Chapter 4 MATERIAL AND METHOD 4. MATERIAL AND ...shodhganga.inflibnet.ac.in/bitstream/10603/44229/11/11...4.2 Evaluation of Quality Control Parameters for Raw Material. 1, 2 4.2.1

Chapter 4 MATERIAL AND METHOD

Bhavik Ph. D Dissertation 98

4. MATERIAL AND METHOD

4.1 Collection of Raw Material

‘Bhunimbadi churna’ consists of ingredients; whole plant of Chirayata (Swertia

chirata), seeds of Indrajav (Holarrhena antidysenterica), rhizome of Sunthi (Zingiber

officinale), fruit of Marica (Piper nigrum), fruit of Pippali (Piper longum), rhizome of

Nagarmoth (Cyperus rotundus), rhizome of Katuki (Picrorrhiza kurroa), root of

Chitrak (Plumbago zeylanica) and stem bark of Kada chhal (Holarrhena

antidysenterica). Raw materials of all ingredients were collected from local market of

Vadodara, India. The plant parts were identified and authenticated by Dr. M. S.

Jangid, Botany Department, Sir P. T. Science College, Modasa and voucher

specimens of the same were deposited in the college.

Plant parts were individually dried and homogenized to make fine powder passing

through 85# and packed separately in air tight container to protect from light and

moisture for future uses. Bhunimbadi churna was prepared by mixing ingredients in

following proposition and also packed in air tight container for future uses (Table 4.1)

Table 4.1: Composition of Bhunimbadi churna

Sr.

no

Plant name in Sanskrit Botanical source Part used Quantity (g)

1 Chirayata (Bhunimba) Swertia chirata Whole plant 11.66

2 Indrajav Holarrhena antidysenterica Seed 11.66

3 Sunthi Zingiber officinale Rhizome 11.66

4 Marica Piper nigrum Fruit 11.66

5 Pippali Piper longum Fruit 11.66

6 Nagarmoth Cyperus rotundus Rhizome 11.66

7 Katuki Picrorrhiza kurroa Rhizome 11.66

8 Chitrak Plumbago zeylanica Root 23.32

9. Kada chhal Holarrhena antidysenterica Stem bark 186.56

All the reagents and instruments used in standardization and evaluation of antioxidant

and antidiabetic activity of Bhunimbadi churna were facilitated by Shri B. M. Shah

College of Pharmaceutical Education and Research, Modasa.



4.2 Evaluation of Quality Control Parameters for Raw Material. 1, 2

4.2.1 Organoleptic parameters: Organoleptic parameter like colour, odour and

taste of all powdered raw materials were carried out. These parameters helped

in visual identity of raw materials.

4.2.2 Microscopy study: For microscopic study, 5g of the drug sample was taken,

powdered. The powdered material was taken on an 85 mesh sieve and allowed

in slow running water for washing away the minerals. The materials were

cleared, wash with distilled water and mounted in glycerin, then observed

characters.

4.2.3 Physicochemical parameters

a) Moisture content/Loss on drying: Placed accurately weighed 2-3g prepared

air-dried raw material in a tarred evaporating pan dish, closed the cover gently,

process was started the to heat the sample by halogen light until it was reach a

steady reading. The reading was in percentage moisture content by using

Citizen Moisture analyzer.

b) Determination of extractive matter

i. Alcohol soluble extractive: Air dried 5g coarsely powdered material

was placed in a glass-stopper conical flask and macerated with 100ml

alcohol of specified strength in closed flask for 24hrs. It was shaken

frequently for the first 6hrs and allowed to stand for 18hrs. Filtered

rapidly to minimize the loss of solvent, transferred 25ml filtrate to

tarred flat-bottomed dish and evaporated to dryness on water bath. It

was dried at 105˚C, cooled in desiccators for 30min and weighed

without delay. The percentage of alcohol-soluble extractive was

calculated with reference to air-dried drug.

ii. Water soluble extractive: Air dried 5g coarsely powdered material

was placed in a glass-stopper conical flask and macerated with 100ml

chloroform-water in closed flask for 24hrs. It was shaken frequently

for the first 6hrs and allowed to stand for 18hrs. Filtered rapidly taking

care not to lose any solvent and transferred 25ml filtrate to tarred flat-

bottomed dish and evaporated to dryness on water bath. It was dried at

105˚C, cooled in desiccators for 30 minutes and weighed without



delay. The percentage of water-soluble extractive was calculated with

reference to air-dried drug.

c) Determination of ash

i. Total ash: Accurately weighed 2g air dried raw material and placed in

a previously ignited and tarred platinum crucible. The material was

spread in an even layer and ignited it by gradually increasing the heat to

450ºC in Muffle furnace until it was white, indicating the absence of

carbon. It was cooled in desiccators and weighed. If carbon free ash

cannot be obtained in this manner, the crucible was cooled and residue

was moisten with 2ml water or a saturated solution of ammonium

nitrate. It was dried on a water bath or on a hot plate and ignited to

constant weight. The residue was allowed to cool in desiccators for

30min and weighed without delay. The percentage of total ash was

calculated with reference to air-dried plant material.

