R－A型甜菊糖甙的研制

以甜菊（ＳｔｅｖｉａｒｅｂａｕｄｉａｎａＢｅｒｔｏｎｉ）的干叶为原料生产的甜菊糖甙是一种食用天然甜味剂。提高甜菊糖甙（Ｓｔｅｖｉｏｓｉｄｅｓ）中优质甜味成分Ｒ－Ａ（甜菊Ａ３甙：ＲｅｂａｕｄｉｏｓｉｄｅＡ）的含量比例是生产中急待解决的课题。本文以Ｒ－Ａ型和Ｓｔ（甜菊甙Ｓｔｅｖｉｏｓｉｄｅ）型甜菊叶为原料,采用Ａ和Ｂ两种方法进行甜菊糖甙的提取,通过Ｂ法获得Ｒ－Ａ含量比例高的甜菊糖甙即Ｒ－Ａ型甜菊糖甙,为甜菊糖甙产品优质化提供了新方法、新技术     

仙茅根茎中的配糖体

从仙茅（ Ｃｕｒｃｕｌｉｇｏ ｏｒｃｈｉｏｉｄｅｓＧａｅｒｔｎ） 根茎中分离到７ 个化合物, 经光谱分析推定其化学结构分别为： ２, ６ － 二甲氧基苯甲酸（Ａ） ; 苔黑酚葡萄糖甙（Ｂ） ; 仙茅素Ａ （Ｃ） ; 仙茅甙（Ｄ） ; ２４ｓ, ３β, １１α, １６β, ２４ － 四羟基环阿尔廷醇－ ３ － Ｏ－ α－ Ｌ－ 鼠李吡喃糖基（１ →２） － β－Ｄ－ 葡萄吡喃糖甙（Ｅ） ; ２４ｓ, ３β, １１α, １６β, ２４ － 四羟基环阿尔廷醇－ ３－ Ｏ－ β－ Ｄ－ 葡萄吡喃糖基（１ →２） － β－ Ｄ－ 葡萄吡喃糖甙（Ｆ） 和胡萝卜甙（Ｇ） 。Ｆ为一新甙。     

鞭打绣球中的苯丙素甙和环烯醚萜甙

从鞭打绣球（ＨｅｍｉｐｈｒａｇｍａｈｅｔｅｒｏｐｈｙｌｌｕｍＷａｌｌ．）（玄参科）的全草中分离到２个新的苯丙素甙,命名为鞭打绣球甙Ａ和Ｂ（ｈｅｍｉｐｈｒｏｓｉｄｅＡａｎｄＢ）,２个已知的苯丙素甙,ｐｌａｎｔａｍａｊｏｓｉｄｅ和ｐｌａｎｔａｉｎｏｓｉｄｅＤ,以及３个已知的环烯醚萜甙,ｇｌｏｂｕｌａｒｉｃｉｓｉｎ,ｇｌｏｂｕｌａｒｉｎ和ｉｓｏ－ｓｃｒｏｐｈｕｌａｒｉｏｓｉｄｅ．通过化学和光谱分析,鞭打绣球甙Ａ和Ｂ的结构分别鉴定为２－（３－羟基－４－甲氧基苯基）乙基０－β－Ｄ－葡萄吡喃糖基（１→３）－４－Ｏ－反式阿魏醚基－β－Ｄ－葡萄吡喃糖试和２－（３,４－二羟基苯基）乙基Ｏ－［６－Ｏ－乙醚基－β－Ｄ－葡萄吡喃糖基（１→３）］－４－Ｏ－反式咖啡醚基－β－Ｄ－葡萄吡喃糖甙．     

赬桐的化学成分

应用正反相硅胶柱层析、中压柱层析、制备性薄层层析等手段,从桐（Ｃｌｅｒｏｄｅｎｄｒｕｍｊａｐｏｎｉｃｕｍ）中分离得到４个苯丙素甙类成分,根据光谱数据及化学方法,鉴定为马蒂罗甙（１）,单乙酰马蒂罗甙（２）,贞桐甙Ａ（４）以及阿克甙（３）,其中,贞桐甙Ａ是一新化合物。同时还得到２２,２３一二氢菠甾醇、豆甾醇、２５,２６一去氢豆甾醇,乌索酸,丁二酸酐和小麦黄素等。     

