2015 Volume 21 Issue 4 Pages 563-571
This study investigated the antioxidative and antiglycative abilities of flesh and peel extracts of Chinese purple yam. Among five fractions of purple yam extracts, the ethyl acetate (EA) fraction exerted the best antioxidant activity. Peel (P-EA) and flesh EA extracts (F-EA) exhibited excellent antiglycation ability by efficiently suppressing different stages of the glycation cascade, and the effect was mainly due to their antioxidant activities. The cytoprotective effect of F-EA and P-EA on methylglyoxal induced oxidative damage in HepG2 cells was also determined, and the underlying mechanism was the strengthened antioxidant defense system in HepG2 cells. Peel extract exerted better bioactivity than flesh extract, which indicated that purple yam peel, a kind of kitchen waste that is always overlooked, might serve as a promising therapeutic natural product for the prevention of diabetes or other advanced glycation end products-related diseases.
The non-enzymatic reaction between the aldehyde group of sugars with a free amino acid (mainly lysine and arginine) of a protein, lipid or DNA can lead to the reversible formation of a Schiff base which undergoes rearrangement to form relatively stable Amadori products, known as early glycation products. Amadori products further undergo complicated chemical rearrangements (oxidations, reductions, and hydrations), and form highly reactive intermediate products α-dicarbonyls or oxoaldehydes, including 3-deoxyglucosone (3-DG) and methylglyoxal (MGO) (Baynes and Thorpe, 1999), which can react with amino, sulfhydryl, and guanidine functional groups in proteins (Lo et al., 1994). These reactions result in the formation of denatured, brownish, cross-linked proteins called advanced glycation end products (AGEs), which are major factors responsible for the complications of diabetes and aging (Gugliucci, 2000).
Normally, AGEs form and accumulate with time in many tissues of the body at a constant but slow rate, and are responsible for many age-related pathologies. However, in diabetic patients, hyperglycemia contributes to increased glycation and AGE formation by up to four times (Alikhani et al., 2005) with high production of free radicals capable of damaging proteins, lipids, and DNA. Thus diabetics suffer from premature onset of various age-related complications and a higher risk of oxidative damage.
Aminoguanidine (AG), with strong scavenging activities against reactive carbonyl species, is the first compound designed to inhibit AGE formation. However, it cannot be used for therapeutic purposed because of its high toxicity in many patients (Thornalley, 2003). Compared with synthetic drugs, natural products have been demonstrated to be relatively safe for human consumption. In this regard, interest and demand for dietary supplements derived from plants are increasing, instead of drugs with unpleasant side-effects.
The plant kingdom possesses abundant unexplored sources of potentially useful antidiabetic drugs. Targeting oxidative stress and the glycation cascade concerning diabetes, the evaluation of antioxidant and antiglycation properties of some natural extracts might provide effective and safe therapeutic approaches for preventing the development of diabete mellitus and its complications. Chinese yam has a long cultivation history in China. It has been regarded as a tonic food and traditional Chinese medicine for the treatment of diabetes, diarrhea, asthma, and other ailments for more than 2000 years. Purple yam, which belongs to the species of Dioscorea alata L., is characterized by amaranth flesh that is rich in pigments. Multiple biological effects of yam tubers have been well documented, including ameliorating insulin resistance (Kim et al., 2012), antioxidant and anti-inflammatory activities (Chiu et al., 2013). However, as a kind of kitchen garbage, yam peels are always overlooked, although they might contain more polyphenols and flavonoids than flesh and exhibit good antioxidant activity (Kim et al., 2010). It has been demonstrated that yam peels present considerably higher antioxidant activities than flesh (Chung et al., 2008). Thus, in the present study, both purple yam flesh and peels were used for the evaluation of antioxidant and antiglycation properties.
