狗尾草平脐蠕孢NY1菌株的次级代谢产物分析

为进一步研究和利用具有杂草生防潜力的狗尾草平脐蠕孢Bipolaris setariae菌株NY1,对其次级代谢产物进行了分离与结构鉴定。从菌株NY1的大米发酵的乙酸乙酯提取物中分离到7个化合物,通过波谱分析将其分别鉴定为:邻苯二甲酸二异壬酯,麦角甾-5,7,22-三烯-3β-醇,5α,8α-过氧化麦角甾-6,22-二烯-3β-醇,6-甲基苯-1,2,4-三酚,3-脱羟基蛇孢菌素A,邻羟基苯甲醛,蛇孢菌素I。其中3-脱羟基蛇孢菌素A是一种植物毒素,而具有抗癌、抗菌作用的蛇孢菌素I的产量较高,具有工业化生产的潜力。     

外源因子对桦褐孔菌发酵产桦褐孔菌醇的影响

食药用真菌因其丰富的天然活性物质成为具有开发潜力的药物来源。桦褐孔菌Inonotus obliquus作为一种珍稀的药用真菌,因其对糖尿病、消化系统疾病、心血管疾病、肝病和癌症等疾病有良好的治疗效果而受到广泛关注。桦褐孔菌醇（inotodiol）是桦褐孔菌特有的一种羊毛甾烷型三萜类化合物,具有多种抗癌活性。本文的主要目的是研究外源因子的添加对桦褐孔菌液态发酵产桦褐孔菌醇的影响,以及对桦褐孔菌醇合成途径中酶活的影响。结果表明：最佳外源因子是香叶醇,最佳添加浓度和添加时间分别为0.02%（V/V）和第144小时。发酵结束时（240h）桦褐孔菌醇的产量为27.89mg/L是对照组（9.23mg/L）的3.02倍。通过对比添加香叶醇后桦褐孔菌醇的产量变化以及合成途径中4种酶（法尼基焦磷酸合酶、角鲨烯合酶、角鲨烯环氧化酶和羊毛甾醇合酶）的活性变化,对香叶醇的作用机制进行了初步探究。研究结果表明添加香叶醇后,4种酶活性均较对照组有显著的提高,与此对应的桦褐孔菌醇产量也显著增加,说明这4种酶在桦褐孔菌醇合成途径中起到了积极的作用。     

根霉3078的代谢产物的研究

从根霉3078菌丝体的甲醇提取物中分离得到9个化合物,通过波谱分析,鉴定为5α,8α-表二氧-(20S,22E,24R)-麦角甾-6,22-二烯-3β-醇(1)、甘油醇-1-单油酸酯(2)、4-羟基苯乙酮(3)、4-羟基苯乙酸(4)、(20S,22E,24R)-麦角甾-7,22-二烯-3β,5α,6β三醇(5)、(S)-3-羟基-3-苯基丙酸(6)、胸腺嘧啶(7)、尿嘧啶(8)和腺苷(9)。     

罗汉松内生真菌Pestalotiopsis heterocornis的代谢产物研究

从罗汉松内生真菌Pestalotiopsis heterocornis的发酵培养液中分离得到10个代谢产物,应用质谱、核磁共振等现代波谱学方法鉴定为jesterone(1),hydroxy-jesterone(2),ambuic acid(3),6β-羟基-豆甾-4-烯-3-酮(4),(24S)-麦角甾-5-烯-3β,7α-二醇(5),7,22-二烯-3β,5α,7β-三羟基-麦角甾醇(6),麦角甾-7,22-二烯-3-酮(7),(4E,8E,2S,3R,2'R)-N-2'-羟基棕榈酰-9-甲基-4,8-sphingadienin(8),鲨肝醇(9),棕榈酸(10)。以上化合物均为首次从内生真菌P. heterocornis的代谢产物中分离得到,化合物4,6,7为首次从拟盘多毛孢属真菌代谢产物中分离得到。     

