利用毛细管电泳法分析甜菊糖苷的含量

本文介绍了一种用毛细管区带电泳法筛选甜菊糖苷突变体的有效方法。根据实验结果,优化的电泳条件为60mmol/LTris-硼酸缓冲液(pH8.0),柱温30℃,工作电压25kV。优化条件下,甜菊苷(Stevioside)迁移时间的R.S.D为0.45%(15次),且在7.45×10     

高效液相色谱法测定甜菊糖苷中甜菊醇的含量

建立高效液相色谱法测定甜菊糖苷中甜菊醇的含量。色谱条件:色谱柱为Phenomenex C18(4.6 mm×250 mm,5μm);流动相为乙腈-水(50∶50);流速1.0 mL/min;检测波长213 nm;柱温30℃;进样量10μL。线性范围为1.046μg/mL~52.3μg/mL(r=0.9997),加标平均回收率为96.60%,RSD为0.51%(n=6)。本方法准确度高、精密度高、重复性好、简捷易操作,可以作为甜菊糖苷中甜菊醇含量的测定方法。     

甜菊不同叶龄细胞结构及其甜菊糖甙含量分布的研究

本文报道甜菊(Stevia rebaudiana Bertoni)不同叶龄细胞结构与甜菊糖苷含量分布。应用电镜技术观察表明,现蕾期成叶细胞内具有内含物丰富的巨大液泡,这些内含物呈大小不一的颗粒或小泡。应用差速离心法,对甜菊成叶的叶肉细胞进行亚细胞分离,并对各部分进行甜菊糖苷的提取与微量测定。结果表明,甜菊糖苷主要存在于12000g的上清液(这部分主要包括液泡内含物和可溶性细胞质)。结合细胞结构和细胞化学研究结果,表明细胞质是合成UDPG的主要场所,在甜菊糖苷合成中具有重要作用。对不同叶龄叶片甜菊糖苷测定表明,现蕾期成叶的甜菊糖苷含量最高。从甜菊不同叶龄细胞结构和甜菊糖苷含量测定结果,现蕾期是甜菊叶片收割的最适时期。     

一株快速转化甜菊苷的细菌鉴定、产酶及转化特性

【目的】本研究鉴定了一株筛选的甜菊苷特异降解菌、优化了该菌产β-葡萄糖苷酶的条件以及研究了该菌对甜菊苷的转化特性。【方法】经16SrRNA基因序列测序和系统发育学分析,结合形态学特征确定该菌株的系统发育地位。用单因素及多因素分析探讨了其对甜菊苷的降解,通过液质联用检测了降解产物。【结果】菌株-J2与巨大芽孢杆菌的16SrRNA基因序列相似性达到100%,结合形态学特征,鉴定该菌为巨大芽孢杆菌(Bacillus megaterium)。在玉米淀粉4%、豆粕粉1%、硫酸镁0.04%、pH7.0、37℃、220r/min、接种量10%、培养36h的条件下,该菌产β-葡萄糖苷酶活力为779.68U/mL。甜菊苷转化的结果表明:3d可将10mg/mL甜菊糖溶液中甜菊苷转化74%,使莱鲍迪甙A和甜菊苷的比例(RA/SS)由转化前的0.38上升至0.99,RA的相对量增加160.5%,5d时转化完全。转化产物经液质联用鉴定为甜菊双糖甙。【结论】确定菌株-J2为巨大芽孢杆菌(Bacillus megaterium),该菌对甜菊苷具有高效、特异的转化能力,为首次报道的新型、安全菌株。     

甜菊苷及其衍生物的生物活性研究进展

甜菊苷是一种常用天然甜味剂,属于四环二萜糖苷类。药理学研究表明,甜菊苷及其水解产物甜菊醇、异甜菊醇和甜菊双糖苷等具有降血糖、降血压、抗炎、抗肿瘤、止泻、抗菌和免疫调节等多种生物活性。综述甜菊苷、甜菊醇、异甜菊醇、甜菊双糖苷及相关衍生物的生物活性研究进展。     

甜菊醇糖苷生物合成关键基因的导入和鉴定分析

甜叶菊（Stevia rebaudiana Bertoni）生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。     

一种作用于花青素和甜菊醇的甜菊糖基转移酶的基因克隆和功能分析

本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53．2KDa。与常见的糖基转移酶基因比较,相似性达44％以上,工具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。     

