Gibberella intermedia CA3-1羟基化去氢表雄酮的工艺

利用Gibberella intermedia CA3-1对甾体化合物去氢表雄酮(DHEA)进行C7α-羟基化反应研究。用单因素实验的方法考察接种量、装液量、转速、有机溶剂助溶、投料浓度以及底物投加时间对产物7α-羟基去氢表雄酮(7α-OH-DHEA)生成的影响。最终确定最适工艺条件:接种量6%,装液量30 m L(250 m L三角瓶),转速220 r/min,体积分数6%丙二醇助溶,接种时即添加底物,底物DHEA质量浓度1 g/L,转化时间36 h。采用优化后的转化条件,7α-OH-DHEA摩尔得率为72.34%。     

细胞色素P450诱导剂对Gibberella intermedia CA3-1双羟化去氢表雄酮的影响

3β,7α,15α-三羟基雄甾-5-烯-17-酮(7α,15α-diOH-DHEA)是女用口服避孕药\"优思明\"主要成分屈螺酮的关键中间体。为了提高目的产物7α,15α-diOH-DHEA的摩尔得率,研究不同细胞色素P450酶(CYP450)诱导剂对Gibberella intermedia CA3-1转化去氢表雄酮(DHEA)生成7α,15α-diOH-DHEA生物转化过程的影响。结果发现:己烷、DHEA和苯可以显著提高7α,15α-diOH-DHEA的摩尔得率,其中苯是最佳诱导剂。在以上工作基础上,建立了如下的诱导过程:将进入对数生长期(接种后24 h)的种子液以10%的接种量接种到新鲜的转化培养基中,转化12 h后添加体积分数0.8%的苯进行诱导,然后在对数生长期结束时(转化24 h)将8 g/L的底物加入转化培养基进行转化。采用以上诱导条件,在5 L发酵罐中进行了生物转化。与不添加苯的工艺相比,CYP450的浓度提高了43%,7α,15α-diOH-DHEA的摩尔产率提高到68.7%±1.37%,同时转化周期缩短了8 h,这为7α,15α-diOH-DHEA的工业化生产奠定基础。     

1-辛基-3-甲基咪唑二氰铵盐/硫酸铵双水相体系分离纯化银杏黄酮

建立了由亲水性离子液体1-辛基-3-甲基咪唑二氰铵盐([C8mim][N(CN)2])和(NH4)2SO4形成的双水相萃取体系并应用于银杏黄酮的分离纯化研究。研究了盐浓度、体系温度、pH值、NaCl量等因素对银杏黄酮萃取效率的影响;并对下相中无机盐进行回收。体系由18.52%[C8mim][N(CN)2],25.93%(NH4)2SO4构成,加入1.5 mmol NaCl,在室温下进行萃取时萃取效率最佳,在最佳的条件下[C8mim][N(CN)2]/(NH4)2SO4体系对银杏黄酮的萃取效率达96.73%。与传统的双水相体系相比,该体系的萃取效率高,粘度低,同时(NH4)2SO4的回收率达90.54%。[C8mim][N(CN)2]/(NH4)2SO4双水相体系是一种很好的分离纯化银杏黄酮的方法。     

离子液体-微波辅助提取黄芩饮片中四种黄酮类成分(英文)

采用HPLC法建立黄芩饮片中黄芩苷、汉黄芩苷、黄芩素、汉黄芩素四种黄酮类化合物同时测定的方法;并且优选离子液体-微波辅助提取(IL-MAE)黄芩饮片中四种黄酮类成分的最佳工艺。优选的色谱分离条件为:Aglient TC-C18(250×4.6 mm,5.0μm)色谱柱,流动相为0.4%磷酸水(A)和甲醇(B),梯度洗脱程序为0~10min,40%~50%B;10~20 min,50%B;20~30 min,50%~60%B;30~40 min,60%~80%B;50 min,80%B,检测波长280 nm。优选的最佳的黄芩饮片IL-MAE条件为:以溴化-1-丁基-3-甲基咪唑盐为溶剂,固-液比为1∶100,提取时间为3 min,微波功率为264 W;将该工艺所得四种成分的含量与采用中国药典(2010版)所收载的"回流提取法"所得含量进行对比,IL-MAE显著地提高了黄芩饮片中四种黄酮类成分的含量并且缩短了提取时间。研究结果表明,所建立的HPLC分析方法灵敏、准确,可用于黄芩饮片的质量控制;所优选的黄芩饮片的IL-MAE简单,高效,快速,无污染。     

