In vitro,in vivo and in silico anti-hyperglycemic inhibition by sinigrin

Objective:To evaluate the anti-hyperglycemic potential of sinigrin using in ritro.in silico and in riro streptozotocin(STZ) induced hyperglycemic zebrafish model.Methods:The in vitro enzyme inhibition assay was carried out to determine the IC_(50) value against α-glucosidase and α-amylase.in silico molecular docking was performed against both enzymes with PyRx tool and simulations were performed using GROMACS tool.Hyperglycemia was induced in zebrafishes using three intraperitoneal injections on alternating days for one week at 350mg/kg of STZ.Hyperglycemic fishes were treated intraperitoncally with 50.100 and 150 mg of sinigrin/kg of body weight for 24 h and glucose levels were measured.Results:The sinigrin showed very strong inhibition against α-glucosidase and α-amylase with 0.248 and0.00124 μM while reference drug acarbose showed IC_(50) value of 73.0700 and 0.0017 μM against α-glucosidase and α-amylase,respectively.Kinetic analysis revealed that sinigrin has the mixed type mode of inhibition against α-glucosidase.Molecular docking results revealed its strong binding affinity with α-glucosidase(-10.00 Kcal/mol) and α-amylase(-8.10 Kcal/mol).Simulations graphs confirmed its stability against both enzymes.Furthermore.in hyperglycemic zebrafishes most significant(P<0.001) reduction of glucose was occurred at150 mg/kg.moderate significant reduction of glucose was observed at 100 mg/kg and no any significant reduction of glucose was measured at 50 mg/kg.Conclusions:It can be evident from the present results that sinigrin has potent anti-hyperglycemic activity and it may prove to be effective treatment for the hyperglycemia.

QM/MM Study on Enzymatic Mechanism in Sinigrin Biosynthesis

As the major and abundant type of glucosinolates(GL)in plants,sinigrin has potential functions in promoting health and insect defense.The final step in the biosynthesis of sinigrin core structure is highly representative in GL compounds,which corresponds to the process from 3-methylthiopropyl ds-GL to 3-methylthiopropyl GL catalyzed by sulfotransferase(SOT).However,due to the lack of the crystallographic structure of SOT complexed with the 3-methylthiopropyl GL,little is known about this sulfonation process.Fortunately,the crystal structure of SOT 18 from Arabidopsis thaliana(At SOT18)containing the substance(sinigrin)similar to 3-methylthiopropyl GL has been determined.To understand the enzymatic mechanism,we employed molecular dynamics(MD)simulation and quantum mechanics combined with molecular mechanics(QM/MM)methods to study the conversion from ds-sinigrin to sinigrin catalyzed by AtSOT18.The calculated results demonstrate that the reaction occurs through a concerted dissociative mechanism.Moreover,Lys93,Thr96,Thr97,Tyr130,His155,and two enzyme peptide chains(Pro92-Lys93 and Gln95-Thr96-Thr97)play a role in positioning the substrates and promoting the catalytic reaction by stabilizing the transition state geometry.Particularly,His155 acts as a catalytic base while Lys93 acts as a catalytic acid in the reaction process.The presently proposed concerted dissociative mechanism explains the role of At SOT18 in sinigrin biosynthesis,and could be instructive for the study of GL biosynthesis catalyzed by other SOTs.

Variation in Plant Morphology and Sinigrin Content in Ethiopian Mustard(Brassica carinata L.)

Ethiopian mustard(Brassica carinata) is mainly grown as a leafy vegetable and oilseed crop. Sinigrin is predominant glucosinolate in the leaves of Ethiopian mustard. It is hydrolyzed by enzyme myrosinase to produce bioactive product specifically allyl-isothiocyanates. Due to sinigrin content, Ethiopian mustard has a mild flavor and it is a healthy addition to human diet. However, sinigrin concentration depends on genotype.Understanding variations of plant morphology and sinigrin content may be the first step for crop improvement. According to the analyses of various plant characteristics among ninety nine Ethiopian mustard accessions, we found significant differences in plant height, chlorophyll content, and petiole length. In addition, sinigrin content was determined using ultra-performance liquid chromatography tandem quadrupole mass spectrometers/tunable ultraviolet detector(UPLC-TQS/TUV). We found a significant difference in sinigrin content among leaves of 94 Ethiopian mustard accessions. Except for petiole length, the sinigrin content was negatively correlated with leaf area, leaf width, number of primary branches, and plant height. This information will help Ethiopian mustard breeders in screening breeding lines for new accessions.

