高效液相色谱法测定桦褐孔菌4种成分含量及不同部位含量比较

目的 评价桦褐孔菌药材质量，建立高效液相色谱(HPLC)法测定桦褐孔菌药材中4种成分含量，并比较不同部位4种成分含量的差异。方法 采用HPLC法测定桦褐孔菌中栓菌酸、桦褐孔菌醇、麦角甾醇、羊毛甾醇含量，并对桦褐孔菌药材黑褐色外壳和内部黄色菌肉中4种成分含量进行比较。色谱柱为Agilent 5 TC-C₁₈(250 mm×4.6 mm, 5μm),柱温30℃;以乙腈-水为流动相，梯度洗脱，流速为1.0 mL·min^-1;检测波长为203,282 nm;进样量：20μL。采用SPSS23.0版统计软件对不同产地桦褐孔菌药材进行聚类分析，对内外两部位含量进行统计学分析。结果 4种成分在各自浓度范围内线性关系良好(r≥0.999 7);平均加样回收率分别为101.79%,102.96%,101.12%和101.37%,RSD分别为2.35%,2.25%,2.27%和2.13%(n=6);精密度、重复性、稳定性(24 h)实验RSD均<3.0%(n=6);23批桦褐孔菌药材中栓菌酸、桦褐孔菌醇、麦角甾醇、羊毛甾醇含量范围分别为0.383 0～3....     

桦褐孔菌药材的薄层色谱鉴别和HPLC指纹图谱建立及化学模式识别分析

目的 建立桦褐孔菌药材的薄层色谱鉴别方法和高效液相色谱（HPLC）指纹图谱，并结合化学模式识别分析评价桦褐孔菌药材的质量。方法 采用薄层色谱法对桦褐孔菌药材中的栓菌酸、桦褐孔菌醇进行定性鉴别；建立桦褐孔菌药材的HPLC指纹图谱，根据《中药色谱指纹图谱相似度评价系统（2012版）》确定共有峰并进行相似度评价；采用SPSS 23.0软件和SIMCA14.1软件对22批桦褐孔菌药材的指纹图谱数据进行化学模式识别分析（聚类分析、主成分分析和正交偏最小二乘法-判别分析）。结果 薄层色谱鉴别结果显示，供试品色谱和对照品色谱在相应位置上显相同颜色斑点。22批桦褐孔菌药材的HPLC指纹图谱共有10个共有峰，相似度为0.942～0.995；指认出3号峰为栓菌酸，4号峰为桦褐孔菌醇，9号峰为麦角甾醇，10号峰为羊毛甾醇。聚类分析结果显示，S1～S15、S19、S21、S22聚为第1类，S16～S18、S20聚为第2类。主成分分析结果显示，综合得分排名前4位的样品分别为S17、S18、S16、S20。正交偏最小二乘法-判别分析结果显示，以变量重要性投影（VIP）值>1为标准，筛选出4号峰（桦褐孔菌醇，...     

冬虫夏草菌丝体水提醇沉物体外抗肿瘤活性研究

目的 研究冬虫夏草菌丝体水提醇沉物的体外抗肿瘤活性及其机制。方法 采用热水浸提-醇沉法得到水提物,高效液相色谱仪测定水提醇沉物中腺苷的量;采用MTT法测定其抗肿瘤活性,利用流式细胞仪结合碘化丙锭染色法检测其对细胞周期的抑制。结果 实验表明水提醇沉物能抑制人肝癌HepG2细胞株及大细胞肺癌NCI-H460细胞株的增殖,并呈浓度相关性;半数抑制浓度（IC₅₀）值分别为（1.49±0.19）和（1.67±0.27）mg/mL。细胞周期分析表明,水提醇沉物分别阻滞HepG2及NCI-H460细胞周期于G₂/M期、S期,并可诱导上述两种细胞发生凋亡。结论 冬虫夏草水提醇沉物通过阻滞HepG2及NCI-H460细胞周期循环,诱导其凋亡,从而表现出良好的增殖抑制活性。为深入研究冬虫夏草菌丝体水提醇沉物抗肿瘤的机制提供了实验证据。     

