunoglobulin profile and CD4

+ count of male

diabetic rats challenged with Klebsiella pneumoniae were evaluated in a total of sixty-four mature albino rats,

weighing 150-250 grams. The rats were separated into eight groups of eight rats each. The groups were as follows:

A- rats fed on rat feed, B-rats fed on rat feed and soyabean meal, C-diabetes-induced rats feed on rat feed, D-

diabetes-induced rats fed on rat feed and soyabean meal, E-rats fed on rat feed but challenged with Klebsiella

pneumoniae, F- rats fed on rat feed and soyabean meal challenged with Klebsiella pneumoniae, G-diabetes induced

rats fed on normal rat feed but challenged with Klebsiella pneumoniae and H-diabetes-induced rats fed on rat feed

and soyabean meal but challenged with Klebsiella pneumoniae. The study lasted for 28days. At the end of 28 days,

the rats were bled and blood collected, processed and the serum obtained used for assessment of immunoglobulin

profile and CD4+ T-cell count. Immunoglobulin profile was carried out using immunoturbidimetric assay with

Randox(R) kit, while CD4+ T-cell count was done using CY-flow machine. Results showed that there were significant

increase (P< 0.05) in serum lgA, 1gM, 1gG levels and CD4+ T-cell of diabetic rats fed on normal feed and soyabean

meal when compared with their counterparts fed with only normal feed. Results of the study suggest that soyabean

diet could assist in boosting the serum immunoglobulin levels and CD4+ cell of diabetic rats and hence protecting the

rats from K. pneumoniae infection.

KEY WORDS Diabetes mellitus, Immunoglobulins, CD4

+-T-cell, K. pneumoniae and Soyabeans.

INTRODUCTION

Diabetes mellitus, often referred simply as

diabetes, is a syndrome characterized by

disordered metabolism and abnormally high

fasting blood sugar (hyperglycaemia) resulting

from insufficient levels of the hormone, insulin or

its insensitivity (Tierney et al., 2002). It is a chronic

disease characterized by elevated blood glucose

levels and disturbances in carbohydrate, fat and

protein metabolism (Sky, 2000, Rother, 2007). The

World Health Organization, recognizes three main

form of diabetes mellitus type I , type II and

gestational diabetes (occurring during pregnancy)

(WHO, 1999).

Type I diabetes mellitus formerly called juvenile

onset or insulin dependent diabetes mellitus

(IDDM) is characterized by loss of the insulin










Pag

e56

4

producing beta cell of the islets of langerhans of

the pancreas, leading to a deficiency of insulin.

The main cause of this loss of β- cell is a T-cell

mediated autoimmune attack (Rother, 2007).

There is no known preventive measure which can

be taken against type I diabetes. While type 2

diabetes mellitus, also known as non-insulin

dependent diabetes mellitus (NIDDM), is due to

insulin resistance or reduced insulin sensitivities

combined with reduced insulin secretion

(Rosenbloom and Silverstain, 2003). Furthermore,

gestational diabetes mellitus (GDM) resembles

type 2 diabetes in several respects, involving a

combination of relatively inadequate insulin

secretion and responsiveness (WHO, 1999). In

Nigeria, diabetes mellitus has emerged as one of

the biggest health problems and its prevalence is

increasing at an alarming rate (Unadike, 20120).

Soyabean, is a plant of the family leguminoseae.

Legumes are noted for their relatively high protein

content when compared with cereals, tubers,

fruits and vegetables. Soyabean contains about

40% protein, 18% fat and has a high concentration

of minerals such as calcium, iron etc (Anosike et

al., 2007).

Infections, no matter how mild, have adverse

effects on nutritional status. The significance of

these effects depends on the nutritional status of

the individual, the nature and duration of the

infection and the diet during the recovery period

(Scrimshaw and Sangiovanni, 1997). Furthermore,

it is not surprising that protein deficiency is so

consistently observed to interfere with resistance

to infection because most immune mechanisms

are dependent on cell replication or the

production of active protein compounds.

However, because protein cannot be synthesized

without a balance of essential amino acids,

experimental amino acid deficiencies have the

same effect as protein. Essentially all forms of

immunity have been shown to be affected by

protein-energy malnutrition in young children,

depending on the severity of the protein

deficiency relative to energy. The effects include

impaired antibody formation (Pretorius and De

Villiers, 1962, Reddy and Srikantia, 1964 and

Reddy et al., 1977), decreased serum

immunoglobulin (Aref et al., 1977 and Watson et

al., 1987), decreased thymic function (Watts,

1969, Purtilo and Connor, 1975 and Chandra et

al., 1982) and splenic lymphocytes, delayed

cutaneous hypersensitivity (Simythe et al., 1971

and Chandra, 1974), decreased complement

formation (Sythe et al., 1971, Suskind et al.,

1976), decreased interferon and effects on

nonspecific mechanisms that include anatomic

barriers and secretory substances such as lysomes

and mucus (Scrimshaw and Sangiovanni, 1997).

In this study, the effect of soyabean diet on the

serum immunoglobulin profile (i.e. lgM, lgG and

lgA) and CD4+ count of male diabetic rats

challenged with Klebsiella pneumoniae (a Gram-

negative bacteria) was evaluated.

MATERIALS

Chemical used: All the chemical used in this

research were of analytical grade.

Animal used: Mature apparently healthy male

albino rats weighing 150-250 grams were

procured from the Animal House of Faculty of

Veterinary Medicine, University of Nigeria,

Nsukka campus. The animals were allowed to

acclimatize for fourteen days before the

commencement of the study.

Soyabean meal: Rats on soyabean meal were

given 9.6g/kg body weight of soyabean powder

dispersed in water by intubation each morning

before the animals were fed for the day.









Pag

e56

5

Rat feed: The normal rat feed used in this study is

pelleted growers mash of Vital(R) feed. It was

produced by Grand Cereals and Oil Mills, Jos,

Nigeria.

Pathogenic Organism: Klebisella pneumoniae

was used as the pathogenic organism in this

study. Its stock was procured from the

Department of Veterinary Microbiology, Faculty

of Veterinary Medicine, University of Nigeria,

Nsukka.

METHODS

Experimental Design

Sixty-four mature male albino rats, weighing 150-

250grams were used in the study. The rats were

separated into eight groups of eight rats each and

kept in eight different standard cages as follows:

Groups Treatment

A Rats fed on rat feed

B Rats fed on rat feed and soyabean meal

C Diabetes-induced rats fed on rat feed

D Diabetes-induced rats fed on rat feed and soyabean meal.

E Rats fed on rat feed but challenged with Klebsiella pneumoniae

F Rats fed on rat feed and soyabean meal challenged with Klebsiella pneumoniae

G Diabetes-induced rats fed on normal rat feed but challenged with Klebsiella pneumoniae

H Diabetes-induced rats fed on rat feed and soyabean meal but challenged with Klebsiella

pneumoniae

All the protocols as approved by Institutional

Animal Ethics Committee (IAEC) were observed in

the study. At the end of the 28days, the rats were

bled from the retro-bulbar plexus of the medical

canthus of the eye. The blood samples were

collected into EDTA tubes and plain sample

bottles. The EDTA acted as anticoagulant. The

blood samples in the EDTA bottles were used for

CD4+ T-cell counts, while the blood sample

collected in the plain tubes without EDTA were

allowed to stand for 30 minutes and centrifuged

at the speed of 3,000 revolutions per minutes for

10minutes. The serum was extracted and used for

lgA, lgG and lgM assays using standard

biochemical methods.

Induction of Diabetes

Diabetes was induced in the rats by injecting

180mg/kg body weight of alloxan monohydrate

intraperitoneally in 0.9% w/v NaCl (normal saline)

after fasting the rats for 12hours. Seven days after

the alloxan administration, the rats were fasted

for 12hours and their blood glucose level was

measured. Rats having blood glucose level of

400mg/dl and above were selected and used as

diabetic rats in this study. Rats that were

described as fasted were deprived of food for at

least 12hours but were allowed free access (ad

libitum) to drinking water.

Pathogen Challenge

The rats were challenged with Klebsiella

pneumoniae by intraperitioneal administration of

0.4ml of the bacterial suspension containing 108

c.f.u/ml in 0.9% (w/v) normal saline on the 22nd

day of the study.

Proximate Analysis

The proximate composition of soyabean and the

normal animal feed used in the study were

evaluated. Their protein content were determined

by micro khfelldal method as was described by









Pag

e56

6

AOAC (2000) and Changsam (2003), while their

ash, moisture and fats contents were determined

by gravimetric method as described by AOAC

(2000). Crude fiber were determined by

gravimetric method as described by James (1995).

The total carbohydrate were then estimated using

the formula as was given by Bemiller (2003).

Immunoglobulin Profile and CD4+ T-cell

Determination

The serum lgm, lgA and lgG were determined

using immunoturbidimetric assay (Whicher et al.,

1983) with Randox(R) kit, while CD4+ count was

carried out using CY Flow SL Green Machine.

RESULTS

The result of proximate analysis for the soyabean

powder used in this study (Fig.1) indicated that its

moisture content was 7.40%, protein 48.22%,

fibre 3.80%, fat 15.90%, ash 4.60% and

carbohydrate content 20.08%. Also, the result of

proximate analysis of normal feed (pelleted

growers mash of vital(R) feed) used in this study

(Fig. 2.) showed that the feed’s moisture content

was 16.75%, fat 5.80%, ash 7.25%, fibre 2.60%

and protein 17.95% and crbohydrate content was

49.65%.

Furthermore, when the different groups of rats

were challenged with Klebsiella pneumoniae, it

was observed that only the group G with diabetic

rats fed with normal feed came down with the

infection. This was indicated by the sickly

appearance of the rats in the group and mortality

of half of the rats in the group G before the end of

the study.

Fig. 1: Soyabean proximate composition

Fig.3.1: Result of soyabean proximate analysis

Moisture

6%

Protein

48%

Fibre

4%

Fat

16%

Ash

5%

Carbohydrate

21%









Pag

e56

7

Fig. 2: Normal feed proximate composition

Fig. 3: Mean serum IgM concentration for different groups of rats used

Fig.3.2:Result of normal feed proximate analysis

Moisture

17%

Ash

7%

Fat

6%

Fibre

3%Protein

18%

Carbohydrate

49%

Figure 3.4.1:Results of mean serum IgM concentration for different rat

groups used

120.55133.75

79.21

123.39

182.64

208.41

62.49

163.14

0

50

100

150

200

250

A B C D E F G H

Groups of albino rat used

IgM

co

nc.(

mg

/dl)

Series1









Pag

e56

8

Fig. 4: Mean serum IgA concentration for different groups of rats used

Fig. 5: Mean serum IgG concentration for different groups of rats used

Fig.3.4.2:Results of mean serum IgA concentration for different rat groups used

169.61

181.63

124.12

167.04

188.71

224.43

132.72

193.67

0

50

100

150

200

250

A B C D E F G H

Groups of rat used

IgA

co

nc

.(m

g/d

l)

Series1

Figure 3.4.3:Results of mean serum IgG concentration for different rat

groups used

597.96 627.26

311.57

502.94

757.55

899.31

413.11

612.92

0

200

400

600

800

1000

1200

A B C D E F G H

Groups of rats used

IgG

co

nc.(

mg

/dl)

Series1









Pag

e56

9

Fig. 6: Mean serum CD4

+ concentration for different groups of rats used

The result of the mean serum immunoglobulin M

(IgM) for different groups of rats used in the study

(Fig 3) indicated that the mean serum IgM of

groups E, F and H were significantly higher

(P<0.05) than that of the control (group A). The

result (Fig. 3) also showed that there were

significant reductions (P<0.05) in the serum lgM

concentrations of diabetic rats fed on normal feed

only, group C (79.21+ 20.73 mg/dl) and diabetic

rats fed on normal feed but challenged with

Klebsiella pneumoniae, group G

(62.49+22.96mg/dl) when compared to control,

group A (120.55 + 9.29mg/dl). Fig.3 indicated that

the mean serum lgM of diabetic rats fed on

soyabean diet (group D) was significantly higher

(P<0.05) than that of diabetic rats fed on normal

feed. Result (Fig.3) also showed that the mean

serum lgM of diabetic rats fed on soyabean diet

challenged with klebsiella pneumoniae (group H)

was significantly higher (P< 0.05) than that of

diabetic rats fed on normal feed but challenged

with Klebsiella pneumoniae (group G). Fig.3 also

indicated that the mean serum IgM of normal rats

fed on soyabean diet (group B) and normal rats

fed on soyabean diet but challenged with

Klebsiella pneumoniae (group F) were higher than

their counterparts fed on normal feed (groups A

and E respectively). The increases were

statistically not significant (P >0.05).

The result of the mean serum lgA concentrations

for the different groups of rats used in this study

(Fig. 4) revealed that the lgA levels of normal rats

fed on normal rat feed and soyabean meal but

challenged with klebsiella pneumoniae, group E

(188.71 + 15.09mg/dl) and diabetic rats fed on

normal rat feed and soyabean meal but

challenged with Klebsiella pneumoniae, group H

(193.67 + 19.43 mg/dl) were significantly higher

(P<0.05) than that of the control, group A (169.61

+ 17.53mg/dl). Fig.4 further indicated that the

mean serum lgA concentration of diabetic rats fed

on normal feed, group C (124.12+ 25.21 mg/dl)

was significantly lower (P<0.05) than those of the

Figure 3.5:Results of mean CD4 count for different

groups of albino rats used

320331.25

161.75

226.5211.75

219.75

99.75

125

0

50

100

150

200

250

300

350

400

A B C D E F G H

Groups of albino rats used

CD

4 (c

ells

/mic

rolit

re)

Series1









Pag

e57

0

control, group A (169.61 + 17.53mg/dl) and those

of the diabetic rats fed on normal feed and

soyabean meal, group D (167.04 + 18.61mg/dl).

Fig.4 also indicated that the mean serum lgA of

rats fed on soyabean but challenged with

Klebsiella pneumoniae (group F) and diabetic rats

fed on soyabean but challenged with Klebsiella

pneumoniae ( group H) were significantly higher

(P<0.05) than their counterparts fed on normal

feed (group E and G respectively).

The result of the mean serum lgG concentrations

for the different groups of rats used in this study

(Fig.5) showed that the mean serum lgG

concentrations of normal rats fed on normal feed

but challenged with Klebsiella pneumoniae, group

E (757.55 + 63.22mg/dl) and normal rats but also

challenged with Klebsiella pneumoniae group F

(899.31 + 56.23mg/dl) were significantly higher

(P< 0.05) than that of the control, group A (599.96

+45.06mg/dl). Fig.5 also revealed that the serum

mean lgG concentration of diabetic rats fed on

normal feed, group C (311.57+ 53.36mg/dl) was

significantly reduced (P<0.05) when compared to

the control, group A (597.96 + 45.06mg/dl) and

to that of the diabetic rats fed on normal rat feed

and soyabean meal, group D (502.94+

41.54mg/dl). Figure 5 further indicated that the

mean serum lgG of rats fed on soyabean diet

challenged with Klebsiella pneumoniae (group F)

and diabetic rats fed on soyabean diet challenged

with Klebsiella pneumoniae (group H) were

significantly higher (P< 0.05) than their

counterparts fed on normal feed only (groups E

and G respectively). The result also revealed that

there was no significant difference (P< 0.05)

between the mean serum lgG of rats fed on

soyabean diet (group B) and that of rats fed on

normal feed only (group A).

The result of CD4+ cell count for the different

groups of albino rats used in this study as shown

in Fig.6 indicated that the mean CD4+ cell count of

normal rats fed on normal feed and soyabean

meal, group B (331-25 + 22.50 cells/µl) and

normal rats fed on soyabean diet but challenged

with Klesibiella pneumoniae (group F) were

significantly not different (P>0.05) from their

counterparts fed on normal feed only (groups A

and E respectively). However, with the exception

of group B, the mean CD4+ count of all the other

groups were siginificantly lower (P< 0.05) than

that of the control, group A. Moreover, the results

shown in Fig.6 indicated that there were

significant reduction (P<0.05) in CD4+ count of all

the groups challenged with Klebsiella pneumoniae

when compared with their counterparts not

challenged with the pathogen. The results also

showed that the mean CD4+ counts of diabetic rats

fed on normal rat feed and soyabean meal, group

D (226.50+ 21.41 cells/µl) was significantly higher

than that of the diabetic rats fed on only normal

feed, group C (161.75+ 28.74 cell/µl).

DISCUSSION

Over the years, most investigations on diabetes

have concentrated on the effect of various diets or

herbal extracts on glucose levels without

adequate consideration of their effect on the

immune response. However, the effect of

soyabean diet on some immune markers (lgG, lgA,

lgM and CD4+cells) of diabetic rats were evaluated

in this study.

The results of the immunoglobulin profile

indicated significant increase (P< 0.050 in the lgM

concentrations (Fig.3) of normal rats fed on

normal rat feed but challenged with Klebsiella

pneumoniae (group E), normal rats fed on normal

feed and soyabean meal challenged with

Klebsiella pneumoniae (group F) and diabetic rats

fed on normal feed and soyabean meal but

challenged with Klebsiella pneumoniae when

compared to the control group (group A). This









Pag

e57

1

suggested that the presence of Klebsiella

pneumoniae (antigen), the state of health of the

rats and the nutritive value of the soyabean diet

used in the study could have likely been the reason

for the increased lgM antibodies. This observation

corroborated Edelman (1973) observation that in

active immunity, the effective contact with

antigens causes the host to produce antibodies in

response to the antigens. When the lgM

concentration of diabetic rats fed on normal rat

feed (group C) was compared to the control, a

significant reduction (P<0.05) was observed in

group C, suggesting that diabetes also causes

depletion of the lgM level, thereby reducing

immune competence in the rats. However, when

the lgM concentration of diabetic rats fed on

normal feed (group C) was compared with those

of their counterparts fed on normal feed and

soyabean meal (Group D), it was observed that

the reduction in lgM concentration in group C was

restored to normalcy with the incorporation of

soyabean diet in group D, an observation that

could be attributed to soyabean’s high protein

composition with its high content of essential

amino acids. These amino acids could have helped

in boosting the plasma proteins which could have

led to increase in the IgM concentration, since

immunoglobulins are synthesized by plasma cells.

This observation is in accord with McGee and

McMurray (1977) report that soyabean (protein)

could help in boosting immunoglobulin

concentration in the intestine of mice.

Immunoglobulins are proteins. So the increase in

IgM concentration observed in the diabetic rats

fed on soyabean diet (group D) when compared to

that of the diabetic rats fed on normal feed (group

C) could be as a result of high essential amino

acids present in the soyabean used.

Also, the results of IgA and IgG concentrations for

normal rats fed on normal feed challenged with

Klebsiella pneumoniae (group E), normal rats fed

on normal feed and soyabean meal challenged

with Klebsiella pneumoniae (group F) and diabetic

rats fed on normal feed and soyabean meal but

challenged with Klebsiella pneumoniae (group H)

indicated significant increases (P<0.05) in IgA and

IgG concentrations when compared with control

group A (Fig. 4 and 5). This also suggested that

the production of more antibodies (IgA and IgG) in

both diabetic and non-diabetic rats (except in

group G) as soon as they were challenged with

Klebsiella pneumoniae in order to protect the rats

from invading antigen could have been affected

by the presence of Klebsiella pneumoniae, the

state of health of the rats (i.e. whether diabetic or

non-diabetic) and the nutritive value of the diet

(soyabean) used. This observation is also in

consonance with the report that the presence of

antigen could trigger off the production of

antibodies as protective measure of the host

against invading antigen (Edelman, 1973).

It was also observed that more IgM, IgA, and IgG

antibodies were synthesized in the groups in

which soyabean meal was incorporated into their

diet when compared with their counterparts fed

on only normal feed. This suggests that soyabean

diets could have helped in boosting their

immunoglobulin levels, due to the presence of

essential amino acids in the soyabean. These

essential amino acids could have helped in

boosting the immunoglobulin level. This

observation also corroborates McMurray (1977)

which stated that protein (essential amino acids)

helps in boosting immunoglobulin concentration

in the intestine of mice. Furthermore, the IgM, IgA

and IgG concentrations of diabetic rats fed on

normal feed (group C) were significantly reduced

(P<0.05) when compared to the control, an

indication that diabetes causes depletion of

immunoglobulin levels. This could be due to the

diabetics’ inability to utilize blood glucose for

energy generation. Muscle proteins are therefore









Pag

e57

2

metabolized for energy generation, leading to loss

of muscle amino acids which could result to the

shrinking of the muscles. The depletion of the

muscle’s amino acids could have caused the

decrease in the immunoglobulin levels, since

immunoglobulins are synthesized from amino

acids. However, it was observed that the reduced

immunoglobulin concentrations in group C was

restored almost to normal level in diabetic rats fed

on Soyabean diet (group D). The suspicion is that

the essential amino acids in soyabean could have

been replenished the lost amino acids for energy

generation. The replenished essential amino acids

from Soyabean Diet could have assisted in

boosting the immunoglobulin concentration in the

rats fed with Soyabean supplemented diet, since

immunoglobulins are proteins.

Moreover, Fig.6 indicated that with the exception

of normal rats fed on normal rat feed and

soyabean meal (group B), that the mean CD4+

count of all other groups (groups C, D, E, F, G and

H) were significantly lower (P< 0.05) than the

control (group A). This suggests that diabetes and

the presence of pathogen (Klebsiella pneumoniae)

could have caused reduction in CD4+ cells. The

utilization of muscle protein for energy generation

could have brought about the depletion of

essential amino acids in the diabetic rats. This in

turn could have caused the decreased CD4+ cell

count in diabetic rats as observed, since CD4+ cells

are produced from essential amino acids. Also

results in Fig.6 showed that both diabetic and

non-diabetic rats challenged with Klebsiella

pneumoniea but fed on soyabean incorporated

diet had higher CD4+ count than their counterparts

fed on only normal rat feed. This is because

soyabean is rich in essential amino acids. These

essential amino acids could have helped in

replenishing the lost muscles and tissues amino

acids during energy generation in diabetic rats fed

in soyabean diet. The replenished essential amino

acids in diabetic rats fed on Soyabean

supplemented diet could have boosted their CD4+

cell count when compared to that of the diabetic

rats fed on normal feed. This should also be

expected considering the high nutritive value of

Soaybean (Fig.1) with high percentage of protein,

minerals and vitamins including antioxidants

(Regel et al., 2000; Anosike et al., 2007; and

Villegas and Gao, 2008). The above observation

also corroborates the report that decreased

dietary intake of antioxidants could lead to a

concomitant decrease in CD4+ T-cells (Oguntibeju

et al., 2005 and 2006). Since the CD4+ T-cells,

perform a central and coordinating role in the

immune response (Hughes et al., 1997; Vajpayee

et al., 2009), and Soyabean diet helped in raising

the CD4+ cells in diabetic rats as observed in this

study, Soyabean diet therefore could help in the

general boosting of the immune system of

diabetics.

CONCLUSION

This study revealed significant increases (P<0.05)

in serum IgA, IgM and IgG levels and CD4+ T-cells

concentration of diabetic rats fed with normal

feed supplemented with soyabean meal (group D)

and the rats infected with K. pnemoniae and fed

with normal feed and supplemented with

soyabean meal (group H) when compared with

their counterparts fed with only normal feed

(groups C and F). Moreover, it was observed that

when various groups of rats were challenged

intraperitoneally with 0.4ml of Klebsiella

pneumonia suspension containing 108 c.f.u/ml,

establishment of infection and high mortality rate

of the infected rats were observed in diabetic rats

fed with normal feed only (group G), whereas no

mortality was observed in diabetic rats fed with

normal rats feed supplemented with soyabean

meal (group H). These results suggest that

soyabean diet could be useful in the general









Pag

e57

3

boosting of the immune system and in the general

management of diabetes mellitus.

SUGGESTIONS FOR FURTHER STUDIES

It is suggested that further studies be carried out

as follows:

Inducing diabetes that could last for more

than 3 months in order to appreciate the

effect of soya protein more, in chronic

complications of diabetes.

Feeding the animals with soyabean diet for

more than 3 months (i.e. longer period), since

the longer the animal is exposed to the diet,

the more the effect of the diet could be

discovered.

Using more than two different pathogenic

organisms that are infectious to albino rats in

subsequent study, to confirm these results.

ACKNOWLEDGEMENT

We are grateful to the following for assistance

rendered towards the success of this research

work: Prof. L.S.U. Ezeanyika and Prof. I.N.E.

Onwurah, both from Department of Biochemistry,

University of Nigeria, Nsukka. Also to Prof. J. I.

Ihedioha, Prof. S.V.O Shoyinka and Prof. K. F.

Chah, all from the Faculty of Veterinary Medicine,

University of Nigeria, Nsukka. We will also not

forget the Head of Hematological Section of

Nnewi Teaching Hospital, Mr. A. Ozoabia, who

assisted us in carrying out CD4+ count with their

machine.

REFERENCES o Anosike, C.A., Ezeanyika, L.U.S. and Obidoa, O. (2007).

