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 spinosa Roxb., an Indian medicinal

Saikat Sen1*, Biplab De2, N Devanna3, Raja Chakraborty4

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 (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-

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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 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 () 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 & 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. spinosa using different in vitro and ex vivo dried extract. models. The total phenolic and total flavonoid contents in the 2.4.2 Determination of total flavonoid content extracts were also estimated to determine the relationship A preliminary test for flavonoids was performed using between the free radical scavenging activity and the the lead acetate, ferric chloride, ammonium hydroxide re- polyphenolic content. agent, and the results were positive. Therefore an attempt was

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Saikat Sen, et al. /Chinese Journal of Natural Medicines 2013, 11(2): 147−157 made to determine the total flavonoid content quantitatively. 2.5.4 Nitric oxide radical scavenging assay An aliquot of 0.5 mL of sample solution (1 mg·mL−1) was Nitric oxide, spontaneously generated from an aqueous mixed with 0.1 mL of 10% aluminum nitrate, 0.1 mL 1 solution of SNP at physiological pH, can interact with oxy- mol·L−1 potassium acetate, and 4.3 mL of 80% ethanol. The gen to produce nitrite ions, which can be quantified by the reagents were thoroughly mixed and allowed to stand for 40 Griess Illosvoy reaction. Four mL of extract solution at dif- min at room temperature; the absorbance in the supernatant ferent concentrations were added to test tubes containing 1.0 was measured at 415 nm [22]. A yellow coloration of the mix- mL of SNP solution (25 mmol·L−1), and incubated at 37 ºC ture indicated the presence of flavonoids. Quercetin was used for 2 h. After incubation, 2.0 mL of the incubated solution to plot a standard calibration curve, and the results were ex- was removed and mixed with 1.2 mL Griess reagent. The pressed as quercetin equivalents in milligram per gram (mg absorbance of chromophore that formed during diazotization QE/g) of dried extract. of the nitrite with sulfanilamide, and subsequent coupling 2.5 In vitro antioxidant and radical scavenging activity with naphthylethylenediamine dihydrochloride, was taken 2.5.1 DPPH radical scavenging activity immediately at 570 nm. A control experiment was also car- One mL of a solution of DPPH in methanol (0.1 ried out in a similar manner, using distilled water in place of mmol·L−1) was mixed with 3.0 mL of extract in various con- the extract solution [26]. Ascorbic acid was used as a standard centrations during 30 min at room temperature in the dark, antioxidant. The experiment was performed (in triplicate) and and the absorbance was recorded at 517 nm [23]. The scav- the percentage scavenging activity was calculated using enging activity of the extracts was estimated based on the equation (1). percentage of DPPH radical scavenged (I%) using equation 2.5.5 Hydrogen peroxide scavenging activity (1): The hydrogen peroxide-scavenging ability of the extracts [27] I% = [(Acontrol – Asample)/Acontrol] × 100 was estimated according to the method of Bozin et al . For −1 this purpose, a solution of H2O2 (40 mmol·L ) was prepared Where Asample is the absorbance of a sample solution, and in phosphate buffer (pH 