林业科学  2015, Vol. 51 Issue (2): 112-120   PDF    
DOI: 10.11707/j.1001-7488.20150214
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文章信息

刘超, 史正琴, 向伟, 黄先智, 徐立, 兰俊
Liu Chao, Shi Zhengqin, Xiang Wei, Huang Xianzhi, Xu Li, Lan Jun
桑枝1-脱氧野尻霉素的提取及其含量与α-葡萄糖苷酶抑制活性的相关性分析
1-Deoxynojirimycin Extraction from Ramulus Mori and Relationship Analysis between Its Content in Extracts and α-Glucosidase Inhibitory Activity
林业科学, 2015, 51(2): 112-120
Scientia Silvae Sinicae, 2015, 51(2): 112-120.
DOI: 10.11707/j.1001-7488.20150214

文章历史

收稿日期:2014-04-23
修回日期:2014-05-22

作者相关文章

刘超
史正琴
向伟
黄先智
徐立
兰俊

桑枝1-脱氧野尻霉素的提取及其含量与α-葡萄糖苷酶抑制活性的相关性分析
刘超1, 史正琴1, 向伟1, 黄先智1, 徐立1, 兰俊2    
1. 西南大学生物技术学院 重庆 400715;
2. 厦门绿洲环保产业股份有限公司 厦门 361004
摘要:【目的】 1-脱氧野尻霉素(DNJ)是一种降血糖功效较好的天然活性产物,在桑树中的含量相对较高。对超声-微波联合辅助提取桑枝中DNJ的工艺进行优化,以获得较高提取率;同时,分析桑枝水提物中DNJ含量与α-葡萄糖苷酶抑制活性的相关性,以获得其中DNJ对α-葡萄糖苷酶抑制活性的贡献程度。这可能为桑枝中DNJ的提取、分离及桑枝α-葡萄糖苷酶抑制剂的极性分布等研究提供一定的依据。【方法】 利用9-芴甲氧羰酰氯对DNJ进行衍生化,弥补其本身不具有紫外吸收特性的缺点,利用高效液相色谱在254 nm波长下进行定性定量检测;单因素试验分别求得微波处理时间、超声波处理时间和液料比的优化范围,以响应曲面法对超声-微波联合辅助提取DNJ三因素的平方和、均方、P值、R2等参数进行优化,获得二次多项回归方程和相应的最优提取条件;在此基础上利用系统溶剂法萃取浓缩的水提物,计算石油醚、乙酸乙酯、正丁醇和水4种极性部分的α-葡萄糖苷酶抑制活性(通过酶标仪在415 nm波长处测定α-葡萄糖苷酶对4-硝基苯-α-D吡喃葡萄糖苷的分解率来计算)与DNJ的含量,并对二者之间的关系进行分析。【结果】 用0.05 moL·L-1 的盐酸溶液提取,微波处理9.60 min、超声波处理30.89 min和液料比46.47 mL·g-1时达到最优条件,此时DNJ的产率为0.205%,结果检验值与优化值的拟合率达99.27%,相对于对照组增长了10.86%;正丁醇部分DNJ的含量最高,乙酸乙酯次之,水部分仍含有少许,石油醚部分为零,说明正丁醇的萃取效果较好;正丁醇部分的抑制曲线趋势与乙酸乙酯部分相近,水部分与石油醚部分相近,且各萃取部分之间的IC50都差异显著,结合DNJ的含量分布,说明桑枝中具有α-葡萄糖苷酶抑制活性的物质间有一定的极性差异;根据各萃取部分中DNJ的含量,从其半抑制浓度中换算出DNJ的浓度,即为"其中DNJ的浓度",正丁醇部分"其中DNJ的浓度"与DNJ标准品的IC50差异不显著,说明此部分α-葡萄糖苷酶抑制活性物质几乎尽是DNJ;水部分虽含有少量的DNJ,但其活性却相对更小,可能其中含有具备提高α-葡萄糖苷酶活性的多糖等物质,或一些糖类等黏性物质影响了DNJ与酶的结合;乙酸乙酯部分"其中DNJ的浓度"比DNJ标准品的IC50小,可能是由于此部分含有个别黄酮类等对DNJ具有活性增强/协同作用,或其中含有一些游离生物碱、香豆素或黄酮苷元等物质具备相似活性;石油醚部分不含DNJ,却有一定的α-葡萄糖苷酶抑制活性,说明桑枝中含有一些极性小的脂溶性化合物具备α-葡萄糖苷酶抑制活性。【结论】 微波处理初步粉碎的新鲜桑枝,既有干燥作用又有辅助提取效果,而后用超声波辅助浸提可提高DNJ的提取效率;桑枝中主要的α-葡萄糖苷酶抑制是DNJ,除此外还可能含有一些极性相对较小的活性成分。这些结果有利于桑枝DNJ及其他降血糖活性成分的进一步研究。
关键词1-脱氧野尻霉素    超声-微波联合辅助提取法    响应曲面法    α-葡萄糖苷酶抑制活性    桑枝    
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. College of Biotechnology, Southwest University Chongqing 400715;
2. Oasis Sources Environmental Protection Industry Ltd. Xiamen 361004
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    

