Objective: The main aim of this study was to determine the in vitro antioxidant activity of Iris kashmiriana ethanolic leaf extract and also total phenol and flavonoid content was evaluated.Methods: Total phenol content (TPC) was determined by Folinâ€“Ciocalteu method, total flavonoid content (TFC) was estimated by aluminum trichloride spectrophotometer method. Furthermore, antioxidant activity was revealed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydrogen peroxide (H2O2) scavenging activity, and reducing power assay.Results: The ethanolic leaf extract of I. kashmiriana showed TPC of 13.25Â±0.57 Î¼g/100 Î¼g gallic acid equivalents and TFC of 33.61Â±3.37 Î¼g/100 Î¼g rutin equivalents. The DPPH assay revealed IC50 of 0.418 mg/ml and for H2O2 radical scavenging IC50 was 0.476 mg/ml for the plant extract while as reducing power assay revealed concentration-dependent absorption values which clearly determine the antioxidant property of plant.Conclusion: From the results, it is apparent that I. kashmiriana ethanolic leaf extract possessed potential antioxidant activity which can be used to cure wide range of diseases

Alia, Aparna

Khalid, Shah

Pn, Shrivastava

Rather, Muzafar Akbar

Sheikh, Muzafar Ah

English

Innovare Academic Sciences: E-Journals

Vol 10, Issue 8, 2017Online - 2455-3891 Print - 0974-2441EVALUATION OF IN VITRO ANTIOXIDANT ACTIVITY AND ESTIMATION OF TOTAL PHENOL AND FLAVONOID CONTENT OF ETHANOLIC LEAF EXTRACT OF IRIS KASHMIRIANASHAH KHALID1*, APARNA ALIA2, SHRIVASTAVA PN1, MUZAFAR AKBAR RATHER1, MUZAFAR AH SHEIKH31Department of Botany, Pest Control and Ayurvedic Drug Research Lab, Vidisha, Madhya Pradesh, India. 2Department of Botany, Rajeev Gandhi College, Shahpura, Bhopal, Madhya Pradesh, India. 3Department of Zoology, Government Degree College, Ganderbal, Jammu and Kashmir, India. Email: khalidbotanica@gmail.comReceived: 04 March 2017, Revised and Accepted: 15 May 2017ABSTRACTObjective: The main aim of this study was to determine the in vitro antioxidant activity of Iris kashmiriana ethanolic leaf extract and also total phenol and flavonoid content was evaluated.Methods: Total phenol content (TPC) was determined by Folin–Ciocalteu method, total flavonoid content (TFC) was estimated by aluminum trichloride spectrophotometer method. Furthermore, antioxidant activity was revealed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, hydrogen peroxide (H2O2) scavenging activity, and reducing power assay.Results: The ethanolic leaf extract of I. kashmiriana showed TPC of 13.25±0.57 µg/100 µg gallic acid equivalents and TFC of 33.61±3.37 µg/100 µg rutin equivalents. The DPPH assay revealed IC50 of 0.418 mg/ml and for H2O2 radical scavenging IC50 was 0.476 mg/ml for the plant extract while as reducing power assay revealed concentration-dependent absorption values which clearly determine the antioxidant property of plant.Conclusion: From the results, it is apparent that I. kashmiriana ethanolic leaf extract possessed potential antioxidant activity which can be used to cure wide range of diseases.Keywords: Iris kashmiriana, Total phenol content, Total flavonoid content, 1,1-diphenyl-2-picrylhydrazyl, Hydrogen peroxide.INTRODUCTIONPlants are the natural source of medicine and have been used since time to cure various ailments or diseases. The medicinal property of the plants is due to the presence of various bioactive constituents such as alkaloids, phenols, flavonoids, terpenoids, saponins, triterpenes, and tannins. The biological activities of such compounds are being investigated through different screening techniques [1]. It is commonly accepted that medicines derived from plant products are safer than their synthetic counterparts [2]. The ingestion of vegetables, fruit and herbs is coupled with the prevention of several bactericidal, antiviral, anti-inflammatory, analgesic and anticarcinogenic disorders, and due to their antioxidant activities [3,4]. The plant products such as fruits, leaves, seeds, and