LWT - Food Science and Technology 44 (2011) 1745e1751
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In vitro synergistic anti-oxidant activities of solvent-extracted fractions from Astragalus membranaceus and Glycyrrhiza uralensis Minghua Li a,1, Yan Xu a,1, Wenjian Yang a, Jinkui Li a, Xiaoyan Xu a, Xing Zhang a, Fangtian Chen a, Dapeng Li a, b, * a b
College of Food Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, PR China State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2010 Received in revised form 22 February 2011 Accepted 24 February 2011
Our study estimated the in vitro anti-oxidant activities of the solvent-extracted fractions from Astragalus membranaceus (AME), Glycyrrhiza uralensis (GU) and the combination of them (AG), using 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging assay and ferric reducing anti-oxidant power (FRAP) assay. Different solvent extracts have different anti-oxidant activities. As to the herb pair (AG), the highest anti-oxidant capacity and total phenolic/flavonoid content were observed in the ethyl acetate fraction, which were exhibited in different solvent fractions of the individual herbs. The ethyl acetate extract of herb pair (EAeAG) showed significantly higher anti-oxidant capacity than the theoretical sum of two single herbs, and presented better cytoprotection and induced higher activity of anti-oxidant enzymes than the single herbs. Moreover, there’s a linear correlation between the increment of total phenolics/flavonoids and anti-oxidant capacities, especially between polyphenol content and TEAC values (correlation coefficient ¼ 0.9*). These indicated that the phenolic and flavonoid may be the main compounds which caused the synergistic anti-oxidant capacity and cytoprotection in the combination of AME and GU. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Herb pair Synergistic effect Antioxidant activity Cell viability
1. Introduction Oxidative damage, induced by superfluous free radicals, is suggested to be the causes of aging, cancer, cardiovascular diseases, neurodegenerative diseases and inflammation in humans (Stadtman, 1992). Free radicals are chemical species and contain one or more unpaired electrons, so they are highly unstable and tend to cause damage to other molecules by extracting electrons in order to attain stability. Ali et al. (2008) found that many antioxidants can help organisms deal with oxidative stress. Gerber et al. (2002) also reported that food rich in antioxidants played an essential role in preventing cardiovascular diseases and cancers. Thus, it is important to search for natural anti-oxidant sources among plants to be used as food additives and increase the anti-oxidant intake in the diet.
* Corresponding author. College of Food Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, PR China. Tel.: þ86 0538 8242876; fax: þ86 0538 8242850. E-mail address:
[email protected] (D. Li). 1 First two authors contributed equally to this work. 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.02.017
In recent years, various studies have indicated that traditional Chinese herbs possessed a wide variety of natural antioxidants, such as phenolic acid, flavonoids and tannins, which had a more potent anti-oxidant activity than common dietary (Li, Wong, Cheng, & Chen, 2008). Astragalus membranaceus (root of Astragalus, known as Huangqi in Chinese) is one of the best-known natural traditional Chinese herbs, with biological properties including immunomodulation, anti-aging, and anti-tumour effects (Cho & Leung, 2007). Recent studies revealed that the flavonoids of Radix Astragali showed strong anti-oxidant activity (Fan, Wu, Gong, Zhou, & Hu, 2003) and pharmacological properties, which were applied to impair the barrier function induced by hypoxia (Fan et al., 2003). Gancao (in Chinese) is the name applied to the roots and stolons of some Glycyrrhiza species as well as a tonic medicine for thousands of years (Nomura, Fukai, & Akiyama, 2002). The Glycyrrhiza roots have been used as flavoring for a long time, as well as demulcents and expectorants in western countries. Takagi and Ishii (1967) indicated that Glycyrrhiza uralensis was also useful for detoxification, anti-inflammation and anti-viral, and the flavonoid-rich fraction from the extract of G. uralensis has been used as an antiulcer medicine.