ii. Acid insoluble ash: Add 25ml of dil. HCl in the platinum crucible

containing the total ash, covered with a watch glass and boiled gently

for 5min. The watch glass was rinsed with 5ml hot water and added this

liquid to the crucible. The insoluble matter was collected on an ashless

filter-paper (Whatman 541) and washed with hot water until the filtrate

was neutral. The filter paper containing the insoluble matter was

transferred to the original crucible. It was dried on a hot plate and

ignited to for 15min at a temperature not exceeding 450ºC in Muffle

furnace. The residue was allowed to cool in desiccators for 30min, and

weighed without delay. The percentage of acid insoluble ash was


iii. Water soluble ash: Add 25ml water in the crucible containing total

ash, and boiled for 5min. The insoluble matter was collected in a

crucible or on an ashless filter paper and washed with hot water. The

filter paper containing the insoluble matter was transferred to the

original crucible and ignited in a crucible for 15min at a temperature not

exceeding 450ºC in Muffle furnace. The residue was allowed to cool in

desiccators for 30min and weighed without delay. The weight of this



residue was subtracted from the weight of total ash. The percentage of

water soluble ash was calculated with reference to air-dried plant

material.

4.2.4 Determination of pH3: pH meter was calibrated using buffer of 4, 7 and 9

pH. 10g raw material powder was dissolved in 100ml demineralized water and

measured pH. The pH of a solution was determined potentiometrically by

using glass electrode (reference electrode).

4.3 Evaluation of Quality Control Parameters for Churna.3, 4

4.3.1 Description: The general appearance, its visual identity is essential for

consumer acceptance and for control of lot-to-lot uniformity of Churna.

4.3.2 Bulk density: About 50g churna was weighed and filled into graduated

cylinder of densitometer. Initially volume was measured as Initial volume.

Bulk density is calculated using following equation.

4.3.3 Tapped density: About 50g churna was weighed and filled into graduated

cylinder of densitometer. Measure the volume initially as bulk volume. Put the

cylinder on the densitometer and set the parameter for 100 tapping. Measure

the volume and further tapping was done until constant volume was obtained.

Tapped density is calculated using following equation.

4.3.4 Hausner ratio: About 50g churna was weighed and filled into graduated

cylinder of densitometer. Measure the volume initially as bulk volume (Initial

volume). Put the cylinder on the densitometer and set the parameter for 100

tapping. Measure the volume and further tapping was done until constant

volume was obtained. Measure the volume, it was called final volume.

Hausner ratio was calculated using following equation.



4.3.5 Carr’s index: Carr’s index was calculated using following equation.

Flow property of powder using scale of Carr’s index (Compressibility index) and

Hausner ratio is given in Table: 4.2.

Table 4.2: Scale of flow ability

Flow Character Carr’s index Hausner Ratio

Excellent < 10 1.00-1.11

Good 11-15 1.12-1.18

Fair 16-20 1.19-1.25

Passable 21-25 1.26-1.34

Poor 26-31 1.35-1.45

Very poor 32-37 1.46-1.59

Very very poor >38 >1.60

4.3.6 Angle of repose: The fixed funnel and the free standing cone method was

employed. A funnel secured with its tip at 2.5cm (H) height, taken above the

graph paper that was placed on flat horizontal surface. Churna was carefully

poured through the funnel until the apex of the conical pile just touched the tip

of the funnel. Angle of repose is calculated using following equation.

Where; α = Angle of repose, H = Height of pile of powder, R= Redius of the conical pile.

Flow properties and corresponding Angles of Repose is given in Table 4.3.

Table 4.3: Flow properties and related angles of repose

Flow property Angle of Repose (º)

Excellent 25-30

Good 31-35

Fair-aid not needed 36-40

Passable-may hang up 41-45

Poor-must agitate, vibrate 46-55

Very poor 56-65

Very very poor >66



4.3.7 Determination of pH: About 10g churna was dissolved in 100ml

demineralized water and pH was measured. The pH of a solution was

determined potentiometrically by using glass electrode as a reference

electrode.

4.3.8 Determination of moisture content: 2g churna placed in a tarred evaporating

pan dish, closed the cover gently start the process to heat the sample by

halogen light until it was reach a steady reading. The reading was given in

percentage moisture content by using Citizen Moisture analyzer.

