Evaluation to the antioxidant activity of total flavonoids extract from persimmon (Diospyros kaki L.) leaves

Food and Chemical Toxicology 49 (2011) 2689–2696

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Evaluation to the antioxidant activity of total flavonoids extract from persimmon (Diospyros kaki L.) leaves Lijun Sun, Jianbao Zhang, Xiaoyun Lu, Liyu Zhang, Yali Zhang ⇑ Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, PR China

a r t i c l e

i n f o

Article history: Received 18 March 2011 Accepted 14 July 2011 Available online 23 July 2011 Keywords: Persimmon leaves Flavonoids Antioxidant activity Radical-scavenging activity

a b s t r a c t Persimmon leaves are commonly consumed as beverages, but are also used as a popular folk medicine in China. The purpose of this work is to assess the antioxidant activity of an extract of total flavonoids from persimmon leaves (TFPL). The effect of TFPL on total antioxidant activity, reducing power, 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical scavenging, superoxide anion ( O 2 ) radical scavenging, hydroxyl (OH ) radical scavenging and metal chelating activities was examined. We found that TFPL possesses considerable amounts of flavonoids (192 lg catechin equivalent/g of extract). The effect of this extract in total antioxidant activity, scavenging activity of superoxide anion and hydroxyl radical, reducing power and iron chelating activity was significantly better than that of rutin. However, the effect of TFPL in free radical scavenging of DPPH was significantly not as good as than rutin. In addition, TFPL significantly decreased the level of reactive oxygen species (ROS) and malondialdehyde (MDA), while increasing the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in MC3T3E1 cells in a dose-dependent manner. In conclusion, TFPL possess potent antioxidant and free radical scavenging activities. These antioxidant activities could contribute, at least in part, to the traditionally claimed therapeutic benefits of persimmon leaves. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Oxidative stress is a result of imbalance between the antioxidant defence system and the formation of reactive oxygen species (ROS). It is believed to damage cell membranes and DNA, as well as membrane lipid peroxidation with subsequent decreases in membrane fluidity (Finkel and Holbrook, 2000; Melov et al., 2000). Oxidative damage may cause cell injury, death and exacerbate the development of several age-related chronic diseases including cancer, Alzheimer’s disease, Parkinson’s disease and heart disease (Raouf et al., 2000). Therefore, antioxidants are considered to be important nutraceuticals with many health benefits. Antioxidants are widely used in the food industry as potential inhibitors of lipid peroxidation (Scherer and Godoy, 2009). However, many synthetic antioxidants used in foods, such as butylated hydroxyanisole and butylated hydroxytoluene, may accumulate in the body, resulting in liver damage and carcinogenesis (Valentao et al., 2002; Luo and Fang, 2008). For this reason, more attention has been paid to natural non-toxic antioxidants in an effort to protect the human body from free radicals and retard the progress of many chronic diseases.

⇑ Corresponding author. Tel.: +86 029 82668463. E-mail address: [email protected] (Y. Zhang). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.07.042

Persimmon (Diospyros kaki L.) is a kind of plant native to China, used traditionally for many medicinal purposes, including the treatment of paralysis, frostbite, and burns, and to stop bleeding (Matsuo and Ito, 1978). Flavonoid oligomers, tannins, phenols, organic acids, chlorophyll, vitamin C, and caffeine are found in persimmon leaves (Matsuo and Ito, 1978; Jo et al., 2003). The leaves are commonly used for tea in Asia. Previous studies have shown that persimmon leaves have beneficial effects on haemostasis, constipation, hypertension, apoplexy and atherosclerosis (Kotani et al., 2000; Matsumoto et al., 2002; Tanaka et al., 2003; Sakanaka et al., 2005). In particular, flavonoid aglycones in persimmon leaves, such as catechin, kaempferol, and quercetin, reportedly possess strong antioxidant activities by acting as oxygen radical scavengers and metal chelators (Morel et al., 1993; Birt et al., 2001). Chemically, flavonoids and isoflavonoids are one-electron donors. They are derivatives of conjugated ring structures and hydroxyl groups that have the potential function as antioxidants in cell culture in vitro, or in cell free systems. The previous studies suggest that the flavonoids present in persimmon leaves could contribute to the health benefits attributed to persimmon (Bei et al., 2005, 2009; Lee et al., 2006). The balance between osteoclastic bone resorption and osteoblastic bone formation maintains bone mass at a homeostatic steady state. The imbalance that occurs when bone resorption is greater than bone formation can lead to skeletal diseases, including