oils possess antioxidant property as they contain flavonoids, phenolics, tannins, coumarins, lignans, curcumanoids, xanthons, and terpenoids [5]. Phenols and flavonoids have been shown to acquire an essential antioxidant activity toward free radicals [6]. Rancidity of lipid and lipid containing is reduced with the use of antioxidants. They reduce the formation of toxic oxidation, maintain nutritional quality and enhance the life of food products [7].Therefore, there is a growing demand day by day of the substances exhibiting antioxidant properties and which are part of human and animal food or used as specific preventative pharmaceutical product [8]. Hence, keeping in view the importance of antioxidants, this study, we planned to determine the in vitro antioxidant activity of ethanolic leaf extract of Iris kashmiriana.METHODSSample collection and authenticationI. kashmiriana shoot was collected in the month of August (Village Lar, District Ganderbal, Jammu and Kashmir, India). Dr. Akhtar H. Malik (Curator, Centre for Biodiversity and Taxonomy, University of Kashmir) identified and authenticated the plant. A voucher specimen, bearing No. 2079 KASH was submitted to the same department for future reference.Preparation of sampleThe leaves were detached from the plant, washed firstly with tap water to remove foreign matter and finally were washed with distilled water twice. Leaves were shade dried for about 2 weeks and then powdered with the help of electric grinder (Philips). The powder was packed in the polythene bags for further use, and the percentage loss was determined.Extraction procedureIn the Soxhlation process, 20 g of leaf powder was extracted using 90% ethanol as a solvent for 24 hrs with the help of Soxhlet Assembly (J-Sil, 50/42, Borosil glass). The extract was filtered and subjected to vacuum rotary evaporator (Scientech) to obtain a semi-solid extract. The extract was subjected to organoleptic evaluation, and the percentage yield of the extract was calculated, stored at −4°C.Phytochemical analysisThe crude leaf extract of I. kashmiriana was subjected to preliminary phytochemical analysis for presence or absence of bioactive constituents, according to standard procedures [9,10].Determination of total phenol content (TPC)Folin–Ciocalteu method [11] was used to determine the TPC. Calibration was done with a standard gallic acid calibration curve. Different concentrations gallic acid (10-100 µg/ml) was prepared in methanol. Test sample (100 µg/ml) was prepared in methanol. A volume of 0.5 ml test sample was added to 2 ml Folin–Ciocalteu © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i8.18268Research Article310Asian J Pharm Clin Res, Vol 10, Issue 8, 2017, 309-312 Khalid et al. reagent (1:10 in Deionized Water). Neutralization of mixture was brought by adding 4 ml of sodium carbonate solution (7.5% w/v). The final solution was incubated at room temperature for 30 minutes with intermittent shaking to develop color. At 765 nm, absorbance of colored solution was taken using double beam spectrophotometer (ultraviolet-visible analyst - 0001) using methanol as blank. Standard curve gallic acid was prepared, and line of regression was obtained (Fig. 1). TPC of the herb was calculated and expressed as µg/mg gallic acid equivalent.Determination of total flavonoid content (TFC)Aluminum trichloride spectrophotometer method [12] was used to determine TFC of plant, using rutin as standard. Various concentrations of rutin (10-100 µg/ml) were prepared in methanol. Furthermore, test sample (100 µg/ml) was prepared in methanol. A volume of 0.5 ml of test sample was diluted with 2 ml distilled water. 