Journal of Pharmacognosy and Phytochemistry 2017; 6(2): 10-16

E-ISSN: 2278-4136
P-ISSN: 2349-8234 Standardization of marketed cystone tablet: A herbal
JPP 2017; 6(2): 10-16
Received: 01-01-2017 formulation
Accepted: 02-02-2017

Kiran A Wadkar
Dept. Of Pharmacognosy,
Kiran A Wadkar, Manish S Kondawar and Sachin G Lokapure
Appasaheb Birnale College of
Pharmacy, South Shivaji Abstract
Nagar,Sangli Maharashtra, The Present study aims to standardize Cystone tablet based upon chromatographic and spectral studies.
India. The spectral data and HPTLC fingerprint of ethanolic extract of Cystone formulation could be used as a
valuable analytical tool in the routine standardization of tablet to check the batch to bath variation. The
Manish S Kondawar ethanolic extract of Cystone tablet were compared by TLC, HPTLC and HPLC analysis to evaluate the
Dept. of Pharmacognosy,
presence of catechin employing Toluene: Ethyl acetate: acetone (2:4:4 v/v/v), as a mobile phase
Appasaheb Birnale College of
Pharmacy, South Shivaji Nagar,
respectively. The Rf values (0.80) for catechin in both sample and reference standard were found
Sangli Maharashtra, India. comparable under UV light at 366 nm respectively. The high performance thin layer chromatography
method developed for quantization was simple, accurate and specific.The present standardization
Sachin G Lokapure provides specific and accurate tool to develop qualifications for identity, transparency and reproducibility
Dept. Of Pharmacognosy, of biomarkers catechin tablet formulation.
Appasaheb Birnale College of
Pharmacy, South Shivaji Keywords: Standardization, cystone tablet, Markers
Nagar,Sangli Maharashtra, India
1. Introduction
Ayurveda is considered by many scientists to be the oldest healing science. In Sanskrit,
Ayurveda means “The Science of Life.” Ayurvedic knowledge originated in India more than
5,000 years ago and is often called the “Mother of All Healing” [1]. Ayurveda translates into
knowledge (Veda) of life (Ayur) and is one of the oldest and still widely practiced medical
systems in the Indian subcontinent [2]. The concept of Ayurvedic medicine is to promote
health, rather than to fight disease, and Ayurveda in daily life aims at maintaining harmony
between nature and the “individual” to ensure optimal health [1]. Ayurveda contains 8 branches
of sciences and 10 different diagnostic tools based on tridosha theory (three humours of body).
Ayurveda comprises of various types of medicines including the fermented forms namely
arishtas (fermented decoctions) and asavas (fermented infusions). These are regarded as
valuable therapeutics due to their efficacy and desirable features.
Bergenia ligulata belongs to family saxifragaceae. Pashanbheda, Pashana, Zakhmehayat,
Asmaribheda, Ashmabhid, Ashmabhed, Nagabhid, Upalbhedak, Parwatbhed and Shilabhed are
the common name of Bergenia ligulata. It is called Stone breaker becauses it dissolv slabs.
Rhizome is the medicinalally used part of this. The plant Bergenia ligulata is main botanical
source of Pashanbheda drug which is used in indigenous system of medicine [3-7].

2. Material and methods

2.1 Chemicals
Chloroform, formic acid, ethyl acetate, toluene were purchased from Merck, India. Methanol
and ethanol of analytical reagent grade (Merck, Darmstadt, Germany) were used. catechin
reference standard were purchased from Sigma–Aldrich GmbH, Germany. All other solvents
and chemicals were of the highest analytical grade.

2.2 Apparatus
All the solvents purchased from E. Merck and S.D. Fine Chemicals, Mumbai. All solvents
used for extraction, TLC and HPTLC studies were distilled before use. Solvents used for UV
and IR studies were of spectroscopy grade. Solvents used for HPLC analysis were of HPLC
grade. Precoated silica gel GF-254 plates procured from E. Merck, Mumbai were used for
TLC and HPTLC studies. The UV spectra were recorded on a JASCO V 530
Correspondence spectrophotometer. The FT IR spectra were recorded on JASCO FT IR 410. Atron HPTLC
Kiran A Wadkar
Dept. Of Pharmacognosy, system consisting of Sparylin spotting, elite-miniluminascence photo documentation and
Appasaheb Birnale College of CAMAG scanner. The HPLC analyses were done on a TOSOH–CCPM system. All the results
Pharmacy, South Shivaji Nagar, are obtained by repetition of the each experiment at least three times.
Sangli Maharashtra, India
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Journal of Pharmacognosy and Phytochemistry