斑嗜蓝孢孔菌弱极性成分GC-MS分析

目的:采用GC-MS法对斑嗜蓝孢孔菌弱极性成分进行分析。方法:使用乙醇提取、石油醚淋洗的方法获得嗜蓝孢孔菌弱极性成分,利用气相色谱-质谱法分离并分析其化学成分,采用GC峰面积归一化法处理得到各组分百分含量。结果:鉴定出29种成分,其成分最多的是甾类和萜类化合物,含量较丰富的有酒酵母甾醇、麦角甾醇、麦角甾-7,22-二烯-3β-醇、麦角甾-7、22-二烯-3-酮、麦角甾-5,24(28)-二烯-3β-醇、菌甾醇、谷甾醇、羊毛甾醇、羽扇豆烯酮、麦角甾-4,6,8(14),22-四烯-3-酮、羽扇醇和白桦脂醇等。结论:本测定结果为研究斑嗜蓝孢孔菌的药效与化学成分的相关性提供了一定的研究基础。     

UPLC-QTOF-MS结合主成分分析法考察胡芦巴盐制前后的化学成分差异

目的 利用超高效液相-四极杆-飞行时间串联质谱( UPLC-QTOF-MS ) 结合多元统计分析筛选并鉴定胡芦巴和盐胡芦巴的差异性成分。方法 采用 C₁₈反相色谱柱0.5% 乙酸-乙腈梯度洗脱,在正离子模式下采集质谱数据,应用Simca-P等软件进行主成分分析 (PCA) 和偏最小二乘判别分析（PLS-DA）,筛选胡芦巴与盐胡芦巴的差异性成分。通过质谱提供的精确分子量、碎片离子并结合文献数据鉴定PLS-DA分析中VIP值＞1.5的差异性成分的结构。结果 利用准分子离子、二级质谱图、特征碎片离子及其他碎片离子等信息,并参考相关文献报道鉴定了胡芦巴与盐胡芦巴中的24个差异性成分,其中包括7个黄酮碳苷类化合物,15个甾体皂苷类化合物以及1个生物碱类化合物即胡芦巴碱和1个脂肪族化合物即亚麻酸乙酯。通过比较各化合物在胡芦巴和盐胡芦巴样品的质谱中的响应值,盐制后黄酮碳苷类成分响应升高,甾体皂苷中的呋甾烷醇型甾体皂苷响应降低,而螺甾烷醇型/异螺甾烷醇型甾体皂苷响应升高。结论 盐制促进了胡芦巴中黄酮碳苷类成分的溶出,并使胡芦巴中皂苷类成分发生了结构转化。     

白首乌C21甾体苷诱导肝癌细胞凋亡的作用及其机制

研究白首乌C₂₁甾体苷对肝癌实体瘤细胞的凋亡作用及其机制。建立肝癌实体型(Heps)小鼠移植性肿瘤模型，随机分为模型组和C₂₁甾体苷各用药组，连续灌胃，10 d后脱颈椎处死小鼠，进行抑瘤率计算，对肿瘤组织进行电镜下观察，采用免疫荧光(AO/EB)测定肿瘤细胞凋亡，免疫组化染色检测bcl-2基因的表达。C₂₁甾体苷(10，20和40 mg·kg^-1)对小鼠移植性肝癌Heps有抑制作用，3个剂量组的抑瘤率分别为34.79%，47.08%和50.23%。C₂₁甾体苷(10，20和40 mg·kg^-1)可增加肿瘤细胞的凋亡，电镜下可见凋亡的形态学改变，并出现凋亡小体；免疫组化结果显示，bcl-2基因的表达与模型组比较明显降低(p<0.01)，但不同于凋亡结果的是高剂量组的阳性面积表达比中剂量略高。降低高表达的bcl-2基因从而促进肝癌细胞的凋亡，可能是C₂₁甾体苷抗肝癌的机制之一。     

3′(5α-3β,17β-双羟基-雄甾烷-17α)-丙酸内酯(Ⅴ)的微生物转化——螺旋内酯甾(Ⅱ)的合成

3′-(5α-3β,17β-双羟基-雄甾烷-17α)-丙酸内酯(Ⅴ)经诺卡氏菌(Nocardia sp.)转化获得6个化合物(Ⅱ和Ⅵ～Ⅹ),其中螺旋内酯甾(Ⅱ)为其主要产物(～50%)。Ⅷ～Ⅹ是尚未被鉴定的羟基化合物。化合物(Ⅴ)用诺卡氏菌在氮源贫乏的培养基中转化主要生成△⁴-3-酮化合物,而在氮源丰富的培养基中转化则主要为△^1,4-3-酮的产物。     

