Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China

ARTICLE IN PRESS JOURNAL OF FOOD COMPOSITION AND ANALYSIS Journal of Food Composition and Analysis 21 (2008) 219–228 www.elsevier.com/locate/jfca

Original Article

Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China Xiaoli Liua, Mouming Zhaoa,, Jinshui Wanga, Bao Yangb, Yueming Jiangb a

College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, People’s Republic of China b South China Botanical Garden, Chinese Academy of Sciences, Guangzhou Leyiju 510650, People’s Republic of China Received 28 October 2007; received in revised form 29 October 2007; accepted 30 October 2007

Abstract The phenolic contents of methanolic extracts of emblica (Phyllanthus emblica L.) fruit from six regions in China were measured in this work. The antioxidant activities of these extracts were also evaluated. Total phenolic content was ranged from 81.5 to 120.9 mg gallic acid equivalents (GAE)/g and the flavonoid content was varied from 20.3 to 38.7 mg quecetin equivalents (QE)/g, while proanthocyanidin content was ranged from 3.7 to 18.7 mg catechin equivalents (CE)/g. Among all the methanolic extracts analyzed, the Huizhou sample exhibited a significantly higher phenolic content than other samples (Po0.05). The antioxidant activities were evaluated by in vitro experiments using scavenging assays of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, and superoxide anion radicals, chelating ability of ferrous ion, reducing power, and inhibition capability of Fe (II)-induced lipid peroxidation, respectively. The Huizhou sample was found to have the strongest antioxidant activities in scavenging DPPH radicals, superoxide anion radicals, and had the highest reducing power, while the Chuxiong sample showed the best performance in chelating iron and inhibiting lipid peroxidation. Furthermore, the Chuxiong sample exhibited a stronger inhibition activity of the hydroxyl radicals compared with other samples. The high correlation coefficient was existed between the phenolic content and superoxide anion radical scavenging activity, but no significant correlation was found between the former and hydroxyl radical scavenging activity. Methanolic extracts of emblica fruit from some selected regions exhibited stronger antioxidant activities compared to those of the commercial compounds (quercetin and BHA). It might be considered as a potential plant source of antioxidants. r 2007 Elsevier Inc. All rights reserved. Keywords: Phyllanthus emblica; Emblica officinalis; Indian gooseberry; Amla berry; Total phenolic; Flavonoid; Proanthocyanidin; Antioxidant activity

1. Introduction Emblica (Phyllanthus emblica L.) as an euphorbiaceous plant is widely distributed in subtropical and tropical areas of China, India, Indonesia, and Malaysia. Emblica fruit is well accepted by consumers for its special taste. It has abundant amounts of vitamin C and superoxide dismutase (Verma and Gupta, 2004), and is used in many traditional medicinal systems, such as Chinese herbal medicine, Tibetan medicine and Ayurvedic medicine (Zhang et al., 2000). Emblica fruit is reported to have hypolipidemic (Anila and Vijayalakshmi, 2000; Jacob et al., 1988; Mathur et al., 1996; Thakur et al., 1988) and hypoglycemic activities Corresponding author. Tel./fax: +86 20 87113914.

E-mail address: [email protected] (M. Zhao). 0889-1575/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2007.10.001

(Abesundara et al., 2004; Anila and Vijayalakshmi, 2000), and acts as a important constituent of many hepatoprotective formulations available (Antarkar et al., 1980; De et al., 1993; Panda and Kar, 2003). It is also used as antimicrobial agent (Dutta et al., 1998; Godbole and Pendse, 1960; Rani and Khullar, 2004), anticancer (Jeena et al., 2001; Zhang et al., 2004), and antiinflammatory agent (Asmawi et al., 1993; Lampronti et al., 2004; Perianayagam et al., 2004), and can improve the metalinduced clastogenic effects (Biswas et al., 1999; Dhir et al., 1990). Free radicals play an important role in some pathogenesis of serious diseases, such as neurodegenerative disorders, cancer, liver cirrhosis, cardiovascular diseases, atherosclerosis, cataracts, diabetes and inflammation (Aruoma, 1998). Compounds that can scavenge free radicals

