Qualitative and Quantitative Analysis of Phytochemicals of Cyperus corymbosus Rottb Rhizome

The present study was aimed to analyze phytochemicals present in rhizome of Cyperus corymbosus Rottb. The rhizome of the plant was collected from their natural habitat from the river bank of Cauvery, Mandya and Mysore, India. Dried rhizome powder was subjected to sequential solvent extraction with increasing polarity. Various solvent extracts of rhizome found to contain different phytochemicals to various extents. Acetone and methanol extracts were rich in phenols, flavonoids, alkaloids, saponins, terpenoids, and Tannin. Cardiac glycosides recorded highest in ethyl acetate extract and water extract was rich in saponins and phlobatannins. Anthraquinones was found equally distributed in ethyl acetate, acetone, and methanol extracts. Quantitative analysis revealed that methanol extract contains the highest total phenol (0.615 mg GAE/mg extract) and flavonoid content (0.23 mg QCTE/mg extract) was highest in acetone extract, and total Tannins content (0.089 mg TAE/mg extract) was found highest in the methanol extract. Further, isolation of phytochemicals present in different solvent extracts of C. corymbosus rhizome will pay the way for significant application in medicinal and pharmaceutical industries.


INTRODUCTION
Plant-derived secondary metabolites are highly diverse in their structure and function. Though these secondary metabolites are not involved in the primary function, their timely expression enhances the fitness of plants in nature. These plant metabolites provide either protection against various pest and pathogens or climatic stress or even help for pollination or even to improve their adaptation abilities. Today, phytochemicals are widely used in medicines for their high effectiveness and low toxicity 1 . The phytochemicals also have tremendous application in agriculture, cosmetics and food too, apart from medicine 2,3,4 . The potential of these infinite pools of metabolites is yet to be explored.
Among the several phytochemical sources, the family Cyperaceae and genus Cyperus was one of the vital one, which possess highly valuable phytochemicals. Among them, C. rotundus is a multivalent drug plant possessing phytochemicals of pharmacological properties like, analgesic, antibacterial, antidiarrheal, antidiabetic, anti-inflammatory, antioxidant, antipyretic, antisaturative, appetizer, diaphoretic, digestant, lactodepurant, thirst relieving and tranquilizing effect 5,6,7,8,9 . The C. rotundus studied for phytochemicals revealed the presence of sesquiterpenes, flavonoids, phenylpropanoids, phenolic acids, alkaloids, and saponins 10 . The C. rotundus rhizomes analysis revealed the presence of alkaloids, carbohydrates, glycosides, steroids, saponins, resins, tannins and phenols in its various solvents extracts 10 . The total oligomeric flavonoids endowed in rhizome extract of C. rotundus exhibits a broad spectrum of biological properties such as antimicrobial, antioxidant, antimutagenic, antigenotoxic, anticancer and neuroprotective properties 11 .
One of the problems that existed with C. rotundus is availability of biomass for large scale extraction. On the other hand, several related species with high biomass producing capability exist. But a detailed scientific study is required to use these plants as an alternative to C. rotundus. One such neglected plant which produces higher biomass compared to C. rotundus is C. corymbosus. Hence, in the present study, we performed preliminary work on the phytochemicals of C. corymbosus rhizome.

MATERIAL AND METHODS Collection and identification of plant materials
Cyperus corymbosus found growing naturally on the river banks of Cauvery in and around Mandya and Mysuru districts of Karnataka were collected during January 2015 and one kg of rhizome samples were collected (Fig. 1). The plant specimen was identified by Dr. Sampath kumara, K. K., taxonomist, lecturer in Biology, Govt. P. U. College, Davangere.
Further, the plant identity was authenticated by sending the herbarium to Botanical Survey of India (BSI), Howrah, West Bengal, India. Extraction of phytochemicals from the rhizome Surface of rhizomes was cleaned by washed under tap water, blot dried and used for further process. Surface dried rhizomes are cut into small pieces (4-6 mm) and dried at 45°C until a constant weight was attained. The dried samples were coarse powdered in a mechanical blender and stored at the dry and cool place until further use. The powdered samples were taken in timble and used for extraction using Soxhlet apparatus with different solvents with increasing polarity [hexane (0), benzene (3.0), ethyl acetate (4.3), chloroform (4.4), acetone (5.4), methanol (6.6) and water (9.0)]. The extracts are concentrated using Rota vapor and used for further studies.

