Antioxidant Activity of Polysaccharides in Yam Bulbils and Their Hypoglycemic Effect in Diabetic Mice

Xiao LIANG, Yueqin HUANG, Jianping CHEN*, Pengwei HAO

1. Chinese PLA General Hospital, Beijing 100853, China;

2. Department of Chemistry and Chemical Engineering, Huainan Normal College, Huainan 232001, China;

3. College of Science, Anhui University of Science Technology, Huainan 232001, China

Yam bulbils refer to the axillary buds of Dioscorea opposita Thunb[1]. They are commonly known as “Shanyao Dan”,which are oval or elliptical and in the diameter of 0.4-2.0 cm.The outer skin of yam bulbils is pale yellow and has fine wrinkles. In the center of tops, there are hard stem scars. Their section is gray to brown. The yam bulbils have light smell and are not bitter, but taste sticky. They mainly contain starch,polysaccharides (including mucoid substances and glycoproteins), proteins, free amino acids and other active ingredients[2]. Yam bulbils are a kind of herb with high medicinal value,and they are edible and have high medical value[3]. Yam bulbils have rich resources, and their yield can reach 3 000 -6 000 kg/hm2. Only a small proportion of yam bulbils are used for cooking and breeding, and most of them are discarded as waste. In order to further enhance the development value of yam bulbils and increase the added value of yam industry, the antioxidant activity and hypoglycemic effect of polysaccharides in yam bulbils were studied in this paper,thereby laying theoretical basis for the comprehensive development and utilization of yam bulbils.

Materials and Methods

Materials

The tested yam bulbils were purchased from Jiaozuo City, Henan Province.The ICR mice,of SPF grade and in weight of 20-25 g, were purchased from the Beijing Weitong Lihua Laboratory Animal Technology Co.,Ltd.

Methods Polysaccharides in yam bulbils

The crushed yam bulbils were soaked in hot water at 60-80 ℃for 5 h. The extract was collected and concentrated by reducing pressure to an appropriate volume.The proteins in the ex-tract were removed by the Sevag method, and then the crude polysaccharides were precipitated with ethanol.

Modeling of experimental animal and treatments A total of 80 ICR mice were selected. They were first bred in test environment for 4 d. After fasted for 12 h,the mice were injected once with 150 rag/kg of alloxan into their abdomen. They were bred for another 3 d. After a 6-h fasting, the fasting blood glucose in mice was determined. The mice with fasting blood glucose higher than 10 mmol/L were selected as diabetic mice.

The ICR male mice were divided into 5 groups, including normal control group, diabetic model group and three polysaccharides dose treatment groups. There were 10 mice in each group. In the three polysaccharides dose treatment groups, 10, 20 and 30 rag/kg of polysaccharides from yam bulbils were given to mice respectively by gavage.In the normal control group and diabetic model group, sameamount distilled water was given to the mice by gavage.The gavage was carried out once a day and lasted for 30 consecutive days. The body weights and fasting blood glucose in mice were determined at different times. The glucose tolerance in mice was determined after the test ended. After a 6-h fasting,the mice were supplied with 25 g/kg of glucose by oral administration,and then, the blood glucose contents in mice were determined 0, 1 and 2 h after the administration respectively[4].

Reducing power of polysaccharides in yam bulbils Certain amounts(2.5 ml) of sample solutions at different concentrations were added to tubes.Subsequently, a certain amount (2.5 ml) of phosphate buffer (0.2 mol/L, pH 6.6) and a certain amount (2.5 ml) of 1%potassium hexacyanoferrate solution were added to each of the tubes.After bathed in water at 50 ℃for 20 min,the tubes were cooled rapidly and added with certain amounts(2.5 ml)of 10% trichloroace-tic acid solution. After the mixing, the tubes were centrifuged at 5 000 r/min for 10 min.Subsequently,certain amounts (2.5 ml)of supernatants were removed to other new tubes. A certain amount (2.5 ml)of distilled water and a certain amount(0.5 ml)of 0.1%ferric chloride solution were added to each of the new tubes in turn. After a complete mixing, the mixtures were stood for 10 min. With distilled water as the reference solution, the absorbances of the mixtures were determined at a wavelength of 700 nm.The larger the absorbance is,the stronger the reducing power is[5].