2.3 Procurement of drug mentioned in protocol for testing. Acid insoluble ash and
Commercially available brand (M/S Himalaya Pharma) of sulphated ash were done by method mention in AP;
Cystone was procured from local market.
3.4 Loss of weight on drying at 105 °C
2.4 Standardization using physicochemical parameters Loss of weight on drying (LOD) at 105 °C was done by
The sample of Cystone was analyzed for various parameters method mention in AP.
such as Organoleptic evaluation, Physical evaluation,
Moisture content etc. 3.5 pH value
pH value of 1% solution and pH value of 10% solution was
2.5 Physiochemical parameters determined as per the method mentioned in physiochemical
Organoleptic properties standardization of Unani Medicine part IV.
Appearance, color, smell, and taste were evaluated.
3.6 Weight variation
2.6 Friability test Twenty tablets were selected randomly from selected batch
Friability test apparatus Roche's friabilator (Labinda mod. no. and weighed individually. Average weight was calculated,
1020) was used for determination of friability of tablet. This and individual weights were compared to average weight. If
device subjected the tablet to the combined effect of abrasion not more than 2 tablets are outside the percentage limit,
and hock in a public chamber and dropping the tablets at a tablets meet the USP test (USP weight variation test).
height of 6 inches in each revolution. Weighed tablets were
placed in friabilator revolving at 25 rpm for 100 revolutions. 3.7 Preparation of extracts
Tablet was de-dusted using a soft muslin cloth and weighed. 100 gm of the Cystone tablet powdered material was extracted
with 250ml methanol and ethanol ina soxhlet extractor at a
F = (W1− W2/W1) × 100
temperature of 45-50 0C for 48 hours. The extract obtained
Where; (W1= Initial weight of tablets, W2= Final weight of was then concentrated under reduced pressure using rotary
tablets) evaporator which concentrates bulky solution down to small
volumes, without bumping, at temperatures between 30 and
2.7 Tablet hardness test 40 0C.
Randomly three tablets were pickup and they were
individually tested for the hardness by Monsanto hardness 3.8 Qualitative chemical examination
tester (Shital scientific industries Sr. no. 11012010) in terms The ethanolic and metnanolic extracts were qualitatively
of kg/cm. evaluated by chemical tests and TLC studies for the presence
of various phytoconstituents like alkaloids, carbohydrates,
2.8 Disintegration test saponins, phenolic compounds and tannins, phytosterols and
Disintegration testing apparatus (Thermonik: Mod. no. TD anthraquinone glycosides [8].
20S) was used for determination of disintegration time.
3.9 Isolation of Catechin by pre-para tine TLC
2.9 Uniformity of diameter This extract was dissolved in methanol and resulting solution
Diameter of three randomly selected tablets was measured was used for preparative thin layer chromatography. For that
individually using a Vernier Caliper (UTTAR, IME type 6 purpose TLC plates of size 15.2×20.2 were used. Better
inch/15 cm) and expressed in mm. resolution of catechin was obtained in the solvent system of
Toluene: Ethyl acetate: acetone (2:4:4 v/v/v).
3 Extractive value (Soxhlet apparatus) The developed preparative TLC was showed in [Figure no. 2].
3.1 Successive extractive value The Rf value of catechin was calculated. After proper
The coarse powder of QT was extracted successively using resolution, the spot of catechin was observed in UV chamber.
soxhlet apparatus with different solvent, in increasing order of The developed preparative TLC in UV chamber was showed
polarity, petroleum ether → benzene → chloroform → in [Figure no. 3].Using sharp pointer the spot was isolated.
ethanol. 10 g powdered drug was taken and subjected to The isolated catechin was subjected for phytochemical test.
successive extraction with each solvent for 6 h. The extracts The retention factor is defined as the distance travelled by the
were filtered using filter paper (Whatman No. 1) and dried on solute divided by distance travelled by the solvent.
water bath. The extractive values were determined with
reference to the weight of the drug taken (w/w). The 4. HPTLC studies
procedure was repeated 3 times to calculate mean extractive HPTLC fingerprint of ethanolic extract was recorded at 366
values. nm. Ethanolic extract were subjected to HPTLC studies to
develop fingerprints using same conditions as used for TLC.
3.2 Non successive extractive value
The coarse powder of QT was extracted separately in different 5. Spectral studies
solvent (water, ethyl alcohol and petroleum ether) using UV, IR and fluorescence spectra were recorded for extract.
soxhlet apparatus. 10 g powdered drug was taken and UV spectra were recorded in ethanol. IR spectra were
subjected to separate extraction with each solvent. The recorded of neat sample.
extracts were filtered using filter paper (Whatman No. 1) and
evaporate on water bath. Extractive values were determined 6. HPLC studies
with reference to drug taken (w/w). Isolated compound indicated presence of Catechin which is
reported to be a major active component of Cystone
3.3 Ash value formulation. Extract was analyzed by HPLC using following
Total ash and water soluble ash were done by method conditions:

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Journal of Pharmacognosy and Phytochemistry

Column: C18 (25 cm×4.6 mm, i.d.), 10πm 7.2 Physical evaluation: [9-12]
Mobile phase: methanol: water (60:40) 7.2.1 Determination of solvent extractive values
Detection: at 254 nm Solvent extractive value is the amount of active constituent in
Flow rate: 1 ml/min a specified weight of medicinal plant material when extracted
with specific solvent. The extraction of any crude drug with a
7. Results and discussion particular solvent yields a solution containing different
Standardization of Cystone as per pharmacopoeia was carried phytoconstituents. The composition of these phytoconstituents
out based on the physicochemical parameters [16]. The in that particular solvent depends upon the nature of the drug
marketed sample of Cystone was found to pass all the and solvent used.
pharmacopoeial tests (Table 1). The solvent extractive value can be determined by following
methods to measure the water soluble extractive, alcohol
7.1 Organoleptic evaluation soluble extractive and hexane soluble extractive value using
Appearance: Circular uncoated tablet (slightly biconvex); water, ethanol and hexane as solvent for extraction
Colour: Yellow; Smell: Rosy; Taste: Aromatic and bitter; respectively.
Texture: Hard.
The mean value of friability (%), hardness (kg/cm), 7.2.2 Determination of water soluble extractive value
disintegration time (minutes) and diameter (mm) of QT. Were For the determination of water soluble extractive value, 5 g of
determined and the values are depicted in [Table 1]. Cystone tablet powdered materialwas weighed and taken into
the conical flask in which 100ml of water was added
Table 1: Organoleptic evaluation of Cystone tablet. separately and allowed to macerate for 24 hours, shaking
frequently for first 6 hours then allowing to stand for next 18
Parameter Mean
hours, which was then filtered rapidly and 25ml of the filtrate
Friability (%), 0.08 ± 0.0053
was allowed to evaporate to dryness in a tared 250ml beaker
Hardness (kg/cm), 8.1±0.082 at1050C. The difference in weight of the beaker is an
Disintegration time (minutes) 25.80±0.453 indication of water soluble extractive value of that drug with
Diameter 12±0.1 respect to the amount of drug (5 g) taken for extraction. The
obtained results are tabulated in [Table no. 2].

Table 2: Water soluble extractive value of Cystone tablet. *n=3

Wt. of Mean water soluble Water soluble extractive
Sample ± SD
sample (g) extractive value* (g) value (% w/w)
Cystone tablet 5 0.64 12.8 0.0143

7.2.3 Determination of alcohol soluble extractive value hours, which was then filtered rapidly and 25ml of the filtrate
For the determination of alcohol soluble extractive value, was allowed to evaporate to dryness in a tared 250ml beaker
5gof Cystone tablet powdered materialwas weighed and taken at1050C. The difference in weight of the beaker is an
into the conical flask in which 100ml of ethanol was added indication of alcohol soluble extractive value of that drug with
separately and allowed to macerate for 24 hours, shaking respect to the amount of drug (5g) taken for extraction. The
frequently for first 6 hours, then allowing to stand for next 18 obtained results are tabulated in [Table no. 3].