冬虫夏草人工繁育品与野生品基于甾醇特征图谱的比较研究

目的：对冬虫夏草人工繁育品与野生品进行比较研究,建立基于甾醇类成分的特征图谱,并比较麦角甾醇含量的差异,为该药材的质量监管提供参考。方法：以10批冬虫夏草人工繁育品、17批野生品以及5批凉山虫草为研究对象,采用超高效液相色谱法,以麦角甾醇为对照品,选用XBridgeC18色谱柱（100 mm×2.1 mm,1.6 μm）;流动相为甲醇-水（95：5）,检测波长282 nm,流速0.3 mL·min^-1,柱温30℃,采用CHEMPATTERN化学计量学软件对野生品色谱图进行处理,生成特征图谱,并计算所有批次的相似度。结果：17批野生冬虫夏草、10批冬虫夏草人工繁育品与野生品对照图谱之间的相似度均大于0.9,相似度良好;而5批凉山虫草相似度均未超过0.8,相似度较低。10批人工繁育品的平均含量为0.121%±0.031%;17批野生品的平均含量为0.115%±0.045%;5批凉山虫草的平均含量为0.468%±0.038%。冬虫夏草人工繁育品和野生品基于甾醇的特征图谱以及麦角甾醇含量基本一致;与凉山虫草有显著性差异。结论：本研究有助于冬虫夏草的真伪鉴别,同时为冬虫夏草人工繁育资源开发以及合理使用提供科学依据。     

知母中的两种新呋甾皂苷

目的研究知母根茎的化学成分。方法采用水煎提取、大孔吸附树脂SP825柱色谱、反相C₁₈柱色谱等进行分离，并通过化学手段和光谱分析(FAB-MS，¹H NMR，13C NMR，¹H-¹H COSY)鉴定其化学结构。结果从知母根茎中分离得到6种甾体皂苷，分别鉴定为：(25S)-26-O-β-D-吡喃葡糖基-22-羟基-5β-呋甾-2β，3β，26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-β-D-吡喃半乳糖苷(知母皂苷N，1)，知母皂苷E_l(2)，(25S)-26-O-β-D-吡喃葡糖基-22-甲氧基-5β-呋甾-2β，3β，26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-β-D-吡喃半乳糖苷(知母皂苷O，3)，知母皂苷E₂(4)，(25R)-26-O-β-D-吡喃葡糖基-22-羟基-5α-呋甾-2α，3β，26-三醇-3-O-β-D-吡喃葡糖基-(1→2)-［β-D-吡喃木糖基-(1→3)］-β-D-吡喃葡糖基-(1→4)-β-D-吡喃半乳糖苷(purpureagitosid，5)，marcogenin-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside (6)。结论化合物1和3为新化合物，命名为知母皂苷N和知母皂苷O，化合物5为首次从知母中分离得到。     

肿瘤化学治疗的研究 ⅩⅨ.N,N-双(2-氯乙基)腙化合物的合成

合成了睾丸素、甲基睾丸素和其他一系列羰基化合物的N,N-双(2-氯乙基)腙化合物(Ⅱ)、(Ⅲ)、(Ⅳ)和(Ⅴ_1-10),以进行抗肿瘤试验。它们的制备,系由N,N-双(2-氯乙基)肼盐酸盐(Ⅰ)与相应的羰基化合物,在碱存在或不存在的醇溶液中反应而得。此外又自17-氢基Δ⁵孕-3醇,与对双(2-氯乙基)氨基苯甲醛缩合,制得了另一甾体氮芥化合物——17-[对双(2-氯乙基)氨基苯亚甲基]氨基-Δ⁵-孕-3醇(Ⅵ)。     

In vitro antitumor activity and structure characterization of ethanol extracts from wild and cultivated Chaga medicinal mushroom, Inonotus obliquus (Pers.:Fr.) Pilát (Aphyllophoromycetideae)

Inonotus obliquus (Pers.:Fr.) Pilát has been traditionally used as a folk remedy for treatment of cancers, cardiovascular disease and diabetes in Russia, Poland, and most of the Baltic countries, but natural reserves of this fungus have nearly been exhausted. This study was designed to investigate the artificial cultivation of I. obliquus and the antitumor activity of its tissues. The ethanol extract of cultivated sclerotium had the highest cell growth inhibitory rate (74.6%) as determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 78% of the bags produced sclerotia and only 6.17 g/bag of sclerotium was obtained. Extracts of the cultivated fruiting body showed 44.2% inhibitory activity against tumor cells. However, the yield was as high as 18.24 g/bag, and 98% of the bags produced fruiting body. The results of gas chromatography-mass spectroscopy (GC-MS) showed that similar compounds were extracted from the wild and cultivated samples. The principal compounds observed were lanosterol, inotodiol, and ergosterol. Their percentages of the mass fraction were 86.1, 59.9, and 71.8% of the total, for the wild sclerotium, cultivated sclerotium, and cultivated fruiting body, respectively. Ergosterol was found to be much higher (27.32%) in cultivated fruiting body. We conclude that cultivated fruiting body of I. obliquus obtained by inoculation of the substrate with spawn mycelium of the fifth generation could serve as an ideal substitute for the wild I. obliquus.     

