A PRELIMINARY PHYSICO CHEMICAL ASSAY OF GOKSURA GRANULES – A PILOT STUDY

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.


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 reestablishing the equilibrium of Doshas 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).
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.

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.

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. 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 twentyfour 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 ( 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.