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Intracellular Antioxidant Detoxifying Effects of Diosmetin on 2,2Azobis(2-amidinopropane) Dihydrochloride (AAPH)-Induced Oxidative Stress through Inhibition of Reactive Oxygen Species Generation Wenzhen Liao, Zhengxiang Ning, Luying Chen, Qingyi Wei, Erdong Yuan, Jiguo Yang,* and Jiaoyan Ren* College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China ABSTRACT: The intracellular antioxidant activities of diosmetin were evaluated by cellular antioxidant activity (CAA) assay, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis assay and cupric chloride (CuCl2)induced plasma oxidation assay. The results showed that diosmetin exhibits strong cellular antioxidant activity (EC50 = 7.98 μmol, CAA value = 58 μmol QE/100 μmol). It was also found that diosmetin treatment could effectively attenuate AAPHinduced erythrocyte hemolysis (91.0% inhibition at 100 μg/mL) and CuCl2-induced plasma oxidation through inhibition of intracellular reactive oxygen species (ROS) generation. Diosmetin could significantly restore AAPH-induced increase of intracelluar antioxidant enzyme (SOD, GPx, and CAT) activities to normal levels, as well as inhibit intracellular malondialdehyde (MDA) formation. Thus, the intracellular antioxidant detoxifying mechanism of diosmetin is associated with both nonenzymatic and enzymatic defense systems. KEYWORDS: diosmetin, cellular antioxidant activity, oxidative hemolysis, plasma oxidation

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INTRODUCTION Diosmetin (3′,5,7-trihydroxy-4′-methoxyflavone) is bioflavonoid found in citrus fruits, spearmint, and legume leaves1 that has antibacterial activity against Helicobacter pylori(1),2 Staphylococcus aureus,3 and Bacillus subtilis.4 In addition, diosmetin also exhibits antimutagenic, antioxidant, and anti-inflammatory activities.5,6 Recent evidence had revealed that diosmetin possessed immunoregulating activity; for example, it could modulate macrophage colony-stimulating factor (M-CSF) induced proliferation and reduced the production of transforming growth factor beta 1 (TGF-β1), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) in mouse macrophages.7 Other studies in cancer cell lines and animal models indicated that diosmetin had strong cancer chemopreventive and antiproliferative abilities.8−12 The latest study also demonstrated that diosmetin could significantly enhance adenosine triphosphate (ATP) levels in liver and kidney cells.13 One factor underlying the pathology of many diseases, including cancer, diabetes, and coronary artery disease, is an increase in oxidative stress;14−16 this suggests that the numerous beneficial properties of diosmetin may be related to its antioxidant activity. However, although the extracellular antioxidant activity of diosmetin has been extensively studied, there is little information on its antioxidant properties within cells. This is significant because extracellular assays mostly rely on chemical methods that do not take the metabolism and bioavailability of antioxidants into account. Therefore, tests of diosmetin’s intracellular antioxidant activity, which address issues of uptake, distribution, and metabolism, are urgently needed. The cellular antioxidant activity (CAA) assay is a newly developed approach that quantifies the antioxidant capacity of bioactive compounds in cell cultures.17 The CAA assay © 2014 American Chemical Society

represents a marked improvement over traditional chemical antioxidant activity assays because it simulates cellular biochemical processes, including cellular uptake and metabolism. Therefore, the CAA assay should allow a better understanding of how antioxidants behave under physiological conditions. To date, little information about the evaluation of the antioxidant activity of diosmetin by CAA has been done. One of the main causes of intracellular oxidation is the presence of reactive oxygen species (ROS); for example, ROS can react with unsaturated fatty acids in phospholipids to form lipid peroxides, leading to damage of the cell membrane. The production and destruction of ROS is normally maintained in a steady-state balance by inherent cellular defense systems, including enzymatic and nonenzymatic antioxidants.18,19 However, oxidative stress will occur either when there is an excess of ROS or when the intrinsic defenses are damaged. In cell models, oxidative stress can be induced by many reagents, for example, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH).20,21 Thus, an AAPH-induced erythrocyte hemolysis assay can visually reveal biologically relevant radical-scavenging ability by measuring ROS and malondialdehyde (MDA) levels, as well as activities of intracellular antioxidant enzyme activities (superoxide dismutase, SOD; glutathione peroxidase, GPx; and catalase, CAT). Up to now, little information about the protective effects of diosmetin on AAPH-induced erythrocyte hemolysis is available. A variety of studies suggested that cupric chloride (CuCl2) was a pro-oxidant agent, which could cause lipid oxidation. Received: Revised: Accepted: Published: 8648

