[Downloaded free from http://www.phcogres.com on Wednesday, May 12, 2021, IP: 223.186.91.179] Pharmacogn. Res. ORIGINAL ARTICLE A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products www.phcogres.com | www.phcog.net In vitro Antioxidant Potential of Euclea crispa (Thunb.) Leaf Extracts Chella Perumal Palanisamy, Devaki Kanakasabapathy1, Anofi Omotayo Tom Ashafa Phytomedicine and Phytopharmacology Research Group, Department of Plant Sciences, University of the Free State, QwaQwa Campus, Phuthaditjhaba 9866, South Africa, 1Department of Biochemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India ABSTRACT leum ether extract Background: Euclea crispa is a South African medicinal plant belonging • The fresh E. crispa leaves displayed high content of enzymatic and nonenzy‑ to the family Ebenaceae. Objectives: The objective of this study was to matic antioxidants. analyze the in vitro antioxidant activity of different extracts of E. crispa leaves. Materials and Methods: 2, 2‑diphenyl‑1‑picrylhydrazyl (DPPH) radical scavenging assay, reducing power assay, ferric reducing antioxidant power (FRAP) assay, hydroxyl scavenging assay, and nitric oxide scavenging assay were used to analyze free‑radical scavenging activity. The superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), total reduced glutathione (TRG), and estimation of vitamin C assays were carried out to analyze the enzymatic and nonenzymatic antioxidants on a fresh leaf of E. crispa. Results: The DPPH radical scavenging assay (135.4 ± 0.7 µg/ml), hydroxyl scavenging assay (183.6 ± 0.9 µg/ml), and nitric oxide scavenging assay (146.2 ± 1.3 µg/ml) showed the significant half maximal inhibitory concentration (IC50) values in ethanolic extract when compared to the ethyl acetate, chloroform, and petroleum ether extract of E. crispa leaves. Further, the ethanolic extract exhibited good reducing power assay and FRAP assay showed (the maximum absorption of 0.79 and 0.68 at 500 µg/ml, respectively) when compared to other solvent extracts. The fresh E. crispa leaves possess high content of enzymatic and nonenzymatic antioxidants Abbreviations Used: DPPH: 2, 2‑diphenyl‑1‑picrylhydrazyl, FRAP: Ferric such as SOD (41.3 ± 0.34 units/mg protein), CAT (124 ± 0.54 µmole of reducing antioxidant power, SOD ‑ Superoxide dismutase, CAT: Catalase, H2O2 consumed/min/mg protein), GPX (261.2 ± 0.42 µg of glutathione GPX: Glutathione peroxidase, TRG: Total reduced glutathione, ROS: Reactive oxidized/min/mg protein), TRG (42.3 ± 0.16 µg/mg protein), and estimation oxygen species, DNA: Deoxyribonucleic acid, EDTA: Ethylenediaminetetraacetic of vitamin C (185 ± 0.39 µg/mg) assays. Conclusion: Based on the results acid, TCA: Trichloroacetic acid, TBA: Thiobarbituric acid, NED: Naphthyl obtained from this study, it can be concluded that the E. crispa leaves can ethylenediamine dihydrochloride, IC50: Half maximal inhibitory concentration. be used for the preparation of antioxidative therapeutic agents. However, further studies are necessary to substantiate the current findings. Access this article online Key words: Euclea crispa leaves, enzymatic, free‑radical scavenging Website: www.phcogres.com activity, nonenzymatic antioxidant activity, solvent extractions Correspondence: Quick Response Code: SUMMARY Dr. Anofi Omotayo Tom Ashafa, Department of Plant Sciences, University of the • E. crispa leaf extracts have a variety of secondary metabolites, particularly alkaloids, steroids, flavonoids, tannins, phenols, glycosides, saponins, and Free State, Qwaqwa Campus, terpenoids. Phuthaditjhaba 9866, South Africa. • The ethanolic extract of E. crispa leaves possess significant free radical scav‑ E‑mail: [email protected] enging activity when compared with ethyl acetate, chloroform, and petro‑ DOI: 10.4103/pr.pr_123_17 INTRODUCTION constituents has resulted in broad research on naturally occurring antioxidants which can deactivate highly reactive free radicals.[4] In this Reactive oxygen species (ROS), such as singlet oxygen, superoxide respect, flavonoids, alkaloids, terpenoids, and phenolic compounds anion, hydroxyl radical, and hydrogen peroxide, are frequently which are usually found in medicinal plants have been reported to generated as byproducts of biological reactions or from exogenous have high antioxidant activity as well as multiple biological effects.[5] factors. Every molecule of ROS leads to oxidative damaging effects Currently, the over‑the‑counter synthetic antioxidants might be unsafe on living cells including DNA if excess ROS are not eliminated by if used over a prolonged period and its toxicity has also been criticized. It the antioxidant system.