响应面法优化酶解辅助-超声提取菥蓂中的黑芥子苷

为从菥蓂种子中高效提取高得率的目的活性成分黑芥子苷,采用酶解辅助-超声提取法,并利用高效液相色谱测定其含量。在单因素试验的基础上,以酶添加量、酶解温度和酶解时间为关键因素,以黑芥子苷得率为响应值,应用Design-Expert V11软件,Box-Behnken Design响应面法优化菥蓂种子中黑芥子苷酶解辅助-超声提取工艺;并与单独超声、索氏提取2种提取法的黑芥子苷得率进行比较。结果表明,采用Agilent HCC18柱(25 mm×4.6 mm×5μm),流动相A和B分别为甲醇和0.5%磷酸水溶液,检测波长215 nm,柱温25℃,流速1 mL·min^(-1),进样量10μL,在等度洗脱10 min下,获得的分离效果和峰型较好。以筛选出的碱性蛋白酶为酶制剂,最佳提取工艺条件为:酶解温度49℃,酶用量2.90%,酶解时间18 min;在此条件下,菥蓂种子中黑芥子苷得率为2.679 mg·g^(-1),经方差分析与理论值(2.687 mg·g^(-1))差异不显著,但显著高于单独超声提取(2.036mg·g^(-1))和索氏提取(1.702 mg·g^(-1))(P<0.05)。优化得到的菥蓂中黑芥子苷酶解辅助-超声提取工艺,方法简单、节能、环保、高效,目的活性成分得率高,可应用于菥蓂中黑芥子苷的生产实践,为菥蓂的深度开发奠定了理论基础。

近红外漫反射光谱法测定川藏道地药材菥蓂子中黑芥子苷含量

提出了用近红外光谱技术快速测定川藏道地药材菥蓂子中指标成分黑芥子苷含量的方法。对黑芥子苷含量在1.962%～3.917%范围的不同来源的菥蓂子,根据其在12500～3600cm-1的近红外吸收光谱,采用偏最小二乘法(PLS)建立了校正模型,比较了光谱不同预处理方法对校正结果的影响。当采用最大最小归一化法时能最有效地提取光谱的信息,此时校正集相关系数(r)为0.9898,校正集标准偏差(SEC)为0.0233,预测集标准偏差(SPE)为0.1112。该方法快速简便,适合于重要指标成分的快速分析。

Plackett-Burman设计和响应面法优化芥子中硫苷的超声波提取工艺

通过预试验和扫描电镜法分析得出超声波提取可提高芥子中硫苷的提取率,进而采用Plackett-Burman(PB)试验和响应面法研究芥子中硫苷的超声波提取最佳工艺条件,并以硫苷的提取率作为响应值。在单因素试验的基础上,采用PB试验从影响超声波提取的因素中筛选出超声功率和乙醇浓度两个显著因素,在此基础上,通过最陡爬坡试验逼近最大硫苷提取率区域,然后通过中心组合设计试验对显著因素进行优化,得出最佳超声波提取工艺条件为:仪器(超声波细胞粉碎机)参数为超声功率300 W、超声工作时间4 s(间歇时间6 s),提取总时间25 min(即超声150次),液料比15:1(mL/g)和乙醇体积分数48%。在此条件下,硫苷提取率为2.902%,与预测值2.894%相对误差为0.276%。这表明PB试验结合响应面法可以很好地优化芥子中硫苷的超声波提取工艺。