Sterol biosynthesis in Pneumocystis: unique steps that define unique targets

Pneumocystis lacks ergosterol, and several antimycotics that bind ergosterol in fungal membranes or inhibit its synthesis are ineffective against Pneumocystis pneumonia. The organism synthesizes C(28) and C(29) Delta(7) 24-alkylsterols, 24-alkyllanosterol derivatives, and Delta(5) 24-alkylsterols, which may be produced by modifying scavenged Delta(5) sterols. Mammals cannot desaturate C-22 and alkylate C-24 of sterols, thus, these processes are particularly attractive targets for antifungal drug development. Recent data indicate that C-22 desaturation is not, but C-24 alkylation is an attractive target in P. carinii. The P. carinii S-adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) has unique properties; it prefers lanosterol as its sterol substrate.     

气-质联用研究氟康唑对白念珠菌甾醇生物合成的抑制作用

目的　为抗真菌药物作用机理的研究提供有力的工具。方法　白念珠菌经药物作用后提取未皂化脂 (NSLs) ,其中的甾醇组分经衍生化后GC MS分析 ,测定各组分的结构和含量。结果　经氟康唑作用的真菌 ,CYP5 1酶受抑制 ,使细胞膜内羊毛甾醇和2 4 (2 8) 亚甲基 2 4 ,2 5 二氢羊毛甾醇累积 ,后者更为明显 ,而麦角甾醇合成受阻。结论GC MS分析获满意的效果 ,可对抗真菌药物阻断真菌麦角甾醇合成通路所引起各甾醇组分的含量变化进行研究。     

Polysaccharides from Inonotus obliquus sclerotia and cultured mycelia stimulate cytokine production of human peripheral blood mononuclear cells in vitro and their chemical characterization

Inonotus obliquus is an edible and medicinal mushroom to treat many diseases. In the present study, polysaccharides and fractions were isolated and purified by DEAE-52 and Sephadex G-200 chromatography from I. obliquus wild sclerotia, culture broth and cultured mycelia under submerged fermentation. The extracts and fractions could significantly induce the secretion of TNF-α, IFN-γ, IL-1β, and IL-2 in human peripheral blood mononuclear cells (PBMCs) and showed no toxicity to PBMCs. The stimulation effect of the six extracts and eight fractions on the four-cytokine production was dose-dependent. Sclerotial polysaccharides were more effective in the four-cytokine production at 150 μg/ml while exopolysaccharides and endopolysacchrides showed a much better effect on IL-1β production at 30 μg/ml. Purified fractions from exopolysaccharides and endopolysaccharides were more effective than the fraction from sclerotia in most cytokine production. These heteropolysaccharide-protein conjugates mainly contained glucose, galactose, and mannose. Protein content, molecular weight, monosaccharide molar ratio, and anomeric carbon configuration differed from each other and had effects on the cytokine induction activity of the polysaccharides to some extent.     

桦褐孔菌提取物诱导人胃癌BGC-823细胞凋亡的研究

目的:探讨桦褐孔菌提取物对肿瘤细胞周期的影响及其抗癌的作用机制。方法:将不同浓度(20、40、80mg·L-1)桦褐孔菌提取物作用于人胃癌BGC-823细胞48h,用碘化丙啶染色法检测凋亡细胞和细胞周期分布。采用免疫细胞化学染色法检测药物处理前、后Ki-67抗原的表达。结果:3种浓度桦褐孔菌提取物处理48h后细胞凋亡率分别为11.57%、20.97%、31.22%,阻滞细胞周期于S期,并使Ki-67阳性表达率下降,且具有浓度依赖性。结论:桦褐孔菌提取物可抑制人胃癌BGC-823细胞的生长,细胞主要阻滞于S期,并能诱导部分细胞凋亡。     

桦褐孔菌多糖的提取工艺优化

目的研究桦褐孔菌多糖提取工艺优化。方法以多糖含量为指标,在单因素研究基础上,采用4因素3水平的响应面分析法对桦褐孔菌多糖的提取工艺进行优化研究,并检测重金属砷的含量。结果桦褐孔菌多糖提取的优化工艺条件为：乙醇浓度30％、提取温度95℃、提取时间2．5h、液料比30：1。在该条件下,粗多糖得率为33．6％,样品中多糖含量为6．177％。桦褐孔菌多糖实际提取含量为5．993％,重金属含量降低。结论优化工艺提取率高,快速,重金属含量低,可用于桦褐孔菌的多糖提取。     