May 20, 2014 July 22, 2014 July 30, 2014 July 30, 2014 dx.doi.org/10.1021/jf502359x | J. Agric. Food Chem. 2014, 62, 8648−8654

Journal of Agricultural and Food Chemistry

Article

evaluated as the inhibition of erythrocyte hemolysis based on the method described by Cheung et al.25 with some modifications. Blood samples were collected from normal male adult volunteers under 30 years of age. Erythrocytes were separated from plasma by centrifugation at 1200g for 10 min at 4 °C and washed three times with PBS (pH 7.4). An aliquot of 0.2 mL of a 20% erythrocyte suspension in PBS was mixed with 0.2 mL of PBS (absorbance A) or diomestin (absorbance B) at different concentrations. The mixture was incubated at 37 °C for 20 min with gentle shaking, after which 0.4 mL of 200 mM AAPH was added, and the incubation continued at the same temperature for another 2 h. Finally, the reaction solution was diluted with 8 mL of PBS and centrifuged at 1200g for 10 min at 4 °C. The absorbance of the supernatant was measured at 540 nm. To achieve complete hemolysis, 8 mL of ultrapure water was added to the mixture, which was then centrifuged at 1200g for 10 min at 4 °C, and the absorbance of the supernatant was determined at 540 nm. The percentage of hemolysis inhibition was calculated as

Although copper (Cu) is an essential component of many enzymes and participates in various physiological processes, Cu releases from proteins can facilitate the formation of hydroxyl radicals and lead to lipid peroxidation.22 Thus, the CuCl2induced plasma oxidation assay can be used as a biologically relevant method for evaluating the antioxidant capacity of bioactive compounds. In this research, the CuCl2-induced plasma oxidation assay was first applied to investigate the intracellular antioxidant capacity of diosmetin. In the present study, the intracellular antioxidant properties of diosmetin were investigated using the CAA, AAPH-induced erythrocyte hemolysis, and copper-induced plasma oxidation assays. The results showed that diosmetin exhibited potent antioxidant activity via both nonenzymatic and enzymatic mechanisms.

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% hemolysis inhibition = (1 − A /B) × 100%

MATERIALS AND METHODS

Materials. Diosmetin, AAPH, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma Co. (St. Louis, MO, USA). An MDA kit was purchased from Nanjing Jiancheng Institute of Biotechnology (Jiangsu, China). Kits for the determination of SOD, cellular GPx, CAT, ROS, and bicinchoninic acid (BCA) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), and phosphate-buffered saline (PBS, pH 7.4) were obtained from Gibco Life Technologies (Grand Island, NY, USA). Human hepatocellular carcinoma (HepG2) cells were obtained from the Medical College of Sun Yat-Sen University (Guangzhou, China). Heat-inactivated fetal bovine serum (FBS) was purchased from Sijiqing Co. Ltd. (Hangzhou, China). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). CAA Assay. CAA of Diosmetin. The intracellular antioxidant capacity of diosmetin was assessed using the CAA assay as described16 with some modifications. Briefly, HepG2 cells were grown in DMEM with 10% FBS, 100 μg/mL streptomycin, and 100 units/mL penicillin.23 During the logarithmic growth phase, cells (6 × 105/ mL) were seeded into a 96-well microplate (100 μL/well) and incubated for 24 h in a fully humidified atmosphere in a 5% CO2, 37 °C incubator. The culture medium was then removed, and the cells were washed with 200 μL of PBS and incubated for an additional 2.5 h with medium that contained diosmetin and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (final concentration = 25 μM). The culture medium was then removed, and the cells were washed with 200 μL of PBS, after which 100 μL of new medium (HBSS plus 10 mM HEPES) containing 600 μM AAPH was added to each well. The 96-well microplate was placed in a Fluoroskan Ascent microplate fluorometer (Thermo Electron Corp., Vantaa, Finland), and the fluorescence was monitored every 5 min for 1 h (ex/em, 488/525 nm). Each plate included triplicate blank wells, which contained cells treated without oxidant. Quantification of CAA. The blank and initial fluorescence values were subtracted, and the CAA value of each diosmetin concentration was calculated as the integration of the area below the curve of fluorescence versus time, according to the following equation:24 CAA (units) = 1 − (∫ SA/∫ CA), where ∫ SA is the integration of the area under the sample fluorescence versus time curve and ∫ CA refers to the integration of the area under the control curve. The 50% effective concentration (EC50) for diosmetin was determined from the median effect plot of log( fa/f u) versus log(dose), where fa is the effective fraction of the treatment (CAA units) and f u is the ineffective fraction (1 − CAA units) of the treatment. The EC50 was presented as the mean ± SD and calculated for triplicate data sets. EC50 values were expressed as micromoles of quercetin equivalents (QE) per 100 μmol of diosmetin; the EC50 value for quercetin was obtained from four separate experiments. Assay for Erythrocyte Hemolysis Mediated by Peroxyl Free Radicals. The intracellular antioxidant activity of diosmetin was

