A new lanostane-type triterpene from the fruiting bodies of Ganoderma lucidum

A new lanostane-type triterpene, named ganoderic acid LM 2 ( 5 ), was isolated from the fruiting bodies of Ganoderma lucidum . Its structure was characterized as (23S) 7 g , -dihydroxy-3, 11, 15-trioxo-5 f -lanosta-8, 24-dien-26-oic acid by 1D- and 2D-NMR spectra. In addition, a known triterpene, ganoderic acid l ( 4 ), was obtained. Both of them exhibited potent enhancement of ConA-induced mice splenocytes proliferation in vitro .     

灵芝子实体的化学成分研究

目的 研究灵芝（Ganoderma lucidum）子实体中的化学成分。方法 用硅胶柱色谱法及Sephadex LH-20柱色谱法进行分离纯化，依据理化性质和光谱数据鉴定化合物结构。结果 从灵芝的乙酸乙酯提取物中分离得到10个化合物，分别被鉴定为：灵芝烯酸B（ganoderenic acid B，1）、灵芝酸A（ganoderic acid A，2）、甲基灵芝酸A（methyl ganoderate A，3）、甲基灵芝酸B（methyl ganoderate B，4）、灵芝酸E（ganoderic acid E，5）、甲基灵芝酸E（methyl ganoderate E，6）、灵芝酸Q（ganoderic acid C3，7）、灵芝酸DM（ganoderic acid DM，8）和灵芝酸G（ganoderic acid G，9），2．（2’-hydroxydocosanoylamino）octadocane-1，3，4-triol（10）。结论 化合物10为新化合物。     

Metabolism and pharmacokinetics in rats of ganoderiol F,a highly cytotoxic and antitumor triterpene from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The metabolism of ganoderiol F (GF), a cytotoxic and antitumor triterpene from Ganoderma lucidum, by intestinal bacteria and its pharmacokinetics in rats were investigated by using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). GF was converted to ganodermatriol by anaerobic incubation with bacterial mixtures from rats and humans. This metabolite was detected in rat feces, but not in plasma and urine, after oral administration of GF. The fate of GF after oral (p.o.) and intravenous (i.v.) administration to rats was examined in pharmacokinetics studies. Plasma samples pretreated by solid-phase extraction were quantified by HPLC/MS/MS over a GF concentration range of 1.25–100 ng/ml (S/N = 5). The intra- and interday precision (CV%) was below 8% and accuracy was within the range of 95.9–103.6% for all samples. The range of recovery ratios was 89.2–98.2%. After the administration of GF at 0.5 mg/kg i.v., the plasma concentrations of GF quickly declined and the elimination half-life values (t _1/2α and t _1/2β) were about 2.4 and 34.8 min. On the other hand, the elimination half-life values (t _1/2α) after p.o. administration of GF at doses of 20 and 50 mg/kg were 14.4 and 143.3 min for the former, and 18.6 and 114.6 min for the latter. The AUC_0–t value was 11.17 (ng/ml) h at a GF dose of 0.5 mg/kg i.v., but 49.4 and 111.6 (ng/ml) h at GF doses of 20 and 50 mg/kg p.o., respectively, indicating that the AUC_0–t value is proportional to the administered oral doses. The estimated absolute bioavailability of GF in rats was F = 0.105.     

赤芝子实体中一个新的三萜化合物(英文)

赤芝 (Ganoderma lucidum) 是我国及东亚地区常用的民间草药, 具有良好的治疗及保健作用。本文对赤芝子实体的化学成分进行了研究, 从中分离并鉴定了两个羊毛甾烷型三萜类化合物, 其中化合物1为新化合物, 鉴定为 (24S)-羊毛甾-7-氧-8-烯-3β, 24, 25-三醇, 并命名为灵芝三醇 M (1); 化合物2为灵芝酸ε 。     

Lucidimine A-D,four new alkaloids from the fruiting bodies of Ganoderma lucidum

Abstract

Four new polycylic alkaloids, lucidimine A–D, were isolated from the fruiting bodies of Ganoderma lucidum. Their chemical structures were established based on 1D and 2D NMR data as well as HREIMS/HRESIMS analyses.     

