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Capparis decidua (Forsk.) Edgew (Capparidaceae) is a wild, self-growing shrub mainly found in the Northern parts of India, mostly in the hot arid regions of western Rajasthan. Besides having many socioeconomic and ecological importance, nearly all parts of this plant possess one or many medicinal properties. The green fruits, locally known as Kair, are used as a vegetable (in food preparations like ‘panchkuta’), in pickles and fully ripe fruits are also consumed fresh as they are sweet in taste (Baloda & Bangarwa, 2010).

Capparis decidua is a part of many traditional herbal medicines to cure asthma, cough, toothache, malaria, inflammation, intermittent fevers, rheumatism and swelling (Singh & Singh, 2011; & Mann et al., 2013). C. decidua is also well known for its antibacterial, antifungal (Keymanesh et al., 2009; & Gull et al., 2015), antihemolytic (Vaishnav et al., 2015) and antioxidant properties (Zia-Ul-Haq et al., 2011). The fruits and the seeds are claimed to possess antidiabetic and diuretic properties as well as they are used to cure cholera, dysentery and urinary tract disorders (Rathee et al., 2010; & Zia-Ul-Haq et al., 2011). Antiarthritic potential of Capparis decidua stem, root and leaves on rats have been reported by Kumar et al. 2017. The green small sized immature fruits possess antihelminthic properties (kills intestinal worms) and used to cure many digestive tract disorders like constipation and piles acting as a laxative (Goyal & Grewal, 2003; Joseph & Jini, 2011; & Singh et al., 2011). C. decidua fruit extract possesses hypolipidemic (Yadav et al., 1997; & Chahlia, 2009) and antiatherosclerotic properties (Purohit & Vyas, 2006). 
            The chemical makeup of a plant determines its uses in the medicinal or pharmacological field. Several phytochemical and pharmacological investigations have been carried out on C. decidua to evaluate various Sterols, alkaloids, fatty acids, flavones, oxygenated heterocyclic constituents etc. found in the plant (Sharma & Kumar, 2008; Rathee et al., 2010; & Shad et al., 2014). An isothiocyanate glucoside was also reported from different parts of this plant (Juneja et al., 1970). The nutritional value of flowers and fruits of C. decidua has also been reported (Chauhan et al., 1986; Dahot et al., 1993; & Alrasheid et al., 2018). Pharmacological properties have been depicted by different extracts of this plant (Chishty & Monika, 2016). The plant extract has also been reported for its activity as sedative and Central Nervous System depressant (Goyal et al., 2009). Anti-inflammatory activity of the extracts of the fruits of C. decidua has been experimentally proven on mice (Zhou et al., 2010). Methanol and water extracts of C. decidua stems were reported to possess hepatoprotective activity (Ali et al, 2011). C. decidua extracts have been reported to be more potent than C. spinosa extracts in terms of their anti-microbial activity (Gull et al., 2015). Owing to the presence of a saga of phenomenal compounds present in C. decidua, its economic importance has increased proportionately and it can be regarded as a miracle tree (Nazar et al., 2020).
            Zia-Ul-Haq et al. 2011 studied the proximate composition of tocopherols, sterols, glucosinolate and total phenolic content in the extract of aerial parts of C. decidua and have reported its antidiabetic and antioxidant activity. Work on changes in the total phenolic content and total antioxidant activity of kair fruits during different stages of maturity was reported by Kumar, 2018.
            The present study emphasizes on preliminary phytochemical screening for qualitative examination, estimation of proximate values of total phenol content (TPC), total flavonoid content (TFC) and quantitative estimation of rutin and quercetin in the methanolic extract of fruits of Capparis deciduausing RP-HPLC technique.