Qualitative phytochemical analysis
The bioactive constituents present in different solvent extracts of rhizome was qualitatively analysed following standard procedures as described by Harborne 12 , Trease and Evans 13 and Sofowara 14 . Alkaloids: 8 ml of HCl (1%) was added to plant extracts (100 mg), mixed, warmed and filtered. The filtrate (2 ml) was treated with Dragendorff's reagents. The solution was observed for the formation of brownish red precipitate, which specifies the presence of alkaloids. Flavonoids: Dilute ammonia (5 ml) (1%) was added to plant extract (100 mg), mixed well and few drops of concentrated sulphuric acid was added. The solution was observed for the formation of yellow coloration which specifies the presence of flavonoids. Saponins: 5 ml of distilled water was added to 200 mg of plant extracts, mixed and filtered. 500 µl of filtrate was withdrawn to a test tube and makeup to 5 ml with distilled water. The test tube was shaken vigorously for 2 min. the test tube was observed for the formation of stable foam which specifies the presence of saponins. Phenols: The different solvent extracts (100 mg) were added to a test tube and mixed with 2 ml of distilled water. To this few drops of aqueous ferric chloride (10%) solution was added. The solution was observed for the formation of green, purple, deep blue or black colour shows the existence of phenols. Terpenoids: Plant extracts (100 mg) was mixed with 2 ml chloroform. To this solution few drops of concentrated sulfuric acid was carefully added. The solution was observed for the development of a layer of the reddishbrown coloration which specifies the presence of terpenoids in extracts. Anthraquinones: Plant extracts (500 mg) is boiled 6 ml of HCl (1%) and filtered. To the filtrate, benzene (5 ml) was added and shaken well, and benzene layer was removed. To this solution 3 ml of NH 4 OH (10 %), and observed for the formation of pink or violet or red colour in alkaline phase with specifies the presence of anthraquinones. Cardiac glycosides: Plant extracts (5 ml) was added to premixed solution containing glacial acetic acid (2 ml) and one drop of ferric chloride (FeCl 3 ). To this solution, 1 ml concentrated Sulphuric acid was added. The solution was observed for formation of brown ring at the interface which specifies the presence of deoxysugar of cardenoloides. Similarly, formation violet and brown ring specify the presence of cardiac glycosides. Phlobatannins: Plant extracts (200 mg) dissolved in water (2 ml) was boiled with (few drops) 1% aqueous hydrochloric acid. The solution was observed for the formation of red precipitate, thus specifying the presence of phlobatannins. Tannins: Plant extracts (250 mg) was dissolved in distilled water (10 ml) and filtered. To this solution few drops of aqueous Iron chloride (FeCl 3 ) solution (1%) (Few drops) was added. Development of intense green, purple, blue, or black colour specifies the presence of tannins. Quantitative analysis of phytochemicals Cyperus corymbosus rhizome extracts (RE) were dissolved in a common solvent, methanol except water extract which was dissolved in water to prepare stock solutions of 2 mg/ml. Further double dilutions were made to obtain different concentrations.

Estimation of Total Phenols
The total phenol was estimated, according to Abirami et al. 15 , with minor modification. The Gallic acid was used as standard. 10 µl of different dilutions of sample was taken in a 5 ml test tube and 50 µl of Folin-Ciocalter reagent (FC reagent) (1 mol/L) was added, mixed. To this solution, 2 ml of distilled water was added, mixed well and incubated for 3 minutes. Further, 500 µl of 20 % sodium carbonate solution was added and mixed well by vortexing. The reaction mixture was then incubated for 40 min in the dark and absorbance was read at 765 nm with UV-vis spectrophotometer. The total phenolic content of different solvent extracts of plant was expressed equivalents of Gallic acid (mg GAE/mg extract).

