桃拟茎点霉对孕酮的生物转化研究

研究了一株桃拟茎点霉（Phomopsis amygdale）对孕酮的生物转化,并对该转化菌株的形态学和分子生物学进行鉴定,转化产物经分离纯化后,运用核磁共振（NMR）、质谱（MS）、红外光谱（IR）分析进行结构鉴定。该菌株转化孕酮得到2个转化产物,转化产物分别为15β-羟基孕酮和6β,15β-二羟基孕酮。结果表明,桃拟茎点霉具有转化甾体的能力,对孕酮进行生物转化的主要产物是15β-羟基甾体衍生物。     

一株黄芪内生真菌甾醇类代谢产物的分离和鉴定

目的:研究分离自夏季黄芪根部的内生真菌AR-17-S03的次级代谢产物。方法:采用组织学的方法从恒山黄芪中分离内生真菌,真菌的鉴定采用形态学法结合分子生物学法,采用硅胶柱色谱法、薄层层析制备色谱(pTLC)、Sephadex LH-20凝胶柱色谱法和半制备HPLC色谱法分离内生真菌代谢产物,采用质谱法、核磁共振法结合物化性质鉴定分离得到的9个化合物。结果:经形态学结合分子生物学鉴定该菌株黑曲霉,为9个甾醇类化合物鉴定为麦角甾醇、过氧化麦角甾醇、胆固醇、啤酒甾醇、23-甲基麦角甾-7,22-二烯-3β,5α,9α-三醇、5α,8α-表二氧麦角甾-6,9(11),22-三烯-3β-醇、5α,8α-表二氧-23-甲基麦角甾-6,22-二烯-3β-醇、麦角甾-4,6,8(14),22-四烯-3-酮、3β,5α-二羟基麦角甾-7,22-二烯-6-酮。结论:其中,23-甲基麦角甾-7,22-二烯-3β,5α,9α-三醇、5α,8α-表二氧-23-甲基麦角甾-6,22-二烯-3β-醇、麦角甾-4,6,8(14),22-四烯-3-酮是首次从黄芪内生真菌中分离得到。     

药食两用小蓟中细胞毒活性成分解析

本课题组前期研究发现,小蓟乙醇提取石油醚萃取部位具有较好的抑制多种人肿瘤细胞毒活性的作用,现对其中强活性亚组分的化学成分进行研究,挖掘发挥细胞毒活性的功能因子。在体外抑制细胞毒活性指导下,应用多种色谱技术进行分离纯化,采用波谱学方法进行结构鉴定,并结合文献数据对照确定化合物结构。从石油醚的1个活性组分中共分离鉴定了5个甾体化合物,分别为:3β-羟基豆甾-5-烯-7-酮、豆甾-22-烯-3β,5α,6β-三醇、6β-羟基-豆甾-4-烯-3-酮、6β-羟基-豆甾-4,22-二烯-3-酮和6β-羟基-芸苔甾-4-烯-3-酮。5个化合物均为首次从蓟属植物中发现;除化合物α-豆甾-22-烯-3β,5α,6β-三醇外,其余4个化合物对不同人肿瘤细胞均具有一定的抑制活性。     

降解植物甾醇产9α-羟基雄烯二酮工程菌株构建及发酵工艺优化

9α-羟基雄烯二酮(9α-OH-AD)作为一种不可替代的甾体药物中间体,是合成多种皮质类甾体激素类药物的重要原料。本研究通过在产雄甾-4-烯-3,17-二酮(AD)的菌株Mycobacterium sp.TFZ中表达3-甾酮-9α-羟基化酶(KSH)基因kshA和kshB,获得了一株可直接转化植物甾醇生产9α-OH-AD的工程菌株Mycobacterium sp.TFZ3215,然后以9α-OH-AD产量为评价指标,对油水两相转化体系中添加乳化剂吐温的种类和含量进行优化。研究结果表明:与出发菌株Mycobacterium sp.TFZ相比,工程菌Mycobacterium sp. TFZ3215发酵液中9α-OH-AD的含量由1.7%提高到94.7%,AD的含量由80.1%减少到3.4%。进一步通过在油水两相发酵体系中添加2 g/L的吐温-80后,9α-OH-AD的产量由1.69 g/L增加到7.53 g/L,提高了3.4倍。本研究成功构建了高产9α-OH-AD的工程菌株,并对油水两相发酵体系进行了优化,9α-OH-AD的产量达到7.53 g/L,具有极好的产业化前景。     