[1] ROS plays a major role in the formation of is generally assumed that frequent use of plant‑derived phytochemicals chronic and degenerative diseases including cancer, autoimmune, inflammatory, cardiovascular, neurodegenerative diseases, and aging. This is an open access journal, and articles are distributed under the terms of the Radical scavenging antioxidants are mainly significant in protecting cells Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which from the injury of free radical.[2] Thus, antioxidants with free‑radical allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. scavenging activities may have enormous significance in the prevention and therapeutics of diseases in which oxidants or free radicals are For reprints contact: [email protected] implicated.[3] Recent investigations have shown that varieties of bioactive substances Cite this article as: Palanisamy CP, Kanakasabapathy D, Tom Ashafa AO. are present in medicinal plants which are widely used in the prevention In vitro antioxidant potential of Euclea crispa (Thunb.) leaf extracts. Phcog Res and management of various diseases. The demand for natural food 2018;10:296-300. 296 © 2018 Pharmacognosy Research | Published by Wolters Kluwer - Medknow [Downloaded free from http://www.phcogres.com on Wednesday, May 12, 2021, IP: 223.186.91.179] CHELLA PALANISAMY, et al.: Antioxidant potential of Euclea crispa leaves extracts may contribute to shift the stability in the direction of sufficient Nitric oxide radical scavenging assay antioxidant status. As a result, attention to natural antioxidants, in The nitric oxide was generated by sodium nitroprusside and particular, those from plant origin has deeply amplified in recent measured.[11] The reaction mixture contained 10 mM SNP, phosphate years.[6] buffered saline (pH 7.4), and various doses (100–500 µg/ml) of the Euclea crispa is an afrotropical plant species, commonly known as the test solution in a final volume of 3 ml. After incubation for 150 min at blue guarri (Eng.); bloughwarrie (Afr.); motlhaletsogane (Setswana); 25°C, 1 ml sulfanilamide (0.33% in 20% glacial acetic acid) was added to and iDungamuzi, umGwali (isiZulu). It is a hardy and evergreen plant 0.5 ml of the incubated solution and allowed to stand for 5 min. Then, that usually forms a dense stand of shrubs or grows to tree size. It is 1 ml of naphthyl ethylenediamine dihydrochloride (NED) (0.1% w/v) [7] widespread and common in the interior regions of Southern Africa. was added, and the mixture was incubated for another 30 min at 25°C. This plant is used conventionally against a wide range of ailments such as The pink chromophore generated during diazotization of nitrite ions gonorrhea, leprosy, scabies, diarrhea, and wound infections and previous with sulfanilamide and subsequent coupling with NED was measured reports have shown that the plant possessed antibacterial and antifungal spectrophotometrically at 540 nm against a blank sample. All tests were [8] activities. Therefore, the main aim of this study is to evaluate in vitro performed triplicate. antioxidant potential of E. crispa leaves extracts since this aspect of medicinal potential of this valuable plant have not been reported in any Hydroxyl radical scavenging assay scientific literature. The hydroxyl radical scavenging activity ofE. crispa leaf extracts was MATERIALS AND METHODS measured.[12] All the solutions were freshly prepared. To 1 ml reaction mixture contained, 2‑deoxy‑2‑ribose (2.8 mM); KH PO ‑KOH Plant collection 2 4 buffer (20 mM, pH 7.4); FeCl3 (100 µM); ethylenediaminetetraacetic Fresh leaves of E. crispa were collected from Qwaqwa campus, University acid (EDTA) (100 µM); H2O2 (1.0 mM); ascorbic acid (100 µM); and of the Free State, South Africa during April 2017 and identified by Prof. various concentrations (100‑500 µg/ml) of the test sample. After AOT Ashafa. The plant sample was authenticated at University of the incubation for 1 h at 37°C, 0.5 ml of the reaction mixture was added Free State herbarium with herbarium collection of Taylor and Van Wyk, to 1 ml of 2.8% trichloroacetic acid (TCA), then 1 ml 1% aqueous TBA 1994 with reference number: 6404000‑400. Collected plant leaves were was added, and the mixture was incubated at 90°C for 15 min for the washed under running tap water to remove contaminants and foliar odor to develop the color. After cooling, the absorbance was measured at debris, air dried, powdered, and stored in airtight container at 4°C for 532 nm against an appropriate blank solution. All tests were performed further studies. in triplicates. Percentage of inhibition was evaluated by comparing the test and blank solutions. Preparation of extract Using exhaustive