A PRELIMINARY
PHYSICO CHEMICAL ASSAY OF GOKSURA
GRANULES – A PILOT STUDY
Thirunavukkarasu
MS*, Galib1, Shukla VJ2, Harisha CR3, Thakar
AB4, Baghel MS5
* Corresponding Author: PhD
Scholar, Department of Kayachikitsa, IPGT&RA, Gujarat Ayurved University,
Jamnagar, E-mail ID: ayurthiru@yahoo.co.in
1 Lecturer,
Department of Rasa Shastra, IPGT&RA, GAU, (galib14@yahoo.co.in)
2Head,
Department of Pharmaceutical Chemistry IPGT&RA,
GAU, (drvivekshukla@yahoo.co.in)
3
Head, Department of Pharmacognosy,
IPGT&RA, GAU, (harishkumar33@ymail.com)
4 Reader,
Department of Panchakarma, IPGT&RA, GAU, (anup_thakar@yahoo.com)
5
Director, IPGT&RA, Gujarat Ayurved University, Jamnagar, (baghelayu@rediffmail.com)
Abstract
Tribulus terrestris (Linn) of Zygophyllaceae family commonly known as Goksura is
used in Ayurvedic system of medicine. It is distributed through out tropical and warm temperate regions of the world. Commonly
it is known as Caltrops, Puncture vine. The Fruits of Goksura are considered as Diuretic,
Aphrodisiac; used in Urolithiasis, Sexual dysfunctions and Infertility. As the
powder is not palatable, its form has been converted in to granules form and
attempts were made to evaluate its physico chemical profile. Pharmacognostically
authenticated Tribulus terrestris was used for the
preparation of granules and it was analyzed through qualitative and
quantitative analysis of Physico – chemical parameters. Fingerprints of Thin
Layer Chromatography (TLC) and High-Performance Thin Layer chromatography study
(HPTLC) were also developed.
Keyword: Tribulus
terrestris, Zygophyllaceae, Goksura,
Phytochemical Analysis, Chromatography, Ayurveda.
Introduction:
In
the description of Shodasakala Chikitsa
(1) (Sixteen Pre-requisite Qualities), Ayurvedic
classics have given special importance to the Physician’s ability to understand
the rationale behind the utility of natural components in modifying the disease
conditions and in re-establishing the equilibrium of Doshas (Humour), Dhatus
(Tissues) and Mala’s (Waste Products) or in other words, maintaining the
Health.
Acharya Charaka, explained Goksura under Madhuraskandha dravya(2) (Group
of drugs possessing sweet taste),
Mutravirechana gana (Group of drugs with Diuretic properties), Svayathuhara gana (Group of drugs with
Anti inflammatory properties) and Anuvasanopaga gana(3) (Group of drugs
used in preparation of unctuous enema), where as Acharya Susruta included this drug under Vidarigandhadi gana, Virataruvadi gana(4) (Group of drug commencing from Vidarigandha and Virataruvadi), and Kantak panchamula(5) (Group of five thorny drugs whose roots are
used).
Tribulus
terrestris (Zygophyllaceae) is a procumbent annual or perennial herb with many
spreading slender branches, the immature portions covered in a fine silky
hair.(6) It is commonly known as Caltrops, Puncture vine and it is
distributed through out tropical and warm
temperate regions of the world.
(7)
Literature survey reveals the presence of Chlorogenin, diosgenin and its
acetate, gitogenin, astragalin, dioscin, gracillin, hecogenin, ruscogenin,
trillin, furostanol glycoside, spirosterol saponin and a dihydroxy
spirosteroidal sapogenin, trigogenin-3-diglucorhamnoside (terrestroside F),
saponins C & G, kaempferol, kaempferol-3-glucoside,
kaempferol-3-rutinoside, kaempferol-3-b-D- (6”-p-coumaroyl) - glucoside, glucose, rhamnose, rutin, harmine,
neogitogenin, quercetin, reducing sugars, campesterol, b-sitosterol, stigmasterol,
amino acids, alkaloids harmine, harmaline, harman and tetrahydroharmine;
neotigogenin, amino acids (root), Tigogenin, Hecogenin and Gitogenin in
hydrolysed extracts of fruits, roots and leaves.(8) Fruits of Goksura
are used as Diuretic(9), Aphrodisiac(10) and indicated in Urolithiasis(11), Sexual dysfunction as
well as Infertility(12).
In the description of
Chatuspada (Four Limbs of
Treatment), Drugs have the second most importance in treating the
diseases and also the drug should possess the good qualities in it(13). So,
proper identification and standardization of the drug is essential. Each and
every drug has its own physical and chemical characteristics that help for
separating it from other closely related drugs. Hence physicochemical studies
of a particular drug by making use of various parameters help in standardizing
the drug and validate it. Chromatographic techniques were adopted for the
separation of active moieties present in the formulation. Therefore, an attempt
has been made to standardize Goksura
granules, an Ayurvedic medicine based on their TLC and HPTLC fingerprint
profile.