桑(Morus alba)枝是我国传统中药,其药用功效在历代药典中多有记载。我国是蚕桑业大国,每年夏伐和冬季重剪都会产生上千万吨的桑枝条,远远大于其作为生药材的需求量,但多余部分被烧掉,造成了极大的资源浪费和严重的环境污染。现代植物化学研究表明,桑树中含有生物碱、黄酮、多糖等多种降血糖活性物质(赵骏等,2000原爱红等,20042006),尤其是1-脱氧野尻霉素(DNJ)(Toshiyuki et al., 2007Liu et al., 2011Kwon et al., 2011)。近年来,随着天然健康的价值回归,桑树活性物质展现出了可被应用于食品、医药、保健、、美容等方面的巨大潜能,呈现出古树新花的局面(徐立等,2013赵全成,2013刘万顺,2012毛绍春,2013陈琴,2012)。因此,研究桑树中降血糖活性物质,对阐述桑树作为中药材的药理作用、提高蚕桑经济效益和促进产业升级越来越具有现实意义。

与传统提取方法相比,微波辅助和超声波辅助提取技术具有效率高、简单、快速及环保等优点(袁忠海等,2003买买提江·依米提等,2010Wang et al., 2008Wang et al., 2007Xu et al., 2013),目前已应用于食品、医药、化学等领域,特别在中药活性成分提取中显现出了巨大的优势。响应曲面法(response surface methed,RSM)是一种重要的试验设计与分析方法,以低投入和高效率为指标,对试验各重要参数进行优化(Montgomery,2004)。Huang等(2010)在优化提取灵芝多糖的试验中认为超声波和微波在辅助提取上有互补作用。因而,用响应曲面法对超声-微波联合辅助提取桑枝DNJ的参数进行合理优化,也可能大幅度提高桑枝DNJ的提取效率。

DNJ和桑枝提取物的α-葡萄糖苷酶抑制活性研究较为常见(Asano et al., 1994Abdel et al., 2005Zhang et al., 2014),但DNJ含量与桑枝水提物的降糖活性的相关性、降糖活性物质在桑枝水提物中的极性分布情况方面的研究却鲜见报道。本试验通过分析桑枝水提物4个萃取部分的DNJ含量与α-葡萄糖苷酶的抑制活性的关系,一定程度上可为促进桑树天然活性物质的开发利用和提升蚕桑产业的经济附加值提供理论基础。

1 仪器与试剂

高效液相色谱,美国Waters公司;酶标仪,美国 BIO-RAD公司;微波炉,格兰仕微波炉电器有限公司;数控超声波清洗器,昆山市超声仪器有限公司;纯水仪,法国 Millipore公司;电子天平,上海精天电子仪器有限公司;200 g摇摆式高速万能粉碎机,温岭市林大机械有限公司;旋转蒸发仪,巩义市予华仪器有限责任公司。

α-葡萄糖苷酶(α-Glucosidase)、1-脱氧野尻霉素(1-Deoxynojirimycin,DNJ)、4-硝基苯-α-D吡喃葡萄糖苷(4-Nitrophenyl-α-D-glucopyranoside,pNPG)、9-芴甲氧羰酰氯(9-Fluorenylmethyl Chloroformate,FMOC-CL)和乙腈均购自Sigma-Aldrich公司。红果2号果桑桑枝于2013年3月采自重庆九园桑树种植专业合作社。