Table 3: Alcohol soluble extractive value of Cystone tablet. *n=3

Wt. of Mean alcohol soluble Alcohol soluble
Sample ± SD
sample (g) extractive value* (g) extractive value (% w/w)
Cystone tablet 5 0.48 9.6 0.01155

7.2.4 Determination of hexane soluble extractive value was allowed to evaporate to dryness in a tared 250ml beaker
For the determination of hexane soluble extractive value, 5g at105 0C. The difference in weight of the beaker is an
of Cystone tablet powdered materialwas weighed and taken indication of hexane soluble extractive value of that drug with
into the conical flask in which 100ml of hexane was added respect to the amount of drug (5 g) taken for extraction. The
separately and allowed to macerate for 24 hours, shaking obtained results are tabulated in [Table no. 4].
frequently for first 6 hours then allowing to stand for next 18 In the determination of all extractive values, the percentages
hours, which was then filtered rapidly and 25ml of the filtrate were determined with respect to the air dried material.

Table 4: Hexane soluble extractive value ofCystone tablet. *n=3

Wt. of Mean hexane soluble Hexane soluble extractive
Sample ± SD
sample (g) extractive value* (g) value (% w/w)
Cystone tablet 5 0.55 11 0.00577

7.2.5 Determination of ash values variation of the weight of ash from sample to sample is very
The ash of any organic material is composed of their non- small and any marked difference indicates a change in quality.
volatile inorganic components. Controlled incineration of The ash value can be determined by three different methods to
crude drug results in an ash residue consisting of an inorganic measure the total ash, the acid insoluble ash and the water
material (metallic salts and silica) in certain drugs, the percent soluble ash.

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Journal of Pharmacognosy and Phytochemistry

7.2.6 Determination of total ash content carbon free ash was not obtained thus the cooled crucible was
The total ash was determined by placing 2 g of Cystone tablet removed and the residue was moistened with about 2ml of
accurately powdered material into the tared crucible. Then water, dried on water bath, and ignited to constant weight.
Cystone tablet sample was placed in muffle furnace and the The residue was allowed to cool in desiccator for 30 minutes.
sample was ignited by gradually increasing the heat up to Content of total ash was calculated with reference to the
500-6000C until it is white, indicating the absence of carbon. amount of Cystone tablet taken. The obtained results are
The crucible was kept in desiccator and weighed. Initially tabulated in [Table no. 5].

Table 5: Determination of total ash content of Cystone tablet. *n=3

Sample Wt. of sample (g) Mean total ash content*(g) Total ash content (% w/w) ± SD
Cystone tablet 2 0.25 12.5 0.010

7.2.7 Determination of acid insoluble ash value washed with hot water, then ignited in muffle furnace at about
The total ash which was obtained in the previous step was 450 0C for 30 minutes, cooled in desiccator and weighed. The
boiled with 25ml of 2 M HCl for 5 minutes, then it was percent of acid insoluble ash was calculated with reference to
filtered through ash-less filter paper, the insoluble matter was the amount ofCystone tablet taken. The obtained results are
collected in a previously tared crucible, then the residue was tabulated in [Table no. 6].

Table 6: Determination of acid insoluble ash value of Cystone tablet. *n=3

Sample Wt. of sample (g) Mean acid insoluble ash value* (g) Acid insoluble ash value (% w/w) ± SD
Ash 1 0.05 5 0.011

7.2.8 Determination of water soluble ash not exceeding 4500C separately. Then the weight of this
In the crucible containing total ash, 25ml of water was added residue was subtracted from the weight of total ash. Finally
and boiled for 5 minutes. The insoluble matter was collected the content of water soluble ash with reference to the amount
on an ash less filter paper. This was then washed with hot of Cystone tablet taken was calculated. The obtained results
water and ignited in a crucible for 15 minutes at a temperature are tabulated in [Table no. 7].

Table 7: Determination of water soluble ash value of Cystone tablet. *n=3

Sample Wt. of sample (g) Mean water soluble ash value* (g) Water soluble ash value (% w/w) ± SD
Ash 1 0.04 4 0.0101

7.2.9 Determination of moisture content atmospheric pressure at room temperature for specific period
Method of determination of moisture content includes the loss of time.
on drying. It can be carried out either by heating at 1000- The moisture content of Cystone tablet was found to be 6.66
1050C or in a desiccator over phosphorus pentoxide under w/w. The obtained results are tabulated in [Table no. 8].