The highly reactive intermediate of the glycation cascade, MGO, can move across plasmalemma and mitochondrial membrane to attack different molecular targets and induce glycation of multiple proteins in the cytosol, mitochondria, and other vesicles (Wang et al., 2009). Many studies revealed that MGO can induce apoptosis and dysfunction in various cell lines (Sharma et al., 2014; Wang et al., 2014; James et al., 2014). Besides, MGO can also impair insulin signaling and insulin action in the pancreatic beta cell line INS-1E (Fiory et al., 2011). However, to our knowledge, the effect of MGO on the antioxidant defense system in HepG2 cells has not been investigated. Thus, in the present study, 1 mM MGO was utilized to induce oxidative stress in HepG2 cells. The protective effects of both purple yam flesh and peel extracts were further evaluated by analyzing the cellular glutathione (GSH) content, total antioxidative capacity (TAOC) and activities of superoxide dismutase (SOD) and catalase (CAT).
Chemicals and reagents Purple yams were obtained from Zhejiang Province, China, 2,4,6-Tripyridyl-s-triazine (TPTZ), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), phenazine methosulfate, NADH-2Na, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), gallic acid, Folin Ciocalteu's phenol reagent, bovine serum albumin (BSA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), AG, sodium azide, D-glucose, D-fructose, MGO (40% aqueous solution), nitro-blue tetrazolium (NBT), Girard-T regeant, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), dimethyl sulfoxide (DMSO) and N-acetyl-L-cysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA), PBS, Hank's balanced salt solution (HBSS), fetal bovine serum (FBS), and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO (BRL Life Technologies, Grand Island, NY, USA). All other chemical reagents were of analytical grade and purchased from Beijing, China. Assay kits for GSH, SOD, CAT, TAOC, and protein were obtained from Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, China.
Preparation of Chinese purple yam extracts In brief, 1 – 2 mm-thick layers from the outer surface of intact yam tubers were collected for the preparation of peel extracts. The remaining inner tissues of the tubers were cut into slices to prepare flesh extracts. Both parts were freeze-dried (Four-Ring Science Instrument Plant, Beijing Co., Ltd., Beijing), ground to a fine powder, and passed through a 60 mesh sieve. The powdered plant material was extracted by ultrasonication three times with 80% ethanol for 30 min at room temperature. The combined extracts were concentrated under reduced pressure and yielded a crude extract (CE). The evaporated CE was suspended in water and extracted sequentially using n-hexane (HX), ethyl acetate (EA), and n-butanol (BT), the remaining water (RW) solution was also regarded as a fraction. The four fractions were concentrated using a rotary and then freeze-dried. Before use, all the dried samples were dissolved in DMSO.
Determination of total phenolic content The total phenolic contents in different fractions of purple yam extracts were determined using the Folin Ciocalteu colorimetric method (Slinkard and Singleton, 1977). In brief, 0.5 mL of diluted extracts and 2.5 mL of Folin Ciocalteu reagent (diluted 1:10, v/v) were reacted with 2 mL of 7.5% (v/v) Na2CO3 solution at 30°C for 90 min. Solution absorbance at 765 nm was measured using a microplate reader (SpectraMax M2e, USA). The total phenolic content was expressed as gallic acid equivalents (mg gallic acid/g dried extract).
Ferric reducing antioxidant power (FRAP) assay The FRAP assay was described by Pulido et al. (2000) with minor modifications. In brief, 0.2 mL of diluted extract was mixed with 1.8 mL of FRAP reagent, which was freshly prepared as a mixture of 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3·6H2O at a ratio of 10:1:1. The absorption of the reaction mixture was measured at 595 nm after being left in the dark at 37°C for 8 min. The FRAP value was expressed as FeSO4·7H2O equivalents (mg FeSO4·7H2O /g dried extract).
