Premilinary Phytochemical Study of Different Extracts of One Medicinal Plant Cissus quadrangularis

Herbal plants are the most valuable resources to prevent from many illness and to treat the many disorders in humans. Cissus quadrangularis is a one of the traditional medicine and belonging to vitaceae family. In Ayurvedha medicinal system this plant is used to treat many diseases like diabetes, snake bites, rheumatic pain, cardiovascular diseases. Cissus quadrangularis used as anticancer properties against MG63 human osteocarcinoma cells. Stem of the Cissus quadrangularis is an edible vegetable and the plant commonly called as ‘bone setter’. Our present study was analyzed the primary phyto constituents present the different solvents of Cissus quadrangularis and antioxidant activity. Three different extracts aqueous, methanol and ethanol were used to find out the phytoconstituents and antioxidant properties. The results were reveals that the primary and secondary phytoconstituents. Flavonoids and tannins were found to be higher in ethanol extracts, legnings, saponins was high in methanol extracts. Antioxidant study also reveals ethanol extracts shows more antioxidant activity than other two solvents.


INTRODUCTION
Plants are the valuable sources of food and medicine for the prevention and treatment of human aliments [1]. Since from ancient times, Cissus quadrangularis is easily available medicinal plant. It is a perennial climber, belonging to vitaceae family. It is commonly called as veldt grape or Devil's Backbone and 'bone setter'. This plant considered as a native plant for many tropical countries and found all over the world. The distribution of the plant more in tropical countries like india, srilanka, South Africa as 13 genera and 800 species. Especially in India 8 genera and 63 species have been identified [2]. Appearance of the plant stem resembles the shape of the bones and joints in our body. The stem of the plant is edible vegetable. All parts of this plant have important medicinal properties, and used to cure piles, bone fracture, weight loss , muscular pains, antiulcer, antimicrobial, anti hemorrhoidal, swelling, scurvy, gout, disease in ear and nose bleeding, diabetes [3]. Fresh shoot paste is used in burns and wounds. Cisus quadrangularis stem extract have the high impact on loss of body weight by inhibiting the oxidation of LDL cholesterol and by lowering the blood glucose on obese patients [4]. Ethanol extract of Cissus quadrangularis possess more antioxidant and anti cancer activity against bone cancer [5]. The medicinal properties of flavonoids,Tannins, proteins, phenolics, lignings, carbohydrates and saponins are found important secondary metabolites in Cissus quadrangularis extracts. Ethanol extract of CQ act as antioxidant, anticancer, antifungal and antiviral [6]. The current study was analyzed for phytochemical analysis and antioxidant activity of Cissus quadrangularis.

Sample Collection
Cissus quadrangularis stem were collected from erode. The collected plant material was identified and authenticated by Botanical survey of India, southern regional centre, Coimbatore. Voucher No. BSI/SRC/5/23/2020/Tech/799. The plant was identified and authenticated by agricultural university. Coimbatore. The voucher no. The stems bars were isolated from the whole plant and the separated stems were air dried under the shade. About 2 kg of dried samples was powered by using maceration method. The powdered samples were stored for this study.

Preparation of Extracts
The powder samples were subjected to extraction by hot percolation method with aqueous methanol and ethanol solvents in soxhlet apparatus. Each solvent extraction was carried out for 24 hrs. After the process the extracts were concentrated by rotary evaporator and stored at 4 o C. The stored extracts were used for further study [6].

Phytochemical Analysis
Phytochemical screening was carried out for the aqueous, methanol, and ethanol extracts of Cissus quadrangularis to evaluate the presence of phytochemicals such as phenolic groups, alkaloids groups, proteins, carbohydrates, fat and olis, legnins, tannins, quercetin.

Test for Alkaloids
Mayer's test was done to find out the alkaloids. In brief, 5 mg samples were taken in the fresh test tube, 1% HCL were added into sample. The solution was heated. Appearance of slight Red colour indicate the presence of alkaloids [7].
Wagners test -5 mg samples were added in the test tube and 0.5 ml of wagner's reagent were mixed with that sample and shaken gently. Appearance of slight brown colour will indicate the presence of alkaloid [7].

Test for Flavonoids
Shinoda test -5 mg extracts were pored into the test tube and minimal quality of magnesium were top up with in the solution and add few drops of concentrated H2SO4. The solution in the test tube pink in colour it indicate the presence of flavonoids [8].
Lead ethanoate test -Take 5mg of extract in the test tube, add lead ethanoate, 1-2 ml. The obtained solution gives buff coloured, it will indicate the flavonoid content(Test for Sodium hydroxide -1 ml of 10% of H2O2 were added with 5 mg of extract. The colour appearance of yellow colour after addition of 1 ml of diluted HCl, if the presence of alkaloids the colour should be changed from yellow to colourless after adding 2 ml of diluted HCL [8].

Test for Tannins
Test for Ferric chloride -Minimal amount (5 mg) of extract were mixes with 0.5 ml of ferric chloride. Appearance of black colour precipitate indicate the presence of tannins.
Gelatine test one of the main test done to identify tannins. 5 mg of sample were added into gelatin. After that 1 ml of H2O wre poured into it. Presence of white precipitate will indicate the tannins [9].