一株木防己拟盘多毛孢的鉴定及其次生代谢产物分析

目的:研究分离自葡萄状枝瑚菌Ramariabotrytis子实体的16072314菌株分类学地位及其次生代谢产物成分。方法:采用形态学观察结合ITS序列分析法对16072314菌株进行菌种鉴定;利用硅胶柱色谱、凝胶柱色谱、制备薄层色谱和半制备高效液相色谱对16072314菌株固体发酵产物的化学成分进行分离纯化,根据理化性质及波谱数据分析鉴定化合物。结果:经鉴定16072314菌株为木防己拟盘多毛孢Pestalotiopsis cocculi;从16072314菌株的代谢产物中分离纯化了8个化合物,分别鉴定为7-羟基香豆素,阿魏酸,麦角甾醇,对羟基苯甲醛,对羟基苯乙醇,齐墩果酸,β-谷甾醇,过氧麦角甾醇。结论:8个化合物均为首次从木防己拟盘多毛孢中分离得到,阿魏酸为首次从拟盘多毛孢属中分离得到。本研究为拟盘多毛孢属的真菌资源利用和开发奠定了基础。     

大王棕内生真菌次级代谢产物的研究

通过高效液相色谱法,正相硅胶柱色谱法、凝胶柱色谱法等分离方法对大王棕内生真菌的次级代谢产物进行分离纯化,再应用NMR和MS等现代波谱分析方法对其进行结构鉴定,从中首次分离鉴定了6个化合物,分别为1’,2’-epoxy-3’,4’-didehydropenicillide.;丁二酸;3β-羟基-5α,8α-过氧化麦角甾-6,22-二烯;ergosta-7,22-dien-3β,5α,6α-triol;3β-羟基-麦角甾-5,7,22-三烯;4-羟基苯乙酸。     

莱菔子化学成分的分离与鉴定

目的：研究莱菔子化学成分,探索生品莱菔子的物质基础。方法：采用大孔吸附树脂HP-20、聚苯乙烯型大孔吸附树脂（MCI gel）、ODS、硅胶及LiChroprep RP-18等色谱技术进行分离纯化,运用光谱学方法对得到的化合物进行结构鉴定。结果：从莱菔子甲醇提取物中分离鉴定了16?个化合物：顺-13-二十二碳烯酸（1）、豆甾-4-烯-3-酮（2）、（22E,24R）-麦角甾-5,22-二烯-3β-醇（3）、豆甾-5-烯-3β-醇（4）、反-阿魏酸甲酯（5）、反-芥子酸甲酯（6）、β-豆甾醇-3-O-β-D-葡萄糖苷（7）、α-D-吡喃半乳糖基-（1-6）-α-D-吡喃葡萄糖基-（1-2）-β-D-呋喃果糖苷（8）、β-D-呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（9）、β-D-（3-芥子酰基）呋喃果糖基-α-D-葡萄糖苷（10）、β-D-（3-芥子酰基）呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（11）、β-D-（3,4-二芥子酰基）呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（12）、异鼠李素-3-O-β-D-葡萄糖苷（13）、异鼠李素-3,4’-O-β-D-二葡萄糖苷（14）、异鼠李素-3-O-β-D-葡萄糖-7-O-α-L-鼠李糖苷（15）、3’-O-甲基-（－）-表儿茶素-7-O-β-D-葡萄糖苷（16）。结论：化合物2、3、7～10、13～16为首次在该植物中得到。     

杨桃病原真菌分离鉴定及代谢产物研究

为了解杨桃病原真菌的种类、生物学特性和代谢产物类型,本研究通过组织分离法对杨桃果实发病部位进行病原真菌分离,通过形态学结合基于ITS序列分析的分子鉴定方法对真菌进行鉴定,并对其生物学特性进行探究,使用液相色谱分离纯化菌株代谢产物,利用核磁共振谱确定代谢产物结构。结果表明：4株病原真菌分别是木贼镰刀菌（Fusarium equiseti）、新拟盘多毛孢属（Neopestalotiopsis sp.）、裂褶菌（Schizophyllum commune）和枝状枝孢菌（Cladosporium cladosporioides）。这4株菌均可导致杨桃果实发病。进一步从木贼镰刀菌Fusarium equiseti发酵产物中分离得到4种化合物,分别为交链孢酚、1,4,7-三羟基-3,9-二甲氧基-6H-苯并[c]色烯-6-酮、红粉苔酸和7-羟基-4-甲基-1(3H)-异苯并呋喃酮。生物学特性的研究结果表明,4株病原真菌的最适生长温度在25～28℃,在pH4～10的范围内均能生长。     