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Traditional Chinese herbs are generally applied in the form of multi-herb formulas in medical treatments and as dietary supplements (Takagi & Ishii, 1967). It is noteworthy that traditional Chinese herb pair (TCHP) is the basic unit in traditional Chinese formulas, which presents significantly better pharmacological efficacy than individual herbs (Sun, Wei, Wu, Gui, & Wang, 2007; Yang et al., 2009). In addition, our previous study also found that ethanol extract of AMEeGU (the combination of Astragalus membranaceus and Glycyrrhiza uralensis) had stronger free radical scavenging capacity compared with their theoretical sum (Yang et al., 2009). In order to investigate the specific mechanism of this synergy, the anti-oxidant activities of different polarity extracted fractions as well as the correlation between the antioxidant capacity increments and the total phenolic/flavonoid increments were further determined. Moreover, the protective effects on H2O2 induced oxygen damage in MRC-5 cell were also studied to elucidate the possible mechanism of the synergistic effect.
2. Materials and methods 2.1. Chemicals and equipments 1,1-Diphenyl-2-picryl hydrazine (DPPH), TPTZ (2,4,6-tripyridyl-striazine), Trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxylicacid, a water soluble homologue of vitamin E), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), FolineCiocalteau reagent (FCR), gallic acid, rutin (0.95 g/g) and vitamin E (VE) were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, U.S.A.). All other chemical reagents were of analytical grade and obtained from Shanghai Chemical Reagent Co. Ultravioletevisible (UVevis) spectroscopy was performed on a Scinco S3100 spectrophotometer (Scinco Co., LTD., Seoul, South Korea). Inverted microscope (TE2000) was provided by Nikon, Tokyo, Japan.
2.2. Plant material Astragalus membranaceus (AME) and Glycyrrhiza uralensis (GU) were obtained from Shijiazhuang Pharmaceutical Group of China and stored at room temperature. 2.3. Preparation of herb extract The powder (100 g) of single herbs (AME, GU) was extracted with 200 mL of ethanol by heat reflux for 2 h, which was repeated twice. Then the extracts were combined, concentrated by rotary vacuum evaporation and obtained the residue (yield 17.89 g/100 g and 33.53 g/100 g, respectively). Then the residue was re-suspended in 250 mL water (OH) and successively extracted with the equivalent volume of n-hexane (HX), chloroform (CF), ethyl acetate (EA), and n-butanol (NB); giving five fractions for each herb extract (HXeAME, CFeAME, EAeAME, NBeAME, and OH for AME; and HXeGU, CFeGU, EAeGU, NBeGU, and OH for GU). The crude extracts were stored at 20 C until use. In order to research the synergistic effect of the herb pair, a powder mixture containing equal proportions of AME and GU (50 g:50 g) was extracted according to the above method with the yield of 18.97 g/100 g. The residue obtained was then redissolved with 250 mL water and successively extracted as stated above. Various solvent fractions of herb pair were labeled as HXeAG, CFeAG, EAeAG, NBeAG, OHeAG. The crude extracts were stored at 20 C until use.
2.4. Determination of the anti-oxidant activity 2.4.1. FRAP assay The ferric reducing anti-oxidant power (FRAP) was performed as previously described by Benzie and Strain (1996) with slight modification. The fresh FRAP reagent was prepared daily by mixing acetate buffer (300 mmol/L, pH 3.6), TPTZ solution (10 mmol/L in 40 mmol/L HCl) and 20 mmol/L FeCl3$6H2O solution in proportions of 10:1:1. The mixture was incubated at 37 C for several minutes. 0.1 mL samples of various crude extracts dissolved in ethanol were added directly to 3.9 mL of FRAP reagent. The absorbance of the reaction mixture was then measured at 593 nm after 10 min. The calibration curve was constructed with aqueous solutions of FeSO4$7H2O (100e1000 mmol/L), and the results were expressed as mmol Fe (Ⅱ)/g dry weight of herb extract. 