Estimation of Total Flavonoids
The flavonoid content was estimated, according to Abirami et al. 15 with minor modification. The rhizome extracts was mixed with AlCl 3 (2 %) in methanol in 1:1 ratio. The reaction mixture was incubated for 15 min at 30°C. The absorbance of the samples were read at 415 nm. The total flavonoid content was expressed as equivalents of Quercetin (mg QCTEs/mg extract).

Estimation of Tannins
The tannin content was determined according to Muthukumar et al. 16 . 100 µl of each solvent extract of rhizome were taken and made up to 7 ml with distilled water. To this solution, potassium ferric cyanide (8 mM) and ferric chloride (20 mM) prepared in 0.1 M hydrochloric acid was added. The contents were mixed and absorbance was measured at 700 nm. The Tannic acid (TA) was used has standard. The Tannin content was expressed equivalents of TA (mg TAE/mg extract).

Statistics
The data obtained from the experiments were statistically analyzed by subjecting data's to Analysis of Variance (ANOVA) using SPSS V21 software (SPSS Inc., Chicago IL). The significant difference between the means was compared using the highest significant difference (HSD) as obtained by Tukey's-b test at p < 0.05 level.

RESULTS AND DISCUSSION
The herbal traditional medicine is the oldest medicinal practice of the world. The WHO in 1985 estimated that 80% of the world inhabitants were dependent primarily on traditional medicines for their primary health care facilities 17 . The crude drug was prepared from the rhizomes of C. corymbosus used for birth control processes in indigenous medicines 18 .

Identification of plant materials and its solvent extractions
One Kg of plant specimens was collected from the river bank of Cauvery in Mandya and Mysore districts of Karnataka. Upon processing, it yielded 400 gm dried coarse powder. The rhizomes powder was subjected to phytochemical extractions using different solvents of increasing polarity (Fig. 2). The different solvent extracts were then subjected to further analysis. Qualitative phytochemical analysis Presence of alkaloids, carbohydrates, phenols, glycosides, phytosterols, etc. in ethanolic extracts of leaves of C. rotundus was reported by Elezabeth and Arumugam 19 . Several early researchers recorded the presence of flavonoids, tannins, glycosides, monoterpenes, sesquiterpenes, sitosterol, saponins, terpenoids, starch and proteins in C. rotundus rhizome 20,21,22,23,24 . In our study, Different solvent extracts of rhizome found to contain different phytochemicals to various extents. Acetone and methanol extracts were found positive for all the metabolites. They were rich in alkaloids, flavonoids, saponins, phenols, terpenoids, and Tannin. Cardiac glycosides recorded highest in ethyl acetate extract, and water extract was rich in saponins and phlobatannins. Anthraquinones was found equally distributed in ethyl acetate, acetone, and methanol extracts (Table 1).

Quantitative analysis of phytochemicals
Among the different extracts, Methanol extract was found rich in total phenol content (0.615mg GAE/mg extract) followed by Acetone extract (0.577mg GAE/mg extract). Water extract was found to contain least total phenol content of 0.127 mg GAE/mg extract (Fig. 3). Similarly, Acetone and Methanol extracts were also found to contain the highest flavonoid content of 0.23mg QCTE/mg extract and 0.17 mg QCTE/mg extract, respectively (Fig. 4). Highest of 0.089 mg TAE/mg extract of tannin was recorded in Methanol extract followed by Acetone extract (0.0689 mg TAE/mg extract) (Fig 5). In all the three cases, the benzene extract was found least in metabolites. Bashir et al. 25 reported antioxidant activity from flavonoids in different parts of C. rotundus L. by using methanol and ethanol solvents extraction. The total flavonoids content of C. rotundus rhizome solvents extracts varied from 7.196 to 200.654 µg Quercetin (QE)/mg, where acetone extract showed the higher value of total flavonoids content 10 .
Flavonoids, tannins, and polyphenols were identified in these extracts. Further, the extracts were found to decrease the ear oedema in mouse, abdominal contractions in mice, the peripheral analgesic activity of extracts significantly enhance lymphocyte proliferation. It also gives clues that analgesic, anti-inflammatory, antioxidant, and immunomodulatory effects of the extracts may be attributed to the presence of flavonoid, tannin, and polyphenol contents 26 .