茶果皮多糖理化性质、体外抗氧化活性及对α-葡萄糖苷酶的抑制作用

In vitro antioxidant activity and potential inhibitory action against α-glucosidase of polysaccharides from fruit peel of tea ( Camellia sinensis L.) # # 茶果皮多糖理化性质、体外抗氧化活性及对α-葡萄糖苷酶的抑制作用 1 2 first-author 1 1 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zdxp@zju.edu.cn †E-mail:zdxp@zju.edu.cn zdxp@zju.edu.cn 3 1 1 Department of Tea Science, Zhejiang University, Hangzhou 310058, China Department of Tea Science, Zhejiang University, Hangzhou 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 310053, China College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 310053, China The conditions for extracting polysaccharides from tea ( Camellia sinensis L.) fruit peel (TFPPs) were studied. Three parameters (temperature, time, and liquid/solid ratio) affecting the extraction of TFPP were optimized using response surface methodology (RSM). Under the optimized conditions, the yield of TFPP was predicted to be 4.98%. The physicochemical properties, in vitro antioxidant activities, and inhibitory effects on α-glucosidase of fractionated TFPPs (TFPP-0, TFPP-20, TFPP-40, and TFPP-60) were investigated. We found that the TFPPs were all acid protein-bound heteropolysaccharides, although with different chemical compositions. They had not only remarkable scavenging activity on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and reducing activity, but also excellent inhibitory potential against α-glucosidase in vitro . Our results suggest that tea fruit peel could be treated as a potential bioresource for the development of polysaccharide antioxidants. 研究目的 2 利用响应面优化茶果皮多糖（TFPP）提取条件，用乙醇分级分段得到4个多糖组分（TFPP-0、TFPP-20、TFPP-40和TFPP-60），并研究其理化性质、抗氧化活性和对α-葡萄糖苷酶抑制作用，为综合高效利用茶果皮多糖资源提供理论基础。 创新要点 2 1. 首次将茶果皮作为一种潜在生物资源研究；2. 首次研究茶果皮多糖这一功能成分；3. 将工艺优化、理化性质和生物活性结合研究。 研究方法 2 三因素三水平响应面设计（见表1），傅里叶转换红外光谱法分析茶果皮粗多糖的功能团结构（见图3），高效液相色谱法检测单糖组分（见表2），2,2′-氨基-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐（ABTS）自由基清除法（见图 4a）和铁离子还原能力法（FRAP）（见图4b）分析茶果皮多糖抗氧化活性。 重要结论 2 1. 茶果皮多糖是一种水溶性的酸性杂多糖蛋白复合物；2. 乙醇分级是一种有效多糖分离手段；3. 茶果皮多糖具有出色的生物活性；4. 茶果皮资源可以作为一种可再生生物资源进行深度的开发。 Polysaccharides Tea ( Camellia sinensis L.) fruit peel Physicochemical properties Antioxidant activity α-Glucosidase inhibition 茶果皮 多糖 响应面 抗氧化 α-葡萄糖苷酶抑制 Introduction 1 Free radicals are not only closely associated with lipid peroxidation, but also involved in the development of a variety of diseases, including cellular aging, mutagenesis, carcinogenesis, coronary heart disease, diabetes, and neurodegeneration (Moskovitz et al., 2002 2002 ). Antioxidants, which can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction, would therefore seem to be very important in the prevention of these diseases (Praticò and Delanty, 2000 2000 ). However, although synthetic antioxidants are widely used, their side effects are of the public concern (Grice, 1988 1988 ). Thus, there has been increasing interest in finding natural, effective, and safe antioxidants, since they may protect the human body from free radicals and retard the progress of many chronic diseases. A review of the literature shows that polysaccharides derived from plants, such as Pteridium aquilinum (Xu et al., 2009 2009 ), Ficus carica L. (Yang et al., 2009 2009 ), Lycium barbarum L. (Lin et al., 2009 2009 ), and Portulaca oleracea L. (Chen et al., 2009 2009 ) have remarkable antioxidant activity. Meanwhile, the hypoglycemic effect of polysaccharides from plants, particularly the inhibition of postprandial hyperglycemia, has been highlighted recently (Zhu et al., 2013 2013 ). Furthermore, their potential inhibitory action against α-glucosidase has been proposed as one of the main mechanisms responsible for this hypoglycemic effect (Wang et al., 2012 2012 ). Tea ( Camellia sinensis L.) fruit peel is the main byproduct of the production of tea seed