Microbiological hydroxylation of androst-1,4-dien-3,17-dione by Neurospora crassa

Nine hydroxy-derived androstadiene compounds were isolated from the fermentation broth of Neurospora crassa when incubated in the presence of androst-1,4-dien-3,17-dione (ADD; I) for 7 days. Hydroxylations at 6β, 7β, 11α, 14α- positions and 17-carbonyl reduction of the substrate were the characteristics observed in this biotransformation. Their structures were determined by spectroscopic methods as 17β-hydroxyandrost-1,4-dien-3-one (II), 14α-hydroxyandrost-1,4-dien-3,17-dione (III), 6β-hydroxyandrost-1,4-dien-3,17-dione (IV), 11α-hydroxyandrost-1,4-dien-3,17-dione (V), 6β,17β-dihydroxyandrost-1,4-dien-3-one (VI), 7β-hydroxyandrost-1,4-dien-3,17-dione (VII), 14α,17β-dihydroxyandrost-1,4-dien-3-one (VIII), 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), and 11α,17β-dihydroxyandrost-1,4-dien-3-one (X). A new steroid substance, 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), was also characterized during this study. The best fermentation condition was found to be 7-day incubation at 25°C and pH values of 5.0–6.0 in the presence of 0.05 g 100 mL^?1 of the substrate. At a concentration above 0.075 g 100 mL^?1, the biotransformation was completely inhibited.     

Transformations of steroids and the steroidal alkaloid,solanine, by Phytophthora infestans

The following steroids and steroidal alkaloids have been incubated with the blight fungus Phytophthora infestans: androst-4-ene-3,17-dione, cholesterol, cholesteryl acetate, cholesteryl myristate, cholesteryl palmitate,cholesteryl stearate, dehydroisoandrosterone, 6α-hydroxy-androst-4-ene-3,17-dione, 6β-hydroxyandrost-4-ene-3,17-dione, 11α-hydroxyprogesterone, pregnenolone, progesterone, sitosterol, sitosteryl acetate, solanidine, solanine, stigmasterol, stigmasteryl acetate and testosterone. No hydroxylation was observed, but the fungus is able to oxidize alcohol functions at C-3β, C-6α, C-11β and C-17β to carbonyl. In addition, hydrolysis of acetate to hydroxyl at C-3β, and of solanine to solanidine, was observed. The relationship between metabolism and the nature of substitution at C-17β is discussed.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

白花鬼针草中多烯炔类成分的分离及其生物活性研究

为了解白花鬼针草(Bidens pilosa var.radiata)的化学成分,采用多种色谱技术从其提取物中分离多烯炔类成分,并对其生物活性进行研究。结果表明,从白花鬼针草乙酸乙酯提取部位中分离鉴定出4个多烯炔类化合物,分别为5-acetoxy-2-phenylethinyl-thiophene (1)、1-phenylhepta-1,3,5-triyne (2)、5-phenyl-2-(1-propynyl)-thiophene (3)和icthyothereol acetate (4)。体外活性评价结果表明,化合物1～4均具有中等抗MRSA活性,且均对人肝LO_2细胞无毒性。首次为化合物1提供核磁数据并进行结构解析,化合物3、4为首次从该属植物中分离得到。     

铁皮石斛内生真菌次生代谢产物研究

为了解铁皮石斛(Dendrobium officinale)内生真菌Phyllosticta aristolochiicola的次生代谢产物,从该真菌中分离得到15个化合物,经波谱分析分别鉴定为N-methyl-2-pyrolidinone (1)、环-(甘氨酸-L-脯氨酸)(2)、环-(D-丙氨酸-L-脯氨酸)(3)、环-(L-缬氨酸-L-脯氨酸)(4)、环-(L-亮氨酸-L-脯氨酸)(5)、cyclo-(L-Leu-D-4-hydroxyprolinyl)(6)、环-(L-苯丙氨酸-L-脯氨酸)(7)、环-(L-苯丙氨酸-L-4-羟基脯氨酸)(8)、环-(L-酪氨酸-L-脯氨酸)(9)、环-(L-苯丙氨酸-L-亮氨酸)(10)、啤酒甾醇(11)、对羟基苯乙醇(12)、对羟基苯乙酸(13)、(2S,3R)-1-(4-羟基苯基)丁烷-2,3-二醇(14)和(2R,3S)-1-苯基丁烷-2,3-二醇(15)。采用MTS法检测抗肿瘤活性表明,化合物2、10和14对HL-60、A-549、SMMC-7721、MCF-7和SW-480细胞株具有一定的抑制活性。     