Trolox equivalent antioxidant capacity (TEAC) assay TEAC assay was performed using the procedures of Re et al. (1999) A solution of 7 mM ABTS•+ was prepared in 100 mM PBS (pH 7.4) with 2.45 mM potassium persulfate in the dark at ambient temperature for 16 h and diluted to an absorbance of 0.70 ± 0.05 at 734 nm with PBS. A mixture of 980 µL of ABTS•+ solution and 20 µL of samples was left to stand in the dark at room temperature for 6 min after thorough mixing. The absorbance at 734 nm was recorded to calculate the TEAC values. The TEAC results were expressed as Trolox equivalents (mg Trolox/g dried extract).
Evaluation of superoxide anion (O2•-)-scavenging activity The determination of O2•− scavenging activity of purple yam extracts was referred from the method of Ewing and Janero (1995), and adapted for a 96-well plate. In brief, 25 µL of sample, 60 µL of 300 µM NBT and 60 µL of 936 µM NADH·2Na were added the wells and mixed well, followed by 60 µL of 30 µM freshly prepared phenazine methosulfate. After incubation in the dark for 15 min, the absorbance at 540 nm was recorded for the calculation of O2•− scavenging activity which was expressed as IC50.
Evaluation of DPPH-radical-scavenging capacity The DPPH radical-scavenging capacity was determined by a microplate method adapted from Brand-Williams et al. (1995). In brief, 180 µL of 0.2 mM DPPH methanolic solution and 20 µL of sample were mixed in a 96-well plate and allowed to stand in the dark at room temperature for 15 min. The absorbance at 540 nm was measured, and the scavenging capacity of purple yam extracts was expressed as IC50.
Chelating activity on metal ions According to a slightly modified method of Dinis et al. (1994), the chelating activity assay of purple yam extracts was conducted on a 96-well plate. Diluted extracts (100 µL) were mixed with 50 µL of 1 mM FeCl2 for 5 min followed by adding 100 µL of ferrozine (1 mM). The mixture was left to stand for another 10 min. Subsequently, 750 µL of methanol was added and the absorbance at 562 nm was determined spectrophotometrically. The results were expressed as chelating percentage (%).
In vitro glycation of proteins The BSA-glucose/fructose reaction was performed according to the method of Duraisamy et al. (2003) The reaction mixture contained 10 mg/mL BSA, 0.5 M glucose or 0.2 M fructose, 10 – 50 µg/mL F-EA or P-EA and 3 mM sodium azide in 0.1 M sodium phosphate buffer (pH 7.4), and incubated at 37°C for 7, 14, and 21 d. Another reaction system, adapted from Lunceford and Gugliucci (2005), containing 1 mg/mL BSA and 5 mM MGO, was used to evaluate protein glycation induced by MGO. The mixture was prepared in 0.1 M sodium phosphate buffer (pH 7.4) with 3 mM sodium azide and 25 – 150 µg/mL F-EA or P-EA, and incubated at 37°C for 3 d. AG (1 mM) was used as the positive control for both BSA-glucose/fructose and BSA-MGO systems.
MGO -trapping activity of purple yam extracts A reaction system containing 1.25 mM MGO in 0.1 M sodium phosphate buffer (pH 7.4) with 3 mM sodium azide and 25 – 150 µg/mL F-EA or P-EA was applied for the detection of MGO trapping activity. After incubation at 37°C for 3 h, each sample was subjected to α-dicarbonyl compound analysis. As an MGO -trapping agent, AG (150 µg/mL) was used as a positive control.
Spectrophotometric analysis of Amadori products, α-dicarbonyl compounds, and AGEs Amadori products were detected using fructosamine assay (Baker et al., 1994). In brief, 30 µL of glycated material and 100 µL of NBT reagent (0.3 mM) in sodium carbonate buffer (100 mM, pH 10.35) were incubated at room temperature in the dark for 15 min, and the absorbance was read at 530 nm against a blank.
Girard-T assay for detecting α-dicarbonyl compounds was described by Wells-Knecht et al. (1995) In brief, 10 µL of glycated materials was incubated with 10 µL of Girard-T stock solution (500 mM) and 85 µL of sodium formate (500 mM, pH 2.9) at room temperature for 1 h. The absorbance at 290 nm was recorded against a blank.