oil, which has long been recognized as an edible oil of high quality. As a result of the increasing popularity of tea seed oil, the yield of tea fruit peel has also increased. However, most tea fruit peel is discarded as industrial waste, resulting in loss of a valuable resource and environmental pollution. Nowadays, recovery of bioactive compounds, including polysaccharides, from food processing wastes is attracting increasing attention as a potentially cheap and reliable source of new and efficient natural antioxidants (Xu et al., 2012 2012 ; Çam and Aaby, 2010 2010 ). The extraction and bioactivities of polysaccharides from the seeds of Plantago asiatica L. (Ye et al., 2011 2011 ), the fruit shell of Camellia oleifera Abel (Shen et al., 2010 2010 ), and the seabuckthorn pericarp (Ma et al., 2011 2011 ) have been reported. However, to the best of our knowledge, little information is available about the polysaccharides from tea fruit peel TFPPs). The objectives of this study were to optimize the conditions for extracting polysaccharides from tea fruit peel by response surface methodology (RSM), and to fractionate the polysaccharides using graded ethanol precipitation. The physicochemical properties and in vitro activities, including the antioxidant activity and inhibitory potential against α-glucosidase, of the fractionated polysaccharides were also investigated. Materials and methods 1 Materials and experimental design 2 Tea ( Camellia sinensis L.) fruit was collected from the Panban tea garden (Zhejiang, China) in December, 2012. The ground fruit peel was treated with ethanol, followed by water extraction. A central composite design was applied and RSM was used to determine the optimum conditions for water extraction of total polysaccharide from ground fruit peel. After extraction with ethanol, centrifugation, and dialysis to give crude polysaccharides, the extract was further fractionated by ethanol at concentrations of 0%, 20%, 40%, and 60%. Composition analysis 2 Neutral sugar content was measured by the anthrone-sulfuric acid method (Morris, 1948 1948 ) using D-glucose as a standard reference. High-performance liquid chromatography (HPLC) equipment and Fourier transform infrared spectroscopy (FTIR) were used to identify the chemical compositions. Antioxidant capacity and α-glucosidase inhibitory activity 2 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay was carried out according to the method of Cai et al. ( 2004 2004 ). The ferric-reducing antioxidant power (FRAP) assay was performed according to the modified method of Benzie and Strain ( 1999 1999 ). The α-glucosidase inhibitory activity of the tea fruit peel extracts was determined according to the method described by Apostolidis and Lee ( 2010 2010 ) with slight modification. Detailed materials and methods are described in Data S1 S1 . Results and discussion 1 RSM was used to evaluate the effects of multiple factors and their interactions on one or more response variables, and to find the optimum combination of factor levels (Ballard et al., 2009 2009 ). The second-order polynomial model was expressed by the following quadratic equation: $$Y = 4.65 + 0.39{X_1} + 0.26{X_2} + 0.053{X_3} - 0.019{X_1}{X_2} + 0.030{X_1}{X_3} - 0.038{X_2}{X_3} - 0.63X_1^2 - 0.13X_2^2 + 0.12X_3^2$$ , where Y is yield, X 1 is extraction temperature, X 2 is extraction time, and X 3 is liquid/solid ratio. Analysis of variance (ANOVA) analysis of the quadratic regression models for TFPP yield showed that the model was highly significant ( P < 0.01) with an F -value of 11.05 (Table 1 1 ). The P -value of 0.891 for “lack of fit” implied that it was not significantly relevant to the pure error (Jiang et al., 2011 2011 ). The determination coefficient ( R 2 ) and adjusted coefficient ( $$R_{\text{a}}^2$$ ) were 0.9087 and 