ww.sciencedirect.com

d r u g i n v e n t i o n t o d a y 5 ( 2 0 1 3 ) 3 2e3 8

Available online at w

journal homepage: www.elsevier .com/locate/di t

Original Article

Nanosuspension of vasicine from Adhatoda vasica: Isolationand characterization

Jitender Kumar Malik a,*, Akshat Sharma a, Sanjiv Singh b, Sourabh Jain c

a Lakshmi Narain College of Pharmacy, Bhopal 462021, M.P., IndiabTruba College of Pharmacy, Bhopal 462021, M.P., IndiacNRI Institute of Pharmaceutical Sciences, Bhopal 462021, M.P., India

a r t i c l e i n f o

Article history:

Received 27 December 2012

Accepted 12 March 2013

Keywords:

Nanosuspension

Vasicine

Nanoparticles

Nanosized particles

* Corresponding author. Tel.: þ91 9893864455E-mail address: Jitender_malik@hotmail.c

0975-7619/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.dit.2013.03.005

a b s t r a c t

Objective: Vasicine the major alkaloid found in Adhatoda vasica shows better potent bron-

chodilation response compared to theophylline, despite extensive research, this com-

pound not yet been approved or used as a single therapeutic molecule. The major problem

associated with vasicine is its low bioavailability and stability hence no single formulation

of vasicine alone is available in market.

Method: The present study on isolation of vasicine from A. vasica and characterize by

various phytochemical test the isolated vasicine is an attempt for is better formulation

which may be useful as a novel drug delivery system. In the present study, vasicine

nanosuspension was prepared by the solvent/anti-solvent method and stabilized by a

surface active agent.

Result & discussion: The average particle size of the obtained nanoparticles, as estimated by

field emission scanning electron microscope (FESEM) micrographs, is observed to be about

60e80 nm and the particle had spherical morphology. Particle size analysis results shows

that about 50% of particle in nanosuspension were found to be in the range of 8e10 nm and

while the other half is in the range of 210e230 nm. The average particle size distribution is

found to be w115 nm and zeta potential is �6.91 mV.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction Natural products like Quinine, Theophylline, Penicillin G,

Their effectiveness, easy availability, low cost and compara-

tively being devoid of serious toxic effects (time tested) are the

main causes of popularity of herbal medicines. A herbal

remedy is triumph of popular therapeutic diversity.1 Nature

always stands as a golden mark to exemplify the outstanding

phenomenon of symbiosis. Nature has provided the complete

storehouse of remedies to cure all ailments of mankind.2

.om (J.K. Malik).2013, JPR Solutions; Publi

Morphine, Digitoxin, Vincristine, Cyclosporin and Vitamin A,

which are the cornerstones of modern pharmaceutical care,

are all natural products derived from plants. The use of nat-

ural substances, particularly plants, to control diseases is

centuries-old practice that has led to the discovery of more

than half of all “Modern” pharmaceuticals. Documentation on

the use of natural substances for medicinal purposes can be

found as back as 78 A.D. when Dioscorides wrote “De Materia

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Total ash value of sample % ¼ (Z�X) � 100/Y

Z¼weight of the dishþ ash (after complete incineration)

X ¼ weight of empty dish, Y ¼ weight of drug taken

Acid insoluble ash value of the sample % ¼ (A/B) � 100

A ¼ water soluble ash, B ¼ air dried drug

d ru g i n v e n t i o n t od a y 5 ( 2 0 1 3 ) 3 2e3 8 33

Medica” describing many medicinal plants that remain

important in modern medicine, not because they are

continued to be used as crude drug preparations, but because

they serve as the important source of pure chemicals that

have become mainstay of modern therapy. The positive

benefit of extract of two species ofDigitalis purpurea (Fox glove)

and Digitalis lanata were recognized long before the active

constituents were isolated and characterized structurally. The

cardiac glycosides, which include Digoxin, Digitoxin and

Deslanoside, exert a powerful and selective positive inotropic

action on the cardiac muscles. The synthetic local anesthetics

such as Lidocaine, Benzocaine were synthesized to mimic the

nerve blocking activity of cocaine, a natural alkaloid.3

Herbal medicines are the oldest form of healthcare known

to mankind. Herbs had been used by all cultures throughout

history. It was an integral part of the development of modern

civilization. Primitiveman observed and appreciated the great

diversity of plants available to him. The plants provided food,

clothing, shelter, and medicine. Much of the medicinal use of

plants seems to have been developed through observations of

wild animals, and by trial and error. As time passed, each tribe

added the medicinal power of local herbs to its knowledge-

base. Many drugs commonly used today are of herbal origin.