7.4). Different concentrations of Acontrol is the absorbance of the control solution (containing all of the reagents, except the test sample). plant extract in 3.4 mL phosphate buffer (pH 7.4) were added 2.5.2 Superoxide anion radical scavenging activity to 0.6 mL of H2O2 solution. The absorbance value of the Superoxide radical scavenging ability of M. spinosa leaf reaction mixture was recorded spectrophotometrically at 230 extracts and BHA was assayed using the NTB reduction as- nm. Gallic acid was used as the positive control. The per- say method [24]. Samples of different concentration (0.3 mL) centage of H2O2 scavenging (I%) of the examined extracts dissolved in methanol were added into 3.0 mL of reaction was calculated by equation (1). mixture of Tris-HCl buffer (100 mmol·L−1, pH 7.4), which 2.5.6 Reducing power capacity contained 0.75 mL of NBT (300 μmol·L−1) solution, 0.75 mL Briefly, 1.0 mL of different concentrations of the sample −1 of nicotinamide adenine dinucleotide-reduced (NADH) (936 solutions were mixed with 2.5 mL of 0.2 mol·L phosphate μmol·L−1) solution. The reaction was started by adding 0.75 buffer (pH 6.6) and 2.5 mL of 1% (W/V) K3Fe(CN)6. The mL of PMS (120 μmol·L−1) into the reaction mixture. After 5 mixture was incubated for 20 min at 50 ºC, and after incuba- min of incubation at room temperature, when the reaction tion 2.5 mL of TCA (10%) was added. The resulting mixture −1 mixture had reached a stable color, the absorbance was taken was centrifuged at 12 000 r·min for 10 min, if required. An at 560 nm against a blank using a UV-Vis spectrophotometer aliquot of 2.5 mL of supernatant solution was pipetted out (Elico SL 164). The capability of scavenging superoxide and 2.5 mL of distilled water, 0.5 mL of 0.1% (W/V) FeCl3 radical (I%) was calculated using equation (1). solution were mixed to the solution. The absorbance was 2.5.3 Hydroxyl radical scavenging activity recorded at 700 nm using Elico SL 164 UV–Vis spectropho- [28] The colorimetric deoxyribose method was used to assess tometer . The reducing power of ascorbic acid (25−400 −1 the hydroxyl radical scavenging activity of the extracts [25]. µg·mL ) was also determined as a positive control. The test was run in triplicate and averaged. The reacting mixture consisted of 0.2 mL KH2PO4–KOH (100 mmol·L−1), 0.2 mL deoxyribose (15 mmol·L−1), 0.2 mL 2.5.7 Metal chelating ability −1 The reaction mixture contained 0.4 mL of different con- FeCl3 (500 mmol·L ), 0.1 mL ethylene diamine tetra-acetic acid (EDTA) (1 mmol·L−1), 0.1 mL ascorbic acid (1 centration of the extract solution, and 0.05 mL of FeCl2 (2 −1 mmol·L−1), 0.1 mL H O (10 mmol·L−1), and 0.1 mL sample. mmol·L ) solution. The reaction was started by the addition 2 2 −1 The solutions were incubated at 37 ºC for 1 h. After the in- of 0.2 mL of ferrozine (5 mmol·L ) and the total volume of cubation period, 1.0 mL of TBA (1%, W/V) and 1.0 mL of the mixture was adjusted to 4.0 mL with ethanol. The mixture TCA (2.8%, W/V) were added to the mixture. The resultant was shaken vigorously and left standing at room temperature solutions were heated on a water bath at 80 ºC for 20 min, for 10 min. To determine the ferrous ion chelating activity of and the absorbance of the solution was measured at 532 nm. the extracts, the absorbance of the mixture was measured [29] Quercetin was used as the positive control. The scavenging spectrophotometrically at 562 nm . The percentage of in- 2+ activity (I %) was calculated using equation (1). hibition of ferrozine–Fe complex formation (I%) was cal-