2.4.2. DPPH free radical scavenging assay DPPH radical scavenging assay was based on previous study with slight modifications (Brand-Williams, Cuvelier, & Berset, 1995). Briefly, 0.1 mL samples of various crude extracts dissolved in ethanol were added directly to 3.9 mL of DPPH $ solution in ethanol (0.1 mmol/L) and then immediately shaken thoroughly. The absorbance was measured at 517 nm after 30 min at room temperature, and the anti-oxidant capability (AA) was expressed as the percentage of DPPH $ reduced, which was calculated with the following formula: AADPPH%¼((AB AS)/AB) 100, where AS is the absorbance of the DPPH solution after reacting with the sample at a given concentration and AB is the absorbance of the DPPH $ solution with an ethanol blank instead of the sample. The percentage of DPPH $ reduced was plotted against the concentration of each sample, and an SC50 value, which is defined as the concentration of the sample needed to scavenge 50% of the DPPH $, was calculated from the graph. 2.4.3. ABTS free radical scavenging assay The ABTS free radical scavenging ability was carried out using a modified method as described by Re et al. (1999). Potassium persulfate was added into 7 mmol/L of ABTS $ þ and kept for 12e16 h at room temperature in the dark. The ABTS $ þ solution was diluted with ethanol to an absorbance of 0.70 0.02 at 734 nm before analysis. ABTS $ þ solution (3.9 mL) was added to 0.1 mL ethanol or samples of various crude extracts dissolved in ethanol and mixed thoroughly. The absorbance of the reaction mixture was recorded at 734 nm after 15 min at room temperature. A calibration curve was made by absorbance reduction and the concentration of trolox. Results were expressed as TEAC values (trolox equivalent anti-oxidant capacity) (mmol/g). 2.5. Determination of total phenolic and flavonoid contents Total phenolic content was measured using the FolineCiocalteau method (Singleton & Rossi, 1965). Briefly, 0.1 mL of sample was mixed with 1 mL of the FolineCiocalteau reagent (diluted ten-fold) and incubated at room temperature for 5 min, and then 1 mL of 0.1 g/ mL Na2CO3 solution was added to the mixture. The absorbance was measured at 765 nm after 90 min incubation at room temperature, and the results were expressed as gallic acid equivalents (mg GAE/g). Total flavonoid content of the samples was determined according to colorimetric assay reported by Jia, Tang, and Wu (1999) with some modifications. The sample (0.1 mL) was mixed with 0.3 mL of 0.05 g/mL NaNO2 solution in a test tube and incubated for 5 min, and then 0.3 mL of 0.1 g/mL AlCl3 solution was added and incubated for another 6 min. The reaction was terminated by adding 2 mL of 1 mol/L NaOH solution. Absorbance of the mixture was determined immediately at 510 nm. Total flavonoid content was expressed as rutin equivalents (mg RE/g).
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2.6. Determination of protective effect on H2O2 induced oxidative damage in MRC-5 cells 2.6.1. Cell culture MRC-5 cell line was obtained from Shanghai Institute of Cell Biology and incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 100 mL/L fetal bovine serum (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 C, and the medium was changed every other day. 2.6.2. Cell viability assay Cell survival was evaluated by MTT assay as previous described (Yoo, Lee, Lee, Moon, & Lee, 2008), which was based on the reduction of a tetrazolium salt by mitochondrial dehydrogenases in viable cells. The optical density of the formazan formed in the control cells was taken as 100% viability. Data are mean percentages of viable cells versus the respective controls. 2.6.3. SOD, CAT, and GSH-Px activities of MRC-5 cells Cells in logarithmic growth phase were split into six culture flasks for incubating 24 h, and then treated with fresh DMEM medium containing samples for another 24 h (A: control, B: H2O2 (0.2 mmol/L), C: H2O2 þ EAeAME (20 mg/mL), D: H2O2 þ EAeGU (20 mg/mL), E: H2O2 þ EAeAG (20 mg/mL), F: H2O2 þ VE (vitamin E) (10 mg/mL)). After removing the medium, cells were washed and resuspended with phosphate buffer solution. Cells suspension was broken up with ultrasonic cell disruption system (JY92-ⅡYJ, Nanjing Beidi experiment instrument Co., Ltd), and then centrifuged. The supernatants were used for investigating the activities of SOD, CAT and GSH-Px, which were performed with commercially available detection kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). 2.7. Statistical analysis All data were expressed as mean standard deviation. Statistical analysis was performed with the one-way analysis of variance (ANOVA), the Duncan test and the Bivariate Correlations using the SPSS 13.0 software. Values of p < 0.05 were considered to be statistically significant. The synergistic effect was evaluated according to the previous method with slight modifications (González & Nazareno, 2011; Liu, Shi, Colina Ibarra, Kakuda, & Jun Xue, 2008). The theoretical value was calculated as the sum of two single herbs’