Aims &
objectives:
1. Pharmacognostical
Study of
powdered drug – Fruits of Goksura
2. Physico-chemical
analysis of Goksura Granules
Method
of preparation of the Goksura
Granules
The
dried fruits of Goksura were
subjected to pulverizer to get fine powder. Equal quantity of sugar was taken
and syrup was prepared by adding sufficient quantity of water in mild flame
with constant stirring till syrup reaches the Tantumathvam (thread like) stage. Then Goksura powder was added to the sugar syrup and mixed thoroughly to prepare a homogeneous blend. The blended mass was sieved through a 40# sieve to obtain granule
form and
kept it for drying in room temperature.
Materials & methods:
Plant Material: The dried fruits of Tribulus terrestris were collected from the
Pharmacy, Gujarat Ayurved University, Jamnagar. The fruits were pulverized and sieved through # 80 and fine powder was collected.
The powder was subjected to powder microscopy.
Pharmacognostical Study: Morphological,
Organoleptic and Microscopic study of the powdered drug was done as per the guidelines
of Ayurvedic pharmacopoeia of India(6) at
Department of Pharmacognosy, I.P.G.T & R.A, Jamnagar.
The powder was converted in to granules at the Pharmacy, Gujarat
Ayurved University, Jamnagar.
Physico - chemical study: Goksura granules were analyzed by using, qualitative and
quantitative parameters at Pharmaceutical Chemistry Laboratory of I. P. G.T
& R. A., Gujarat Ayurved University, Jamnagar.
RESULTS & DISCUSSION:
Pharmacognostical
Study:
Organoleptic
characters: The
powder was greenish yellow in color, has pleasant odor and sweet and bitter in
taste.
Powder Microscopy: The dried powder was mounted in the distilled water to detect the
unicellular Trichomes, Starch grains, Stratified Fibers, Oil
globules, Prismatic crystals of calcium
oxalate. When stained with Phloroglucinol and Conc. Hcl, Lignified cells with
polygonal cells of mesocarp cells, Lignified
Parenchyma, Stone cells were observed. (Plate No. 1). All the microscopic
characteristics identified were equivalent to the standard profile. (6)
Physico - chemical Study:
Organoleptic
Characters:
The characters of the sample are tabulated in table no.2
Physico-chemical parameters: The granules were
evaluated for physico chemical parameters like Total Ash Value, loss on drying,
pH value, Sugar estimation (Total sugar, Reducing Sugar, Non -
Reducing Sugar),
Acid soluble and water-soluble extractive values. The results were placed at
table no.3
The
Common parameters mentioned for Goksura
in Ayurvedic Pharmacopoeia of India are total ash, pH
Value,
water and alcohol soluble extractives (6). On its basis the parameters like
total ash content, water and methanol soluble extractives etc., were selected.
Presence of more moisture content in a sample can create preservation problem.
Hence loss on drying was also selected as one of parameters. Since, the sample
was in the form of granules has the possibility of containing significant
quantity of sugar, hence Sugar estimation was considered as another parameter.
Total sugar was found to be 50.73 % w/w suggesting presence of considerable
amount of sugar in the sample. The water-soluble extractive and methanol
soluble extractive values were found to be 51.6 % and 27 % respectively,
indicating considerable amount of polar compounds in the sample.
Qualitative
Test of Goksura Granules: The methanol extract
of the sample was analyzed qualitatively for different functional groups.
Details are placed at table no.4.
Thin layer chromatography:
Methanol Extract:
Granules
weighing 5 gm are taken with 100 ml of alcohol kept for twenty-four hours.
Filtrate was prepared and evaporated till it gets dried in a flat-bottomed
shallow dish and concentrated on water bath to volume
of requirement.
TLC is mentioned as a primary tool for identification as part of
monographs on all medicinal plants. (14) Alkaloid fraction
was used for the spotting of the TLC plate (Silica gel G Precoated plates).
Then the spotted TLC was run with the solvent systems (Toluene (8 ml), Ethyl
acetate (2 ml), Glacial acetic acid (0.5 ml)) separately. And
the resulting TLC pattern was viewed under long wave ultra violet light at 366
nm or Short wave ultra violet light at 254 nm (Table no.5). Then after spraying
with the Anisaldehyde Sulphuric acid reagents and drying
in a hot air oven and the number of spots viewed under daylight (Table no.6).
(Plate No.2)
TLC
of alcoholic extract of drug on silica get "G" plate using Toluene (8
ml): Ethyl acetate (2 ml): Glacial acetic acid (0.5 ml) shows five spots Under
366 nm U.V. at hRf 15, 43, 50, 72 and 97. Where as in 254 nm three zones
visible at hRf 15, 72, 97. On running mobile phase over stationary phase, well
distributed, distinct, clear spots were observed without clumping.