HPLC determination of four triterpenoids in rat urine after oral administration of total triterpenoids from Ganoderma lucidum

A sensitive and simple high-performance liquid chromatography (HPLC) method was applied for the quantitative determination of four major triterpenoids (ganoderic acids C(2), B, K and H) in rat urine after oral administration of total triterpenoids from Ganoderma lucidum. The urine sample was extracted with dichloromethane-ethyl acetate (90:10) after acidification by hydrochloric acid (0.2 mol/ml). Chromatographic separation was achieved on a Zorbax SB-C(18) column (250 mm x 4.6 mm, 5 microm) at 35 degrees C, with a linear gradient of acetonitrile and 0.03% aqueous phosphoric acid (v/v), at a flow rate of 1.2 ml/min. The four triterpenoids and internal standard (hydrocortisone) were detected at a wavelength 252 nm. The within- and between-day assay coefficients of variation for the four triterpenoids in urine were less than 9% and the extraction recovery of this method was higher than 90%. Using this method, the excretion profile of the triterpenoids in rat urine after oral administration of total triterpenoids of G. lucidum was revealed for the first time.     

不同菌种灵芝药材中三萜类成分的对比研究

目的 研究不同菌种灵芝药材中三萜类成分的差异,比较三萜化学成分基于菌种的区别.方法 选定5个西南地区大面积培育的灵芝菌种,在相同条件下培育成灵芝药材;建立灵芝三萜类化学成分的HPLC指纹图谱,运用中药色谱指纹图谱相似度评价软件评价不同菌种灵芝中的三萜成分,结合SPSS软件对共有峰的峰面积进行主成分分析,并采用紫外分光光度法测定不同菌种灵芝子实体中总三萜的含量,综合评价灵芝药材的质量.结果 不同菌种灵芝栽培过程中菌盖发育良好,生长稳定,灵芝总三萜含量为0.99％～2.35％,其中,荣保灵芝1号菌种中灵芝总三萜的含量最高;建立了灵芝三萜类化学成分的HPLC指纹图谱,标定了22个共有色谱峰,采用对照品指认了10个主要色谱峰,不同菌种灵芝间的图谱差异明显,相似度较低,以荣保灵芝为对照品的相似度为0.742 ～0.779;不同菌种灵芝共有峰的峰面积主成分分析,提取了3个主成分,累计贡献率98.05％,荣保灵芝1号菌种灵芝的综合得分最高.结论 菌种的差异性直接影响灵芝中三萜类成分的种类及含量,菌种的优选对保证灵芝的品质至关重要.     

Development of a chromatographic fingerprint for the chloroform extracts of Ganoderma lucidum by HPLC and LC-MS

A new high-performance liquid chromatographic (HPLC) fingerprinting method was developed for the quality control of Ganoderma lucidum. Twenty-nine batches obtained from three different origins in China were used to establish the fingerprint. The constituents of these samples were separated with a Kromasil C(18) column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (100:0.1, v/v) and acetonitrile as mobile phase components at a flow rate of 0.8 ml/min and detector wavelength at 254 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of TCM". There were 19 common peaks in this fingerprint. Eleven of these common peaks were tentatively identified with reference to literature data based on their liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) and UV data. This profile was then successfully used to identify and assess the differences among samples from various origins with the aid of similarity analysis. The diverse similarities among different samples indicated that the quality of G. lucidum was not stable and the products from different areas were inconsistent. All results showed that the developed fingerprint assay was specific and could further serve for quality identification and comprehensive evaluation of G. lucidum.     

Characterization of an alkali-extracted peptidoglycan from KoreanGanoderma lucidum

The biologically active peptidoglycan was purified from the alkali fraction of the fruiting bodies of Ganoderma lucidum and the composition of the peptidoglycan was investigated by conventional analyses. The alkali-extracted peptidoglycan showed differences in chemical compositions from the water-extracted. The alkali-extracted peptidoglycan contained 6.9% protein and 75.9% carbohydrates composed mainly of beta-glucose, mannose, and alpha-glucose. The molecular weight range of the peptidoglycan was determined as 2,000 kDa-17 kDa. The peptidoglycan is considered to be a hybrid molecule of polysaccharide chains covalently bound as a side chain to the polypeptide core.     

Constituents from the fruiting bodies ofGanoderma applanatum and their aldose reductase inhibitory activity

Eight compounds were isolated from the fruiting bodies of Ganoderma applanatum, and were identified as 2-methoxyfatty acids (1), 5-dihydroergosterol (2), ergosterol peroxide (3) 3beta,7beta, 20,23xi-tetrahydroxy-11,15-dioxolanosta-8-en-26-oic acid (4), 7beta,20,23xi-trihydroxy-3,11,15-trioxolanosta-8-en-26-oic acid (5), cerevisterol (6), 7beta,23xi-dihydroxy-3,11,15-trioxolanosta-8,20E (22)-dien-26-oic acid (7), and 7beta-hydroxy-3,11,15,23-tetraoxolanosta-8,20E(22)-dien-26-oic acid methyl ester (8) by spectral analysis. All compounds were isolated for the first time from this fruiting bodies, and their effect on rat lens aldose reductase (RLAR) activity was tested. Among these eight compounds, ergosterol peroxide (3) was found to exhibit potent RLAR inhibition, its IC50 value being 15.4 microg/mL.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5μm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.     