The detection of total AGEs can be generally followed by measuring their characteristic fluorescence using the excitation and emission maxima of 370 and 440 nm, respectively.
Cell culture HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution, and grown at 37°C in the presence of 5% CO2. HepG2 cells were plated into 96-well or six-well plates at a density of 1 × 105 cells/mL. After 24 h, 1 mM MGO with or without extracts was incubated with HepG2 cells for 24 h.
Measurement of ROS by DCFH assay After different treatments, cells were loaded with membrane-permeable, non-fluorescent probe DCFH-DA (5 mM) for 40 min at 37°C in HBSS in the dark. The cells were washed twice with HBSS, and the fluorescence intensity was detected by a microplate reader with excitation and emission wavelengths of 488 and 525 nm, respectively. The fluorescent intensity of the oxidized dichlorofluorescein (DCF) corresponded to the amount of ROS.
Measurement of GSH, SOD, CAT, and TAOC in HepG2 cells Cells were harvested using cell scrapers with PBS buffer, washed once with PBS, and centrifuged for 10 min at 200 g. The supernatant was discarded, and the cell pellets were resuspended with PBS. Homogenates of ultrasonically disrupted cells were obtained to measure the GSH content, TAOC level, and activities of SOD and CAT in HepG2 cells using commercially available kits (Nanjing, Jiangsu, China). A unit activity of SOD is defined as 50% inhibition of the formation of hydroxylamine from O2• radicals per mg protein, and 1 mg protein break down 1 µmol H2O2 per second is defined as a unit activity of CAT. The results were expressed as U/mg protein.
Statistical analysis Statistical significance was analyzed by one-way ANOVA using Origin version 8.5 or SAS system for Windows V8. A p value less than 0.05 was considered to be statistically significant. The results were expressed as the mean ± standard deviation (SD) of at least three individual experiments, and each run was performed in triplicate.
Total polyphenol content (TPC) and antioxidant capacities of purple yam extracts TPCs in different fractions of purple yam extracts are summarized in Table 1. The results show that both peel and flesh extracts contained phenolic compounds in the following order: EA > CE > BT > HX > RW. The EA fraction contained far more polyphenols compared with the other fractions, with polyphenol contents of 479.5 ± 33.1 and 695.1 ± 35.1 mg/g for flesh and peel, respectively. Peels contained more polyphenols than flesh, indicating that peel might possess more bioactive properties than flesh.
| TPCb | FRAP valuec | TEAC valued | O2•−-scavenging activity (IC 50)e | DPPH-scavenging activity (IC 50)e | Chelating activityf | |
|---|---|---|---|---|---|---|
| F-CE | 48.3 ± 4.1 e | 86.5 ± 1.6 gf | 108.1 ± 2.8 f | 457.5 ± 20.8 c | 183.4 ± 5.3 c | 24.3 ± 1.6 e |
| F-HX | 16.5 ± 1.2 ef | 42.3 ± 1.2 gfh | 39.8 ± 1.6 h | 1387.5 ± 110.3 a | 632.0 ± 26.9 b | 32.0 ± 2.2 c |
| F-EA | 479.5 ± 33.1 b | 630.1 ± 19.4 b | 1326.1 ± 17.3 b | 78.2 ± 1.2 g | 13.9 ± 1.1 d | 15.1 ± 1.1 g |
| F-BT | 46.2 ± 2.8 e | 83.9 ± 5.3 gf | 111.8 ± 5.5 f | 333.2 ± 10.6 d | 245.1 ± 32.1 c | 0.0 ± 0.1 i |