4.3.9 Determination of extractive matter.

i. Alcohol soluble extractive: Accurately weighed 5g coarsely powdered of

churna was placed in a glass-stopper conical flask and macerated with 100ml

of the alcohol in closed flask for 24hrs. It was shaken frequently for the first

6hrs and allowed to stand for 18hrs. Filtered rapidly taking care not to lose any

solvent and transferred 25ml filtrate to tarred flat-bottomed dish and

evaporated to dryness on water bath. It was dried at 105˚C, cooled in a

desiccator for 30min and weighed without delay. The percentage of alcohol-

soluble extractive was calculated with reference to air-dried drug.

ii. Water soluble extractive: Accurately weighed 5g coarsely powdered of


Chloroform-water in closed flask for 24hrs. It was shaken frequently for the

first 6hrs and allowed to stand for 18hrs. Filtered rapidly taking care not to

lose any solvent and then transferred 25ml filtrate to tarred flat-bottomed dish

and evaporated to dryness on water bath. It was dried at 105˚C, cooled in a

desiccator for 30min and weighed without delay. The percentage of water


iii. Methanol soluble extractive: Accurately weighed 5g coarsely powdered


of the methanol in closed flask for 24hrs. It was shaken frequently for the first

6hrs and allowed to stand for 18hrs. Filtered rapidly taking care not to lose any

solvent and transferred 25ml filtrate to tarred flat-bottomed dish and


desiccator for 30min and weighed without delay. The percentage of methanol




iv. Chloroform soluble extractive: Accurately weighed 5g coarsely powdered


of the chloroform in closed flask for 24hrs. It was shaken frequently for the

first 6hrs and allowed to stand for 18hrs. Filtered rapidly taking care not to

lose any solvent and transferred 25ml filtrate to tarred flat-bottomed dish and


desiccator for 30min and weighed without delay. The percentage of

chloroform-soluble extractive was calculated with reference to air-dried drug.

4.3.10 Determination of ash.

i. Total ash: Accurately weighed 2g churna was placed in a previously ignited

and tarred platinum crucible. The material was spread in an even layer and

ignited it by gradually increasing the heat to 450ºC until it was white,

indicating the absence of carbon. It was cooled in desiccator and weighed. If

carbon free ash cannot be obtained in this manner, the crucible was cooled and

residue was moisten with 2ml of water or a saturated solution of ammonium

nitrate. It was dried on a water bath or on a hot plate and ignited to constant

weight. The residue was allowed to cool in desiccator for 30min, and it was

weighed without delay. The percentage of total ash was calculated with

reference to air-dried plant material.

ii. Acid insoluble ash: In the platinum crucible containing the total ash, 25ml dil.

HCl was added and covered with a watch glass and boiled gently for 5min.

The watch glass was rinsed with 5ml hot water and added this liquid to the

crucible. The insoluble matter was collected on an ashless filter-paper

(Whatman 541) and washed with hot water until the filtrate was neutral. The

filter paper containing the insoluble matter was transferred to the original

crucible. It was dried on a hot plate and ignited to constant weight. The residue

was allowed to cool in desiccator for 30min, and it was weighed without

delay. The percentage of acid insoluble ash was calculated with reference to

air-dried plant material.

iii. Water soluble ash: In the crucible containing the total ash, 25ml water was

added and boiled for 5min. The insoluble matter was collected in a platinum



crucible or on an ashless filter paper (Whatman541) and washed with hot

water. The filter paper containing the insoluble matter was transferred to the

original crucible and ignited in a crucible for 15min at a temperature not

exceeding 450ºC. The residue was allowed to cool in desiccator for 30min,

and it was weighed without delay. The weight of this residue was subtracted

from the weight of total ash. The percentage of water soluble ash was


4.4 Qualitative Phytochemical Screening 5,6,7

: Qualitative chemical tests for

identifying various phyto-constituents present were carried out for powdered raw

material and also churna as follows.

1) Tests for Alkaloids

i. Dragandroff’s Test (Potassium bismuth iodide solution): Few drops

of Dragendroff’s reagent was added to sample solution, produced

reddish brown precipitate indicated presence of alkaloids.

ii. Mayer’s Test (Potassium mercuric iodide solution): Few drops of

Mayer’s reagent was added to sample solution, produced creamy white

precipitate indicated presence of alkaloids.

iii. Wagner’s Test (Solution of Iodine in Potassium Iodide): Few drops of

Wagner’s reagent was added to sample solution, produced reddish

brown precipitate indicated presence of alkaloids.

2) Tests for Glycosides

i. General Test: Extracted 200mg drug using 5ml 10% H2SO4 and boil

on water bath. Add equal volume of water to the volume of NaOH.

Add 0.1ml Fehling’s A (Copper sulphate in distilled water) and

Fehling’s B (Potassium tartarate and sodium hydroxide in distilled

water) until alkaline (red litmus changes to blue) and heat on water

bath for two minutes. Above solution was added. The quantity of the

red precipitate formed represents the glycoside after acid hydrolysis.

ii. Foam Test: The drug extracted or dry powder had shaken vigorously

with water and observed persistent frothing.