Effect of roasted soyabean (glycine max) diet on the

histology of selected rat tissues. Bio-Research, 5 (2):

237-240.

o AOAC (2000). Official method of analysis of Association

of Official Analytical Chemists. Washington D.C., 16th

Edition. Pp. 12-18.

o Aref, G.H., el-Din, M.k. and Hassan, A.J., (1970).

Immunoglobulins in Kwashiorkor. Journal of Tropical

Medicine and Hygiene, 73:186-191.

o Bemiller, N.J. (2003) Carbohydrate Analysis. Food

Analysis. Third edition. Kluwer Academic publisher, New

York Pp. 143-146.

o Chandra, R.K., Gupta, S. and Singh, H. (1982). Inducer

and suppressor T-cell subsets in protein-energy

malnuitrition: analysis by monoclonal antibodies.

Nutrition Research, 2:21-26.

o Changsam, K.C. (2003). Protein Analysis. Food Analysis.

Third edition kluwer Academic/Plenum publisher, New

York. Pp. 134-135.

o Hughes, M. D., Johnson, V. A. and Hirsch, M. S. (1997).

Monitoring plasma HIV–I RNA levels in addition to CD4+

lymphocyte count improves assessment of antiretroviral

therapeutic response. Annals of Internal Medicine,

126:929-938.

o James, O.K. (1995). Gravimetric method of analysis

employed in twenty different cereals and legumes,

Wayton, U.K.

o Oguntibeju, O. O., Van den Heever, W. M. J. and Van

Schakwyk, F. E. (2005). An analysis of based line dietary

intake of HIV-positive/AIDS patients. South African

Journal of Medical Technology, 19 (2): 3-9.

o Oguntibeju, O. O., Van den Heever, W. M. J. and Van

Schakwyk, F. E. (2006). The effect of a liquid nutritional

supplement on viral load and haematological

parameters of HIV-positive/AIDS patients. British

Journal of Biomedical Science, 63 (3): 134-139.

o Pretorius, P.J. and Villiers, L.S. (1962) Antibody response

in children with Protein Malnutrition. American Journal

of Clinical Nutrition, 10: 379-383.

o Purtilo, D.T. and Connor, D.H. (1975). Fatal infection in

protein-calorie malnourished children with

thymolymphatic atrophy. Archieves of Disease in

Childood 50: 149-152

o Reddy, V. and Srikantia, S.G. (1964). Antibody response

in Kwashiorkor. Indian Journal of Medical Research, 52:

1154-1158.

o Reddy, V., Bhaskaram, C. and Raghuramulu, N . (1977).

Immunological responses in malnourished children.

Indian Journal of Padiatrics, 14: 255- 258

o Regel, F. J., Fraser, G. D., Weeks, E. C. and Greenberg, A.

N. (2000). Dietary phytoestrogen have anti-

inflammatory activity in guinea pig. Proceedings of the

Society for Experiment Biology and Medicine. 223 (4):

372-378

o Rosenbloom, A. and Silverstain, J.H., (2003) Type 2

Diabetes in children and Adolescents: A Clinicians Guide

to Diagnosis, Epidemiology, Pathogenesis, Prevention

and Treatment. American Diabetes Association, U.S.I.,

o Rother, K.I. (2007). “Diabetes Treatment Bridging the

Divide”. The New England Journal of Medicine, 356 (150:

1499-1501.









Pag

e57

4

o Scrimshaw, N.S. and san Giovanni, J.P. (1997).

Synergism of Nutrition, Infection and Immunity.

American Journal of clinical Nutrition, 66:464s-477s.

o Sky, G.M. (2000). Kinetics of insulin secretion underlying

notabolism events in diabetes mellitus. Diabetes

mellitus: A fundamental and clinical text. Lippicott

willians and Wilkins, Plucadelphia Pp. 2-11.

o Smythe, P.M., Brereton-stiles, G.G. and Grace, H.J.

(1971). Thymolymphatic deficiency and depression of

cell mediated immunity in protein-calorie malnutrition.

Lancet, 2: 939-943.

o Suskind, R., Edelman, R., KulapongsP., Pariyanonda, A.

and Sirisinha, S. 91976). Complement activity in Children

with Protein Calorie Malnutrition. American Journal of

Clinical Nutrition, 29: 1089-1092.

o Tierney, I.M., Mcphee, S.J. and Papadakis, M.A. (2002).

Current Medical Diagnosis and Treatment of Diabetes.

Large Medical books. McGraw-Hill, New York.Pp. 1203-

1215.

o Unadike, B.c. (2010). Awareness and knowledge about

diabetes mellitus among nursing students in the Niger

Delta region of Nigeria. Middle East Journal of Nursing,

4(1): 1-8.

o Vajpayee, M., Kaushik, S., Sreenivas, V., Mojumdar, K.,

Mendiratta, N. and Chauhan, K. (2009). Role of immune

activation in CD4+ T-Cell depletion in HIV –I infected

Indian patients. European Journal of Clinical

Microbiology and Infectious Diseases, 28: 69-73.

o Villegas, R. and Gao, Y. T. (2008). Legume and soy food

intake and incidence of

o type 2 diabetes in the Shanghai womens health study.

American Journal of

o Clinical Nutrition. 87 (1): 162-167.

o Watson, B.B, Reyes, M.A. and Mc Murray, D.N., (1978)

Influence of malnutrition on the concentration of lgA,

lysozymes, amylase and aminopeptidase in children’s

tears. Proceedings of the Society for Experiment Biology

and Medicine, 157: 215-219.

o Watts, T. (1969). Thymus weights in malnourished

children. Journal of Tropical Pediatrics, 15:155-158.

o Whicher, J.J., Price, C.P. and Spencer, K. (1983).

Immunoturbidimetric assay. Critical Reviews in Clinical

Laboratory Science, 18: 213-216.

o WHO (World Health Organization) (1999). Department

of Non Communicable Disease Surveillance (1999).

Definition, Diagnosis and Classification of Diabetes

Mellitus and its complications.

*Corresponding Author: Andrew C. Nwaka, Ph.D, Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria. E-mail: [email protected]; Phone: +234 (0) 8037834171.







ReviewArticle

PharmaceuticalSciences


Prodyut Mondal*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e57

5

HERBAL MEDICINES USEFUL FOR THE TREATMENT OF DIABETES IN NORTH-EAST INDIA:

A REVIEW

Prodyut Mondal*, Niroj Bhuyan, Sonjit Das, Mritunjay Kumar, Sudarshana Borah, Kabita Mahato.

Department of Pharmaceutical Sciences, Dibrugarh University. Assam 786004.India.


ABSTRACT Aim: Diabetes mellitus (DM) is a group of metabolic disorder characterized by hyperglycemia, which is associated

with abnormalities in carbohydrate, fat and protein metabolism result in chronic complications. The main

objective of the study to presenting the medicinal plants used in North-East India for anti-diabetic purposes. This

study emphasizes potential sources for the development of new anti-diabetic drugs from indigenous medicinal

plants found in North-East India. Methods: The North-east India is the indigenous for its biodiversity aspect. The

herbal drugs which have the anti-diabetic properties are documented here on the basic of the information of

articles published on the popular journal in different times and based on the information of extensive field survey.

Results: There are 141nos. of medicinal plant have documented. Traditionally most of the plants show promising

anti-diabetic activity. Whole plant and parts of the plant such as bark, leaf, fruit etc. are being used by the ethnic

community to cure diabetes. In compares to the other parts of the plant the leaves are the mostly used by the

traditional people as anti-diabetic purposes of the different plant. Conclusion: The traditionally used medicinal

plants have possessing potent anti-diabetic properties that has not still explored scientifically. The article

summarize the significance of medicinal plants that are used in north-east India as anti-diabetic purposes and the

requisite level of activity as well as toxicity would be considered for further scrutiny to develop the potential drug

molecules.

KEY WORDS Medicinal Plants, anti-diabetic drugs, diabetes mellitus, North East India.

INTRODUCTION

The prevalence of diabetes is rising all over the

world due to population growth, aging,

urbanization and an increase of obesity and

physical inactivity. Unlike in the West, where

older persons are most affected, diabetes in

Asian countries is disproportionately high in

young to middle-aged adults. This could have

long-lasting adverse effects on a nation’s health

and economy, especially for developing

countries. The International Diabetes Federation

(IDF) estimates the total number of people in

India with diabetes to be around 87.0 million by

2030 [1]. Diabetes mellitus is a common and very

prevalent disease affecting the citizens of both

developed and developing countries. It is

estimated that 25% of the world population is

affected by this disease [2]. Diabetes is the

condition where the fasting and post prandial

(after food) blood glucose level become high

(normal 120mg/dl after 2 hours of meal, adult

male) due to mainly improper metabolism of

carbohydrates, fats and proteins. Diabetes

mellitus is a group of syndromes characterized






Pag

e57

6

by hyperglycemia altered metabolism of lipids,

carbohydrates, and proteins and an increased

risk of complicates form vascular disease. Most

patients can be classified clinically as having

either type 1-diabetes mellitus (type IDM,

formerly known as insulin–dependent diabetes

or IDDM) or type 2- diabetes mellitus (type 2DM,

formerly known as non –insulin-dependent

diabetes or NIDDM) [3].

Generally the four types of cells (α, β, γ and Δ)

are in the islets of langerhans gland of pancreas

in a human body. Within these the alpha (α) and

the beta (β) cells take important role to secrete

insulin and glucagon hormone respectively for

the balancing of blood glucose level. In case of

Type I diabetes mellitus (IDDM) there is the

destruction of pancreatic beta cells majority of

cases due to autoimmuno antibodies. This type

of diabetes mellitus is less common to the

population. The International Diabetes

Federation and the World Health Organization

estimate that, worldwide over 100 million

people suffer from type II diabetes and 50% of

those cases are undiagnosed. Over 90% cases of

diabetes mellitus is in Type II (NIDDM) where

generally no loss or moderately loss of β-cell

mass occur; insulin in circulation is low and no β -

cell antibody is demonstrable [4].

Plants have been the basis of many traditional

medicine systems throughout the world for

thousands of years and continue to serve

mankind with new remedies. At present, there is

a worldwide movement or assessing the plant

resources which are of medicinal and economical

value and importance. Researchers are focusing

mainly on ethnobotanical & ethnomedicinal

investigations to fulfill the increasing demand of

herbal products. In the last few decades there

has been an exponential growth in the field of

herbal medicine and these drugs are gaining

popularity both in developing and developed

countries because of their natural origin and less

side effects [5]. WHO estimate that, about 80%

of the population in the developing countries

depends directly on plants for its medicine [6, 7].

WHO listed 20,000 medicinal plants globally, [8]

within these India’s contribution is 15-20% [9]. In

India 2,000 medicinal preparations used are of

plant origin.

Indian has an ancient heritage of traditional

medicine. Indian Materia Medica includes about

2000 drugs of natural origin almost all of which

are derived from different traditional system and

folklore practices. Out of these drugs derived

from traditional system, 400 are of mineral and

animal origin while the rest are of vegetable

origin. The plant based indigenous knowledge

was passed down from generation to generation

in various parts of the world throughout its

history and has significantly contributed to the

development of different traditional systems of

medicine [10]. India has a rich heritage of

traditional medicine and the traditional health

care system have been flourishing for many

centuries. It mainly consists of three major

systems namely the Ayurveda, the Siddha and

the Unani. Lot of efforts has been taken by the

government and private sectors for the

development of the traditional system based on

these three methods.

The North-East region of India (220-290 N; 890-

970 E) comprises the Sikkim and the seven sister

states namely Assam, Arunachal Pradesh,

Nagaland, Meghalaya, Mizoram, Manipur and

Tripura. This region of India has several hill

ranges interspersed with valleys and is by large

sparsely populated. Nearly 40% of the total

geographical area of this region is covered by

evergreen forest. Each state of the North–East

inhabited by number of ethnic tribes

characterized by their native languages, rituals,

costumes and housing pattern. There are more

than 180 major tribal communities of the total

427 tribal communities found in India [11, 12].





Pag

e57

7

The north-east region is also rich in medicinal

plant wealth. Many medicinal plant species

having industrial potential are growing wild in

this region [13]. A large no of people belonging

to various groups of the north-eastern region of

India still practicing their own traditional heath

care systems. The traditional treatment systems

adopted by these ethnic communities are being

used generation wise without any scientific

validation. They have the vast knowledge about

various plants that are used for food and as

medicine.

Since the discovery of insulin several synthetic

oral hypoglycemic drugs (OHDs) are available in

market. But these synthetic drugs are not

sufficient to treat this deadly disease, particularly

type II. Most of these synthetic drugs have some

serious side effects. In this respect our

traditional herbal therapeutic system can act as

an alternative to synthetic drugs. Generally these

phytochemicals have fewer side effects. Many of

them are helpful in preventing the secondary

complications of diabetes [14]. The herbal

medicines are generally cost effective and readily

accessible in compares to synthetic drugs. The

adverse side effects and higher cost of the

existing anti-diabetic drugs necessitate the

search for novel, well tolerated, efficient and

easily available herbal remedies to tackle the

deadly diabetes.

MATERIALS AND METHODOS

Medicinal plants provide a promising bargain

since they are easily accessible and affordable.

The North-eastern communities of India use

traditional herbal medicines for controlling the

disease some of which are reported to be highly

potent and promising. Herbal preparation of

different plant parts such as leaf, root, fruit, bark

and also the whole plant are used by numerous

communities [15,16].The data were collected

based on the available literature published in the

form of journal [14, 17, 18, 19, 20], popular

articles and active field survey. Some medicinal

plants that are used as anti-diabetic purposes

are listed in Table No 1.

Table No– 1. Medicinal Plants used as anti-diabetic purposes in North-East India.

Sl. No Botanical Name & Family Local Name Part Used Preparation to

used

1.

Acacia Concina DC

(Mimosaceae)

Khangthur Leaves The leaves

infusion is

taken orally.

2. Aconiotum hetrophylloides

Ranuculaceae

Bish, Seli, Chandu Dried tuberous roots Decoction of

root is used

3.

Adhatoda vasica Nees

Acanthaceae

Bogabahok Roots Infusion of

roots is used

4.

Aechmea fasciata

Bromeliaceae

Aechmea Whole plant Crushed and

mixed with

water

5.

Aegle marmelos correa]

Rutaceae

Bel Leaves Leaves boiled

with water

6. Ajuga bracteosa Neel-kantha Leaves Leaves boiled





Pag

e57

8

Lamiaceae with water

7.

Albizzia procera Benth

Mimosaceae

Koroi

Siris

Roots, leaves, flowers Leaves juice is

taken orally

8.

Alocacia indica schott

Araceae

Mankachu Rhizomes Dried rhizome

is used

9.

Aloevera tournex.Linn

Liliaceae

Ghrita kumari Leaves Leaves paste is

used

10. Alstonia scolaris R.Br.

Apocynaceae

Satiana Leaves, Bark Leaves paste

and bark

extract is

taken.

11.

Anana scomosus (L) merr

Bromeliaceae

Matikathal Fruits, Whole plant Fruit juice is

used.

12.

Andrographis paniculata

Nees

Acanthaceae

Kalmegh Whole plant Crushed and

boiled with

water

13.

Annonareticulata Linn

Annonaceae

Aatlas Barks Dried barks

decoction is

used

14.

Annona squamosa Linn

Annonaceae

Manga Leaves Leaves boiled

with water

15. Annona reticulata L.

Annonaceae

Atlas

Leaves, Fruits

Leaves, Fruits

juice is used

16. Antidesmaacidum Retz

Euphorbiaceae

Nikhutenga Leaves Leaves juice is

used orally

17. Antocarpus heterophyllus

Linn

Moraceae

Kothal Leaves Leaf juice is

used orally

18. Areca catecheu Linn

Arecaceae

Tamul Nut Dried and

soaked with

water.

19. Argyreia speciosa Linn. F.

Convulaceae

Takoria alu Stem, Leaves Stem, Leaves

paste is used.

20. Artocarpus lokoocha Roxb

Moraceae

Diwatenga Barks Barks infusion

is used

21. Artemisia maritma

Asteraceae

Chinglaibaknag Leaves Boiled leaves

extract

22. Averrhoacarambola Linn

Oxalidaceae

Kardoi Barks Bark infusion is

used.

23. Beta vulgaris L.

Chenopodiaceae

Beet Roots Root juice

taken orally





Pag

e57

9

24. Boenninghausenia albiflora

Rutaceae

Yomri, Nukmam Roots Root juice is

taken orally

25. Bombox malabaricum DC

Bombacaceae

Simolu Roots Mixed with

water or root

juice is used.

26. Bouganvillea spectabilis

Willd.

Nyctaginaceae

Kagaz phul Leaves Leaves paste is

anti-diabetic

but not used by

human.

27. Brassica juncea(L)Czern.

Brassicaceae

Sorioh Leaves, Seeds Seed powder

with milk taken

orally

28. Caesalpinia crista Linn.

Caesalpiniaceae

Lataguti Seeds Crushed

powder is used.

29. Caesalpinia pulcharrima

Caesalpiniaceae

Guletura Flowers Flower juice is

used

30. Cajanus cajan Mill

Papilionaceae

Rahar Leaves Powder leaves

mixed with

water.

31. Calotropis gigantea (L) W.

Aiton.

Asclepiadaceae

Akon Leaves Leaves, Flower

paste is used

32. Canna indica Linn

Cannaceae

Parijat Leaves, Stem Dried leaves

are used.

33. Cannabis sativa Linn

Cannabaceae

Bhang Leaves, stem, flowers Leaves and

stem soaked

with water

34. Carrica papaya Linn

Carricaceae

Amita Seeds Seed powder

mixed with

water

35. Casia alata Linn.

Caesalpiniaceae

Khorpat

Tender leaf Leaves mixed

with water

36. Cassia angastifolia Linn

Caesalpiniaceae

Channa

Leaves Infusion of

leaves is used

37. Cassia fistula L.

Caesalpinaceae

Sunaru Barks Bark powder is

used

38. Cassia occidentalis Linn.

Caesalpinaceae

Bonoriadadol Seeds, stem Seeds powder

is used

39. Cassia sophera Linn

Caesalpinaceae

Bonmadelua Seeds, stem, bark Seeds and bark

powder is used

40. Catharanthus roseus (L.) G.

Don.

Nayantara Whole plant Boiled extract

of leaves,





Pag

e58

0

Apocynaceae

flowers taken

orally

41. Centella asiatica(L.)Urban.

Apiaceae

Manimuni Whole plant Whole plant

juice is taken in

empty stomach

42. Ciceracida Linn

Euphorbiaceae

Holfoli Leaves Infusion of

leaves is used

43. Cichorium intybus

Asteraceae

Kasni Seeds Seeds powder

is used

44. Cinnamomum tamala fr. Nus

Lauraceae

Tezput Bark. roots Bark powder

made infusion

45. Citrus aurantifolia Linn

Rutaceae

Nemu Fruits Fruit juice is

used

46. Clerodendrum infortunatum

L.

Lamiaceae

Vetetita Leaves Leaf paste is

taken orally.

47. Coccinia indica cogn

Cucurbitaceae

Balipoka Fruits, root Fruits juice is

used.

48. Cocos nucifera L.

Arecaceae

Narikol Oil Oil is taken out

and consumed.

49. Coix lacrymajobi

Poaceae

Chaning angouba

(Manipur)

Roots Crushed extract

of roots is used.

50. Colocasiaesculenta(L) Scholl

Araceae

Kolakachu Roots Roots boiled

with water

51. Coptis teeta

Ranuculaceae

Rings,

Mishimetectga

Dried root and

rhizomes.

Roots and

rhizomes juice

is orally taken

in empty

stomach.

52. Coriandrumsativum Linn

Apiaceae

Dhania Leaves Leaves infusion

is used

53. Costus speciosus

(Koeing).Smith

Zingiberaceae

Jamlakhati Rhizomes Rhizome paste

taken oral

54. Curcuma domestica val

Zingiberaceae

Halodhi Leaves, rhizomes Powdered

rhizomes are

used.

55. Curcuma aromatic Salisb

Zingiberaceae

Bonoria Halodhi Rhizomes Powder

rhizome is used

56. Cucumis melo Roxb

Cucurbitaceae

Sal kumura Seeds Seed powder is

used.

57. CynodondactylonPers Dubori Whole plant Crushed and





Pag

e58

1

Poaceae boiled with

water

58. Dalbergia sisco Roxb.

Papilionaceae

Sishu Stem barks Bark powder is

used.

59. Daucas carota Linn

Apiaceae

Gajor Roots Paste or juice is

used.

60. Debregeasia

longiafoliaWedd.

Urticaceae

U-khajing

Manipur

Tubers Thirst of

diabetic patient

61. Dilleniaindica Linn.

Dilleniaceae

Aautenga Fleshy perianth Juice is used.

62. Dioscorea alata Linn.

Diosconeaceae

Kath alu

Rhizome Boiled with

water and

taken.

63. Eichhornia cussipes

F- NF

Kachuripana Whole plant. Crushed

powder

infusion is

used.

64. Enhydra fluctuanas Lour

Asteraceae

Komperek

Manipur

Nodes of the plant Boiling the

nodes of plant

with water

65. Equisetum debile Roxb.

Equisteraceae

Lai-utang

Manipur

Whole plant Boiled with

root of male

Carica papaya.

66. Erythairina indica Linn

Papilionaceae

Moder

Gadela (Assam)

Roots Root powder is

used

67. Eugenia jambolana Linn

Myrtaceae

Kala Jamu Fruits, Barks, Seeds. Fruit juice is

used orally.

68. Euphorbia hirta Linn.

Euphorbiaceae

Gakhirotibon Whole plant Crushed and

made infusion

69. Ficus benghalensis Linn.

Moraceae

Bot Stem, barks Bark infusion or

decoction is

used

70. Ficus religiosa L

Moraceae

Dhup Leaves, Fruits Leaf, fruit taken

orally

71. Flacourita jangomas Lour

Flacourtiaceae

Heitroi

Fruits Raw fruits are

used.

72. Garcinia padunculata Roxb.

Clusiaceae

Borthekera Fruits Fruit juice or

raw fruit is

used.

73. Gloriosa superba Linn. Gloriosa Whole plant Made infusion





Pag

e58

2

Liliaceae and used.

74. Glycine max Merr

Papilionaceae

Soyabeen Seeds Seed soaked

with water.

75. Gmelinaarborea Roxb

Verbenaceae

Gomari Leaves, fruits Leaves juice is

used.

76. Grewia abutelifolia Juss

Tiliaceae

Petuk Fruits

Fruit juice is

used.

77. Heliotropium indicum Linn.

Heliotropiaceae

Hatisur Aerial part

Dried and

infusion is

filtered before

used.

78. Holoptelia integrifolia Planch

Ulmaceae

Holoptelia Barks Bark powder is

used

79. Holorrhena antidysenterica

wall

Apocyanaceae

Kutaz Barks, fruits Fruit juice is

used

80. Hydrocotyle rolundifolia

Roxb

Apiaceae

Soru manimuni Whole plant

Crushed to

make paste.

81. Ichnocarpus frutescent R. Bn.

Apocyanaceae

Syamalota Roots

Roots

decoction is

used

82. Imperata cylindrical Beauv

Poaceae

Ulukher Roots

Root infusion is

used.

83. Ipomoea aquatica Forssk.

Convulaceae

Kalmou Leaves Dried leaf

powder mixed

with Piper

nigrum and

taken orally.

84. Ipomoea batata (L) Lam.

Convulaceae

Mitha alu Leaves Leaf boiled and

juice is taken

orally.

85. Jatropha curcus Linn.

Euphorbiaceae

Bongaliara Leaves, twigs

Leaves juice is

used

86. Jussieua repens

Onagraceae

Ishing-kundo

Leaves, whole plant Boil extract of

the plant is

used.

87. Justicia adhatoda

Acanthaceae

Nongmang-

khaangouba

Leaves Boil the leaves

with the leaf of

Clerodendrun

spihonanthus.

88. Kyllinga triceps Rottb Chumthang Whole plants Boiled extract





Pag

e58

3

Cyperaceae namthibi of the plant.

89. Lawsonia inermis L.

Lythraceae

Jetuka Leaves Leaves juice

mixed with

cow’s milk

taken once in a

week.

90. Leucaena glauca

Mimosaceae

Cialag

Manipur

Leaves Leaves

decoction is

used.

91. Leucas aspera Spreng.

Lamiaceae

Doron Whole plant Stomach along

with

Monopterus

cuchia fish.

92. Lindernia bracheata Linn

Scruphulariaceae

Kachidoria Whole plant Juice used.

93. Litseaglutinosa(lour) CB

Robins

Lauraceae

Haluca Leaves Leaves juice is

used.

94. Ludwigia octovalvis Jacq. Kabo Kaji Whole plant Boiled extract

is used.

95. Madhuka indica Gmel

Sapotaceae

Mohua Barks, Seeds Bark and seed

crushed and

infusion is

used.