| F-RW | 7.4 ± 0.5 f | 8.5 ± 0.5 h | 12.2 ± 0.3 i | 874.5 ± 18.2 b | 1532.7 ± 123.a | 50.4 ± 5.1 a |
| P-CE | 194.8 ± 14.6 c | 144.5 ± 8.5 de | 357.7 ± 8.4 d | 162.2 ± 9.8 f | 47.7 ± 2.7 d | 21.4 ± 0.2 f |
| P-HX | 149.3 ± 13.7 d | 97.3 ± 11.2 ef | 242.3 ± 14.4 e | 237.9 ± 12.2 e | 55.0 ± 0.9 d | 34.1 ± 1.2 b |
| P-EA | 695.1 ± 35.1 a | 534.4 ± 35.3 c | 1578.1 ± 15.5 a | 30.3 ± 0.3 gh | 8.5 ± 0.2 d | 8.5 ± 1.0 h |
| P-BT | 192.4 ± 6.3 c | 162.1 ± 3.0 d | 448.4 ± 9.0 c | 147.9 ± 4.1 f | 43.8 ± 3.3 d | 34.3 ± 3.2 b |
| P-RW | 38.5 ± 2.5 ef | 29.1 ± 1.7 gh | 74.0 ± 2.9 g | 461.1 ± 14.1 c | 225.1 ± 12.9 c | 29.1 ± 0.6 d |
| Troloxg | 2294.3 ± 104.4 a | 5.2 ± 0.1 h | 9.0 ± 0.5 d |
F: flesh; P: peel; CE: crude extracts; HX: n-hexane fraction; EA: ethyl acetate fraction; BT: n-butanol fraction; RW: remaining water fraction
The antioxidant activity of purple yam extracts was evaluated using FRAP, TEAC assays, O2•− and DPPH-scavenging activities, as well as metal chelating assay. Different fractions of flesh and peel extracts shared the same order in TPC, FRAP and TEAC assays. As shown in Table 1, F-EA and P-EA possessed considerably higher FRAP values of 534.4 ± 35.3 and 630.1 ± 19.4, respectively, compared with other fractions, whereas lower than the positive control Trolox. The TEAC values for F-EA and P-EA were 1326.1 ± 17.3 and 1578.1 ± 15.5 mg Trolox equivalent/g dry extract, respectively, indicating that they possessed higher antioxidant activity than Trolox itself in the TEAC assay. Similar to the above assays, F-EA and P-EA showed high potencies on scavenging free radical O2•− and DPPH with IC50 of 78.2 ± 1.2 and 13.9 ± 1.1 for F-EA, respectively, and 30.3 ± 0.3 and 8.5 ± 0.2 for P-EA, respectively. However, a completely different order was observed among the fractions in the metal chelating assay (Table 1). The inhibition rates for F-EA and P-EA were only 15.1 ± 1.1 and 8.5 ± 1.0 at the concentration of 0.5 mg/mL, ranking the second least and least chelating activity among the fractions. Apart from metal chelating activity, all the fractions from peel exerted better antioxidant effect compared with the corresponding fractions in flesh extracts, and almost all of them exhibited a significant difference (p < 0.05).
Antiglycation capacities of purple yam extracts Considering the high antioxidant activity, F-EA and P-EA was chosen to conduct the antiglycation assay. As a product of early stage glycation, Amadori products were detected using fructosamine assay. As shown in Fig. 1, group A represented the Glu/BSA system, and group B represented the Fru/BSA system. A significant inhibition of Amadori products was observed in the F-EA- and P-EA-treated groups in a dose dependent manner, and P-EA exerted significantly better effect than F-EA at higher concentrations (p < 0.05). However, no detectable inhibition was presented in the AG treated group (Figs. 1A-1 and 1B-1). AG is an accepted antiglycation agent that can trap intermediates, such as MGO during the glycation period (Brownlee et al., 1986). However, it is unable to interfere the formation of Amadori products, which is consistent with previous reports (Ardestani and Yazdanparast, 2007).