藏药菥蓂中黑芥子苷对黄嘌呤致小鼠高尿酸作用的研究

采用黄嘌呤致小鼠高尿酸模型,将昆明种小鼠随机分为模型组、空白对照组、阳性组、未灭活水提液(WMHSTY)组、灭活水提液(MHSTY)组、醇提液(CTY)组和水提醇沉浓缩液(STCCNSY)组并分别给药,以提取液对尿酸降低程度与提取液中黑芥子苷含量的关系阐明黑芥子苷与菥蓂治疗痛风作用的关系。结果,MHSTY和WMHSTY对黄嘌呤致小鼠高尿酸模型有显著的降低尿酸作用,两者间无明显差异,但黑芥子苷含量差异显著;CTY中黑芥子苷含量最高,但对本模型无明显作用;STCCNSY中无法检测出黑芥子苷,但对本模型有极显著作用。表明,藏药菥蓂中黑芥子苷的含量与菥蓂对黄嘌呤致小鼠高尿酸模型的作用无明显关系。

油菜种子中烯丙基硫苷的HLPC检测体系建立及其含量测定

实验以油菜种子为材料,建立了其中所含烯丙基硫苷的HPLC检测体系,并对不同油菜种子中烯丙基硫苷的含量进行了测定.结果表明,烯丙基硫苷HPLC分离检测的最佳条件为:流动相:甲醇∶水(0.005mol.L-1KCl)=9∶91;流速:0.5mL.min-1;柱温:30℃;检测波长:230nm.推广品种‘中陇油5’号烯丙基硫苷含量最高,达到8.677μmol.g-1;‘绵杂04-20’种子烯丙基硫苷含量只有0.032μmol.g-1.杂交品系油菜种子中的烯丙基硫苷含量普遍高于推广品种,最高达11.705μmol.g-1.

Identification of MAM1s in Regulation of 3C Glucosinolates Accumulation in Allopolyploid Brassica juncea

Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown.In this study,we genome-widely identified GSLs metabolic genes,indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B.juncea.Eight methythioalkylmalate synthase(MAMs)homologs were identified from B.juncea,in which six MAM1s were located in chloroplast and the other two were not detected with any expression.Furthermore,BjMAM1-4,BjMAM1-5,and BjMAM1-6 displayed higher expression levels in leaves than other tissues.Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation.The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle,suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis.Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs,thereby facilitating the manipulation of aliphatic GSLs content in B.juncea.

菜籽饼脱毒方法的研究进展

菜籽饼是一种利用价值很高的高蛋白资源,但由于菜籽饼中含有硫甙、芥酸、植酸、单宁等有毒物质和抗营养成分,使其应用范围受到了限制。综述了物理法、化学法、生物法等脱毒方法,并对其应用前景进行了展望。

微波炮制芥子的正交试验研究

目的探讨微波加热法和传统清炒法炮制芥子的优劣。方法采用正交设计试验优选微波炮制芥子的最佳工艺。测定芥子水分含量、浸出率、煎出率;用K.H.Bauer方法测定芥子苷的含量。结果微波炮制芥子的最佳工艺条件为中火,加热4 min,药物铺展厚度为2.0 cm;芥子生品、炒品与微波炮制品比较,其有效成分芥子苷含量有显著性差异。结论微波法炮制芥子优于传统炒法,且工艺条件可控。