2 方法 2.1 DNJ的衍生化与HPLC分析

参照Kim等(2003)的方法,将30 μL DNJ标准品溶液或试验中含DNJ的提取液与30 μL 0.4 mol·L-1的硼酸钾缓冲液(pH 8.5)混合于0.5 mL 离心管中,加入60 μL 5 mmol·L-1的FMOC-Cl,25 ℃条件下水浴20 min,再加30 μL 1 mol·L-1的甘氨酸终止反应。用30 μL 0.1%(v/v)乙酸溶液稳定新生成的DNJ-FMOC,并以120 μL 蒸馏水定容。最后将各组提取液过0.22 μm尼龙滤膜,置于4 ℃冰箱待测。每步间都用涡旋仪震荡约10 s以使溶液充分混合。

DNJ的HPLC检测条件为:5 μm C18(4.6 mm×150 mm)反相色谱柱(Waters,Atlantis,Irel and ),流动相为乙腈∶ 0.1%醋酸(v/v)55∶45,流速1 mL·min-1,柱温30 ℃,检测波长254 nm,进样量10 μL,每个样品做3个重复。

2.2 单因素和响应曲面试验设计

选择辅助提取法常用的强度适中功率即微波480 W和超声波350 W(麻明友等,2010李粉玲等,2011张吉祥等,2010Krishna et al., 2014)为试验条件。微波处理前先用水浸湿待提取材料,处理后再用0.05 mol·L-1的盐酸溶液浸提(陈业高,2004张薇等,2012),超声波则直接用于处理桑枝粉末的浸提过程。设计液料比30 mL·g-1、超声波处理时间25 min时微波处理时间,液料比30 mL·g-1、微波处理时间7 min时超声波处理时间,微波处理时间7 min、超声波处理时间25 min时液料比3个单因素试验。HPLC分别检测各处理组的吸光值,并通过DNJ标准曲线方程计算其产率。

通过分析单因素试验结果并结合响应曲面法(Box-Behnken Design),先以微波处理用水浸湿的待提取材料,再用超声波提取浸于0.05 mol·L-1盐酸溶液中的桑枝粉末。独立变量X1(超声波处理时间,min)、X2(微波处理时间,min)和X3(液料比,mL·g-1)的三水平设计见表 1

表 1 DNJ提取率的三因素三水平试验设计 Tab.1 Experimental three-factor, three-level face-centered cube design of DNJ production
2.3 DNJ的系统溶剂法萃取

340 g桑枝晒干粉碎,过60目筛。分别用0.05 mol·L-1 的盐酸溶液(Kim et al., 2003张作法等,2007)以液料比46 mL·g-1浸提,微波-超声波依次辅助提取9.6 min和31 min,过滤,并浓缩成浸膏。蒸馏水溶解,分别用石油醚、乙酸乙酯和正丁醇萃取,并保留剩余的水相部分。4个萃取部分减压浓缩成浸膏,分别用二甲基亚砜采取对半稀释法配制成12.8,6.4,3.2,1.6,0.8,0.4,0.2,0.1,和0.05 mg·mL-1的浓度梯度液,分别测定DNJ含量和α-葡萄糖苷酶抑制活性的IC50值。并用对半稀释法配制120,60,30,15,7.5,3.75,1.875,0.938,0.469,0.234和0.117 μg·mL-1 的DNJ标准液,测定DNJ的α-葡萄糖苷酶抑制活性的IC50值。

2.4 α-葡萄糖苷酶抑制活性检测

结合Kim等(2010)Yang等(2012)的方法,将待测溶剂、α-葡萄糖苷酶(0.5 U·mL-1)及67 mmol·L-1 的磷酸钾缓冲液(pH 6.8)各50 μL混合,充分震荡,37 ℃预保温15 min,加入50 μL 6 mmoL·L-1 pNPG,再37 ℃酶解15 min,加200 μL 0.2 moL·L-1 Na2CO3溶液终止反应,每个处理分别做3个重复。各移取200 μL反应液于96孔板孔中,用酶标仪在415 nm波长下测定吸光值。以如下公式计算抑制率,并用SPSS软件计算相应IC50值:

抑制率(%)=[1-(Abs1- Abs2)/Abs0] × 100。

式中:Abs1是样品组的吸光度;Abs2是不加酶组的吸光度;Abs0是不加样品组的吸光度。

3 结果与讨论 3.1 DNJ标准曲线的测定与HPLC分析

DNJ标准曲线的线性回归方程为y=2 870.6 x-1 709,R2=0.999 4,其线性取值范围是0~240 μg·mL-1。如图 1的色谱所示,DNJ的保留时间约为2.15 min,虽然出峰时间较早,但其峰型明显,适用于DNJ含量的检测。

图 1 DNJ的色谱(a: 标准品;b: 桑枝水提物) Fig. 1 The chromatogram of DNJ (“a” and “b” are the chromatograms of standard substance and extract of ramulus mori respectively)
3.2 单因素试验结果

图 2a 所示,在30 min前DNJ的产率呈上升趋势,而后下降,故选用20,30和40 min作为响应曲面法优化超声波处理时间的3个水平;如图 2b所示,在前9 min内,DNJ的产率随时间的延长而增加,此后呈下降趋势,故选用7,9和11 min作为响应曲面法优化微波处理时间的3个水平;如图 2c 所示,在40 mL·g-1前DNJ的产率随时间的延长急剧增加,而后缓慢下降,结合生产成本和产率,选用30,40和50 mL·g-1作为响应曲面法优化液料比的3个水平。

图 2 单因素试验中超声波处理时间(a)、微波处理时间(b)及液料比与产率(c)的关系 Fig. 2 The relationship between ultrasonic processing time and yield (a), between microwave processing time and yield (b) and between solvent-to-sample ratio and yield (c) in single factor experiment
3.3 响应曲面试验结果 3.3.1 方差分析

超声-微波联合辅助提取DNJ的响应曲面法三因素三水平结果见表 1。利用Design-Expert 软件对试验数据进行多元回归拟合,得二次多项回归方程:

Y=0.19+2.725E-003X1+3.238E-003× X2+0.050X3+1.250E-004X1X2- 3.250E-004X1X3+1.700E-003X2X3 - 0.014X1X1-7.310E-003X2X2- 0.039X3X3

表 2可知,模型P < 0.000 1说明模型的显著置信水平已达99%。液料比的结果差异显著,其线性项和二次项是最具效应的变量。检测系数R2为0.992 0,说明99.20%的变量能用此模型解释,方程模型能很好地反映试验数据。

表 2 回归模型的方差分析 Tab.2 Analysis of variance for the response surface regression model
3.3.2 模型分析

图 3所示的三因素对DNJ产率的三维曲面和等值线可知,超声波处理时间和微波处理时间对DNJ产率的影响大致为随着时间的延长呈先增加、再下降的趋势。这可能是由于超声波的空化效应和微波的热效应破坏了植物细胞的膜壁结构,DNJ随之被浸提出来。但超声波和微波对DNJ的结构也具有破坏作用,当作用时间过长,DNJ被浸提出的速率小于被降解的速率时,DNJ在溶液中的含量就会逐渐下降。随着液料比的增加,DNJ产率几乎一直都在上升,直到约45 mL·g-1时,产率渐渐趋于平缓并开始下降。Design-Expert软件的分析结果表明:微波处理时间9.60 min、超声波处理时间30.89 min、液料比46.47 mL·g-1是最优的辅助提取条件,此时DNJ的产率达0.205%。

图 3 超声波处理时间、微波处理时间及液料比对DNJ产率的三维响应曲面(a)和等值线(b) Fig. 3 Three-dimensional response surface (a) and contour plot (b) for DNJ yield versus ultrasonic processing time, microwave processing time and solvent-to-sampde ratio
3.3.3 结果检验

为了检验最优提取条件的拟合率,本试验以微波处理时间9.6 min、超声波处理时间31 min、液料比46 mL·g-1为操作条件进行验证,此时DNJ产率为0.203 5%(图 4优化组),其与优化值的拟合率达99.68%,说明优化结果可靠。此外本试验又分别设置了对照组(不经超声波和微波处理)、仅用超声波处理31 min组和仅用微波处理9.6 min组(图 4)。结果表明在超声-微波联合辅助提取的过程中,超声波的作用与微波的差异不显著,二者有互补叠加效应。这可能由于微波引起分子电磁振荡产生的热效应和超声波产生瞬时高压的空化效应依次发生,较有利于从内外2层面破坏植物细胞结构。更兼超声波直进流和加速度作用与空化效应一道使DNJ易于脱离细胞内一些结构或化合物的吸附和结合,最终使二者在提取中展现出协同效应。