3) Tests for Flavonoids

i. Shinoda Test: (Magnesium Hydrochloride reduction test): To the

sample solution add few fragments of magnesium ribbon and

concentrated HCl drop wise, pink scarlet or crimson red color appears

after few minutes indicated presence of flavonoids.

ii. Lead Acetate Test: To small quantity of sample, add lead acetate

solution and yellow colored precipitate is formed indicated presence of

flavonoids

4) Test for Tannins

i. FeCl3 Test: The water extract of the sample drug was treated with

alcoholic FeCl3. Deep blue-green or deep blue-black color indicated the

presence of tannins.

ii. Gelatin Solution Test: The water extract of the sample drug was

treated with gelatin solution. White precipitates indicated the presence

of tannins.

5) Tests for Sterols and Triterpenoids

i. Libermann-Burchard Test: Extract was treated with few drops of

acetic anhydride, boiled and cooled, H2SO4 was added from the side of

the test tube, A brown ring at the junction of two layers and the upper

layer turns green indicated the presence of sterols and formation of

deep red color indicated the presence of triterpenoids.

ii. Salkowski’s Test: Chloroform extract was treated with few drops of

H2SO4, shake well and allow to stand for some time, red color

appeared in the lower layer indicated the presence of sterols and

formation of yellow colored lower layer indicated the presence of

triterpenoids.

6) Test for Phenolic Compound

i. FeCl3 Test: Water extract of the sample drug was treated with

alcoholic FeCl3. Blue-green color indicated the presence of phenolic

compound.



ii. Acetic Acid Solution Test: Water extract of the sample drug was

treated with acetic acid solution. Red color indicated the presence of

phenolic compound.

7) Test for Carbohydrates.

i. Molish Test: The extract solution was treated with few drops of

alcoholic -napthol. Add 0.2ml H2SO4 slowly through the sides of the

test tube, purple to violet color ring appeared at the junction indicated

the presence of carbohydrates.

ii. Fehling’s Test: Equal volume of Fehling’s A (Copper sulphate in

distilled water) and Fehling’s B (Potassium tartarate and Sodium

hydroxide in distilled water) reagents were mixed and boiled. Few

drops of extract solution were added and heated a brick red precipitate

of cuprous oxide form, if reducing sugars present.

8) Test for Proteins and Amino acids.

i. Millon’s Test: Extract solution and 2ml Millon’s reagent (Mercuric

nitrate in nitric acid containing traces of nitrous acid) white precipitate

appeared, which turns red upon gentle heating indicated the presence of

proteins and amino acids.

ii. Ninhydrin Test: Extract solution when boiled with 0.2% solution of

Ninhydrin (Indane 1, 2, 3 trione hydrate) produced violet color

indicated the presence of proteins and amino acids.

4.5 Thin Layer Chromatography of Raw Material and Churna With

Laboratory Standard. 8,9

4.5.1 TLC analysis

i. Selection of plate and adsorbent: Pre-coated aluminum plates with

Silica gel 60F254 (Merck, Germany) of 5 x 10cm and 0.2mm thickness,

were used for the detection. The plates were pre-washed by methanol

and activated at 60°C for 5min prior to chromatography.

ii. Sample solution: Extract 4g Churna in 75ml methanol under reflux on

a water-bath for 30min, filter and concentrate to 10ml and carried out

the thin layer chromatography. Preparation of raw material solution



was same method for TLC.

iii. Application of sample: The sample to be separated was applied by

micro capillary prepared by heating and drawing out a melting point

capillary, as a small band (6-8mm broad) of 8-10μl solution/extract

about 1cm from the end of the plate opposite.

iv. Development: The plate was developed in CAMAG glass twin-

through chamber (10-10cm) previously saturated with the solvent for

60min (Temperature 25±2°C, Relative humidity 40%). TLC plate was

developed for distance of 8cm.

v. Visualization: After development of TLC, allow the plate to dry in air

and examine under UV light at 254nm and 366nm, reagent was

sprayed followed by heating at 1100C for about 10min and observe

under visible light.

The mobile phase or solvent system and spraying reagent for all the standards, raw

material as well as churna is given in Table 4.4.

Table 4.4: Mobile phase and spraying reagent for ingredients of Bhunimbadi churna

Sr. no. Sample Solvent System Spraying Reagent

1 S. chirata Toluene : Ethyl acetate : Formic

acid (5:4:1)10

ASR

2 H. antidysenterica

(seed)

Toluene: Ethyl acetate: Diethyl

ether (70: 20: 10)11

Dragendorff reagent

3 Z. officinale Toluene : Ethyl acetate (93:7)11

VSR

4 P. nigrum Toluene : Ethyl acetate (7:3)11

VSR

5 P. longum Toluene : Ethyl acetate (90:10)10

VSR

6 C. rotundus Toluene : Ethyl acetate (9:1)12

VSR

7 P. kurroa Chloroform: Methanol (95:5)13

ASR

8 P. zeylanica Toluene : Ethyl acetate (3:1)10

ASR

9 H. antidysenterica

(stem bark)