Mycobacterium sp. BFZ304转化植物甾醇产9α-羟基雄烯二酮培养基的响应面优化

本研究以Mycobacterium sp.BFZ304作为实验菌株,研究了不同碳源、氮源、磷酸盐等培养基成分对Mycobacterium sp.BFZ304发酵转化植物甾醇生产9α-羟基雄烯二酮的影响。在单因素实验的基础上,以9α-羟基雄烯二酮的产量为衡量指标,采用响应面法优化了转化培养基的组成,并建立了玉米浆、葡萄糖、硝酸钠、磷酸氢二铵变化的二次回归方程,探讨了各因子对9α-羟基雄烯二酮产量的影响。最终确定最适宜的转化培养基为葡萄糖10 g/L、玉米浆40 g/L、硝酸钠6 g/L、磷酸氢二铵0.7 g/L。在此条件下,底物植物甾醇添加量为10 g/L时,9α-羟基雄烯二酮的产量可达到4.86 g/L,底物植物甾醇的转化率比优化前提高了近40%,具有极好的产业化前景。     

石花菜醇提物成分分析

目的：研究石花菜的成分,为其功能食品的研发提供基础数据。方法：采用薄层色谱、液-质谱联用方法分析石花菜醇提物的化学成分的丰富度。通过硅胶柱、高效液相等色谱方法对其进行分离纯化,并用气-质谱联用、核磁结合文献确证化合物结构;并对部分化合物抗氧化活性进行测定。结果：薄层色谱、液-质谱联用分析表明石花菜醇提物中含有丰富的次级代谢产物,且很可能是甾醇类成分。采用气-质谱联用方法从中鉴定到29个化合物（5个为甾醇类化合物）,其中棕榈酸（46.76%）、棕榈酸乙酯（13.82%）、胆甾醇（7.20%）、胆甾-4,6-二烯（4.99%）为含量最高的4个化合物。经色谱分离、核磁鉴定出9个单体化合物,分别为胆甾醇（1）、1,2-二醇植烯（2）、帕格甾醇C(3)、6-羟基-胆甾-4-烯-3-酮（4）、胆甾-5-烯-7-酮（5）、胆甾-5-烯-3β,7α二醇（6）、胆甾-5-烯-3β,7β二醇（7）、胆甾-4-烯-6-酮（8）、3-羟乙基-2-甲基-马来酰亚胺（9）。其中,化合物7清除DPPH自由基和ABTS自由基能力最好,其IC50值分别为180μg/mL和12.2μg/mL。结论：首次系统研究了石花菜醇...     

Fate of endogenous steroid hormones in steer feedlots under simulated rainfall-induced runoff

Steroid hormones pose potential risks to fish and other aquatic organisms at extremely low concentrations. To assess the factors affecting the release of endogenous estrogenic and androgenic steroids from feedlots during rainfall, runoff, and soil samples were collected after simulated rainfall on a 14-steer feedlot under different rainfall rates and aging periods and analyzed for six steroid hormones. While only 17α-estradiol, testosterone, and progesterone were detected in fresh manure, 17β-estradiol, estrone, and androstenedione were present in the surficial soil after two weeks. In the feedlot surficial soil, concentrations of 17α-estradiol decreased by approximately 25% accompanied by an equivalent increase in estrone and 17β-estradiol. Aging of the feedlot soils for an additional 7 days had no effect on estrogen and testosterone concentrations, but androstenedione concentrations decreased substantially, and progesterone concentrations increased. Androstenedione and progesterone concentrations in the surficial soil were much higher than could be accounted for by excretion or conversion from testosterone, suggesting that other potential precursors, such as sterols, were converted after excretion. The concentration of androgens and progesterone in the soil were approximately 85% lower after simulated rainfall, but the estrogen concentrations remained approximately constant. The decreased masses could not be accounted for by runoff, suggesting the possibility of rapid microbial transformation upon wetting. All six steroids in the runoff, with the exception of 17β-estradiol, were detected in both the filtered and particle-associated phases at concentrations well above thresholds for biological responses. Runoff from the aged plots contained less 17α-estradiol and testosterone, but more estrone, androstenedione, and progesterone relative to the runoff from the unaged plots, and most of the steroids had a lower particle-associated fraction.     