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osteoporosis. Oestrogen deficiency causes osteoporosis via increased generation of ROS. Several reports have demonstrated that osteoblast differentiation can be inhibited by oxidative stress or induced by exogenous stimuli such as hydrogen peroxide or xanthine/xanthine oxidase (Mody et al., 2001; Bai et al., 2004). These findings suggest that ROS may represent a critical target for the treatment and/or prevention of osteoporosis. For this reason, antioxidants may prove to be effective therapeutic candidates for osteoporosis (Riggs et al., 2002). If a total flavonoid extract of persimmon leaves (TFPL) is able to prevent free radical damage in osteoblast-like cells in vitro, it should be able to prevent cell death. The prevention of bone loss by the inhibition of oxidative stress and the prevention of osteoblast death in vitro could be considered as groundwork for the therapeutic benefit of TFPL. The clinical experiment is still needed to investigate the therapeutic effects of TFPL administration to humans, especially those at highest risk of osteoporosis, i.e., elderly women. To our knowledge, few investigations have been made of the antioxidant properties of persimmon leaves, although this medicinal plant is widely used by Chinese traditional healers. The aims of this study were to determine the total flavonoid content of persimmon leaves and to evaluate the properties of TFPL using widely accepted antioxidative and free radical-scavenging model systems. Rutin, a lipid-soluble analogue of flavonoids, was used as a reference antioxidant compound (Yang et al., 2008). In addition, the levels of ROS and antioxidant enzymes such as superoxide-dismutase (SOD) and glutathione (GSH-Px) were also determined in MC3T3E1 cells after treatment with TFPL. The results suggest that TFPL could be useful as a potential natural antioxidant in the functional food and pharmaceutical industries. 2. Materials and methods 2.1. Materials Persimmon (D. kaki L. folium) leaves were harvested in Shaanxi Province, China in October 2010, and identified according to the identification standard of the Pharmacopeia of the People’s Republic of China. Persimmon (D. kaki L. folium) leaves were dried in the shade for a week and powdered and passed through 60 mesh sieves. The raw material was stored at 20 °C before extraction. The reagents 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferrozine, Dimethylsulphoxide (DMSO), 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and 2’,7’-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemicals (St Louis,  MO, USA). Assay kits for superoxide anion radical ( O 2 ), hydroxyl radical (OH ), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), protein, and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) was purchased from Gibco BRL (Grand Island, NY, USA). All other reagents were of analytical grade and made in China. 2.2. Preparation of extracts of persimmon leaves Persimmon leaves (50 g) were cut into pieces approximately 2 cm in width and dried. Leaves were soaked in a 70% ethanol solvent (1:10, w/v) for 2.5 h and then placed in an ultrasonic bath and sonicated at 200 kHz at 55 °C for 45 min. Samples were then filtered through a 0.45 lm microporous membrane (Shanghai Wanzi Shiye Co. Ltd., Shanghai, China). The filtrate was collected, and the solid was extracted two more times with the same volume of fresh solvent. Solutions were combined and concentrated to dryness under reduced pressure in a rotary evaporator to yield dried crude total extracts. The extract was added to distilled water and defatted with petroleum ether and ethyl acetate. 2.3. Determination of total flavonoid content The total flavonoid content of the extract was determined by the method described in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, 2005). The 500 ll extract was diluted appropriately and mixed with 1 mL NaNO2 (5%). After standing for 6 min, 1 mL of 10% AlCl3 and 10 mL of NaOH (1 M) were added to the mixture. The mixture was adjusted to 25 mL with 70% ethanol and allowed to rest for 15 min. The absorbance (A) was measured at 510 nm, with 70% ethanol as a blank control. Rutin was used as a reference standard and the total flavonoid content was expressed as rutin equivalents (RE, lg/mg extract). All determinations were performed in triplicate.

2.4. Antioxidant activity assays 2.4.1. Total antioxidant capacity (TAOC) Suitable working standards (0.24, 1.0, 5.0, and 10 mg/mL) were prepared by dissolving the TFPL in distilled water. Aliquots (0.30 mL) were mixed with 3 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped with aluminium foil and incubated at 95 °C for 90 min (Umamaheswari and Chatterjee, 2008). The tubes were cooled to room temperature and absorbance was measured at 695 nm against a blank. Ascorbic acid was used as a standard. Total antioxidant capacity was expressed as equivalents of ascorbic acid (Raghavan et al., 2003). 2.4.2. Radical scavenging activity 2.4.2.1. DPPH radical scavenging activity. The ability of the TFPL to scavenge the DPPH free radical was assayed according to the method of Shimada et al. (1992). Briefly, a 0.1 mM solution of DPPH in 100% MeOH was prepared. To 1 ml of this solution was added 4 ml of sample solution in 40% MeOH at different concentrations (1–160 lg/mL). The mixture was shaken vigorously and incubated for 15 min in the dark at room temperature until stable absorption values were obtained. The reduction of the DPPH radical was measured by continuously monitoring the decrease in absorption at 517 nm. In the control, 40% MeOH was substituted for samples. Lower absorbances of the reaction mixture indicated higher free radical scavenging activity. The DPPH radical scavenging activity was calculated by the following equation:

Scavenging effect ð%Þ ¼ ð1  Asample517 =Acontrol517 Þ  100 The EC50 value is the concentration of the sample required to scavenge 50% of the DPPH free radical. 2.4.2.2. Superoxide anion radical scavenging activity. The measurement of superoxide anion scavenging activity was based on the method described by Nishikimi et al. (1972) with slight modifications. Superoxide radicals were generated in the PMS– NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 lM NADH (adenine dinucleotide), 72 lM NBT (nitroblue tetrazolium), and 30 lM PMS (phenazine methosulphate). Varying concentrations of samples ranging from 25 to 400 lg/mL were added to the PMS–NADH system for free radical scavenging. These reaction mixtures were incubated at room temperature for 5 min before the absorbance was read at 560 nm against the blank. In the control, the sample was substituted with Tris–HCl buffer. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The ability of TFPL to scavenge superoxide radical was calculated using the following equation:

Scavenging effect ð%Þ ¼ ð1  Asample560 =Acontrol560 Þ  100 EC50 value (mg/ml) is the concentration at which the scavenging activity is 50%. 2.4.2.3. Hydroxyl radical scavenging activity. The hydroxyl radical scavenging activity of samples of TFPL was measured using a modified Smirnoff and Cumbes’ (1989) method. Hydroxyl radicals were generated in a solution of 2 mM EDTA–Fe (0.5 mL), 3% H2O2 (1 mL), and 360 lg/mL crocus in 4.5 mL sodium phosphate buffer (150 mM, pH 7.4). Samples at concentrations ranging from 25 to 400 lg/mL were incubated for 30 min at 37 °C and hydroxyl radicals were detected by monitoring absorbance at 520 nm. In the control, the sample was substituted with distilled water and sodium phosphate buffer replaced H2O2. The capability of hydroxyl radical scavenging was calculated using the following equation:

Scavenging effect ð%Þ ¼ ð1  Asample520 =Acontrol520 Þ  100 EC50 value (mg/ml) is the concentration at which the scavenging activity is 50%. 2.4.3. Reducing power The reducing power of TFPL was determined using the method of Yen and Chen (1995). Briefly, 0.13 mL of different concentrations of samples (25–400 lg/mL) suspended in phosphate buffer (0.2 M, pH 6.6) were mixed with 0.125 mL of potassium ferricyanide (1%, w/v) and incubated at 50 °C. At 20 min, 0.125 mL of trichloroacetic acid (10%, w/v) was added to the mixture to terminate the reaction. The solution was mixed with 1.5 ml of ferric chloride (0.1%, w/v), and the absorbance was measured at 700 nm. An increased absorbance of the reaction mixture indicated increased reducing power. 2.4.4. Chelating activity on ferrous ions The chelating activities of test compounds on Fe2+ were estimated based on the decrease in the maximal absorbance of the iron (Fe2+)–ferrozine complex assayed following previously reported methods (Dinis et al., 1994), with some modifications. Briefly, 1.0 ml of test compounds dissolved in ethanol were incubated with 0.5 ml of FeCl24H2O (1.0 mmol/L). The reaction was initiated by the addition of 1 ml of ferrozine (5.0 mmol/L), and the total reaction volume was adjusted to 4 ml with ethanol. After the mixture had reached equilibrium (10 min), the

L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696 absorbance at 562 nm was read. The negative control was prepared without the test compound, and EDTA was used as the positive control. The chelating activity of the test compound on Fe2+ was calculated as follows:

Chelating activity ð%Þ ¼ ðAControl562 =ASample562  1Þ  100

2.5. Antioxidant activities of TFPL in MC3T3-E1 cells 2.5.1. Cell culture and treatment Murine osteoblast MC3T3-E1 cells were maintained in DMEM supplemented with 10% heat-inactivated fatal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified 5% CO2 incubator at 37 °C. The medium was changed every 2 days. For experimental treatments, TFPL was added to the cultured cells. After 24 h of co-culturing, the cells were harvested, counted, and used for further assays. 2.5.2. Cell viability The cell survival rate was quantified using a colorimetric MTT assay to measure mitochondrial activity in viable cells. This method is based on the conversion of MTT to formazan crystals by mitochondrial enzymes. Briefly, cells seeded at a density of 5  104 cells/well in a 96-well plate were left to adhere overnight. MTT was prepared at 2.5 mg/mL in PBS. Aliquots (20 ll) of the MTT stock solution were pipetted into each well and the plate was incubated at 37 °C in a humidified 5% CO2 incubator. After 4 h, the medium was removed and DMSO (200 ll) was added to each well to dissolve the formazan. After 10 min, the optical density of each well was measured at 570 nm by spectrophotometry.