where A refers to the absorbance A and B refers to absorbance B as metioned above. Plasma Oxidation Assay. Plasma samples were obtained from blood by centrifugation at 1200g for 10 min at 4 °C and then diluted 40-fold with PBS and stored at 4 °C until use. Then 0.2 mL of plasma was mixed with 0.2 mL of PBS or diomestin at different concentrations and incubated at 37 °C for 20 min with gentle shaking. Subequently, 0.4 mL of 200 μM CuCl2 was added as oxidant and the incubation continued for another 2 h. Levels of conjugated dienes were determined every 10 min by measuring absorption at 245 nm. Scanning Electron Microscopy (SEM). Erythrocyte imaging was carried out using a scanning electron microscope (Hitachi TM3000, Hitachi Ltd., Tokyo, Japan). A film consisting of a single layer of red blood cells was made by spreading the treated blood samples onto newly split mica and settled with 2.5% glutaraldehyde, after which the film was placed on the specimen holder and images of blood samples were acquired. Determination of Intracellular ROS Level. A ROS assay kit with DCFH-DA as a fluorescent probe was used to determine the relative levels of intracellular ROS. Treated red blood cells were collected by centrifuging at 1200g for 10 min at 4 °C, washed three times with PBS, and then incubated at 37 °C for 20 min in the presence of 10 μM DCFH-DA. DCFH-DA is transformed to the fluorescent compound 2′,7′-dichlorofluorescin (DCF) within cells. The fluorescence intensity of cells was measured using a Fluoroskan Ascent microplate fluorometer (ex/em, 488/525 nm). MDA, GPx, SOD, and CAT Assays. Red blood cells were collected and washed as described above. Cells were then resuspended in 600 μL of cold ultrapure water and stored at −80 °C before determination. A BCA protein assay kit and microscale MDA kit were used to monitor the protein and MDA contents, respectively. The Cellular Glutathione Peroxidase Kit, Total Superoxide Dismutase Assay Kit, and Catalase Assay Kit were used to measure the activities of GPx, SOD, and CAT according to the manufacturer’s instructions. Statistics. Experiments were carried out in triplicate, and all data are reported as the mean ± standard deviation (SD). The significant differences between the means of parameters were calculated by Duncan’s multiple-range test (p < 0.05) using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

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RESULTS AND DISCUSSION Cellular Antioxidant Activity of Diosmetin. The intracellular antioxidant activity of diosmetin was evaluated using the CAA assay, which was regarded as a more biologically relevant assay than commonly used chemical antioxidant activity assays.26 For the CAA assay, the membrane-permeable intracellular probe DCFH-DA was deacetylated to DCFH by cellular esterases in HepG2 cells. DCFH was then trapped within the cells and oxidated by peroxyl radicals generated from 8649

dx.doi.org/10.1021/jf502359x | J. Agric. Food Chem. 2014, 62, 8648−8654


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Figure 1. Inhibitory effects of diosmetin on peroxyl radical-induced oxidation of DCFH in HepG2 cells (A) and EC50 plots for inhibition of peroxyl radical-induced DCFH oxidation by diosmetin (B).