藏药菥蓂子的定性、定量方法研究

目的:建立藏药菥蓂子的定性与定量控制方法。方法:采用薄层色谱、高效液相色谱分别鉴定及测定15批菥蓂子中的黄酮类成分异牡荆素、当药黄素和硫代葡萄糖苷类成分黑芥子苷。两类成分薄层色谱鉴别的固定相分别为聚酰胺薄膜、高效硅胶GF254,展开剂分别为三氯甲烷-甲醇-冰醋酸(11∶1∶1,V/V/V)、乙酸乙酯-甲醇-三乙胺(4∶5∶0.5,V/V/V)。在异牡荆素、当药黄素含量测定的色谱条件中,色谱柱为CAPCELL PAK MGⅡC18,流动相为乙腈-0.4%冰醋酸溶液,梯度洗脱,流速为1.0 mL/min,检测波长为336 nm;在黑芥子苷含量测定的色谱条件中,色谱柱为CAPCELL PAK MGII C18,流动相为乙腈-0.02 mol/L四丁基硫酸氢铵水溶液(15∶85,V/V,pH 6),流速为1.0 mL/min,检测波长为227 nm。结果:在薄层色谱鉴别图谱中,供试品分别检测出与异牡荆素、当药黄素及黑芥子苷对照品相应的斑点;异牡荆素、当药黄素、黑芥子苷检测质量浓度线性范围分别为1.26~79.00、1.21~75.38、12.80~640.00μg/mL(r均≥0.999 5),检测限分别为0.09、0.12、0.15μg/mL,定量限分别为0.39、0.43、0.54μg/mL;精密度、稳定性(24 h)、重复性试验的RSD均≤2.0%(n=6),加样回收率分别为99.1%、97.0%、98.1%,RSD分别为1.9%、1.8%、1.8%(n=6)。15批菥蓂子药材中异牡荆素、当药黄素、黑芥子苷含量范围分别为0.013~0.090、0.020~0.130、18.92~40.75 mg/g。结论:建立的质量控制方法操作简便,重复性及稳定性良好,可用于藏药菥蓂子的质量控制。

黑芥子苷通过p38/JNK MAPK信号通路对阿霉素心脏毒性的保护作用

目的:探讨黑芥子苷对阿霉素诱导的心脏毒性的作用及机制。方法:10周龄雄性C57/BL6小鼠随机分为对照组、黑芥子苷组、阿霉素组和黑芥子苷+阿霉素组。阿霉素组和黑芥子苷+阿霉素组给予单次腹腔注射阿霉素(15 mg/kg),对照组和黑芥子苷组给予单次腹腔注射生理盐水(15 mg/kg),黑芥子苷组和黑芥子苷+阿霉素组小鼠同时用黑芥子苷5 mg/kg灌胃,每2天1次,共3次。7 d后超声心动图检测心脏功能,Western blot检测心肌中凋亡相关蛋白和MAPK信号通路相关蛋白的表达水平。结果:黑芥子苷组与对照组小鼠超声心动图各指标、凋亡相关蛋白表达水平和MAPK相关蛋白表达水平的差异无统计学意义。与对照组相比,阿霉素组小鼠LVEDD、LVESD均明显增大,LVEF和LVFS均明显减小,Bax表达水平、p38和JNK的磷酸化水平明显升高,Bcl-2表达水平明显降低(P均<0.05)。与阿霉素组相比,黑芥子苷+阿霉素组小鼠LVEDD、LVESD均明显减小,LVEF和LVFS均明显增大,Bax表达水平、p38和JNK的磷酸化水平均明显降低,Bcl-2表达水平明显升高(P均<0.05)。结论:黑芥子苷通过p38/JNK MAPK信号通路减少心肌细胞凋亡,减轻阿毒素诱导的心脏毒性。

十字花科植物与食用菌共培养通过根系分泌物促进菌丝生长

以十字花科植物芥菜为供体,6种食用菌(金针菇、凤尾菇、香菇、杏鲍菇、黑木耳、银耳)为受体,通过共培法,研究了芥菜及其化感物质黑芥子苷对食用真菌菌丝生长的影响.结果表明,芥菜对杏鲍菇、黑木耳和银耳菌丝生长无明显促生作用,但可以显著促进金针菇、凤尾菇和香菇菌丝的生长,从而有利于缩短菌株生长周期.进一步的试验发现不是因为芥菜幼苗产生的挥发物,而是其根系分泌物对菌丝生长有促进作用.将十字花科芥菜根系分泌的主要化感物质黑芥子苷加入培养基用来培养食用菌,当浓度等于1 mg·mL^(-1)时对金针菇、凤尾菇和香菇菌丝生长促生效果最佳.