离子液体-微波辅助提取黄芩饮片中四种黄酮类成分(英文)北大核心CSCD

采用HPLC法建立黄芩饮片中黄芩苷、汉黄芩苷、黄芩素、汉黄芩素四种黄酮类化合物同时测定的方法;并且优选离子液体-微波辅助提取（IL-MAE）黄芩饮片中四种黄酮类成分的最佳工艺。优选的色谱分离条件为：Aglient TC-C18（250×4.6 mm,5.0μm）色谱柱,流动相为0.4%磷酸水（A）和甲醇（B）,梯度洗脱程序为0-10min,40%-50%B;10-20 min,50%B;20-30 min,50%-60%B;30-40 min,60%-80%B;50 min,80%B,检测波长280 nm。优选的最佳的黄芩饮片IL-MAE条件为：以溴化-1-丁基-3-甲基咪唑盐为溶剂,固-液比为1∶100,提取时间为3 min,微波功率为264 W;将该工艺所得四种成分的含量与采用中国药典（2010版）所收载的＂回流提取法＂所得含量进行对比,IL-MAE显著地提高了黄芩饮片中四种黄酮类成分的含量并且缩短了提取时间。研究结果表明,所建立的HPLC分析方法灵敏、准确,可用于黄芩饮片的质量控制;所优选的黄芩饮片的IL-MAE简单,高效,快速,无污染。     

分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达(简报)

我们在以往研究中,引入选择性增强体液免疫效应的新型分子佐剂C3d,成功构建了重组避孕疫苗hCGB-C3d3,通过免疫Th2型优势的 BALB/c小鼠和,Th1型优势的C57BL/6小鼠,显示分子佐剂C3d在不同品系小鼠均使免疫效应从Th1型细胞免疫向Th2型体液免疫偏倚。     

用聚乙二醇(PEG)／无机盐双水相体系从枯草杆菌发酵液中提取a-淀粉酶的研究

本文报道分别用聚乙二醇(PEG)／磷酸盐和PEG／(NH·)2SO4双水相体系从枯草轩菌发酵液中提取a-淀粉酶。研究了PEG的平均分子量、PEG浓度,成相的盐的浓度和Nacl的浓度对a-淀粉酶和蛋白质的分配系数以及相比的影响,确定了最佳的操作条件。实验表明在180%PEG 1500／10％磷酸盐／0．05M NaCl的体系中,a-淀粉酶的分配系数为6.62,蛋白质分配系数为1．14,相比为2．5,a-淀粉酶总收率为94．3％,在16％PEG 1500／12％(NH₄)₂SO₄／O．05M NaCl体系中,a-淀粉酶分配系数为82,蛋白质分配系数为5．2,相比为O．92,酶收率为99％,结果表明用PEG／无机盐双水相体系直接从含有菌体的发酵液中提取a-淀粉酶是可行的。     

强啡肽A_(1-13)对谷氨酸诱导的大鼠C6胶质瘤细胞肿胀的影响

目的和方法 :探讨脑水肿发病的细胞机制 ,采用 [3H]OMG摄取的方法测定细胞含水量 ,观察DynA对谷氨酸诱导的大鼠C6胶质瘤细胞肿胀的影响。结果 :①给予 0 .5 ,1.0 ,10 .0mmol/L的谷氨酸作用 1h均可引起细胞的含水量增加 ;②DynA可显著降低谷氨酸诱导肿胀的大鼠C6胶质瘤细胞含水量 ;③κ阿片受体拮抗剂nor BNI可阻断DynA1-13 降低谷氨酸诱导肿胀的大鼠C6胶质瘤细胞含水量的作用。结论 :谷氨酸可诱导大鼠C6胶质瘤细胞肿胀 ;DynA1-13 可通过激活κ阿片受体抑制谷氨酸诱导的大鼠C6胶质瘤细胞肿胀     

三羟基雄甾烯酮高转化菌株的选育及工艺优化

以能将去氢表雄酮 (DHEA) 转化为三羟基雄甾烯酮 (7α,15α-diOH-DHEA) 的赤霉菌Gibberella intermedia CA3-1为出发菌株,对其进行了诱变选育及工艺优化,以期提高转化效率。采用0.12 mg/mL亚硝基胍诱变处理赤霉菌孢子悬液30 min,以350 μmol/L酮康唑为最低抑制浓度进行抗性平板筛选,最终获得一株遗传稳定性较好的高产菌株M-10,其产物摩尔得率达到70.2%,为出发菌株的1.2倍。通过摇瓶转化工艺的优化,确定了突变菌株的最适转化工艺。在优化的转化工艺下,当底物DHEA投料浓度为5 g/L时,7α,15α-diOH-DHEA摩尔得率提高到75.6%,较原始菌株提高了31.3%。     

Ionic liquids in biotechnology: applications and perspectives for biotransformations