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Saikat Sen, et al. /Chinese Journal of Natural Medicines 2013, 11(2): 149−157 culated using equation (1). as inhibition percentage of erythrocyte hemolysis (I%) using 2.5.8 Ferric thiocyanate (FTC) method equation (1). The FTC method was used to determine the total anti- 2.7 Statistical analysis oxidant activity of the extracts according to the method de- The data in triplicate were subjected to analysis of vari- scribed by Liu and Yao [30]. Briefly, a reaction solution con- ance (ANOVA) and expressed as mean ± SEM (n = 3). A taining the extract sample (200 µg) in ethanol (4.0 mL), 2.5% one-way analysis of variance followed by Dunnett’s multiple linoleic acid in ethanol (4.0 mL), phosphate buffer (8.0 mL, comparison tests was used for the data analysis, using SPSS 0.05 mol·L−1, pH 7.0), and distilled water (4.0 mL) was (Statistical Package for Social Sciences) version 10.0 soft- placed in a screw cap tube and thoroughly mixed. The reac- ware. A level of P < 0.05 was used as the criterion for statis- tion solution was incubated in the dark at 40 ºC. Aliquots (0.1 tical significance. mL) were drawn from the incubation reaction mixture and mixed with 75% ethanol (9.7 mL) and 30% ammonium thio- 3 Results cyanate (0.1 mL). Exactly 3 min after the addition of 0.1 mL Total phenolic content and total flavonoid content were −1 20 mmol·L ferrous chloride in 3.5% hydrochloric acid to determined from the calibration curves of gallic acid (Y = the reaction mixture, the absorbance of the red color was 0.004 4 x + 0.031, R2 = 0.999 5), and quercetin (Y = 0.028 8x measured at 500 nm every 24 h until the absorbance of the + 0.005 8, R2 = 0.999 1), respectively. The total phenolic and control reached a maximum. The control and standard were total flavonoid contents among the different extracts are pre- prepared as the sample. Linoleic acid mixture without the sented in Table 1. The results showed that methanol extract addition of sample was used as the control; α-tocopherol at possessed the highest phenolic [(90.08 ± 0.44) mg GAE/g of the same concentration served as the reference antioxidant. dry material] and flavonoid components [(58.50 ± 0.09) mg 2.6 Ex vivo antioxidant activity QE/g of dry material], followed by the ethyl acetate extract, 2.6.1 Lipid peroxidation assay while the petroleum ether extract contained very limited Wistar rats (200−250 g) were fasted for 16 h, and sacri- polyphenolic compounds. ficed by decapitation. The homogenate (5.0%, W/V) of liver tissues was prepared in phosphate buffered saline. An aliquot Table 1 Total phenolic and flavonoid contents of M. spinosa of 1.0 mL of homogenate was mixed with 100 μL of sample leaf extracts (mean ± SEM, n =3) solution and incubated for 2 h at 37 °C. Then, 1.0 mL of TCA Total phenolic content Total flavonoid content (15%, W/V) and 1.0 mL (0.67%, W/V) of TBA were added to Extracts (mg GAE/g of dry (mg QE/g of dry material) material) the mixture. This mixture was warmed in boiling water bath Methanol for 15 min and cooled. The final volume of the solution was 90.08 ± 0.44 58.50 ± 0.09 extract made up to 5.0 mL by adding deionized water and then cen- Ethyl acetate −1 55.83 ± 0.46 38.02 ± 0.07 trifuged at 2 800 r·min for 10 min. The absorbance of the extract [31] supernatant solution was recorded at 532 nm . The control Petroleum 36.83 ± 1.02 18.09 ± 0.08 sample was prepared without extracts. The inhibition of lipid ether extract peroxidation in percent (%) was calculated according to equation (1), where, Asample is the absorbance of a extract in M. spinosa leaf extract showed a concentration-response presence of liver homogenate and Acontrol is the absorbance of relationship in DPPH scavenging activity. An increase in the control solution (containing all of the reagents except the concentration is synonymous with an increase in scavenging test sample). capacity. As the positive control, ascorbic acid showed high −1 2.6.2 Oxidative hemolysis inhibition assay scavenging activity with IC50 of (3.3 ± 0.01) µg·mL . The −1 Oxidative erythrocyte hemolysis inhibition assay was IC50 of the methanol extract was (16.4 ± 0.41) µg·mL , fol- −1 performed using rat blood as per the procedure of Su et al. lowed by the ethyl acetate with IC50 of (17.8 ± 0.38) µg·mL , [32-33] with some modifications . Under mild anesthesia, a and petroleum ether extract with IC50 of (22.0 ± 0.43) blood sample was obtained from rat eyepit in heparinized µg·mL−1 (Table 2). tubes, and 5% erythrocyte suspension in phosphate buffered Superoxide anion reduces the yellow dye (NBT2+) to saline (PBS, 10 mmol·L−1, pH 7,4) was prepared which was generate the blue formazan, which is determined spectropho- used for the assay. Briefly, 0.5 mL of erythrocyte suspension tometrically at 560 nm. Extracts are able to inhibit the forma- was mixed with 0.5 mL extract solution (0.02−0.16 mg·mL−1) tion of blue NBT. All of the extracts effectively scavenged −1 and 0.05 mL of H2O2 (100 mmol·L ). The mixture was in- the superoxide anion generated by the system at concentra- −1 cubated at 37 ºC for 60 min, and 4.2 mL of distilled water tions in the range of 20−160 µg·mL . The IC50 of BHA, and was added after incubation. The solution was centrifuged at 1 the methanol, ethyl acetate, and petroleum ether extracts were 000 r·min−1 for 10 min, and the absorbance of the supernatant found to be (23.8 ± 0.89), (35.9 ± 0.19), (51.9 ± 0.28), and was read at 415 nm. The control sample was prepared with- (56.0 ±0.27) µg·mL−1, respectively (Table 2). At a concentra- out extract. The protective effect of the extract was calculated tion of 160 µg·mL−1, the scavenging activity of the methanol