Table 1. EC50 Value and CAA Value of the Inhibitory Effect of Diosmetin on AAPH-Induced DCFH Oxidation at Different Concentrations (2−10 μmol)

b

compound

EC50 (μmol)

quercetin diosmetin EGCG myricetin luteolin kaempferol

4.626 7.98 15.8 15.4 23.1 6.31

± ± ± ± ± ±

0.272 0.003 0.4 0.5 1.0 0.21

Figure 2. Protective effects of diosmetin on AAPH-induced erythrocyte hemolysis assay (A) and CuCl2-induced accumulation of conjugated dienes in plasma (B). Erythrocytes were pretreated with different concentrations (12.5−100 μg/mL) of diosmetin for 25 min prior to AAPH (200 mM) treatment for 2 h; the positive group was treated with 200 mM AAPH only. Plasma was pretreated with different concentrations (0.5−8 μg/mL) of diosmetin for 10 min prior to CuCl2 (200 μM) treatment for 2 h. The positive group was treated with 200 μM CuCl2 only.

CAA (μmol QEb/100 μmol diosmetin) 58.0 33.1 36.8 32.3 81.1

± ± ± ± ±

0.019 1.017 3.817 0.917 2.717

known phytochemicals such as kaempferol, myricetin, epigallocatechin gallate (EGCG) and luteolin.16 The EC50 value of diosmetin was 7.98 ± 0.20 μmol, and the CAA value of diosmetin was 58.0 ± 0.02 μmol QE/100 μmol diosmetin, which is higher than those of EGCG (33.1 ± 1.0 μmol of QE/100 μmol), myricetin (36.8 ± 3.8 μmol of QE/ 100 μmol), and luteolin (32.3 ± 0.9 μmol of QE/100 μmol), and lower than that of only kaempferol (81.1 ± 2.7 μmol of QE/100 μmol), as reported by Wolfe et al.,17 indicating that diosmetin had strong cellular antioxidant activity. This high activity probably results from the chemical structure of the diosmetin molecule (Figure 1B). It has three hydroxyl groups, one each attached to C3′, C5, and C7. Of these, the hydroxyl group attached to C3′ has the most positive effect on the free radical scavenging activity of diosmetin.30 The 2,3-double bond conjugated with a 4-oxo group in the C ring of diosmetin also contributes significant activity against oxidation by H2O2.29 Attenuation of Erythrocyte Hemolysis and Plasma Oxidation by Diosmetin. The antioxidant ability of

QE, quercetin equivalents.

AAPH to form a fluorescent substance, DCF. The level of fluorescence was proportional to the degree of oxidation. The reduction of cellular fluorescence compared to the control group indicates the antioxidant activity of the tested samples.27−29 The kinetics of DCFH oxidation induced by AAPH are shown in Figure 1A. It was found that diosmetin significantly inhibited this oxidation process in a dose-dependent manner as indicated by the obvious decrease of the fluorescence level. The EC50 for the inhibitory action of diosmetin on DCFH oxidation was calculated from the median effect curve plotted for each concentration (Figure 1B). On the basis of the fitting curves, the CAA value of diosmetin was determined (Table 1). Quercetin was used as a standard, as it has already been shown previously to have high CAA capacity compared to other 8650



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Figure 4. Inhibitory activity of diosmetin on AAPH-induced ROS overexpression in human erythrocytes. Erythrocytes were pretreated with different concentrations (12.5−100 μg/mL) of diosmetin for 30 min prior to AAPH (200 mM) treatment for 2 h; the positive group was treated with 200 mM AAPH only.