秦艽中的环烯醚萜甙成分

秦艽为著名传统中药。中青海西宁产秦艽的甲醇提取物的水溶性部分分离到１个新裂环烯醚萜甙,命名为秦艽甙Ａ,２个已知的环烯醚萜甙即龙胆苦甙和哈马甙以及２个甾醇甙,胡萝卜甙和β－谷甾醇－３－Ｏ－龙胆糖甙。这些化合物的结构通过红外光谱、紫外光谱、核磁共振波谱（包括－维氢醇－３－Ｏ－龙胆糖甙。这些化合牧的结构通过红外光谱、紫外光谱、核磁共振波谱（包括一维氢谱、碳谱、氢－氢相关谱、碳－氢相关谱、远程碳－氢相关谱     

慈溪麦冬甙A和B的结构

从浙江慈溪产中药麦冬（Ｏｐｈｉｏｐｏｇｏｎ ｊａｐｏｎｉｃｕｍ）中分离得到２个新的Ｃ２７甾体甙－－慈溪麦冬甙Ａ（２）和Ｂ（３）以及已知甙ｏｐｈｉｏｇｅｎｉｎ３－氧－α－Ｌ－鼠李糖吡喃基（１→２）「β－Ｄ－木糖吡喃基（１→３）」－β－Ｄ－葡萄糖吡喃甙（２）和ｏｐｈｉｏｇｅｎｉｎ ３－氧－α－Ｌ－鼠李糖吡喃基（１→２）「β－Ｄ－木糖吡喃基（１→３）」「β－Ｄ－葡萄糖吡喃基（１→４）」－β－Ｄ－葡萄糖吡     

贞桐的化学成分

用正反相硅胶柱层析、中压柱层析、制备性薄层层析等手段,从贞桐中分离得到４个苯丙素甙类成分,根据光谱数据及化学方法,鉴定为马蒂罗甙（１）,单乙酰马蒂甙（２）,贞桐甙Ａ（４）以及阿克甙（３）,其中,贞桐甙Ａ是一新化合物。同时还得到２２,２３－二氢菠甾醇,豆甾醇,２５,２６－去氢豆甾醇,乌索酸,丁二酸酐和小麦黄素等。     

单条草的皂甙成分

从单条草（ＬｙｓｉｍａｃｈｉａｃａｎｄｉｄａＬｉｎｄｌ）全草中分离出３个皂甙类化合物,经波谱分析并结合化学方法鉴定为：报春花素Ａ３ＯβＤ吡喃木糖基（１→２）βＤ吡喃葡萄糖基（１→４）［βＤ吡喃葡萄糖基（１→２）］αＬ吡喃阿拉伯糖甙（１）、原报春花素Ａ３ＯβＤ吡喃木糖基（１→２）βＤ吡喃葡萄糖基（１→４）［βＤ吡喃葡萄糖基（１→２）］αＬ吡喃阿拉伯糖甙（ｌｙｓｉｋｏｉａｎｏｓｉｄｅ,２）和α菠甾醇葡萄糖甙（３）。其中１是新化合物,命名为单条草甙（ｃａｎｄｉｄｏｓｉｄｅ）。     

印楝愈伤组织的化学成分

从印楝（Ａｚａｄｉｒａｃｈｔａ ｉｎｄｉｃａ Ａ．Ｊｕｓｓ）的愈伤组织中,共分离鉴定出５个化合物,经理化性质和波谱分析分别确定为：柳杉酚⑴,豆甾醇⑵,软酯酸－１－甘油酯⑶,豆甾醇－３－Ｏ－β－Ｄ－吡喃葡萄糖甙⑷,豆甾醇－３－Ｏ－β－Ｄ－吡喃葡萄糖甙－６’－十六烷酸酯⑸。     