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extract was 84.95%, while BHA produced a 82.53% scav- spinosa leaves. The IC50 value was higher for the methanol ex- enging effect at a concentration of 120 µg·mL−1. tract [(24.1 ± 0.33) µg·mL−1] than the other two extracts, but −1 In the hydroxyl radical scavenging assay, the degradation was lower than the standard drug [IC50 (20.8 ± 0.78) µg·mL ] +3 of deoxyribose by Fe -ascorbic acid-EDTA-H2O2 system (Table 2). was significantly decreased by all of the extracts of M. The methanol extract exhibited superior NO• scavenging

Table 2 Antioxidant activities of the leaves extracts of Mayna spinosa by using different in vitro models (mean ± SEM, n =3) −1 IC50/(µg·mL ) Extracts/ Standard DPPH Superoxide Hydroxyl Nitric oxide Hydrogen Fe2+ chelating radical radical radical radical peroxide ability Methanol extract 16.4 ± 0.41 35.9 ± 0.19 24.1 ± 0.33 23.7 ± 0.09 126.8 ± 2.92 117.2 ± 1.01 Ethyl acetate extract 17.8 ± 0.38 51.9 ± 0.28 41.6 ± 0.38 47.5 ± 0.18 282.0 ± 4.01 123.0 ± 1.07 Petroleum ether extract 22.0 ± 0.43 56.0 ± 0.27 55.4 ± 0.60 94.4 ± 0.29 323.0 ± 4.34 207.5 ± 1.82 Ascorbic acid 3.3 ± 0.01 23.0 ± 0.08 BHA 23.8 ± 0.89 α-Tocopherol 107.0 ± 1.23 Quercetin 20.8 ± 0.78 Gallic acid 65.0 ± 1.32 P < 0.05 vs positive control group