96. Mangifera indica Linn

Anacardiaceae

Aam Fruits Fruit juice or

raw fruit is

used.

97. Meliaazadirachta Linn

Meliaceae

Mohaneem Leaves

Leaves juice is

used.

98. Mentha arvensis

Lamiaceae

Nungshi-hidak Plant part Mixed with

honey.

99. Meyna spinosa Roxb.

Rubiaceae

Lam-heibi Fruits Boiled extract

of fruits

100. Mimosa pudica Linn.

Mimosaceae

Nilazibon Whole plant Whole plant

boiled with

water.

101. Mirabilis jalapa L.

Nyctaginaceae

Gopal godhuli Roots Root juice is

taken.

102. Moringaoleifera Linn

Moringaceae

Sogina Barks, flowers, leaves Barks infusion

is used.

103. Mormordica charantia Linn

Cucarbitaceae

Tita-Karela Leaves, fruits

Leaves and fruit

juice is used





Pag

e58

4

orally.

104. Morus indica Linn

Moraceae

Nuni Tender leaves Eat with any

roasted fish

105. Mucunapruriens DC.

Papillionaceae

Bandarkakura Roots

Roots powder

soak with

water

106. Murraya koeningii Sprang

Rutaceae

Narasingha Leaves Leaves juice is

used

107. Musa paradisiacal Linn

Musaceae

Kashkol Flowers, fruist

Fruit juice is

used.

108. Nelumbo nucifera Gaertn

Nelambaceae

Podumful Tender leaves Crushed and

pinch of

powder taken

with water.

109. Nictanthus arbor tristis Linn

Oleaceae

Sewali Leaves, flowers Young Leaf

juice, flower

paste is taken

orally.

110. Nigella sativa

Ranuculaceae

Keman Seeds

Seeds powder

is used.

111. Ocium sanctum L.

Lamiaceae

Tulsi Leaves Leaf powder

taken with

honey to treat

Diabetes.

112. Osbeckiane palensis Hook

Melastomaceae

Photkola

(Boga)

Tender shoot

Crushed

powder is used

113. Oxalis corniculata L.

Oxalidaceae

Tengesi Aerial part 100 gm aerial

part are

wrapped with

banana leaf

and after

roasted in

firewood is

given with salt

once daily 3

days interval.

114. Parkia timoriana (A.DC)

Mimosaceae

Yongchak

Barks Decoction of

bark with

Centella

asiatica and

Ficus

glomerata





Pag

e58

5

fruits is used.

115. Peristrophe fera C.B. Clarke

Acanthaceae

Ishing langthrei Leaves or whole

plants

Extract of the

plant is used

116. Phologocanthus tubiflorus

Nees.

Acanthaceae

Sang-chi Barks Decoction of

bars with

decoction of

Zingiber

officinale

117. Phyllanthuse emblica Linn

Euphorbiaceae

Amlakhi Fruits Fruit juice or

raw frit is taken

orally.

118. Phyllanthus niruri Linn

Euphorbiaceae

Bonamlokhi Whole plant

119. Phyllanthuse urinaria Linn Heikruman Leaves or whole plant Boiled extract

is used orally.

120. Plumeria acuminata

Apocyanaceae

Sun-champa Barks Powder bark

decoction is

used.

121. Portulaca oleraceae

Portulaceae

Kulfa Whole plant Crushed

powder is used.

122. Pouzolzia zeylanica (L) Benn

Urticaceae

Borali bakua Whole plant

Decoction is

used

123.

PremnalatifoliaRoxb

Verbenaceae

Agnimantha Leaves, Barks

Leaves juice is

used.

124. Psidium guyava Linn

Myrtaceae

Madhuri Fruits, leaves Leaves infusion

is used.

125. Swertia chirata L

Gentianaceae

Chirata Whole plant Whole plant

extract is

consumed.

126. Saraca indica Linn

Caesalpiniaceae

Ashok Fruits

Fruit juice is

used.

127. Scleria terristris (Linn)

Cyperaceae

Thangjou

Plant part Boiled extract

of the plant

part is used.

128. Sesamum orientale

Pedaliaceae

Senum seeds Seeds.

129. Sesbaniaseban (Linn) Merr.

Fabaceae

Chuchurangkei Leaf or tender shoot

Boiled extract

of leaves.

130. Smilax lanceifolia Roxb.

Smilaceae

Kwa manbi Roots Boiled extract

of root.

131. Solanum indicum Linn.

Solanaceae

Bhakuritita Fruits Fruit juice is

used.





Pag

e58

6

132. Spondias mangifera wild

Anacardiaceae

Amara Fruist Raw fruit is

used.

133. Sterculia villosa Roxb

Starculiaceae

Udal Roots

Root infusion is

used.

134. Syzigium cumini (Linn.)

Myrtaceae

Jamhei

Seeds

Boiled extract

of seeds.

135. Tabernaemontanadivericota

(L)

R. Br.

Apocynaceae

Kothal pool Leaves and flowers Fresh flower

soaked with

water.

136. Terminalia chebuta Roxb.

Combretaceae

Selekha

Fruits Fruit juice is

used.

137. Thevetia peruviana (pers)

Merill

Apocyanaceae

Halodhia-korobi

Utonglei

Barks

Powder bark is

used.

138. Inospora cordifolia Miers

Menispermaceae

Sidhilota Leaves, bark Powder bark

infusion is

used.

139.

Trigonella foenumgraceum

Linn

Papilionaceae

Mithi

Seeds

Seeds soaked

with water and

taken in the

morning.

140. Vinca rosea Linn

Apocyanaceae

Nayantora Leaves

Leaves chewed

at morning or

juice is used.

141. Zanthoxylum armatum DC.

Rutaceae

Muthrubi

Leaves and roost

Roots and

leaves

decoction is

used.

RESULT AND DISCUSSION

Herbs have been used for healing purposes and

to promote wellness since from the ancient

times and are not categorized as medicines but

treated as food since they are natural products.

Nowadays, herbal medicines, health and dietary

supplements are flooding the markets. The use

in the right way provides effective and safe

treatment for many ailments and the

effectiveness is mostly subjective to the Patient

[21]. One of the major advantages of the herbal

drug is that it is cost effective and easily

affordable. In compares to the synthetic drugs

they became less toxic or least side effects. From

the ancient times the people of the north-

eastern part of India have been using the herbal

medicines for curing the diabetes. Most of the

plant has possessing prominent activity in this

literature there are 141 nos. of plants have

mentioned from the North-east India that are

used for anti-diabetic purposes. The figure 1

shows the percentage use of plant part of the





Pag

e58

7

herbal medicine. Leaves: 29%, Whole plants:

15%, Roots: 10%, Fruits and Seeds: 19%,

Rhizomes and tubers 13%, Bark: 13%, and

Flowers: 4%.

Figure 1: It shows the percentage of plant parts is used for anti-diabetic purposes.

The ethnomedicinal information from the above

table and figure (Tab-1 & Fig-1) shows that the

peoples of this region are too much habituated

for the use of herbal drugs in diabetes. The

ethnomedicinal knowledge emphasizes the

significance of the crude drugs. The north-

eastern communities have the ethnic knowledge

to use the anti-diabetic plant. They use these

plants generaously without any scientific

modification. So it is utmost necessary to screen

these traditional medicines scientifically for

proper investigation into their use. It is necessary

to keep in mind that most of the medicinal

plants have number of allied species that are

morphologically similar and are difficult to

distinguish. Plant parts used in preparations are

usually collected by common people without any

attention to botanical authentication. So, there is

the possibility of incorporation of wrong plant or

parts of the plant with less activity or without

any activity. Therefore, it is very essential to

evaluate the botanical identity and quality of the

medicinal plants used in the traditional

preparation. Pharmacognostic studies are usually

adopted for standarization and quality

evaluation of botanical drugs.

CONCLUSION

The North-East India is stands for it is glory of

eminent bio-diversity aspect and the ethnic

communities of this region provide the footstep

for the traditional knowledge. By the

advancement of the concept of living peoples of

the ethnic communities practicing less

dependency to their traditional knowledge. So

the knowledge which is generously rolled, facing

a critical barrier for advancement, gradually. So

it will be the aim to documented of the ethnic

knowledge and preserved thereof.

Differentnatural products with anti-diabetes

activity have been described in the literature.

There is a need for a multidisciplinary approach

to develop potentially effective drugs. Within the

list many of the medicinal plants have possessing

potent anti-diabetic properties that has not still

0

5

10

15

20

25

30

35

PERCENTAGE OF PLANT PART

PERCENTAGE OF PLANT PART





Pag

e58

8

explored scientifically. The article summarize the

significance of medicinal plants that are

traditionally used in north-east India as anti-

diabetic purposes and the requisite level of

activity as well as toxicity would be considered

for further scrutiny to develop the potential drug

molecule.

ACKNOWLEDGEMENTS

The authors gratefully thanks to Dr.

KamaruzZaman (Asst. Professor) Department of

Pharmaceutical Sciences, Dibrugarh University,

Dibrugarh, Assam-786004, India, for his kind

advice to the present work.

REFERENCES [1] A Ramachandran, AK Das, SR Joshi, CS Yajnik, S Shah, KM

Prasanna Kumar, Current Status of Diabetes in India

and Need for Novel Therapeutic Agents: Special Issue

On Human GLP1 Analougues Volume 58.

[2]Maiti R, Jana D, Das UK, Ghosh D.Antidiabelic effect of

aqueous extract of seed of Tamarindusindicain

streptozotocin induced diabetic rats. J Ethnopharmacol

2004, 92, 85-91.

[3]TiinamaijaTuomi., Type I and Type II diabetes,

International Journal of Pharmacy and Pharmaceutical

Sciences, 2005; 34: 3214-3218.

[4]Tripathi KD. Essentials of medical pharmacology,

6thediton, Jaypee Brothers Medical Publishers (P) Ltd.

New Delhi: 254-255.

[5]Grover J K., Yadav S. & Vats V., Medicinal plants of India

with anti-diabetic potential, Journal of

Ethnopharmacology, 2002; 81: 81.

[6]Pareek, S.K., Medicinal plants in India: Present status and

future prospect. In prospect of medicinal plants

(edsGautam P. L. et al), Indian society for plant genetic

Resources, New Delhi, 1998; 5-14.

[7] Mukhopadhaya, S., Conservation, protection and

biodiversity of medicinal plants. In Prospects of

medicinal plants (edsGautam P. L. et al), Indian society

for plant genetic Resources, New Delhi, 1998; 15-28.

[8] Gupta R. and Chadha, K. L., Medicinal and aromatic plant

in India. In advance in Horticulture, Medicinal and

aromatic plants (eds. Chadha K.L. and Gupta R.),

Malhotra Publishing House, New-Delhi, 1995; 1-44.

[9]Singh, H.B., Alternative source for some conventional

drug plants of India, Ethnobotany and ethnomedicinal

plants of Indian subcontinent (edsMaheshwari J.K.,),

Science Publishers, Jodhpur, India, 2000; 63-78.

[10] ZamanKamaruz, Pharmacognostical and phytochemical

investigations on some anti-malarial herbal drugs of

north-east India: Department Of Pharmaceutical

Sciences, Dibrugarh University, Assam; 2010; 2-3.

[11]Sajem A.L., Rout. J and Nath. M. Traditional tribal

knowledge and status of some rare and endemic

medicinal plant of North Cacher Hills district of Assam,

North-east India. Ethnobotanical Leaflet, 2008; 12: 261-

275.

[12] Kala C.P., Ethnomedicinal botany of the Apatani in the

Eastern Himalayan Region of India. Journal of

Ethnobiology&Ethnomedicine, 2005; 1:11.

[13] Bhagabati AK, Kalita MC and Barua S. (eds). In:

Biodiversity of Assam: Status stretagy and action plan

for conservation, EBH Publishers, Guwahati (India) on

behalf of Assam Science Society, 2006; 60-62.

[14]Sarmah PC. Ethno Antidiabetic Plants Of Assam;

International Journal of Applied Biology and

Pharmaceutical Technology: Volume: 2: Issue-4: Oct -

Dec -2011.

[15] Bora U., Sahu A., Saikia A.P., Ryakala V. K.,andGoswami

P., Medicinal plants used by the people of north-east

India for curing malaria. Phytotherapy Research, 2007;

21:800-804.

[16] Kumar. Sudhir., The medicinal Plants of North-East

India. Scientific Publishers (India), Jodhpur 2002;

22,59,63,82.

[17]Barukial J.,Sarmah J.N., Ethnomedicinal Plant used by

the people of Golaghat district, Assam India. Int. J. Med.

arom.Plants. Vol. 1, No. 3: December 203-211.

[18] Chakravarty S., Kalita CJ. An Investigation On Anti

Diabetic Medicinal Plants Used By Villagers In Nalbari

District, Assam, India. IJPSR (2012), Vol. 3, Issue 06.

[19]Khan Md. H., Yadav PS., Anti-diabetic plants used in

Thoubal district of Manipur, Northeast India. Indian

Journal of traditional knowledge; Vol.9 (3), July 2010;

510-514.

[20] Das T, Mishra SB., Saha D., Agarwal S., Ethnobotanical

Survey of Medicinal Plants Used by Ethnic and Rural

People in Eastern Sikkim Himalayan Region. African

Journal of Basic & Applied Sciences 4 (1): 16-20, 2012.

[21]G.B. Kavishankar, N. Lakshmidevi, S. Mahadeva Murthy,

H.S. Prakash, S.R. Niranjana. Diabetes and medicinal

plants-A review. Int J Pharm Biomed Sci 2011, 2(3), 65-

80





Pag

e58

9

*Corresponding Author: Prodyut Mondal* Department Of Pharmaceutical Sciences Dibrugarh University, Assam 786004. India.



ResearchArticle

BiologicalSciences


VidyaPai*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e59

0

MULTIPLE Β LACTAMASE ENZYMES PRODUCING CLINICAL ISOLATES OF GRAM NEGATIVE

BACTERIA IN A TEACHING HOSPITAL

VidyaPai*, Sunil Rao P, Bhaskaran Nair

Department of Microbiology, Yenepoya Medical College, Nithyanandanagar, Mangalore-575018, India.

*Corresponding AuthorEmail:

ABSTRACT Context: Gram negative bacilli producing Beta lactamases have been increasingly reported worldwide and

infections with such bacteria are difficult to treat. It is also not unusual to find a single isolate that expresses

multiple beta lactamase enzymes further complicating the treatment options. Aims: The present study was

designed to investigate the coexistence of different beta lactamase enzymes in clinical isolates of gram negative

bacilli. Material and Methods: A total of 321 isolates of gram negative bacilli obtained from various clinical

specimens were included in the study. Antimicrobial susceptibility testing was performed for all the isolates in

accordance with CLSI guidelines. All bacterial strains were tested for ESBL, Amp. C & MBL production. Statistical

analysis used: Descriptive statistics was used and the percentage of ESBL, Amp C and MBL carrying gram negative

bacilli isolates were calculated. Results: ESBL production was seen in 100 (31.1%) isolates with maximal incidence

in Citrobacter species (52.1%), followed by P. aeruginosa (30.4%). Amp C production was detected in 67 (27.8%)

isolates with highest percentage (25.4%) among non-fermenters. Conclusions: Early detection of these multiple β

lactamase producing isolates in a routine laboratory could help to avoid treatment failure, as often such isolates

show a susceptible phenotype in routine sensitivity testing. Unless strict measures to limit the indiscriminate use of

cephalosporins and Carbapenems in the hospitals are undertaken, the multiple β lactamase producing pathogens

would spread with no treatment options left to treat nosocomial infections with such pathogens.

KEY WORDS ESBL, Amp C, MBL.

INTRODUCTION

Gram negative bacilli account for the majority of

bacterial pathogens isolated from clinical

specimens.1The incidence of infections due to

Gram negative bacilli resistant to β lactam agents

has increased in recent years. Till 2006, ESBL

production by GNB was considered as the most

important threat to clinical therapeutics. 2, 3.This

led to a parallel increase in the usage of β

Lactam/ β lactamase inhibitor combinations,

monobactams and carbapenems. Eventually, in

the last few years, reports from worldwide show

resistance to these drugs as well. 4, 5

The resistance to monobactams and

carbapenems is due to the production of Amp.C

and Metallo beta lactamases respectively. The

genes coding for these β lactamases are carried

on plasmids, facilitating rapid spread between

micro-organisms and often are co-expressed in

the same isolate.6 The treatment options for

such infection are limited and hence of great

concern. Hence the present study was designed

to investigate the presence of different classes of

β lactamase enzymes in the clinical isolates of

gram negative bacilli.





Pag

e59

1

MATERIAL AND METHODS

The study was conducted in the department of

microbiology of our medical college hospital. A

total of 321 consecutive, non-duplicate isolates

of gram negative bacilli obtained from various

clinical specimens were included in the study.

The isolates were characterized by using

standard microbiological techniques.7

Antimicrobial susceptibility testing was

performed for all the isolates by using the

commercially available discs [Himedia, Mumbai,

India] in accordance with CLSI guidelines.8 The

antibiotics which were tested include,

Piperacillin 100µg ([PIP), Ceftazidime 30 µg

(CAZ), Imipenem 10 µg (IPM), Ciprofloxacin 5 µg

(CIP), Gentamycin 10 µg (GEN), Amikacin 30 µg

(AK) and Aztreonam 30 µg (ATM). Quality control

was achieved using standard strains of E.coli

ATCC 25922 and Pseudomonas aeruginosa ATCC

27853.

All bacterial strains were tested for ESBL, Amp. C

& MBL production by the following methods.

ESBL detection method: ESBL status of the

isolates was detected by combined disc diffusion

using Cefotaxime 30 µg [CTX] &Ceftazidime 30

µg [CAZ] disc alone and in combination with

Clavulanic acid [CEC & CAC] as per CLSI

recommendations.

Amp C detection method: The isolates were

tested for Amp C production by the disc

antagonism test. A test isolate [with a turbidity

equipment to that of 0.5 McFarland standard)

was spread over a Mueller Hinton agar plate.

Cefotaxime 30µg (CTX) and Cefoxitin 30 µg (Fox)

discs were placed 20mm. apart from centre to

centre. Isolates showing blunting of CTX zone of

inhibition adjacent to the Fox disc were taken as

Amp C producers.

MBL detection method: The isolates were

screened for the presence of MBLs by the

combined disc test (CDT). Two Imipenem 10 µg

discs were placed on the surface of an agar plate

with bacterial inoculum and 5 µl EDTA was

added to one of them to obtain a concentration

of 750 µg. The zones of inhibition of IPM alone

and IPM-EDTA were compared after 16-18 hours

incubation in air at 35oC. An increase in zone size

of >7mm was taken as positive.

Descriptive statistics was used and the

percentage of ESBL, Amp C and MBL carrying

gram negative bacilli isolates were calculated.

RESULTS

A total of 321 bacterial isolates were included in

the study. Table 1 depicts the different bacterial

species tested and their resistance pattern.

ESBL production was noticed in 100 (31.1%)

isolates with maximal incidence in Citrobacter

species (52.1%, n=12), followed by P. aeruginosa

(30.4%, n=32).

Amp C production was detected in 67 (27.8%)

isolates. Majority of P.aeruginosa strains (28.4%,

n=30) produced Amp C β lactamases. imipenem

resistance was seen in a mere 11(30.9 %) strains,

whereas resistance to ciprofloxacin was seen in

108 (95.5 %) strains (Table 2).

Co-production of Amp C β lactamase and ESBL

was seen in 17.1% (n=55) strains and MBL

production was detected in 11 isolates. Co-

production of Amp C and Metallo β lactamases

was found in 1.2% (n=4) isolates with maximal

occurrence among Acinetobacter species (2.3 %)

isolates. (Table 3)





Pag

e59

2

Table 1: Resistance pattern of bacterial isolates:

Isolate No. PIP CIP GEN AK ATM CAZ IPM

E.coli 60 40 39 60 14 9 19 1

Klebsiella species 72 72 30 24 16 12 23 4

Citrobacter species 23 23 23 23 23 2 14 1

Proteus species 18 2 6 4 -- -- 1 --

Pseudomonas species 105 92 84 75 53 34 32 3

Acinetobacter 43 41 35 40 31 10 11 2

Total 321 270 217 226 137 67 100 11

Table 2: ESBL & Amp C production in bacterial isolates.

Clinical isolate No. of

isolates

ESBL producers (%) Amp C

producers

Both ESBL &

Amp c

E.coli 60 16 (26.6%) 9(15%) 5 (8.3%)

Klebsiella species 72 20(27.7%) 11 (15.2%) 8(11.1%)

Citrobacter species 23 12 (52.1%) 2(8.6%) 6 (26.0%)

Proteus species 18 1(5.5%) - (-) - (-)

Pseudomonas species 105 32 (30.4%) 30 (28.4%) 21 (20.0%)

Acinetobacter 43 11 (25.5%) 9 (20.9 %) 9(20.9%)

Total 321 100 (31.1%) 67 (20.8%) 55(17.1%)

Table 3: MBL & Amp C production

MBL producers Amp C Both MBL + Amp C

E.coli 1 (1.6) 9(15%) -

Klebsiella species 4 (5.5) 11 (15.2%) -

Citrobacter species 1(4.3) 2(8.6%) -

Proteus species -- (-) - (-) -- ( - )

Pseudomonas species 3(2.8) 30 (28.4%) 2 (1.9)

Acinetobacter 2(4.6) 9 (20.9 %) 2 (2.3)

Total 11(3.4) 67 (20.8%) 4(1.2)





Pag

e59

3

DISCUSSION

Gram negative bacterial isolates show a

multiplicity of resistance mechanisms. ESBL

producing strains of GNB have emerged as a

major problem in hospitalized on well as

community based patients.9 The incidence of

ESBL in major hospitals of India has been

reported as high as 60%-80%.10,11 Their

prevalence worldwide has been non-uniform. US

hospitals have reported 40% of

Klebsiellapneumoniae isolates as ESBL producers

whereas reports from Taiwan show 94% of

Klebsiella species as ESBL producers. 12,13

In our study 31.1% of total GNB included,

showed ESBL production with the highest

incidence in Citrobacter species (52.1%) followed

by P.aeruginosa (30.1%). Higher percentages of

ESBL producing GNB were shown by other

studies 14,15whereas reports from Chennai 16 and

Hyderabad 17 show lower percentages of ESBL

producers.

Shortly after ESBLs, Amp C β lactamase emerged

which were resistant to 3rd generation

Cephalosporin including β-lactam/ β lactamase

inhibitor (in contrast to ESBL) but sensitive to 4th

generation cephalosporins. In 2003, 20.7% Amp

C producers were reported from Delhi, 18 37%

from Chennai.19 The numbers of Amp C

producers has been increasing over the years.

In our study 27.8% of GNB isolates showed Amp

C production with highest percentage (25.4%)

among non-fermenters. Some hospitals have

reported high percentage (up to 80%) of Amp C

producers.20

The only β lactam active against Co-Amp C and

ESBL producers are Carbapenems, however,

recently resistance to Carbapenems has been

increasing, which is mostly due to production of

MBL.21 Our findings showed 3.4% of the bacterial

isolates produced MBLs and 1.2% strains

produced both Amp C and MBLs.

Carbapenemases have been reported in E.coli,

Klebsiella species. Pseudomonas species and

Acinetobacter species from different parts of the

globe. The percentage various widely with some

centres reporting low figures (48% in

Acinetobacter) where as others showing upto

80% (Acinetobacter species). 22 Lower resistance

to imipenem in our centre may probably due to

the reserved use of thus drug.

An interesting finding was that 2 isolates were

sensitive to imipenem by routine disc diffusion

method but showed MBL production by CDT

(IPM-EDTA).

These carbapenem susceptible isolates carrying

hidden MBL genes, may spread unnoticed and

may lead to untoward infection control

problems. As there is no single method proven

as ideal method for MBL detection in all the

isolates, we used the CDT, which is

recommended by CLSI and proven by many

other studies.

CONCLUSION

The present study emphasizes the prevalence of

gram negative bacilli producing β lactamase

enzymes of diverse mechanisms. Early detection

of these multiple β lactamase producing isolates

in a routine laboratory could help to avoid

treatment failure, as often such isolates show a

susceptible phenotype in routine sensitivity

testing. Unless strict measures to limit the

indiscriminate use of cephalosporins and

Carbapenems in the hospitals are undertaken,

the multiple β lactamase producing pathogens

would spread with no treatment options left to

treat nosocomial infection with such pathogens.

REFERENCES 1. Eisenstein BI, Zaleznik DF. Enterobacteriaceae. In:

MandellGL, Bennett JE, Dolin R, editors. Principles and

practiceof infectious diseases, 5th ed. Philadelphia, Pa:

ChurchillLivingstone; 2000 p. 2294-310.





Pag

e59

4

2. Livermore DM. b-lactamase mediated resistance and

opportunities for its control. J

AntimicrobChemother1998; 41 (Suppl D): 25-41.

3. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of

extended spectrum beta lactamase producing gram

negative bacteria in a tertiary care hospital. Indian J

Med Res 2002; 115: 153-7.