甜菊糖苷积累与其生物合成基因表达的关系

为探究甜叶菊叶片中甜菊糖苷积累与其合成途径上关键基因表达的关系,本研究分别检测了鑫丰3号苗期不同冠层和3个不同品种甜叶菊(守田3号、江甜3号、谱星1号)收获期混合叶片样品中多种糖苷的含量,同时定量检测对应样品中甜菊糖苷合成关键基因的表达水平。结果显示,总糖苷在鑫丰3号顶叶中最高,底叶中最低,而多数检测基因(6/8)表达水平也在顶叶最高底叶中最低;单一糖苷甜菊苷在顶叶中积累最高,而其催化酶编码基因Sr UGT74G1表达也在顶叶中最高;莱鲍迪苷A则在底叶中积累最多,其催化酶编码基因Sr UGT76G1表达水平也在底叶中表达最高。3个品种相比,总糖苷和莱鲍迪苷A的积累在江甜3号中最高,谱星1号中最低;甜菊苷的积累在守田3号中最高,江甜3号中最低,但基因表达水平与糖苷积累趋势一致的类似结果并未在不同品种间出现。由此可知,甜菊糖苷合成基因的表达水平可以影响总糖苷的积累,且在同一甜叶菊品种中单一糖苷合成调控基因的表达水平可以反映其调控的单糖苷的积累量。     

阿魏酸在缺氧条件下促进人脐静脉内皮细胞血管形成的实验研究

目的:研究阿魏酸(ferulic acid,FA)在缺氧条件下对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖、迁移和管腔样结构形成的影响。方法:原代培养人脐静脉内皮细胞,在缺氧实验条件下,细胞被分为7组,即1个对照组和6个实验组。对照组采用1%酒精处理,实验组用不同浓度(1×10~(-8)、1×10~(-7)、1×10~(-6)、1×10~(-5)、1×10~(-4)及1×10~(-3) mol/L)的阿魏酸处理。分别采用MTS法、划痕法、Matrigel法分析不同浓度阿魏酸处理对人脐静脉内皮细胞的增殖、迁移和管腔样结构形成的影响。结果:缺氧条件下,浓度为1×10~(-6)~1×10~(-4)mol/L的阿魏酸处理能明显促进HUVECs的增殖(P0.05),以1×10~(-5) mol/L处理的效果最好(P0.01);与对照组相比,1×10~(-6)mol/L(P0.05)、1×10~(-5) mol/L(P0.01)及1×10~(-4) mol/L(P0.01)阿魏酸处理均能明显促进HUVECs横向迁移,以1×10~(-5) mol/L处理迁移的细胞数量最多;1×10~(-8)~1×10~(-4) mol/L阿魏酸处理能不同程度地促进HUVECs管腔样结构的形成,以1×10~(-5) mol/L处理形成管腔样结构的数量最多(P0.01)。结论:阿魏酸在缺氧条件下能促进人脐静脉内皮细胞的增殖、迁移和管腔样结构形成。     

甜菊糖苷的应用及生物合成研究进展*

甜菊糖苷是一种从甜叶菊叶片中提取的高甜度、零热量甜味剂,可用作食品添加剂。近年来,甜菊糖苷在国内外市场需求量剧增,引起了广泛关注。概述国际上对甜菊糖苷的安全性研究及评价,从经济价值角度分析甜菊糖苷的市场需求及应用前景,总结甜菊糖苷作为食品甜味添加剂的应用以及其在抗糖尿病、抗心脏纤维化、抗菌等保健功能方面的最新研究成果。综述甜菊糖苷的生物合成研究进展,重点介绍甜菊糖苷的微生物体从头合成以及生物催化低甜度糖苷生成高甜度、口感更优的甜菊糖苷,探讨提高甜菊糖苷产量的关键因素,为高端甜味剂绿色合成工艺的研究与开发提供理论依据。     

Highly sensitive spectrofluorimetic determination of rutin based on its activated effect on a multi‐enzyme redox system

A highly sensitive and simple spectrofluorimetric method for the determination of rutin, based on its activated effect on a haemoglobin‐catalysed reaction, was developed. Under optimum conditions, the concentration of rutin was linear, with decreased fluorescence (ΔF) of the system under optimal experimental conditions. The calibration graph was linear in the range 1.0 × 10^–7–3.0 × 10^–5 mol/L, with a detection limit of 7.0 × 10^–8 mol/L. The relative standard deviation (RSD) was 3.26% for 11 determinations of 1.0 × 10^–5 mol/L. This method was used for the determination of rutin in pharmaceuticals with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

Potassium permanganate–acridine yellow chemiluminescence system for the determination of fluvoxamine,isoniazid and ceftriaxone

Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 10^?9 to 1 × 10^?6 mol/L of fluvoxamine; 2 × 10^?8 to 8 × 10^?6 mol/L of ceftriaxone and 5 × 10^?8 to 4 × 10^?5 mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Flow injection–chemiluminescence determination of amoxycillin using potassium permanganate and formaldehyde system

It was found that amoxycillin can react with potassium permanganate in an acidic medium to produce chemiluminescence, which is greatly enhanced by formaldehyde. The optimum conditions for this chemiluminescent reaction were studied in detail using a flow‐injection system. The experimental results indicate that, under optimum conditions, the chemiluminescence intensity is linearly related to the concentration of amoxycillin in the range 5.48 × 10^?8–2.74 × 10^?6 mol[sol ]L, with a detection limit (3σ) of 4.1 × 10^?8 mol[sol ]L. The relative standard deviation was 1.0% at 1.1 × 10^?6 mol[sol ]L amoxycillin (n = 11 measurements). This method has the advantages of high sensitivity, fast response and ease of operation. The method was successfully applied to the determination of amoxycillin in raw medicines and capsules. Copyright © 2005 John Wiley & Sons, Ltd.     