图 4 不同处理条件的结果比较 Fig. 4 Comparison results of different processing conditions
3.4 α-葡萄糖苷酶抑制活性分析

萃取液减压浓缩后,得石油醚部分浸膏0.723 g,乙酸乙酯部分浸膏82.989 g,正丁醇部分浸膏63.225 g,水部分浸膏1.823 g。通过HPLC检测得到各萃取部分的DNJ含量如图 5所示,正丁醇部分的含量最高,乙酸乙酯次之,水部分仍含有少许,石油醚部分不含DNJ。这说明正丁醇的萃取效果最好,建议作为DNJ萃取试验的一个参考。

图 5 不同萃取部分DNJ含量比较 Fig. 5 DNJ content of different extraction parts

DNJ标准品及4个萃取部分对α-葡萄糖苷酶的抑制率曲线如图 6所示,正丁醇部分的抑制曲线趋势与乙酸乙酯部分相近,而水部分与石油醚部分相近。根据各萃取部分中DNJ的含量,从其半抑制浓度中换算出DNJ的浓度,即为“其中DNJ的浓度”,结果见表 3,各萃取部分之间的IC50都差异显著。正丁醇部分“其中DNJ的浓度”与DNJ标准品的IC50差异不显著,说明此部分降血糖活性物质几乎尽为DNJ;水部分虽含有少量的DNJ,但其活性却相对更小,可能是由于其中含有一些糖类等黏性物质将DNJ一定程度上束缚在了一些萃取后剩余的固体颗粒物上,使其与酶的作用减弱,或其中含有具备提高α-葡萄糖苷酶活性的多糖等物质(Li et al., 2013),二者理论上都可以相对削弱提取液中DNJ的活性,进而提高此部分的IC50值;乙酸乙酯部分“其中DNJ的浓度”比DNJ标准品的IC50小,可能是由于此部分含有个别黄酮类等对DNJ具有活性增强/协同作用(Kim et al., 2013王芳等,2005),或其中含有一些游离生物碱、香豆素或黄酮苷元等物质具备相似活性(Asano et al., 1994Ito et al., 1984Jun et al., 1997谭永霞,2009王竹等,2008王斯慧等,2012叶菲等,2002);石油醚部分不含DNJ,却有一定的α-葡萄糖苷酶抑制活性,说明桑枝中含有一些极性小的脂溶性化合物具备α-葡萄糖苷酶抑制活性。虽然桑树中抑制α-葡萄糖苷酶活性成分主要为多糖类、生物碱类、黄酮类等物质(马燕燕等,2010),但其中像β-胡萝卜素、桑色素、山奈酚等(许延兰等,2008邹宇晓等,2005)具脂溶性性质的游离甾体、三萜等化合物也被报道了降血糖活性,但其机制是否为抑制α-葡萄糖苷酶活性尚不太清楚,故石油醚部分具体作用成分尚难以推测。

图 6 DNJ(a)及4个萃取部分(b)的抑制活性 Fig. 6 Inhibition curves of DNJ (a) and four extraction parts (b)
表 3 检测样品对α-葡萄糖苷酶的半抑制浓度(IC50) Tab.3 IC50 for α-glucosidase of DNJ and test samples
4 结论

从本文的研究结果来看:经过响应曲面法优化超声-微波辅助提取桑枝DNJ,其产率相对于对照组增长了10.86%,建议DNJ提取时待提鲜材料可直接用微波处理(既起干燥作用又有辅助提取效果)而后用超声波辅助浸提;正丁醇对DNJ具有较好的萃取效果,但其沸点较高,不易蒸发回收,而乙酸乙酯萃取效率稍差,挥发性却较好,便于回收,因而建议不同研究可根据试验的侧重方向选取合适的萃取溶剂;桑枝水提物中的主要α-葡萄糖苷酶抑制剂为DNJ,但还含有一些极性较小的脂溶性化合物也对α-葡萄糖苷酶具有抑制作用,同时推测桑枝中也可能含增强α-葡萄糖苷酶活性的物质。本试验分别从提取、萃取及活性方面对桑枝DNJ进行了研究和分析,为DNJ的提取和桑枝降糖物质的利用提供了一定的理论参考。

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