4. Navaneeth BV, Sridaran D, Sahay D, Belwadi MRS. A

preliminary study on metallo b-lactamase producing

Pseudomonas aeruginosain hospitalized patients.

Indian JMed Res 2002; 116: 264-7.

5. Chitnis SV, Chitnis V, Sharma N, Chitnis DS. Current

status of drug resistance among gram-negative bacilli

isolated from admitted cases in a tertiary care center. J

AssocPhysiciansIndia2003; 51: 28-32.

6. Chatterjee SS, Karmacharya R, Madhup SK, Gautam V,

Das A, Ray P. High prevalence of co-expression of

newer β-lactamases (ESBLs, Amp-C-β-lactamases, and

metallo-β-lactamases) in gram-negative bacilli. Indian J

Med Microbiol 2010; 28:267-8

7. Crichton P.B. Enterobacteriaceae: In: Mackie and

McCartney Practical MedicalMicrobiology, 14th ed.

Collee J.G, Fraser A.G, Marmion B.P, Siminous A,

editors.(Churchill Livingstone: New York) 1996. 361-4.

8. Performance standards for Antimicrobial Disc

Susceptibility Tests, Clinical and Laboratory Standards

Institute. Vol.29 No.3, Jan 2009.

9. An update on newer [beta]-lactamases Ind J Med Res

Nov, 2007.

10. Hansotia JB, Agarwal V, Pathak AA, Saoji AM. Extended

spectrum beta-lactamase mediated resistance to third

generation cephalosporins in Klebsiellapneumoniae in

Nagpur, central India. Indian J Med Res 1997; 105 :

158-61.

11. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of

extended spectrum beta lactamase producing Gram

negative bacteria in a tertiary care hospital. Indian J

Med Res 2002; 115 : 153-7.

12. Jacoby GA, Munoz-Price LS: The new beta lactamases.

N Eng J Med 2005; 352: 380-91.

13. Yan JJ, Hsueh PR, Chang FY, Shyr JM, wan JH, Liu YC,

Chuang YC, Tsao SM, Wu HH, Wang LS, Lin TP, Wu HM,

Chen HM, Wu JJ: Extended spectrum beta lactamases

and Plasmid mediated AmpC enzymes among clinical

isolates of E. coli and Klebsiellapneumoniae from seven

medical centres in Taiwan. Antimicrob Agents

Chemother, 2006; 50 (5):1861-1864.

14. Rajini E, Sherwal BL, Anuradha . Detection of Extended-

Spectrum β-lactamases in AmpC β-lactamase-

Producing Nosocomial Gram-negative Clinical Isolates

from a Tertiary Care Hospital in Delhi Vol. 4, No. 6

(2008-01 - 2008-02)

15. Mathur P, Kapil A, Das B, Dhawan B: Prevalence of

Extended spectrum beta lactamase producers in a

tertiary care hospital. Ind J Med Res.2002; 115:153-7.

16. Kumar MS, Lakshmi V, Rajagopalan R: Occurrence of

Extended spectrum beta lactamases among

Enterobacteriaceaespp isolated at a tertiary care

institute. Ind J Med Microbiol. 2006; 24 (3):208-211.

17. Menon T, Bindu D, Kumar CPG, Nalini S,

ThirunarayanMA:Comparision of double disc and three

dimensional methods to screen for ESBL producers in a

tertiary care hospital. Ind J Med Microbiol. 2006; 24

(2):117-120.

18. Manchanda V, Singh NP: Occurrence and detection of

AmpC β lactamases among gram negative clinical

isolates using a modified three dimensional test at

Guru TeghBahadur Hospital, Delhi, India. J

AntimicrobChemother, 2003; 51: 415-418.

19. Subha A, Devi VR, Ananthan: AmpC β lactamase

producing multidrug resistant strains of Klebsiellaspp

and E.coli isolated from children under five in Chennai.

Ind J Med Res.2003; 117:13-18.

20. Woodford N, Reddy S, Fagan EJ: Wide geographic

spread of diverse acquired AmpC β lactamases among

E. coli and Klebsiellaspp in the UK and Ireland. J

AntimicrobChemother, 2007; 59(1):102-5.

21. Livermore DM, Woodford N: Carbapenemase (2000) A

problem in waiting? CurrOpinMicrobiol 3:489-95

22. Shobha KL, Lenka PR, Sharma M K, Ramachandra L ,

Bairy I .Metallobetalactamase production among

Pseudomonas species and Acinetobacter species in

coastal Karnataka. Journal of Clinical and Diagnostic

Research [serial online] 2009 October [cited: 2009

October 5]; 3:1747-1753.





Pag

e59

5

*Corresponding Author: Dr.VidyaPai, Professor, Dept. Of Microbiology, Yenepoya Medical College, Nithyanandanagar, Mangalore-575018.



Review Article



B. Venkateswara Reddy*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e59

6

A REVIEW ON PARENTERAL PRODUCTION TECHNOLOGY

B. Venkateswara Reddy1*, B.Rasmitha Reddy1, K.Navaneetha1, V.Sampath Kumar2

1St.Pauls College of Pharmacy, Turkayamjal, Ranga Reddy (Dist), A.P-501510 2 Sree Datta Institute of Pharmacy, Ibrahimpatnam, Ranga Reddy (Dist), A.P.

ABSTRACT The main objective of this paper is to facilitate the area planning, utilities, environmental control for production of

parenteral. Compare to other dosage forms parenterals are efficient. This gives quick onset of action and provides

a direct route for achieving the drug effect within the body. So by producing these under necessary requirements we

can yield better economic and therapeutical performance.

KEY WORDS Area Planning, change rooms, environmental control, personnel flow.

INTRODUCTION:

Parenteral preparations are sterile, pyrogen-free

liquids (solutions, emulsions, or suspensions) or

solid dosage forms containing one or more active

ingredients, packaged in either single-dose or

multidose containers. They are intended for

administration by injection, infusion, or

implantation into the body.

The dosage form for conveying a drug by means

of injection through the skin or mucous

membranes. Parenteral drugs are administered

directly into the veins, muscles or under the skin

or more specialized tissues such as the spinal

cord. Circumvented the highly efficient first line

body defense that is skin and mucus membrane.

Thus they should be free from microbial

contamination and should have high purity

Preparations such as vaccines, human blood and

products derived from human blood, peritoneal

dialysis solutions, and radioactive

pharmaceuticals require special formulation,

methods of manufacture, or presentation

appropriate to their particular use and may not

comply with certain parts of this monograph.

TYPES:

There are four main forms of parenteral

preparations:

Injections,

Intravenous infusions (large volume

parenterals),

Powders for injections, and

Implants.

Certain injections and intravenous infusions may

be presented in the form of sterile concentrated

solutions, which must be suitably diluted before

use.

FACILITIES REQUIRED FOR PARENTERAL

PRODUCTION:

PRODUCTION:

Parenteral preparations may contain excipients

such as solvents, suspending agents, buffering

agents, substances to make the preparation

isotonic with blood, stabilizers, or antimicrobial

preservatives. The addition of excipients should

be kept to a minimum. When excipients are used,

they should not adversely affect the stability,

bioavailability, safety, or efficacy of the active









Pag

e59

7

ingredient(s), or cause toxicity or undue local

irritation. There must be no incompatibility

between any of the components of the dosage

form.

Water for injections is used as the vehicle for

aqueous injections. It should be freshly distilled

by the process described under "Aqua pro

Injection", be free from carbon dioxide, and

comply with Test for bacterial endotoxins.

Sterilization at this stage may be omitted,

provided that the solution or preparation is

immediately sterilized upon finalization. For non-

aqueous injections, fixed oils of vegetable origin

are used as vehicles.

Unless otherwise specified in the individual

monograph, sodium chloride or other suitable

substance(s), may be added to an aqueous

solution for injection in order to render the

preparation isotonic.

Figure: 1 Overview of manufacturing process

Figure: 2 Flow of materials

Types of sterile products processing:

1 Terminally sterilized

→ prepared, filled and sterilized

2 Sterilized by filtration

3 Aseptic preparations

Manufacture of sterile preparations:-

1. Terminally sterilized: - usually involves filling

and sealing product containers under high-quality

environmental conditions. Products are filled and

sealed in this type of environment to minimize the

microbial and particulate content of the in-

Planning & scheduling









Pag

e59

8

process product and to help ensure that the

subsequent sterilization process is successful. In

most cases, the product, container, and closure

have low bio-burden, but they are not sterile. The

product in its final container is then subjected to

a sterilization process such as heat or irradiation.

2. Sterilization by Filtration:-

Previously sterilized container are taken.

Filters having nominal pore size 0.22 μm

or less are used for filtration

Remove bacteria and moulds but Not

viruses & Mycoplasmas

Double filter layer or second filtration

No fiber shedding or asbestos filters

Filter integrity testing

3. Aseptic Preparation: - In an aseptic process,

the drug product, container, and closure are first

subjected to sterilization methods separately, as

appropriate, and then brought together. Because

there is no process to sterilize the product in its

final container, it is critical that containers be

filled and sealed in an extremely high-quality

environment Before aseptic assembly into a final

product, the individual parts of the final product

are generally subjected to various sterilization

processes. Any manual or mechanical

manipulation of the sterilized drug, components,

containers, or closures prior to or during aseptic

assembly poses the risk of contamination and

thus necessitates careful control.

Note: - In area occupied by personal, the air must

be exchanged with the frequent intervals. Fresh

outside or recycled air must be first filtered to

remove particulate matter and then HEPA filters

are used to get CLASS-100 air systems.

GMP Requirements for Sterile Products

▶ Specific points relating to minimizing risks of

contamination.

– Microbiological

– Particulate matter

– Pyrogen

General Requirements

▶ Production in clean areas

▶ Airlocks for entry

– Personnel entry.

– Material entry

▶ Separate areas for operations

– Component preparation

– Product preparation

– Filling

– Sealing etc…

▶ Level of cleanliness

▶ Filtered air

▶ Air classification: Grade A, B, C and D.

▶ Laminar air flow:

– Air speed (horizontal versus vertical flow)

– Number of air changes

– Air samples

▶ Conformity to standards

▶ Work station and environment

▶ Barrier technology and automated systems.

Space requirements:-

[QUANTITATIVE LAYOUT OF PARENTERAL MANUFACTURING]

Function

Area

Square meter Percentage

Production 11,094 45.1

Warehouse 7,606 30.9

Utility 1,716 4.1

Quality control 1,716 7.0

Administration 1,018 4.1









Pag

e59

9

Maintenance 1,014 4.5

Employee services 1,014 4.1

Security 39 0.9

Total 24,607 100.0

Table: 1 space requirements

AREA PLANING AND ENVIRONMENTAL

CONTROL:-

Area planning may be addressed by functional

groups ground this critical area with particular

attention given to maintaining cleanliness.

The goal of the designer is to group

manufacturing operations so that the flow to

people, product, and components proceeds in the

direction of successive steps of increasing

cleanliness likewise, the flow of waste materials

and products must be thoroughly separated from

the flow of clean personnel and product in order

to prevent contamination.

Functional groupings:-

Warehousing:-

o Basic warehousing functions include

receiving, shipping, and in-process

storage.

o Receiving areas include unpacking,

sampling and incoming quarantine.

o Shipping includes quarantine prior to

shipment.

o The storage of spare parts, air filters,

change parts, water treatment chemicals,

office supplier, laboratory supplies,

janitorial supplies, uniforms, an so on

may be handled as central storage or

individually by department.

o Finished product and certain raw

materials need special environmental

storage conditions, such as, temperature

and humidity control.

o The first and most basic warehouse

function is received and holds incoming

materials.

Warehouse space is usually of greater height than

production areas, is less rigidly controlled from an

environmental and sanitation stand point, and

usually has a relatively high density of flammable

materials. Thus a separate but adjoining area

separated by a firewall is usually the best

arrangement.

Administrative areas:-

Administrative area planning requires careful

analysis of the direct and indirect administrative

requirements of a particular plant.

Successively higher levels of supervision are

usually provided successively larger office areas.

Some offices are individual, while some are

grouped in an “open area concept”.

The relative location of administrative areas

demands particular attention. For the necessary

to maintain production, a close proximity is

desirable. Any other support offices should be

separated from the production area because,

production area contaminations can be related to

people. The reduction of numbers of people will

reduce the challenge to the plant cleanliness.

Many of the “fringes” normally associated with

administrative areas-plants, flowers, closets,

outside windows are potential contamination

sources. Finally the traffic of visitors, vendors,

employment application, and so on, who are not

particularly acquainted with pharmaceutical

discipline can be reduced.

Requirements are related more to the

relationship between the plant and the company

or corporate headquarters. These indirect

administrative requirements will usually include

functions not directly related to plant operation,









Pag

e60

0

such as company or corporate management and

staff functions.

Environmental control zone grouping:-

1st. Zones as per the c GMP:- 1st. Zones as per Gazette of India

Figure: 3 environmental control zone groups

ZONES AS PER GAZZETE OF INDIA:

White zone:-Final step (filling of

parenteral)

Grey zone:-weighing, Dissolution &

filtration.

Black zone:-Storage, Worst area from

contamination view point

a) Have a per-cubic-particle count of not

more than 100 in a size range of 0.5

micron and larger through the entire

work area upstream of the work

piece.

b) Be supplied at the point of use as

specified in section 212.77.

The layout of the plant must be carefully

developed in coordination with the needs of the

HVAC system.

Zone-7:-filling line:-

The walls of the filling area are the last physical

barrier to the ingress of contamination, but within

the filling area a technique of contamination

control known as laminar flow may be considered

as the barrier to contamination.

Zone-6:-filling area:-

Zone 6 is a distinct zone of the controlled

environment area for an aseptic filling process but

may not be distinct zone for non-aseptic filling

processes.

Figure: 4 Aseptic filling

Zone 7:- Filling line

Zone 6:- Filling area

Zone 5:- Weighing, mixing &

transfer area.

Zone 4:- Clean area

Zone3:-General production

Zone 2:- Warehouse

Zone 1:- Exterior

BLACK

GRAY

GRAY

WHITE









Pag

e60

1

Non asepting filling, followed by terminal sterilization, normally requires less rigid environmentalcontrol.

Figure: 5 Non aseptic filling

Zone-5:-weighing, mixing, and transfer area:-

Zone5 encompasses those activities of “weighing,

mixing, filling or transfer operations” addressed

by c GMP section 212.81 which are not handled

as zone 6 but which require a controlled

environment.

Zone-6:-clean area:-

Activities in these may include washing and

preparations of equipment or accumulation and

sampling of filled product.

Zone-3:-general production and administration

area:-

The third zone of environmental control is formed

by the periphery of the general production area.

Openings into the area are usually well sealed and

large enough for only essential material-handling

equipment and personnel.

Zone-2:-plant exterior:-

The environmental with in which a plant located

is first environmental control zone. It is a base

point from which to work in determining the

requirements for the various control barriers.

Management actions to control zone 1

might include the maintenance of sterile areas

around the facility where weeds, insects and

rodents are controlled or eliminated.

2. WALL & FLOOR TREATMENT:

The design of filling areas or more generally,

controlled environment areas involves attention

to many seemingly minor details. The basic

cleanlability requirement includes smooth,

cleanable walls, floors, ceilings, fixtures, and

partition exposed columns, wall studs, bracing,

pipes, and so on are unacceptable. The need for

cleanability also eliminates the open floor system

commonly used in the microelectronics industry

for laminar airflow rooms. The goal of the

designer, when creating the details for the

architectural finishes and joining methods, is to

eliminate all edges or surfaces with in the room

where dirt may accumulate.

All inside walls must be finished; common

methods of finish are block, plaster, or gypsum

board. Concrete block walls are sturdy and easily

constructed. The porosity of concrete block walls

can be reduced by coating with block filler prior

to painting. But even filled concrete block walls

have a surface texture that is not conductive to

cleaning. Painted concrete block walls are

particularly susceptible to peeling if they are

subjected to moisture as from leakage or rain on

the backside.

Use of ceramic-faced block can overcome the

surface finish problems of concrete block. Epoxy









Pag

e60

2

paint is normally used to increase the durability

and impermeability of the surface.

When gypsum board is used, an epoxy point

system is normally employee to create a surface

that is resistant to cleaning compounds.

Gypsum board is not an acceptable surface for

use in powder-filling operations without

incorporating an additional surface coating or

vapor barrier. By itself, gypsum is susceptible to

vapor barrier. By itself, gypsum is susceptible to

vapor migration which presents problems in a low

humidity controlled area.

To overcome the surface weaknesses of most

walls, various heavy coverings are available.

A few spray on and brush on coatings have

provide a much harder and more durable surface

than gypsum, but are still relatively economical to

install and do not present the installation

difficulties of vinyl sheeting.

The use of modular systems has increased

substantially in the last few years that provide a

much harder and more durable surface than

gypsum, but are still relatively economical to

install and do not present the installation

difficulties of vinyl sheeting.

The use of modular wall systems has increased

substantially in the last few years because they

arrive at the construction site prefinished and are

much faster. Selection of floor materials poses a

particularly difficult problem since they must be

durable, and easily cleaned and sanitized. To

achieve good floor results, the application must

be matched to the particular characteristics of the

floor system.

Hardeners may be added to concrete to increase

to surface hardness by as much as a factor of 3,

greatly improving the floor’s resistance to

scratching and dusting and are available in colors

to improve the appearance of the floor.

A sealed concrete floor is therefore not

acceptable for use in controlled areas with in a

parental filling plant because of the potential for

cracking of the soil beneath the concrete when

laid as a coating over a cured concrete surface.

The plants in many parenteral plants are

constructed of epoxy terrazzo.

Finally, the floor is sealed with several coats of

urethane to protect the surface finish. The result

is a very attractive floor that is extremely impact

and abrasion-resistant. A third general type of

floor is composed of large sheets vinyl or

polyvinylchloride laid on a concrete base floor

and “welded” together with heat or sealed at the

seams with cement. Selection of compatible

material-handled equipment whets and for floors

will reduce floor damage. All floors in areas where

water can accumulate should toward one or more

drain points.

3. LIGHTNING FIXTURES:

Lighting fixtures should be reduced flush with the

ceiling. Since most lighting fixtures are not tightly

sealed, the diffuser should be sealed integrally

with the ceiling, and the lamps changed from

outside the room. Either recessed or surface

mounted fixtures can be used. Special “wash-

down” fixtures are well sealed, but protrude

obtrusively into the room and have clips and

sealing lips which are difficult to sanitize. Areas

having a full HEPA ceiling obviously cannot

accommodate recessed lighting fixtures. In these

areas, fixtures are of a special “teardrop” shape

which minimizes disruption to the laminar airflow

pattern.

4. CHANGE ROOMS:

Personnel access to all controlled areas should be

through change rooms. Change rooms concepts

and layouts vary from single closet size rooms to

expensive multi-room complexes.

Entrance to a change area is normally through

vestibules whose doors are electrically

interlocked so that both cannot be opened

simultaneously, thus maintaining the necessary









Pag

e60

3

air pressure differential to prevent the entry of

airborne contamination. Upon entry into the

change room wash sinks are provided for

scrubbing hands and forearms. Although

commercial hands are often used, they may

create undesired airflow patterns and may

circulate particular laden air. Special filtered

driers are available to minimize the creation of

particulate contamination. Further control may

be achieved by using filtered and heated

compressed air for drying to reduce further

particular potential. In some facilities, a foamed

type of alcohol is dispensed on the hands, which

then evaporates. This is used to eliminate need

for tap water and sinks in the gowning rooms,

since these can be a potential source of

contamination. After hands are dry, garments are

taken from dispensers and donned while moving

across a dressing bench. As a final gowning step,

aseptic gloves are put on and sanitized. Exit from

the change room to the controlled area is, like

entrance, through an interlocked vestibule.

Depending on the degree of disrobing required,

separate gowning facilities facilities may be

provided for men and women.

Separate “degowning” rooms are provided where

the clean room garments can be discarded prior

to leaving the controlled zone.

Figure: 6 Change room

5. PERSONNEL FLOW:-

The movement of personnel should be planned

during the design of individual plant areas. Each

individual production area may have a smooth

and efficient personnel flow pattern, a

discontinuous or crowded pattern may develop

when several individual production area plants

are combined. The separation of people and

products is greatly facilitated by the use of the

third dimension. Security concerns about

personnel flow may include minimizing access to

controlled substances and minimizing the

personnel traffic in or near work areas where

controlled substances are handled.

The flow of material and personnel through

corridors are inefficient and unsafe paths for

moving materials, particularly if heavy forklifts

are required.

Parenteral plants, like any other plant have

visitors and the degree of access to be granted

must be determined. A glassed mezzanine or

balcony provides absolute solution yet may give

an excellent view of the processes, but may not

be adaptable for single-floor layouts.









Pag

e60

4

Figure: 7 personnel flow

Discontinuous and crowded flow patterns can

decrease production efficiency, increase security

problems, and increase the problems of

maintaining a clean environment. Personnel flow

path from zone to zone must be such that access

to higher level of cleanliness is only through

change rooms, gowning rooms, locker rooms, or

other areas as may be required to prepare the

personnel for the cleaner area.

6. UTILITIES AND UTILITY EQUIPMENT

LOCATION:-

Utilities:-

Piping system in particular, must be initially and

often periodically cleaned and serviced. Exposed

overhead piping is not acceptable from a

cleanliness or contamination standpoint since it

collects dirt, is difficult to clean and may leak.

Buried or concealed pipe may require

unacceptable demolition for cleaning or repair.

Whenever possible, major utility distribution

services should located outside of clean areas.

The actual utility connections are distributed with

in the plant, building codes usually require that

their distribution systems be exposed and not

buried with in walls or ceilings.

Utilities equipment location:-

Public utilities require space for metering. In

addition to meeting, electrical power system

require for switchgear and transformers. Water

systems usually require treatment to ensure

consistent quality. Plant generated utilities

typically require steam boilers, air compressors,

and distillation, the typical “boiler room”

approach. Although a central location minimizes

distribution problems and minimizes service

distribution distances. Proper equipment

maintenance is difficult in foul weather, especially

winter. Heavy equipment may damage the roof-

structure, particularly if the equipment location

requires numerous penetrations through the roof

which, coupled with equipment vibration, will

invariable lead to leakage. A mezzanine

equipment platform eliminates the problems of

operation in a harsh environment and roof

loading.

MATERIALS:

The selection of materials for a piping system

depends on the product tube handled, the

product purity desired, material cost, and

installation cost.

Carbon steel:

Carbon steel pipe, manufactured according to

ASTM standard A53 of A106 is commonly

available in various schedules or wall thicknesses.

The standard schedule is number 40. Common

uses include water, compressed air, oil, nitrogen,

steam and steam condensate.

Copper:

Copper is commonly used for water and

compressed air piping because of easy

1 3

2 4

× Design √ Design

1

3 4









Pag

e60

5

installation. Either type of K or type L, tubing is

available in annealed form, making it more

flexible. Copper has a smooth surface finish

compared to that of carbon steel and is relatively

resistant to corrosion. Copper loses strength

rapidly at higher temperatures and is not

recommended for steam use.

Type 304 Stainless Steel:

It contains approximately 18% chromium and 8%

nickel, being nonmagnetic and non-harden able.

Type 304 is a good general purpose alloy for

pharmaceutical applications where pitting

corrosion is not a problem.

Type 316 Stainless Steel:

It is similar to type304 except that type 316 has 2-

4% higher nickel content. 2% less chromium and

has 2-3% molybdenum.

The molybdenum gives type 316 improved

resistances to pitting corrosion as compared to

type 304 and slightly improved general corrosion

resistance.

Both type 304 and 316 stainless steel are

susceptible to intergranular corrosion adjacent to

welded areas

Type 316L piping is typically used for distribution

of water for injection, clean steam, deionized

water, compressed air to be used in controlled

environmental areas and or product transfer

piping.

Plastics:

Plastic piping has been used in drain lines and

chemical treatment systems. Additionally, some

companies have used poly vinyldene fluoride

(PVDF) piping for dematerialized water. This poly

fluroplastic has an advantage in that a system is

constructed by thermal fusion of the joints rather

than welding.

Surface finish:

Surface finish specifications after refer to 3-A

sanitary standards. According to these standards

a product contact surface should be polished to a

number 4 finishes, a finish obtained by polishing

with a 150-grit sanding belt. In addition to

mechanical polishing, electro polishing has been

used to improve further the surface finish of

stainless steel.

The electro polished surface exhibits somewhat

better corrosion resistance than mechanically

polished surfaces.

Joining techniques:

Piping system can be joined by threading, welding

or clamping. Threaded connections are common

for non-electrical applications where iron pipe

may be used.Sanitary tubing is welded by using an

automatic fusion welding machine that fuses the

two sections of tubing together, using an electric

current and a purge of inert gas on the inside of

the tubing to yield a high quality weld. The quality

of the weld is checked internally by the use of a

video boroscope.

Following the welding, the piping is passivated

with nitric acid to form an oxide layer on the

inside of the pipe, thereby providing increased

corrosion resistance.