葛藤与葛根中异黄酮类成分的比较

分析比较了野葛〔Ｐｕｅｒａｒｉａｌｏｂａｔａ（Ｗｉｌｄ．）Ｏｈｗｉ〕的藤（葛藤）和根（葛根）的主要异黄酮类活性成分。从葛藤中首次分离得到３个化合物,经化学方法和光谱鉴定,证实为大豆甙元（Ａ）、大豆甙（Ｂ）和葛根素（Ｃ）。采用双波长薄层扫描法测定葛藤与葛根中上述３种异黄酮化合物的含量,葛藤中大豆甙元、大豆甙和葛根素的含量分别为０．１９５％,３．９３３％和２．４８１％;葛根中则分别为０．０５９％,０．７１４％和４．３１５％。研究结果为葛藤新药源的开发利用提供了科学依据     

Rebaudioside A Enhances LDL Cholesterol Uptake in HepG2 Cells via Suppression of HMGCR Expression

Background:Rebaudioside A is one of the major diterpene glycosides found in Stevia had been reported to possess anti-hyperlipidemic effects. In this study, we explore the potential cholesterol-regulating mechanisms of Rebaudioside A in the human hepatoma (HepG2) cell line in comparison with simvastatin.Methods:Cells were incubated with Rebaudioside A at several concentrations (0-10 µM) to determine the cytotoxicity by the MTT assay. Cells were treated with selected dosage (1 and 5 µM) in further experiments. Total cellular lipid was extracted by Bligh and Dyer method and subjected to quantitative colorimetric assay. To illustrate the effect of Rebaudioside A on cellular lipid droplets and low-density lipoprotein receptors, treated cells were subjected to immunofluorescence microscopy. Finally, we investigated the expression of experimental gene patterns of cells in response to treatment.Results:In this study, cytotoxicity of Rebaudioside A was determined at 27.72 µM. Treatment of cells with a higher concentration of Rebaudioside A promotes better hepatocellular cholesterol internalization and ameliorates cholesterol-regulating genes such as HMGCR, LDLR, and ACAT2. Conclusion:In conclusion, our data demonstrated that Rebaudioside A is capable to regulate cholesterol levels in HepG2 cells. Hence, we proposed that Rebaudioside A offers a potential alternative to statins for atherosclerosis therapy.Key Words: Rebaudioside A, Anti-hypercholesterolemia, Lipid droplets, Low-density lipoprotein, HMGCR     

Efficient enzymatic production of rebaudioside A from stevioside

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30?h with 2.4?mM stevioside, 7.2?mM sucrose, and 0.006?mM UDP was 78%.     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

Effect of Rebaudioside A, a diterpenoid on glucose homeostasis in STZ-induced diabetic rats

Rebaudioside A (Reb A), a major constituent of Stevia rebaudiana, was recently proposed as an insulinotropic agent. The aim of this investigation was to evaluate the antihyperglycemic effect of Reb A on the activities of hepatic enzymes of carbohydrate metabolism in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in adult male Albino Wistar rats, weighing 180-200 g, by a single intraperitoneal injection at a dose of STZ (40 mg/kg body weight). Diabetic rats showed significant (P<0.05) increase in the levels of plasma glucose and glycosylated hemoglobin and significant (P<0.05) decrease in the levels of plasma insulin and hemoglobin. Activities of gluconeogenic enzymes such as glucose-6-phosphatase and fructose-1,6-bisphosphatase were significantly (P<0.05) increased while hexokinase and glucose-6-phosphate dehydrogenase were significantly (P<0.05) decreased in the liver along with glycogen. Oral treatment with Reb A to diabetic rats significantly (P<0.05) decreased blood glucose and reversed these hepatic carbohydrate metabolizing enzymes in a significant manner. Histopathology changes of pancreas confirmed the protective effects of Reb A in diabetic rats. Thus, the results show that Reb A possesses an antihyperglycemic activity and provide evidence for its traditional usage in the control of diabetes.     