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protection against dextran sodium sulfate-induced colitis by dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat

In this study the anti-oxidant effect of DHEA and 7alpha-hydroxy-DHEA against oxidative stress induced by colitis was investigated in vivo in rats. The two steroids were intraperitoneally injected once daily (50 mg/kg body weight) for 7 days before the induction of colitis that was effected by a daily treatment of 5% (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days. This was quantified by the evidence of weight loss, rectal bleeding, increased wall thickness, and colon length. The inflammatory response was assessed by neutrophil infiltration after a histological examination and myeloperoxidase (MPO) activity measurement. Two markers of oxidative damage were measured in colon homogenates after the onset of DSS treatment: protein carbonyls and thiobarbituric acid-reacting substances. The colonic metabolism of corticosterone by 11beta-hydroxysteroid dehydrogenases types 1 and 2 (11beta-HSD) was investigated in control and treated animals. Results indicated that colitis caused a decrease in body weight and colon length. Severe lesions were observed in the colon with a reduced number of goblet cells which contained less mucins. The lesions were associated with increased MPO activity and oxidative damage. Colonic inflammation down and up regulated the 11beta-HSD2 and 11beta-HSD1, respectively. Treatments by DHEA and 7alpha-hydroxy-DHEA attenuated the inflammatory response when MPO activity decreased; but this did not increase the colonic oxidation of corticosterone into 11-dehydrocorticosterone. Both DHEA and 7alpha-hydroxy-DHEA exerted a significant anti-oxidant effect against oxidative stress induced by colitis through reducing the oxidative damage to proteins and lipids. This resulted in a moderate increase in the amount of colonic mucus. Both DHEA and 7alpha-hydroxy-DHEA may prove useful in the prevention or treatment of colitis.     

Dynamic structure–function relationships in enzyme stabilization by ionic liquids

The stability of α-chymotrypsin and Candida antarctica lipase B (CALB) in two ionic liquids (i.e. 1-ethyl-3-methyl-imidazolium, bis[(trifluoromethyl)sulfonyl]imide [emim] [NTf₂], and butyl-trimethylamonium bis[(trifluoromethyl)sulfonyl]imide [btma] [NTf₂]) has been studied. Both enzymes were strongly stabilized by the ionic liquids, the respective half-life times increasing 96 and 1660 times, with respect to those obtained in classical organic solvents such as 1-propanol and hexane, respectively. The stabilization of both enzymes by ionic liquids may be related to the associated structural changes of proteins that they can be observed by both fluorescence and circular dichroism spectroscopic studies.     

Rapid synthesis of long chain fatty acid esters of steroids in ionic liquids with microwave irradiation: Expedient one-pot procedure for estradiol monoesters

We report the rapid synthesis (1 min) in high yield of fatty acid ester (FAE) derivatives of several steroids under microwave irradiation in an ionic liquid (IL). An expedient regioselective hydrolysis at C-3 of estradiol diesters is also reported.     

Antioxidant effects of dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat colon, intestine and liver

This study examined in healthy male Wistar rats the in vivo antioxidant effect of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-DHEA administered by intraperitoneal injections (50 mg/kg body weight) for 2 or 7 days. Markers of oxidative damage to lipids (thiobarbituric acid-reacting substances, TBARS) and to proteins (protein carbonyls) were assessed in colon, small intestine, and liver homogenates. DHEA and 7alpha-hydroxy-DHEA caused a decrease in body weight. DHEA treatment significantly increased liver, colon, and small intestine cell weights. After 7 days, DHEA exerted an antioxidant effect in all organs studied. In the colon, oxidative damage protection was accompanied by a goblet cell proliferation and increase in acidic mucus production. After 2 days, the antioxidant effect of 7alpha-hydroxy-DHEA was mainly observed in the liver. Nonprotein sulfhydryl groups (mostly glutathione levels) were altered by DHEA in the liver whereas they remained unchanged after 7alpha-hydroxy-DHEA treatment. The results indicate that in healthy animals, DHEA exerts a protective effect, particularly in the colon, by reducing the tissue susceptibility to oxidation of both lipids and proteins. This effect was not limited to a specific tissue, whereas the metabolite 7alpha-hydroxy-DHEA exerted its antioxidant effect towards the two markers of oxidative damage earlier than DHEA, and mainly in the liver.     

NMR evidence for the participation of triflated ionic liquids in glycosylation reaction mechanisms

A systematic low-temperature NMR study of a glycosylation reaction was performed in the presence of different ionic liquids and acidic catalysts. The influence of the triflate anion derived from [emim][OTf] on the stereochemistry of the glycosylation products was evaluated.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.