Valuing:

A typical ball valve as ported ball that is rotated

90° to regulate flow. A diaphragm valve, control

flow by compressing a diaphragm against a wire

placed across his direction flow. A number of new

valves came into the market recently to deal with

the limitations of existing valves. One of the best

is pinch valve. The pinch valve is a cylindrical valve

that is modulated by pinching the inner tubing

wall of the valve.

Utility services connection arrangements:

Utilities must be carefully connected to avoid

stagnant areas and to avoid difficult to clean areas

just as would be done for the utility distribution

system. To minimize contamination potential,

typical utility arrangements and typical service

connections should be defined during planning.

Utilities can be arranged so that the service

connections enter a room vertically upward,









Pag

e60

6

horizontally, and vertically downward, with

various advantages and disadvantages. Vertical

upward service connections, with connections

under machinery, create a very neat appearance,

a low full unobstructed machine access, and

require only short connection lengths. Horizontal

service connections are often used in single level

facilities to avoid floor excavation during

equipment relocation or utility maintenance.

Horizontal service do limit machine access, create

some congestion, and may necessarily be longer

than vertical service connections. Vertical

downward services create a visually cluttered

appearance and may restrict access to the

working surface of equipment. This type of

connections may also be undesirable if laminar

flow coverage of the equipment is necessary.

7. Engineering and maintenance:-

From an engineering stand point, even a location

outside the plant can serve well if access to the

production area by engineers for field wok is not

too difficult often particularly in small or less

complex plants, maintenance or other plant

service functions such as utilities or combined

with engineering, making an in-plant location

desirable. Although often associated with

engineering, maintenance is a unique and distinct

function.

Maintenance responsibilities cover all areas of

the plant and can generally be grouped into two

categories: Plant maintenance and production

maintenance.

Production maintenance is a direct production

support function and includes all the routine and

recurring operating maintenance work.

Production maintenance facilities are usually

minimal, often only a place to store a tool box,

and seldom have more than a small workbench.

Plant maintenance operations, in contrast, are

more diverse. They vary from heavy maintenance

on production equipment to cosmetic work on

the building exterior and often include plant

service functions such as sanitation, ground

sweeping, or waste disposal.

Facilities required are extensive and mostly

include provisions for equipment cleaning.

Disassembly major rebuilding of equipment and

painting. These operations can present a

contamination risk to pharmaceutical operations

and must be isolated.

Although maintenance requires access to all parts

of a plant, it must be located to be able to receive

and handle cumbersome and bulky groups.

An absolute must is that the plant maintenance

shop be located so that its personnel have easy

access to major plant utilities and service

equipment.

Types of containers:

1. Ampoules: They are intended for single use

only; ampoules are opened by breaking the glass

at a score line on the neck. Because glass particles

may become dislodged during ampoule opening,

the product must be filtered before it

administered. Because of their unsuitability for

multiple-dose use, the needs to filter solutions

before use and other safety considerations have

markedly reduced ampoule use.

2. Vials: are glass or plastic containers are closed

with a rubber stopper and sealed with an

aluminum crimp.

Advantages over ampoules:

They can be designed to hold multiple

doses (if prepared with a bacteriostatic

agent).

It is easier to remove the product.

They eliminate the risk of glass particle

contamination during opening.

3. Prefilled syringes -These designed for quickest

administration and maximum convenience. Drugs

administered in an emergency (e.g., atropine,









Pag

e60

7

epinephrine) may be available for immediate

injection when packaged in prefilled syringes.

4. Infusion solutions are divided into two

categories: small volume parenteral (SVP), those

having a volume of 100 ml; and large volume

parenteral (LVP), and those having a volume of

100 ml or greater. Infusion solutions are used for

the intermittent or continuous infusion of fluids

or drugs.

LIST OF EQUIPMENTS (as per schedule-M):

The following equipment's is recommended:

a) Manufacturing area: -

1. Storage equipment for ampoules, vials bottles

and closures.

2. Washing and drying equipment.

3. Dust proof storage cabinet

4. Water still.

5. Mixing and preparation tanks or other

containers.

6. Mixing equipment where necessary.

7. Filtering equipment.

8. Hot air sterilizer.

b) Aseptic filling and sealing rooms -

9. Benches for filling and sealing.

10. Bacteriological filters.

11. Filling and sealing unit under laminar flow

work station.

c) General Room.

12. Inspection table.

13. Leak testing table.

14. Labeling and packing benches.

15. Storage of equipment including cold storage

and refrigerators if necessary.

An area of minimum sixty square meters

partitioned into suitable sized cubicles with air

lock arrangement, is recommended for the basic

installation.

EQUIPMENTS:

Sterile Garment Cabinet:

Made up of Stainless steel.

Ensure a clean storage space by making

use of UV disinfectant and heating

through IR lamps.

These cabinets may be designed in

horizontal air flow system and clean air

through HEPA filters

Syringe Filling Machine:

Figure: 8 Syringe Filling Machine

Characteristics:

o Barrier isolators

o In-process check weighing

o Filling: rotary piston pumps.

o Volume: 0.2 to 29 ml

o All types of syringe including glass, plastic can

be filled.

o Filling Rate: 300 to 600 syringes in a minute.









Pag

e60

8

Ampoule Washing Machine:-

Figure: 9 Ampoule washing machine

Process:

Water is sprayed onto the ampoules.

Turned to an angle of 180 degree with

their mouth downward to remove water.

Finally the ampoules are filled with

compressed air to remove residual water.

Certain machines have a high

temperature zone meant for killing any

bacteria.

Washing cycle:-

1st wash - Recycled Water (WFI)

2nd wash - Compressed Air

3rd wash - DM Water

4th wash - Compressed Air

5th wash - Water for Injection (WFI)

6th wash - Compressed Air

Vial Filling Machine:-

Figure: 10 Vial filling machine

Fill vials and bottles

Liquids, viscous material and suspensions and

powders.

Unique patented system for filling liquid

products in sterile conditions.

Global solution: preparation and sterilization

of components, handling, sterile filling,

process control and vial laser etching.

More than 15 years of proven reliability in

sterile filling.









Pag

e60

9

PROCESS:-

The machine comprises of an intake

section which loads the vials.

Transferred through an intermittent

transport section.

Liquid filling section which fill the vials

with predetermined quantity.

Finally the filled and rubber stoppered

vials are released and discharged.

Main Advantages:-

Vial is closed and protected throughout

the process.

Vial is opened in the final filling stage in a

controlled environment with horizontal

laminar flow.

No need for dry heat tunnel sterilization

as it is carried out in an autoclave.

Sterilization and depyrogenation

combined with a HWFI washing cycle and

an autoclave cycle. No need for a dry heat

tunnel.

SIP System:

For in-line sterilization of various

processing equipments.

Handling various biological solutions and

mixtures requires cleaning and sterilizing

these equipments from time to time as

they are susceptible to contamination.

Proper SIP integration with

pharmaceutical equipment is very

important for the overall success of the

operation.

CONCLUSION

The parenteral route of administration is the most

effective route for the delivery of the active

pharmaceutical substances with narrow

therapeutic index, poor bioavailability especially

for those drugs, prescribed to unconscious

patients.

The present article describes that area planning,

facilities, design, construction and manufacturing

of sterile products. It is more impartment to

produce good quality of parenteral. Parenterals

are the pyrogen free liquids these are

manufactured and stored according to cGMP

guidelines. Proper area, environmental control,

personnel observation will gives excellent

parenteral products and attain their described

therapeutic effect.

REFERENCES 1. Industrial pharmacy (sterile products) by Leon Lachman,

657-659

2. Good manufacturing practices for pharmaceuticals 6th

edition, Joseph D.Nally, page no-37-113

3. Pharmaceutical science by Remington, 21th edition,

vol.1, page.no-814-828

4. American Journal of Hospital Pharmacy, Vol. 38, Issue 8,

1144-114710. Dispensing for pharmaceutical students;

5. www.fda.gov.

6. Drugs & Cosmetics Act 1940.

7. www.GMP.online.coms

8. www.ispc.org

9. www.whqlibdoc.who.org

10. www.dwscientific.co.uk

11. www.pharmamachines

12. www.pharmamachines.com









Pag

e61

0

*Corresponding Author: B. Venkateswara Reddy St.Pauls College of Pharmacy, Turkayamjal, Ranga Reddy (Dist), A.P-501510







Review Article

BiologicalSciences


A. Anita Margret*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e61

1

MARVELOUS MEDICINAL MUSHROOMS

G.Hepzibah Beulah1, A. Anita Margret2*and Jeyakumar Nelson3 1Assistant Professor, Department of Zoology, Rani Anna Government College,

Tirunelveli- 627 012, Tamilnadu, India Phone: 91-96-77-565578 2Assistant Professor, Department of Biotechnology, Bishop Heber College,

Tiruchirappalli-620 017, Tamilnadu, India. 3Unit of Microbiology, Faculty of Medicine, AIMST University, Semeling-08100,

Bedong, Kedah DarulAman, Malaysia.


ABSTRACT The significance of high-quality nutrition has long been known in relation to preventing numerous human health

problems and the palatability of healthy foods are increasingly appreciated. Mushrooms are the heterotrophic

macro fungi and their nutritional properties made them a better-quality dietary food. Its products bestow

improvement of the human immune system and thereby known to increase disease resistance. Identification of

new compounds from mushrooms which can aid to develop nutritional supplements needs to be encouraged.

Medicinal mushrooms have plenty of novel bioactive compounds that are significant for enhancement of human

life. In current scenario, global utilization of these mushrooms gained importance in glimpse and bloom of its vital

role in modern medicine. Though several mushrooms have been studied for their usage in medicinal purpose, this

review provides insight into prospective implications of medicinal mushroom applications.

KEY WORDS Macro fungi, Nutritional property, Medicinal mushrooms, Mushroom research, Health benefits, Therapeutic

usage.

INTRODUCTION

Humans are constantly searching worldwide for

the development of new therapeutic agents

especially from natural sources. Nature has

blessed mankind with diverse biota that creates

an opportunity for potential findings. The same

strategy can be more easily achieved using

microbial origin. Beneficial microorganisms have

valuable sources of compounds which influences

human health either by direct or indirect ways.

Mushrooms are a special group of macro fungi

and are premier recyclers on the planet.

According to recent estimates, mushrooms

constitute at least 12,000 species in the

ecosystem and 2,000 species are reported as

edible among the known. About 35 edible

mushroom species are commercially cultivated

and nearly 200 species were collected from wild

and used for medicinal purposes.

After the discovery of the first wonder drug,

Penicillin from filamentous fungi much more

attention has been carried out in therapeutic

usage of fungus especially from medicinal

mushrooms. The fruiting body of mushroom

contains excellent source of nutrients, high

proteins, low in calories, rich in minerals, fibers,

essential amino acids and vitamins are

considered as a vegetable meat. The modern

cultivation technology has been implicated for

economic growth of edible mushrooms. With an






Pag

e61

2

intensive advanced research in mushroom

biology there is growing awareness about the

utilization of high quality nutritional and

medicinal mushrooms. The bioactive ingredients

found in medicinal mushrooms are natural,

simpler and make them easily ingestible in body

system. At present, there is an enormous

covenant of public interest in the use of

medicinal mushrooms for health and healing.The

future challenge is to correlate the significance

of these mushrooms in healing the dreadful

diseases of mankind.

Mushroom research and development: Present

scenario

Edible mushrooms are widely used for industrial

purposes like other microorganisms for the

production of valuable substance. Mushroom

biotechnology deals with the principles of

mushroom biology and bioprocess technology to

develop safe mushroom medicinal products.

Though, the progress of mushroom technology

has achieved tremendous improvement that

allowed the scientist to address various issues

raised for the new developmental approach. In

order to increase the utility of mushroom, it is

necessary to develop the following areas of

concern.

1. Production strain selection

2. Techniques in strain improvement

3. Optimization of growth parameter

4. Post harvesting technology

5. Marketing surveillance

Significant increases in the medicinal mushroom

research and development were initiated

especially during last four decades. The current

scenario of mushroom production is fully

exploited using standard cultivation

methodology. About 5 million tons of

mushrooms are now commercially cultivated in

more than 100 countries. China is the major

producer of edible mushroom and accounts for

over 64% of global production.

It is generally recognized that in order to

maintain high-yielding strains, the techniques

employed in mushroom breeding should be

frequently modified and improved by new

findings, particularly in the field of Microbial

biotechnology and Genetics. Development of

economic cultivation technology, selection of

disease resistance strains and formulation of

cheap substrates has been studied in detail and

recommended to mushroom growers to achieve

the high yield target. As a result of this current

research, 25% of the yield is increased in

mushroom production.

Mushrooms are not only considered as a

nutritious protein-rich food but also serves as a

potential source for producing pharmaceutical

and nutraceutical compounds. Assuming that the

proportion of useful mushrooms among the

unknown shall be only 5 % and may be

thousands of mushroom species yet to be

discovered having possible benefits to human

kind. However, valuable health benefits could be

obtained from many edible and even non-edible

mushrooms. Hence, there is a need to evaluate

novel bioactive compounds which has drawn

more attraction of researchers now days. The

research area in mushroom science especially on

medicinal mushrooms is now focused on

characterization of such pharmaceutically

important novel compounds. From a medical

point of view, we have now realized the

importance and benefits of mushrooms usage. In

order to meet our future challenges, more

innovative approaches in successful

development of medicinal mushroom is

essential. If research is geared up in right

direction, certainly mushroom technology will

bloom revealing the magic of health and healing.





Pag

e61

3

Health benefits of Medicinal mushrooms

Traditionally, mushroom has been used as a

delicacy for many centuries throughout the

world. It has been shown that constant intake of

either mushrooms or mushroom nutraceuticals

(dietary supplements) can make people healthy

and fit. It is conventionally used in many

countries particularly in Asian countries like

China, Japan and India to indulgence common

diseases such as atherosclerosis, hepatitis,

hyperlipidemia, diabetes, dermatitis and cancer.

Due to the presence of its high protein, fiber

with low fat contents which aid the dietician

choice as a food for health related problems.

Mushrooms such as Lingzhi

(Ganodermalucidum), Shiitake (Lentinulaedodes)

and Yiner (Tremellafuciformis) have been used

by traditional healers. The common bioactive

compound present in the mushroom includes

polysaccharides, triterpeniods, glycoprotein and

antibiotics. In particular, the presences of

polysaccharides have been proved as potential

antitumor and immune modulating properties.

There have been a number of studies suggesting

the possible role of these mushrooms with

immune modulating, anti-diabetic, anti-tumor,

anti-viral, and anti-inflammatory activities.

Nutraceutical attributes of Mushrooms

Edible mushrooms are good source of protein

rich food, enriched minerals,B-complex vitamins,

riboflavin, niacin, thiamin, folic acid, pantothenic

acid, vitamins C and D. Mushrooms can be

served as potential prebiotics which contains

carbohydrates like chitin, hemicellulose, Beta-

glucans, mannans, xylans and galactans which

enhance the immune function as well as improve

digestion.The nutritional benefits of edible

Oyster mushroom have high protein, low fat and

also a natural source of bioactive compound

lovastatin, a chemical used in pharmaceutical

drugs to reduce the total cholesterol level in

human and as an important food supplement for

patients suffering from hypercholesterolemia.

Grifolafrondosahas more protein content, a high

proportion of unsaturated fatty acids, vitamin

including B1, B2,C, D, Niacin and minerals.

Shiitake has rich in several anti-oxidants, anti-

tumor substances and cardiovascular benefits.

Dried and whole mushroom have been utilized in

traditional medicine since long time. The

mushroom, G.lucidumhas 400 different bioactive

compounds which were extracted from the

fruiting body, mycelia and spores. The bio active

compounds especially polysaccharides, organic

germanium, triterpenoids and ganoderic essence

shows a significant health benefit. It reduces the

incidence of tumor and also recommended as a

chemo preventive agent against cancer.

Numerous reports suggested its anti-viral, anti-

inflammatory and immunomodulating

activities.The extract of this mushroom protect

DNA damage related with free radicals and

radiation.Novel bioactive molecules isolated

from Shiitake known as lentinan (“Elixir of Life”)

has been licensed as an anti-cancer drug by

Japanese FDA which has proved to heal on bowel

cancer, liver cancer, stomach cancer, ovarian

cancer and lung cancer. Its immune modulating

activity shows increased host resistance to

bacterial and viral infections. Lenthionine, a

cyclic organosulfur compound of Shiitake

mushrooms has antimicrobial activity.

The mushroom, Flammulinavelutipeshas a major

polysaccharide named flammulin which is

proved to have an effective anti-tumor property.

Frequent consumption of Enoki mushroom

reduces cancer rate in the community of the

Nagano city in Japan was recorded. The

experiment of this mushroom extract also

proved that it can be used for the treatment of

liver diseases and gastric ulcer. Grifolan, a

polysaccharide from Maitake mushroom

(Grifolafrondosa) has been commercialized as a





Pag

e61

4

chemo preventive agent against cancer.

Moreover, it improves the body immune

system by activates helper T cells, cytotoxic T

cells, natural killer cells and macrophages in the

treatment of breast, liver and lung cancer. It has

anti-diabetic properties and reduces some side

effects of anti-cancer drugs such as hair loss,

pain, nausea etc.

Anti-cancerous properties and chemo preventive

activity in Agaricus mushroom was confirmed by

The Japanese Cancer Association and the

Japanese Pharmacological Society.

Hericiumerinaceusis used for the treatment of

gastric and esophageal carcinoma and ingestion

of this mushroom extending the life of cancer-

ridden patients. Tremellafusiformis extracts are

used to protect liver cells from radiation

damage. Anti-inflammatory, anti-viral and

antioxidant activities had also been reported

from Pleurotus mushroom. Bioactive compounds

which aid fibrinolysis were isolated from variety

of medicinal mushrooms which substantiate

their significant role in treating cardiovascular

diseases. Chitosan have been extracted from

several mushrooms such as Pleurotusostreatus,

Agaricushortensis and Lycoperdonperlatum

which exhibit attractive role in multiple industrial

applications, pharmacological, biomedicine and

cosmetic fields. All these research has strongly

recommended that medicinal mushrooms can be

used as a potential source for the development

of new therapeutic agents.

CONCLUSION

The increasing global population in the twenty-

first century demands high quality food with

health care. Mushroom has been recognized as

an alternative potential source of food and

medicine to overcome the needs. More research

in this area, may lead to the development of new

cultivation technology using mushroom

biotechnology hence, larger quantities of novel

mushrooms can be massively harvested. There is

an urgent need to study the medicinal

importance of wild mushrooms and it creates an

opportunity for scientists to elucidate the active

principle behind these mushrooms. Besides

research, extension activities for exploration and

establishment of medicinal mushroom farms

need to be initiated. Furthermore, a healthy diet

formulation can be generated which provides

bioactive ingredients that promote human

health and healing potential. According to the

father of Medicine, Hippocrates, “Let food is

your medicine and medicine is your food”, it may

be concluded that mushrooms are the most

potent, natural immune force ever discovered

and hence it can be considered as a priceless

asset for human welfare.

REFERENCES [1] Aida F.M.N.A., Shuhaimi M., Yazid M. and Maaruf A.,

Mushroom as potential sources of prebiotics: a review.

Trends in Food science and Technology. (20):567-575,

(2009).

[2] Douglas L. C. and Sanders. M. E., Probiotics and

prebiotics in dietetics practice. Journal of the American

Dietetic Association, (108): 510-521, (2008).

[3]Gao Y., Zhou. S.H., Chen G., Dai X. and Ye J., A phase I-II

study of a Ganodermalucidum (Curt.:Fr.) P. Karst.

Extract (ganopoly) in patients with advanced cancer.

International. Journal of Medicinal Mushrooms. 4(3):

207–214, (2002).

[4]Hobbs C., Medicinal Mushrooms: an Exploration of

Tradition, Healing and Cultures; 1995 Kidd P. The use of

mushroom glucan and proteoglucans in cancer

treatment; Alternative Medicine Review .5(1):4-

27(2000)

[5]Khor E. and Lim L.Y., Implantable applications of chitin

and chitosan, Biomaterials. 24(13): 2339–2349 (2003).

[6]Nanba H. and Kubo K., Effect of Maitake D-fraction on

cancer prevention. Annals of the New York Academy of

Sciences, (833): 204-207(1997).

[7]Sanchez C., Modern aspects of mushroom culture

technology. Applied. Microbiology Biotechnology.

(64):756-762(2004).

[8]SeonJoo Yoon, MyeongAe Yu, Yu RyangPyun, Jae Kawn

Hwang, Djong Chi Chu, Lekh Raj Juneja and Paulo A.S.

Mourao., The nontoxic mushroom Auriculariaauricula





Pag

e61

5

contains a polysaccharide with anticoagulant activity

mediated by anti-thrombin. Thrombosis Research.

(112):151-158(2003).

[9]Stamets P., MycoMedicinals, an informative booklet on

medicinal mushrooms. Mycomedia, (3): Olympia,

Washington (1999).

[10] Synytsya A., Mickova k., Jablonsky I., Spevacek and

Erban V., Glucans from fruit bodies of cultivated

mushrooms Pleurotusostreatus and Pleurotuseryngii:

structure and potential prebiotic activity. Carbohydrate

Polymers. (10):1016. 2008.

[11]Wasser S.P., Medicinal mushrooms as a source of

antitumor and immunomodulating polysaccharides.

Applied Microbiology and Biotechnology, (60):258-

274(2002).

[12]Zang M., Cui S.W, Cheung P. C. K. and Wnag Q.,

Antitumor polysaccharides from mushrooms:a review

on their isolation. Trends in Food Science Technology,

(18):4-19(2007).

*Corresponding Author: A. Anita Margret* Assistant professor, Department of Biotechnology, Bishop Heber College, Tiruchirappalli- 620 017. Tamil Nadu, South India E-mail: [email protected] Phone: 91-97-87-395808 / 00-91-431-2770136/2770158/2772345 Fax: 00-91-431-2770293



ResearchArticle

BiologicalSciences


Nirmala Rajesh Naidu*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e61

6

EFFECT OF LONG TERM ADMINISTRATION OF ALUMINIUM CHLORIDE ON

OXIDATIVE STRESS AND ACETYLCHOLINESTERASE ACTIVITY IN RAT BRAINS 1*Nirmala Rajesh Naidu, 2ShankarBhat, 3Urban Dsouza

1*PhD Scholar, Physiology Department, Yenepoya Medical College,

University Road, Deralakatte, Mangalore 575018 2Professor & HOD, Physiology Department, Yenepoya Medical College,

University Road, Deralakatte, Mangalore 575018. 3Professor, physiology department,KVG medical college,Sullia


ABSTRACT Oxidative modifications are the hallmark of oxidative imbalance in the brain of individuals with Alzheimers,

Parkinsons and Prion diseases and their respective animal models. The aim of the research was to study the

impact of aluminum chloride (AlCl3) administration in drinking water (7mg/kg body weight) and D-

galactose(i.p).The results revealed that the levels of lipidperoxidation were significantly increased, while the

activities of superoxide dismutase (SOD) as well as reduced glutathione (GSH) content were significantly

decreased in the brains of rats. Additionally, brain acetylcholinesterase (AChE) activities were significantly

increased. It can be concluded that Al-induced neuronal oxidative stress and inhibition of the antioxidant system

and enzyme activities could be the mechanisms of AlCl3 neurotoxicity. The stained samples were examined by

means of light microscope for histological changes. Histological examinations showed clumpy of cell neurons, or

reduced pyramidal cells and scanty neurofibrillary tangle which was an indication of neurodegeneration in the

treated groups when compared to the control. It was however, concluded that the oral administration of

aluminium chloride could induce brain damage which may impair memory and learning as seen in Alzheimer

disease.These results suggest that AlCl3, enhances oxidative stress in the brain, thereby disturbing the antioxidant

defense of rats. Increased oxidative stress could be one of the mediating factors in the pathogenesis of AlCl3,

toxicity in the brain.

KEY WORDS Aluminium chloride, Acetylcholinesterase, Hippocampus, Oxidative stress.

INTRODUCTION

Aluminum (Al) has the potential to be neurotoxic

in humans and animals. It is present in many

manufactured foods and medicines and is also

added to drinking water for purification

purposes (1). Al is widely used in antacid drugs,

as well as in food additives and tooth paste (2).

Environmental pollution with different aluminum

containing compounds, especially those in

industrial waste expose people to higher than

normal levels of Al (3). Particulate matters

distributed by cement – producing factories

contain, high amount of Al, and animals and

populations residing in the vicinity are exposed

to the pollution (4). Although aluminum has

been implicated in Alzheimer's disease,

Parkinsonism, Dementia complex and causes

extensive damage to the nervous system, to date

the mechanism of Al neurotoxicity has not been

fully elucidated (5). In recent researches,






Pag

e61

7

aluminum has been reported to accelerate

oxidative damage to biomolecules like lipid,

protein and nucleic acids (6.).