0.8265, respectively, indicating the model equation was adequate for predicting the extraction yield of TFPP within the range of experimental variables. The significance of the regression coefficients was examined by F -test. Accordingly, the extraction temperature ( X 1 ), extraction time ( X 2 ), and a quadratic term of extraction temperature ( $$X_1^2$$ ) were significant ( P < 0.01), while other variables were not ( P > 0.05). P < 0.05 SS: sum of square; DF: degree of freedom; MS: mean square; X 1 : extraction temperature; X 2 : extraction time; X 3 : liquid/solid ratio Source SS DF MS F -value Pr > F Model 4.370 9 0.490 11.050 0.0004 a a X 1 1.520 1 1.520 34.700 0.0002 a a X 2 0.670 1 0.670 15.260 0.0029 a a X 3 0.028 1 0.028 0.640 0.4421 X 1 X 2 2.846×10 −3 1 2.846×10 −3 0.065 0.8041 X 1 X 3 7.268×10 −3 1 7.268×10 −3 0.170 0.6926 X 2 X 3 0.012 1 0.012 0.270 0.6148 $$X_1^2$$ 1.110 1 1.110 25.270 0.0005 a a $$X_2^2$$ 0.050 1 0.050 1.130 0.3122 $$X_3^2$$ 0.042 1 0.042 0.960 0.3497 Residual 0.440 10 0.044 Lack of fit 0.100 5 0.021 0.310 0.8901 Pure error 0.340 5 0.067 Cor total 4.800 19 Fitted response surface plots for the model were established, with the shape of the contour plots indicating the significance of interactions between variables. A circular contour plot suggests that the interaction between the related variables is negligible, while an elliptical contour plot indicates that interaction is significant (Muralidhar et al., 2001 2001 ). The response surface plots and contour plots (Fig. 1 1 ) were generated using Design-Expert, and describe the interaction between extraction time and temperature on TFPP yield under a constant liquid/solid ratio of 20 (v/w). Solid-liquid extraction is a mass transport phenomenon in which solid contained in a matrix migrates into a solvent brought into contact with the matrix (Shi et al., 2005 2005 ). Corrales et al. ( 2009 2009 ) hypothesized that the extraction temperature affects the mass transport phenomenon by inducing changes in diffusion coefficients. In this study, both the extraction time and extraction temperature had a significant impact on yields. A slight increase in the TFPP yield was observed with a longer extraction time. The peak yield of TFPP was reached around 75 °C, and higher or lower temperatures led to an inferior output. Liquid/solid ratio was set to 20 (v/w) Based on the model, the optimum extraction conditions for the maximum predicted yield of TFPPs (4.98%) were a liquid/solid ratio of 20 (v/w), an extraction time of 90 min, and an extraction temperature of 70 °C. The yield of polysaccharides extracted from the fruit peel was considerably higher than that extracted from seeds of Plantago asiatica L. (2.47%) (Ye et al., 2011 2011 ) and lower than that extracted from seeds of Arachis hypogaea (8.28%) (Shen et al., 2010 2010 ), indicating that tea fruit peel could be used as a resource for development of polysaccharide-enriched extracts. The crude TFPP was further fractionated using ethanol at concentrations of 0%, 20%, 40%, and 60%. The chemical compositions of the fractionated TFPPs are presented in Table 2 2 . Notably, TFPP-Crude contained the most protein (14.25%) and uronic acid (68.96%). TFPP-60 had the highest content of neutral sugar (23.00%), but the lowest content of uronic acid (46.42%). Monosaccharide composition analyzed by HPLC (Fig. 2 2 ) revealed that all the fractions contained seven monosaccharides (rhamnose, mannose, glucose, galactose, arabinose, xylose, and fucose) with different molar ratios. The TFPPs were a kind of seromucoid multi-mixture composed of multiple components (Table 2 2 and Fig. 2 2 ), which may create differences in primary or even high level structure, and affect their bioactivities (Srivastava and Kulshreshtha, 1989 1989 ). a–e Means (±SD) with the same letter