Test for Oil
Test for Stain -Minimal amount of sample were taken in the whatman filter paper. Roll the paper with that sample. Deposition of oil on the paper will indicate the presence of oil [9].

Test for Saponins
Saponins were identified with the help of Form test. 5 ml of distilled water were added with minimal amount of sample. The test tube were rotated well for proper mixing till foam was observed [10].

Test for Glycoside
Libermann's test -2 ml of chloroform and 2 ml of acetic acid were mixed with minimal amount of sample in the test tube. After get cooled, 1 ml of concentrated sulfuric acid were mixed with that solution. Colour changes wre observed from violet to green. Green colour indicate the presence of glycoside [8].
Salkowski's test -2 ml of chloroform were mixed with 1 ml of extract. Additionally 2 ml of concentrated H 2 SO 4 were added and shaken gently. A reddish brown colour will indicate the presence of glycoside [8].

Steroid Test
About 5 ml of extract were added with 1 ml of chloroform then one or two drops of concentrated sulfuric acid and acetic acid was poured in it. The green colour indicate the presence of steroids [8].

Test for Proteins
Biuret's testabout 5 ml of extract were mixed with some drops of biuret's reagent. The solution were mixed well and allowed to heat 1-5 minutes.
Appearance violet colour will indicate the presence of proteins.
Million's test -2 ml of Million's reagent were added with 5 ml of extract. The solution was warmed for 5 min. Appearance of red colour precipitation will indicate the presence of proteins [8].

Test for Lignins
Extracted sample materials was oven dried at 24 h before acid hydrolysis. 350 g of extracts were taken in a glass beaker along with 72% sulfuric acid 3 ml were added. The mixer were shaken at room climate for 1 h. After this pre-hydrolysis stage, the medium were diluted by adding 84 ml of distilled water. The reaction mixer was heated to boiling the reaction was maintained under reflux for 4 h. At the end of the process, the flask cooled to room temperature and the content was filtered by using whatman filter paper no1. Lignin retained with 100 ml of distilled water and dried in an oven at 103°C for 48 h [9]. Same procedure was done for all the three solvents.

Test for Phenolic Compounds
The sample were dissolved in the mixture of alcohol and 1 drop of neutral ferric chloride.The obtained solution intense colour indicates the presence of phenolic compounds [8].

Carbohydrates Test
Benedict's test -few drops of benedict's reagent were taken in a test tube and 5 mg extract were added and obtained solution was heated. Reddish brown precipitate will indicate the presence of the carbohydrates [11].
Molisch's test -Minimal amount of extract were taken in test tube, 1 ml of Molisch's reagent were added into it. The solution were shaked well. 2 ml of concentrated H2SO4 were poured carefully along the sides of the test tube. Appearance of a violet ring at the interface indicated the presence of carbohydrates [11].

Test for Antioxidant
The antioxidant activity of the all the three extracts were evaluated according to the procedure by prieto et al. In brief, 2 ml of extract was taken at dissimilar concentrations (100,200,300,400 and 500 µg) and mixed with 1 ml of standard reagent 0.6 M sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate. Then reaction mixture was incubated at 95 o C for 90 min. Absorbance of all the samples was measured at 635 nm [12]. The same test was repeated for all the three solvents.

Phytoconstituents Screening
Phytochemical analysis discovered the presence of many important phytochemical constituents such as glycosides, flavonoids, saponins, tannins, aminoacids, proteins, fat and oil, lignins (

DPPH free radical scavenging
NazninAra and HasanNur method were used for the present study. The effects of these extract to scanvenge DPPH radical were determined by this method. The absorbance were read at 518 nm.
Antioxidant activity (%)= Abs control -Abs sample/ Abs controlx100 Abs control-Optimal density of control Abs sample-Optimal density of sample extract.

FRAP assay
Total antioxidant activity for this study were measured by ferric reducing antioxidant power assay of Benzie and stain. The FRAP assay, is presented as a novel method for analyzing antioxidant power. Absorbance at 593 nm were measured. FRAP value of sample (µM)= change in absorbance of sample from 0 to 5 mints/change in absorbance of standard from 0 to 5 mints x FRAP value of standard (1000 µM) [12].

Antioxidant study
Cissus quadrangularis have been identified to possess a lot of antioxidant properties which enable their extracts and active principles such as oxidative stress, free radical scavenging activities [11]. In this study three different solvents extracts were investigated for their radical scavenging activity and their results were shows in Fig. 1. Methanol extracts contains more Lignins + + +

(+) -present; (-) -absent
phenolics and flavonoids. This secondary phytochemicals have more antioxidant properties. Ethanol and aqueous extracts shows relatively low antioxidant activity when compare with the methanol extracts. Saponins act as a protective agent for the plant such that it is called plant protector, it also contains more antioxidant properties [13]. Cissus quadrangularis can act on the lipid peroxidation to lower their levels of enzymes having antioxidant properties.

Discussion
Cissus quadrangularis extracts contains more useful and powerful phytoconstituents, they are used to treat bone related many diseases and anti diabetic drug [14]. Recently the same extracts were used for infertility study purpose [15]. Due to high amount of antioxidant properties, the plant Cissus quadrangularis extracts were used for infertility study [16]. The exact mechanism behind the extracts need to be explored.

RESEARCH SIGNIFICANCE
The study highlights the efficacy of "Ayurvedha" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.