Toluene: Ethyl acetate: Diethyl

ether (70: 20: 10)11

Dragendorff reagent

ASR= Anisaldehyde Sulphuric acid reagent VSR = Vanillin Sulphuric acid reagent



4.6 HPTLC of Raw Material and Churna with Marker Compound.14,15

High Performance Thin Layer Chromatography (HPTLC), the simplest separation

technique, gives better precision and accuracy with extreme flexibility for various

steps (stationary phase, mobile phase, development technique and detection). HPTLC

technique is useful to identify and to check the purity of raw herbal extracts as well as

finished product. Hence forth it is very useful tool in standardizing process of raw

herbal and finished products. HPTLC was carried out using a Camag microlitre

Hemilton syringe 100μl HPTLC syringe, Band wise with Camag Automatic TLC

Sampler-4 automatic spotting device, Camag twin trough chamber, Camag TLC

Scanner-3, winCATS integration software, pre-coated aluminum sheet with Silica Gel

60F254 (Merck, Germany), 0.2mm thickness.

4.6.1 Steps involved in HPTLC analysis

A. Selection of plate and adsorbent: Pre-coated aluminum plates with Silica

Gel 60F254 (Merck, Germany) of 20x10cm and 0.2mm thickness, were used

for the detection. The plates were pre-washed by methanol and activated at

60°C for 5min prior to chromatography.

B. Sample and standard solution: The sample was prepared by extracting 1g

coarsely powdered drug with 100ml methanol for 24hrs by cold extraction

method. The extracts were filtered by Whatmann542 filter paper to prepare

10mg/ml solutions. For preparation standard, 10mg standard mangiferine, 6-

gingerol, piperin and conessine were dissolved in 10ml of methanol and make

up to 10ml in standard volumetric flask to prepare stock solution (1mg/ ml).

C. Application of sample: Sample application is the most critical step for

obtaining good resolution for quantification in HPTLC. The automatic

application devices are preferable. Most recent automatic device ‘CAMAG

Automatic TLC Sampler-4’ spotting device was used to apply a band of 6mm

width with 0.01-0.5µg/µl concentration of standard solution.

D. Development: The plate was developed in CAMAG glass twin-through

chamber (20-10cm) previously saturated with the solvent for 60min

(Temperature 25±2°C, Relative humidity 40%). The development distance

was 8cm. Subsequently scanning was done. The mobile phase or solvent

system for all the standards and churna, which is given in the Table 4.5



Table 4.5: Solvent system for marker compound, plants and Bhunimbadi churna.

Sr.

no.

Sample Marker

compound

Solvent System

1 S. chirata Mangiferine Ethyl Acetate: Methanol: Water:

Formic Acid (10:1:1:0.5)16,17

3 Z. officinale 6-gingerol n-hexane: Ethyl acetate (50:50)18

4 P. nigrum and

P. longum

Piperine Toluene: Ethyl acetate: Formic acid

(3:1:0.1)19

5 H. antidysentrica Conessine Toluene: Ethyl acetate: Diethyl amine

(7.0:2.0:1)20

6 Bhunimbadi

churna

-------------- Toluene : Ethyl acetate : Formic acid

(5 : 1.5 :0.5)21

E. Detection: The plate was scanned at UV 366nm and 254nm using CAMAG

TLC Scanner-3. Rf value and peak area of separated compounds were

recorded.

4.7 Heavy Metal Analysis

A. Preparation of samples: Accurately weighed 0.5g churna was taken in

digestion vessels and nitric acid was added in fuming hood. Keep it for pre-

digestion till devoid of fume or gases for 5-10min. All vessels were arranged

in turnable disc for digestion using Microwave accelerated reaction system

(MARS) by CEM corporation instrument. After digestion open the vessels by

uncapping safely to release gas. Filter the contents of vessels by using

Whatman filter paper in a 25ml volumetric flask and the volume was made-up

with double distilled water using double distilled water as a blank. The

standards of Lead (Pb), cadmium (Cd), arsenic (As) and mercury (Hg) were

prepared as per the protocol in the manual and the calibration curve was

prepared for each of them.

B. Detection: Samples were analyzed for the presence of Pb, Cd, As and Hg

using Labindia Inductively coupled plasma-optical emission spectroscopy

(ICP-OES).22



4.8 Microbial Analysis.1, 2

4.8.1 Total viable aerobic count

A. Preparation of sample: Dissolved 10g churna being examined in buffered

sodium chloride peptone solution pH 7.0 and adjust volume 100ml with same

medium.

B. Examination of sample: Total viable aerobic count in the sample was

examined by using the plate count method by Digital colony counter by

Chemiline.

C. For bacteria: Petri dishes of 10cm diameter were used; 1ml pretreated

mixture was added to each dish and 15ml liquefied casein soyabean digest

agar at a temperature not more than 45˚C. Two Petri dishes for each sample

were prepared using the same dilution and incubated at 30- 40˚C for 3days.

The number of colonies form was calculated using the digital colony counter.

Results were calculated using plate with the greatest number of colonies but

taking 300 colonies per plate as the maximum consistent with good evaluation.