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values in every experiment multiplying 0.5 for their proportion in herb pair was 0.5:0.5, and the actual value was obtained in our experiment. When the two values present significant differences (p < 0.05), the synergistic effect is sure to exist in the mixture. 3. Results and discussion 3.1. Total anti-oxidant power of herbs’ extracts measured by FRAP assay The FRAP assay is a simple method to measure the anti-oxidant activity of samples. As shown in Table 1 and Fig. 1, different solvent extracts of AME, GU and AG presented a wide range of differences in ferric reducing anti-oxidant power. Of the various solvent fractions tested, the chloroform extracts of AME (CFeAME) and GU (CFeGU) presented the highest ferric reducing anti-oxidant power. However, compared to other solvent fractions, the highest ferric reducing anti-oxidant power was observed in the ethyl acetate extracts of AG (EAeAG), which was also significantly higher than the theoretical sum value of the constituent herbs (p < 0.05). Interestingly, after combining the two herbs, the highest ferric reducing anti-oxidant power had been shown in the more polar extract, as follows: EAeAG > CFeGU > CFeAME. 3.2. The DPPH free radical scavenging ability of herbs’ extracts As shown in Table 1, the ethyl acetate extracts of GU (EAeGU) and AG (EAeAG) had the highest DPPH free radical scavenging efficiency compared to other solvent fractions, which was similar to the previous study (Park et al., 2007). The ethyl acetate fraction of AG was the most effective fraction and the free radical scavenging effect of EAeAG was significantly better than that of other fractions (p < 0.05) (Fig. 2). Besides, it is noteworthy that the SC50 of different solvent fractions of AG was much lower than that of the theoretical sum of the constituent herbs (AeDPPH < TeDPPH), but excepting for the water fraction (OHeAG). 3.3. The ABTS free radical scavenging ability of herbs’ extracts As shown in Table 1, all the solvent extracts of GU and AG had the higher TEAC values comparing with the extracts of AME. EAeAG had the highest ABTS free radical scavenging efficiency among the five extracts (HXeAG, CFeAG, EAeAG, NBeAG, OHeAG), and significantly differences were observed among them (p < 0.05).
Table 1 Comparison the contents of phenolics, flavonoids and anti-oxidant activity of the extractions from the herbs and herb pair. Values are the mean standard deviation (n ¼ 3). Plant material
FRAP value (mmol/g)
HXeAME CFeAME EAeAME NBeAME OHeAME HXeGU CFeGU EAeGU NBeGU OHeGU HXeAG CFeAG EAeAG NBeAG OHeAG
0.326 0.454 0.333 0.112 0.144 0.651 1.123 0.990 0.170 0.035 0.526 0.707 1.266 0.314 0.039
0.011 0.010 0.006 0.002 0.008 0.032 0.065 0.044 0.021 0.002 0.030 0.037 0.052 0.036 0.001
DPPH SC50a (mg/mL) 37.251 16.736 24.544 47.276 52.946 1.436 1.208 1.156 5.475 64.038 8.163 2.706 1.660 6.882 69.806
0.061 0.051 0.175 0.554 0.073 0.137 0.129 0.065 0.015 4.548 0.425 0.057 0.024 0.101 1.026
TEAC value (mmol/g) 2.030 6.718 7.055 2.425 1.501 88.647 69.306 51.725 14.614 1.373 19.053 37.994 67.625 9.369 1.416
0.136 0.213 0.136 0.202 0.110 0.474 0.302 0.087 0.122 0.064 0.337 0.558 0.313 0.247 0.079
Total phenolics (mg GAE/g) 18.654 33.456 27.646 9.094 6.864 134.768 121.861 163.502 44.408 8.905 49.715 71.606 106.414 43.132 6.881
0.109 0.483 0.113 0.088 0.065 5.894 4.008 5.312 0.502 0.428 0.468 0.215 3.461 0.792 0.326
Total flavonoids (mg RE/g) 17.878 12.486 7.048 2.642 0.830 62.517 66.546 60.285 18.770 3.601 17.198 32.193 54.794 21.100 2.893
0.212 0.173 0.871 0.246 0.081 0.468 0.218 0.807 0.101 0.099 0.111 0.321 0.808 0.222 0.048
The residue from ethanol extracts of single herbs of Astragalus membranaceus (AME) and Glycyrrhiza uralensis (GU) was re-suspended in water (OH) and successively extracted with the equivalent volume of n-hexane (HX), chloroform (CF), ethyl acetate (EA), and n-butanol (NB), and obtained five fractions for each herb extract (HXeAME, CFeAME, EAeAME, NBeAME, OHeAME; and HXeGU, CFeGU, EAeGU, NBeGU, and OHeGU). The herb pair of Astragalus membranaceus and Glycyrrhiza uralensis (AG) was extracted according to the above method, and obtained different solvent extracts (HXeAG, CFeAG, EAeAG, NBeAG, and OHeAG). a The SC50 value is defined as the sample concentration needed to scavenge 50% of the DPPH.