are not significantly different ( P > 0.05). The yield of TFPP is expressed as dried weight percent; the compositions of protein, neutral sugar, and uronic acid are expressed as weight percent; and the others are expressed as mole percent Sample 2 Yield (%) 2 Protein (%) 2 Neutral sugar (%) Uronic acid (%) TFPP-Crude 2 1.07±0.21 e 2 14.25±0.11 a 2 15.69±0.52 b 68.96±2.25 a TFPP-0 2 69.16±0.35 a 2 13.27±0.09 b 2 12.07±0.49 c 65.52±1.12 b TFPP-20 2 4.00±0.02 d 2 3.62±0.11 e 2 11.74±2.79 c 56.31±0.34 c TFPP-40 2 14.80±0.19 b 2 6.29±0.06 c 2 13.65±0.34 bc 51.72±2.08 d TFPP-60 2 12.04±0.04 c 2 5.68±0.02 d 2 23.00±0.12 a 46.42±1.09 e 8 8 Sample Rhamnose (%) Mannose (%) Glucose (%) Galactose (%) Arabinose (%) Xylose (%) Fucose (%) 8 TFPP-Crude 1.03 0.66 0.54 2.25 1.63 0.19 3.70 TFPP-0 1.17 0.84 0.77 1.72 1.15 0.22 4.13 TFPP-20 0.12 0.38 0.09 1.51 0.91 0.54 6.45 TFPP-40 0.07 1.03 0.36 3.21 1.91 0.24 3.19 TFPP-60 0.27 0.84 0.96 2.86 3.07 0.44 1.56 (a) Standard mixture; (b) TFPP-Crude; (c) TFPP-0; (d) TFPP-20; (e) TFPP-40; (f) TFPP-60. Peak 1: rhamnose; Peak 2: mannose; Peak 3: glucose; Peak 4: galactose; Peak 5: arabinose; Peak 6: xylose; Peak 7: fucose FTIR is typically used for the qualitative measurement of organic functional groups, especially O–H, N–H, and C=O (Qian et al., 2009 2009 ). All four polysaccharides demonstrated absorption near 3400 and 3930 cm −1 , the stretching vibration regions of O–H and saturated C–H, respectively (Fig. 3 3 ). The prominent absorption bands of the four polysaccharides in the range 1000–1200 cm −1 suggested that they all had a pyranose ring (Ding et al., 2010 2010 ). A stretching peak appeared at around 1620 cm −1 with a weak peak at around 1418 cm −1 , suggesting the presence of carboxyl groups. A small absorption peak at about 890 cm −1 could be associated with β-glycoside linkages between the sugar units. This may suggest that the TFPPs belong to β-type heteropolysaccharides with a pyran group. However, a difference that appeared at nearly 1500 cm −1 (Figs. 3a and 3b 3a and 3b ) was the absorption of the stretching vibration of C=O, indicating the existence of uronic acid, which strengthened our conclusion from the results in Table 2 2 that TFPP-Crude and TFPP-0 contained the most uronic acid. The ABTS assay is often used in evaluating the total antioxidant ability of single compounds or complex mixtures from various plants, where an electron transfer process is involved (Huang et al., 2008 2008 ). In the present study, the scavenging ability of the TFPPs on ABTS free radicals was determined (Fig. 4a 4a ). The scavenging ability of the TFPPs correlated positively with their increasing concentrations, but was significantly weaker than that of butylated hydroxytoluene (BHT). At a concentration of 1000 μg/ml, the order of scavenging abilities of all tested samples was: TFPP-Crude (90.72%) > BHT (87.55%) > TFPP-0 (77.68%) > TFPP-60 (53.81%) > TFPP-40 (44.27%) > TFPP-20 (35.67%). The results suggest that the TFPPs had a strong scavenging activity on ABTS. Also, as the ABTS model can be used to assess the scavenging activity for both polar and non-polar samples (Kaviarasan et al., 2007 2007 ), the strong scavenging activity of TFPP-Crude might be attributable to its total content of protein, neutral sugar, and uronic acid (98.90% TFPP-Crude, 90.86% TFPP-0, 75.10% TFPP-60, 71.67% TFPP-20, and 71.66% for TFPP-40). Data are expressed as mean±SD ( n =3) FRAP measures the antioxidant effect of substances in the reaction medium by their reducing ability, which is considered as the ability of a natural antioxidant to donate electrons (Shi et al., 2009 2009 ). The metal chelating activity of polysaccharides increased with increasing concentrations used in the test (Fig. 4b 4b ). The chelating ability decreased in the order BHT > TFPP-60 > TFPP-40 > TFPP-Crude > TFPP-20 > TFPP-0 at the concentration of 1000 μg/ml. The results suggest that the TFPPs had a strong reducing ability on FRAP, and that the strong reducing ability of TFPP-60 might be attributable to its high content of neutral sugar. It has been recognized that metal chelating ability could be involved in antioxidant activity and may affect other functions that contribute to antioxidant activity. The antioxidant activity of plant polysaccharides may be influenced by lots of factors, such as their structure, molecular weight, and chemical composition (Chen et al., 2008 2008 ). For example, hydroxyl groups of monosaccharides in a polysaccharide are considered to be a significant factor in affecting free radical scavenging ability, due to their potential to donate hydrogen (Tian et al., 2011 2011 ). Many studies have revealed that oxidative stress is associated with a wide range of diseases (Finkel and Holbrook, 2000 2000 ; Rains and Jain, 2011 2011 ). Thus, the antioxidant activity of bioactive compounds can be considered as an important index by which their potential benefits for human health can be evaluated (Wang et al., 2011 2011 ). The chelating effect of TFPPs on ferrous ions may to some extent affect the other activities of scavenging free radicals to protect organisms against oxidative damage (Ye et al., 2011 2011 ). α-Glucosidase, a key enzyme for carbohydrate digestion, has been recognized as a therapeutic target for modulation of postprandial hyperglycemia, the earliest metabolic abnormality in type 2 DM (Krentz and Bailey, 2005 2005 ). Previous reports have shown that polysaccharides from a wide range of food or medical plant sources could be effective α-glucosidase inhibitors (Kwon et al., 2008 2008 ; Shibano et al., 2008 2008 ; Kim et al., 2010 2010 ). In this study, the inhibitory effects of TFPPs on α-glucosidase were investigated. All four TFPPs exhibited much stronger inhibitory effects on α-glucosidase than did acarbose at a concentration higher than 250 μg/ml (Fig. 5 5 ). The α-glucosidase inhibitory effect of TFPP-0 increased rapidly from 61.0% to 93.8% as the concentration increased from 62.5 to 500.0 μg/ml. The half maximal inhibitory concentration (IC 50 ) values of TFPP-40, TFPP-60, TFPP-20, and acarbose estimated from Fig. 5 5 were 126.9, 452.5, 483.3, and 870.2 μg/ml, respectively. This indicates that the inhibition effects of TFPPs on α-glucosidase were notably better than that of acarbose. TFPP-0 showed the strongest inhibition of α-glucosidase, with a maximum of 97%, which was mainly attributable to its high contents of protein and uronic acid (Table 2 2 ). Our results indicated that TFPPs could act as both antioxidants and α-glucosidase inhibitors, and have potential to be used in the prevention of type 2 DM. Further studies on the inhibitory effect of TFPPs on α-glucosidase in in vivo models are in progress. Data are expressed as mean±SD ( n =3) Based on the results obtained, we conclude that tea fruit peel is a potentially valuable renewable bioresource for the development of polysaccharide antioxidants. TFPPs were found to be acid protein-bound heteropolysaccharides, and not only to possess remarkable scavenging activity on ABTS and reducing activity, but also to exhibit excellent inhibitory potential against α-glucosidase in vitro . Protein, neutral sugar, and uronic acid are suggested to have at least some positive effects on the biological behavior of TFPPs. Further studies on the purified TFPPs should be carried out to help illustrate more clearly the structure/activity relationship of the polysaccharides. References E Apostolidis , C Lee . In vitro potential of Ascophyllum nodosum phenolic antioxidant-mediated α-glucosidase and α-amylase inhibition . 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