4.8.2 Total yeast and mould count: Procedure was used as described in the test for

bacteria using Chloramphenicol yeast glucose agar (CYGA) in place of casein

soyabean digest agar and incubate the plates at 20-25˚C for 5days, unless a more

reliable count was obtained in shorter time. Results were calculated using the plates

with not more than 100 colonies by using colony counter.

4.8.3 Test for Escherichia coli

Dissolved 10g churna in 90ml fluid casein digest-soya lecithin-Polysorbate 20

medium to get 1:10 dilution. 10ml mixture was added in 50ml nutrient broth in sterile

screw-capped container, shake, allowed to stand for 1hr and shake again and

incubated at 35-37˚C for 24hrs. After incubation, 1ml was added to 5ml of Mac-

Conkey broth and incubated at 36-38˚C for 48hrs. If the contents of the tube show

acid and gas indicated the possible presence of E. coli. Alternative test of an

inoculating loop, Streak a portion from the enrichment culture (obtained in the

previous test) on the surface of Mac-Conkey agar medium. Covered and invert the

dishes and incubated at 45˚C for 24hrs. Growth of red, generally non-mucoid colonies

of Gram negative rods, sometimes surrounded by a reddish zone of precipitation,

indicated the presence of E. coli.



4.8.4 Test for Salmonella species

Dissolved 10g churna in 90ml fluid casein digest-soya lecithin-Polysorbate 20

medium (1:10 Dilution). 10ml of mixture was added in 100ml nutrient broth in sterile

screw-capped container, shake, allowed to stand for 4hrs and shake again and

incubated at 35-37˚C for 24hrs. After incubation, 1ml enrichment culture to each of

the two tubes containing (A) 10ml selenite F broth and (B) tetrathionate bile brilliant

green broth and incubated at 36-38˚C for 48hrs. From each, streak looped on Bismuth

sulphate agar and Xylose lysine deoxycholate agar media and incubated the plates at

36˚-38˚C for 24hrs. Well developed, black or green in Bismuth sulphate agar and red

with or without black centers colonies in Xylose lysine deoxycholate agar indicated

the presence of Salmonella species.

4.9 Evaluation of In-vitro Antioxidant Activity

A. Instruments: UV spectrophotometer (Shimadzu-UV-1601), Centrifuge

Machine (Eltek-research centrifuge-TC-4100D).

B. Chemicals: All chemicals used for the study were purchased from SD-fine

chemicals; India and all other reagent used were of analytical grade.

C. Preparation of stock solution of churna: 10mg churna was dissolved in

10ml methanol to get 1000µg/ml solution. From the stock solution different

concentrations were prepared in methanol and used for antioxidant studies.

D. Preparation of standard stock solution of ascorbic acid: Ascorbic acid used

as standard for the study and its stock solution was prepared in the

concentration of 1mg/ml in methanol. It was prepared freshly and used

immediately for the study. Different concentration eg.10, 20, 40, 60, 80,

100μg/ml were prepared in methanol from the stock solution and used for

antioxidant studies.

E. Statistical analysis: Experimental data were analyzed using Excel (Microsoft

Inc.) and SPSS version 17.0 software. Significant differences between samples

were analyzed using analysis of variance (ANOVA) and Dunnett using

GraphPad as Instat 3 software multiple-range test (P< 0.05). Data obtained

were reported mean ± standard deviation.

4.9.1 DPPH radical scavenging activity 23,24,25,26

A. Chemicals: α- α diphenyl β picryl hydrazyl (DPPH) and methanol.



B. Principle: The antioxidant reacts with stable free radical, DPPH and converts

it to 1, 1- Diphenyl-2-Picryl Hydrazine. The free radical scavenging activity of

the product extract, based on the scavenging activity of the stable (DPPH) free

radical was determined.

C. Procedure: Churna extract (0.1 ml) and different concentrations viz. 10, 20,

40, 60, 80, 100μg/ml of standard ascorbic acid solution was added to 3ml

0.004% DPPH solution in methanol. An equal amount of methanol and DPPH

served as control. After 30min incubation in the dark, absorbance was

recorded at 517nm, and the percentage inhibition activity was calculated using

following equation.

% Inhibition of antioxidant activity = [(A0-A1)/A0] ×100,

Where;

A0 is the absorbance of the control, and

A1 is the absorbance of the extract/standard.

The antioxidant activity of the extract was expressed as IC50. The IC50 value was

defined as the concentration (μg/ml) of extracts that inhibits the formation of DPPH

radicals by 50%. All the tests were performed in triplicate and the graph was plotted

with the average of three observations.

4.9.2 Scavenging of hydrogen peroxide 27

A. Chemicals: Hydrogen peroxide and phosphate buffer saline.

B. Principle: The ability of extract of churna to scavenge hydrogen peroxide was

determined.

C. Procedure: 20mM H2O2 solution was prepared in phosphate buffer saline (pH

7.4). Different concentrations of churna extract and 10, 20, 40, 60, 80,

100μg/ml standard ascorbic acid in methanol solution (1ml) were added to 2ml

H2O2 solution. Absorbance of H2O2 at 230nm was determined after 10min



against a blank solution containing phosphate buffer without H2O2. Separate

blank sample was used for back ground subtraction for each concentration.

