Comparitive Pharmacognostical and Phytochemical Evaluation of Brihat and Laghu

Laghu Gokshur (Tribulus terrestris) and Brihat Gokshur (Pedalium murex) are well known drugs used in Ayurveda as diuretics. These are classified under mishrak varga as ‘Dashmoola’ in Ayurveda and in chemotaxonomy as Saponin glycosides. The objective of the work is to find out the diagnostic tool to identify the two varieties of Gokshur. The powder was studied for macroscopic, microscopic and physicochemical parameters. For HPTLC Stationary phase was Pre-coated silica gel GF 254 and mobile phase was Toluene: Ethyl acetate: Formic acid (7:2:1 v/v/v). The plate was scanned and quantified at 254 nm for Diosgenin.Results shows that microscopic characters like trichomes, stomata and crystals show some difference in the two varieties while physicochemical parameters show difference in extractive values. Phytochemical screening also shows similar findings. HPTLC analysis carried out using Diosgenin as reference standard revealed the presence of steroidal Saponin “Diosgenin”. Quantitative estimation for Saponins found marked variation in the two varieties, where Laghu gokshur had 16% of Saponins while Brihat gokshur had 13% Saponins. The study can be used as a diagnostic tool for identification of these two varieties of Gokshur.


INTRODUCTION
Brihat Gokshur Pedalium murex Linn.and Laghu Gokshur Tribulus terrestris Linn.are the plants under the mishrak varga, Dashmoola having diuretic property.Both plants are categorized under Glycosides steroidal Saponins.The chemical constituent of the plants includes Sapogenins, Tannins, Glycosides etc.Among these steroidal Saponins is the main active principle which is responsible for the diuretic activity of the plants.
P.murex Linn. is a branched succulent herb which grows up to 40 cm in height.Leaves simple, opposite, fleshy, serrate or crenate; flowers bright yellow, solitary and axillary.Fruits 4 angled with horizontal spines from the angles.T.terrestris Linn. is a annual or rarely perennial hirsute, prostrate herb with many slender spreading branches, one to two feet or more in length and about 0.05 inch in thickness, bearing unequal pairs of abruptly pinnate leaves and small solitary stalked yellowish flowers or spiny fruits at each node.The entire plant has a faint aroma and hence named as 'Ikshuu gandhika ( Al-Ali et al.,. 2003 ).Diosgenin is a steroidal Sapogenin possessing estrogenic and antitumor properties found in both the species.The pharmacological property of a steroidal Saponins has been described including their hypocholesterolemic, antidiabetic and antioxidant activities (Trease and Evans, 1996).
In the present work, comparative Pharmacognostical, phytochemical profiles of whole plants of Tribulus terrestris and Pedalium murex w.s.r to the steroidal Saponin "Diosgenin''were studied.
Present study aims to carry out detailed Pharmacognostical and Phytochemical evaluation of Laghu Gokshur and Brihat Gokshur which includes macroscopic, microscopic and physicochemical analysis including HPTLC fingerprinting.In various Ayurvedic literature it has been mentioned that Laghu gokshur can be used as substitute for Brihat gokshur.Keeping this view under consideration the work was designed to find the characteristic feature where these two varieties shows similar characters.

Collection of plants
The plants Brihat Gokshur and Laghu Gokshur were collected from surrounding areas of Jamnagar.The plants were washed, dried, powdered and taken for further study.

Macroscopic characters
Macroscopic details of the respective parts are noted by usual observation and with the help of flora, botanical texts.In the description, general condition of the drug color, odour, taste, texture etc. are noted.Drugs can be identified with the aid of the above, only in entire condition (Trease and Evans, 1996).

Microscopy characters
Transverse sections of root of both the plants had been taken and photomicrography had been done after proper mounting and staining.Powders of Panchanga of both the samples were studied microscopically for their characters (Trease and Evans, 1996).

Physicochemical parameters
Physicochemical study includes the parameters such as Loss on drying, Ash value, Acid insoluble ash, Extractive values in water and methanol, pH according to the various textual references (Trease and Evans, 1996).

Histochemical Tests
Some Histochemical tests were performed to detect various primary & secondary plant metabolites and cellular contents by using different reagents (Khandelwal , 2008).