Table 8: Determination of moisture content of Cystone tablet. *n=3

Sample Wt. of sample (g) Mean moisture content * (g) Moisture content (% w/w) ± SD
Cystone tablet 1.5 0.10 6.66 0.0115

7.2.10 Loss on drying recorded and loss on drying was expressed as percent w/w.
Loss on drying is the loss of mass expressed as percent w/w.
To estimate the loss on drying 2-5 g of air dried drug is 7.2.11 Determination of moisture content by Analytical
accurately weighed in dried and tared flat weighing bottle. Moisture Balance
The substance is dried to a constant mass or for the prescribed Method of determination of moisture content includes the loss
time as specified. on drying. It can be carried out by using Analytical Moisture
1.5g of sample of Cystone tablet was accurately weighed in Balance (Sartorius Moisture Balance Model -MA 150). The
separate dried and taredpetridish. Sample Cystone tablet was difference in weight before and after heating was recorded
placed in an oven, and drying of sample was carried out at and loss on drying was expressed as percent w/w. The
1050C until a constant mass of the sample was not observed. obtained results are tabulated in [Table no. 9].
The difference in weight before and after heating in oven was

Table 9: Determination of moisture content of Cystone tablet by Analytical Moisture Balance. *n=3
Sample Wt. of sample (g) Mean moisture content * (g) Moisture content (%LR) Loss of Residue (%R)
Cystone tablet 2 0.051 2.60 97.47

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Journal of Pharmacognosy and Phytochemistry

Ethanolic and Metahnolic extract of Cystone formulation

were subjected to qualitative chemical investigation. A
summary of the qualitative analysis is given in Table 10.

Table 10: Qualitative chemical evaluation of extracts

Serial Extract Extract
Phytoconstituents
number Ethanol Methanol
1 Alkaloids + +
2 Steroids - -
3 Flavonoids + +
4 Saponins - -
5 Tannins + +
6 Cardiac glycoside - -
7 Anthraquinone glycoside - -
8 Coumarins glycoside - -
(+) Indicates presence, (−) indicates absence. The chemical
evaluation reported here is for the entire extract and not for the
individual spot.
Fig 2: Developed preparative TLC in UV chamber. ( UV long wave)
After isolation process the crude dried extract was obtained. Isolated catechin were evaluated by chemical and spectral
This ethanolic extract was dissolved in methanol and resulting methods to study the nature of the components. The color
solution was used for preparative thin layer chromatography. characteristics in UV and visible light revealed presence of
For that purpose TLC plates of size 15.2×20.2 were used. flavones and/or flavonol in isolated catechin. (Table 3).
Better resolution of catechin was obtained in the solvent
system of Toluene: Ethyl acetate: acetone (2:4:4 v/v/v). Table 11: TLC studies of isolated catechin of extract.
The developed preparative TLC was showed in [Figure no. 1].
Color
The Rf value of catechin was calculated. After proper Sr No Catechin Rf Indication
characteristics
resolution, the spot of catechin was observed in UV chamber. Flavones and /or
The developed preparative TLC in UV chamber was showed 1 Standard 0.80 Black
flavonol
in [Figure no. 2].Using sharp pointer the spot was isolated. 2 Isolated 0.80 Dark Blue Isoflavones
The isolated catechin was subjected for phytochemical test.
The retention factor is defined as the distance travelled by the Spectral studies confirmed the results of TLC studies. UV and
solute divided by distance travelled by the solvent. IR spectra of isolated catechin gave characteristic peaks
indicating presence of Flavones. The UV, IR spectra and
HPTLC fingerprints of isolated catechin can be used for
routine standardization of Cystone tablet (Table 4).

Table 12: Spectral data of isolated fractions

Sr. No Isolated Catechin UV IR (cm -1)
1 I 245, 254 3412, 3238, 3505, 1144

8. Fingerprint analysis of ethanolic extract of Cystone

formulation by HPTLC [13]
HPTLC fingerprint analysis was carried out on ethanolic
extract of Cystone formulation with solvent system i.e.
Toluene: Ethyl acetate: Acetone (2:4:4 v/v/v) using Atron
HPTLC system consisting of Sparylin spotting, elite-
miniluminascence photo documentation and CAMAG
scanner. The chromatogram obtained was studied under 366
Fig 1: TLC study of Isolated and Standard Catechin. nm (Figure. 3).