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have great potential in ameliorating these diseases (Kirakosyan et al., 2003). It is reported that phenolic compounds in plants possess strong antioxidant activity and may help to protect cells against the oxidative damage caused by free radicals (Kahkonen et al., 1999). Due to the presence of the conjugated ring structures and hydroxyl groups, many phenolic compounds have the potential to function as antioxidants by scavenging superoxide anion (Robak and Dryglewski, 1988), singlet oxygen (Husain et al., 1987), and lipid peroxy radicals (Torel et al., 1986), and stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species (Shahidi and Wanasusdara, 1992). It was reported that emblica has a strong antioxidant activity (Bafna and Balaraman, 2004; Anila and Vijayalakshmi, 2003; Jose and Kuttan, 1995), which may be partially due to the existence of flavonoids and several gallic acid derivatives including epigallocatachin gallate (Anila and Vijayalakshmi, 2002; Sabu and Kuttan, 2002). In the present study, the antioxidant activities of methanolic extract of emblica fruit from six regions in China were evaluated in terms of reducing power, activities of scavenging DPPH radicals, superoxide anion radicals and hydroxyl radicals, capability of inhibition of Fe (II)-induced lipid peroxidation and ability of chelating ferrous ions. The contents of phenolics of these extracts were also determined. The results will be helpful to understand this fruit and significant for industrial development.

2. Materials and methods 2.1. Plant materials Fresh fruits of emblica were gathered from six regions in China: Guangzhou (Guangdong province), Huizhou (Guangdong province), Haikou (Hainan province), Liuzhou (Guangxi province), Chuxiong (Yunnan province), and Putian (Fujian province). The weather of the sampling regions was subtropical monsoon climate for Liuzhou and Chuxiong, subtropical oceanic climate for Guangzhou, Huizhou, and Putian, and tropical oceanic climate for Haikou. These samples were picked at each commercial harvest time and selected for the same uniformity of maturity by evaluating the color and taste. The fruits were stored at 4 1C till analysis.

2.3. Preparation of methanolic extract Fresh emblica fleshs were air-dried in an oven at 40 1C for 8 h and then were pulverized to 0.2–0.4 mm particle in a cutting mill. Powder (10 g) was extracted for 24 h with 100 mL of methanol in a glass conical flask using a shaker at 25 1C and filtrated through 0.45 mm filter paper. The residue was then extracted twice with 100 mL methanol as described above. The combined methanolic extracts were concentrated at 40 1C, using a rotary evaporator under low pressure. The residue was freeze-dried and then stored in a amber colored air-tight containers at 4 1C, prior to further use. 2.4. Determinations of total phenolics, flavonoid, and proanthocyanidin contents The total phenolic content (TPC) was determined by the Folin–Ciocalteu method (Singleton et al., 1999). The extract (0.1 g) was dissolved with methanol and up to 100 mL, and then the extract solution (0.5 mL) was mixed with 0.5 mL of the Folin–Ciocalteau reagent and 0.5 mL of 100 mg/mL sodium carbonate (added 2 min after the Folin–Ciocalteau reagent). After initial mixing the opaque flasks were allowed to stand for 2 h. The optical density of the blue-colored samples was measured at 765 nm. The total phenolic contents were expressed as mg gallic acid equivalent (GAE)/g dry basis. Flavonoid contents were measured using a modified colorimetric method (Jia et al., 1999). Extract solution (0.25 mL, 1 mg/mL) was added to a test tube containing 1.25 mL of distilled water. Sodium nitrite solution (5%, 0.075 mL) was added to the mixture and maintained for 5 min. Then, 0.15 mL of 10% aluminum chloride was added. After 6 min, 0.5 mL of 1 M sodium hydroxide was finally added. The mixture was diluted with 0.275 mL of distilled water. The absorbance of the mixture at 510 nm was measured immediately in comparison to a standard curve prepared by quercetin. The flavonoid contents were expressed as mg quecetin equivalent (QE)/g dry basis. Contents of proanthocyanidins was determined by the procedure of Sun et al. (1998). Five hundred microliters of extract solution was mixed with 3 mL of 4% vanillin– methanol solution and 1.5 mL hydrochloric acid. The mixture was allowed to stand for 15 min. The absorbance was measured at 500 nm, while the final result was expressed as mg catechin equivalent (CE)/g dry basis. 2.5. Determination of DPPH radical scavenging activity