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metabolite with anti-oxidative, anti-inflammatory and anti-carcinogenic effects. Rutin can also reduce the fragility of blood vessels found in haemorrhagic disease and hypertension in humans (Oomah et al., 1996). In this study, rutin was used as positive control. Flavonoids are very important constituents of plants because of the scavenging ability conferred by their hydroxyl groups. The flavonoids may contribute directly to anti-oxidative action. It is known that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans when up to 1 g daily is consumed from a diet rich in fruits and vegetables (Tanaka et al., 1998). Flavonoid compounds from plants are known to be good natural antioxidants. However, the activity of synthetic antioxidants was often observed to be higher than that of natural antioxidants (Ningappa et al., 2007). Flavonoid compounds at certain concentrations markedly slowed down the rate of conjugated diene formation. Interest in phenolics is increasing in the food industry because of their ability to retard oxidative degradation of lipids, thereby improving the quality and nutritional value of foods (Aneta et al., 2007). 3.2. Antioxidant activities

2.5.3. Characterisation of oxidative status Cellular ROS levels were measured by the dichlorofluorescein assay, as described by Zuo and Clanton (2002). Accumulations of intracellular ROS can be detected using DCFH-DA, which crosses cell membranes and is hydrolysed by intracellular esterases to non-fluorescent DCFH. In the presence of ROS, DCFH is oxidised to the highly fluorescent dichlorofluorescein (DCF), which is readily detected in a fluorescent microplate reader. The cells (105 cells/well) were incubated with 100– 500 lg/mL of a test sample for 24 h. Subsequently, 200 mM ethanol was added to the cells, which were then incubated with 25 lM DCFH-DA for an additional 30 min. The fluorescence intensity of the cells was measured on a fluorescence microplate reader (BioTek Instruments, Winooski, VT) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Activities of SOD, CAT, MDA and GSH-Px were determined spectrophotometrically using commercially available assay kits following the manufacturer’s protocols. The level of lipid peroxidation was indicated by the amount of MDA in the cells as assayed using the thiobarbituric acid reaction (TBARS). Values of MDA were expressed as nmol/ml of cells. Protein concentration was measured using the BCA protein assay kit (Pierce) with BSA as standard. The activity of CAT was determined by measuring the decrease in absorbance at 405 nm due to the degradation of H2O2. The results of this enzymatic assay were reported in units of CAT activity per milligram of protein (U/mg protein), where 1 U of CAT is defined as the amount of enzyme that decomposes 1 mmol of H2O2 per second. The activity of SOD was determined spectrophotometrically at 550 nm, indicating the inhibition of the oxidation of oxymine by the xanthine/xanthine oxidase system. The results of this enzymatic assay were reported in units (U) of SOD activity per ml (U/ml) of cells. The activity of GSH-Px was assayed by quantifying the rate of oxidation of glutathione to oxidised glutathione by H2O2. One unit of GSH-Px was defined at 412 nm as the amount that reduced the level of GSH by 1 lmol/L in 1 min/mg protein (U/mg protein).

It has long been recognised that naturally occurring substances in higher plants have antioxidant activity. Among those substances, the flavonoids that are widely distributed in plants have the ability to scavenge free radicals, superoxide and hydroxyl radicals by single-electron transfer (Havsteen, 2002; Le et al., 2007; Leman’ska et al., 2001). An antioxidant exerts its antioxidant activity through various mechanisms, including chelating ferrous iron, degrading peroxide, and scavenging free radicals. In our experiments, the antioxidant and radical scavenging activity of TFPL has been investigated by assessing their effect on TOAC, DPPH radical scavenging activity, superoxide anion removal, hydroxyl radical trapping potential, reducing power and metal chelating ability. We speculate that the antioxidant profiles of TFPL and rutin are similar, but may be quantitatively different, perhaps associated with their similar but different chemical structures.