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Interestingly, the TEAC values of the five solvent extracts of the herb pair (AG) were higher than the theoretical sum of those of the constituent herbs (Fig. 3), which were similar to that of DPPH assay and FRAP assay. So the results indicated that the synergistic antioxidant effect existed in the process of combining AME and GU and could be evaluated comprehensively using these three different antioxidation methods. 3.4. Total phenolic and flavonoid contents Phenolic and flavonoids compounds are important antioxidants in many plants, so the content of them was measured to further study the synergistic effect. Table 1 showed that the contents of total phenolics and flavonoids had a wide range of differences in different solvent extracts. Total phenolic and flavonoid contents of various AME extracts were lower than those of GU and AG. Besides, total phenolic and flavonoid contents of EAeAG extract were obviously higher than those of other four AG extracts, which was in consistent with the previous study (Yen, Wu, Lin, Cham, & Lin, 2008). Moreover, our results also showed that total phenolic and flavonoid contents of EAeAG fraction were significantly higher compared with the theoretical sum of that of the constituent herbs, which indicated that ethyl acetate (EA) may be the appropriate solvent to achieve more available anti-oxidant components (Figs. 1e4). 3.5. The correlation between the phenolic/flavonoid increments and the increases of anti-oxidant capacity Previous studies reported that there was a correlation between the phenolic content and the antioxidation capacity (Andrzej, Dawidowicz, & Baraniak, 2006; Kumaran & Karunakaran, 2007). In our previous study, a significant correlation was also found between the total anti-oxidant activity and the phenolic/flavonoid
Fig. 1. Comparison the ferric reducing anti-oxidant power of the traditional Chinese herb pair (TCHP) and the theoretical sum of that of the constituent herbs. Values are mean standard deviation (n ¼ 3). The FRAP (ferric reducing anti-oxidant power) value is defined as mmol ferrous iron equivalents per g of extract; A-FRAP means the actual value of the ferric reducing anti-oxidant power we tested; T-FRAP means the theoretical value of the ferric reducing anti-oxidant power, and was calculated by the sum of two single herbs’ FRAP values multiplying 0.5 for their proportion in herb pair was 0.5:0.5. The residue from ethanol extract of herb pair of Astragalus membranaceus and Glycyrrhiza uralensis (AG) was re-suspended in water (OH) and successively extracted with the equivalent volume of n-hexane (HX), chloroform (CF), ethyl acetate (EA), and n-butanol (NB), and obtained five fractions (HXeAG, CFeAG, EAeAG, NBeAG, The FRAP value of CFeAG. The FRAP and OHeAG). - The FRAP value of HXeAG. The FRAP value of OHeAG. Identical value of EAeAG. The FRAP value of NBeAG. letters above the bars indicate no significant difference (p < 0.05).