Therefore, the present study was carried out to

investigate the alteration in biochemical

parameters including free radicals, enzymes

activities and histopathological alterations

induced by AlCl3 in brain tissue of rats.

METHOD AND MATERIAL

Animals

Adult wistar rats weighing between 180-200g of

either sex were procured from the central animal

house, Yenepoya University, Mangalore. The

protocol was approved by the Institutional

Animal Ethics Committee (CPCSEA- registration

no 347/ CPCSEA) and was carried out in

accordance with the Indian National Science

Academy Guidelines for the use and care of

animals. Animals were acclimatized to laboratory

conditions at room temperature prior to the

experiments. The rats were acclimatized for one

week in the animal house facility. They were

housed in polypropylene cages at an ambient

temperature of 25±1°C with a natural dark-light

cycle. They had free access to standard pellet

diet and water given ad libitum. All experiments

were conducted in the forenoon (9:30 AM to

1:00 PM).

Treatment group

Rats were divided into two group of six animals

each:

a) Control group was orally administered

distilled water for 90 days

b) Treatment group was orally

administered Aluminium chloride

(7mg/kg body weight) dissolved in

distilled water for 90 days and intra

peritoneal injection of D-galactose

(84mg/kg body) (7).

EXPERIMENTAL PROCEDURE:

Preparation of the Tissue Homogenate:

Brain tissues were washed with cold saline and

dried. Each of these tissues was separately

transferred to a glass homogenizer containing

10ml of 10mM cold phosphate buffer saline (PBS

- pH 7.4). The tissues were homogenized using

an electrical homogenizer (Remi 8000 RPM). The

unbroken cells and cell debris were removed by

centrifugation at 3000 RPM for 10 minutes by

using Remi C 24 refrigerated centrifuge (-4°C).

The obtained supernatant was used for the

biochemical estimations.

Estimation of Lipid Peroxidation

Lipid peroxidation was estimated according to

the method of Kartha and Krishnamurthy. (8).

This assay is based upon the reaction of TBA with

malondialdehyde (MDA) which is one of the

aldehyde products of lipid peroxidation.

Estimation of GSH

GSH was estimated by Beaulter et al (9) and

glutathione content was expressed as (g/gm

protein).

Estimation of SOD

SOD was estimated by the technique explained

by Fridovich(10). The activity was expressed as

unit/ mgprotein

Protein Estimation

Protein content of the tissue samples was

determined by Lowry et al method (11)

Histological Study

Brain Specimen used for histological study was

fixed in neutral formalin for a week at room

temperature dehydrated and embedded in

paraffin wax. The paraffin section were cut at

20m thickness and stained with hematoxylin and

eosin.

Statistical Analysis

The Biochemical data were subjected to one way

ANOVA followed by Turkey-Kramer multiple

comparison post hoc test, using Graph Pad Instat





Pag

e61

8

(version 3.00 for windows). A value of less than

0.05 has been taken as significant.

RESULTS

Effect of Aluminium chloride on selected

biochemical parameters in rat brain is present in

Table 1. Administration of Aluminium chloride

and D-galactose to rats for 3 months resulted in

statistically significant increase in lipid

peroxidation and elevation of AchE activity in

comparison with control. Aluminium group

exhibited significant reduction in SOD activity as

well as GSH content compared to control.

The staining shows that there were typical

neuropathological changes in the hippocampus

of AD model rat. In the control group the

neurons were full and arranged tightly, the

nuclei were light-stained. By comparison in the

model group rat, the cytoplasm of neurons were

shrunken, the nuclei were side-moved and dark-

stained, neurofibrillary degeneration and neuron

loss were observed in hippocampus.

Table 1: Brain antioxidants/oxidants system in rats treated with oral AlCl3 and D-galactose (i.p)

Treatment LP nmol/mg

tissue protein

GSH

nmol/mg

tissue protein

SOD

nmol/mg

tissue protein

Acetylcholinesterase

activity

(μmole enzyme/mg

protein/min)

Control group 0.4960±0.10 0.4030±0.04 0.65±0.03 4.84±0.06

Aluminum

receiving group 1.2594±0.07 0.3375±0.016 0.28±0.02 8.46±0.89

Statistical

significance ***P‹0.001 **P‹0.01 ***P‹0.001 ***P‹0.001

Statistical significance test done by ANOVA followed by Turkey-Kramer multiple comparisons test

Values: Mean ± SD. *Significance of the results: p < 0.001.

Figure 1:Microscopic study of hippocampus in mouse brain. Grossly (x40). Histological sections of

brain were stained with hematoxylin& eosin (H&E). Control (A). Exposed rat to 7mg/kg/day AlCl (3)

during 3 months (B)





Pag

e61

9

Figure 2:Microscopic study of cerebral cortex in mouse brain. Grossly (x40). Histological sections of

brain were stained with hematoxylin & eosin (H&E). Control (A). Exposed rats to 7 mg/kg/day AlCl(3)

during 3 months (B).

DISCUSSION

In this study, during three months observation of

rats receiving aluminium chloride, decreases in

water and food intake and transient diarrhoea

occurred, which resulted inlowering of final body

mass of animals in comparison to the controls

(differences statistically significant). The rats’

body mass after three months of the study was

322±21g in the control group and 269±23g in the

investigated group. In the course of the

experiment no changes were observed in the

behavior of animals. In humans, chronic

exposure to aluminium ions may result in mood

changes, dysmnesia, convulsions, muscular

weakness, and pathological fractures of bones.

Aluminium accumulates mainly in bones, spleen,

liver and lungs [3, 4, 7, 8]. In our study the

content of aluminium was not investigated in the

mentioned organs. Assessment of harmful effect

of aluminium ions was based on the analysis of

selected biochemical parameters. Statistically

significant increase of brain lipid peroxidation

and decrease of reduced glutathione, SOD and

AchE in animals receiving aluminium chloride is

of interest.

While aluminum is not a transition metal and

cannot initiate peroxidation, many studies have

looked for a correlation between aluminium

accumulation and oxidative damage in the brain.

It has been pointed out in in-vitro studies that

aluminium significantly accelerates iron-

mediated lipid peroxidation under acidic and

neutral conditions (12). The elevation of brain

lipid peroxidation in this study suggests the

participation of free-radical- induced oxidative

cell injury in mediating the toxicity of AlCl3 as

proposed by other studies (13); it is reported

that the neurotoxicity of AlCl3 was due to the

increase in brain lipid peroxidation as a result of

being AlCl3 able to cross the blood–brain barrier

as an L-glutamate complex and it deposits in a

rat’s brain (14).

As oxidative damage is mediated by free

radicals, it was necessary to investigate the

status of endogenous antioxidant enzymes like

superoxide dismutase and glutathione, which are

the first line of defense against free radical

damage under oxidative stress conditions (15).

In our study, chronic administration of

aluminium chloride resulted in marked oxidative

stress as indicated by increases in lipid

peroxidation, as well as decreases in reduced

glutathione, superoxide dismutase, compared to

the control groups is in accordance with what

has been previously reported by (16).

The decreased activity might have resulted from

the oxidative modification of genes that control

these enzymes. Under the oxidative stress





Pag

e62

0

conditions, SOD presents the first line of defense

against superoxide as it converts the superoxide

anion to H2O2 and O2 (17). It also has an

important role in detoxifying superoxide radical

to H2O2, which is then converted to H2O by CAT

and GSH at the expenditure of GSH. Therefore

the increased lipid peroxidation may be

interpreted here by an inhibition of SOD and GSH

activities and other antioxidants in the brain

tissue leading to membrane damage and neuron

death (17).

In the present study, aluminium may have

altered the cellular redox state by inhibiting the

enzymes involved in antioxidant defense which

functions as blockers of free radical processes as

postulated (18). The results are in accordance

with Nehru B, Anand P (18) who observed

significant decrease in the activities of SOD in

cerebrum, cerebral hemisphere and brain stem

after Al exposure.

Glutathione in its reduced form is the most

abundant intracellular antioxidant and is

involved in direct scavenging of free radicals or

serving as a substrate for the glutathione

peroxidase enzyme that catalyzes the

detoxification of H2O2. It is also known that SOD

and catalase are protective enzymes and both

function in very close association for the

detoxification of highly reactive free radicals.

Cholinergic neurons are positive markers for the

evolution of memory and related disorders

affecting acetylcholine and resulting in

decreased activity of acetylcholinesterase and

choline acetyl transferase [19]. Recent findings

suggested that administration of aluminium was

found to increase acetylcholinesterase in mouse

brain [20]. We also demonstrated that chronic

administration of aluminium to rats significantly

increased acetylcholinesterase

The hippocampus and the cerebral cortex are

the key structures of memory formation.

Because the hippocampus is especially

indispensable in the integration of spatial

information, a decline in learning ability may be

induced by the deterioration of hippocampal

function [20].

In this study the brains of experimental animals,

studied by optical microscopy, displayed a

massive cellular depletion in the hippocampal

formation with neurofibrillary degeneration. We

observed numerous ghost-like neurons with

cytoplasmic, nuclear vacuolations and necrosis of

the cerebral cortex which are form of neuro-

degeneration which can be due to the

accumulation of Aluminium in these regions [21].

Other experimental protocols have provided

evidence that Al can accumulate in hippocampus

and cortex [22]. Evidence for stronger glia

activation was observed in Al-exposed animals,

indicative of an acceleration of pathological and

inflammatory events by Al [23,]. Inflammatory

responses are known to play an important role in

neurodegenerative disease such as AD [24].

Recently, it has also been suggested that there

may be an important link between Al, oxidative

stress, inflammation and AD [25]. This is

supported by our data and by other studies

indicating that Al facilitates iron-induced

oxidative stress in vitro [26], this may be the

cause for Al-induced learning and memory

deficits observed before severe

neurodegeneration can be identified. This action

may also be the basis for Al as a putatively

contributing factor in AD.

CONCLUSION

The result summarized here indicates that

chronic ingestion of aluminium chloride leads to

oxidative stress which is a hallmark of oxidative

imbalance in the brain of individuals with

Alzheimer’s. All our observation in the present

study provides conclusive evidence that the

aspects of Aluminium toxicity to human beings





Pag

e62

1

increase the risk of occupational hazard with

particular reference to neurological diseases.

REFERENCE 1. Newairy A.S., Salama A.F., Hussien, H.M. and Yousef,

M.I. (2009): Propolis alleviates aluminium-induced lipid

peroxidation and biochemical parameters in male rats.

Food ChemToxicol, 47(6):1093-8, (2009)

2. Abbasali K.M., Zhila T. and Farshad N.: Developmental

Toxicity of aluminum from High Doses of AlCl3 in Mice.

The Journal of Applied Research, 5: 575-579, (2005)

3. Kloppel H., Fliedner A. and Kordel W. :Behaviour and

endotoxicology of aluminium in soil and water. Review

of the scientific literature. Chemosphere. 35: 353-363,

(1997)

4. Shehla K.F., Prabhavathi P.A., Padmavathi P. and Reddy

P.P.: Analysis of chromosomal aberrations in men

occupationally exposed to cement dust. Mutat Res.,

490: 179-186. (2001)

5. Niu Q. Yang Y., Zhang Q., Niu P., He S., Di Gioacchino

M. AndBoscolo P.: "The relationship between Bcl-2

gene expression and learning & memory impairment in

chronic aluminum-exposed rats. Volume 12, Number 3,

163-169, (2007)

6. Jyoti A., Sethi P. and Sharma D.: Bacopamonniera

prevents from aluminium neurotoxicity in the cerebral

cortex of rat brain. Journal of Ethnopharmacology ,111,

56–62, (2007)

7. Pan Rui, Qiu sheng, Lu Da-Xiang and Dong Jun.

Curcumin improves learning and memory ability and its

neuroprotective mechanism in mice. Chin Med J.,

121(9):832-839,(2008)

8. Med Of Aryreiaspeciosa in Mice.journal of Health

Science,53(4) 382-388..1963, 61; 882-890,(2007)

9. kartha R, Krishnamurthy S. factors affecting in vivo lipid

peroxidation in rat brain homogenates. Ind. J.

Physiol. Pharmacol. 22(1); 44-52, (1978)

10. Charles Beauchamp and Irwin Fridovich. Superoxide

dismutase: Improved assays and an assay applicable to

acrylamide gel. Analytical Biochemistry., 276-287,

(1971)

11. Oliver H. Lowery, Nira J., Rosebrough, A.Lewis Farr, and

Rose J. Randall.Protein measurement with the folin

phenol reagent .265-274, (1971)

12. Ohyashiki T, Karino T, Suzuki S, Matsui K. Effect of

aluminum ion on Fe2_-induced lipid peroxidation in

phospholipid liposomes under acidic conditions. J

Biochem . 120:895–900, (1996)

13. Anane R, Creppy EE Lipid peroxidation as pathway of

aluminium cytotoxicity in human skin fibroblast

cultures: prevention by superoxide dismutase_

catalase and vitamins E and C. Hum ExpToxicol 20:477–

481, (2001)

14. Deloncle R, Huguet F, Babin P, Fernandez B, Quellard

N, Guillard O . Administration of aluminum L-glutamate

in young mature rats: effects on iron levels and lipid

peroxidation in selected brain areas. ToxicolLett

104:65–73, (1999)

15. Benzi G, Marzatico F, Pastoris O, Villa RF. Relationship

between aging, drug treatment and the cerebral

enzymatic antioxidant system. ExpGerontol 24:137–

148, (1989)

16. Flora SJS, Mehta A, Satsangi K, Kannan GM, Gupta M.

Aluminum- induced oxidative stress in rat brain:

response to combined administration of citric acid and

HEDTA. Comp BiochemPhysiol C ToxicolPharmacol

134:319–328, (2003)

17. Fridovick I Superoxide radical: an endogenous toxicant.

Annu Rev PharmacolToxicol 23:239–253, (1975)

18. Nehru B, AnandP. Oxidative damage following chronic

aluminiumexposure in adult and pup rat brains. J Trace

Elem Med Biol .19:203–208. (2005)

19. Dua R, Gill KD. Aluminium phosphide exposure:

implications on rat brain lipid peroxidation and

antioxidant defence system. PharmacolToxicol 89:315–

319, (2001)

20. Platt, B., G. Fiddler, G., Riedel and Z. Henderson.

Aluminium toxicity in the rat brain histochemical and

immunocytochemical evidence Brain Res. Bull., 55:

257-267, (2001)

21. Buraimoh, S.A. Ojo, Hambolu and S.S. Adebisi. Effects

of Oral Administration of Aluminium Chloride on the

Histology of the Hippocampus of Wistar Rats. Current

Research Journal of Biological Sciences 3(5): 509-515,(

2011)

22. Adebayo AdekunleBuraimoh. Effects of Aluminium

Chloride Exposure on the Histology of the Cerebral

Cortex of Adult Wistar Rats. Journal of Biology and Life

Science. Vol. 3, No. 1, 87-113, (2012)

23. Yokel, R.A. and J.P. O'Callaghan. An aluminum induced

increase in GFAP is attenuated by some chelators.

Neurotoxicol. Teratol., 20: 55-60, (1998)

24. Rogers, J.P., S. Webster, L.F. Lue, L. Brachova W.H.

Civin, M. Emmerling, B. Shivers, D. Walker and P.

Mcgeer,. Inflammation and Alzheimer’s disease

pathogenesis. Neurobiol. Aging, 17: 681-686, (1996)

25. Campbell, A. and S.C. Bondy . Aluminum induced

oxidative events and its relation to inflammation: a

role for the metal in Alzheimer's disease. Cell. Mol.

Biol., 46: 721-730,( 2000)

26. Xie, X.X., M.P. Mattson, M.A. Lovell and R.A. Yokel.

Intraneuronalaluminium potentiates iron- induced





Pag

e62

2

oxidative stress in cultured rat hippocampal neurons. Brain Res., 743: 271-277, (1996.)

*Corresponding Author: Mrs. Nirmala Rajesh Naidu Yenepoya Medical College,University Road, Deralakatte, Mangalore 575018 Phone: +91 824 2204668/69/70, Fax: +91 824 2204667 E-Mail: [email protected]



Research Article



CHANDRA K SEKHAR*et al Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e62

3

A NEW UV-METHOD FOR DETERMINATION OF BORTEZOMIB

IN BULK AND PHARMACEUTICAL DOSAGE FORM

CHANDRA K SEKHAR*1, P.SUDHAKAR1, G.RAMESH REDDY2, P.VIJAYA BABU3, N.LINGA SWAMY4

*Quality Control, Bio-Leo Analytical Labs India Pvt Ltd, IDA, Prasanthi Nagar, Kukatpally,

Hyderabad, India *Corresponding Author Email: [email protected]

ABSTRACT A new simple easy UV-spectrophotometric method was developed for the estimation of Bortezomib in bulk and

dosage form. The maximum absorption was found to be at 270 nm. Methanol was used as a diluent. The Calibration

curve was linear over the concentration range of 3.5-21.0 µg/ml. The propose method was validated as per the ICH

guidelines parameters like Linearity, precision, accuracy, robustness and ruggedness. The method was accurate,

precise, specific and rapid found to be suitable for the quantitative analysis of the drug and dosage form.

KEY WORDS Method development and validation, Bortezomib, UV, Spectrophotometric.

1. INTORDUCTION

[(1R)3methyl1({(2S)3phenyl2[(pyrazin2ylcarbonyl

)amino]propanoyl}amino)butyl]boronic acid.

There is no official UV-method for the

Bortezomib. As per literature survey a few

methods have been reported the estimation of

Bortezomib individually 1-5. With this present

proposed method Bortezomib estimates easy,

simple and economical by UV-method in bulk and

pharmaceutical formulation.

2. MATERIAL AND METHODS

2.1 Spectrophotometric Conditions

Shimadzu UV-Vis double beam

spectrophotometer provided with matched

10mm quartz cuvettes equipped with UV-probe

software from shimadzu corporation, Japan was

employed in the study. AR grade methanol and

Class-A glassware purchased from E.Merck Co;

Mumbai, India were used in the study.

2.2 Drug Samples

The reference samples were obtained from M/s.

Bio-Leo Analytical Labs India Pvt Ltd, Hyderabad,

India, the formulation samples were purchased

from local market.

2.3 Preparation of stock and working standard

solution of Bortezomib

About 3.5mg of Bortezomib was weighed

accurately on Sartorius semi micro balance

model-CPA225D and transfers in to 25ml

volumetric flask the solution was sonicated and

the resulting solution was diluted with the

methanol to get a working standard solution of

140 µg/ml.

2.4 Sample Preparation

Weighed accurately equivalent to 3.5 mg of

sample transferred to 25ml volumetric flask make

up to the mark with methanol sonicated and

filtered through 0.45µ membrane filter paper.

Further dilute 10ml to100 ml with methanol.










Pag

e62

4

2.5 Linearity and Construction of Calibration

Curve

Linearity of the peak area response was

determined by taking measurement at Six

concentration prints (6 replicates at each point)

working standard dilution of Bortezomib in the

range of 3.5-21µg/ml. The drug monitored at 270

nm and the corresponding spectrums were

obtained. Form these chromatograms the mean

peak areas were calculated and a plot of

concentration over the peak absorbance was

constructed. This regression equation was later

used to estimate the amount of Bortezomib in

pharmaceutical dosage form. A representative

spectrum presented in fig.1


The present study was aimed at developing a

simple economical precise and accurate UV

method for the analysis of Bortezomib in bulk

drug and in pharmaceutical dosage form.

Methanol was used as a diluent. A good linear

relationship (r2 = 0.993) was observed for

Bortezomib. The regression concentration and

absorbances are given in Table 1 & 2. When test

solutions were analyzed by the proposed method

for finding out intra and inter-day variation, low

co-efficient of variation was observed.

High recovery values obtained from the dosage

form by the proposed method indicates the

method is accurate. The drug content in tablets

was quantified using the proposed analytical

method are given in Table 3.

The deliberate changes in the method have not

much affected the results. This indicated the

robustness of the method. The lowest value of

LOD and LOQ as obtained by the proposed

method by calculated using 3.3xstdev/slope for

LOD and 10xstdev/slope for LOQ. The standard

solution of the drug was stable up to 24 hrs as the

difference in percent assay during the above

period is within limit system suitability

parameters were studied with six replicates

standard solution of the drug and the calculated

parameters are within the acceptance criteria.

The system precision was established by six

replicate of the standard solution containing

analytes of interest. The values of relative

standard deviation were found within the limit,

indicating the repeatability of the method. The

relative standard deviation was found within the

limit, indicating the injection repeatability of the

method. The results were presented in Table 4.

The diluted preparations of marketed tablets

were injected in duplicate and the results were

calculated and presented in Table 5.

Hence it can be concluded that the proposed UV

method is simple economical sensitive and

reproducible for the analysis of Bortezomib in

bulk and in pharmaceutical dosage form.

Table 1: Optical characteristics of the proposed method

Parameter Value

Absorption Maxima(nm) 270

Beer’s law 0.993

Regression equation (Y=mX+c) Y=0.025x+0.023

Slope(m) 0.025

Intercept(c) 0.023

LOD(µg/ml) 0.09939

LOQ(µg/ml) 0.3012









Pag

e62

5

Table 2: Calibration data of the proposed method

Bortezomib

Conc

(mcg/ml)

Mean Area

3.500 0.123

7.000 0.201

10.500 0.307

14.000 0.381

17.500 0.456

21.000 0.535

Table 3 : Accuracy data (Triplicate values at 50,100 &150 percent levels)

Amount taken

(µg)

Amount found

(µg)

Percent Recovery Percentage of mean

recovery

Bortezomib

7.0 7.10 101.42 101.42

10.5 10.45 99.52 99.52

17.5 17.42 99.54 99.54

*Each value is a mean of three readings

Table 4: Precision Study

S.No. Abs

1 0.307

2 0.306

3 0.306

4 0.307

5 0.305

6 0.306

avg 0.306167

stdev 0.000753

%RSD 0.246

Table 5: Assay Results

Drug Amount present/ml % of Assay

Bortezomib 3.52 mg 100.57









Pag

e62

6

Figure 1: UV-spectrum of Bortezomib (10.5mcg/ml).

Figure 2: Linearity of Bortezomib

ACKNOWLEDGEMENT

The authors are thankful to M/s BIO-LEO

ANALYTICAL LABS INDIA PVT.LTD, HYDERABAD for

providing a gift samples and laboratory facilities,

India for encouragement.









Pag

e62

7

REFERENCES 1. S.Venkat Rao, M.Srinivasa Rao, G.Ramu and

C.Rambabu, UV Visible spectrophotometric

determination of Bortezomib in its bulk and formulation

dosage forms, DER PHARMACIA LETTRE, 2012, 4(3):

720-727.

2. C.Rambabu, S.Venkat Rao, G.Rambabu and M.Ganesh,

Estimation of Bortezomib in bulk and its pharmaceutical

dosage forms by using a novel validated accurate RP-

HPLC, International journal of pharmacy and

pharmaceutical sciences, 2011, 3(3): 303-305.

3. Scott E Walker, Debbie Milliken and Shirley law, Stability

of Bortezomib reconstituted with 0.9% sodium chloride

at 4 and 23 degrees, Can J Hosp Pharm, 2008, 1(1):14-

20.

4. Kasa Srinivasulu, Mopidevi narasimha naidu, kadaboina

rajasekhar, murki veerender, and mulukutla venkata

suryanarayana, Development and validation of stability

indicating LC method for the assay and related

substances determination of a proteasome inhibitor

Bortezomib, Chromatography Research International,

2012, doi:10.1155/2012/801720.

5. Venkataramana M, K Sudhakar babu, and SKC Anwar, A

validated stability-indicating UFLC method for

Bortezomib in the presence of degradation products

and its process related impurities, J chromatograph

Separat techniq, 2012, doi.org/10.4172/2157-

7064.100017

*Corresponding Author: CHANDRA K SEKHAR, Bio –Leo Analytical Labs India Pvt Ltd, IDA, Prasanthi Nagar, Kukatpally, Hyderabad, India. Email: [email protected]








Research Article



K.Swathi*and M. Sarangapani Int J Pharm Bio Sci www.ijpbs.com or www.ijpbsonline.com

Pag

e62

8

SYNTHESIS AND SCREENING OF BIOLOGICALLY SIGNIFICANT INDOLE DERIVATIVES

FOR ANTICONVULSANT ACTIVITY

K.Swathi*and M. Sarangapani

Medicinal Chemistry Laboratory, U.C.P.Sc., Kakatiya University

Wararagal-506009, Andhra Pradesh, India *Corresponding Author Email: [email protected]

ABSTRACT In the present work, some new 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-diones and 5-Hydroxyindole 3-

semicarbazone 2-ones were prepared from 5-hydroxy isatin. The structures of the products were characterized by

IR, NMR, MASS Spectral studies. All the compounds were examined for anticonvulsant activity by maximal

electroshock seizure (MES) and pentylenetetrazole (PTZ) induced convulsion method. These compounds were also

evaluated for their neurotoxicity study by rotorod method. Some of these compounds showed good anticonvulsant

activity when compared with standard drug Phenytoin and all the compounds showed less neurotoxicity when

compared with standard drug Diazepam.