纤维素酶辅助提取桦褐孔菌多糖的研究

目的优化桦褐孔菌提取工艺,提高桦褐孔菌多糖得率。方法首先对桦褐孔菌多糖提取的主要影响因素纤维素酶用量、酶解温度、溶剂倍量、酶解时间、pH值进行单因素条件筛选。然后采用正交设计法对影响纤维素酶辅助提取多糖的条件进行优化。结果最佳提取条件为:料液比1:40、酶解时间60 min、酶解温度50℃、加酶量0.15 g、酶解pH=5.0。结论纤维素酶对桦褐孔菌多糖进行辅助提取,相比传统的热水浸提,能够显著提高桦褐孔菌多糖的提取率,比水浸提高29.07%,是一种较为有效的提高多糖得率的方法。     

Effect of hypocholesterolemic agents on central nervous system cholesterol biosynthesis—I: Zuclomiphene

The hypocholesterolemic agent zuclomiphene was found to inhibit sterol formation from [2-¹⁴C]mevalonic acid in cell-free preparations of developing rat brain. This inhibition was found if zuclomiphene were added to an incubation of normal brain. If the animals were pretreated for several weeks with the drug, but the brain tissue then incubated without the drug being added, the same reduction in sterol biosynthesis was seen. Incubation of brain tissue from pretreated animals, with addition of drug to the incubation as well, had reduced sterol biosynthetic activity, but the total labeled neutral lipid fraction was greater than without drug addition to the incubation. Intracerebral injection of [2-¹⁴C]mevalonic acid into zuclomiphene-treated and control animals resulted in the isolation of much less labeled cholesterol from the drug-treated animal brains. Thin-layer Chromatographic and radioactivity-monitored gas—liquid Chromatographie analysis of the labeled free sterol fractions indicated more labeled lanosterol and zymosterol and less labeled desmosterol in the drug-treated brains. Approximately 20 per cent of the total free sterol radioactivity derived from the drug-treated brains had the mobility of C^Δ5,7₂₇-C^Δ8,14₂₇ sterols on silver nitrate thin-layer chromatography plates. On radioactivity-monitored gas-liquid chromatography the radioactive material derived from this thin-layer region was found to be contained in only one sterol. It had a retention time greater than C^Δ8,14₂₉ but less than would be expected for C^Δ8,14,24₂₉. Intraperitoneal administration of [3-¹⁴C]-D-(?)-β-hydroxybutyrate to zuclomiphene and corresponding controls resulted in lower [¹⁴C]sterol content in brain, spinal cord and liver of the treated animals. Silver nitrate thin-layer chromatography of the [3-¹⁴C]-d-(?)-β-hydroxybutyrate labeled brain and spinal cord free sterol fractions again indicated increased radioactivity in the C^Δ5,7₂₇?C^Δ8,14₂₇ sterol region. One of the short-term effects of zuclomiphene pretreatment in the central vervous system seems to be blockage of sterol biosynthesis shortly after lanosterol formation.     

Validation of a method for the determination of sterols and triterpenes in the aerial part of Justicia anselliana (Nees) T. Anders by capillary gas chromatography

An accurate and sensitive method, combining soxhlet extraction, solid phase-extraction and capillary gas chromatography is described for the quantitative determination of one triterpene (lupeol) and three sterols (stigmasterol, campesterol and beta-sitosterol) and the detection of another triterpene (alpha-amyrin) from the aerial part of Justicia anselliana. This is the first method allowing the quantification of sterols and triterpenes in this plant. It has been fully validated in order to be able to compare the sterol and triterpene composition of different samples of J. anselliana and therefore help to explain the allelopathic activity due to these compounds. This method showed that the aerial part of J. anselliana contained (292+/-2)mg/kg of lupeol, (206+/-1)mg/kg of stigmasterol, (266+/-2)mg/kg of campesterol and (184+/-9)mg/kg of beta-sitosterol.     

Studies on the constituents of the higher fungi of Korea(XI)

From the carpophores ofCoriolus sanguineus Fr., a bright-red, wood-rotting fungus, which were collected at Suwon, an antibiotic component was isolated as red crystal and identified as cinnabarin by UV and IR spectra. The antibiotic activities against five bacteria and three fungi were determined. It showed considerably high activity againstBacillus subtilis ATCC 6633 andBacillus maceraus and also against all the three fungi tested. And from this fungus a sterol was isolated and identified as ergosterol by cochromatographing with four authentic sterols.