contents of AME, GU and AG ethanol extracts (Yang et al., 2009). However, whether the synergistic effect in anti-oxidant activities of the different solvent-extracted fractions is related to the contents of total phenolics/flavonoids is still unknown. Therefore, the correlation between the phenolic/flavonoid increments and the increases of anti-oxidant capacity in the process of combining the two single herbs was further investigated. As shown in Fig. 5, the correlation coefficients between the total phenolic/flavonoid increments of different extracts and anti-oxidant activities investigated by three antioxidation methods (PCDPPH ¼ 0.5/0.6, PCTEAC ¼ 0.9*/0.8, PCFRAP ¼ 0.7/0.6) were higher than 0.5. These suggested that the phenolic/flavonoid increments of various extracts were closely related to the synergistic effect of anti-oxidant activities. Besides, there was significant linear correlation between the phenolic increments and TEAC values increments with the correlation coefficient of 0.9*, which indicated that the phenolic substances played an important role in the ABTS free radical scavenging ability of AG solvent-extracted fractions. 3.6. Protective effect on H2O2 induced oxidative damage in MRC-5 cells 3.6.1. Cell viability assay In MTT assay, it was found that the cell viability of H2O2 (0.2 mmol/L) treated group was significantly lower than that of control group (p < 0.05) (Table 2). H2O2 plays a pivotal role among a great variety of ROS, and could diffuse freely in and out of cells to cause lipid peroxidation and DNA damage in cells (Barbouti, Doulias, Nousis, Tenopoulou, & Galaris, 2002) which may be the major reason for the decrease in cell viability of H2O2 group. The groups treated with the ethyl acetate extract from AME, GU, AG have higher cell viability than H2O2 group, but have no significant difference with VE group (10 mg/mL) (p < 0.05). Moreover, there were no significant differences in cell viability between the group treated with the ethyl acetate extract from herb pair (AG) and the groups treated with the ethyl acetate extract from the two
Fig. 2. Comparison the DPPH $ scavenging activity of the traditional Chinese herb pair (TCHP) and the theoretical sum of that of the constituent herbs. Values are mean standard deviation (n ¼ 3). The SC50 value is defined as the sample concentration needed to scavenge 50% of the DPPH; A-DPPH means the actual value of the DPPH $ scavenging activity; T-DPPH means the theoretical value of the DPPH $ scavenging activity, and was calculated by the sum of two single herbs’ SC50 values multiplying 0.5 for their proportion in herb pair was 0.5:0.5. The samples of HXeAG, CFeAG, EAeAG, NBeAG, and OHeAG refer to the legend in Fig. 1. - The SC50 value of The SC50 value of CFeAG. The SC50 value of EAeAG. The SC50 value of HXeAG. The SC50 value of OHeAG. Identical letters above the bars indicate no NBeAG. significant difference (p < 0.05).
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single herbs (AME, GU) (p < 0.05), which exhibited a synergistic cytoprotection between EAeAME and EAeGU. 3.6.2. Effect of herbs’ extracts on SOD, CAT and GSH-Px activities of MRC-5 cells SOD, CAT and GSH-Px are important endogenic anti-oxidant enzymes in organisms for evaluating the effects of free radical scavenging activity and guarding against superoxide toxicity (Zhu et al., 2004). Results suggested that H2O2 significantly reduced the activities of Cu/Zn-SOD, Mn-SOD, CAT and GSH-Px compared with the control group (p < 0.05), which were remarkably alleviated when supplemented with the ethyl acetate extract from AME, GU, AG (Table 2). Moreover, the ethyl acetate extract from GU can protect the activity of Mn-SOD, CAT and GSH-Px better than that from AME, which may due to the higher anti-oxidant activity of EAeGU than that of EAeAME (Table 1). Besides, it’s noteworthy that much higher activity of Cu/Zn-SOD and CAT in the group treated with the herb pair (EAeAG) was observed than that of the single herbs (EAeAME, EAeGU), and the activity of GSH-Px of the group treated with EAeAG excelled the theoretical sum of EAeAME and EAeGU, which suggested that a synergistic effect might also exist between EAeAME and EAeGU in cell experiments. Furthermore, the activity of SOD in the group of EAeAG presented no significant difference with the positive control (VE þ H2O2), but the activity of GSH-Px in the group of EAeAG was significantly higher than that of the positive control, which suggested that EAeAG possessed good ability in protecting cells from oxidative stress. H2O2 is one of important ROS and can produce oxygen radicals and hydroxyl radicals, which may destroy the balance between the prooxidant and anti-oxidant molecules. But various anti-oxidant enzymes will protect cells from oxidative stress. For example, GSH-Px as well as CAT can catalyze the reduction of H2O2, and SOD can promote the destruction of superoxide radicals to protect oxygen-metabolizing cells from the harmful effect (Li, Li, Qian, Kim, & Kim, 2009; Zhu et al., 2004). So the herbs’ extracts can alleviate the oxidative damage induced by H2O2 in the way of protecting the activity of
Fig. 3. Comparison the ABTS $ radical scavenging activity of the traditional Chinese herb pair (TCHP) and the theoretical sum of that of the constituent herbs. Values are mean standard deviation (n ¼ 3). The TEAC (trolox anti-oxidant capacity) value is defined as mmol trolox equivalents of per g extract; A-TEAC means the actual value of the ABTS $ radical scavenging activity; T-TEAC means the theoretical value of the ABTS $ radical scavenging activity, and was calculated by the sum of two single herbs’ TEAC values multiplying 0.5 for their proportion in herb pair was 0.5:0.5. The samples of HXeAG, CFeAG, EAeAG, NBeAG, and OHeAG refer to the legend in Fig. 1. - The TEAC value of HXeAG. The TEAC value of CFeAG. The TEAC value of EAeAG. The TEAC value of NBeAG. The TEAC value of OHeAG. Identical letters above the bars indicate no significant difference (p < 0.05).