KEY WORDS Synthesis, 5-[2(3)-dialkyl amino alkoxy] Indole 2, 3-diones, 5-Hydroxyindole 3-semicarbazone 2-ones,

Anticonvulsant activity.

1. INTRODUCTION

Epilepsy, one of the common neurological

disorders, is a major public health problem,

affecting around 4% of individuals over their

lifespan. About 20-30% of the epilepsy patients

are resistant to the available medical therapies.

This fact warrants the investigation for new

antiepileptic drugs.

Isatin is an endogenous compound isolated in

1988 and reported [1] to possess a wide range of

central nervous system activities. Surendranath

pandya [2] et al. reported the synthesis and

anticonvulsant activity of some novel n-

methyl/acetyl, 5-(un)-substituted isatin-3-

semicarbazones. In the last few years, Isatin

derivatives have been discovered which show

potential hypnotic [3], antibacterial [4-6] and

MAO inhibitory [7] activity.

It is evident from the literature survey that Isatin

derivatives, isatin semicarbazone derivatives and

dialkylamino alkyl derivatives showing more

promising central nervous system and

anticonvulsant activities. Keeping in view of

these two molecular moieties viz., 5-hydroxy

isatin (Resembles serotonin) and dialkylamino

alkyl (Resembles NT), it is our endeavor to bring

such important moieties into a single molecular

frame as a model for molecular conjunction by

appropriate synthetic routes and to screen them

for anticonvulsant activity and neurotoxicity.

2. MATERIALS AND METHODS

The compounds were mostly synthesized by

conventional methods and described in

experimental selection and also by the methods

established in our laboratory.






Pag

e62

9

2.1. Chemicals

Leptazole, Diazepam, Dialkylaminoalkylhalides,

semicarbazidehydrochloride purchased from

Sigma- Aldrich Chemicals Private Limited,

Hyderabad, India. p-amino phenol,

hydroxylamine hydrochloride, sodium sulfate

were purchased from Merck Chemicals Private

Limited, Hyderabad, India.

2.2. Chemistry

Solvents were dried or distilled before use.

Melting points were obtained on a Thoshniwall

melting point apparatus in open capillary tubes

and are uncorrected. The purity of the

compounds were ascertained by TLC on silica gel

–G plates(Merck).Infrared spectra(IR) were

recorded with KBR pellet on a Perkin-Elmer BX

series, Infrared spectrophotometer. Mass

spectra were recorded by the direct inlet

method on Thadmam-mass-quantam API 400H

mass spectrophotometer.1H NMR spectra were

recorded on Brucker spectrospin 400 MHz

spectrophotometer in DMSO-d6. 5-hydroxy Isatin

was synthesized from p- amino phenol by using

Sandmayer[8] method It consists in the reaction

of aniline with chloral hydrate and

hydroxylamine hydrochloride in aqueous sodium

sulfate to form an isonitrosoacetanilide, which

after isolation, when treated with concentrated

sulfuric acid, furnishes isatin in >75% overall

yield.

2.3. Preparation of 5-Hydroxyindole 3-

semicarbazone 2-one

5-Hydroxyisatin was heated under reflux in

methanol containing two or three drops of acetic

acid with semicarbazide hydrochloride for half

an hour. The product thus separated was filtered

and purified by recrystalization from suitable

solvent. (Yield 89%, m.p.270oc)

2.4 Preparation of 5-[2(3)-dialkyl amino alkoxy]

Indole 2,3 diones and 5-Hydroxyindole 3-

semicarbazone 2-ones

A mixture of 5-hydroxyisatin/5-Hydroxyisatin-3-

semicarbazone (0.01 Moles) and dialkylamino

alkylhalide (0.01 Moles) placed in 10% alcoholic

potassium hydroxide and this mixture was

stirred at room temperature for 6 hours .The

alcohol was reduced to half of its volume and

cooled. The product separated was filtered,

washed with small portions of cold alcohol

repeatedly and dried .It was purified by

recrystalisation from hydro alcoholic mixtures to

get a crystalline solid. Similarly other 5-Hydroxy

Isatin derivatives as shown in Scheme 1 were

prepared and their melting points were

determined in Open capillary tubes using

Toshniwall melting point apparatus and are

uncorrected. Purity of the compounds was

checked by TLC. The physical data of the title

compounds were presented in Table 1. The

compounds were characterized by spectral data.

2.5. Spectral data

The compounds have been characterized by the

spectral data IR, PMR and Mass.

IR spectrum (KBr) of compound (III) exhibited

absorption bands (cm-1) 3421.47 (OH), 1630.08

(C = O), 1548 (Ar,C=C), 1282(C-O-C), 883.85-

579.8 (Ar). 1H NMR (300 MHz, DMSO-d6): 13.3 (s,

1H, OH), 10.36(s, 1H,-CONH), 6.65-7.29(m, 3 H,

Ar-H). Mass spectrum of compound III showed

molecular ion (M+) base peak at m/z (164.1).

Compound (IIIa) showed characteristic IR peaks

at 3276(NH), 1651.96 (C=O), 1569.82 (Ar, C=C),

1276(C-O-C), 807.93(Ar). 1H NMR (300 MHz,

DMSO-d6):10.36(s, 1H,-CONH ), 7.01-7.29(m,3

H,Ar-H),3.2 (T,2H,O-CH2 s) ,2.9 (T,2H,N-CH2), 1.36

(S,6H,N-(CH3)2).Mass spectrum of compound IIIa

showed molecular ion (M+) base peak at m/z

231 (100%).It also shows peak at m/z (71) may

be due to the fragmentation of the alkyl chain

from the molecule ion.





Pag

e63

0

Scheme 1: Synthetic protocol of the title compounds.





Pag

e63

1

Table I: Physical data of 5-[2(3)-dialkyl amino alkoxy] Indole 2,3 diones and

5-Hydroxyindole semicarbazone 2- ones

S.No Compound R R1 N X M.F %

YEILD M.P

M.Wt

1 IIIa CH3 H 1 O C12H14N2O3 91% <320 234

2 IIIb C2H5 H 1 O C14H18N2O3 86% <320 262

3 IIIc CH3 H 2 O C13H16N2O3 93% <320 248

4 IIId CH3 CH3 1 O C13H16N2O3 85% <320 248

5 IIIe CH

3

CH3

CH2

H 1 O C16H24N2O3 81.8% <320 292

6 IVa CH3 H 1 NNHCONH2 C13H17N5O3 92% <320 291

7 IVb C2H5 H 1 NNHCONH2 C15H21N5O3 83% <320 319

8 IVc CH3 H 2 NNHCONH2 C14H19N5O3 92% <320 365

9 IVd CH3 CH3 1 NNHCONH2 C14H19N5O3 86% <320 365

10 IVe CH

3

CH3

CH2

H 1 NNHCONH2 C17H27N5O3 82% <320 349

Compound (IIIb) showed characteristic IR peaks

at 3274(NH), 1681.53 (C=O), 1570.21 (Ar ,C=C),

1243(C-O-C), 845.51(Ar). 1H NMR (300 MHz,

DMSO-d6): 10.25(s, 1H,-CONH ), 7.03-7.45(m,3

H,Ar-H),2.99 (T,2H,O-CH2 s) ,2.72 (T,2H,N-CH2)

,1.24 (S,10H,N-(C2H5)2).

Mass spectrum of compound IIIb showed

molecular ion (M+) base peak at m/z 263

(100%).It also shows peak at m/z (71) may be

due to the fragmentation of the alkyl chain from

the molecule ion.

Compound (IIIc) showed characteristic IR peaks

at 3274(NH), 1651.96 (C=O), 1579.72 (Ar, C=C),

1266(C-O-C), 805.91(Ar). 1H NMR (300 MHz,

DMSO-d6):10.46(s, 1H,-CONH ), 7.21-7.49(m,3

H,Ar-H),2.84 (T,2H,O-CH2) , 2.51 (M,2H, CH2),2.48

(T,2H,N-CH2), 1.25 (S,6H,N-(CH3)2).Mass

spectrum of compound IIIc showed molecular

ion (M+) base peak at m/z 247 (100%).

Compound (IIId) showed characteristic IR peaks

at 3257(NH), 1679.64 (C=O), 1546.86 (Ar, C=C),

1245(C-O-C), 812.71(Ar). 1H NMR (300 MHz,

DMSO-d6):10.51(s, 1H,-CONH ), 7.12-7.42(M,3

H,Ar-H),2.76 (M,2H,O-CH2) , 2.45 (T,3H,

R1=CH3),2.31 (M,1H,N-CH), 1.44 (S,6H,N-

(CH3)2).Mass spectrum of compound IIId showed

molecular ion (M+) base peak at m/z 247 (100%).

Compound (IIIe) showed characteristic IR peaks

at 3257(NH), 1689.46 (C=O), 1576.34 (Ar, C=C),

1228(C-O-C), 814.53(Ar). 1H NMR (300 MHz,

DMSO-d6):10.26(s, 1H,-CONH ), 7.34-7.51(m,3

H,Ar-H),2.96 (T,2H,O-CH2 s) ,2.82 (T,2H,N-CH2),

1.35 (S, 2H,N-CH) ,1.21 (D,12H,Csss -(CH3)2).Mass

spectrum of compound IIIe showed molecular

ion (M+) base peak at m/z 291 (100%).It also

shows peak at m/z (71) may be due to the

fragmentation of the alkyl chain from the

molecule ion.

Compound (IVa) showed characteristic IR peaks

at 3276(NH), 1651.96 (C=O), 1569.82 (Ar, C=C),

1276(C-O-C), 807.93(Ar). 1H NMR (300 MHz,

DMSO-d6): 10.36(s, 1H,-CONH ), 7.01-7.29(m,3

H,Ar-H),3.2 (T,2H,O-CH2 s) ,2.9 (T,2H,N-CH2), 1.36

(S,6H,N-(CH3)2).Mass spectrum of compound IVa


291 (100%). The mass spectrum shows its base

peak at m/z 77 (100%) may be due to the

fragmentation of the semicarbazone from the

molecule ion.





Pag

e63

2

Compound (IVb) showed characteristic IR peaks

at 3274(NH), 1681.53 (C=O), 1570.21 (Ar ,C=C),

1243(C-O-C), 845.51(Ar). 1H NMR (300 MHz,

DMSO-d6):10.25(s, 1H,-CONH ), 7.03-7.45(m,3

H,Ar-H),2.99 (T,2H,O-CH2 s) ,2.72 (T,2H,N-CH2)

,1.24 (S,10H,N-(C2H5)2), 7.41-7.46(d,2H, NH2)

,11.36(S,1H, NH).Mass spectrum of compound

IVb showed molecular ion (M+) base peak at m/z

317 (100%). The mass spectrum shows its base

peak at m/z 77 (100%) may be due to the

fragmentation of the semicarbazone from the

molecule ion.

Compound (IVc) showed characteristic IR peaks

at 3274(NH), 1651.96 (C=O), 1579.72 (Ar, C=C),

1266(C-O-C), 805.91(Ar). 1H NMR (300 MHz,

DMSO-d6):10.46(s, 1H,-CONH ), 7.21-7.49(m,3

H,Ar-H),2.84 (T,2H,O-CH2) , 2.51 (M,2H, CH2),

7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH),2.48

(T,2H,N-CH2), 1.25 (S,6H,N-(CH3)2). Mass

spectrum of compound IVcshowed molecular ion

(M+) base peak at m/z 363 (100%).

Compound (IVd) showed characteristic IR peaks

at 3257(NH), 1679.64 (C=O), 1546.86(Ar ,C=C),

7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH) 1245(C-O-

C), 812.71(Ar). 1H NMR (300 MHz, DMSO-d6):

10.51(s, 1H,-CONH ), 7.12-7.42(M,3 H,Ar-H),2.76

(M,2H,O-CH2) , 7.41-7.46(d,2H, NH2) ,11.36(S,1H,

NH), 2.45 (T,3H, R1=CH3),2.31 (M,1H,N-CH), 1.44

(S,6H,N-(CH3)2).Mass spectrum of compound IVd


363 (100%).

Compound (IVe) showed characteristic IR peaks

at 3257(NH), 1689.46 (C=O), 1576.34 (Ar, C=C),

1228(C-O-C), 814.53(Ar). 1H NMR (300 MHz,

DMSO-d6): 10.26(s, 1H,-CONH ), 7.34-7.51(m,3

H,Ar-H),2.96 (T,2H,O-CH2 s) , 7.41-7.46(d,2H,

NH2) ,11.36(S,1H, NH),2.82 (T,2H,N-CH2), 1.35 (S,

2H,N-CH) ,1.21 (D,12H,Csss -(CH3)2).Mass

spectrum of compound IVe showed molecular

ion (M+) base peak at m/z 347 (100%). The mass

spectrum shows its base peak at m/z 77 (100%)

may be due to the fragmentation of the

semicarbazone from the molecule ion.

3. Pharmacology

3.1. Anticonvulsant Activity

Materials: Normal saline, test compounds,

Leptazole, stop watch, phenytoin.

Animals: Swiss mice.

3.1.1. Maximal electroshock seizure (MES)

method [9]

Method: The anticonvulsant activity was studied

by Maximal Electroshock Induced Convulsion

method by using electro-convulsometer. Healthy

male mice weighing between 20-25g were fasted

for overnight and divided into groups of six

animals each. The test compounds suspended in

normal saline were administered at a dose of

100 mg/kg body weight i.p. The control group

animals received only vehicle (Normal saline).

The test started 30 min after i.p. injection.

Maximal seizures were induced by the

application of electrical current to the brain via

corneal electrodes. The stimulus parameter for

mice was 50 mA in a pulse of 60 Hz for 200 ms.

Abolition of the hind limb tonic extensor spasm

was recorded as a measure of anticonvulsant

activity; results are presented in Table 2.





Pag

e63

3

Table 2: Anticonvulsant activity and neurotoxicity study of

5-[2(3)-dialkylamino alkoxy] Indole 2,3-diones

S.No Compound

Animals protected in % Neurotoxicity

(%) MES induced Convulsions

Chemically induced convulsions

1 IIIa 65.54 60.9 7

2 IIIb 58.61 64.6 18

3 IIIc 48.42 56.6 6.7

4 IIId 41.5 49.8 3.4

5 IIIe 37.46 44.83 4.6

6 IVa 65.18 71.2 7

7 IVb 66.68 65.77 5

8 IVc 54.76 44.3 6.2

9 IVd 38.18 40.3 7.8

10 IVe 37.44 33.9 7

11 Phenytoin 100 89.36 -

12 Control 0 0 2

13 Diazepam - - 88

Number of animals n=6, The compounds were tested at adose of 100mg/kg (b.w)

3.1.2. Pentylenetetrazole (PTZ) method [10]

Materials: Normal saline, test compounds,

Leptazole, stop watch, phenytoin.

Animals: Swiss mice.

Method: The anticonvulsant activity was studied

by using Leptazole (Pentylene tetrazole) as a

chemical convulsion inducer. Healthy male mice

weighing between 20-25g were fasted for

overnight and divided into groups of six animals

each. The animals were injected with Leptazole

(80mg/kg) given intraperitonially. Those animals

which show convulsions were selected for the

experiment. The test compounds suspended in

normal saline were administered at a dose of

100 mg/kg body weight i.p. The control group

animals received only vehicle (Normal saline).

The Leptazole is again given in the same dose

and the time taken for convulsions to start was

noted, results are presented in Table 2 and

Figure 1.

3.1.3. Neurotoxicity study [11]

Healthy male mice weighing between 20-25gm

were fasted for overnight and divided into

groups of six animals each. Turn of the rotating

rod, select an appropriate speed (25 rpm), and

place the animal one by one on the rotating rod.

A normal mouse (untreated) generally falls off

within 3-5 min. Test compound dissolved in

saline were administered, intraperitonially in a

dose of 100 mg/kg. The control group received

saline only. One group of animals was

administered diazepam as a standard (i.p 4





Pag

e63

4

mg/kg). After 30 minutes, repeated the

experiment as done earlier noted the fall of time

of animals before and after test compounds and

diazepam treatment respectively, results are

presented in Table 2 & Figure 2.

Figure 1: Anticonvulsant activity of 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-dions

and 5-Hydroxyindole 3-semicarbazone 2-ones.

Figure 2: Neurotoxicity study of the 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-diones and

5-Hydroxyindole 3- semicarbazone 2-ones





Pag

e63

5

4. RESULTS AND DISCUSSIONS

Physical data TLC, IR, 1H NMR and mass spectra

confirmed the structures and purity of the

synthesized compounds. All the title compounds

decomposed before melting. All the synthesized

compounds were evaluated for their in vivo

anticonvulsant and skeletal muscle relaxant

activity. It was observed that compounds IVa,

IIIa, IVb, IIIb, exhibited more promising

anticonvulsant activity. Among the test

compounds IIIc, IVc, IIId, IVd, IIIe, IVe were

found to be next in the order of reducing the

duration of convulsions. Compounds with

dimethyl amino and diethyl amino ethyloxy

group at c5 of Isatin showed more protection

against Maximal Electroshock Seizure (MES)

induced convulsions where as compounds IIIa,

IIIb and IVa, IVb exhibited more protection

activity against chemically (Leptazole)induced

convulsions. 5-[2(3)-dialkylamino alkoxy] isatin -

3-semicarbazones showing more activity

compared to 5-[2(3)-dialkylamino alkoxy] isatins

.All the test compounds showed less (<20%)

neurotoxicity (Skeletal muscle relaxant activity)

when compared with Diazepam.

5. CONCLUSION

A new series of five 5-[2(3)-dialkyl amino alkoxy]

Indole 2,3 dione derivatives were synthesized by

reacting 5-hydroxyindole 2,3 dione with 2-N,N di

alkylamino alkyl halides.Evaluation of these

compounds as anticonvulsant and skeletal

muscle relaxant activity revealed that the

compounds IVa(R=CH3), IVb(R=C2H5), IIIa(R=CH3)

and IIIb(R=C2H5) with a dimethyl and diethyl

amino ethyl chain derivatives was found to be

relatively superior in anticonvulsant activity and

other compounds(IIIc, IVc, IIId, IVd, IIIe, IVe ) are

next in the order of activity.All the compounds

showed less neurotoxicity compared to

Diazepam.

ACKNOWLEDGEMENTS

The First author would like to thank the CSIR,

New Delhi for providing financial support.

Authors are thankful to Principal University

College of Pharmaceutical Sciences, Kakatiya

University for providing facilities.

REFERENCES 1. K.Bhattacharya Salil, K.Mitra Shankarand

B.AcharyaSatya, J.Psychopharmacol,5, 202(1991).

2. S.N. Pandeya and A. Senthil Raja, J. Pharm. Sci., 5(3),

275 (2002).

3. Pandeya SN, Yogeeswari P and Stables jp.Eur J Med

Chem.35, 879-86 (2000).

4. A.K. Padhy, S.K. Sahu, P.K. Panda, D.M. Kar and P.K.

Misro, Indian J. Chem., 43B, 971 (2004).

5. A. Raviraj, Kusanur, Manjunath Ghate and Manohar,V.

Kulkarni, J. Chem. Sci., 116(5), 265 (2004).

6. S. Gupta, Raman, S.N.Vikas, Srivastava, Asian J. chem.,

16(2), 779-783 (2004).

7. B. Gringberg, L. Imazylis and M. Benhena, Chemija, 2,

87 (1990).

8. C.S.Marvel and G.S.heirs,Organic synthesis

Collect.,1,327(1941).

9. R.L. Krall, J.K. Penry, B.G. White, H.J. Kupferberg & E.A.

Swinyard, Epilepsia., 19, 409(1978).

10. H.Gerhard Vogel(Ed), Drug Discovery and Evaluation of

Pharmacological assays, IInd Edition 487 (2002).

11. H.Gerhard Vogel(Ed), Drug Discovery and Evaluation of

Pharmacological assays, IInd Edition 398s (2002).





Pag

e63

6

*Corresponding Author: K.Swathi* Medicinal Chemistry Laboratory, U.C.P.Sc., Kakatiya University, Wararagal-506009, A.P,India. Email: [email protected]


International Journal of Pharmacy and Biological Sciences INSTRUCTIONS TO AUTHORS


ABOUT JOURNAL International Journal of Pharmacy and Biological



innovative research works in the field of

Pharmaceutical and Biological Sciences includes:

Pharmaceutics

Polymer sciences

Biotechnology

Pathology

Novel Drug Delivery Systems

Pharmaceutical Biotechnology

Microbiology

Cytology

Nanotechnology

Biomaterial Sciences

Cell Biology

Immunobiology

Pharmacology

Natural Chemisty

Biochemistry

Pharmacognosy

Pharmacoinformatics

Bioinformatics

AnalyticalChemistry

BioPharmaceutics

Molecular Biology

Medicinal Chemistry

Hospital and Clinical Pharmacy

Neurobiology

Pharmacy Practice

Pharmacokinetics

Pharmacogenomics

Therefore, the corresponding author is requested to mention the branches which come under either pharmaceutical or biological sciences for their manuscript in their cover letter.

SUBMISSION OF MANUSCRIPT

Authors are encouraged to submit their manuscript

electronically through online submission or an

Email address, [email protected] along

with a covering letter preferably by the

corresponding author or first author. Each

manuscript will be provided with a manuscript ID

by IJPBS automatic system.

COVER LETTER During submission of the article, a cover letter

should be included having

Authors full address and telephone/fax

number.

The type of article (Research or Review)

along with the title and the type of branch

(for branch kindly see “Aim and Scope” of

this journal) under which the article to be

published should be mentioned.

The corresponding author should mention

the undertaking that if any animal studies

carried was in accordance with their country

or institutional ethical committee and also

state that the manuscript has not been

published elsewhere (except in the form of

an abstract or as part of a published lecture

or academic thesis).

Please refer "Model Cover Letter" of this journal to

get an idea.

MANUSCRIPT STATUS Track Your Manuscript: through online system

You can track the status of your manuscript through

the online production process by entering

Manuscript ID in that field.

For the submission of revised manuscript and

queries regarding manuscript status or any other

enquiries, please contact us at

[email protected],[email protected]

m With referring your manuscript ID

RESEARCH ARTICLES Manuscript should be type written in 12 font size,

double-spaced, with margins of at least 2 cm on all

sides. Pages should be numbered consecutively,

starting with the title page and the matter arranged

in the following order: Title page, Abstract,

Keywords, Sections (Introduction, Materials and

Methods, Results and Discussion, Conclusions),

Acknowledgements, References, Tables and

Figures along with caption and legends.

Research articles should have more than 15 pages

and Review articles in the range of 15-30 pages,

inclusive of illustrations. Short communications

should have not more than 4 Pages inclusive of

illustrations.

Title Page:

Title page contains title of the manuscript in bold

face, title case (font size 14), names of the authors

in normal face, title case (font size 12) followed by

the address of authors in normal face, title case

(font size 12). Names of the authors should appear

mailto:[email protected],[email protected]


Ijpbs-INSTRUCTIONS TO AUTHORS

ii

as initials followed by surnames. Full names may be

given in some instances to avoid confusion.

Followed by the author names, please provide the

complete postal address or addresses with pin code

number of the place(s), where the research work

has been carried out. If the publication originates

from several institutes, the affiliation of each author

should be clearly stated by using superscript Arabic

numbers after the name and before the institute.

The author to whom correspondence should be

directed must be indicated with an asterisk. At the

bottom left corner of this page, please mention

“*Corresponding Author” and provide telephone

number and fax number of the research

institution/college and functional Email address of

the corresponding author to whom all

correspondence (including galley proofs) is to be

sent.

Sections:

Manuscripts should be divided into the following

sections:

Titles (normal face, upper case) and subtitles in

each section (bold face, lower case):

Abstract:

An abstract not exceeding 250 words (for Short

Communications between 60 and 80 words) should

be provided typed on a separate sheet. Abstract

should include aims, methods, results and

conclusion.

Keywords:

Up to 4-6 keywords must be provided in

alphabetical order. These keywords should be typed

at the end of the abstract.

Introduction:

It should be a concise statement of the background

to the work presented, including relevant earlier

work, suitably referenced. It should be started in a

separate page after keywords.

Materials and Methods:

It shall be started as a continuation to introduction

on the same page. All important materials and

equipments, the manufacturer’s name and, if

possible, the location should be provided. The main

methods used shall be briefly described, citing

references. New methods or substantially modified

methods may be described in sufficient detail. The

statistical method and the level of significance

chosen shall be clearly stated.

Results and Discussion:

The important results of the work should be clearly

stated and illustrated where necessary by tables

and figures. The statistical treatment of data and

significance level of the factors should be stated

wherever necessary. The discussion should deals

with the interpretation of results, making the

readers to understanding of the problem taken and

should be logical. The scope of the results, which

need to be further explored, could also be dealt.

Digital files are recommended for highest quality

reproduction and should follow the following

guidelines.

300dpi or higher sized to fit journal page

JPEG, GIF, TIFF and PDF formats are preferred)

Acknowledgement (if any)

Conclusions:

Concisely summarizes the principal conclusions of

the work and highlights the wider implications. This

section should not merely duplicate the abstract.