2.6. Statistical analysis

3.2.2. Radical scavenging activity It is well known that antioxidants can seize the free radical chain of oxidation and form stable free radicals, which do not initiate or propagate further oxidation. The DPPH radical was extensively used to evaluate the free-radical scavenging capacity of antioxidants (Halliwell and Gutteridge, 1999). Scavenging of the DPPH radical is related to the inhibition of lipid peroxidation. DPPH radical involves a hydrogen atom transfer process (Kaviarasan et al., 2007). Our results found that both TFPL and rutin were effective at reducing the stable radical DPPH to the yellow-coloured diphenylpicrylhydrazine, indicating that these extracts are active in DPPH radical scavenging (Fig. 2(A)). TFPL had significant scavenging effects with increasing concentrations in the range of 12.5– 500 lg/ml. However, the scavenging effect of TFPL was significantly lower than that of rutin. At 200 lg/ml, TFPL and rutin exhibited 68.73% and 89.13% inhibition, respectively, and the EC50 values were 96.36 and 41.52 lg/ml for TFPL and rutin, respectively (Table

The data in triplicate were subjected to analysis of variance (ANOVA) and expressed as mean ± standard deviation (n = 6). Analysis of variance and the difference among samples were determined by Duncan’s multiple-range test using the Statistical Analysis System (SAS release 6.12) program. A level of p < 0.05 was used as the criterion for statistical significance.

3. Results and discussion 3.1. Total flavonoid content The total flavonoid content of TFPL was expressed as rutin equivalents in mg/g of extracts. The TFPL extracts contained 192 ± 9.6 mg/g total flavonoids. Because flavonoids are responsible for antioxidant activity, the high amount of total flavonoids in the extract suggests that the extract possesses an antioxidant activity in vitro (Lotito and Frei, 2004). Rutin is a flavonol glycoside plant

3.2.1. Total antioxidant activities Total antioxidant activities reflect the capacity of a nonenzymatic, antioxidant defence system. In the phosphomolybdenum method, molybdenum VI (Mo6+) is reduced to form a green phosphate/Mo5+ complex at acidic pHs. High absorbance values indicate that the sample possesses significant antioxidant activity. As shown in Fig. 1, total antioxidant activities of TFPL samples are superior to rutin and the effects are concentration-dependent. The TAOC of TFPL is close to that of rutin at a dose of 400 lg/mL.

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L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696 Table 1 Effect of total flavonoids from persimmon leaves on different radical scavenging activities. Samples

TFPL Rutin

EC50 (lg/mL)a DPPH radical scavenging activity

Superoxide radical scavenging activity

Hydroxyl radical scavenging activity

96.36 ± 2.63 41.52 ± 1.96

41.58 ± 0.21 130.58 ± 3.58

70.64 ± 3.22 111.23 ± 4.37

a EC50 is a measure of radical scavenging activity being the concentration required to inhibit 50% free radical activity.

Fig. 1. Total antioxidant activities of total flavonoids from persimmon leaves. ⁄ p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same concentration group.

Fig. 2. Radical-scavenging activity of total flavonoids from persimmon leaves (TFPL). Radical-scavenging activities were assessed by measuring the DPPH scavenging activities (A), superoxide anion scavenging activities (B), and hydroxyl scavenging activities (C). ⁄p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same concentration group.

1). The different concentrations of TFPL (12.5, 25, 50, 100, 200, 250 and 500 lg/ml) showed antioxidant activities in a dose dependent manner (14.48%, 21.15%, 32.06%, 51.28% 68.73%, 75.62% and 91.59% inhibition, respectively) in the DPPH radical scavenging