increased from 0 to 100 μg/mL. At a dose of 100 μg/mL, the inhibition rate of diosmetin on erythrocyte hemolysis was 91.0%, which was comparable to that of vitamin C, a wellknown antioxidant with an inhibition rate of 96.9% at 1 mg/mL (data not shown). Using SEM, we examined the morphological effect of oxidation on the red cell membranes with or without the addition of diosmetin. The normal biconcave discoid shape of erythrocytes can be clearly seen in Figure 3A. After treatment with AAPH, the erythrocyte lost its normal profile and showed a spiny configuration on its surface due to the severe damage of the cell membrane (Figure 3B). However, this damage was dramatically reduced in cells pretreated with 100 μg/mL diosmetin before the addition of AAPH (Figure 3C). This result confirmed the protective effect of diosmetin on AAPHinduced morphological changes in the red cell. Lipoprotein oxidation in plasma has great pathological significance on atherosclerosis and hyperlipemia. Oxidation of low-density lipoprotein (LDL) is thought to be a great contributing factor to the development of atherosclerosis, whereas oxidation of high-density lipoprotein (HDL) may lead to hypercholesterolemia.31 In a further test of the antioxidant activity of diosmetin, we used the plasma oxidation assay, where plasma is treated with CuCl2, which can induce the formation of conjugated dienes. As shown in Figure 2B, the concentration of conjugated dienes increases over time. Diene accumulation, a hallmark of lipid peroxidation, was induced when plasma was treated with 200 μM copper(II) ions, as indicated by the high absorbance value at 245 nm. Diosmetin at different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 μg/mL) could significantly decrease copper-induced diene accumulation, which showed indirect protection of plasma from oxidative damage. Again, dose dependence could be demonstrated. Diosmetin Inhibits AAPH-Induced ROS Generation in Erythrocytes. The antioxidant protection of erythrocytes by diosmetin is considered to be due to its effect on intracellular ROS generation.32 To further test this, intracellular ROS generation was quantified using DCFH-DA, a fluoresceinlabeled dye that can cross the plasma membrane into the cytoplasm, where it is hydrolyzed by nonspecific esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is

Figure 3. SEM micrographs of erythrocyte samples: (A) normal erythrocyte; (B) AAPH-treated erythrocyte; (C) erythrocyte preincubated with diosmetin prior to AAPH treatment.

diosmetin was further examined using erythrocyte hemolysis and plasma oxidation assays. In the erythrocyte hemolysis assay, AAPH, which can decompose at 37 °C to generate an alkyl radical, is used as an initiator. In the presence of oxygen, these alkyl radicals will be converted to peroxyl radicals that can cause lipid peroxidation and loss of erythrocyte membrane integrity, which could ultimately lead to hemolysis. Therefore, the rate of inhibition of hemolysis is an indirect way to measure the antioxidant activity of diosmetin. As shown in Figure 2A, erythrocyte hemolysis induced by AAPH was effectively attenuated by diosmetin in a dose-dependent manner: the inhibition rate was enhanced as diosmetin concentration 8651



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Figure 5. Changes in MDA content (A) and enzyme activities of SOD (B), GPx (C), and CAT (D) in erythrocytes. Erythrocytes were pretreated with different concentrations (12.5−100 μg/mL) of diosmetin for 30 min prior to AAPH (200 mM) treatment for 2 h; the positive group was treated with 200 mM AAPH only. (E) Possible intracellular antioxidant detoxifying mechanisms of diosmetin that attenuate AAPH-induced oxidative stress through inhibition of ROS generation.

oxidized to DCF, a highly fluorescent compound. Therefore, inhibition by diosmetin of AAPH-induced ROS generation can be measured by the ability of the diosmetin to prevent the oxidation of DCFH and, as a result, the formation of DCF. As shown in Figure 4, the DCF fluorescence intensity of

erythrocytes exposed to AAPH (200 mM) was much higher than that of the control, demonstrating that AAPH treatment produced a large amount of ROS. However, cells pretreated with diosmetin (12.5, 25, 50, and 100 μg/mL) exhibited apparently lower DCF fluorescence intensity in a dose8652



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intracellular antioxidant enzyme (GPx, SOD, and CAT) activities. The particular chemical structure of diosmetin, for example, the hydroxyl group attached to C3′ and the 2,3double bond conjugated with a 4-oxo group in the C ring, contributes significantly to its antioxidant activity. Diosmetin effectively attenuated AAPH-induced oxidative stress in erythrocytes and plasma by inhibition of intracellular ROS generation. It also significantly decreased copper-induced diene accumulation and protected plasma from oxidative damage. Because oxidative stress is involved in the pathogenesis of a number of chronic diseases including heart disease, cancer, type 2 diabetes, and inflammatory diseases,35 our results demonstrated that diosmetin could be good candidate for the treatment of diseases in which ROS overproduction is the etiological factor. It can be developed either as functional foods or as complementary medicines.