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Journal of Pharmacognosy and Phytochemistry

The standard HPTLC chromatogram Standard Catechin and

Isolated Catechin showed two peaks with Rf value 0.76 and
0.82. (Figure. 4 and 5)

9. Characterization of extract of Cystone formulation by

HPLC
The HPLC analysis of standard Catechin and Isolated
Catechin shows peaks with retention times of 2.773 and 2.773
min, respectively, (Figure. 6 and 7).

Fig 3: HPTLC fingerprint of Cystone formulation along with marker

compounds.

1. CS-Catechin Standard, 2. IR- Isolated from Rhizome of Fig 6: HPLC profile of standard Catechin.
berginia ligulata, 3. IF- Isolated from Cystone formulation, 4.
Cys F- Cystone Formulation Extract.

Fig 7: HPLC profile of Isolated Catechin from Formulation.

Fig 4: HPTLC chromatogram of Standard Catechin.
Thus, it can be said that extract contain catechin. It can be the
possible analytical markers for standardization of Cystone
formulation. After quantitative estimation of catechin
specifications can be stated. This should then serve as a
simple, accurate and routine method of analysis for Cystone
formulations.

10. Conclusion
The spectral data and HPTLC fingerprint of ethanolic extract
of Cystone formulation could be used as a valuable analytical
tool in the routine standardization of Cystone formulation to
check the batch to bath variation. Catechin can be used as one
of the appropriate analytical markers for standardization of
Cystone formulation.

11. Acknowledgements
Fig 5: HPTLC chromatogram of Isolated Catechin. This work is financially supported by University Grants
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Journal of Pharmacognosy and Phytochemistry

Commission, New Delhi under Major Research Project [Grant

No.42-708/2013(SR)]. Authors are also thankful to Principal
D. D. Chougule of Appasaheb Birnale college of Pharmacy
Sangli for providing the necessary facilities to carry out this
work.

12. References
1. Rastogi S. Building bridges between Ayurveda and
modern science. International journal of Ayurveda
research. 2001: 1(1):41-42. 2.
2. Valiathan MS, Thatte U. Ayurveda: The time to
experiment. International journal of Ayurveda research.
2001; 1(1):3-4.
3. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian
Medicinal Plants. C.S.I.R., New Delhi, 1956.
4. Yaginuma A, Murata K, Matsuda H, β-Glucan, Bergenia
ligulata. as cosmetics ingredient. Fragrance J, 2003;
31:114-119.
5. Panda H. Medicinal plant cultivation and their uses.
National Institute of Industrial Research, 2002.
6. Dush B, Kashyap L. Herbal plants in kidney stone. In
Materia Medica of Ayurveda. Concept Publishing Co.
New Delhi, 1979, 89.
7. Panday G. Medicinal Plants of Himalaya. Sri Satguru
Publications. A Division of Indian Books Centres. Delhi.
India, 1995.
8. Km Ruby, Jaya Dwivedi, Rajani Chauhan. Pashanbheda
A Golden Herb of Himalaya: A Review, International
Journal of Pharmacy Review & Research. 2012; 2(2):97-
105.
9. Umashankar D, Chandra Reddy, Amrik S Chawla. High
Pressure Liquid Chromatographic Determination of
Bergenin and (+) -Afzelechin from Different Parts of
Paashaanbhed (Bergenia ligulata Yeo) Phytochemical
Analysis. 1999; 10:44-47.
10. Dharmender R, Madhavi T, Reena A, Sheetal A.
Simultaneous Quantifi cation of Bergenin, (+)-Catechin,
Gallicin and Gallic acid; and quantifi cation of -
Sitosterol using HPTLC from Bergenia ciliata (Haw.)
Sternb. Forma ligulata Yeo (Pasanbheda). Pharm Anal
Acta. 2010; 1:104.
11. Kondawar MS, Kamble KG, Mali DS. Quantitative
estimation of Gallic acid and Ascorbic acid in a marketed
herbal medicine: Triphala Churna by High Performance
Thin Layer Chromatography, International Journal of
Pharm Tech Research. 2011; 3(3):1593-1599.
12. Khandelwal KR. Practical Pharmacognosy Techniques
and Experiments, 9th ed., Nirali Prakashan, 2002
13. Indian Pharmacopoeia, Ministry of health, Govt. of India.
4th edition. 1996; 114.

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