2.2. Chemicals Nitroblue tetrazolium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), phenazine methosulfate (PMS), etylenediamine tetraacetic acid (EDTA), catechin, quercetin, and ferrozine were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.

The free radical scavenging activity was determined by the method of Shimada et al. (1992) and Yang et al. (2006). The methanolic extracts were dissolved with methanol to prepare various sample solutions at 200, 100, 80, 60, 40, 20, 10, and 5 mg/mL. Each extract solution (2 mL) was mixed with 1 mL of methanolic solution containing DPPH radicals, with a final concentration of 0.2 mM DPPH. The

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mixture was shaken vigorously and maintained for 30 min in dark. The absorbance was measured at 517 nm. The absorbance of the control was obtained by replacing the sample with methanol. Quercetin and BHA were used as standard reference. The scavenging activity was calculated using the formula, scavenging activity (%) ¼ [(A517 of controlA517 of sample)/A517 of control]  100.

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The mixture was heated for 15 min on a boiling water bath and then cooled. The absorbance was measured at 532 nm. The absorbance of the control was determined by replacing the sample with methanol. Quercetin and BHA were used as positive control. The scavenging activity on hydroxyl radical was calculated as follows, scavenging activity (%) ¼ [1A532 of sample/A532 of control]  100.

2.6. Determination of reducing power The reducing power of the emblica extracts was determined according to the method of Oyaizu (1988). Extract solution (2 mL), phosphate buffer (2 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2 mL, 10 mg/mL) were mixed, and then incubated at 50 1C for 20 min. Trichloroacetic acid (2 mL, 100 mg/mL) was added to the mixture. A volume of 2 mL from each of the above mixtures was mixed with 2 mL of distilled water and 0.4 mL of 0.1% (w/v) ferric chloride in a test tube. After 10-min reaction, the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated a high reducing power. 2.7. Determination of superoxide anion radical scavenging activity The assay for superoxide anion radical scavenging activity was based on a riboflavin-light-NBT system (Beauchamp and Fridovich, 1971). The reaction mixture contained 0.5 mL of phosphate buffer (50 mM, pH 7.6), 0.3 mL riboflavin (50 mM), 0.25 mL PMS (20 mM), and 0.1 mL NBT (0.5 mM), prior to the addition of 1 mL methanolic extract solution. Reaction was started by illuminating the reaction mixture with different concentrations of the methanolic extracts using a fluorescent lamp. After 20 min of incubation, the absorbance was measured at 560 nm. The absorbance of the control was determined by replacing the sample with methanol. Quercetin and BHA were used as positive control. The percent inhibition of superoxide anion generation was calculated using the following formula, scavenging activity (%) ¼ [(A560 of controlA560 of sample)/A560 of control]  100.

2.9. Inhibition of lipid peroxide formation induced by Fe2+-ascorbate system Lipid peroxidation assay of rat microsomal was carried out as reported earlier by Sabu and Kuttan (2002). The emblica extract solution (100 mL) at various concentrations was incubated for 1 h at 37 1C with 0.5 mL reaction solution containing 0.1 mL of rat liver homogenate (25%; w/v) in Tris–HCl buffer (40 mM, pH 7.0), 100 mL of KCl (30 mM), 100 mL of ferrous iron (0.16 mM) and 100 mL of ascorbic acid (0.06 mM). The lipid peroxide formed was measured by TBARS (Ohkawa et al., 1979). The absorbance of the organic layer was measured at 532 nm. The percent inhibition of lipid peroxidation was determined by comparing the results of the test compounds with that of control sample without the extract solution. Quercetin and BHA were used as positive control.