Thin Layer Chromatography of alcoholic extract of Goksura Granules
after spraying Anisaldehyde Sulphuric acid followed by heating and then
visualized in day light shows 7 prominent spots at hRf 22, 40, 54, 65, 77, 86, 97
High Performance Thin layer
chromatography:
Methanol extract of Goksura
Granules were spotted on precoated silica gel GF 60254 aluminium plate as 5mm
bands, 5mm apart and 1cm from the edge of the plates, by means of a Camag
Linomate V sample applicator fitted with a 100 μL Hamilton syringe.
Toluene (8 ml), Ethyl acetate (2 ml), Glacial acetic acid (0.5 ml) (v/v) (20ml)
was used as a mobile phase. The development distance was 6.4 cm (development
time 30 min.). After development, Densitometric scanning was performed with a
Camag T.L.C. scanner III in reflectance absorbance mode at 254 nm and 366 nm
under control of win CATS software (V 1.2.1 Camag) (Fig No.1). The slit
dimensions were 6 mm x 0.45 mm and the scanning speed was 20 mm s-1 (Table
no.7). Then the plate was sprayed with Anisaldehyde Sulphuric acid followed by
heating and then visualized in day light shows 4 prominent spots (Table no. 8).
Visual observation under UV showed multiple spots, but on
analyzing under densitometer all the spots were not detected. This may be due to the limitations of the
integrative system Savitsky – Golay 7 with following specifications like Slope
– 5; Minimum height – 10AU; Minimum area – 50 AU and Maximum height was 990 AU.
However, chromatogram shows 7 prominent spots at hRf 3, 16, 42, 61, 68, 84, 98 in short wave uv 254 nm and 2 prominent spots at hRf 3, 98 in
long wave uv 356 nm.
HPTLC of Methanolic extract of Goksura Granules after
spraying Anisaldehyde Sulphuric acid followed by heating and then visualized in
day light shows 4 prominent spots at hRf
3, 46, 71, 78.
CONCLUSION:
The
plant Tribulus terrestris is used
from the ancient time for its medicinal values and most of the Ayurvedic
formulations prescribed for various diseases have Goksura as one of the ingredients.
Most of the formulations are clinically effective but cumbersome for the
patient compliance. In the present study, the granule form was evaluated for pharmaceutical feasibility and for patient
palatability.
The
plant Tribulus terrestris was
identified and authenticated phamacognostically and
was used as a unique ingredient. The formulation namely, Goksura Granules were subjected to phytochemical, physico-chemical,
TLC and HPTLC studies. It is inferred that the formulation meets the minimum
qualitative standards as reported in the API at a preliminary level.
On the basis of our observations and experimental results, we
created method of preparation of Goksura
granules for the first time and are economical in terms of time and machinery.
This study may be used as reference standard in the further quality control
researches. Further studies may be carried out based on identification and
separation of active ingredients with the help of Biomarkers like Protodioscin.
ACKNOWLEDGEMENTS
The authors wish to thank Prajapati P.K, Director of Pharmacy,
Department of Rasashastra, IPGT&RA, Gujarat Ayurveda University, Jamnagar
for his sincere guidance in the preparation of granule.
REFERENCES:
1.
Agnivesa,
Charaka Samhita, Acharya Jadavji Trikamji, 5th Edition, Choukambha publication,
Varanasi, 2001; 64
2.
Ibid;
284
3.
Ibid;
33
4.
Sushruta,
Sushruta Samhita, Acharya Jadavji Trikamji, 8th edition, Chaukhambha
orientalia, Varanasi, 2005; 164.
5.
Ibid;
169.
6.
Anonymous,
The Ayurvedic Pharmacopoeia of India, Part- I, Vol I, 40, 1st Edition, 2001,
Ministry of Health And Family Welfare, Department of AYUSH (Government of
India).
7.
Holm
et al, The world’s worst weeds: Distribution and biology. East-West
Center/University Press of Hawaii. 1977; 467
8.
Data Base On Medicinal Plants Used In Ayurveda, Central Council
For Research In Aurveda & Siddha, Volume 3, New Delhi, 2002; 229
9.
Al-Ali M, Wahbi S, Twaij
H, Al-Badr A., Tribulus terrestris:
preliminary study of its diuretic and contractile effects and comparison with
Zea mays. J Ethnopharmacol 2003 Apr;8 5(2-3):257-60
10.
Ivan
A. Ross, Medicinal Plants of World (2001), Ind.J.surg., 1976; 38, 12
11.
Anand R et al, Activity of certain fractions of Tribulus
terrestris fruits against experimentally induced urolithiasis in rats.
Indian J Exp
Biol. 1994 Aug; 32(8): 548-52.
12.
Gauthaman K et al. "Aphrodisiac properties of Tribulus Terrestris extract
(Protodioscin) in normal and castrated rats". Life Sciences. 71.12 (2002):
1385-96.
13.
Agnivesa,
Charaka Samhita, Acharya Jadavji Trikamji, Choukambha publication, Varanasi,
2001; 61
14.
Eike
Reich et al, TLC for the Analysis of
Herbal Drugs - A Critical Review of the Status and Proposal for Improvement of
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