MATERIALS AND METHODS

Sample collection
Fruit samples were collected from the fully grown shrubs of Capparis decidua from the Forest Ecology Experimental Field of Arid Forest Research Institute, Jodhpur. Collection was done in the months of April and May (summer season) in the years 2016, 2017 and 2018.
Chemicals used
All the chemicals used in the experiment were of Analytical grade. HPLC grade Methanol, Ortho-Phosphoric Acid, Triethylamine (TEA) and Pure water was procured from Merck. Standards (high-purity i.e. >95% pure) viz. Quercetin, Rutin, Gallic acid were procured from SIGMA. 
Sample extraction
Fruits of C. decidua were air-dried and ground using a mixer grinder. The crude powdered fruits (100 g) were exhaustively extracted in methanol using Soxhlet apparatus for about 48 Hrs. Then they were dried and made solvent free by using Rotary Vacuum Evaporator (make- EYELA). This sample extract was ready for further analysis.
Experimental
Phytochemical screening
Various standard methods (Harborne, 1998; Brunton, 1995; & Evans, 2009) were used to qualitatively detect the presence or absence of specific secondary metabolites in the fruit extract.
Test for Terpenoids: 5 ml of the sample extract was taken, to which 2 ml of CHCl3 was added in a test tube. Then 3 ml of concentrated sulfuric acid was carefully added to the reaction mixture. No interface of reddish-brown coloration confirmed the absence of terpenoids.
Test for Sterols (Salkowski’s test): 2ml of extract was mixed with 2 ml of chloroform. Then, 2 ml of concentrated Sulphuric acid was added, which formed a layer and a reddish/brown color interface was observed which reflects positive results for the presence of sterols.
Test for alkaloids: A reddish-brown or orange red precipitation after the addition of few drops of Wagner’s reagents or Dragendorff’s reagent is considered as a positive test for alkaloids.
Test for flavonoids: Sodium hydroxide test and Shinoda test were used for the presence of flavonoid. Precipitation of yellow coloration formed after the addition of 2ml of 10% aqueous sodium hydroxide solution indicates the presence of flavonoids. This yellow color turns into colorless upon addition of dilute hydrochloric acid to it. In case of Shinoda test, when few pieces of magnesium chips are added along with 2 drops of concentrated HCl in sample extract appearance of a red or pink color marks the presence of flavonoids.
Test for Tannins: Brownish green or a blue-black coloration obtained from the addition of 2-3 drops of 5% ferric chloride solution to the extract confirms the presence of tannin.
Test for Phenolic compound: Three drops from a freshly prepared mixture containing equal amount of 1% ferric chloride solution and 1% potassium ferrocyanide was added to fruit extract. After filtering this solution, presence of a bluish-green color of the solution indicate positive results.
Test for Saponins (Froth test): 2.5 ml of extract was added to 10 ml of distilled water in a test tube. Then, this test tube was closed with cap and shaken vigorously for 30 second and allowed to stand still for about half an hour. No froth formation observed indicates the absence of saponins.
Test for Anthraquinones: To the extract added 1ml benzene, mixed with 1ml of 10% ammonia. No pink, red or violet color develops, which indicates the absence of anthraquinones.
Test for Quinones and Coumarins:  Addition of 10% NaOH into the test sample did not give blue green or red precipitation that confirms the absence of quinones whereas no yellow color developed from the same test indicates the absence of coumarin.
Total Phenol Content (TPC)
The total phenol content was determined using the Folin-Ciocalteu’s assay (Donald Mc et al., 2001) with Gallic acid as a reference standard. In this method, 0.5 ml of the plant extract was taken in a 10 ml volumetric flask, to which 1.5 ml of Folin- Ciocalteu’s reagent (FCR) which was pre-diluted to 1:10 v/v was added, five minutes later 1.5 ml of 7% sodium carbonate solution was added. The final volume was made up to 10 ml with distilled water and allowed to stand for 90 minutes at room temperature. Absorbance of the sample as well as standard was measured against the blank at 750 nm using a UV-Vis spectrophotometer (make- SPEKOL, 2000). The experiments were performed in triplicates to give more precise results. The values were expressed as mean ± standard deviation in terms of phenol content, mg GAE (Gallic acid equivalent) per g of dry weight (DW basis) of sample extract using a linear equation based on the calibration curve of standard Gallic acid with R2 (Regression coefficient) value of 0.9941.
Total Flavonoid Content (TFC)
Total flavonoid content was determined using Aluminium chloride method (Olajire et al., 2011), quercetin was taken as a reference standard. 1ml of the test sample and 4 ml of distilled water was added to a volumetric flask of 10 ml volume. To this, 0.3 ml of 5 % Sodium nitrite was added. After 5 minutes, 0.3 ml of 10% Aluminium chloride was added. Then, after 6 minutes, 1ml of 1 M Sodium hydroxide was added to the reaction mixture. Immediately the test solution was filled with distilled water to make final volume to 10 ml. Absorbance of the sample was measured against the blank using a UV-Vis spectrophotometer at 510 nm. Values were expressed as mean ± standard deviation in terms of flavonoid content, mg QE (Quercetin equivalent) per g of dry weight (DW basis) of sample extract based on the calibration curve of standard Quercetin with R2 (Regression coefficient) value of 0.9939.
Quantitative estimation of Quercetin and Rutin

A Waters HPLC system equipped with a Waters 2998 PDA detector and Gradient Pump Control Module with two pumps (WATERS, 515) was used for RP-HPLC analysis. Chromatographic separation was managed, chromatograms were recorded, and data were processed with the EMPOWER V.2.0 software. Chromatographic separations were carried out by using a Spherisorb ODS2 column (250 mm, 5 µm particle size). The column was operated at a constant temperature of 25°C. The volume of the extract being injected in the manual Rheodyne injector was 20 μL. The flow rate was 1 mL/min, and gradient elution was used. The mobile phase consisted of Methanol, o-phosphoric acid, TEA, Water. Standard Quercetin and Rutin solutions of concentration 100 ppm each separately were prepared in HPLC grade methanol and filtered through 0.45 micron HPLC filters before injecting into the WATERS HPLC system.

Programming Gradient method for HPLC:

RESULTS AND DISCUSSION

Phytochemical screening of Capparis decidua fruits

The methanolic extract yield obtained was 32.52±2.34 g per 100 g of crude powdered fruit sample. Qualitative analysis was done for phytochemical screening using the standard methods. The presence of Phenolic compounds, alkaloids, flavonoids, sterols and tannin was detected in the fruit extract of C. decidua.

Table 1: Phytochemical screening of Capparis decidua fruits