2.10. Ability of chelating ferrous ions The Fe2+-chelating ability of the emblica extract was measured by the ferrous iron–ferrozine complex method (Decker and Welch, 1990). The extracts were dissolved with methanol to prepare various sample solutions at 10, 8, 6, 4, and 2 mg/mL. The reaction mixture containing 2 mM FeCl2 (0.05 mL) and 5 mM ferrozine (0.2 mL) and the extract solution (0.8 mL) at various concentrations was mixed and then incubated for 10 min at 2572 1C. The absorbance of the reaction was recorded at 562 nm. The absorbance of the control was determined by replacing the sample with methanol. Quercetin and EDTA was used as positive control. The ability of the extract to chelate ferrous ion was calculated using the equation described above for DPPH.

2.8. Determination of hydroxyl radical scavenging activity Hydroxyl radical scavenging activity of the emblica extracts was assayed by the method of Halliwell and Gutteridge (1981). The reaction mixture contained 500 mL of 2-deoxyribose (2.8 mM) in potassium phosphate buffer (50 mM, pH 7.4), 200 mL of premixed ferric chloride (100 mM) and EDTA (100 mM) solution (1:1; v/v), 100 mL of H2O2 (200 mM) without or with the extract solution (100 mL). The reaction was triggered by adding 100 mL of 300 mM ascorbate and incubated for 1 h at 37 1C. A solution of TBA in 1 mL (1%; w/v) of 50 mM NaOH and 1 mL of 2.8% (w/v; aqueous solution) TCA was added.

2.11. Statistical analysis Data were presented as mean7standard deviation (S.D.) of three determinations. Statistical analyses were performed using a one-way analysis of variance. Differences were considered significant at Po0.05. The EC50 values were calculated by linear regression analysis. It was defined as the effective concentration of sample to obtain 50% antioxidant or metal chelating activity. Results were calculated by employing the statistical software (SPSS, version 13.0, SPSS Inc., Chicago, USA).

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3. Results and discussion 3.1. Extraction yield The yield of methanolic extracts of emblica fruit is shown in Table 1. The extraction yield of these samples from six various regions varied from 21.0% to 39.4% with a decreasing order: Huizhou4Chuxiong4Liuzhou4 Guangzhou4Putian4Haikou.

3.2. Total phenolics, flavonoid, and proanthocyanidin contents Phenolic compounds have been proved to be responsible for the antioxidant activity of emblica fruit (Kumar et al., 2006; Sabu and Kuttan, 2002; Anila and Vijayalakshmi, 2002). The amounts of total phenolics, flavonoids, and proanthocyanidins in emlica extracts were measured in this study. These extracts were found to have various phenolic levels (Table 2), ranging from 81.5 to 120.9 mg GAE/g. The Huizhou sample had the highest content of total phenolics, followed by the Chuxiong sample. The lowest content was observed in the Liuzhou sample. The flavonoid contents of these emblica extracts are shown in Table 2, as quercetin equivalents (QE). Compared to the total phenolics, the Chuxiong sample had the highest content of flavonoids

Table 1 Yields of methanolic extracts of emblica fruit from six regions in China Samples

Province

Collection date

Yield (%)a

Guangzhou Putian Haikou Liuzhou Huizhou Chuxiong

Guangdong Fujian Hainan Guangxi Guangdong Yunnan

05/10/2005 06/11/2005 20/9/2005 16/10/2005 11/11/2005 19/10/2005

24.272.3 23.173.0 21.071.7 26.372.5 39.471.2 35.573.4

a

Each value was expressed as the mean7S.D. (n ¼ 3).

Table 2 Contents of total phenolics, flavonoids, and proanthocyanidins in methanolic extracts of emblica fruit from six regions in China Samples

Guangzhou Putian Haikou Liuzhou Huizhou Chuxiong

Total phenolics (mg GAE/g, dry basis) 98.170.4 92.771.8 82.672.1 81.573.7 120.972.1 111.172.7

Ca D E E A B

Flavonoids (mg QE/g, dry basis)

Proanthocyanidins (mg CE/g, dry basis)

27.471.7 27.170.9 20.371.6 22.971.7 35.671.1 38.773.6

4.670.3 3.770.8 8.170.7 6.171.3 18.770.7 12.372.0

C C D E B A

C C D E A B

Means with different letters within a column were significantly different at Po0.05. a Each values was presented as the mean7S.D. (n ¼ 3).