dependent manner, indicating that diosmetin inhibited ROS generation. As a control, cells treated with diosmetin without AAPH addition did not show significantly different DCF fluorescence, implying that diosmetin itself did not cause ROS generation in normal erythrocytes. Taken together, these results confirm that diosmetin can reduce intracellular oxidative stress. Diosmetin Prevents AAPH-Induced MDA Accumulation and Modulates Intracellular Antioxidant Enzyme (GPx, SOD, and CAT) Activities. AAPH-induced ROS generation can cause lipid peroxidation and result in the release of MDA. MDA is involved in tumor promotion, cellular metabolism disruption, and cell membrane dysfunction due to its high reactivity. Excess MDA can ultimately lead to disruption of cellular metabolism.32 As seen in Figure 5A, the MDA level in erythrocytes was significantly increased from 0.25 to 0.87 nmol/mg protein after treatment with 200 mM AAPH for 2 h, indicating the occurrence of lipid peroxidation caused by AAPH-induced oxidative stress. For the cells incubated with different concentrations of diosmetin (12.5, 25, 50, and 100 μg/ mL), the level of MDA in erythrocytes was decreased, especially at the high diosmetin concentration (100 μg/mL), where the MDA concentration declined almost to that of the control. For cells incubated with diosmetin without AAPH supplementation, the MDA level was comparable to that of the control, showing that diosmetin itself does not induce MDA formation. Thus, lipid peroxidation caused by ROS generation was efficiently inhibited by diosmetin. Major radical scavenging antioxidant enzymes in the human body include SOD, GPx, and CAT. These enzymes constitute an intracellular defense system, which can coordinate the elimination of free radicals through a series of chain reactions. Changes in the activities of these antioxidant enzymes may be related to the cell’s antioxidant response. The intracellular level of these antioxidants is crucial for maintaining a steady state of superoxide radicals and hydrogen peroxide.33,34 SOD can catalyze the dismutation of the highly reactive superoxide anion O2•− into O2 and hydrogen peroxide (H2O2), which is less reactive. CAT converts the H2O2 generated within cells to H2O and O2 and prevents the conversion of H2O2 into more active species, such as hydroxyl radicals (•OH), which can ultimately lead to cell death. As for GPx, it catalyzes the reaction of hydroperoxides with glutathione (GSH), thereby protecting cells from oxidative damage. As shown in panels B, C, and D of Figure 5, the activities of SOD, GPx, and CAT in erythrocytes were significantly increased after 200 mM AAPH treatment for 2 h, showing that AAPH treatment activated the enzymatic antioxidant defense systems in erythrocytes. The cellular antioxidant system depends on quenching of O2•− by SOD and further protection by CAT to convert H2O2 to H2O (Figure 5E). Pretreatment with diosmetin provided effective control of red cell antioxidant defense systems as indicated by the lower GPx (Figure 5B), SOD (Figure 5C), and CAT (Figure 5D) levels. This was particularly the case for high (100 μg/mL) diosmetin treatment, which maintained enzyme activities at normal levels. Thus, diosmetin can effectively attenuate AAPH-induced oxidative stress in erythrocytes, mainly through the inhibition of ROS generation, as illustrated in Figure 5E. In conclusion, in a cell-based CAA assay, diosmetin efficiently attenuated AAPH-induced oxidative stress in human erythrocytes. Diosmetin prevented the generation of intracellular ROS, inhibited the formation of MDA, and regulated

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AUTHOR INFORMATION

Corresponding Authors

*(J.Y.) Phone: (+86) 20-87112594. E-mail: [email protected]. cn,. *(J.R.) Phone: (+86) 20-87112594. E-mail: [email protected]. Funding

This study was supported by the National Key Technology R&D Program of China in the 12th five-year period (No. 2012BAD33B11), the Fundamental Research Funds for the Central Universities (No. 2014ZZ0063 and 2013ZZ0061), and Guangdong Natural Science Funds for Distinguished Young Scholars (No.S2013050013954). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express thanks to Professor Alan Tunnacliffe, Department of Chemical Engineering and Biotechnology, University of Cambridge, for improvement of writing.

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