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SOD, CAT and GSH-Px. Furthermore, polyphenols and flavonoids were important natural antioxidants, which could inhibit the generation of reactive oxygen species or scavenge free radicals and prevent H2O2-induced cell injury by enhancing the activity of original natural antioxidants such as SOD, CAT and GSH-Px (Appiah et al., 2009; Zhu et al., 2004), so the increment of the polyphenols and flavonoids in combining the two individual herbs (AME, GU) may be the primary reason for the synergistic cytoprotection effect. In our study, it was found that various solvent extracts of the herb pair (AG) presented significantly better anti-oxidant activity compared with the theoretical sum of those of the constituent herbs (Figs. 1, 2 and 3). Furthermore, as for the different solvent fractions of AG, much higher anti-oxidant capacities were shown in the higher polar (EA) fraction (Figs. 1, 2 and 3, 4), but which were
Fig. 4. Comparison total phenolic/flavonoid content of the traditional Chinese herb pair (TCHP) and the theoretical sum of that of the constituent herbs. Values are mean standard deviation (n ¼ 3). (A) Comparison total phenolic content of the TCHP and the theoretical sum of that of the constituent herbs; (B) Comparison total flavonoid content of the TCHP and the theoretical sum of that of the constituent herbs; RVP means the actual value of total phenolic content; TVP means the theoretical value of total phenolic content, and was calculated by the sum of two single herbs’ phenolic contents multiplying 0.5 for their proportion in herb pair was 0.5:0.5; RVF means the actual value of total flavonoid content; TVF means the theoretical value of total flavonoid content, and was calculated by the sum of two single herbs’ flavonoid contents multiplying 0.5 for their proportion in herb pair was 0.5:0.5. The samples of HXeAG, CFeAG, EAeAG, NBeAG, and OHeAG refer to the legend in Fig. 1. (A)：- The total phenolic content of HXeAG; The total phenolic content of CFeAG; The total The total phenolic content of NBeAG; The total phenolic content of EAeAG; phenolic content of OHeAG. (B): - The total flavonoid content of HXeAG; The total The total flavonoid content of EAeAG; The total flavonoid content of CFeAG; flavonoid content of NBeAG; The total flavonoid content of OHeAG. Identical letters above the bars indicate no significant difference (p < 0.05).
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Fig. 5. The correlation between the increments of total phenolics, flavonoids and antioxidant activity (A) Comparison the increment of total phenolics and anti-oxidant activities; (B) Comparison the increments of total flavonoids and anti-oxidant activities; PC stands for Spearman correlation coefficient. (A): C The correlation between the increment of total phenolics and DPPH $ scavenging activity; B The correlation between the increment of total phenolics and ABTS $ radical scavenging activity; ; The correlation between the increment of total phenolics and ferric reducing antioxidant power. (B): C The correlation between the increment of total flavonoids and DPPH $ scavenging activity; B The correlation between the increment of total flavonoids and ABTS $ radical scavenging activity; ; The correlation between the increment of total flavonoids and ferric reducing anti-oxidant power.
