To investigate the phytochemical constituents present in the Glycyrrhiza glabra stolon and root and estimation of the total phenolic content in ethanolic and aqueous extract. The amount of total phenols was analyzed using a spectrophotometric technique, based on Folin Ciocalteau reagent using gallic acid as standard. The standard curve equation was y=0.007x+0.186 and R2=0.992. The presence of alkaloids, carbohydrates, flavonoids, glycosides, tannins, proteins, phenols, sterols, and resins was revealed by the qualitative examination of Glycyrrhiza glabra stolon and root. The phenolic content in alcoholic and aqueous extract was found to be 244.85 and 232.0 mg/g gallic acid equivalent (GAE) respectively.
Keywords: Phytochemical screening, Total phenolic content, Spectrophotometer, Glycyrrhiza glabra, Gallic acid.

INTRODUCTION

Asl-us-soos is the dried unpeeled stolon and root of the plant Glycyrrhiza glabra (Anonymous, 2007). The word Glycyrrhiza has been derived from two Greek words “ Glykys” means sweet and “Rhiza” means root, thus the meaning of Glycyrrhiza is sweet root .Common name of Glycyrrhiza glabra Linn is liquorice , belonging to the family Leguminosae (Chopra et al., 2002).
G. glabra is grown in Mediterranean countries like South Europe, Asia, Egypt, Turkistan, Iran, and India. In India it is cultivated in Baramulla, Srinagar, Jammu, Dehradun, Delhi and South India. It is mostly found in the sub-tropical and warm climate (Ross, 2003).
Asl-us-soos is a tall, up to 2m high perennial herb or under shrub of pea family. Leaflets are oval, leaves multifoliate, imparipinnate; flowers in axillary spikes, papilionaceous, lavender to violet in colour; pods compressed, containing reinform seeds. Root system is taproot, which is soft, fibrous, and has a bright yellow interior, harvested for medicinal purposes (Ross, 2006; Bakshi, 2001).
The rhizomes are considered to possess an expectorant (Kataria, 2013), carminative((Anonymous, 2007), antimicrobial (Anagha et al., 2014), antiasthmatic, antidiuretic, antihyperlipidemic (Ross, 2006), anticonvulsant (Ambawade, 2002), antiviral (Ovais Omer et al., 2014), anti-inflammatory(Harwansh et al., 2011), antimutagenic( Sharma et al. , 2014) and antioxidant (Sharma and Agrawal, 2014; Lakshmi and Geetha, 2011) activity. Apart from this the roots are sweet, refrigerant, emetic in large dose, tonic, mild laxative, aphrodisiac, haemostatic, hypotensive and hepato protective (Sharma and Agrawal, 2014; Al-Razzuqi et al., 2012; Anonymous, 2007; Chopra et al., 2002). They are also useful in sore throat, cough, bronchitis (Bakshi et al., 2001; Kataria et al., 2013), ulceration of urinary tract, pharyngitis, epilepsy and anaemia(Harwansh et al., 2011; Trease and Evans, 2009; ).
The chemical studies have shown that Glycyrrhiza glabra Linn contains triterpene, saponins, flavonoids, polysaccharides, pectins, simple sugars, amino acids, mineral salts, and various other substances (Obolentsevaet al. 1999). A triterpenoid compound Glycyrrhizin is responsible for the sweet taste of Glycyrrhiza glabra Linn rootwhile its yellow color is due to the flavonoid content of the plant, which includes liquiritin and a chalcone isoliquiritin (Yamamura et al., 1992). The isoflavones glabridin and hispaglabridins A and B give significant antioxidant activity to G. glabra (Vaya, 1997). The contains glabridin and glabrene which possess estrogen-like activity (Tamir et al., 2001).Thus keeping in mind the medicinal properties of Asl-us-soos (Glycyrrhiza glabra Linn), in the present paper the Preliminary phytochemical analysis was carried out for the identification of carbohydrates, proteins & amino acids, glycosides, alkaloids, saponins, polyphenols, phytosterols, flavonoids and resins(Trease and Evans , 2009) and attempts were made to estimate the total phenolic content in ethanolic and aqueous extracts of Glycyrrhiza glabra Linn.