Isolation and structural characterisation of five new and 14 known metabolites from the commercial starfish Archaster typicus

From the commercially available starfish Archaster typicus, five new (1–5) and 14 known (6–19) metabolites were isolated and identified. Detailed 1D and 2D NMR spectroscopic data, including ¹H, ¹³C, DEPT, HSQC, HMBC, and NOESY, established the structures of the new metabolites as sodium 5α-cholesta-9(11),24-dien-3β,6α,20β-triol-23-one 3-sulphate (1), sodium 5α-cholesta-9(11)-en-3β,6α,20β-triol-23-one 3-sulphate (2), sodium (25R)-5α-cholestane-3β,4β,6α,8,14α,15β,26-heptaol-15-sulphate (3), sodium (25R)-5α-cholestane-3β,6α,8,14α,15β,26-hexaol 15-sulphate (4), and sodium cholest-25(27)-ene-3β,4β,5α,6α,7β,8β,14α,15α,24,26-decanol 6-sulphate (5). Other spectroscopic techniques, including IR, ESI–MS, and HR-ESI–MS, were also adopted to further confirm the structures of the metabolites. These five steroids (1–5) are reported in nature for the first time. All of the steroids found in A. typicus (1–12) were tested for anticancer activities against MDA-MB-435 and Colo205 tumour cells. However, only sodium 5α-cholesta-9(11)-en-3β,6α,20β-triol-23-one 3-sulphate (2) and 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol (6) exhibited weak activities.     

两相发酵系统中提取分离雄烯二酮的研究

分枝杆菌B696在构建的油水两相发酵系统中具有较高的降解植物甾醇侧链成为雄烯二酮(4-AD)的能力,但如何提高产物的提取率和纯度是目前要解决的难点之一。采用不同的预处理方式及萃取、结晶等操作对提取率的影响进行研究,确定了产物4-AD 的较适提取工艺条件为50℃加热处理10min,甲醇萃取油相,萃取温度15℃,萃取比3:1,结晶温度10℃,时间15h。此工艺下4-AD 提取率为76.61%,产品纯度达98.24%。     

白鱼腐败细菌的分离与鉴定

通过对不同温度条件下白鱼（Anabarilius）菌落数量的统计发现, 与37 ℃相比,25 ℃培养条件更有利于白鱼携带细菌的生长,也更容易导致白鱼的腐败。通过对分离自腐败的白鱼中21 株细菌的16S rDNA序列分析后,最终确定得到了11 株不同的菌株。同时结合生化鉴定结果,将这些菌种分属7 个不同的属：2 株摩根氏菌（Morganella）,3 株变形杆菌（Proteus）,2 株漫游球菌（Vagococcus）,1 株葡萄球菌（Staphylococcu）,1 株泰瑞芽孢杆菌（Terribacillus）,1 株赖氨酸芽孢杆菌（Lysinibacillus）,1 株透明颤菌（Vitreoscilla）。其中摩根氏菌和变形杆菌同属肠杆菌科,是腐败白鱼的优势菌,可能是白鱼的特定腐败菌 （specific spoilage organisms,SSO）。     

Natural occurrence of steroid hormones in food

The natural occurrence of the sex steroid hormones progesterone, testosterone, 17β-estradiol and estrone in food was investigated in a survey of the German market basket. The main metabolic precursors, intermediates and metabolites (pregnenolone, androstenedione, hydroxyprogesterone, dehydroepiandrosterone (DHEA), dihydrotestosterone, androsterone, 17α-estradiol and estriol) were also included in the investigation. Particular attention was paid to DHEA, which is said to have anti-aging properties. Analysis was carried out by gas chromato-graphy-mass spectrometry (GC-MS). The steroid patterns of pork, meat products, fish and poultry resemble those known for beef. Milk and milk products reflect the hormone profile of female cattle with high amounts of progesterone, which accumulates with increasing milk fat content. Milk products supply about 60–80% of ingested female sex steroids. Eggs are a considerable source of any of the investigated steroids and contribute to the nutritional hormone intake in the same order as meat and fish (10–20%). In vegetable food no estrogens could be detected. Plants supply testosterone in the same order as meat and milk products (20–40%) though. They contain considerable amounts of hormone precursors as well (contribution to DHEA supply: about 80%). In comparison to the human daily production of steroid hormones the nutritional supply (about 10 μgd⁻¹ progesterone, 0.05 μg d⁻¹ testosterone, 0.1 μg d⁻¹ estrogens, 0.5 μg d⁻¹ DHEA) is insignificant.     