−1 activity, with an IC50 of (23.7 ± 0.09) µg·mL , than the ethyl 0.684 ± 0.007, 0.486 ± 0.004, and 0.453 ± 0.003, respectively −1 acetate and petroleum ether extracst (Table 2). The methanol at 50 µg·mL to 1.135 ± 0.005, 0.746 ± 0.002, and (0.697 ± −1 extract produced similar activity to that of the standard 0.005 at 800 µg·mL , respectively. −1 All of the extracts interfered with the formation of fer- ascorbic acid [IC50 (23.0 ± 0.08) µg·mL ]. rous and ferrozine complex suggesting that the extracts have All of the extracts were able to neutralize H2O2 in a significant (P < 0.05) chelating activity. The chelating activ- concentration-dependent manner [IC50 of (126.8 ± 2.92) ity of the extracts increased with concentration. The IC µg·mL−1] for the methanol extract, (282.0 ± 4.01) µg·mL−1 50 values of the iron chelating activity for the methanol, ethyl for the ethyl acetate extract, and (323.0 ± 4.34) µg·mL−1 for acetate, and petroleum ether extracts were (117.2 ± 1.01), the petroleum ether extract). Relatively slight neutralization (123.0 ± 1.07), (207.5 ± 1.82) µg·mL−1, respectively, while the of hydrogen peroxide, exhibited by the ethyl acetate and pe- IC value for α-tocopherol was (107.0 ± 1.23 µg·mL−1 (Ta- troleum ether extracts, could be explained partially by the 50 ble 2). relatively low polyphenolic content. The effects of the various solvent extracts of leaves of M. In the reducing power capacity assay, the extracts cause spinosa in preventing peroxidation of linoleic acid are shown the reduction of the Fe3+/ferricyanide complex to the ferrous in Fig. 2. The experiment was continued for 10 days until the form, which can be observed by measuring the formation of formation of peroxides was stopped because of the Perl’s Prussian blue at 700 nm. Fig. 1 shows the dose-re- non-availability of linoleic acid. The oxidation of linoleic sponse curves for the reducing powers of the extracts from M. acid was reduced in the presence of the extracts. The results spinosa leaves. The sequence for the reducing power was clearly indicate that all of the extracts exhibited significant (P ascorbic acid > methanol extract > ethyl acetate extract > < 0.05) antioxidant activity. The methanol extract produced petroleum ether extract. The reducing power of the methanol, better activity then α-tocopherol during the incubation time. ethyl acetate, and petroleum ether extracts increased from

Fig. 1 Reducing power ability of different leaf extracts from M. spinosa at different concentrations. Results are from Fig. 2 Antioxidant activity of M. spinosa leaf extracts by the triplicate measurements FTC method. Results are from triplicate measurements

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The ethyl acetate extract also showed a significant antioxi- rhosis, nephrotoxicity, cancer, and aging, etc. Antioxidant dant effect, though the petroleum ether extract showed the substrates from plants may offer resistance against the oxida- least effect compared to the other two extracts. tive stress by scavenging free radicals, inhibiting lipid per- Malondialdehyde (MDA) is a cytotoxic product formed oxidation, and by other mechanisms [1, 34]. Natural antioxidant during lipid peroxidation. The extracts significantly reduced defense systems protect biomolecules against ROS/RNS the MDA formation in a concentration dependent manner (Fig. induced damage, and re-establish or maintain ‘redox homeo- 3). The IC of rutin, and the methanol, ethyl acetate, and 50 stasis’. This protective effect can be enhanced by the use of petroleum ether extracts were (75.9 ± 0.92), (76.1 ± 0.69), antioxidant micronutrient (vitamins C and E, β-carotene), and (118.1 ± 0.93), and (120.2 ± 0.88) µg·mL−1, respectively. by non-nutrient ingredients like phenolic and flavonoid The methanol extract produced similar activity to rutin. At compounds from plants [1, 3, 5]. Thus the present study was 160 µg·mL−1 the methanol extract produced 86.36% inhibi- undertaken with the aim to carry out a systematic compara- tion, while rutin showed a 91.53% scavenging effect. tive antioxidant study on different extracts of M. spinosa