Study on the bioactivity of steviol and isosteviol in stevia (Stevia rebaudiana Bertoni)

Steviol and isosteviol are diterpene metabolites that are produced from a shared biosynthesis pathway with gibberellins in stevia leaves. These compounds share a chemically similar structure with gibberellins. An experiment was conducted to find the bioactivity properties of steviol and isosteviol in stevia. During vegetative growth, external solutions of steviol and isosteviol (1.6 × 10^?5 and 1 × 10^?5 M, respectively) were sprayed on stevia shoots and then the stevia growth and metabolites were assessed. The results showed that the stevia leaf growth was reduced by external application of steviol. The leaf growth was not affected by external isosteviol while the stevia height and stem dry weight significantly increased. The antioxidant capacity and steviol glycosides content were increased by external steviol. Our results implicated that the isosteviol bioactivity is similar to that of gibberellin hormone. On the other hand, steviol induces the antioxidant system and stimulates the steviol glycosides biosynthesis in stevia leaves.     

Sensitive determination of 2‐methoxyestradiol in pharmaceutical preparations and serum samples using flow injection chemiluminescence

A rapid and sensitive flow injection chemiluminescence (FI–CL) method is described for the determination of 2‐methoxyestradiol (2ME) based on enhancement of the CL intensity from a potassium ferricyanide–calcein system in sodium hydroxide medium. The optimum conditions for the CL emission were investigated. Under optimized conditions, a linear calibration graph was obtained over the range 1.0 × 10^‐8 to 1.0 × 10^‐6 mol/L (r = 0.998) 2ME with a detection limit (3σ) of 5.4 × 10^‐9 mol/L. The relative standard deviation (RSD) for 5.0 × 10^‐7 mol/L 2ME was 1.7%. As a preliminary application, the proposed method was successfully applied to the determination of 2ME in injection solutions and serum samples. The possible CL mechanism was also proposed. Copyright © 2013 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence determination of diazepam by oxidation with N‐bromosuccinimide

A rapid and sensitive flow‐injection chemiluminescence (FI–CL) method is described for the determination of diazepam based on its reaction with N‐bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy‐transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10^?6 to 2.0 × 10^?4 mol/L with a detection limit of 5.0 × 10^?7 mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10^?5 mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence method for the determination of chloramphenicol based on luminol–sodium periodate order‐transform second‐chemiluminescence reaction

A new chemiluminescence (CL) reaction was observed when chloramphenicol solution was injected into the mixture after the end of the reaction of alkaline luminol and sodium periodate or sodium periodate was injected into the reaction mixture of chloramphenicol and alkaline luminol. This reaction is described as an order‐transform second‐chemiluminescence (OTSCL) reaction. The OTSCL method combined with a flow‐injection technique was applied to the determination of chloramphenicol. The optimum conditions for the order‐transform second‐chemiluminescence emission were investigated. A mechanism for OTSCL has been proposed on the basis of the chemiluminescence kinetic characteristics, the UV‐visible spectra and the chemiluminescent spectra. Under optimal experimental conditions, the CL response is proportional to the concentration of chloramphenicol over the range 5.0 × 10^?7–5.0 × 10^?5 mol/L with a correlation coefficient of 0.9969 and a detection limit of 6.0 × 10^?8 mol/L (3σ). The relative standard deviation (RSD) for 11 repeated determinations of 5.0 × 10^?6 mol/L chloramphenicol is 1.7%. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence determination of ofloxacin using the Ru(bpy)2(CIP)2+−Ce(IV) system and its application

This paper reports a flow‐injection chemiluminescence method for the determination of ofloxacin (OFLX) using the Ru(bpy)₂(CIP)²⁺–Ce(IV) system. Under the optimum conditions, the relative CL intensity was proportional to the concentration of OFLX in the range 3.0 × 10^–8–1.0 × 10^–5 mol/L and the detection limit was 4.2 × 10^–9 mol/L. The proposed method has been successfully applied to the determination of ofloxacin in pharmaceuticals and human urine. The chemiluminescence mechanism of the system is also discussed. Copyright © 2008 John Wiley & Sons, Ltd.