桦褐孔菌提取物抗氧化活性研究

以乙酸乙酯和甲醇为提取剂,采用索氏提取法,剩余残渣采用热水浸提,最终得到桦褐孔菌不同极性提取物,对其DPPH自由基、羟基自由基及超氧阴离子自由基清除活性作用进行了研究,确定桦褐孔菌的抗氧化能力,为深入研究和开发桦褐孔菌功能性食品奠定理论基础。实验结果表明桦褐孔菌具有较好的抗氧化活性,其中乙酸乙酯提取物的DPPH自由基清除率、羟基自由基清除率和超氧阴离子自由基清除率均高于其他两组分及BHT,桦褐孔菌提取物有望成为功能性食品组分中合成抗氧化剂的天然替代品。     

激发子对桦褐孔菌多酚合成的诱导及其生化机制初探

旨在研究真菌激发子对桦褐孔菌多酚合成的影响,采用液体摇瓶培养方式,将真菌激发子加入到桦褐孔菌液体培养物中,检测桦褐孔菌次级代谢产物的积累,以及真菌激发子对桦褐孔菌一氧化氮合酶(NOS)活性的影响。结果表明,适量添加真菌激发子能够提高桦褐孔菌的代谢速率,提高多酚类化合物的合成,胞内和胞外多酚最高可达102.15 mg/L和311.91 mg/L,并能显著提高桦褐孔菌NOS的活性,其活性最高可达151.93 U/mg prot,发酵液一氧化氮(NO)含量最高可达256.28μmol/L。这表明,真菌激发子能够有效激活桦褐孔菌的次级代谢产物合成途径。     

pH对桦褐孔菌(Phaeoporus obliquus)H011菌株深层培养抗氧化特性的影响

研究了p H(4.0~7.0)对桦褐孔菌(Phaeoporus obliquus)H011菌株深层培养菌丝体和滤液甲醇提取物抗氧化活性、自由基清除能力及抗氧化成分的影响。结果表明:p H显著影响桦褐孔菌菌丝体和滤液甲醇抽提物的抗氧化能力、自由基清除能力、总酚和多糖产量;当p H为6.0时,滤液甲醇提取物的抗氧化能力、对自由基的清除能力最强,显著高于其他组(P0.05);当p H为6.0时,菌丝体甲醇抽提物多酚产量和多糖产量达到最高,分别是189.9和47.2 mg·g-1,均分别显著高于其他组的含量(P0.05),桦褐孔菌菌丝体和滤液甲醇抽提物的抗氧化能力、自由基清除能力与总酚、多糖产量有关;深层培养的抗氧化能力比子实体高。因此,可通过桦褐孔菌深层培养生成抗氧化剂来取代固体培养。     

一氧化氮对桦褐孔菌抗氧化酚类化合物积累的影响

本研究探讨了NO对深层培养的桦褐孔菌积累抗氧化酚类化合物的影响。在桦褐孔菌的培养基中分别加入0.01mmol/L、0.1mmol/L以及1mmol/L的NO供体硝普钠,并测定总酚的含量及其抗氧化活性。发酵产物的抗氧化活性以清除DPPH和羟自由基活力表示。加入0.1mmol/L的硝普钠可使桦褐孔菌胞内外酚类化合物分别达到最高水平67mg/g和677mg/L。不同培养时期产生的胞内外酚类化合物都具有抗氧化活性,尤其是添加0.1mmol/L硝普钠的菌丝体。因此NO可用于上调深层发酵培养的桦褐孔菌酚类化合物的积累。     

石灰菌代谢产物的化学研究(Ⅰ)

从石灰菌(Lactarius hysginus Fr.)提取物的中性部分,分离得到六个化合物,经物理常数和光谱分析,鉴定为麦角甾醇(A)、硬脂酸甲脂(B)、N-苯基-2-萘胺(C)、24E-麦角甾7,22-二烯-6-酮一3β,5α-二醇(D)、庚酰胺(E)、24E-麦角甾-7.22-二烯-3β,5α,6β-三醇(F),其中化合物D作为天然产物尚未见文献报道。     