Scavenging ability of polysaccharides in yam bulbils against DPPH·A certain amount(2 ml)of sample solution was mixed with a certain amount(2 ml)of DPPH solution(0.000 1 mol/L,dissolved in 95% ethanol). After placed in shadow at room temperature for 20 min,the mixture was centrifuged at 1 000 r/min for 10 min, and the absorbance (Di)of supernatant was determined at 517 nm. For the blank group,a certain amount(2 ml)of sample solution was mixed with a certain amount(2 ml)of 95%ethanol.The absorbance (Dj)of the mixture was determined at 517 nm. For the control group,a certain amount (2 ml)DPPH solution was mixed with a certain amount (2 ml) of distilled water, and the absorbance (Dc) of the mixture was determined at the wavelength of 517 nm. The same-volume distilled water and 95%ethanol was treated as blank for zeroing.The scavenging rate was calculated according to the following formula:

Scavenging ability of polysaccharides in yam bulbils against·OH

In the sample group, certain amounts of phosphate buffer (0.4 mol/L, 1 ml,pH 7.4), phenanthroline solution (2.5 mmol/L, 1 ml), ferrous sulfate solution(2.5 mmol/L, 1 ml), H2O2(0.02 mol/L,0.5 ml) and sample solution (1 ml)were mixed together. In the injury group, the sample solution was replaced by 1 ml of distilled water.In the blank group, the sample solution and H2O2were replaced by 1.5 ml of distilled water. The mixtures were all bathed in water at 37 ℃for 1 h, and then their absorbances at 536 nm were determined rapidly[7]. The scavenging rate of polysaccharides against·OH was calculated according to following formula:

Wherein, D0refers to the absorbance of the ·OH system added with sample solution and H2O2; D1refers to the absorbance of the·OH system added with sample solution added with sample solution but without H2O2; D2refers to the absorbance of the·OH system added with H2O2but without sample solution.

Blood glucose determination The blood in angular vein of experimental mice was collected, and for the determination of fasting blood glucose, the venous blood of mice fasted for 6 h was collected. And then the serum samples were prepared,and the blood glucose contents were determined with the glucose oxidase-peroxidase method.

Data analysis The test data were expressed as mean±standard deviation. The one-way ANOVA was conducted using Minitab 16,and the comparison among treatment groups was performed using Duncan's multiple range tests.

Results and Analysis

Reducing power of polysaccharides in yam bulbils

Reducing power is an important indicator reflecting the antioxidant capacity of certain substance, and the strength of reducing power is an important parameter for antioxidant activity of polysaccharides. The absorbances of polysaccharides samples from yam bulbils at the wavelength of 700 nm can indirectly reflect the size of the anti-oxidative capacity of polysaccharides. As shown in Fig.1, with the increase of polysaccharides concentration, the absorbances were increased gradually, and the reducing power of polysaccharides was also trended to be increased; when the polysaccharides concentration was 5 mg/ml, the absorbance was highest,and the reducing power was also strongest.

Scavenging ability of polysaccharides in yam bulbils against DPPH·

Fig.2 showed that the polysaccharides solutions at different concentrations all had a certain scavenging effect on DPPH·, and the scavenging rate was increased with the increase of concentration. When the yam bulbil extract concentration was 4.0 mg/ml,the scavenging rate of DPPH· was 91.15% . The regression analysis showed that the relationship between scavenging effect of polysaccharides from yam bulbils on DPPH · and polysaccharides concentration was in line with a linear equation model:

Table 1 Effect of polysaccharides from yam bulbils on fasting blood glucose in mice of each treatment group

Scavenging ability of polysaccharides in yam bulbils against·OH

Hydroxyl radical is the most reactive oxygen in body, and its excessive accumulation can cause a variety of pathological changes. Polysaccharides can provide hydrogen atom,which can combine with hydroxyl radical to form water, thereby scavenging free radicals. As shown in Fig.3, the polysaccharides systems at different concentrations all had a certain scavenging effect on·OH, and the scavenging effect was enhanced with the increase of polysaccharides concentration. When the polysaccharides concentration was 4.0 mg/ml, the scavenging rate of ·OH reached 89.06%.

Effect of polysaccharides in yam bulbils on fasting blood glucose in mice

Table 1 showed that there were no significant differences in fasting blood glucose among control group,model group and treatment groups before the modeling. After the modeling,the blood glucose content in the normal control group was all significantly lower than those in the other groups.After the therapy, the blood glucose contents in mice of the three treatment groups were all reduced, and the decrement was more obvious in the high dose(30 mg/kg)treatment group.It suggests that the polysaccharides from yam bulbils can effectively reduce the blood glucose levels in diabetic mice.