HPLC测定菥蓂子中的黑芥子苷

目的采用HPLC测定菥蓂子中的黑芥子苷。方法色谱柱为DiamonsilTMC18柱(250 mm×4.60 mm,5μm),流动相为乙腈-20 mmol.L-1四丁基硫酸氢铵(pH6)水溶液(20∶80),流速1 mL.min-1,检测波长227 nm,柱温30℃。结果黑芥子苷0.2543～10.17μg与峰面积的线性关系良好(r=0.9999),平均回收率为98.41%,RSD=0.77%,10批药材中黑芥子苷含量的平均值为3.26%。结论所用方法简便、准确、重复性好,可作为菥蓂子药材的质量控制方法。

黄芥子文火微炒和研粉使用的化学内涵

为了探讨炒制方式和炒制时间对黄芥子中主要活性成分芥子苷和芥子碱硫氰酸盐的影响,采用高效液相色谱法,即AlltimaTMC18色谱柱（4.6 mm×250 mm,5μm）,流动相乙腈-0.1%磷酸溶液梯度洗脱,芥子苷检测波长227 nm,芥子碱硫氰酸盐检测波长326 nm,流速1.0 m L·min^-1,柱温35℃,测定黄芥子生品和不同炒制时间的制品水煎液中芥子苷和芥子碱硫氰酸盐的含量。结果发现芥子苷和芥子碱硫氰酸盐的含量随着炒制时间的增加呈现先上升后下降的趋势,0-2 min药材和粉末中芥子苷质量分数增加了9.65%,356.10%;芥子碱硫氰酸盐质量分数增加了12.82%,3.41%;炒制2 min后芥子苷和芥子碱硫氰酸盐含量达到最高,2-7 min药材和粉末中芥子苷质量分数降低了80.35%,82.09%;芥子碱硫氰酸盐质量分数降低了14.29%,17.54%,表明炒制时间对黄芥子中芥子苷和芥子碱硫氰酸盐的含量影响较大,药材粉碎后芥子苷才易与芥子酶在水溶液中充分接触发生酶解反应。建议黄芥子炒制使用时应采用文火微炒;工业生产相关水煎法制备的黄芥子产品可直接生品投料。

高效液相色谱-外标法定量芥蓝中芥子油苷

研究了利用2-丙烯基芥子油苷外标法定量芥蓝中芥子油苷的可行性,并对芥蓝中芥子油苷种类及含量进行报道。结果表明,2-丙烯基芥子油苷在14.2U硫酸酯酶的脱硫下,经过高效液相色谱检测,峰面积与浓度呈正比,建立回归方程:y=3E-07x+0.001,相关系数达到0.9994。回收及对比实验表明,2-丙烯基芥子油苷外标法定量的结果准确可信,可以代替昂贵的内标苯甲基芥子油苷。经HPLC/ESI-MS鉴定和外标法定量,在芥蓝菜薹中存在11种芥子油苷,其中脂肪族7种,吲哚族4种。脂肪族和吲哚族芥子油苷分别占总量的63.41%和36.59%。含量最高的为3-丁烯基芥子油苷,最低的为4-甲硫基丁基芥子油苷。

油菜愈伤组织中烯丙基硫苷的积累和某些外源碳氮化合物对其含量的影响

油菜愈伤组织在培养中生长34d的增殖量最大,增殖倍数为7.08倍,烯丙基硫苷的含量在第6天最高;外源L-缬氨酸明显促进烯丙基硫苷的积累,其浓度为2和4mmol·L-1时烯丙基硫苷含量分别达到98.5和97.40mg·g-1(DW);蔗糖最有利于油菜愈伤组织的生长和其中烯丙基硫苷的积累,浓度以30g·L-1的蔗糖为最佳。

罗汉菜籽中硫甙的分离与分析

目的用高速逆流色谱分离制备罗汉菜籽中的目标组分并对其进行鉴定。方法运用单因素实验设计考察高速逆流色谱分离罗汉菜籽中硫甙的最优条件,采用UHPLC/QTOF-MS分析鉴定出该组分。结果得出高速逆流色谱法分离纯化罗汉菜籽成分的最优分离条件:以正丁醇-乙腈-10%硫酸铵水溶液=1:1:2(v/v)为溶液体系,流速5 m L/min,检测波长229 nm,转速850 r/min,上样量150.0 mg,分离制备2次,可得到纯度为65.9%的目标组分,经分析鉴定出该组分为黑芥子甙。结论制备出的黑芥子甙纯度较高,性质稳定,因此高速逆流色谱可以实现罗汉菜中硫甙的分离制备。