Wide-Bore Capillary Gas Chromatographic Method for Quantification of Cholesterol Oxidation Products in Egg Yolk Powder

A wide-bore capillary gas chromatographic (GC) method was developed for quantification of cholesterol oxidation products in egg yolk powder. Baseline separations were achieved between 7α- and 7β-hydroxycholesterol, cholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide and 7-ketocholesterol chromatographed as the trimethylsilylated sterols. Excellent response linearity was obtained for each cholesterol oxidation product, permitting reliable quantification. Recoveries from freeze-dried egg yolk spiked with various levels of cholesterol oxidation products ranged from 78.2 to 95.1%. The coefficients of variation compared favorably to those for packed column GC methods.     

Confirmation of the progesterone receptor as an efficient marker of treatment with 17β-estradiol in veal calves

Under current European Union legislation the use of anabolic steroids in food-producing livestock is banned because of their long-term adverse effects on human health. We examined the expression profile of the immunohistochemical marker progesterone receptor in veal calves’ sex accessory glands following experimental administration of anabolic compounds. The aim was to confirm the accuracy of the immunohistochemical approach in the detection of the over-expression of the progesterone receptor induced by the administration of sexual steroids at low levels (17β-estradiol and nandrolone alone or in combination). A total of 217 male veal calves were randomly divided into four groups: group A (104 calves) treated with 17β-estradiol (5 mg/head; 4 weekly injections); group B (20 calves) treated with nandrolone (50 mg/head; 4 weekly injections); group C (20 calves) treated with the association of the two steroids (5 mg estradiol + 50 mg nandrolone; 4 weekly injections); and group K (73 calves) kept as a control. All the sexual accessory glands were collected at the slaughterhouse (15 days after the last administration) and subjected to immunohistochemical staining with anti-progesterone receptor antibody. All the calves treated with 17β-estradiol alone or in association with nandrolone (groups A and C) showed strong positivity, while nandrolone-treated calves and controls (groups B and K) gave negative results to the immunohistochemical investigation. The statistical analysis showed that the progesterone receptor is a significant predictor of 17β-estradiol treatment alone or in association with nandrolone (p < 0.001): the immunohistochemical study resulted in 100% sensitivity (CI = 95%: 97.1–100%) and specificity (CI = 95%: 95.1–100%) for prostate and 99% sensitivity (CI = 95%: 95.6–100%) and 100% specificity (CI = 95%: 95.1–100%) for bulbo-urethral glands. The data confirm that this innovative biological approach offers a reliable tool to enhance the efficacy of the histological test to detect illegal treatments with estrogens alone or in association with androgens.     

细菌纤维素合成菌的筛选、鉴定及其产物结构和性质表征

该研究从腐败葡萄中筛选、分离细菌纤维素（BC）合成菌株,对其进行分子生物学鉴定,并对其产物的结构与性质进行表征。结果表明,筛选分离得到一株细菌纤维素合成菌株,该菌株的16S rRNA基因测序结果表明其属于醋酸菌属（Acetobacter sp.）,编号为ZX C6-15。扫描电镜（SEM）和傅立叶变换红外光谱（FT-IR）分析结果表明,菌株ZX C6-15产膜具有典型的3D纳米纤维状结构且具有典型细菌纤维素吸收峰。凝胶渗透色谱（GPC）、X-射线衍射（XRD）分析结果表明,菌株产膜重均分子质量（Mw）为14.1×104 kDa,数均分子质量（Mn）为32.3×104 kDa,分散度为2.28,结晶度指数为96%,吸水率达298%。该菌株合成细菌纤维素具有优良的材料性质,为后续培养条件优化及规模生产奠定基础。