Indeed, about 25 percent of the prescription drugs dispensed

in the United States contain at least one active ingredient

derived from plant material. Some are made from plant ex-

tracts; others are synthesized to mimic a natural plant com-

pound.4 Nanosuspension is a submicron colloidal dispersion

of drug particles which is stabilized by surface active agents. A

pharmaceutical nanosuspension is defined as very finely

dispersed solid drug particles in an aqueous vehicle meant for

either oral and topical use or parenteral and pulmonary

administration. The particle size distribution of the solid

particles in nanosuspensions is usually less than one micron

with an average particle size ranging between 200 and 600 nm.

Nanoparticles are polymeric colloidal carriers of drugs while,

nanosuspension is a drug maintained in the required crys-

talline state with reduced particle size, leading to an increased

dissolution rate and therefore improved bioavailability. The

increase in dissolution rate of nanosized particles (particle

size 1e100 nm) is related to an increase in the surface area and

consequently the dissolution velocity.5 Nanosuspension in-

creases the surface area and concentration gradient which

lead to a much more pronounced increase in the dissolution

velocity as compared to a micronized product. Furthermore,

the saturation solubility is increased as well. Another possible

explanation for the nanosuspensions with increased satura-

tion solubility is the creation of high energy surfaces when

disrupting the more or less ideal drug microcrystal to nano-

particles. Dissolution experiments can be performed to

quantify the increase in the saturation solubility of a drug

when formulated into a nanosuspension.6Variety of reasons

has been cited for the need to study medicinal plants. Most of

the traditional knowledge about medicinal plants in India was

in the form of oral knowledge that had been lost with persis-

tent invasions and cultural adaptations. There is a prevalence

of using plants and plant based products in various contem-

porary and traditional systems of medicines, without any

written documentation or regulation. Therefore, it is essential

that such uses of natural products be documented and studied

for systematic regulation and wide-spread application.7e13

The leads for a significant number of modern synthetic

drugs have originated from isolated plant ingredients. It is

essential that research on phytochemistry of plants, which

are used extensively in traditional medicines, is carried out

systematically. Several considerations make the use of me-

dicinal plants desirable.14 Their low cost, while the new syn-

thetic drugs are becoming increasingly inaccessible to the vast

majority of people, Research has confirmed the presence of

therapeutically active compounds such as alkaloids, glyco-

sides and others, justifying a many good practices of folk

medicine, They have few, if at all, harmful side effects and

hence their direct administration offers little risk of causing

iatrogenic (drug induced) disorders, unlike the modern syn-

thetic drugs.15,16

2. Experimental

2.1. Selection and collection of plant

The selection of a plant is the most critical aspect of the work.

The fresh leaves of Adhatoda vasica were collected from upper

lake, Shymala Hills Bhopal in December e 2010. Thereafter

washed for removal of dust, foreign material etc and after

then shade dried at room temperature.

2.2. Authentication of A. vasica

Leaves collected form A. vasica plant were authenticated by

preparing a herbarium sheet of A. vasica by Dr. Anil Kumar

Certification Officer of MFP-(Trade & Development) Federa-

tion, Bhopal.

2.3. Determination of ash value

2.3.1. Total ash valueAccurately weighed 2e3 g of air-dried leaves were taken in a

tared silica dish and incinerated at a temperature at 450 �C for

1 h (until free from carbon), cooled and weighed.

2.3.2. Determination of acid insoluble ashAbout 0.3 g of ash was weighed and the ash was boiled with

25 ml of 2 M hydrochloric acid for 5 min. Later it was filtered

using ash less filter paper andwashedwith hotwater followed

by drying and igniting the sample and allowed to cool in

desiccators and then weighed.

d r u g i n v e n t i o n t o d a y 5 ( 2 0 1 3 ) 3 2e3 834

2.4. Fluorescence analysis of the drug

The powder was subjected to fluorescence analysis for the

detection of the presence of compounds, which are fluores-

cent in nature. The fluorescence of powders of aerial and root

part were observed in day light and in UV light (254 nm &

365 nm). The powdered drugs were treated with different

solvents in the glass slides. The solvents used were, 1 N HCl,

1 N HNO3, 1 N H2SO4, CH3COOH, 1 N NaOH, 2 N NaOH, Meth-

anolic NaOH, I2, 1 N KOH, Aqueous KOH, Methanolic KOH,

alcohol as such, acidic alcohol and basic alcohol.

