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Total Phenolic, Total Flavonoid Content, and Antioxidant Capacity of the Leaves of Meyna Spinosa Roxb., an Indian Medicinal Plant

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Total Phenolic, Total Flavonoid Content, and Antioxidant Capacity of the Leaves of Meyna Spinosa Roxb., an Indian Medicinal Plant Chinese Journal of Natural Chinese Journal of Natural Medicines 2013, 11(2): 0149−0157 Medicines doi: 10.3724/SP.J.1009.2013.00149 Total phenolic, total flavonoid content, and antioxidant capacity of the leaves of Meyna spinosa Roxb., an Indian medicinal plant 1* 2 3 4 Saikat Sen , Biplab De , N Devanna , Raja Chakraborty 1Department of Pharmacology, Creative Educational Society’s College of Pharmacy, Kurnool 518218, Andhra Pradesh, India; 2Department of Pharmaceutical Science, Assam University, Silchar 788011, Assam, India; 3Oil Technological Research Institute, JNTU Anantapur, Anantapur 515001, Andhra Pradesh, India; 4Department of Pharmaceutical Chemistry, Creative Educational Society’s College of Pharmacy, Kurnool 518218, Andhra Pradesh, India Available online 20 Mar. 2013 [ABSTRACT] AIM: The objective of the present study was to determine the total phenolic and total flavonoid contents, and to evalu- ate the antioxidant potential of different leaf extracts of Meyna spinosa Roxb. ex Link, a traditional medicinal plant of India. METHODS: Free radical scavenging and antioxidant potential of the methanol, ethyl acetate, and petroleum ether extracts of Meyna spinosa leaves were investigated using several in vitro and ex vivo assays, including the 2, 2-diphenyl-picrylhydrazyl radical scaveng- ing, superoxide anion scavenging, hydroxyl radical scavenging, nitric oxide radical scavenging, hydrogen peroxide scavenging activity, metal chelating assay, and reducing power ability method. Total antioxidant activity of the extracts was estimated by the ferric thiocy- anate method. Inhibition assay of lipid peroxidation and oxidative hemolysis were also performed to confirm the protective effect of the extracts. Total phenolic and total flavonoid contents of the extracts were estimated using standard chemical assay procedures. RESULTS: Methanol extracts showed the highest polyphenolic content and possessed the better antioxidant activity than the other two extracts. Total phenolic and total flavonoid contents in the methanol extract were (90.08 ± 0.44) mg gallic acid equivalents/g and (58.50 • ± 0.09) mg quercetin equivalents/g, respectively. The IC50 of the methanol extract in the DPPH , superoxide anion, hydroxyl radical, nitric oxide radical, hydrogen peroxide scavenging activity and metal chelating assays were (16.4 ± 0.41), (35.9 ± 0.19), (24.1 ± 0.33), (23.7 ± 0.09), (126.8 ± 2.92), and (117.2 ± 1.01) µg·mL−1, respectively. The methanol extract showed potent reducing power ability, total antioxidant activity, and significantly inhibit lipid peroxidation and oxidative hemolysis which was similar to that of standards. CONCLUSION: The results indicated a direct correlation between the antioxidant activity and the polyphenolic content of the ex- tracts, which may the foremost contributors to the antioxidant activity of the plant. The present study confirmed that the methanol extract of Meyna spinosa leaves is a potential source of natural antioxidants. [KEY WORDS] Meyna spinosa; Antioxidant; Phenolic; Flavonoid; Methanol extract [CLC Number] R962; R917 [Document code] A [Article ID] 1672-3651(2013)02-0149-09 tightly regulated by different enzymatic and non-enzymatic 1 Introduction antioxidant mechanisms. At low or moderate concentration The recent abundant evidence suggests that oxidative ROS/RNS play a positive role in energy production, phago- stress is one of the primary factors in the development of cytosis, regulation of cell growth, cell signaling, and the syn- degenerative diseases, as well as in the normal process of thesis of biologically important compounds, but overproduc- aging [1-2]. In living systems, reactive oxygen species (ROS) tion of free radicals or reactive species can lead to oxidative [3-4] and reactive nitrogen species (RNS) are generated during stress . Free radicals are able to oxidize biomolecules and normal physiological and biochemical processes, and are may cause protein oxidation, DNA damage, and lipid per- oxidation in living cells leading to mutagenic changes, tissue damage. and cell death [3-5]. Several synthetic drugs, such as butylated hydroxytoluene (BHT) and butylated hydroxyl [Received on] 14-Feb.