红缘拟层孔菌发酵菌丝体对小鼠免疫调节的研究

红缘拟层孔菌是具有良好抗肿瘤活性的药用真菌,其抗肿瘤的主要有效成分为3-乙酰氧基-8,24-羊毛甾二烯-21-酸。以红缘拟层孔菌发酵菌丝体及3-乙酰氧基-8,24-羊毛甾二烯-21-酸单体为实验材料,研究了两者对小鼠免疫功能的调节作用,然后测试免疫功能的4个指标来判断红缘拟层孔菌发酵菌丝体和3-乙酰氧基-8,24-羊毛甾二烯-21-酸单体在免疫系统中的影响。结果表明,与对照组比较,红缘层孔菌组的吞噬率、血清溶血素水平、淋巴细胞转化率和吞噬指数均有显著提高;在小鼠淋巴细胞转化实验中,3-乙酰氧基-8,24-羊毛甾二烯-21-酸在低浓度时对小鼠淋巴细胞转化具有良好的促进作用,但随浓度的增加,促进作用逐渐减弱(P0.05)。因此,红缘层孔菌发酵菌丝体具有良好的增强小鼠免疫功能的作用。     

Production of antioxidant and antitumor metabolites by submerged cultures of <Emphasis Type="Italic">Inonotus obliquus</Emphasis> cocultured with <Emphasis Type="Italic">Phellinus punctatus</Emphasis>

While Inonotus obliquus produces a diverse range of bioactive metabolites in its natural habitats, it accumulates less in its submerged cultures. We show here that coculture of I. obliquus with Phellinus punctatus resulted in less production of mycelial biomass but an increased accumulation of phenolic compounds, melanins, and lanostane-type triterpenoids. Metabolites increased in production by coculture include phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, inoscavin B, davallialactone, methyl davallialactone, foscoparianol D, 21,24-cyclopentalanosta-3β,21,25-triol-8-en, lanosta-7,9(11),23-triene-3β,22,25-triol, and inotodisaccharide and melanins. Metabolites from coculture also showed an increased potential for scavenging free radicals and inhibiting the proliferation of HeLa 229 cells. Davallialactone, methyl davallialactone, and minor phenolic components are the major contributors for scavenging DPPH and hydroxyl radical in monoculture, and phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, methyl davallialactone, foscoparianol D, and inotodisaccharide are those for scavenging the tested radicals in coculture. Lanostane-type triterpenoids indicated limited roles in scavenging free radicals. Nearly all the detected metabolites correlate positively with inhibiting proliferation of HeLa 229 cells. Thus, coculture of I. obliquus with other fungi seems to be a cost-effective strategy for upregulating biosynthesis of bioactive metabolites.     

Prolonged-release gonadotrophin-releasing hormone analogue implants enhance oocyte final maturation and ovulation, and increase plasma concentrations of sulphated C21 steroids in North Sea plaice

Reproductively mature female plaice were implanted with or without 50 μg of gonadotrophin-releasing hormone analogue (GnRHa), suspended in either coconut oil or methacrylate resin. The weight of the GnRHa-treated fish increased significantly (due to hydration of the oocytes) and reached a peak between 10 and 14 days. The fish produced several batches of eggs, which were consistently bigger than those produced by control fish. Plasma concentrations of free 17β-oestradiol and glucuronidated testosterone rose briefly (4 days) in response to the GnRHa, but then fell continuously till the end of the experiment (20 days). Plasma concentrations of sulphated 5β-pregnane-3α,17,20β-triol and 5β-pregnane-3β,17,20β-triol (which are putative metabolites of 17,20β-dihydroxy-4-pregnen-3-one, the oocyte maturation-inducing steroid) increased significantly at 4 days and reached a peak between 12 and 16 days. Concentrations were still very elevated on day 20. Plasma concentrations of sulphated 3α,17,21-trihydroxy-5β-pregnan-20-one showed a slight increase on day 4 but did not change thereafter. There was a highly significant difference in the amounts of GnRHa released into the bloodstream by the two methods of administration on day 4. However, this was not matched by significant differences in the concentrations of any of the steroids.     