(38.7 mg QE/g), while the lowest content (20.3 mg QE/g) was observed in the Haikou sample. The proanthocyanidins contents of these emblica extracts varied from 3.7 to 18.7 mg CE/g (Table 2). However, the highest content of proanthocyanidins was found in the Huizhou samples but a lowest content in the Putian sample. The various levels of phenolics in these emblica extracts could be partly due to the differences in growing conditions. Under field conditions, the phenolic compositions of plant tissues vary considerably with seasonal, genetic, and agronomic factors (Hilton and Palmer-Jones, 1973). In addition, a large variability at various stages of maturation and growing conditions such as temperature and rainfall are known to affect the contents of phenolic compounds (Zheng and Wang, 2001). 3.3. DPPH radical scavenging activity DPPH is a stable nitrogen-centered free radical, and its color changes from violet to yellow when is reduced by either the process of hydrogen- or elctrondonation. Substances to perform this above reaction can be considered as antioxidants and therefore radical scavengers (Brand-Williams et al., 1995). The DPPH radical scavenging activity was known to correlate well with the inhibitory capacity of lipid peroxidation of a test compound (Rekka and Kourounakis, 1991). As shown in Table 3 and Fig. 1, all of these emblica extracts showed appreciable free radical scavenging activities. Among these samples tested in this study, the Huihou sample had the strongest radical scavenging activity (EC50 11.23 mg/mL) while the Liuzhou sample exhibited the lowest radical scavenging activity (EC50 45.44 mg/mL). 3.4. Ability of chelating ferrous ion Ferrous ion, commonly existed in food systems, is well known as an effective pro-oxidant (Hsu et al., 2003). Polyphenols can chelate pro-oxidant metal ions, thus preventing free radical formation from these pro-oxidants (Kris-Etherton et al., 2002). The ability of chelating ferrous ion of the emblica extracts was presented in Table 3 and Fig. 2. The Chuxiong sample had the highest chelating ferrous ion capability (EC50 3.62 mg/mL). 3.5. Superoxide anion radical scavenging activity Although superoxide anion is a weak oxidant, it gives rise to the generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to the oxidative stress (Dahl and Richardson, 1978; Meyer and Isaksen, 1995). In this study, the superoxide anion radical scavenging activities of these emblica extracts are showed in Table 3 and Fig. 3. The EC50 values, indicating superoxide anion radical scavenging activity, ranged from

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Table 3 EC50 values of methanolic extracts of emblica fruit from six regions in China using four various antioxidat assays EC50 value (mg/mL)a

Samples Guangzhou Putian Haikou Liuzhou Huizhou Chuxiong Standards Quercetin BHA

DPPH

Superoxide

Hydroxyl radical

Lipid peroxidation

14.7170.5 18.5270.7 28.3971.1 45.4471.0 11.2370.9 11.3870.1

28.971.0 25.871.4 32.070.8 34.271.1 12.170.9 16.970.4

104.873.3 67.671.9 94.770.8 64.671.6 84.271.1 46.471.3

75.772.5 83.674.2 102.171.3 68.475.1 68.875.8 4873.2

6.670.5 33.370.2

13.170.5 2.870.1

97.074.1 66.570.3

83.375.2 8.272.4

Each value was expressed as the mean7S.D. (n ¼ 3). a EC50 value was determined to be the effective concentrations at which DPPH, superoxide, hydroxyl radicals were scavenged by 50%, respectively. The EC50 value was obtained by interpolation from linear regression analysis.