2.5. Loss on drying

Accurately weighed dried powdered leaves of plant were

taken in a tarred glass bottle and the initial weight of material

was taken. Then the sample was heated at 105 � 1 �C in an air

oven and then weighed. This procedure was repeated until a

constant weight was obtained.

Loss on drying (%) ¼ (loss in weight/weight of the drug in

g) � 100

2.6. Extracts values

2.6.1. Water soluble extract2 g of accurately weighed air-dried powdered material were

taken in a glass stopper flask and macerated with 100 ml of

water. Then it was shaken frequently for 6 h in a shaker and

then allowed to stand for 18 h. After that 10 ml of filtrate was

evaporated to dryness in a tarred flat-bottomed and transfer

to Petri dish and dried at 105 �C, and cooled in desiccators. The

percentage of water-soluble extract was calculated with

reference to air-dried drug.

2.6.2. Alcohol soluble extract2 g of accurately weighed air-dried powdered material were

taken in a glass stopper flask and macerated with 100 ml of

ethanol. Then it was shaken frequently for 6 h in a shaker and

then allowed to stand for 18 h. After that 10 ml of filtrate was

evaporated to dryness in a tarred flat-bottomed and transfer

to Petri dish and dried at 105 �C, and cooled in a desiccators.

The percentage of ethanol-soluble extract was calculatedwith

reference to air-dried drug.

2.6.3. Hexane soluble extracts2 g of accurately weighed air-dried powdered material were

taken in a glass stopper flask and macerated with 100 ml of

hexane. Then it was shaken frequently for 6 h in a shaker and

then allowed to stand for 18 h. After that 10 ml of filtrate was

evaporated to dryness in a tarred flat-bottomed and transfer

to Petri dish and dried at 105 �C, and cooled in desiccators. The

percentage of hexane-soluble extract was calculated with

reference to air-dried drug.

2.6.4. Chloroform soluble extract2 g of accurately weighed air-dried powdered material were

taken in a glass stopper flask and macerated with 100 ml of

water. Then it was shaken frequently for 6 h in a shaker and

then allowed to stand for 18 h. After that 10 ml of filtrate was

evaporated to dryness in a tarred flat-bottomed and transfer

to Petri dish and dried at 105 �C, and cooled in a desiccators.

The percentage of water-soluble extract was calculated with

reference to air-dried drug.

2.7. Qualitative chemical investigation

Various extracts of leaves of A. vasica were subjected to

qualitative chemical tests to know the presence of

phytoconstituents.

2.7.1. Test for alkaloids2.7.1.1. Mayer’s test. Mercury (II) chloride 1.358 g was dis-

solved in 60ml of water. 5 g of potassium iodide was dissolved

in 10 ml water. Both solutions were mixed and made up to

100 ml with distilled water. When few drops of Mayer’s re-

agent were added to few ml of alkaloid extract, it gives cream

colour precipitate which confirms the presence of alkaloid.

2.7.1.2. Dragendorff’s test. Solution of bismuth nitrate (0.17 g)

in alcohol (2 mL) and water (8 mL). Solution B: KI (4 g) in

alcohol (10 mL) and water (20 ml). Mix solutions A and B and

dilute to 100 mL with water. When few drops of Dragendorff’s

reagent were added to few ml of alkaloid extract it gives red-

dish brown precipitate which confirms the presence of

alkaloid.

2.7.1.3. Wagner’s test. Iodine (1.27 g) and potassium iodide

(2 g) is dissolved in 5 ml of water and made to 100 ml with

distilled water to a few ml of plant extract, add few drops of

Wagner’s reagent by side of test tube .A reddish brown pre-

cipitate confirms the presence of alkaloid.

2.7.1.4. Hager’s test. To a fewml of extract 1 or 2ml of Hager’s

reagent (saturated aqueous solution of picric acid) are added

yellow precipitate indicates the presence of alkaloid.

2.7.1.5. Tannic acid test. To a few ml extract add few drops of

10% Tannic acid solution buff colour precipitate were appear

confirms the presence of alkaloid.

