WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Neeru et al. SJIF Impact Factor 6.041 Volume 5, Issue 6, 1884-1891 Research Article ISSN 2278 4357 DETERMINATION OF TOTAL PHENOLIC CONTENT, TOTAL FLAVONOID CONTENT AND TOTAL ANTIOXIDANT CAPACITY OF DIFFERENT EXTRACTS OF ROYLEA ELEGANS WALL (AERIAL PARTS) Neeru 1*, Shilpi Kashyap 1, Esha Vatsa 2, Jitendra Singh 3 and Ankush Sundriyal 4 1 Himachal Institute of Pharmacy, Paonta Sahib District Sirmour. 2 Manav Bharti University, Solan, Himachal Pradesh. 3,4 SBSPGI, Balawala Dehradun. Article Received on 13 April 2016, Revised on 01 May 2016, Accepted on 22 May 2016 DOI: 10.20959/wjpps20166-7002 *Corresponding Author Neeru Himachal Institute of Pharmacy, Paonta Sahib District Sirmour. ABSTRACT Petroleum ether, chloroform, ethyl acetate, methanol and water s of Roylea elegans (aerial parts) was screened for assessing bioactive phytochemical constituents. The preliminary phytochemical screening using the standard phytochemical tests detected the presence of alkaloids, steroids, flavonoids, terpenoids and phenols in different solvent s. Total phenolic and flavonoid contents along with total antioxidant capacity of the ethyl acetate of Roylea elegans (aerial parts) was evaluated to explore the reliable and potential sources of novel natural antioxidants. The ethyl acetate showed high flavonoid content. Due to high flavonoid content it showed better antioxidant activity. KEYWORDS: Roylea elegans, Total phenolic content, total flavonoid content, antioxidant activity. INTRODUCTION In nature plants gives more products which are useful for human beings. These natural products are used for several diseases and also used as antioxidant. Plants give secondary metabolites which are used for several diseases. These secondary metabolites are alkaloids, glycosides, terpenoids, flavonoids, phenolic compound and tannins. These secondary metabolites are produced from various parts of plants like stem, leaves, flowers, fruits and www.wjpps.com Vol 5, Issue 6, 2016. 1884
roots. These secondary metabolites are biologically active and show better activity. Flavonoid and phenolic content shows better antioxidant activity. Roylea elegans a plant belonging to family lamiaceae shows better antioxidant activity due to presence of good flavonoid content. Roylea elegans is a shrub of monotypic genus. The family lamiaceae contain 22 species. Plant Roylea elegans belonging to family lamiaceae. Leaves part of this plant (decoction) is traditionally used as a bitter tonic and also as a febrifuge. It is also used as a tonic in contusions. Leaves are used in skin disease and fever. The leaves of Roylea elegans contains various phytoconstituents like betulin, beta-sitosterol, beta-amyrin, glucose, sti gmasterol, cetyl alcohol, and palmitic, stearic, oleic, gallic, oxalic, fructose, arabinose and tartaric acids. MATERIALS AND METHODS chemicals and reagents All the chemicals which were used in study are of analytical grade and purchased from the Himedia Lab. Pvt. Ltd, Rankem. Plant selection and identification The plant material (Roylea elegans) investigated in the present study was collected from Nauni, near Solan, Himachal Pradesh. The plant material was identified and authenticated at the Herbarium of Council of Scientific and Industrial Research - National Institute of Science Communication and Information Resources (CSIR-NISCAIR), Delhi vide reference no. NISCAIR/RHMD/Consult/2014/2781/160. Preparation of plant The dried aerials parts of plant were collected. Then aerial parts of the plant were pulverized through a mechanical grinder. Powder material was dried in hot air oven at moderate temperature. The Powdered material was subjected to successive Soxhlet ion by solvents in increasing order of polarity viz. Petroleum ether, chloroform, ethyl acetate then macerate with water for 24hour. The was concentrated and stored at 4 0 c until further use in the experiment (Chatwal 2011). Phytochemical evaluation of the www.wjpps.com Vol 5, Issue 6, 2016. 1885
The preliminary phytochemical screening of the aerial parts mainly done for the evaluation of the various phytoconstituents such as steroids, tannin, Alkaloids, Flavonoids and glycosides were present in the aerial parts of different s of R. elegans. Determination of total phenolics content The Total Phenolic content of of R. elegans was estimated according to the method described in Ayurvedic Pharmacopoeia of India. The total phenol content was expressed in milligrams of gallic acid equivalents per gram of. Prepare a stock solution (1mg/ml) of the in methanol. From the stock solution, take suitable quantity of the into 25ml volumetric flask and add 10 ml of water and 1.5ml of folin ciocalteau reagent. Keep the mixture for 5min and then add 4ml of 20% sodium carbonate solution and make up to 25ml with double distilled water Keep the mixture for 30min and record absorbance at 765nm. Calculate percentage of total phenolics from calibration curve of gallic acid prepared by using the above procedure and express total phenolics as percentage of gallic acid. [5] Total phenolic content was calculated with the help of standard curve equation and the formula was below Total phenolic content (%w/w)= GAE V D 10-6 100 W Where, GAE Gallic acid equivalent (µg/ml) V Total volume of sample (ml) D Dilution factor W Sample weight (gm) Determination of total flavonoid content The total flavonoid content of of R. elegans was estimated according to the aluminium chloride method as follows: The aluminium chloride method was used for the determination of the total flavonoid content of the sample s. Rutin was used to make the calibration curve. Ten milligrams of rutin was dissolved in 80% ethanol and then diluted to 25, 50 and 100 μg/ml. The standard solutions (0.5 ml) were prepared by mixing 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. The absorbance of the reaction mixture was measured after 30 min at 415 nm. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. [11] www.wjpps.com Vol 5, Issue 6, 2016. 1886