Conclusions and Discussion

The oxidative damage caused by free radicals is related to the pathogenesis of many diseases. Human can reduce the free radical level in body through the proper intake of substances with antioxidant activity,preventing lipid peroxidation. Lipid peroxidation is closely related to aging, and even can induce many diseases[8].The results of this study show that the polysaccharides from yam bulbils have significant antioxidant activity and strong reducing power,and when their concentration was 4.0 mg/ml, the scavenging rates of DPPH·and ·OH were 91.15% and 89.06%,respectively.

The mechanism of alloxan inducing diabetes has been well known. Alloxan can selectively destroy pancreatic β cells,resulting in reduced secretory cell number,swelling cells and increased vacuoles in pancreas islets.In the destruction of β cells in pancreas islets, free radicals play a vital role[9].This study finds that the polysaccharides from yam bulbils can significantly reduce blood glucose contents in alloxan-induced diabetic mice, which may be related to increased secretion of insulin, improved cellular functions in damaged β cells and scavenging of excess free radicals.

Using the chemical model, this study finds that the polysaccharides from yam bulbils have relatively strong antioxidant and hypoglycemic effect,and they provide a new resource for development of safe, effective, nutritious and natural antioxidants and blood glucose-reducing agents, which will not only improve farmers’ income,but also turn yam bulbils into treasure.

[1]ZHAO B(赵冰).New cultivation technology of Dioscorea opposita thumb(山药栽培新技术)[M]. Beijing: Golden Shield Press(北京: 金盾出版社),1998.

[2]DU HQ(都恒青).Arrangement and study on common Chinese herbal varieties(Northern China) (Second Volume) (常用中药材品种整理和质量研究(北方篇):第二册)[M]. Beijing: Beijing Medical University & Chinese Peking Union Medical College Associated Press (北京: 北京医科大学中国协和医科大学联合出版社),1995.

[3]SUN F (孙锋), GU WY (谷文英), DING XL (丁霄霖).Study on extraction technology of polysaccharide from Dioscorea opposita Thunb(山药粗多糖的提取工艺)[J]. Journal of Food Science and Biotechnology (食品与生物技术学报),2006,25(3):79-83.

[4]VOGEL HG. Pharmacology Laboratory Manual: New Drug Discovery and Pharmacological Evaluation (药理学实验指南: 新药发现和药理学评价)[M].Beijing:Science Press(北京: 科学出版社),2001:699-700.

[5]LUO ZY (罗祖友), YAN FW (严奉伟),XUE ZH(薛照辉),et al.The anti-oxidative effect of polysaccharide from Ampelopsis grossedentata (藤茶多糖的抗氧化作用研究)[J].Food Science(食品科学),2004,25(11):291-295.

[6]ZHA XQ,WANG JH,YANG XF.Antioxidant properties of polysaccharide fractions with different molecular mass extracted with hot-water from rice brain[J].Carbohydrate Polymers, 2009, 78 (3):570-575.

[7]JIN M (金鸣),CAN YX (蔡亚欣),LI JR(李金荣), et al. 1, 10-phenanthroline-Fe2+oxidative assay of hydroxyl radical produced by H2O2/Fe2+(邻二氮菲-Fe2+氧化法检测H2O2/Fe2+产生的羟自由基[J]. Progress in Biochemistry and Biophysics (生物化学与生物物理进展),1996,23(6):553-555.

[8]ZHU SY (朱淑云), DONG Y (董英),ZHANG HH (张海晖),et al.Enzymatic hydrolysis of milk thistle meal protein and antioxidation of hydrolysates (水飞蓟粕蛋白的酶解及其酶解物抗氧化活性研究)[J]. Chinese Cereals and Oils Association (中国粮油学报),2011,26(2):68-72.

[9]YANG SC(杨锁成),MENG J(孟杰),XU ZC (胥志才). Mechanism of preventive and therapeutic effect of Xiaokekangtai on experimental diabetes in rats (消渴康泰对大鼠实验性糖尿病防治作用的机理研究)[J]. Chinese Journal of Traditional Medical Science and Technology(中国中医药科技),2000,7(2):77-78.