Effects of Postnatal Trimethyltin or Triethyltin Treatment on CNS Catecholamine,GABA, and Acetylcholine Systems in the Rat

Abstract: The effects on brain neurochemistry of two neurotoxic tin compounds, trimethyltin (TMT) hydroxide and triethyltin (TET) sulfate, were examined. Long-Evans rats were treated with TMT hydroxide (1 mg/kg, i.p.) on alternate days from day 2 to 29 of life. These treatments caused a weight deficit of 10–20% by the time the animals were killed on day 55 by head-focused microwave irradiation. These TMT treatments are known to cause severe neuronal loss in the hippocampus and lesser damage in other brain regions. Accordingly, the concentration of γ-aminobutyric acid (GABA) was decreased in the hippocampus; however, acetylcholine and choline concentrations were unaffected. These data suggest that TMT-induced effects on GABA systems are greater than that due simply to generalized neuronal loss. The TMT treatments also caused a significant decrease in dopamine concentrations in the striatum, but did not alter the concentrations of dihydroxyphenylacetic acid or homovanillic acid, the acidic metabolites of dopamine. Conversely, concentrations of dopamine and norepinephrine in the brain stem and norepinephrine in the cerebellum were not altered. Despite reports in the literature of TMT-induced neuronal damage in areas of the cortex, no effects on GABA, acetylcholine, or choline levels were found in the cortical areas examined, or in the hypothalamus. TET sulfate (0.3 mg/kg/day) was administered for 6 consecutive days of every week during days 2–29 of life. This dose is lower than that needed to cause intramyelin edema, yet it does result in long-term behavioral changes. Despite this, no changes in the concentration of any of the measured neurotransmitters or their metabolites were detected. In concert, these data demonstrate that neurochemical methods should not be used as neurological “screens,” but rather to define specific mechanisms suggested by detailed behavior, pharmacological, and/or physiological studies.     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.     

Increased placental concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in pregnant rats treated with human chorionic gonadotropin

Pregnant rats were injected intrajugularly with 2500 i.u. human chorionic gonadotropin (HCG) toward the end of gestation (days 18-19) and 7.0 pmoles of tritiated 25-hydroxyvitamin D3 [( 3H]25(OH)D3) the following day. They were sacrificed ten to 24 hours later. [3H]25(OH)D3 and the in vivo produced [3H]24,25-dihydroxyvitamin D3 [( 3H]24,25(OH)2D3) in lipid extracts from maternal serum, kidneys, placenta and fetal tissues were separated by Sephadex LH-20 chromatography, and high performance liquid chromatography (HPLC). HCG treatment of pregnant rats increased significantly 25(OH)D3 levels in the placenta and kidneys and 24,25(OH)2D3 level in the placenta. Fetal metabolites levels were unaffected by HCG treatment. Serum and kidney levels of 25(OH)D3 and 24,25(OH)2D3 in pregnant rats were significantly lower than in non-pregnant rats. Serum and kidney levels of both metabolites in non-pregnant female rats treated with HCG did not differ from the untreated controls. HCG may, therefore, be involved in regulation of fetoplacental vitamin D metabolism.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

Metabolism of progesterone by chorionic cells of the early sheep conceptus invitro

Progesterone-4-¹⁴C was extensively metabolized during incubation with dispersed trophoblast prepared from chorionic membranes of the 21-day sheep conceptus. Of the metabolites formed, 17,20α-dihydroxypregn-4-en-3-one, 20α-hydroxypregn-4-en-3-one, 20(β-hydroxypregn-4-en-3-one, 5α-pregnane-3α,17,20α-triol, 5β-pregnane-3ga, 17,20α-triol, 5β-pregnane-3g,20α-diol, 3β-hydroxy-5α-pregnan-20-one, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-5β-pregnan-3-one, 5α-pregnane-3,20-dione and 5β-pregnane-3,20-dione were identified. These findings indicate that the sheep conceptus acquires extensive steroid metabolizing capability very early in pregnancy.