leaves using different in vitro and ex vivo methods, and to find any correlation between the antioxidant activity and total phenolic, total flavonoid contents of the extracts. DPPH is a stable free radical, when antioxidant reacts with DPPH• the electron is paired off and the DPPH solution is decolorized. The scavenging activity of the antioxidant or the bleaching of the color stochiometrically depends on the number of electrons taken up [1, 5]. The strong scavenging capacity of the extracts of M. spinosa leaves on DPPH• was possibly due to the hydrogen donating ability of the poly- phenolic compounds present in the extracts. The biological toxicity of superoxide depends on its ca- Fig. 3 Inhibition of lipid peroxidation in liver tissues of rat pacity to inactivate iron-sulfur clusters containing enzymes, by M. spinosa leaf extracts and rutin ( mean ± SEM, n = 3) that are important in several metabolic pathways. Superoxide is a highly reactive molecule which can produce a highly The results showed that erythrocyte hemolysis was ef- reactive hydroxyl radical and initiate lipid peroxidation and fectively inhibited by the M. spinosa leaf extracts at concen- thereby cause tissue damage. Superoxide anions also act as a −1 trations of 20−160 µg·mL . The methanol extract showed precursor of reactive species like hydrogen peroxide and the highest activity, and at concentrations of 20, 80 and 160 singlet oxygen [29, 35]. The present results suggest that M. −1 µg·mL , the inhibition rates were 37.67%, 64.15%, and spinosa leaves have a potent superoxide radical scavenging 85.03%, respectively (Fig. 4). The IC50 of ascorbic acid, and effect, which possibly renders them as promising antioxi- the methanol, ethyl acetate, and petroleum ether extracts were dants. (45.3 ± 0.08), (36.0 ± 0.06), (60.8 ± 0.10), and (139.0 ± 0.48) Hydroxyl radical is most reactive oxygen species, caus- −1 µg·mL , respectively. ing lipid peroxidation and enormous biological damage. It is a potent cytotoxic agent, able to attack and damage almost every molecule found in living tissues [36-37]. Therefore the scavenging of hydroxyl radical by extracts may provide a significant protection to biomolecules against free radicals. Nitric oxide is a gaseous free radical and is considered an important pleiotropic mediator of physiological processes like smooth muscle relaxant, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity. Although nitric oxide is considered as relatively less reactive, its metabolic producs,t such as peroxynitrite, formed after reacting with oxygen, is extremely reactive and can induce

toxic reactions, including thiol group oxidation, protein tyro- Fig. 4 The inhibition of M. spinosa leaf extract on erythro- sine nitration, lipid peroxidation, and DNA modification [20, cyte hemolysis in rat blood (mean ± SEM, n = 3) 33-34] . The nitrite scavenging ability of extracts further ex- pands the role of this plant as a potent antioxidant. 4 Discussion Hydrogen peroxide is a non-free radical species, and can Oxidative stress has been implicated in the causation of directly inactivate different enzymes, usually by the oxidation ailments such as diabetes, cardiovascular diseases, liver cir- of essential thiol groups. H2O2 is able to cross cell mem-