苦槛蓝叶的黄酮类成分及其荔枝霜疫霉抑制活性研究

为了解苦槛蓝(Myoporum bontioides)的化学成分,采用色谱分离法从叶中分离得到11个化合物,分别鉴定为：5, 7, 3?-三羟基-4?-甲氧基黄酮(1)、3, 5, 7, 4?-四羟基-3?-甲氧基黄酮(2)、5, 7, 4?-三羟基-3?, 5?-二甲氧基黄酮(3)、木犀草素(4)、山奈酚(5)、鼠李黄素(6)、5, 7-二羟基二氢黄酮(7)、7, 4?-二羟基二氢黄酮(8)、5, 7, 3?, 4?-四羟基二氢黄酮(9)、5-O-乙酰基-3, 7, 3?, 4?-四羟基二氢黄酮(10)和7-甲氧基香橙素(11)。除化合物4、7、11之外,其他化合物均为首次从苦槛蓝叶中分离得到。菌丝生长速率法测试表明化合物4、7~9和11对荔枝霜疫霉菌具有较好的抑菌活性。     

Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum

The strain of Acremonium strictum PTCC 5282 was applied to investigate the biotransformation of androst-1,4-dien-3,17-dione (I; ADD). Microbial products obtained were purified by preparative TLC and the pure metabolites were characterized on the basis of their spectroscopic features (¹³C NMR, ¹H NMR, FTIR, MS) and physical constants (melting points and optical rotations). The 15α-Hydroxyandrost-1,4-dien-3,17-dione (II), 17β-hydroxyandrost-1,4-dien-3-one (III), androst-4-en-3,17-dione (IV; AD), 15α-hydroxyandrost-4-en-3,17-dione (V), 15α,17β-dihydroxyandrost-1,4-dien-3-one (VI) and testosterone (VII) were produced during this fermentation. Formation of the 15α,17β-dihydroxy derivative of ADD is reported for the first time during steroid biotransformation. The bioconversion reactions observed were 1,2-hydrogenation, 15α-hydroxylation and 17-ketone reduction. From the time course profile of this biotransformation, ketone reduction and 1,2-hydrogenation were observed from the first day of fermentation while 15α-hydroxylation occurred from the third day. Optimum concentration of the substrate, which gave the maximum bioconversion efficiency, was 0.5 mg ml⁻¹ in one batch. The highest yield of the microbial products recorded in this work was achieved within the pH range 6.5–7.3 and at the temperature of 27 °C.     

黄皮种子中酰胺类生物碱及其杀线虫活性研究

为了解黄皮种子中的酰胺类生物碱及其杀线虫活性,运用多种色谱学及波谱学方法分离并鉴定了10个酰胺类生物碱,分别为:N-甲基桂皮酰胺(1),clausenalansamide A(2),3-dehydroxy-3-methoxyl-clausenalansamide A(3),clausenalansamide B(4),黄皮新肉桂酰胺B(5),N-(2-苯乙基)肉桂酰胺(6),2′-dehydroxy-2′-oxo-clausenalansamide B(7),lansamide-7(8),homoclausenamide(9),1,5-dihydro-5-hydroxy-1-methyl-3,5-diphenyl-2H-pyrrol-2-one(10)。其中,化合物3,7,10为新天然产物。首次对黄皮种子中的酰胺类生物碱2~8进行全齿复活线虫致死活性的测试,发现所测化合物均有致死活性,其中,化合物2,3,5和8有较强的致死活性,且均优于阳性对照除线磷,可为相关农药的研发提供科学依据。     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

喙果皂帽花根的化学成分研究

为了解喙果皂帽花(Dasymaschalon rostratum)根的化学成分,采用多种色谱技术从其根的乙醇提取物中分离得到8个化合物。通过波谱数据分析,分别鉴定为:花旗松素(1)、(+)-儿茶素(2)、毛蕊异黄酮(3)、(-)-丁香树脂酚(4)、(-)-皮树脂醇(5)、lirioresinol A(6)、N-反式肉桂酸酰对羟基苯乙胺(7)和羟基酪醇(8)。所有化合物均为首次从该植物中获得。化合物1~8均无明显抗菌活性。     

Two new steroidal saponins from Dioscorea panthaica

Two new furostanol saponins, 3-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl]-26-O-β-d-glucopyranosyl-25(R)-furosta-5,22(23)-dien-3β,20α,26-triol (1), 3-O-[β-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-26-O-β-d-glucopyranosyl-20(R)-methoxyl-25(R)-furosta-5,22(23)-dien-3β,26-diol (2) were isolated from the Dioscorea panthaica along with five known steroidal saponins (3–7). The structures of the new saponins were determined by detailed analysis of spectral data (including 2D NMR spectroscopy). The inhibitory activities of the saponins against α-glucosidase were investigated, gracillin (4) and 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-26-O-β-d-glucopyranosyl-25(R)-furosta-5,20(22)-dien-3β,26-diol (5) were found to exhibit potent activities with IC₅₀ values of 0.11 ± 0.04 mM and 0.09 ± 0.01 mM.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β?triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

The hydroxylation of steroidal ring D lactones by Cephalosporium aphidicola

The microbiological hydroxylation of 17a-oxa-d-homo-5α-androstane-3,17-dione, the 3β-alcohol and the 13α-methyl analogue by Cephalosporium aphidicola takes place predominantly at C-7α in contrast to other hydroxylations by this organism.     