2.8. Extraction procedure

The shade dried leaves of A. vasica family Acanthaceae were

reduced to fine powder (#40 size mesh) and around 500 g of

powder was sieve through a metal sieve of pore size 0.8 mm

and then extracted overnight in 95% ethanol (5 timesw/v) on a

rotary shaker at 26 �C at 100 rpm. Ethanol was evaporated, (not

fully) to leave it as syrup. This extract was treatedwith 2 NHCl

(pH 2e3) and then basified with ammonia up to 9 pH. The

extract was filtered through Whatman No.1 filter paper and

then washed with chloroform (3�) and chloroform layer was

separated from other layer through separating funnel. The

extract was then concentrated at 50 �C under reduced pres-

sure in rotary evaporator and dried powder was collected then

in a beaker and crystallized with minimum quantity of hot

ethanol to obtain white crystal which were collected in

Eppendorf tubes.

Fig. 1 e The UV spectrum of vasicine at 281 nm.

d ru g i n v e n t i o n t od a y 5 ( 2 0 1 3 ) 3 2e3 8 35

2.9. Isolation of vasicine by preparative thin layerchromatography

The extract was subjected for preparative TLC with reference

to standard solution to obtain pure vasicine. The fraction with

reference to standard is subjected to preparative TLC plate

and run in mobile phase 1,4 dioxane:Methanol:Tolue-

ne:Ammonia in a ratio of (5:2:2:1) and after running a mobile

phase the spot appear which matches to standard. This

sample was scratched out and collected in a beaker then

dissolved in chloroform and was later filtered using What-

mann filter paper. The pure drug came out in the filtrate while

the silica remained on the filter paper. The chloroform dis-

solved pure drug was allowed to evaporate and the dried

fraction was collected as a pure compound which was

confirmed by using characterization techniques such as UV,

HPLC, FT-IR, 1H NMR.

2.10. Chromatographic analysis

It was done by using one reference standards namely, vasicine

95% purchase from Natural Remedies, Bangalore and plant

extract were applied on one 5 � 10 cm precoated silica gel G60

F254 Merck aluminum plate. The sample was prepared by

taking 10 mg/ml of plant methanolic extract and 10 ml of this

solution was applied on the plate, and simultaneously 1 mg/

ml of standard marker solution (purchased form market) was

prepared and from that 10 ml was applied on plate as the

standard.

2.11. Spectral characterization of vasicine isolated fromA. vasica

The spectral analysiswas performed on vasicine isolated from

a compound A. vasica to conform that the compound is vasi-

cine. The isolated compound was dissolved in methanol and

UV analysis was carried out on VARIAN UV spectrophotom-

eter. High Performance Liquid Chromatography (HPLC) one of

the most used analytical technique involving mass-transfer

between stationary and mobile phase. HPLC utilizes a liquid

mobile phase to separate the components of a mixture. The

stationary phase can be a liquid or a solid phase. These

components are first dissolved in a solvent, and then forced to

flow through a chromatographic column under a high pres-

sure. In the column, the mixture separates into its

Table 1 e Phytochemical screening.

Chemical tests Ethanolextracts

Petroleumether

Aqueousextract

Test for alkaloids

Mayer’s test þve �ve þve

Wagner’s test þve �ve þve

Hager’s test þve �ve þve

Dragendorff test þve �ve þve

Tannic Acid test þve �ve þve

The presence and absence phytoconstituents in Adhatoda vasica

(þve ¼ presence, �ve ¼ absence).

components. The amount of resolution is important, and is

dependent upon the extent of interaction between the solute

components and the stationary phase. The stationary phase is

defined as the immobile packing material in the column. The

interaction of the solute with mobile and stationary phases

can bemanipulated through different choices of both solvents

and stationary phases. FT-IR stands for Fourier Transform

Infrared, the preferred method of infrared spectroscopy. In

infrared spectroscopy, IR radiation is passed through a sam-

ple. Some of the infrared radiation is absorbed by the sample

and some of it is passed through (transmitted). The resulting

spectrum represents the molecular absorption and trans-

mission, creating a molecular fingerprint of the sample. Like a

fingerprint no two unique molecular structures produce the

same infrared spectrum. This makes infrared spectroscopy

useful for several types of analysis. The proton nuclear

Fig. 2 e HPTLC plate of Adhatoda vasica; a [ standard

vasicine in methanol, b [ Adhatoda vasica methanolic

extract, Rf [ 0.70.

Fig. 3 e The HPLC graph of vasicine.

Table 2 eHPTLC of Adhatoda vasica extract with referenceto pure vasicine.