Total flavonoid content was calculated with standard curve equation and the formula Total flavonoid content (% w/w)= RE V D 10-6 100 W Where, RE Rutin equivalent (µg/ml) V Total volume of sample (ml) D Dilution factor W Weight of sample (gm) Antioxidant activity DPPH DPPH will be assayed as described by Elizabeth and Rao. The reaction mixture contained 1.0 ml of 0.3mM DPPH in 50 ml of methanol will be added with concentrations ranging from 20-100 μg/ml.the mixture of DPPH in methanol used as positive control and methanol alone served as blank. When DPPH reacts with antioxidants in the sample, it was reduced and the colour changed from deep violet to light yellow and measured at 517 nm. Quercetin will be used as a reference standard. Scavenging activity (%) = [A517 (control) A517 (sample)/ A517 (control)] X 100. [4] RESULT AND DISCUSSION Table 1: Phytochemical screening of different s of Roylea elegans Wall. (aerial parts) S. no. Phytochemical tests Pet. Ether Chloroform Ethyl acetate Methanol Water 1. Carbohydrate - - - - - 2. Protein - - - - - 3. Amino acid - - - + + 4. Fats + - - - - 5. Steroid - - + - - 6. Triterpenoides + - - - - 7. Glycosides - - - - - 8. Flavonoids - - + + + 9. Alkaloids - + - - + 10. Tannins - - - - + Total Phenolic Content Table 2: Standard curve of gallic acid S. no. Concentration(µg/ml) Absorbance www.wjpps.com Vol 5, Issue 6, 2016. 1887
1. 0.1 0.292 2. 0.2 0.329 3. 0.4 0.402 4. 0.8 0.614 5. 1.6 0.869 Figure 1: Standard curve of gallic acid Table 3: Total phenolic content in different s S. no. Extracts Total phenolic content (%w/w) 1. Petroleum ether 0.0887 2. Chloroform 0.267 3. Ethyl acetate 0.416 4. Methanol 0.565 5. Water 0.737 Figure 2: Total phenolic content Total Flavonoid Content Table 4: Standard curve of rutin www.wjpps.com Vol 5, Issue 6, 2016. 1888
S. no. Concentration (µg/ml) Absorbance 1. 25 0.103 2. 50 0.180 3. 100 0.344 Figure 3: Standard curve of Rutin Table 5: Total flavonoid content in different s. S. no. Extracts Total flavonoid content (%w/w) 1. Petroleum ether 7.5 2. Chloroform 33.43 3. Ethyl acetate 64.37 4. Methanol 18.75 5. Water 11.56 Figure 4: Total flavonoid content Total Antioxidant Capacity Table 6: DPPH scavenging activity www.wjpps.com Vol 5, Issue 6, 2016. 1889
% Inhibition S. Concentration Petroleum Ethyl no. (µg/ml) Rutin Chloroform Methanol Water ether acetate 1. 40 28.73 0.74 2.98 1.49 12.31 0.37 2. 60 49.25 6.34 4.85 10.44 19.02 3.35 3. 80 55.59 11.56 7.46 17.16 21.64 20.89 4. 100 63.43 15.67 10.82 29.10 24.25 29.85 Figure 5: DPPH scavenging activity CONCLUSION In our present study total phenolic content of in different s were showing that water has high phenolic content and total flavonoid content of different showing ethyl acetate has high. Total antioxidant activity of different e s was showing that ethyl acetate has high antioxidant power due to high flavonoid content. REFERENCES 1. Benzie, I. F. F. & Strain, J. J.,The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power : The FRAP assay, Analytical Biochemistry, 1996; 70 76. 2. Bhandari, S. L. Bisht, S. S. Bisht, N. S. (Department of Botany, H.N.Bal University, Campus Pauri,(Garhwal), Micro propagation of Roylea elegans Wall. an important medicinal herb, Journal of Medicinal and Aromatic Plant Sciences v, 2008; 30(4): 370-374. 3. Dobhal, M. P. & Joshi B. C., 1979. Chemical investigations of Roylea elegans Wall. Part I, Herba Pol., 1979; 95-97. 4. Elizabeth, K. & Rao, M. N. A., 1990. Oxygen radical scavenging activity of curcumin. Int J Pharmaceut, 1990; 237-240. www.wjpps.com Vol 5, Issue 6, 2016. 1890
5. Indian Pharmacopoeia. I.P, Vol I. Ministry of Health and Family Welfare, Government of India, Published by the Indian Pharmacopoeia Commission, Ghaziabad, India, 2010; 82-83: 139 & 201. 6. Khandelwal, K.R. Practical pharmacognosy, 19 th edition, 2008; 9: 149-156, 158. 7. Ranganayaki S. Gusain P. S. Singh A. K. The chemical constituents of leaves of Roylea elegans. J Sci Res Plants & Medicines, 1985; 5-9. 8. Sai Khan, M. S. Howard, L. R. Miller, J. C. antioxidant activity and total phenolics in Solanum, J Food Sci, 1995; 60(2): 341-343. 9. Upadhyay, G. Kamboj, P. Malik, J. International Journal of Research in Pharmaceutical and Biomedical Sciences, 2011; 2(4). 10. Upadhyay, G. Maurya, H. Chauhan, N. Traditional claim of Roylea elegans Wall. On liver disorder., 2012. 11. Woisky, R. and Salatino, A. Analysis of propolis: some parameters and procedures for chemical quality control. J. Apic. Res., 1998; 37: 99-105. www.wjpps.com Vol 5, Issue 6, 2016. 1891