The positive role of steviol glycosides in stevia (Stevia rebaudiana Bertoni) under drought stress condition

Steviol glycosides (SVglys) are a group of diterpenoids mainly present in the leaves of stevia (Stevia rebaudiana Bertoni). An experiment was conducted to find the functional role of SVglys compounds in stevia affected by drought stress. In this study, a liquid blend of SVglys (200 ppm) was sprayed on stevia plants grown in well-watered (90% field capacity) and drought-stress conditions (45% field capacity) and then the morphological traits and metabolites were evaluated. It was observed that leaf losses caused by drought stress were stopped through external application of SVglys and consequently the harvest index of stevia was increased. Metabolite analysis of stevia leaves showed that the total SVglys content was significantly decreased due to drought stress, but was compensated by external application of SVglys. Among the SVglys, Rebaudioside A responded more to external SVglys. A slight promotion in total antioxidant activity of stevia leaves was observed when external SVglys was applied. The glucose availability in stevia leaves was increased by external application of SVglys but only in well-watered plants. According to our findings, it can be concluded that in stevia, SVglys may have a positive function in drought stress tolerance by exerting a protective role under such conditions.     

Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi-enzyme system

Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi-enzyme reaction system with S. rebaudiana UDP-glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two-step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side-product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wild-type UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l⁻¹ (1.6 times of wild-type UGTSL2) rebaudioside D from 20 g l⁻¹ stevioside after reaction for 24 h. This system is useful for large-scale rebaudioside D production and expands our understanding of the pathways involved in its synthesis.     

Effect of two plant growth retardants on steviol glycosides content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni)

Two greenhouse experiments were conducted to study the effect of two plant growth retardants, Chlorocholine chloride (CCC) and Paclobutrazol (PBZ), on growth, Steviol glycosides (SVglys) content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni). Five concentrations of CCC (0, 250, 500, 750 and 1,000 ppm) and PBZ (0, 6, 12, 18 and 24 ppm) with three replications were applied to Stevia plants as treatments based on completely randomized design. CCC was sprayed on Stevia shoots, but PBZ was applied as a drench. The obtained results showed that CCC reduced plant height but improved leaf and stem dry weight, especially with 750 ppm concentration. Total SVgly content and consequently SVglys yield were significantly reduced by CCC application, and 1,000 ppm of CCC concentration was more effective than other treatments. PBZ had no effect on Stevia height while it significantly enhanced stem and dry weight at 12 ppm. Moreover, PBZ remarkably increased total SVglys contents, SVglys yield, and Rebaudioside A/Stevioside ratio. Total antioxidant capacity significantly varied with CCC and PBZ and the highest activity was obtained with 1,000 and 12 ppm of CCC and PBZ, respectively. The results of these experiments indicated that, although CCC and PBZ are plant growth retardants and act as anti-gibberellins, only CCC reduced plant height and SVglys production in Stevia. On the contrary, PBZ at 12 ppm concentration, improved Stevia growth, SVglys production, and antioxidant capacity.     

The glio-toxic mechanism of α-aminoadipic acid on cultured astrocytes

The mechanism of action of the glutamate analogue α-aminoadipic (A A A) acid was investigated in terms of its toxicity to cultured astrocytes. A A A was more toxic to type 1 astrocytes than type 2 astrocytes. Also the higher toxicity of the L-isomer as compared to the D-isomer was seen on type 1 astrocytes but not type 2. The toxicity of A A A can be reduced by co-culture of type 1 astrocytes with microglia. This inhibition may be due to glutamate release by microglia. No such effect is seen for type 2 astrocytes. The major uptake route for A A A by type 1 astrocytes is through the sodium dependent glutamate port. Both isomers of A A A are toxic to dividing astrocytes. The D-isomer appears to be toxic only for mitotic cells. The mechanism of this toxicity is protein synthesis dependent. It is suggested that A A A is toxic to mitotic astrocytes by interference with protein synthesis needed for cell division. D-A A A as opposed to L-A A A may prove a valuable tool for investigation of astrocyte proliferation in development and disease.