assay (Fig. 2(A)). A higher DPPH radical scavenging activity is associated with a lower EC50 value. In this assay, the good antioxidant activity of TFPL on the DPPH radical may be attributed to a direct role in trapping free radicals by donating a hydrogen atom. Superoxide anions play important roles in the formation of ROS such as hydrogen peroxide, hydroxyl radical and singlet oxygen, which induce oxidative damage in lipids, proteins and DNA (Pietta, 2000; Wickens, 2001). It was therefore proposed to measure the comparative interceptive ability of the antioxidant extracts to scavenge the superoxide radical. In our study, superoxide-scavenging activities of extracts were measured by auto-oxidation of hydroxylamine in the presence of NBT (nitroblue tetrazolium). The reduction of NBT in the presence of antioxidants was measured. A decrease in absorbance at 560 nm indicates the consumption of superoxide anion in the reaction mixture. As the data in Fig. 2(B) show, different concentrations of TFPL (6.25–150 lg/ml) exhibited superoxidescavenging activity (13.21%, 32.14%, 41.70%, 54.42%, 76.87% and 87.56% inhibition, respectively). Aliquots of TFPL or rutin (100 lg/ml) exhibited 76.87% and 48.12% inhibition, respectively (Fig. 2(B)). The EC50 value of TFPL on superoxide radical scavenging activity was found to be 41.35 lg/ml, whereas the EC50 value of rutin was found to be 130.58 lg/ml (Table 1). All of the extracts scavenged superoxide radicals in a dose dependent manner. Therefore, when compared to rutin, the superoxide scavenging activity of the extract was high. This could be due to the presence of reactive bioactive constituents and the mixture of other nutrients in the extract. Results were statistically significant (p < 0.05). Based on our results, it appears that TFPL scavenges superoxide radicals by combining with superoxide radical ions to form stable radicals, thus terminating the radical chain reaction (Wang et al., 2009). Hydroxyl radicals are extremely reactive free radicals formed in biological systems and have been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells (Gulcin, 2006). Hydroxyl radicals are very strongly reactive oxygen species, and there is no specific enzyme to defend against them in humans (Liu et al., 2005). Therefore, it is important to discover chemicals with good scavenging capacity for these reactive oxygen species. The hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity (Babu et al., 2001). The scavenging effect of TFPL against hydroxyl radicals was investigated using the Fenton reaction. The % inhibition of TFPL (25–125 lg/ml) on hydroxyl radical scavenging was found to be 30.62%, 38.62%, 52.40%, 57.08% and 77.85%, respectively (Fig. 2(C)). All results showed antioxidant activity in a dose dependent manner. The EC50 values of TFPL and rutin were found to be 70.64 and 111.23 lg/ml (Table 1). The ability of the TFPL extracts to quench hydroxyl radicals seems to be directly related to the prevention of propagation of lipid peroxidation; because TFPL seems to be a good scavenger of active oxygen species, it will thus reduce the rate of the chain reaction. Yen and Hsieh (1995) reported that xylose and lysine Maillard reaction products had scavenging activity on hydroxyl radicals in a dose response manner. This result may be attributed to the combined effects of reducing power, the donation of hydrogen atoms

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and the scavenging of active oxygen. Hagerman et al. (1998) have also explained that high molecular weight and the proximity of many aromatic rings and hydroxyl groups are more important for the free radical scavenging activity of phenolics than are specific functional groups. 3.2.3. Reducing power Reducing power is one mechanism of action of antioxidants and may serve as a significant indicator of potential antioxidant activity (Jayaprakasha et al., 2000). Several studies have indicated that the antioxidant effect is related to the development of reductones (Yen and Duh, 1993). Therefore, in this study, reducing activity was determined based on the ability of extracts or rutin to reduce a Fe3+/ferricyanide complex to form an Fe3+ ferrous complex. The amount of Fe2+ was monitored by measuring the formation of Perl’s Prussian blue at 700 nm (Yang et al., 2010). The dose– response curve for the reducing activity of TFPL and rutin is shown in Fig. 3. In the present study, TFPL showed a significantly higher reducing power than rutin. The data suggest that TFPL has a good ability to donate electrons to reactive free radicals, converting them into more stable products and terminating the free radical chain reaction. 3.2.4. Chelating activity on ferrous ions In addition to initiating lipid peroxidation, metal ions possessing catalytic ability have been correlated with arthritis and cancer incidence (Halliwell et al., 1995). Additionally, ferrous ions are thought to be effective pro-oxidants (Yamaguchi et al., 1998). Because EDTA is a strong chelator of metal, it is used as the standard metal chelator in this study. Ferrous ion chelating activities of TFPL, rutin and EDTA are shown in Fig. 4. Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating agents, complex formation is disrupted, resulting in a decrease in the red colour of the complex. Measurement of colour reduction therefore allows an estimation of the metal chelating activity. In this assay, TFPL and both standard compounds interfered with the formation of ferrous and ferrozine complexes, suggest that they have chelating activity and are able to capture ferrous ion before ferrozine. The absorbance of Fe2+–ferrozine complex was decreased by TFPL in a dose dependent fashion from 2.5 to 50 lg/mL. Although the iron chelating ability of TFPL was moderate, it was significantly stronger than rutin. In addition, the percentages of the metal chelating capacities of 50 lg/mL of TFPL and 6.25 lg/mL of EDTA were 77.38% and 87.34%, respectively. Chelating agents, which form r-bonds with metals, are effective as secondary antioxidants because they reduce redox potential, thereby stabilising the oxidised form of the metal ion.

Fig. 3. Reducing power of total flavonoid extract from persimmon leaves (TFPL). ⁄ p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same concentration group.

Fig. 4. Chelating activity on ferrous ions of total flavonoids from persimmon leaves (TFPL). EDTA was used as positive control. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with EDTA.