Qualitative Study
In qualitative analysis various chemical tests were performed to find out the presence or absence of chemical constituents like Saponins, Alkaloids, Glycosides etc.

Quantitative Estimation
For quantitative evaluation of the Phytoconstituents the assay was carried out for total Saponins content by gravimetric method according to the references (Shaver et al., 1964).

Fluorescence Analysis
The fluorescence analysis of Laghu Gokshur and Brihat Gokshur was done in different solvent extracts.For this chloroform, methanol, water, hydrochloric acid and sodium hydroxide were taken as solvents.The extracts of the plants were prepared in selected solvents.All the extracts were subjected to long U.V 366 nm and short U.V 254 nm to observe the difference in colour (Chase and Patter 1949).

Chromatographic fingerprint Preparation of sample solution
10 gm of plant material was extracted with 5 ml of Methanol.The material was refluxed for half an hour.The extract was filtered and volume was made up to 10 ml to get solution concentration.The procedure was same for both samples (Lambert et al., 1988).

Procedure
Before spotting, the plate was pre-washed with Methanol.Sample solutions were applied to the plate as sharp bands by means of Camag Linomat V sample applicator.The spots were then dried in a current of air.The mobile phase Toluene: Ethyl acetate: Formic acid (7:2:1 v/v/v) was poured into a twin trough glass chamber.Then whole assembly was left to equilibrate for 30 min & the plate was placed in the chamber .The plate was then developed until the solvent front had travelled at a distance of 80 mm above the base of plate.The plate was then removed from chamber & dried in a current of air.Detection was done with Camag TLC scanner 3 at a wavelength 254 nm and 366 nm and sprayed with Anisaldehyde Sulphuric acid reagent afterwards (Lambert et al., 1988).

RESULTS AND DISCUSSION
Organoleptic values of powders of P.murex and T.terrestris showed both similarities and dissimilarities (Table 1).Dissimilar characteristics of the powders like colour and taste can be used as diagnostic values; P. murex powder is brown and bitter while that of T.terrestris is dark green and astringent.Powder of whole plant of T.terrestris and P.murex revealed distinguished characters like stomata, crystals, stone cells, fibers, vessels, trichomes (Table 2; Fig     T.S of P. murex shows presence of cork, cortex, sclerides, stone cells, lignified xylem, and phloem.T.terrestris root showed similar characters with medullary rays (Fig. 2) Histochemical test results showed similar findings in both varieties of Gokshur and no difference was observed (Table 3).Loss on drying of powder of T.terrestris was more similar to that of P. murex (Table 4).No significant difference could be observed in Ash values of powders.More values for total Ash showed the presence of inorganic material such as silica, silicate etc. Extractive values also showed distinct variation.The water soluble extractive value of T.terrestris was more than P. murex powder.Alcohol soluble extractive values were same for both samples.Qualitative phytochemical analysis of whole plant extracts of T.terrestris and P.murex showed similar constituents like Alkaloids, Glycosides, Saponins , Tannins and Flavanoids.Estimation of phytochemical constituents confirmed more amount of Total Saponins in T.terrestris i.e. 16% while P. murex had 13% Total Saponins (Table 4).Fluorescence study of powders of both species also revealed distinguished characters (Table 7).Though in of the reagents the colour characteristics were similar, treatment with ammonium hydroxide, hydrochloric acid, methanol and chloroform showed diagnostic colour for powders of T.terrestris and P.murex when subjected to 254 nm and 366 nm which could be used as diagnostic characters.HPTLC profiles of methanol extracts of T.terrestris and P.murex and showed distinct characteristic with only one common spot with similar Rf value (Table 6, Fig. 3).When the plate was observed at 366nm T.terrestris shows 2 spots while 1 spot was found in P.murex.No spot was detected when plate was observed in short U.V. that is 254 nm.When derivatisation (Anisaldehyde spray reaction) was carried out for identification of Diosgenin in the sample, blue colored spot with Rf value 0.62 was observed in both varieties of Gokshur which were identical to standard Diosgenin spot.The colour reaction shows the presence of Diosgenin in Laghu Gokshur and Brihat Gokshur.Hence, TLC profile could be used as a diagnostic tool to identify the two varieties. 1).