Furanofuran lignans from Vitex negundo seeds

A new furanofuran lignan, vitelignin A (1), together with eight known lignan derivatives, were isolated from the seeds of Vitex negundo. Their structures were identified as (+)-4-oxo-8-hydroxy-2,6-di(3,4-methylenedioxy)phenyl-3,7-dioxabicyclo[3.3.0]octane (1), 4-oxosesamin (2), (+)-sesamin (3), (+)-paulownin (4), 4-hydroxysesamin (5), 4,8-dihydroxysesamin (6), 4-oxopaulownin (7), (+)-2-(3-methoxy-4-hydroxyphenyl)-6-(3,4-methylenedioxy)phenyl-3,7-dioxabicyclo[3.3.0]octane (8), and (+)-pinoresinol (9), respectively, based on extensive NMR and MS spectroscopic studies. Compounds 1, 2, and 7 showed moderate antifungal activity in vitro.     

Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four filamentous fungi

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentous fungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, afforded nine new, and two known metabolites 2–12. The structures of these metabolites were characterized through detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylation of 1 at C-6β, -7β, -11α, -14α, -15β, -16β, and -17α positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11α-hydroxylation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity against the HeLa cancer cell lines, and some interesting results were obtained.     

New Aromatic Esters of Progesterone as Antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17α-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17α-(p-chlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17α-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17α-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1–4 have a weak inhibitory activity on 5α-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114?μM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds.

The binding efficiency of the synthesized steroids 1–4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1–4.     

油茶果生炭疽菌小分子GTP酶Rab7的功能研究

【目的】炭疽病是油茶的一种重要病害,果生炭疽菌是油茶炭疽病的主要致病菌。本文对果生炭疽菌小分子GTP酶Rab7进行研究,为油茶炭疽病的防控治理提供依据。【方法】构建CfRAB7基因敲除载体,通过PEG介导的原生质体转化、抗性筛选和PCR电泳验证获得果生炭疽菌突变体菌株△Cfrab7和互补菌株△Cfrab7/CfRAB7。进一步分析CfRAB7基因敲除突变体△Cfrab7的生长、产孢、附着孢的形成、胁迫应答、液泡融合和致病力等生物学表型。【结果】在PDA和MM培养基上,突变体△Cfrab7的菌落直径显著减小,产孢量和附着孢形成率显著降低,且不能穿透玻璃纸;在10mmol/LH2O2条件下,△Cfrab7生长受到明显抑制;进一步研究发现突变体△Cfrab7液泡无法正常融合,在油茶有伤和无伤的幼叶上均不发病。【结论】CfRAB7基因参与调控果生炭疽菌生长产孢、附着孢形成、H2O2胁迫应答、液泡融合和致病力。     

华石斛化学成分研究

为了解华石斛(Dendrobium sinense)的化学成分,采用多种柱色谱技术从其全草乙醇提取液中分离纯化了10个化合物,经波谱分析分别鉴定为:鼓槌石斛素(1)、2′,4′-二羟基查尔酮(2)、2,5,7-三羟基-4-甲氧基-9,10-二氢菲(3)、4,7-二羟基-2,3-二甲氧基-9,10-二氢菲(4)、2,5-二羟基-3,4-二甲氧基-9,10-二氢菲(5)、2,7-二羟基-3,4,6-三甲氧基-9,10-二氢菲(6)、(E)松柏醛(7)、反式对羟基肉桂酸酯(8)、对羟基苯丙酸甲酯(9)和十二元内环酯(10)。所有化合物均为首次从华石斛中分离得到,其中化合物2、6、7和10对乙酰胆碱酯酶具有一定的抑制活性。     

黄皮果皮中的抗菌活性成分研究

为了解黄皮[Clausena lansium(Lour.)Skeels]果皮中的抗菌活性成分,采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱等方法,从黄皮果皮的乙醇提取物中分离得到10个化合物,经波谱学分析鉴定为:lansine(1)、3-甲酰基咔唑(2)、3-甲酰基-6-甲氧基咔唑(3)、6-甲氧基咔唑-3-羧酸甲酯(4)、(6R,7E,9S)-9-羟基-4,7-巨豆二烯-3-酮(5)、7-羟基香豆素(6)、8-羟基呋喃香豆素(7)、辛黄皮酰胺(8)、对羟基肉桂酸甲酯(9)和胡萝卜苷(10)。其中化合物5和9为首次从黄皮属植物中分离得到,化合物8的碳谱数据是首次报道。用滤纸片琼脂扩散法测定化合物的抗菌活性,结果表明,化合物1、2、3、6、7和9对金黄色葡萄球菌(Staphylococcus aureus)有抑制作用。这有利于更好地开发利用黄皮这一药食两用的水果资源。     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.