0.15 ml of NaNO2 solution (5%) was then added and after 6 minutes, 0.15 ml of AlCl3 (10%) was added, allowed to stand for 6 minutes. To the mixture, 2 ml of NaOH solution (1 mM) was added. Finally, the volume of reaction mixture was made 5 ml by diluting it distilled water and allowed to stand for 15 minutes. Taking methanol as blank, at 510 nm, absorbance of reaction mixture was obtained. TPC was determined from a calibration curve and expressed as µg/mg rutin equivalent (Fig. 2).In vitro antioxidant activity1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay [13]Various concentrations of the extract and the ascorbic acid were made in methanol. To 2 ml of extract or standard, add 1 ml of methanolic 0.1 mM DPPH and the reaction mixture was incubated at 37°C for 20 minutes. Absorbance was measured at 517 nm against blank using double beam spectrophotometer and % inhibition was calculated as:% Inhibition = Ac AsAc−×100Ac and As denotes absorbance of control and test sample, respectively.Hydrogen peroxide (H2O2) scavenging assayH2O2 scavenging activity of I. kashmiriana leaf extract was determined according to standard procedure [14] with slight modifications. Different concentration of the test sample was made in distilled water. 40 mM of H2O2 solution was made in phosphate buffer (pH 7.4). 2 ml of test sample and 0.6 ml of H2O2 were mixed well and incubated at room temperature for 10 minutes and then absorbance was measured against the blank (phosphate buffer without H2O2). Ascorbic acid was used as a standard. Percentage of H2O2 scavenging was calculated as:% Inhibition = Ac AsAc−×100Where Ac is the absorbance of the control and As is the absorbance of plant sample. IC50 was determined from the regression curve of % inhibition.Reducing power assayFerric reducing power assay [15] is used to determine the antioxidant property of plant. Diverse concentrations of test sample were made. 0.5 ml phosphate buffer (0.2 M, pH 6.6) was added to 0.5 ml of different concentrations of test sample, followed by simultaneous addition of 0.5 ml of potassium ferricyanide (0.5 ml, 1% w/v). At 50°C, reaction mixture was incubated for 20 minutes. Then, 1.5 ml of trichloroacetic acid solution (10% w/v) was added to reaction mixture after cooling, to terminate the reaction. Followed by addition of 0.5 ml ferric chloride (0.1% w/v) and absorbance was taken at 700 nm. Absorbance of reaction mixture is directly proportion to the reducing power, which is directly related to the antioxidant property.RESULTSAs, I. kashmiriana leaves were shade dried, the calculated percentage loss was 83.8%. Further, on complete Soxhlation of plant sample, an extractive value of 2.9 g was determined with percentage yield of 14.5%. The extract so obtained was dark green in color, bitter in taste, odor like lemon and was sticky in touch. The preliminary phytochemical analysis of crude extract of I. kashmiriana revealed the presence of almost all phytoconstituents except alkaloids as expressed in Table 1. The quantitative estimation of phenolics and flavonoids revealed ethanolic leaf extract of I. kashmiriana contained 13.25±0.57 µg/100 µg gallic acid equivalent of phenolics while as TFC was 33.61±3.37 µg/100 µg rutin equivalent as indicated in Fig. 3.In vitro antioxidant activity results showed diverse concentrations of plant sample scavenges DPPH in a dose-dependent manner (Fig. 4). Results were expressed as % inhibition and were standardized with the ascorbic acid curve (Fig. 5). The IC50 of I. kashmiriana leaf extract was 0.418 mg/ml while that of ascorbic acid 0.399 mg/ml. However, for H2O2 radical scavenging, IC50 was 0.476 mg/ml and percentage inhibition showed gradual increase, i.e., concentration dependent scavenging activity by the plant sample (Fig. 6). Furthermore, reducing power assay showed steady increase in the absorption values (Table 2), indicating potential antioxidant property of plant extract. The 100 µg/ml concentration showed minimum absorption value of 0.192 nm while as 500 µg/ml concentration corresponded to maximum value of absorption 0.755 nm.DISCUSSIONThe secondary metabolites of the plant such as phenols, flavonoids, alkaloids, and terpenes are known to be biologically active constituents, Fig. 1: Gallic acid standard curveFig. 