The percentage inhibition activity was calculated using following equation.

% Inhibition of antioxidant activity = [(A0-A1)/A0] ×100,

Where;

A0 is the absorbance of the control, and

A1 is the absorbance of the extract/standard.

The antioxidant activity of the extract was expressed as IC50. All the tests were

performed in triplicate and the graph was plotted with the average of three

observations.

4.9.3 Ferric reducing power 33

A. Chemicals: Potassium ferricyanide [K3Fe(CN)6, 98.0% purity], ferric chloride

(FeCl3.6H2O, 97.0%purity), ascorbic acid 98.3%purity, potassium hydrogen

phosphate, sodium hydroxide, trichloro acetic acid.

B. Principle: Ability to produce reduction of functional groups by extract of the

product was determined on the basis of formation of coloured complex with

potassium ferricyanide, tricholoroacetic acid and ferric chloride.

C. Procedure: Different concentrations of churna extract and 10, 20, 40, 60, 80,

100μg/ml standard ascorbic acid solution in methanol (1ml) were mixed with

2.5ml, 0.2M pH 6.6 phosphate buffer and 2.5ml, 1% potassium ferricyanide.

The mixture was incubated at 50ºC for 20min. A portion 2.5ml, 10%

tricholoroacetic acid was added to the mixture, which was then centrifuged at

3000rpm for 10min at room temperature. The upper layer of 2.5ml solution

was mixed with 2.5ml distilled water and 0.5ml, 0.1% ferric chloride and the

absorbance of the reaction mixture indicated increased reducing power. The

absorbance was measured at 700nm. All the tests were performed in triplicate

and the graph was plotted with the average of three observations.

4.10 Antidiabetic Activity by Alloxan Induced Diabetic Rat Model.

4.10.1 Preparation of drug sample: 5g churna was suspended into 100ml, 5%

Tween 80 solution. Dose of churna was calculated on the basis of LD5028,29



4.10.2 Preparation of standard: Gibenclamide tablet (3x5mg) was used as a

standard drug, purchased from local market, made into fine powder with mortar pestle

and dissolved in 30ml water to make 500µg/ml Glibenclamide solution30

.

4.10.3 Animals: Healthy Wistar albino rats of either sex weighing about 150-200gm

were used for the study. The animals were housed in a two rat per polypropylene

cages, maintained under standard conditions of temperature (25±3⁰C), humidity

(30%-70%) and 12:12hr light: dark cycle. Animals were fed free access to standard

pellet diet (Pranav Agro Industries Ltd.) and purified drinking water ad libitum.

Protocols of present study were approved by the Institutional Animal Ethical

Committee (IAEC) of BMCPER, Modasa with item number (IAEC/BMCPER/02/13-

14) constituted after the permission from Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and

Empowerment, Government of India. (Reg. No. 194/CPCSEA 1st June 2001).

4.10.4 Acute toxicity of churna: There is a spectrum of toxicity tests using whole

animals, which evaluate chemical, hazards ranging from carcinogenicity to teratology

and reproduction studies, as well as mutagenicity, neurotoxicity and others. The

studies can be loosely classified as acute, sub acute, sub-chronic or chronic toxicity

tests. The LD50 or acute lethality test is generally performed in rats or rabbits and uses

the doses of chemical at which one-half (50%) test animals can be expected to die.

New approaches to the classic LD50 are currently in practice that incorporates

information from preliminary in-vitro screening tests. The modified LD50 known as

range limit study, uses 6-10 animals instead of 80-100, as was now generally used

only to check the solvency of highly toxic chemicals, such as when screening for

potential chemotherapeutic agents or determining the effective strength of

pesticides.31

Results of LD50 tests help to determine safe dose out of the various doses

given and lethal dose when more than 10% of the animals died.

Nulliparous and non-pregnant 2month old female rats were used for the toxicity

studies. The animals were marked for individual identification and kept in their cages

for at least 3days prior to dosing. The acute oral toxicity study and LD50 of churna

was evaluated by the methodology described in the OECD (Organization for

Economic Co-operation and Development) guideline (Revised Draft Guideline 425)32

for the testing of chemicals.



Animals were fasted for 4hr prior to dosing and dose of each animal was determined

and calculated according to its fasted body weight. The first test animal survived,

then, four other animals were dosed sequentially; therefore, a total five animals were

tested. Animals were observed individually during the first 30minute after dosing,

every 4hr during the first 12hr and thereafter for 3 days. They were fed orally with the

churna, prepared in 5% v/v Tween 80 solution was administered in increasing dose

levels of 100-2000mg/kg body weight.

The animals were observed continuously for 2hr for the following:33

A. Behavioral profile: Alertness, restlessness, irritability and fearfulness.

B. Neurological profile: Spontaneous activity, reactivity, touches response, pain

response and gait.