Inhibitory effects of F-EA and P-EA on the glycation cascade. Results are means ± SD (n = 3). (A), Glu/BSA system; (B), Fru/BSA system. (A-1) and (B-1), Formation of Amadori products: samples were incubated with the Glu/BSA system for 7d and the Fru/BSA system for 3d; (A-2) and (B-2), formation of α-dicarbonyl compounds: samples were incubated with the Glu/BSA and Fru/BSA systems for 14d; (A-3) and (B-3), formation of AGEs: samples were incubated with the Glu/BSA and Fru/BSA systems for 7, 14 and 21d. # p < 0.05 vs. model; * p < 0.05.
Amadori products subsequently degrade into various highly reactive α-dicarbonyl compounds. Our study showed that both F-EA and P-EA could inhibit the formation of dicarbonyl compounds in a dose-dependent manner, and the inhibitory effects were much stronger than the positive control AG at the same concentration of 50 µg/mL (Figs. 1A-2 and 1B-2). Besides, all concentrations of P-EA, except 10 µg/mL in Glu/BSA system, possessed significantly better effect on inhibiting dicarbonyl compounds than F-EA with the same concentrations (p < 0.05).
The fluorescence intensity of AGEs in both Glu/BSA and Fru/BSA systems increased sharply within 21 d of incubation (Figs. 1A-3 and 1B-3). As a reducing sugar, fructose possesses much higher glycation ability, for it has 300 times as many highly reactive chain structures as glucose (Bunn and Higgins, 1981). In the present study, the fluorescence intensity of the Fru/BSA system at 7 d increased by almost seven fold compared with that of Glu/BSA system. This property of fructose also explained why fructose took much fewer time to form Amadori products than glucose. As shown in Figs. 1A-3 and 1B-3, both extracts exhibited significant inhibitory effects on AGE formation in a dose-dependent manner, and also, peel extracts exerted significantly better effect than the flesh at all concentrations during the experiment period (p < 0.05) except 10 µg/mL on day 14 in the Fru/BSA system. The positive control AG exerted much less effectiveness than both F-EA and P-EA at the same concentrations.
We also measured the glycation of proteins by MGO, and explored the effects of purple yam extracts on the inhibition of MGO-mediated protein glycation. As shown in Fig. 2, both extracts presented excellent antiglycation capacities, and their effects were markedly better than the positive control AG at the same concentration. However, when incubated with MGO alone, those extracts possessed poor MGO -trapping activity compared with AG, which is a recognized MGO -trapping agent (Fig. 3). 150 µg/mL AG was able to trap 94% of MGO within 3 h, but for extracts, the maximal trapping rate was only 34% in 150 µg/mL P-EA group. Similar to the above assays, P-EA inhibited MGO-mediated protein glycation and trapped MGO with more potency compared with F-EA, and exerted a significant difference (p < 0.05).
Inhibitory effects of F-EA and P-EA on AGEs formation in the MGO/BSA system. Samples with various concentrations were incubated with the MGO/BSA system containing 1 mg/mL BSA and 5 mM MGO for 3 d. Results are means ± SD (n = 3). * p < 0.05.
MGO -trapping activities of purple yam extracts. Samples with various concentrations were incubated with 1.25 mM MGO for 3 h. Results are means ± SD (n = 3). * p < 0.05.
Antioxidative abilities of purple yam extracts in MGO-treated HepG2 cell model The concentration of MGO and purple yam extracts used in this part are all non-cytotoxicity doses. Compared with the control, incubation of HepG2 cells with 1 mM MGO for 24 h caused a significant increase in ROS production, as indicated by oxidized DCF (p < 0.05) (Fig. 4). ROS production was significantly attenuated by co-incubation with both positive controls AG and NAC (5 mM) (p < 0.05). Purple yam extracts could also effectively mitigate MGO-induced ROS production in a dose-dependent manner.
Measurement of cellular ROS generation by DCFH assay. HepG2 cells were treated with 1 mM MGO with various concentrations of samples for 24 h. AG and NAC were used as positive controls, and their concentrations were expressed as mM. Concentrations of F-EA and P-EA were expressed as µg/mL. Results are means ± SD (n = 6). #p < 0.05 vs. control; *p < 0.05 vs. model.