Acknowledgements:

Acknowledgements as well as information regarding

funding sources may be provided.

References:

Citations of literature within the text must be

presented in numerical order and should be set in

square brackets, thus [1, 12]. The cited literature

are also collected in numerical order at the end of

the manuscript under the heading “References”.

The abbreviated title and the volume number

should appear in italics. Only the papers and books

that have been published or in press may be cited.

Please note that website addresses must not be

included as a reference, but should be inserted in

the text directly after the information to which they

refer.

Please note the following examples:

Journals:

[1]Gregoriadis G., Engineering liposomes for drug

delivery: progress and problems. Trends Biotechnol,

13 (12): 527–537, (1995)

Books:

[1]Joseph R. Robinson and Vincent HL Lee, Ed.

Controlled Drug Delivery Fundamentals and

applications, 2nd Edn, Vol 29, Lippincott Williams’s

publisher:555–561,(1994)

[2] Myers, R.H., Montgomery, D., Response Surface

Methodology, Wiley, New York 1995.


iii

Chapter in a book:

[1] Brown, M.B., Traynor, M.J., Martin, G.P.,

Akomeah, F.K., in: Jain, K.K., Walker, J.M. (Eds.),

Drug Delivery Systems, Humana Press, USA 2008,

pp. 119-140.

For Patent Reference

[1]H. Aviv, D. Friedman, A. Bar-Ilan and M. Vered.

Submicron emulsions as ocular drug delivery

vehicles, U.S. Patent US 5496811, 1996.

Tables:

Should each be typed on a separate page,

numbered in sequence with the body of the text.

Tables should be headed with a short, descriptive

caption. They should be formatted with horizontal

lines only: vertical ruled lines are not required.

Footnotes to tables should be indicated with a), b),

c) etc. and typed on the same page as the table.

Figures:

Should be on separate pages but not inserted within

the text. All figures must be referred to in the text

and numbered with Arabic numerals in the

sequence in which they are cited. Each figure must

be accompanied by a legend explaining in detail the

contents of the figure and are to be typed under the

figures. Graphs and bar graphs should preferably be

prepared using Microsoft Excel and submitted as

Excel graph pasted in Word. Alternatively

photographs can be submitted as JPEG images. Keys

to symbols, abbreviations, arrows, numbers or

letters used in the illustrations should not be

written on the illustration itself but should be

clearly explained in the legend. Avoid inserting a

box with key to symbols, in the figure or below the

figure. All Tables and Figures captions and legends

should be typed on a separate page.

REVIEW ARTICLE

Organization of the review article is at the author’s

discretion and must be at a length of 3000 words

excluding references and abstract. Abstract and key

words are required. Tables, figures, illustrations and

references are to be arranged according to research

papers.

SHORT COMMUNICATIONS

Please add the term “Short Communication” below

the title. A short communication contains a

summary and should not exceed 4 printed journal

pages (Including Tables, Figures and References).

Short communication should contain novel

experimental or theoretical findings in need of

urgent publication.

GALLERY PROOFS Gallery proofs are sent to the designated author

through Email. They must be carefully checked and

returned the revised manuscript within 48 hours of

receipt.

COPYRIGHT Authors are asked to sign a warranty and copyright

agreement upon acceptance of their manuscript,

before the manuscript can be published. The

Copyright form can be downloaded from our

website. Submission of your paper to this journal

implies that the paper is not under submission for

publication elsewhere. Material which has been

previously copyrighted, published, or accepted for

publication will not be considered for publication in

this journal. Submission of a manuscript is

interpreted as a statement of certification that no

part of the manuscript is copyrighted by any other

publisher nor is under review by any other formal

publication. By submitting your manuscript to us,

you agree on IJPBS Publication copyright

guidelines. It is your responsibility to ensure that

your manuscript does not cause any copyright

infringements, defamation, and other problems.

Submitted papers are assumed to contain no

proprietary material unprotected by patent or

patent application; responsibility for technical

content and for protection of proprietary material

rests solely with the author(s) and their

organizations and is not responsibility of the IJPBS

Publications or its Editorial Staff. The main author is

responsible for ensuring that the article has been

seen and approved by all the other authors. It is the

responsibility of the author to obtain all necessary

copyright release permissions for the use of any

copyrighted materials in the manuscript prior to the

submission.

ETHICAL MATTERS:




iv







PUBLICATION POLICY: Papers will normally be published in order of





DECLARATION







publication.

DISCLAIMER “No responsibility is assumed by the publisher” for

any injury and/or damage to persons or property as

a matter of products liability, negligence or

otherwise, or from any use or operation of any

methods, products, instructions or ideas contained

in the material herein. The respective individual

author(s) are responsible for the facts and views

expressed in their articles. “Authors are solely

responsible for their manuscripts and data

published in the journals” and any mistakes in the

published articles. The publisher does not take any

such responsibilities. Copying and reproducing any

parts of the published articles should be duly

acknowledged, otherwise, it will be considered as

an offense as per international Publications law.

CHECKLIST FOR SUBMISSION 1. Have you provided a Title Page?

2. Have you provided an Author Information

section at the end of the paper?

3. Have you provided an Abstract of not more

than 250 words?

4. Have you provide keywords of not more than 3

to 4?

5. Are your Tables denoted by Arabic numerals,

and are they in order as cited in the text?

6. Are your Tables submitting at the end of the

text file?

7. Are your Figures denoted by Arabic numerals,


8. Have all your Figures been submitted text file

or PNG or JPG files?

9. Are your References cited in the required style

of the Journal?

10. Have you obtained permission and submitted

documentation for all Personal

Communications cited?

AUTHOR BENEFITS The benefit of publishing in IJPBS includes:

Fast publication times

Free colour in electronic version

Access free on-line issue of IJPBS on

www.ijpbs.com

A rigorous, fast and constructive peer

review process

Excellent editorial standards

Any-Time, Any-Where access

Faster Review

No need for Hard copy submission

Ease of Contacting the IJPBS: 9912185510.

v

MODEL COVER LETTER (TEMPLATE)

International Journal of Pharmacy and Biological Sciences

Date:

Place:

From

(Name and Address of the corresponding author)

To

The Editor

International Journal of Pharmacy and Biological Sciences (IJPBS)

Sir,

Ref: Title ………………….......................................

Type …………………...................................... (Research/Review/Short communication)

Subject …………………...................................... (Pharmaceutical/Biological Sciences)

Branch …………………...................................... (Branch of subject-Refer author instructions)

In reference to the above title, I as a corresponding author, submit the manuscript for

publication in International Journal of Pharmacy and Biological Sciences. I undertake that animal

study (if any) was taken after the prior approval of country/institutional ethical committee. This

manuscript has not been published or considered for publication by any other journal or elsewhere.

Kindly consider the manuscript for publication in your journal.

Thank you

Corresponding author name and Signature

vi

COPY RIGHT FORM

International Journal of Pharmacy and Biological Sciences I certify that I have participated sufficiently in the conception and design of this work

entitled“……………………………………………………………………………………………………….………………………………………

………………………………………………………………………………………………………………………………………………………………

………..” and the analysis of the data, as well as writing of this manuscript, to take public responsibility

for it. I believe the manuscript represents valid work. I have reviewed the final version of manuscript

and approve it for publication. The manuscript is not published elsewhere, in any language, and is not

under simultaneous consideration by any other journal.

All Authors agree that the contents of the manuscript are confidential and will not be

copyrighted, submitted, or published elsewhere (including the Internet), in any language, while

acceptance by the Journal is under consideration.

Furthermore I attest that I shall produce the data upon which the manuscript is based for

examination by the editors or their assignees, if requested.

I hereby assign and transfer to the [email protected], [email protected] copyright and all

rights under it.

I further confirm that this article has not been published elsewhere, nor is it under

consideration by any other publisher.

SIGNATURE AUTHOR/AUTHORS

1. Signature: _____________________________Date: ______________________________

2. Signature: _____________________________Date: _____________________________

3. Signature: _____________________________Date: ______________________________

4. Signature: _____________________________Date: ______________________________

5. Signature: _____________________________Date: ______________________________

Agreed- Yes / No (Put Tick)

After completion of this form, please email the scanned file of original signed form to

[email protected], [email protected] .





vii

SUBSCRIPTION / MEMBERSHIP APPLICATION FORM

Dear Editor-In-Chief, I/We wish to be Annual member/ Subscriber of International Journal of Pharmacy and Biological Sciences and agree to abide by your rules and regulations

1. Name in full

:

2. Date of Birth:

:

3. Academic Qualifications and experience

:

4. Corresponding Address (Delivery Address)

:

5. Phone and E-Mail address : CURRENT SUBSCRIPTION RATES (Online and Print Edition)

Annual Subscription Fees

India Foreign No. Of Issues SELECTION (Put Tick)

Individual Rs. 4000/- $ 90/ €30 04


BANK DETAILS: Payment Options: Mode of Payment: (Put Tick) i) Online: ii) Bank Deposit: iii) Cash:

I. FOR INTERNATIONAL SUBSCRIBERS Pay through PayPal method: http://ijpbs.com/payment.php (Or)

II. FOR INDIAN SUBSCRIBERS

We offer both Online as well as Offline options to make a payment: You can transfer through online Fund Transfer option Or Direct Bank Deposit.

Account Holder Name GANGADI JAYAPAL REDDY

Bank ICICI BANK LTD

Branch HANAMKONDA

A/C No. 020101503760

IFSC / NEFT / RTGS CODE: ICIC0000201

Place: ____________________ Date: _____________________

(Signature of the applicant) NOTE: Kindly send the scanned copy of Pay-in slip of Subscription Fee to email addresses: [email protected], [email protected]

International Journal of

Pharmacy and Biological Sciences P-ISSN: 2321-3272; E-ISSN: 2230-7605

(Indexed by Chemical Abstracts, DOAJ, Index Copernicus) CODEN: IJPBK3 [Chemical Abstract Services (USA)]

#2-2-83/13, Rajajinagar, Naimnagar, Hanamkonda, Warangal-506 001

Mobile: 099121855 Email: [email protected]

http://ijpbs.com/payment.php


International Journal of Pharmacy and Biological Sciences INSTRUCTIONS TO AUTHORS


ABOUT JOURNAL International Journal of Pharmacy and Biological



innovative research works in the field of

Pharmaceutical and Biological Sciences includes:

Pharmaceutics

Polymer sciences

Biotechnology

Pathology

Novel Drug Delivery Systems

Pharmaceutical Biotechnology

Microbiology

Cytology

Nanotechnology

Biomaterial Sciences

Cell Biology

Immunobiology

Pharmacology

Natural Chemisty

Biochemistry

Pharmacognosy

Pharmacoinformatics

Bioinformatics

AnalyticalChemistry

BioPharmaceutics

Molecular Biology

Medicinal Chemistry

Hospital and Clinical Pharmacy

Neurobiology

Pharmacy Practice

Pharmacokinetics

Pharmacogenomics

Therefore, the corresponding author is requested to mention the branches which come under either pharmaceutical or biological sciences for their manuscript in their cover letter.

SUBMISSION OF MANUSCRIPT

Authors are encouraged to submit their manuscript

electronically through online submission or an

Email address, [email protected] along

with a covering letter preferably by the

corresponding author or first author. Each

manuscript will be provided with a manuscript ID

by IJPBS automatic system.

COVER LETTER During submission of the article, a cover letter

should be included having

Authors full address and telephone/fax

number.

The type of article (Research or Review)

along with the title and the type of branch

(for branch kindly see “Aim and Scope” of

this journal) under which the article to be

published should be mentioned.

The corresponding author should mention

the undertaking that if any animal studies

carried was in accordance with their country

or institutional ethical committee and also

state that the manuscript has not been

published elsewhere (except in the form of

an abstract or as part of a published lecture

or academic thesis).

Please refer "Model Cover Letter" of this journal to

get an idea.

MANUSCRIPT STATUS Track Your Manuscript: through online system

You can track the status of your manuscript through

the online production process by entering

Manuscript ID in that field.

For the submission of revised manuscript and

queries regarding manuscript status or any other

enquiries, please contact us at

[email protected],[email protected]

m With referring your manuscript ID

RESEARCH ARTICLES Manuscript should be type written in 12 font size,

double-spaced, with margins of at least 2 cm on all

sides. Pages should be numbered consecutively,

starting with the title page and the matter arranged

in the following order: Title page, Abstract,

Keywords, Sections (Introduction, Materials and

Methods, Results and Discussion, Conclusions),

Acknowledgements, References, Tables and

Figures along with caption and legends.

Research articles should have more than 15 pages

and Review articles in the range of 15-30 pages,

inclusive of illustrations. Short communications

should have not more than 4 Pages inclusive of

illustrations.

Title Page:

Title page contains title of the manuscript in bold

face, title case (font size 14), names of the authors

in normal face, title case (font size 12) followed by

the address of authors in normal face, title case

(font size 12). Names of the authors should appear




ii

as initials followed by surnames. Full names may be

given in some instances to avoid confusion.

Followed by the author names, please provide the

complete postal address or addresses with pin code

number of the place(s), where the research work

has been carried out. If the publication originates

from several institutes, the affiliation of each author

should be clearly stated by using superscript Arabic

numbers after the name and before the institute.

The author to whom correspondence should be

directed must be indicated with an asterisk. At the

bottom left corner of this page, please mention

“*Corresponding Author” and provide telephone

number and fax number of the research

institution/college and functional Email address of

the corresponding author to whom all

correspondence (including galley proofs) is to be

sent.

Sections:

Manuscripts should be divided into the following

sections:

Titles (normal face, upper case) and subtitles in

each section (bold face, lower case):

Abstract:

An abstract not exceeding 250 words (for Short

Communications between 60 and 80 words) should

be provided typed on a separate sheet. Abstract

should include aims, methods, results and

conclusion.

Keywords:

Up to 4-6 keywords must be provided in

alphabetical order. These keywords should be typed

at the end of the abstract.

Introduction:

It should be a concise statement of the background

to the work presented, including relevant earlier

work, suitably referenced. It should be started in a

separate page after keywords.

Materials and Methods:

It shall be started as a continuation to introduction

on the same page. All important materials and

equipments, the manufacturer’s name and, if

possible, the location should be provided. The main

methods used shall be briefly described, citing

references. New methods or substantially modified

methods may be described in sufficient detail. The

statistical method and the level of significance

chosen shall be clearly stated.

Results and Discussion:

The important results of the work should be clearly

stated and illustrated where necessary by tables

and figures. The statistical treatment of data and

significance level of the factors should be stated

wherever necessary. The discussion should deals

with the interpretation of results, making the

readers to understanding of the problem taken and

should be logical. The scope of the results, which

need to be further explored, could also be dealt.

Digital files are recommended for highest quality

reproduction and should follow the following

guidelines.

300dpi or higher sized to fit journal page

JPEG, GIF, TIFF and PDF formats are preferred)

Acknowledgement (if any)

Conclusions:

Concisely summarizes the principal conclusions of

the work and highlights the wider implications. This

section should not merely duplicate the abstract.

Acknowledgements:

Acknowledgements as well as information regarding

funding sources may be provided.

References:

Citations of literature within the text must be

presented in numerical order and should be set in

square brackets, thus [1, 12]. The cited literature

are also collected in numerical order at the end of

the manuscript under the heading “References”.

The abbreviated title and the volume number

should appear in italics. Only the papers and books

that have been published or in press may be cited.

Please note that website addresses must not be

included as a reference, but should be inserted in

the text directly after the information to which they

refer.

Please note the following examples:

Journals:

[1]Gregoriadis G., Engineering liposomes for drug

delivery: progress and problems. Trends Biotechnol,

13 (12): 527–537, (1995)

Books:

[1]Joseph R. Robinson and Vincent HL Lee, Ed.

Controlled Drug Delivery Fundamentals and

applications, 2nd Edn, Vol 29, Lippincott Williams’s

publisher:555–561,(1994)

[2] Myers, R.H., Montgomery, D., Response Surface

Methodology, Wiley, New York 1995.


iii

Chapter in a book:

[1] Brown, M.B., Traynor, M.J., Martin, G.P.,

Akomeah, F.K., in: Jain, K.K., Walker, J.M. (Eds.),

Drug Delivery Systems, Humana Press, USA 2008,

pp. 119-140.

For Patent Reference

[1]H. Aviv, D. Friedman, A. Bar-Ilan and M. Vered.

Submicron emulsions as ocular drug delivery

vehicles, U.S. Patent US 5496811, 1996.

Tables:

Should each be typed on a separate page,

numbered in sequence with the body of the text.

Tables should be headed with a short, descriptive

caption. They should be formatted with horizontal

lines only: vertical ruled lines are not required.

Footnotes to tables should be indicated with a), b),

c) etc. and typed on the same page as the table.

Figures:

Should be on separate pages but not inserted within

the text. All figures must be referred to in the text

and numbered with Arabic numerals in the

sequence in which they are cited. Each figure must

be accompanied by a legend explaining in detail the

contents of the figure and are to be typed under the

figures. Graphs and bar graphs should preferably be

prepared using Microsoft Excel and submitted as

Excel graph pasted in Word. Alternatively

photographs can be submitted as JPEG images. Keys

to symbols, abbreviations, arrows, numbers or

letters used in the illustrations should not be

written on the illustration itself but should be

clearly explained in the legend. Avoid inserting a

box with key to symbols, in the figure or below the

figure. All Tables and Figures captions and legends

should be typed on a separate page.

REVIEW ARTICLE

Organization of the review article is at the author’s

discretion and must be at a length of 3000 words

excluding references and abstract. Abstract and key

words are required. Tables, figures, illustrations and

references are to be arranged according to research

papers.

SHORT COMMUNICATIONS

Please add the term “Short Communication” below

the title. A short communication contains a

summary and should not exceed 4 printed journal

pages (Including Tables, Figures and References).

Short communication should contain novel

experimental or theoretical findings in need of

urgent publication.

GALLERY PROOFS Gallery proofs are sent to the designated author

through Email. They must be carefully checked and

returned the revised manuscript within 48 hours of

receipt.

COPYRIGHT Authors are asked to sign a warranty and copyright

agreement upon acceptance of their manuscript,

before the manuscript can be published. The

Copyright form can be downloaded from our

website. Submission of your paper to this journal

implies that the paper is not under submission for

publication elsewhere. Material which has been

previously copyrighted, published, or accepted for

publication will not be considered for publication in

this journal. Submission of a manuscript is

interpreted as a statement of certification that no

part of the manuscript is copyrighted by any other

publisher nor is under review by any other formal

publication. By submitting your manuscript to us,

you agree on IJPBS Publication copyright

guidelines. It is your responsibility to ensure that

your manuscript does not cause any copyright

infringements, defamation, and other problems.

Submitted papers are assumed to contain no

proprietary material unprotected by patent or

patent application; responsibility for technical

content and for protection of proprietary material

rests solely with the author(s) and their

organizations and is not responsibility of the IJPBS

Publications or its Editorial Staff. The main author is

responsible for ensuring that the article has been

seen and approved by all the other authors. It is the

responsibility of the author to obtain all necessary

copyright release permissions for the use of any

copyrighted materials in the manuscript prior to the

submission.

ETHICAL MATTERS:




iv







PUBLICATION POLICY: Papers will normally be published in order of





DECLARATION







publication.

DISCLAIMER “No responsibility is assumed by the publisher” for

any injury and/or damage to persons or property as

a matter of products liability, negligence or

otherwise, or from any use or operation of any

methods, products, instructions or ideas contained

in the material herein. The respective individual

author(s) are responsible for the facts and views

expressed in their articles. “Authors are solely

responsible for their manuscripts and data

published in the journals” and any mistakes in the

published articles. The publisher does not take any

such responsibilities. Copying and reproducing any

parts of the published articles should be duly

acknowledged, otherwise, it will be considered as

an offense as per international Publications law.

CHECKLIST FOR SUBMISSION 1. Have you provided a Title Page?

2. Have you provided an Author Information

section at the end of the paper?

3. Have you provided an Abstract of not more

than 250 words?

4. Have you provide keywords of not more than 3

to 4?

5. Are your Tables denoted by Arabic numerals,


6. Are your Tables submitting at the end of the

text file?

7. Are your Figures denoted by Arabic numerals,


8. Have all your Figures been submitted text file

or PNG or JPG files?

9. Are your References cited in the required style

of the Journal?

10. Have you obtained permission and submitted

documentation for all Personal

Communications cited?

AUTHOR BENEFITS The benefit of publishing in IJPBS includes:

Fast publication times

Free colour in electronic version

Access free on-line issue of IJPBS on

www.ijpbs.com

A rigorous, fast and constructive peer

review process

Excellent editorial standards

Any-Time, Any-Where access

Faster Review

No need for Hard copy submission

Ease of Contacting the IJPBS: 9912185510.

v

MODEL COVER LETTER (TEMPLATE)

International Journal of Pharmacy and Biological Sciences

Date:

Place:

From

(Name and Address of the corresponding author)

To

The Editor

International Journal of Pharmacy and Biological Sciences (IJPBS)

Sir,

Ref: Title ………………….......................................

Type …………………...................................... (Research/Review/Short communication)

Subject …………………...................................... (Pharmaceutical/Biological Sciences)

Branch …………………...................................... (Branch of subject-Refer author instructions)

In reference to the above title, I as a corresponding author, submit the manuscript for

publication in International Journal of Pharmacy and Biological Sciences. I undertake that animal

study (if any) was taken after the prior approval of country/institutional ethical committee. This

manuscript has not been published or considered for publication by any other journal or elsewhere.

Kindly consider the manuscript for publication in your journal.

Thank you

Corresponding author name and Signature

vi

COPY RIGHT FORM

International Journal of Pharmacy and Biological Sciences I certify that I have participated sufficiently in the conception and design of this work

entitled“……………………………………………………………………………………………………….………………………………………

………………………………………………………………………………………………………………………………………………………………

………..” and the analysis of the data, as well as writing of this manuscript, to take public responsibility

for it. I believe the manuscript represents valid work. I have reviewed the final version of manuscript

and approve it for publication. The manuscript is not published elsewhere, in any language, and is not

under simultaneous consideration by any other journal.

All Authors agree that the contents of the manuscript are confidential and will not be

copyrighted, submitted, or published elsewhere (including the Internet), in any language, while

acceptance by the Journal is under consideration.

Furthermore I attest that I shall produce the data upon which the manuscript is based for

examination by the editors or their assignees, if requested.

I hereby assign and transfer to the [email protected], [email protected] copyright and all

rights under it.

I further confirm that this article has not been published elsewhere, nor is it under

consideration by any other publisher.

SIGNATURE AUTHOR/AUTHORS

1. Signature: _____________________________Date: ______________________________

2. Signature: _____________________________Date: _____________________________

3. Signature: _____________________________Date: ______________________________

4. Signature: _____________________________Date: ______________________________

5. Signature: _____________________________Date: ______________________________

Agreed- Yes / No (Put Tick)

After completion of this form, please email the scanned file of original signed form to

[email protected], [email protected] .





vii

SUBSCRIPTION / MEMBERSHIP APPLICATION FORM

Dear Editor-In-Chief, I/We wish to be Annual member/ Subscriber of International Journal of Pharmacy and Biological Sciences and agree to abide by your rules and regulations

1. Name in full

:

2. Date of Birth:

:

3. Academic Qualifications and experience

:

4. Corresponding Address (Delivery Address)

:

5. Phone and E-Mail address : CURRENT SUBSCRIPTION RATES (Online and Print Edition)

Annual Subscription Fees

India Foreign No. Of Issues SELECTION (Put Tick)

Individual Rs. 4000/- $ 90/ €30 04


BANK DETAILS: Payment Options: Mode of Payment: (Put Tick) i) Online: ii) Bank Deposit: iii) Cash:

I. FOR INTERNATIONAL SUBSCRIBERS Pay through PayPal method: http://ijpbs.com/payment.php (Or)

II. FOR INDIAN SUBSCRIBERS

We offer both Online as well as Offline options to make a payment: You can transfer through online Fund Transfer option Or Direct Bank Deposit.

Account Holder Name GANGADI JAYAPAL REDDY

Bank ICICI BANK LTD

Branch HANAMKONDA

A/C No. 020101503760

IFSC / NEFT / RTGS CODE: ICIC0000201

Place: ____________________ Date: _____________________

(Signature of the applicant) NOTE: Kindly send the scanned copy of Pay-in slip of Subscription Fee to email addresses: [email protected], [email protected]

International Journal of

Pharmacy and Biological Sciences P-ISSN: 2321-3272; E-ISSN: 2230-7605

(Indexed by Chemical Abstracts, DOAJ, Index Copernicus) CODEN: IJPBK3 [Chemical Abstract Services (USA)]

#2-2-83/13, Rajajinagar, Naimnagar, Hanamkonda, Warangal-506 001

Mobile: 099121855 Email: [email protected]

http://ijpbs.com/payment.php


Documents

Vol 3 issue 1 2013 Part-2