observed in the lower polar (HX, CF) compared with the single herbs. Phenolic and flavonoid were important compounds responsible for anti-oxidant activity, and the amount and structure of phenolic and flavonoid varied in different herbs and when
extracted using different solvents (Cho et al., 2006; Wang et al., 2010), which may be one of the reasons for the changes in antioxidant activity between two herbs and the combination. Furthermore, it was noteworthy that there was a linear correlation between the increments of total phenolics/flavonoids and the increases of anti-oxidant capacities in different polar extracts of the herb pair (Fig. 5), which indicated that the synergistic effect was related to the increases of total phenolic and flavonoid contents (Table 1), especially the phenolic contents. In addition, the ethyl acetate extract of herb pair (AG) presented more potent protection on cytotoxicity than that of single herbs (AME, GU) and showed synergistic effect in inducing the activities of Cu/Zn-SOD, CAT and GSH-Px (Table 2), which might also due to the enhancement of total phenolic and flavonoid contents. These results suggested that the process of combining two herbs increased the dissolution of phenolic or flavonoid substances and subsequently powered the anti-oxidant capacity and cell protective effect of the solvent extracts. Previous studies suggested that both pH and complexation commonly influenced solubilization of the compounds (Li, Tabibi, & Yalkowsky, 1998; Tommasini et al., 2004), which indicated that the increases of total phenolic and flavonoid contents in the combination may be caused by the alteration of the acid-base environment. In addition, another possible source of the synergistic effects was related to the change of matter structure, which could also change the anti-oxidant effect. Rice-Evans, Halliwell, and Lunt (1995) found that the increase in polar groups of gallic acid such as hydroxyl group could improve the anti-oxidant capacity of tea polyphenols. The compound containing gallic acylation, catechol group and so on would also lead to a corresponding enhancement in anti-oxidant capacity (Van Acker et al., 1996, p. 331). Besides, Okuda (1999) reported that the anti-oxidant capacity of tannin substances could be doubled by the condensation polymerization of monomer-dimer to further. These indicated that the increase in polar groups and the polymerization of some substances in the process of combining might contribute to the synergistic effects of the herb pair (AG), which may be another reason leading to the changes in anti-oxidant activity between two herbs and the combination. In our study, the synergistic effects obviously exist in the ethyl acetate extracts, but other solvent extracts present additive or antagonistic effects such as the water extracts. It was found that the phenolic and flavonoid contents of the herb pair (OHeAG) showed little differences with the theoretical sum of those of OHeAME and OHeGU (Fig. 4), which were largely responsible for antagonistic effect in anti-oxidant activity. Besides, ethyl acetate may be the appropriate solvent that can affect pH, complexation as well as chemical structures and then enhance the content of phenolic and flavonoid contents or the anti-oxidant activity in the combination, which need further research in our later work. In conclusion, our results suggested that the ethyl acetate extract of herb pair (EAeAG) showed the synergistic effect significantly in anti-oxidant capacity, and also presented better
Table 2 Effect of EAeAG on the cell viability and the activities of Cu/Zn-SOD, Mn-SOD, CAT and GSH-Px of MRC-5 cells. Values are the mean standard deviation (n ¼ 3). Test group
Cell Viability (%)
Cu/Zn-SOD activity (U/mL)
Control H2O2 VEþ H2O2 EAeAMEþH2O2 EAeGUþH2O2 EAeAGþH2O2 T(EAeAGþH2O2)
100.000a 59.466 91.582 86.373 89.408 88.941 87.891
4.308 1.124 4.165 2.2997 2.264 3.935 2.282
5.765d 6.945ab 5.729bc 9.228ab 7.580ab 7.478b
0.206a 0.064d 0.170ab 0.1838c 0.127c 0.129b 0.156c
Mn-SOD activity (U/mL) 3.345 1.715 2.531 2.051 3.241 2.144 2.646
0.178a 0.110d 0.283bc 0.226cd 0.220ab 0.180cd 0.223abc
CAT activity (U/mL) 3.681 0.2710 2.123 0.452 0.881 1.513 0.666
0.034a 0.0255f 0.156b 0.037ef 0.028d 0.031c 0.032de
GSH-Px activity (U/mL) 6.962 1.582 4.114 3.165 5.696 5.380 4.430
0.028a 0.014g 0.021e 0.029f 0.011b 0.024c 0.020d
aeg Means with different letter in the same row present significantly different (p < 0.05). The ethyl acetate extract of single herbs of Astragalus membranaceus (AME) and Glycyrrhiza uralensis (GU) was expressed as EAeAME and EAeGU, and the ethyl acetate extract of herb pair of Astragalus membranaceus and Glycyrrhiza uralensis (AG) was expressed as EAeAG. T(EAeAGþH2O2) means the theoretical sum of that of the constituent herbs with their proportion being 0.5:0.5 in herb pair, which was calculated by the sum of every anti-oxidant enzyme’ activity of two single herbs’ multiplying 0.5.
M. Li et al. / LWT - Food Science and Technology 44 (2011) 1745e1751
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