MATERIALS AND METHODS

Plant material
The roots of Asl-us-soos (Glycyrrhiza glabra Linn) were procured from the local market of Baradari, Aligarh and identified by the botanical literature available and then authenticated in the pharmacognosy section of the department of Ilmul Advia, AMU, Aligarh.
Phytochemical Evaluation
Glycyrrhiza glabra Linn was qualitatively analyzed for the presence of various chemical constituents, according to the scheme proposed by Bhattacharjee and Das (Bhattacharjee and Das, 1969).
Test for Alkaloids: A drop of Dragendroff?s reagent in the extract was added. The reddish brown precipitate showed the presence of alkaloids.
Test for Carbohydrates / Sugars
Fehling’s solution Test: In the aqueous extract, a mixture of equal parts of Fehling?s solution A and B previously mixed was added and heated. A brick red precipitate of cuprous oxide indicates the presence of reducing sugars.
Molisch’s test: In an aqueous solution, a-naphthol was added. Afterwards, concentrated sulphuric acid was gently poured from the sides of the test tube. A purple colour ring at the junction of the two solutions indicates the presence of the reducing sugars.
Test for Flavonoids: Magnesium ribbon was added to the ethanolic extract of the material followed by drop wise addition of concentrated Hcl. Colour change to pink or red is a confirmatory test for flavonoids.
Test for Glycosides: The test solution is to be filtered and sugar is removed by fermentation with baker?s yeast. The acid is removed by precipitation with magnesium oxide or barium hydroxide. The remaining ethanolic extract contains the glycosides which are subsequently detected by the following methods.
a. The hydrolysis of the solution is to be done with concentrated sulphuric acid and after the hydrolysis sugar is determined with the help of Fehling?s solutions.
b. The Molisch?s test is done for sugar using a-napthol and concentrated sulphuric acid.
Test for Tannins: Ferric chloride solution was added in the aqueous extract of the drug. A bluish-black colour, which disappeared on addition of dilute sulphuric acid followed by a yellowish brown precipitate, shows the presence of tannin.
Test for Proteins:
Millon’s reaction: For the test solution, Millon?s reagent was mixed and white coloured precipitate showed the presence of proteins.
Test for Starch: 0.015 gm of Iodine and 0.015 gm of Potassium Iodide was added in 5 ml of distilled water, 2 ml of this solution formed was added to 2 ml of aqueous test solution, the presence of blue colour indicates the presence of starch.
Test for Phenol: 5–8 drops of 1% aqueous solution of Lead acetate was added to aqueous or ethanolic test solution. The presence of yellow coloured precipitate indicates the presence of phenols.
Test for Sterol/Terpenes: Salkowski reaction: In the test solution of chloroform 2 ml sulphuric acid (concentrated) was mixed from the sides of the test tube. The colour of the ring at the junction of the two layers was observed. A red colour ring indicates the presence of the sterols/terpenes.
Test for Amino Acids: The ethanolic extract was mixed with ninhydrin solution (0.1% in acetone). After heating gently on water bath for few minutes it gives a blue to red-violet colour that indicates the presence of amino acids.
Test for Resins
The test solution was gently heated and acetic anhydride was added in it. After cooling, one drop of sulphuric acid was mixed. A purplish red colour that rapidly changed to violet indicates the presence of the resins.
Preparation of extract
The aqueous and alcoholic extract of the test drug was obtained by refluxing 10 gram powdered drug with150 ml distilled water & absolute alcohol respectively for six hours and then filtered and freeze dried in the lypholizer. Stock solutions prepared at a concentration of1gm/100ml and subjected to spectrophotometric procedures for determination of total phenolic content.
Determination of total phenolic content
Total phenolic content in aqueous and ethanolic extract of Gul-e-Zoofa was estimated by Folin Ciocalteau reagent as method described by Singleton & Rossi (Singleton and Rossi, 1965). Calibration curve (fig 1) was plotted by mixing 1 ml aliquots of 50,100, 150, 200, 250, 300, 350, 400 and 450µg/ml of gallic acid solutions with 5.0 ml of Folin Ciocalteu reagent (diluted tenfold) and 4.0 ml of sodium carbonate solution (75g/l). The absorbance was measured after 30 minutes at 765nm. One ml of aqueous and ethanolic extract (1gm/100ml) was mixed separately with the same reagents as did in construction of Calibration Curve, and after 1 hour the absorbance was measured for the determination of total phenolic compound in both the extracts separately by using formula.
C=C1×V/m
where, C= Total phenolic content in mg/g, in GAE (Gallic acid equivalent)
C1=Conc of gallic acid established from the calibration curve in mg/ml
V=Volume of extract in ml
m=The weight of plant extract in gram

RESULTS

Qualitative analysis of organic chemical constituents of drug
The Phytochemicals present in the drug were identified on the basis of different chemical tests given for various plant constituents (Overtone, 1963; Harborne, 1973) in various extracts, results have been summarized in table-1.

Estimation of total phenolic content
The phytochemical screening of various extracts of Glycyrrhiza glabra was carried out and ethanolic as well as aqueous both the extracts showed the presence of phenolics. So, the amount of total phenolics was determined by Folin Ciocalteu method in terms of gallic acid equivalent in mg/g of the extract.

The phenolic content was calculated with the help of graph (fig 1) & standard curve equation was y=0.007x+0.186. The total phenolic content in ethanolic and aqueous extract was calculated as 244.85 and 232.0 mg/g gallic acid equivalents respectively.

DISCUSSION
Phytochemical screening is helpful to know the chemical constituents present in the drug. On phytochemical screening of the alcoholic extract the alkaloids, carbohydrates, flavonoids, glycosides, tannins, phenols, proteins, saponins, steroids and resins were found to be present. As the presence of phenols was confirmed qualitatively, their amount was determined by Folin Ciocalteu reagent method in terms of gallic acid equivalent in mg/g of the extract. The total phenolic content in ethanolic and aqueous extract was calculated as 244.85 and 232.0 mg/g gallic acid equivalents respectively. The total phenolic estimation will be helpful in development of new drug and standardization of the drug. In addition, this shows that the stolon and root of the plant Glycyrrhiza glabra may possess antioxidant property (Saha and Verma, 2014) as they contain phenols in a sufficient amount.

CONCLUSION
The quantitative analysis of the extracts of the test drug showed the presence of alkaloids, carbohydrates, flavonoids, glycosides, tannins, phenols, proteins, saponins, steroids and resins. The total phenolic content in ethanolic and aqueous extract was calculated as 244.85 and 232.0 mg/g gallic acid equivalents respectively using Folin Ciocalteu reagent successfully.

ACKNOWLEDGEMENT
The authors are grateful to D/o Ilmul Advia and DRS-I (UGC) programme of Department of Ilmul Advia for providing assistance during the study.

CONFLICT OF INTEREST
None declared.

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