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Saikat Sen, et al. /Chinese Journal of Natural Medicines 2013, 11(2): 147−157 branes quickly, and may slowly oxidize a number of cell the M. spinosa leaf extracts can be a fundamental property compounds. H2O2 probably can react with metals ions like through which they can mitigate the initiation and/or propa- Fe2+, Cu2+ to form hydroxyl radicals, and this may be the gation of oxidative stress related diseases. origin of many of its toxic effects [24, 27, 37]. Thus, the abolition The cell like erythrocyte contains high concentrations of of hydrogen peroxide by the extracts is significant for both polyunsaturated fatty acids, molecular oxygen, and ferrous human health and the protection of pharmaceutical and food ions and is believed to be highly vulnerable to oxygen radical system. formation. Erythrocytes are highly susceptible to oxidation; The reducing power capacity of the extracts may provide therefore erythrocytes are a suitable cellular model for inves- a significant indication about the potential antioxidant capac- tigation of the oxidative damage in biomembranes. Oxidant ity of the plant. The reducing properties are generally con- damage of cell films, which may be induced by H2O2, can nected with the presence of reductones. The antioxidant ac- cause erythrocyte hemolysis [22, 32]. Therefore, the positive tion of reductones depends on the breaking of the free radical effect of the M. spinosa leaf extracts may help prevent cell chain by donating a hydrogen atom, or reacting with certain films from lipid oxidation and protect erythrocytes. precursors of peroxide to prevent peroxide formation [24, 30, 35]. The antioxidative effectiveness of natural sources has In this study, the methanol extract of the leaves of M. spinosa been reported to be mostly due to presence of phenolic and showed a promising result in this assay. flanonoid compounds. Polyphenolic compounds have re- The metal chelating capacity of a compound is important ceived extensive attention because of their beneficial physio- since it reduces the concentration of catalysing transition logical role, including antioxidant, antimutagenic, and for metals in lipid peroxidation. It was well established that che- other diseases caused by oxidative stress. The phenolic com- lating agents, which form s-bonds with a metal, are important pounds are very important plant constituents because of their as secondary antioxidants because they reduce the redox scavenging ability due to their hydroxyl groups. Flavonoids potential, thereby stabilizing the oxidized form of the metal are a large group of ubiquitous molecules and possess anti- ion [29, 35]. The ferrous ion increases lipid oxidation through oxidant activities. Their planar structure, number and position the break down of hydrogen and lipid peroxides to reactive of their hydroxyl groups, as well as the presence of the free radicals via the Fenton reaction. It can also accelerate C2-C3 double bond, are important for metal chelation, peroxidation by decomposing lipid hydroperoxides into per- free-radical scavenger capacities, and the inhibition of free oxyl and alkoxyl radicals. These radicals can themselves radical producing enzymes [1, 3, 5, 38-40]. The present study abstract hydrogen and perpetuate the chain reaction of lipid found a correlation between the polyphenolic content and the peroxidation [24, 37]. In this method, ferrozine can quantita- antioxidant activity, as the methanol extract contained the tively form complexes with Fe2+, and in the presence of the highest phenolic and flavonoid content, and exhibited the extracts, the complex formation is inhibited. Therefore, higher antioxidant activity. minimizing ferrous ions by the extracts may afford protection against oxidative damage. 5 Conclusions The FTC method was used to measure the amount of This work revealed that extracts of the leaves of Meyna [30, 35] peroxides produced at the initial stage of lipid oxidation . spinosa contain high levels of phenolics and flavonoids, and This method was used to measure the ability of antioxidants possess significant antioxidant activities. The methanol ex- to scavenge peroxyl radicals through hydrogen donation dur- tract exhibited remarkable free radical scavenging and anti- ing polyunsaturated fatty acid (PUFA) oxidation. Peroxides oxidant activity, which may due to the presence of a high are generated during linoleic acid oxidation, and react with polyphenolic content. The present study also indicates that 2+ 3+ Fe to form Fe . Ferric ions then react with thiocyanate to the possible antioxidant mechanism of the extract may be due produce a red-colored complex, and this complex has a to its hydrogen or electron donating and direct free radical [30] maximum absorbance at 500 nm . It is presumed that the scavenging properties. The antioxidant mechanisms and the msethanol extract owe its higher antioxidant activity due to identification of the antioxidant constituents in the methanol its higher phenolic and flavonoid contents. extract should be further studied to investigate use as a natu- Lipid peroxidation was an oxidative deterioration proc- ral antioxidant. It is also suggested that the plant be viewed as ess of polyunsaturated fatty acids which can induce damage a potential source of natural antioxidants which can provide to genomic and mtDNA, and eventually may lead to unstable precious functional ingredients useful for the prevention of cytological conditions such as apoptosis or tumor generation. diseases related to oxidative stress. Peroxidation of membrane lipids may cause an alteration of the stability of ligand-binding domains on the membrane, Acknowledgements disruption of membrane transport proteins, and deactivation The authors thank C.E.S. College of Pharmacy, Kurnool, of membrane-associated enzymes. Hence, oxidative damage India for providing facilities to carry out this work. We are connected with lipid peroxidation may set off a magnitude of thankful to Dr. B. K. Datta, J. Majumder, and K. Majumdar diseases [31-32]. Therefore, inhibition of lipid peroxidation by for helping in the identification of the plant.

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