2: Rutin standard curve311Asian J Pharm Clin Res, Vol 10, Issue 8, 2017, 309-312 Khalid et al. Table 1: Phytochemical analysis of I. kashmiriana leaf extractPhytochemicals Phytochemical tests Crude leaf extract of I. kashmirianaAlkaloids Mayer’s test -Wagner’s test -Hager’s test -Dragendroff’s test -Terpenoids Salkowski test ++Liebermann Burchard’s test -Flavonoids Lead acetate test ++Alkaline reagent test ++Shinoda test ++Carbohydrates Molisch’s test ++Fehling’s test -Benedict’s test ++Barfoed’s test -Glycosides Keller Kiliani test ++Borntrager’s test ++Legal’s test -Tannins and phenolic compounds FeCl3 test ++Dilute iodine solution test ++Nitric acid test ++Gelatin test ++Saponins Froth test ++Amino acids and proteins Biuret’s test -Millon’s test ++Ninhydrin test ++++: Present, -: Absent, I. kashmiriana: Iris kashmirianaFig. 3: Total phenol content and total flavonoid content of Iris kashmiriana leaf extractFig. 4: 1,1-diphenyl-2-picrylhydrazyl assay of Iris kashmiriana leaf extract Fig. 5: Standard ascorbic acidFig. 6: Hydrogen peroxide scavenging by Iris kashmiriana leaf extract312Asian J Pharm Clin Res, Vol 10, Issue 8, 2017, 309-312 Khalid et al. such as these ingredients possess antioxidant, antifungal, anticancer, and antimicrobial activities [16,17]. Phenolic and flavonoid compounds protect plant and plant parts from oxidative damage and thus provide a better antioxidant option for human beings [18].Ethanolic leaf extract of I. kashmiriana showed the presence of bioactive compounds and possessed remarkable amount of phenolics and flavonoids. Our results revealed that I. kashmiriana extract acquires potential antioxidant activity by donating hydrogen to DPPH (deep violet color) free radical and converted it into α,α- diphenyl-β- picrylhydrazine (colorless). The antioxidant potential is indicated by discoloration [19]. Antioxidant activity is directly related with the amount of phenols present in extract [20]. It has been indicated that phenolic compounds are directly involved in the scavenging activity of free radicals [21,22].H2O2, a weak oxidizing agent, inactivates various enzymes by oxidizing their thiol (–SH) group. However, sometimes it gives rise to very toxic hydroxyl free radical, which is very harmful and lethal to the cells [23]. Free hydroxyl radical is formed by interaction of H2O2 with Fe2+ and Cu2+ inside cell [24]. I. kashmiriana extract showed dose-dependent scavenging activity of H2O2 as stated earlier in the results. Furthermore, the ferric reducing power showed the extract acts as a good reductant as the extract reduces ferric ion of ferric cyanide to ferrous ion (Perl’s Prussian blue). The reducing property of extract or compound depends on the reductants [25]. Similar results were found in Combretum albidum [26] and Piper umbellatum [27] leave extracts. In this study, the results clearly indicated that our plant extract exhibit a significant and potential antioxidant activity.CONCLUSIONThe demand for natural antioxidants is increasing day by day. Our results showed I. kashmiriana contained an excellent quantity of phenolics and flavonoids which bestow antioxidant property to the plant. Hence, the plant can be refined for such chemical compounds to cure different diseases and ailments with no or less side effects.REFERENCES1. Bouchagier P, Efthimiadou A, Katsileros A, Bilalis D, Efthimiadis P. Adverse effect of Bermuda grass on physiological and growth components of cotton. J Agron 2008;7(1):49-55.2. Oluyemi KA, Okwuonu UC, Baxter DG, Oyesola TO. Toxic effects of methanolic exract of Aspilia africana leaf on the estrous cycle and uterine tissues of Wistar rats. Int J Morphol 2007;25:609-14.3. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem 2005;53:1841-56.4. Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils. Food Chem Toxicol 2008;8(46):446-75.5. Jeong SM, Kim SY, Kim DR, Nam KC, Ahn DU, Lee SC. Effect of seed roasting conditions on the antioxidant activity of defatted sesame meal extracts. Food Chem Toxicol 2004;69(5):377-81.6. Bors W, Saran M. Radical scavenging by flavonoid antioxidants. Free Radical Res Commun 1987;2:289-94.7. Maisuthisakul P, Suttajit M, Pongsawatmanit R. Assessment of phenolic content and free radical-scavenging capacity of some Thai indigenous plants. Food Chem 2007;100:1409-18.8. Sarikurkcu C, Arisoy K, Tepe B, Cakir A, Abali G, Mete E. Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. Fruits Turk Food Chem Toxicol 2009;47(10):2479-83.9. Trease GE, Evans WC. Pharmacognosy. Vol. 13. London: Brailliar Tiridel Can Acmillian Publishers; 1989. p. 28-32.10. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. Vol. 14. New Delhi, India: Nirali Prakashan; 2006. p. 593-5.11. Singelton VR, Orthifer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 1999;299:152-78.12. Zhishen J, Mengeheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 1999;64:555-9.13. Adedayo BJ, Oboh G, Oyeleye SI, Ejakpovi II, Boligon AA, Athayde ML. Blanching alters the phenolic constituents and in vitro antioxidant and anticholinesterases properties of fireweed (Crassocephalum crepidioides). J Taibah Univ Med Sci 2015;10(4):419-26.14. Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant cate chins isolated from Chinese green tea. Carcinogenesis 1989;10:1003-8.15. Jain R, Jain SK. Total phenolic contents and antioxidant activities of some selected anticancer medicinal plants from Chhattisgarh State. India Pharmacol Online 2011;2:755-62.16. Kokate KC. Practical Pharmacognosy. 4th ed. Delhi: Vallabh Prakashan; 1997. p. 218.17. Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 2nd ed. London: Chapman and Hall; 1998. p. 54-84.18. Diem-Do Q, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, et al. Effect of extraction solvent on total phenol content total flavonoid content and antioxidant activity of Limnophila aromatic. J Food Drug Anal 2014;22:296-302.19. Molyneux P. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol 2004;26(2):211-9.20. Abdille MH, Singh RP, Jayaprakasha GK, Jena BS. Antioxidant activity of the extracts from Dillenia indica fruits. Food Chem 2005;90(4):891-6.21. Vats V, Grover JK, Rathi SS. Evaluation of antihyperglycemic and hypoglycemic effect of Trigonella foenum-graecum Linn Ocimum sanctum Linn and Pterocarpus marsupium Linn in normal and alloxanized diabetic rats. J Ethnopharmacol 2002;79:95-100.22. Karanjit N, Shrestha UK, Ranjitkar RR. A study on hypoglycemic properties of Pterocarpus marsupium Roxb. Bull Dep Plant Res 2008;30:97-101.23. Halliwell B. Reactive oxygen species in living systems: Source biochemistry and role in human disease. Am J Med 1991;91:14-22.24. Miller MJ, Sadowska-Krowicka H, Chotinaruemol S, Kakkis JL, Clark DA. Amelioration of chronic ileitis by nitric oxide synthase inhibition. J Pharmacol Exp Ther 1993;264(1):11-6.25. Duh PD, Tu YY, Yen GC. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum moifolium Ramat). Lebensm Technol 1999;32:269-77.26. Chandar B, Ramasamy MK. Evaluation of antioxidant, antibacterial activity of ethanolic extract in the leaves of Combretum albidum and gas chromatography-mass septrometry analysis. Asian J Pharm Clin Res 2016;9(4):325-9.27. Geetha S, Irulandi K, Mehalingam P. Evaluation of antioxidant and free radical scavenging activities of different solvent extracts of leaves of Piper umbellatum. Asian J Pharm Clin Res 2017;10(2):274-6.Table 2: The reducing power of I. kashmirianaS. No. Concentration (µg/ml) Absorbance (nm)1 100 0.2292 200 0.3553 300 0.5344 400 0.6895 500 0.755

EVALUATION OF IN VITRO ANTIOXIDANT ACTIVITY AND ESTIMATION OF TOTAL PHENOL AND FLAVONOID CONTENT OF ETHANOLIC LEAF EXTRACT OF IRIS KASHMIRIANA

https://innovareacademics.in/journals/index.php/ajpcr/article/download/18268/12109/99184

EVALUATION OF IN VITRO ANTIOXIDANT ACTIVITY AND ESTIMATION OF TOTAL PHENOL AND FLAVONOID CONTENT OF ETHANOLIC LEAF EXTRACT OF IRIS KASHMIRIANA

Abstract

Similar works

Full text

Available Versions

Innovare Academic Sciences: E-Journals