C. Autonomic profile: Defecation and urination.

The numbers of deaths, if any were recorded after 72hr. LD50 is greater than

2000mg/kg if no animal died.

4.10.5 Induction of diabetes in rats: Alloxan is widely used as an agent for the

development of experimental diabetes to induce selective dysfunctioning of

pancreatic β-cells34

. In vitro studies have shown that Alloxan is selectively toxic to

pancreatic β -cells, leading to the induction of cell necrosis. The cytotoxic action of

alloxan is mediated by reactive oxygen species, with a simultaneous massive increase

in cytosolic calcium concentration, leading to a rapid destruction of β-cells35

.

Wistar albino rats of either sex were used. Normal control group (Group I) was fed

with standard laboratory diet (SLD) and water ad libitum. Rests of the rats were fasted

18hrs and were given a single dose 100mg/kg, b.w, i.p of alloxan monohydrate

(Sigma Ltd., USA) dissolved in 0.9% saline. Since Alloxan could evoke fatal

hypoglycemia as a result of massive insulin release, rats received 20% of glucose

solution for first 6hr then simple tape water was given. The rats were kept for next

24hr with free access of 5% glucose solution to prevent hypoglycemia. After a 48hrs,

Alloxan monohydrate treated rats were tested for the evidence of diabetes by

estimating their blood glucose level by using Accu-Chek Active® blood glucose

monitor and Accu-chek active strips, (Mfg By-Roche). Blood sample was collected

by puncturing retro-orbital plexus through sterile glass capillary.



The rats with sugar level more than 250mg/dl were selected for the experiment.

Animals were maintained for two days in diabetic condition for well establishment of

diabetes. Diabetic control group (Group II) was fed with Standard Laboratory Diet

(SLD) and water ad libitum. Reference standard group (Group III) was treated with

5mg/kg b.w., p.o Glibenclamide in water daily for 14 days. Bhunimbadi churna

treated groups (Group IV) and (Group V) respectively were received dose of

200mg/kg b.w. and 400mg/kg b.w daily for 14 days. Animals were fasted 8hrs prior

to oral dosing, the animals were weighed and the test substances were administered in

a single dose by using the oral cannula. After the dose was administered, food was

withheld in rats. The body weight of experimental animals was regularly monitored

during the experimental period. 36,37

4.10.6 Treatment protocol for the study of antidiabetic activity

The experimental animals were divided into five groups, six animals in each group as

follow.

Group I (Normal control): Animals received only the vehicle (Tap water).

Group II (Diabetic control): Untreated diabetes induced animals.

Group III (Reference Standard): Diabetic induced animals treated with 5mg/kg b.w.,

p.o/day Glibenclamid.

Group IV (Bhunimbadi churna treated): Diabetic induced animals treated with

200mg/kg b.w., p.o/day Bhunimbadi churna.

Group V (Bhunimbadi churna treated): Diabetic induced animals treated with 400

mg/kg b.w., p.o/day Bhunimbadi churna.

All animals except group I were treated with 100mg/kg Alloxan monohydrate to

induced diabetics.

4.10.7 Blood sample collection and analysis

Blood samples were collected from the retro-orbital plexus of 8hr fasted and

anesthetized animals by slight exposure to ether on 1st, 7

th and 14

th days. One drop

was poured on strip and rest of blood was kept aside for 30min for clotting. By

centrifuging the same sample at 6000rpm for 20min, the serum was separated and was

analyzed for biochemical parameters. Blood glucose level was measured in all groups

by GOD-POD method (Glucose oxidase-peroxidase method) using Accu-Chek



Active® blood glucose monitor & Accu-chek active strips, (Mfg By-Roche) in the unit

of mg/dl.

Diabetes mellitus is characterized by hyperglycemia together with biochemical

alterations of glucose and lipid metabolism. Experimental diabetes in animals has

provided considerable insight into physiological and biochemical derangement of the

diabetic state. Insulin deficiency leads to various metabolic aberrations in the animals,

viz. increased blood glucose, decreased protein and albumin content, increased

cholesterol and triglycerides levels, increase in alkaline phosphatase, acid

phosphatase, alanine transaminase and aspartate transaminase activities38

.

Considering these factors the effect of Bhunimbadi churna on biochemical parameters

like; serum total cholesterol by CHOD-PAP method39

, serum triglycerides by GPO

method 40

, serum HDL, serum VLDL, serum LDL, serum total protein and albumin

proteins by Biuret method 41

, serum creatinine and urea in Alloxan induced diabetic

rats were estimated using Erba Transasia auto-analyser in pathology laboratory.

4.10.8 Statistical analysis

Results are presented as Mean ± SEM of six animals. Statistical differences between

the means of the various groups were evaluated using one-way analysis of variance

(ANOVA) followed by Dunnett’s test using GraphPad Instat3 software. The

significance difference if any among the groups at p<0.05 was considered statistically

significant.



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