S. no Values observed Tracks identified

1 Start Rf 0.70

2 Start height 116.5

3 Max Rf 0.75

4 Max % 20.21

5 End Rf 0.80

6 End height 64.8

7 Area 19977.9

d r u g i n v e n t i o n t o d a y 5 ( 2 0 1 3 ) 3 2e3 836

magnetic resonance 1H NMR spectra of vasicine in solvent

CDCL3 instrument (Model Bruker AMX-400 MHz) were recor-

ded. 1H NMR, CDCl3 (ppm) 7.003 (d, 2H AreCH, J ¼ 7.2), 7.129

(m, 2H, AreCH), 5.29275 (m, 1H, OH), 3.6761 (m, 3H, methylene

proton), 2.625 (m, 1H, methylene proton), 2.256 (m, 2H,

methylene proton), 4.811 (m, 1H, methane proton).

2.12. Preparation of nanosuspension of vasicine bysolvent/anti-solvent technique

Nanosuspension of vasicine was prepared through solvent/

anti-solvent method, where solvent was prepared by mixing

5 mg of vasicine dissolved in 5 ml of 1, 4 dioxane with gentle

warming on water bath while the anti-solvent was prepared

by dissolving 1 ml of Triton X 100 in 50 ml of distilled water

with continuous stirring. Then by burette drop by drop solu-

tion of vasicine in 1, 4 dioxane was transferred into an anti-

solvent containing surface active agent and stirred it for

24 h and the nanosuspension was prepared.

2.13. Characterization of nanosuspension of vasicine

2.13.1. Particle size and zeta potential analysisThe mean particle size of the nanosuspension were deter-

mined by photo-correlation spectroscopy with a zeta master

(Malvern Instruments, Worcestershire, United Kingdom),

equipped with the Malvern PCS software (version 1. 27). Every

sample was appropriately diluted with water filtered through

a 0.45 mm mesh, and the reading was carried out at a 90-de-

gree angle in respect to the incident beam.

2.13.2. Field emission scanning electron microscopeThe morphological and size examination of the nanoparticles

was performed at AMPRI formerly RRL, CSIR Bhopal with a

field emission scanning electron microscope (FEI model

Nova� SEM 430 FESEM). The samples were placed on copper

grids and dried under vacuum for viewing by field emission

scanning electron microscope.

Fig. 4 e Field emission scanning electron microscope of nanosuspension.

Fig. 5 e Particle size distribution.

d ru g i n v e n t i o n t od a y 5 ( 2 0 1 3 ) 3 2e3 8 37

3. Result & discussion

Extract value of A. vasica alcoholic, aqueous, hexane, chloro-

form found to be 15%, 33.33% 15%, 5% respectively. The total

ash value of plant materials indicated the amount of minerals

and earthymaterial present in the plantmaterials. The results

showed that the total ash value is 12.02 %w/w. The water

soluble ash value indicates the amount of water soluble

mineral present in the plant material. The water soluble ash

value was found to be 0.78% w/w. Loss on drying of plant

material was found to be 4.8% w/w. The acid insoluble ash

value indicates the amount of mineral which are insoluble in

Fig. 6 e Zeta potential.

