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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (2): 112-120.doi: 10.11707/j.1001-7488.20150214

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1-Deoxynojirimycin Extraction from Ramulus Mori and Relationship Analysis between Its Content in Extracts and α-Glucosidase Inhibitory Activity

Liu Chao1, Shi Zhengqin1, Xiang Wei1, Huang Xianzhi1, Xu Li1, Lan Jun2   

  1. 1. College of Biotechnology, Southwest University Chongqing 400715;
    2. Oasis Sources Environmental Protection Industry Ltd. Xiamen 361004
  • Received:2014-04-23 Revised:2014-05-22 Online:2015-02-25 Published:2015-03-11

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Abstract:

【Objective】 1-Deoxynojirimycin (DNJ) is one of the important natural active substances with good hypoglycemic effect. Mulberry has a relatively high DNJ content. To obtain high extraction rate from ramulus mori, the microwave-ultrasonic assisted technology was optimized. Meanwhile, to get DNJ contribution degree to α-glucosidase inhibitory activity in ramulus mori water extract, the relationship between DNJ content and α-glucosidase inhibitory activity was analyzed. The present study would provide a basis for DNJ extraction, separation, and polar distribution of α-glucosidase inhibitors. 【Method】9-Fluorenylmethyl chloroformate was used as DNJ derivatization reagent to make up for the shortcomings of DNJ that does not hold the UV absorption characteristic. Then, quantitative and qualitative analysises of DNJ had been done at a wavelength of 254 nm by high performance liquid chromatography. The optimal dimension of ultrasonic processing time, microwave processing time and solvent-to-sample ratio were obtained by single factor experiment. The optimized values including sum of squares, mean square, p-value of three-factor of face-centered cube design, regression equation and corresponding optimal extraction condition were gotten by response surface methodology. Based on the results, systematic solvent method was used to extract concentrated extracts. α-Glucosidase inhibitory activity (through the detection of decomposition rate of α-glucosidase to 4-nitrophenyl-α-D-glucopyranoside at a wavelength of 415 nm by microplate reader) and DNJ content were calculated. The relationship between them was also analyzed in four parts extracted by petroleum ether, ethyl acetate, n-butyl alcoho and water in turn. 【Result】Microwave processing time 9.60 min, ultrasonic processing time 30.89 min and solvent-to-sample ratio 46.47 mL·g-1 were found to be the optimal condition for extraction with 0.05 mol·L-1 HCl. The yield was 0.205%. The fitting rate between test value and the optimal value was 99.27%. The test yield of DNJ increased by 10.86% comparing with that of the control group. The DNJ content in n-butyl alcohol part was the highest. The second was found in the ethyl acetate part, and the third was found in the water part. Petroleum ether part had no DNJ. It informed that the extraction effect of n-butyl alcohol was the best. The inhibition curve trend of n-butyl alcohol part was close to ethyl acetate part, and that of water part and petroleum ether part was similar. The 4 parts had apparent difference in IC50 value. It suggested that some differences in polarity existed among α-glucosidase inhibitors in ramulus mori combined with the content distribution of DNJ. DNJ concentration, which was called "DNJ content at this concentration", was calculated from 50% inhibiting concentration based on DNJ content of each extraction part. "DNJ content at this concentration" of n-butyl alcohol part and DNJ IC50 had no apparent difference. It indicated that the α-glucosidase inhibitors of n-butyl alcohol part was almost entirely DNJ. A small amount of DNJ was in water part, but its activity was relatively smaller. It may be due to some components like polysaccharide with the ability of improving α-glucosidase inhibitory activity were possible or some sticky substances that affected the combination of DNJ and enzyme. "DNJ content at this concentration" of ethyl acetate part was smaller than DNJ IC50. This may be because some components like flavone which had enhancement/synergistic function with DNJ or it was possible that some components like alkaloids, coumarins and flavone glycoside etc. had similar activity with DNJ. Petroleum ether part had no DNJ, but had a certain activity. It provided that some other hypoglycemic active compounds with less polarity and lower activity than DNJ were existed in ramulus mori extracts. 【Conclusion】Crushed fresh ramulus mori was treated by microwave, which had both drying effect and assistant extraction effect. Then, extraction was done under ultrasonic conditions. The operation could improve the extraction efficiency of DNJ. The main α-glucosidase inhibitor was DNJ, but there were also some other compounds with less polarity and lower activity than DNJ in ramulus mori. The results are conducive for the further study of DNJ extraction and the composition of hypoglycemic activity compounds in mulberry.

Key words: 1-Deoxynojirimycin, ultrasonic-microwave assisted extraction, response surface methodology, α-glucosidase inhibitory activity, ramulus mori

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