Extracts or compounds with chelating activity are believed to inhibit lipid peroxidation by stabilising transition metals. Accordingly, it is suggested that the low-to-moderate chelating effect of TFPL would be at least partially beneficial in protecting against oxidative damage. Rutin (quercetin-3-rutinoside) is one of the major flavonoids found in a variety of plants. Rutin is receiving increasing attention due to its various health beneficial biological activities, including antioxidative, anti-inflammatory, and anti-hypertensive effects. Yet practical limitations do exist regarding the incorporation and effectiveness of flavonoids as antioxidants in many food systems, possibly due to their poor water solubility (0.125 g/L) (Khalifa et al., 1983). Total flavonoids from persimmon leaves possess a good water-soluble. Therefore, TFPL possess higher antioxidant activities that rutin in water as the solvent test system. As potent antioxidants, flavonoids reportedly possess strong free radical scavenging activities based on their ability to act as hydrogen or electron donors. In addition, some flavonoids may also react directly with radicals to form less reactive products, or else chelate transition metals which could otherwise act as procoxidants in the system (Le et al., 2007). Overall, it is the structural elements making up the flavonoid or flavonoid esters which dictate its antioxidative behaviour, including features like (i) degree and position of hydroxylation, (ii) type and position of additional substituents, (iii) presence of double bonds or conjugation and (iv) glycosylation (Leopoldini et al., 2011). Five flavonoid compounds, kaempferol 3-O-h-D-galactopyranoside (TR), kaempferol 3-O-h-Dglucopyranoside (AS), isorhamnetin 3-O-h-D-glucopyranoside (IS), quercetin 3-O-h-D-galactopyranoside (HY) and quercetin 3-O-h-Dglucopyranosyl-(6!1)-a-L-rhamnopyranoside (RU), isolated from the leaves of persimmon have been reported (Saito et al., 1994; Meng and Xu, 1998). Flavonols and flavones containing a catechol group in ring B are found to be highly active, with flavonols more potent than the corresponding flavones because of the presence of the 3-hydroxyl group. On the contrary, rutin is a large 3-OH-substituted aromatic glycoside groups, so that the whole molecular structure rearrangement, molecular energy is reduced, 30 -OH and 40 -OH activity has weakened more, and its antioxidant activity is also greatly reduced. The presence of C-3 glycoside groups in ring C of rutin diminishes the antioxidant activity (Leopoldini et al., 2011). However, Kayoko et al.’s study shows the galloylated flavonol glycoside mixture had an antioxidant activity approximately twofold stronger than that of the nongalloylated flavonol glycosides (Kawakami et al., 2011). This suggests that further studies of these flavonoids on its pharmaceutical functions and threedimensional structure may be of help in the development of its clinical application.

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3.3. Effect of TFPL on MC3T3-E1 cells 3.3.1. Cell viability We used the MTT assay to study the proliferative activity of TFPL. MTT is reduced by mitochondrial dehydrogenase to produce formazan, an insoluble purple compound. We measured cell proliferative activity in terms of the intensity of the purple colour. As Fig. 5 shows, increasing doses of TFPL are not toxic and do not inhibit the growth of MC3T3-E1 cells. Instead, TFPL promoted the growth rate of the cells. At TFPL doses of 5, 10, and 20 lg/mL, cell viabilities were 98.17%, 99.62%, and 100.32%, respectively, after treatment for 24 h. The cell viability of MC3T3-E1 cells treated with TFPL was significantly superior to the control group. The results indicated that TFPL was not harmful to MC3T3-E1 cells at the tested concentration ranges and markedly facilitated the growth rate of MC3T3-E1 cells at concentrations between 5 and 20 lg/ml. TFPL may act by facilitating glucose uptake and subsequently increasing pyruvate concentrations, leading to increased cellular energy and proliferation. These findings in cells in vitro may prove relevant to protecting against the loss of bone mass and the development of osteoporosis in human subjects. 3.3.2. The levels of ROS and antioxidant enzymes Reactive oxygen species are believed to play an important role in cell death. Drugs that exhibit properties of an antioxidant by regulating the levels of enzymes with antioxidant activity, such as SOD, CAT and GSH, may serve as potential candidates for the treatment of osteoporosis. To elucidate the effect of TFPL on MC3T3-E1 cells, measures of oxidative stress, the ROS level, the MDA level, the GSH level, and the activity of SOD and CAT were measured. As shown in Fig. 6, TFPL had significant effects on ROS levels, MDA content, SOD, CAT and GSH-Px activities in MC3T3E1 cells (p < 0.05). The SOD activities of cells treated with 10 and 20 lg/mL TFPL were significantly higher than the control (p < 0.01). The activity of SOD was significantly higher in cells with 5, 10, and 20 lg/mL TFPL compared to controls (p < 0.05). MDA is an end product of lipid peroxidation, and its content is used to estimate the degree of oxidative damage in cells. Therefore, antioxidant enzyme activities and MDA content are key indicators of antioxidant capacity. It was reported that SOD converts superoxide radicals to hydrogen peroxide, which is subsequently converted to water by CAT and GSH-Px (Wang et al., 2007). Thus, SOD protects cells from the toxicity of superoxide radicals. The enzyme GSH plays an important role in regulating intracellular redox status. An increase in GSH level protects cells against cell death either by removing free radicals or by conjugating them with toxicants (Kong et al., 2009). Elevated GSH levels and SOD activities