d r u g i n v e n t i o n t o d a y 5 ( 2 0 1 3 ) 3 2e3 838

acid and the result was found to be 1.05% w/w. The water

soluble extract value indicated the presence of sugar, acid and

inorganic compound the alcohol soluble extract value indi-

cated the presence of polar constituents like phenols, alka-

loids, steroids glycosides, extract value of hexane indicate the

non-polar secondary metabolites present in the plant. The

presence and absence phytoconstituents in A. vasica by

phytochemical screening shown in Table 1. The fluorescence

analysis of powder drug in day light, U.V light in 254 nm and

365 nm of different chemicals shows different colors. The

powder as such gives light green colour at 254 nm and purple

at 365 nm. The powderwith 1 NHCl gives light brown colour at

254 nm and at 365 nm gives purple colour. The powder with

50%HNO3 gives reddish brown colour at 254 nmand at 365 nm

gives light green. The powder with 50% H2SO4 gives algae

brown colour at 254 nm and at 365 nm gives greenish black

colour. The powder with methanol gives light green colour at

254 nm and at 365 nm gives purple colour. The UV data shows

absorbance of vasicine at 281 nmwhich confirms the presence

of vasicine. HPTLC profile of A. vasica finds 18 tracks in the

plant extract. The presence of vasicine in extract with refer-

ence to standard is confirmed by HPTLC by matching the Rf

value. The mobile phase used in HPTLC is 1, 4 dioxane: Met-

ahnol:Toluene:Ammonia (5:2:2:1) and the Rf ¼ 0.70 of vasicine

were recorded. The HPLC method presented shows an easy

but reliable and precise analysis of vasicine the sharp peak

will appear after 3 min. The mobile phase for HPLC is 100%

methanol and stationary phase column is C-18 result is shown

in Figs. 1e3 and Table 2. The purity assay results shows that

isolated vasicine were >95% pure. The particle size of nano-

suspension observed by the field emission scanning electron

microscope images at different nano scale is in range of

50e80 nm shown in Fig. 4. The particle size analyzer shows

that 50% of nanosuspension are in a range of 8e10 nm while

the other half is in the range of 200e220 nm and the zeta po-

tential of nanosuspension were recorded as �6.91 mV shown

in Figs 5 and 6.

4. Conclusion

Nanosuspension has emerged as an efficient means of

enhancing the aqueous solubility and dissolution rate of

many drugs. The present formulation study of vasicine was

used as an attempt to prepare nanosuspension drug delivery

system by using by solvent/anti-solvent method which was

stabilized by surface active agent. The ideal properties of

nanosuspension are consider it mainly involve smaller parti-

cle size, good entrapment of drug and release of drug. Nano-

suspension of vasicine was successfully prepared and the

average particle size observed was about w110 nm by particle

size analyser and FESEM studies shows that the nanoparticle

was in the range of 40e60 nm.

Conflicts of interest

All authors have none to declare.

Acknowledgement

Authors are thankful to Dr. Anil Kumar MFP-(Trade & Devel-

opment) Federation, Bhopal for authentication of plant and

also thankful to the Director of Regional Research Laboratory,

Bhopal for providing facilities to formulate nanosuspension.

r e f e r e n c e s

1. Ebadi Manuchair. Pharmacodynamic Basis of Herbal Medicine.CRC Press, LLC; 2002:1e129.

2. Tyler Varro E, Brady Lynn R, Robbers James E. Pharmacognosy.8th ed. Mumbai: K.M. Varghese Company; 1988:1e10.

3. Bhanu PS, Sagar, Zafar R. The Indian pharmacist.2003;2(12):13e16.

4. http://www.holisticonline.com/herbal-Med/hol_herb-intro.htm.5. Harborne JB. Phytochemical Methods e A Guide to Modern

Techniques of Plant Analysis. Springer; 1998:1e49.6. Roja G, Heble MR, Sipahimalani AT. Tissue culture of Withania

somnifera: morphogenesis and withanolide synthesis.Phytother; 1991:185e187.

7. Das Chaitali, Poi Rajlakshmi, Chowdhury Asim. HPTLCdetermination of vasicine and vasicinone in Adhatoda vasica.Phytochem Anal. 2001;16:90e92.

8. Srivastava Shaifali, Ram Verma, et al. HPLC determination ofvasicine and vasicinone in Adhatoda vasica with photo diodearray detection. J Liq Chrom Rel Technol. 2001;24(2):153e159.

9. Muller RH, Jacobs C, Kayer O. Nanosuspensions for theFormulation of Poorly Soluble Drugs. In: Nielloud F, Marti-Mestres G, eds. Pharmaceutical Emulsion and Suspension. NewYork: Marcel Dekker; 2000:383e407.

10. Nash RA, Swarbrick J, Boylan JC. Encyclopedia of PharmaceuticalTechnology. 2nd ed. vol. 3. New York: Marcel Dekker;2002:2045e3032.

11. Kokate CK, Purohit AP, Gokhale. Text Book of Pharmacognosy.New Delhi: Nirali Prakashan; 1991. 522,558,594.

12. Mukherjee PK. Quality control of herbal drugs. Bus Horiz;2007:695e697.

13. Quality Control Methods for Medicinal Plant Materials. Geneva:World Health Organization; 1978:357e365.

14. General Guidelines for Methodologies on Research and Evaluation ofTraditional Medicine. Geneva: World Health Organization;2002:415e422.

15. Anonymous. The Wealth of India e A Dictionary of Indian RawMaterials and Industrial Products. New Delhi: CSIR; 1972:31e32.

16. Ahmed Sayeed, Garg Madhukar, Ali Maksood, et al. A phyto-pharmacological overview on Adhatoda zeylanica Medic.Syn A. vasica (Linn.)Nees. Nat Prod Radiance. 2009;8(5):549e554.