The cells have an effective defense system including endogenous antioxidants and antioxidant enzymes to protect and neutralize free radical-induced damage. Interestingly, 1 mM MGO caused a significant increase in GSH compared with the control (p < 0.05) (Fig. 5A). GSH level was further increased after co-incubation with extracts in a dose dependent manner. The positive control AG, which was characterized by MGO-trapping activity, showed no significant elevation in GSH levels compared with the control. As a non-specific antioxidant and GSH precursor, NAC exerted excellent ability in increasing GSH to levels significantly higher than that in the model group (p < 0.05).
Measurement of cellular antioxidant defense system in HepG2 cells. (A), GSH contents; (B), CAT activity; (C), SOD activity; (D) TAOC level. HepG2 cells were treated with 1 mM MGO with various concentrations of samples for 24 h. AG and NAC were used as positive controls, and their concentrations were expressed as mM. Concentrations of F-EA and P-EA were expressed as µg/mL. Results are means ± SD (n = 6). #p < 0.05 vs. control; *p < 0.05 vs. model.
A significant decrease in the activities of antioxidant enzymes CAT (Fig. 5B), SOD (Fig. 5C), and TAOC (Fig. 5D) were observed in the 1 mM MGO-treated group (p < 0.05). However, this was notably ameliorated when co-incubated with samples and positive controls. The cells treated with MGO and F-EA or P-EA dose-dependently enhanced the activities of SOD and CAT, as well as increased TAOC levels in HepG2 cells. It should be noted that P-EA exerted similar effect with F-EA in the above cell experiments at only one half of the concentrations of F-EA, further demonstrated the better bioactivity of purple yam peel than purple yam flesh.
Chinese purple yam was proved to possess excellent antioxidant activity, but poor metal ion chelating ability. AGEs inhibitors act as quenchers of dicarbonyl intermediates, metal ion chelators or antioxidants. Inhibiting the glycation process only by sequestering all free redox metal ions in vivo by chelation is not practical or desirable, so targeting dicarbonyl intermediates quenchers and antioxidants might serve as more practical and effective strategies for inhibiting AGE formation. The present study also demonstrated that P-EA and F-EA possess poor MGO-trapping activity compared with AG, which is a recognized MGO-trapping agent, indicating that the excellent antiglycative activity of purple yam extracts might due to their antioxidant activity. A previous study revealed that no oxidation reaction is involved in the early stage of glycation (formation of Amadori products), but oxidative reactions play a role in the free-radical-mediated conversion of Amadori products to AGEs (Fu et al., 1994). Thus we assumed that the antioxidant activities of purple yam extracts were not the unique mechanism for the inhibition of glycation because they could inhibit both oxidation and non-oxidation periods of glycation. The mechanisms need to be further investigated.
Endogenous and exogenous carbonyls and dicarbonyls are involved in numerous pathogenic processes in vivo, including enhancing inflammatory responses, oxidative stress, and inducing AGE formation (Neade and Uribarri, 2008). Therefore, MGO should act as a target for reducing the undesirable consequences of protein glycation. The present study demonstrated that MGO, the most reactive intermediate during the glycation cascade, could induce oxidative stress in HepG2 cells. The physiological concentrations of plasma MG were reported to be approximately 5 µM in rats (Nagaraj et al., 2002) and 3.3 µM in healthy humans, but were elevated by two- to fourfold in type 2 diabetic patients (Wang et al., 2007). Despite the low physical concentration of MGO, under experimental conditions, considerably higher concentrations have to be utilized to achieve certain effects than those in vivo as a compensation for the short reaction time. Fiory et al. (2011) used 1 mM MGO to investigate the ability of MGO to affect insulin signaling and cell functions in the INS-1E beta cell line. Li et al. (2013) evaluated MGO-induced injury in cultured human brain microvascular endothelial cells with a concentration of 2 mM. In addition, up to 10 mM MGO has been used to investigate its effect on insulin-secreting cells and insulin signaling pathways in rat L6 myoblasts (Riboulet-Chavey et al., 2006). Thus, in this study, 1 mM MGO with no cytotoxicity was used to induce oxidative stress in HepG2 cells.