Fig. 5. Effect of rutin and TFPL on cell viability in MC3T3-E1 cells. MC3T3-E1 cells were incubated at 37 °C in the absence or presence of rutin and TFPL. Cell viability was measured by MTT assay. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with the control group.

Fig. 6. Effect of TFPL on the ROS levels (A), MDA levels (B), SOD activities (C), CAT activities (D), and GSH levels (E) in MC3T3-E1 cells. MC3T3-E1 cells were incubated at 37 °C in the absence or presence of TFPL. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with the control group.

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provide a repair mechanism for oxidised membrane components. The results of this experiment indicated that SOD, CAT, and GSHPx activities in MC3T3-E1 cells treated with 1.25–20 lg/mL TFPL were higher than those of controls. The MDA content of cells was decreased by TFPL additions at the same concentrations. Thus, TFPL in the range of 1.25–20 lg/mL could improve the antioxidant capacity of MC3T3-E1 cells through increasing the activity of enzymes with antioxidant activity. 4. Conclusions In conclusion, the present study has demonstrated that TFPL possesses considerable amounts of flavonoids (192 lg catechin equivalent/g of extract). Rutin, a lipid-soluble analogue of flavonoids, was used as a reference antioxidant compound. The data obtained clearly indicate that the extract possesses potent total antioxidant activity, scavenging superoxide anion and hydroxyl radicals, reducing power and iron chelating activity, at levels superior to rutin. The TFPL was able to scavenge 1,1-diphenyl-2-picrylhydrazyl, although less effectively than rutin. This study found that the treatment of MC3T3-E1 cells with TFPL caused a marked reduction in ROS and MDA production and a rise in GSH level, SOD and CAT activities, suggesting that the protective effect of TFPL may be related to its antioxidant ability. These antioxidant activities could have contributed, at least partly, to the therapeutic benefits of the certain traditional claims for persimmon leaves. In view of the potential use of persimmon leaves in the functional food industry, its therapeutic benefits and bioactive compounds warrant further investigation. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financed by the National Natural Science Foundation of China (30801059, 10972177), by the Scientific and Technical Foundation of Xi’an City (2009, SF09031(3)), by a Project of Basic Research of Xi’an Jiaotong University (08143011), and by a Project of the National Undergraduate Student Innovative Experiment of China. This work was partly supported by Institute of Mitochondrial Biology and Medical, Xi’an Jiaotong University, Xi’an China. References Aneta, W., Jan, O., Renata, C., 2007. Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chemistry 105, 940–949. Babu, B.H., Shylesh, B.S., Padikkala, J., 2001. Antioxidant and hepatoprotective effect of Alanthus icicifocus. Fitoterapia 72, 272–277. Bai, X., Lu, D., Bai, J., Zheng, H., Ke, Z., Li, X., et al., 2004. Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-jB. Biochemical and Biophysical Research Communications 314, 197–207. Bei, W., Peng, W., Ma, Y., Xu, A., 2005. Flavonoids from the leaves of Diospyros kaki reduce hydrogen peroxide-induced injury of NG108-15 cells. Life Sciences 76 (17), 1975–1988. Bei, W., Zang, L., Guo, J., Peng, W., Xu, A., Good, D.A., et al., 2009. Neuroprotective effects of a standardized flavonoid extract from Diospyros kaki leaves. Journal of Ethnopharmacology 126 (1), 134–142. Birt, D.F., Hendrich, Suzanne, Wang, W.Q., 2001. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacology and Therapeutics 90, 157–177. Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics 315, 161–169. Finkel, T., Holbrook, N.J., 2000. Oxidant, oxidative stress and biology of ageing. Nature 408, 239–247. Gulcin, I., 2006. Antioxidant and antiradical activities of L-carnitine. Life Science 78, 803–811.

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