-2012 [*Corresponding author] Saikat Sen: Assistant Prof., Tel: 91- anisole (BHA) are commonly used as antioxidants, but they 9032011182, E-mail: [email protected] have been reported to cause tissue toxicity, cell damage, These authors have no conflict of interest to declare. inflammation, and atherosclerosis in both animals and hu- 2013 年 3 月 第 11 卷 第 2 期 Chin J Nat Med Mar. 2013 Vol. 11 No. 2 149 Saikat Sen, et al. /Chinese Journal of Natural Medicines 2013, 11(2): 149−157 mans [6-7]. Recent findings clearly show that the consumption 2 Materials and Methods of plant foods and natural antioxidant supplements may be 2.1 Chemicals and reagents used to protect the body against various diseases, including 2, 2-Diphenyl-picrylhydrazyl (DPPH), sodium nitro- cancer, cardiovascular, and neurodegenerative diseases. prusside (SNP), phenazine methosulfate (PMS), and nitro Natural antioxidants help the endogenous antioxidant system blue tetrazolium (NBT) were purchased from Sigma Aldrich to reverse oxidative damage or protect oxidative stress in- (Bangalore, India). Hydrogen peroxide (H O ), 2-deoxy- duced deterioration [1, 3, 8]. Phenolic compounds and flavon- 2 2 ribose, trichloro acetic acid (TCA), thiobarbituric acid (TBA), oids have been found to have therapeutic applications against and quercetin were procured from SD Fine Ltd. Mumbai. different diseases caused by oxidative stress, and recently Linoleic acid, ammonium thiocyanate, sodium nitroprusside, several researchers demonstrated the correlation between Folin-Ciocalteau reagent, butylated hydroxyanisole (BHA), polyphenolic compounds and the antioxidant activity of ascorbic acid, α-tocopherol, and gallic acid were obtained plant/extracts [2-3]. Natural antioxidants present in plants have from Sisco Research Laboratories Pvt. Ltd. (SRL), Mumbai. received huge attention in recent years, and there has been a All other chemicals used in the study were obtained commer- worldwide surge towards the identification and use of anti- cially and were of analytical grade. oxidant principles from medicinal plants, which can provide 2.2 Plant material and extraction enormous scope in correcting redox imbalance and prevent Fresh mature whole leaves of Meyna spinosa Roxb. were free radical associated diseases in biological systems. collected in October 2010 from Khowai subdivision of Tri- Meyna spinosa Roxb. ex Link (Rubiaceae) is a spiny, pura, India. The plant was identified by its vernacular name usually a shrub or armed small tree, which can grow up to 8 and later validated by Dr. B.K. Datta, Department of Botany, m. Branches are busy, spines are axillary, straight, sharp, Tripura University, Tripura, India. A voucher specimen 5−40 mm. Leaves are membranous, ovate or elliptic-oblong, (TU/BOT/HEB/SS23072011a) was deposited at the herbar- while flowers crowded into fascicales and have shorter pedi- ium of the Plant Taxonomy & Biodiversity Laboratory, De- cels and petioles. Fruits are yellowish, subglobose drupe, partment of Botany, Tripura University. smooth with persistent calyx lobes [9-10]. The plant is used in 2.3 Preparation of the extracts traditional folk medicines and widely distributed in the North The air dried leaves of M. spinosa were powdered using Eastern, Eastern, and Southern parts of India [9]. Fruits and a mechanical grinder, and used for solvent extraction. The the bark of the plant are used to treat headache [11], while the powdered leaves (250 g) were extracted with methanol using fruits and leaves are beneficial in diabetes, jaundice, and a Soxhlet apparatus. The extract was concentrated to dryness other gastrointestinal disorders[12-14]. Tender leaves, ripe fruits under reduced pressure to yield a dried crude methanol ex- and seeds are useful to cure skin infections and pimples [13, tract. Similarly, the protocol was repeated with ethyl acetate 15-16], the leaf is also prescribed in indigestion and to treat and petroleum ether to obtain crude ethyl acetate and petro- dyspepsia [17]. Fruits are a good source of nutrienst, and are leum ether extracts. The yield of methanol, ethyl acetate and used to cure cough and as a refrigerant traditionally [14, 17]. petroleum ether extract was 15.2%, 13.0%, and 10.7%, W/V respectively. The extracts were then stored at 4 ºC till the The plant is also important for its abortifacient activity; seeds time of use. and fruits are used by several ethnic groups in India to induce 2.4 Determination of the amount of antioxidant compounds abortion [16, 18]. Recently, two compounds were isolated from 2.4.1 Determination of total phenolic content the fruits of M. spinosa which possess antimicrobial activity The amount of total phenolics in the extracts of M. against B. subtilis, K. pneuminiae, E. coli, S. aureus and C. spinosa leaves was measured using the Folin-Ciocalteu re- albicans. One was identified as oleanolic acid which pos- agent method [21]. The ethanol solution of each extract (0.5 sesses the highest antimicrobial activity [19]. Goswami et al. mL, l.0 mg·mL−1) was added into test tubes containing 2.5 (2006) have also reported the antifungal activity of the mL of 10% (V/V) Folin-Ciocalteu reagent and 2.0 mL of so- methanol extract of M. spinosa [20]. However, the phyto- dium carbonate (2%, W/V), and the tubes were shaken thor- chemical profile and the pharmacological activity of the plant oughly. The mixture was incubated at 45 °C for 15 min with leaf material has not been investigated thoroughly. In view of intermittent shaking. Absorbance was measured at 765 nm the number of diseases associated with oxidative stress, and using an Elico SL 164 UV-Vis spectrophotometer. Gallic acid as a review of the literature afforded no detailed reports on was used as standard to obtain a calibration curve (ranging the antioxidant potential of the leaves of M. spinosa, the pre- from 0 to 0.8 mg·mL−1), and the results were expressed as sent study was undertaken to evaluate antioxidant activity of gallic acid equivalents in milligram per gram (mg GAE/g) of the leaves of M.
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