120

GZH PT HK

100

LZH DPPH scavenging (%)

HZH 80

CHX

60

40

20

0

1

2

3 4 5 Extract concentration (µg/mL)

6

7

Fig. 1. DPPH radical scavenging activities of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

12.06 mg/mL for the Huizhou sample to 34.27 mg/mL for the Liuzhou sample. 3.6. Hydroxyl radical scavenging activity The hydroxyl radical is an extremely reactive in biological systems and has been implicated as highly damaging species in free radical pathology, capable of damaging biomolecules of the living cells (Halliwell, 1997; Waling, 1975). As shown in Table 3 and Fig. 4, the Chuxiong sample showed a higher hydroxyl radical scavenging activity than other samples. The lowest hydroxyl radical scavenging activity was found in the Guangzhou sample.

3.7. Reducing power The reducing power has been used as one of important antioxidant capability for medicinal herbs (Duh et al., 1999; Duh and Yen, 1997). The reducing power of these emblica extracts was dose-dependent (Fig. 5). All of these emblica extract samples exhibited high reducing power. Furthermore, the Huizhou sample had a higher reducing power than other samples. 3.8. Determination of capability of lipid peroxidation inhibition Lipid peroxidation (LPO) can inactivate cellular components and plays an important role in oxidative stress in

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120 GZH PT HK

100

LZH HZH CHX

Ferrous ion chelating (%)

80

60

40

20

0 2

4 6 Extract concentration (mg/mL)

10

8

Fig. 2. Abilities of chelating ferrous ion of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

100 GZH PT HK Superoxide radical scavenging (%)

80

LZH HZH CHX

60

40

20

0 5

10

20 40 60 Extract concentration (µg/mL)

80

100

Fig. 3. Superoxide anion radical scavenging activity of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

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100 GZH PT HK 80

LZH

Hydroxyl radical scavenging (%)

HZH CHX 60

40

20

0

20

40

60

80

100

200

Extract concentration (µg/mL) Fig. 4. Hydroxyl radical scavenging activities of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

1.2 GZH 1

PT HK LZH

Absorbance at 700 nm

0.8

HZH CHX

0.6

0.4

0.2

0 20

40 60 80 Extract concentration (µg/mL)

100

200

Fig. 5. Reducing power of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

biological systems. Furthermore, several toxic byproducts from the peroxidation can damage other bio-molecules (Box and Maccubbin, 1997; Esterbauer, 1996). It is well

established that transition of metal ions such as iron and copper stimulate lipid peroxidation through various mechanisms (Halliwell and Gutteridge, 1984). These may

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100 GZH PT HK

80

Inhibition capability (%)

LZH HZH CHX

60

40

20

0

0

20

40

60

80

100

200

Extract concentration (µg/mL) Fig. 6. Capabilities of lipid peroxidation inhibition of methanolic extracts of emblica fruit from six regions in China. Each value was presented as the mean7S.D. of three replicate determinations. GZH, Guangzhou; PT, Putian; HK, Haikou; LZH, Liuzhou; HZH, Huizhou; CHX, Chuxiong.

either generate hydroxyl radicals to initiate the lipid peroxidation process or propagate the chain process via decomposition of lipid hydroperoxides (Braughler et al., 1987). In this study, these emblica extracts inhibited the lipid peroxidation in a concentration-dependent manner (Fig. 6 and Table 3). The highest ability of chelating ferrous ion was observed in the Chuxiong sample, while the Haikou sample had the lowest activity (Table 4). 3.9. Correlations with free radical scavenging activity, antioxidant activity, and phenolic content Emblica extracts with high radical scavenging activity and/or antioxidant ability generally had higher phenolic content with good correlations. In this study, the regression analyses indicated the correlations between free radical scavenging activity, antioxidant activity and phenolic content (Table 5). The EC50 value for DPPH radical scavenging activity was correlated with content of total phenolics (r ¼ 0.8176) and flavonoids (r ¼ 0.7479). However, the poor correlation coefficient was observed between phenolic content and hydroxyl radical scavenging activity. In addtion, no significant correlation was found between ability of chelating ferrous ion and content of total phenolics. 3.10. Comparative study with reference antioxidants The antioxidant capability and scavenging activity of emblica extracts in comparison to the reference substances in this study are shown in Tables 3 and 4. Quercetin was found to exhibit the strongest DPPH radical scavenging