The body has an effective defense system including endogenous antioxidants and antioxidant enzymes to protect and neutralize free radical-induced damage. However, these multifunctional protective systems may not be physically sufficient to counteract the deleterious effects of ROS or pro-oxidants. Thus, antioxidants that can increase GSH levels and the activities of antioxidant enzymes could serve as therapeutic agents to cope with ROS overproduction. In the present study, 1 mM MGO caused significant increase in GSH compared with the control. We supposed that HepG2 cells produced more GSH to eliminate elevated MGO levels, since GSH plays a central role in the degradation of MGO by binding MGO and making it available to the glyoxalase enzymes (Wang et al., 2007). Another speculation is that pro-oxidative circumstances stimulated GSH increase, for GSH is a crucial antioxidant within cells. Žegura et al. (2006) also demonstrated that cellular defense against ROS-midiated DNA damage was strengthened in HepG2 cells by increasing intracellular GSH level. On the other hand, the GSH level was further increased after co-incubated with the excellent antioxidants P-EA and F-EA. However, the positive control, AG, eliminated MGO-stimulated GSH increase probably by trapping MGO promptly.
SOD and CAT are major antioxidant enzymes that comprise primary defense system against oxidative stress (Inal et al., 2001). SOD catalyzes the dismutation of superoxide radical into O2 and H2O2, and CAT catalyzes the decomposition of harmful H2O2 to water and O2. TAOC reflects the non-enzymatic antioxidative capacity against various reactive oxygen and nitrogen radicals. In addition, low TAOC levels indicate oxidative stress or increased susceptibility to oxidative damage (Young, 2001). Purple yam extracts efficiently reversed the decrease in activities of SOD and CAT as well as TAOC level in HepG2 cells induced by MGO, further proved that purple yam could strengthen the antioxidant defense system within cells through their antioxidant activities.
Phenolic compounds have been reported to possess strong antioxidant abilities, and such antioxidant effects might contribute to the inhibition of protein modifications in the glycation process (Wu and Yen, 2005). The main active phenolic acids in purple yam were demonstrated to be sinapic acid and ferulic acid (Fang et al., 2011). Wu et al. (2010) showed that methoxylated phenolic acids including sinapic acid and ferulic acid, have a significantly greater inhibitory effect against ROS and AGE formation among the 12 phenolic acids tested. Thus we assumed that sinapic acid and ferulic acid possibly contributed to the excellent antioxidant and antiglycation activities of Chinese purple yam. Notably, peel extracts possessed better antioxidative and antiglycative activities than flesh extracts, probably because of the higher contents of bioactive components or their synergistic interactions in purple yam peel. The potential mechanism needs to be further investigated.
This study indicated that the EA fraction of Chinese purple yam flesh and peel extracts rich in polyphenols showed both antioxidant and antiglycation behaviors. They could efficiently suppress different periods of glycation cascade in three different glycation systems, which were mainly due to their antioxidant activities. We also demonstrated that MGO, a highly reactive intermediate and precursor of AGEs, could induce oxidative damage in HepG2 cells for the first time, and purple yam extracts cope with MGO-induced oxidative stress by strengthening the antioxidant defense system. Notably, yam peel, a kitchen garbage or by-product of the yam processing industry, possessed better bioactivities than flesh, and showed potential value for exploitation and utilization as a functional food or added ingredient.
Acknowledgments This work is financially supported by the National Key Technology R&D Program in the Twelfth Five-year Guideline of China (Project 2011BAD08B03-01).