Table 4 The abilities of chelating ferrous ion of methanolic extracts of emblica fruit from six regions in China EC50 value (mg/mL)a Samples Guangzhou Putian Haikou Liuzhou Huizhou Chuxiong

4.270.6 4.770.3 6.570.1 3.770.4 4.270.3 3.670.52

Standards Quercetin EDTA

5.970.7 4872.5 (mg/mL)

Each value was expressed as the mean7S.D. (n ¼ 3). a The concentrations of the samples at which ferrous ions were chelated by 50%.

activity (EC50=6.6 mg/mL), whereas BHA showed the highest superoxide radical scavenging activity (EC50=2.8 mg/mL). The DPPH radical savenging activity of quercetin was higher than BHA, which was in agreement with the report of Chen and Ho (1997). For emblica extract samples from six regions, the Chuxiong sample had a higher hydroxyl radical scavenging activity than BHA and quercetin. Furthermore, the Chuxiong sample exhibited higher lipid peroxidation inhibition ability and chelating ferrous ion capability than quercetin, while the Huizhou sample exhibited a stronger DPPH radical scavenging ability than BHA and a higher superoxide radical scavenging activity than quercetin.

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Table 5 Correlation coefficients (r) between the EC50 values of bioactivities and phenolic contents Correlation coefficients (r)

DPPH radical scavenging Hydroxyl radical scavenging Superoxide radical scavenging Lipid peroxidation inhibition Ferrous ion chelating

Total phenolics

Flavonoids

Proanthocyanidins

0.8176 0.0812 0.9396 0.6019 0.4442

0.7479 0.1411 0.9230 0.8017 0.3685

0.4306 0.1670 0.9116 0.3947 0.1640

4. Conclusion The contents of phenolics in relation to antioxidant activities of methanolic extracts of emblica fruit from six regions in China were analyzed. Among these extracts tested, the Huizhou sample had significantly higher content of total phenolics, flavonoids, and proanthocynidins than other samples. Furthermore, all of these extracts of emblica fruit had strong antioxidant activity, and the Chuxiong and Huizhou samples exhibited quite strong antioxidant and radical scavenging activities in comparison to the reference compounds BHA and quercetin. The above results indicated that region could lead to significant differences both in the content of bioactive compounds and their bioactivities. Emblica fruit can be a source of plant antioxidants, with a potential use in food, cosmetics and pharmaceutical fields. The phenolics might be the major active component responsible for the strong antioxidant activity. However, a more detailed investigation between the individual phenolic compounds present in emblica fruit and the antioxidant activities needs to be carried out. Acknowledgment The financial support from Eleventh five-year National Key Technology R&D Program (Nos. 2006BAD27B03 and 2006BAD27B04) was appreciated. References Abesundara, K.J.M., Matsui, T., Matsumoto, K., 2004. a-glucosidase inhibitory activity of some Sri Lanka plant extracts, one of which, Cassia auriculata, exerts a strong anti hyperglycemic effect in rats comparable to the therapeutic drug acarbose. Journal of Agricultural and Food Chemistry 52, 2541–2545. Anila, L., Vijayalakshmi, N.R., 2000. Beneficial effects of flavonoids from Sesamum indicum, Emblica officinalis and Momordica charantia. Phytotherapy Research 14, 1–4. Anila, L., Vijayalakshmi, N.R., 2002. Flavonoids from Emblica officinalis and Mangifera indica— effectiveness for dyslipidemia. Journal of Ethnopharmacology 79, 81–87. Anila, L., Vijayalakshmi, N.R., 2003. Antioxidant action of flavonoids from Mangifera indica and Emblica officinalis in hypercholesterolemic rats. Food Chemistry 83, 569–574. Antarkar, D.S., Ashok, B.V., Doshi, J.C., Athavale, A.V., Vinchoo, K.S., Natekar, M.R., Thathed, P.S., Ramesh, V., Kale, N., 1980. Double-

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