福建灵芝高效薄层色谱鉴别及其在溯源中的应用

目的 本研究旨在建立福建灵芝高效薄层色谱方法,并对比福建产区与其他产区薄层的异同点,为灵芝药材在薄层溯源中的应用提供实验依据。方法 采用Merk HPTLC Silica gel 60预制板,氯仿-乙腈-甲醇-甲酸(13∶1.5∶0.2∶0.2)为展开剂,3%硫酸乙酸乙酯溶液显色,在紫外366 nm下检视,采用对照品比对鉴别薄层色谱条带。使用斑点条带数字化表征,对不同产区样品进行聚类分析。结果 高效薄层色谱共分离灵芝药材中17个条带,用对照品比对鉴定了其中11个成分,包括灵芝烯酸C(Rf₅=0.31)、灵芝酸C2(Rf₆=0.33)、灵芝酸I(Rf₇=0.35)、灵芝酸G(Rf₈=0.41)、灵芝酸A(Rf₉=0.44)、灵芝酸B(Rf₁₀=0.46)、灵芝内酯B(Rf₁₁=0.53)、3β,7β,15β-三羟基-11,15-二羰基-羊毛甾烷-8-烯-24→20内酯(Rf₁₂=0.56)、灵芝烯酸D(Rf     

Characterization and immunostimulatory activity of a polysaccharide from the spores of Ganoderma lucidum

Spores of Ganoderma lucidum contain a large amount of bioactive substances and have a higher bioactivity than the fruit bodies of G. lucidum. However, ingredients from spores are less studied due to the difficulties in collecting the spores and breaking the rigid shell. In this study, a water-soluble polysaccharide named GSG was extracted from the spores of G. lucidum. GSG is characterized to be a branched glucan that contains several different kinds of linkages. It was an effective inducer of MAPKs- and Syk-dependent TNF-α and IL-6 secretion in murine resident peritoneal macrophages. Dectin-1 could recognize GSG and partially mediate its biological activities. Additionally, in vivo administration of GSG potentiated the Con A-induced proliferative response of splenocytes and induced anti-tumor activity against Lewis lung cancer in mice. Therefore, these results suggest that GSG is an effective immunomodulator and may be a promising adjuvant remedy for anti-tumor therapies.     

星点设计-效应面法优化鹿角灵芝微乳超声提取工艺

目的 探讨以微乳超声法同时提取鹿角灵芝中灵芝酸和灵芝多糖的最佳条件。方法 采用星点设计-效应面法,以物料溶剂比(X₁)、超声提取时间(X₂)和超声功率(X₃)作为考察因素,以灵芝酸A、灵芝酸C₂、灵芝多糖含量和提取物得率为考察指标,并对考察因素和考察指标总评归一值进行多元线性回归和二项式拟合,以效应面优选最佳的微乳超声提取工艺,并做验证试验。结果 微乳超声提取鹿角灵芝的最佳条件为物料溶剂比1∶18、超声提取时间59 min、超声功率513 W,此条件下灵芝酸A、灵芝酸C₂、灵芝多糖含量和提取物得率分别为0.647 8,0.108 5,11.20 mg·g^-1和34.28%。结论 微乳超声可同时提取鹿角灵芝中脂溶性和水溶性成分,并且节能、省时、操作简便,为工业化提取鹿角灵芝提供了新途径。     

基于结肠癌HCT116细胞三维培养模型的灵芝组分抗肿瘤活性研究

目的 基于结肠癌HCT116细胞的二维(2D)和三维(3D)培养模型研究灵芝组分(Ganoderma components,Gc)的抗肿瘤作用。方法 采用UPLC-Q-TOF-MS鉴定3种Gc的化学组成;以Matrigel基质胶为基质材料建立体外HCT116细胞3D培养模型,评价Gc1、Gc2、Gc3和5-氟尿嘧啶(5-fluorouracil,5-FU)对2D和3D培养的HCT116细胞增殖的影响,并在HCT116 细胞3D培养模型中分析Gc3对周期、凋亡、耐药、脂代谢及5-FU抗肿瘤活性的影响;采用CCK-8法检测细胞活力;Real-time PCR检测细胞mRNA表达水平;Western blotting检测细胞蛋白表达水平;采用HPLC检测细胞内5-FU含量。结果 从Gc1、Gc2和Gc3中分别鉴定出76,69和17个化合物;与2D培养相比,3D培养的HCT116细胞增殖率更低,周期促进蛋白CDK2、CDK4、CDK6及脂肪酸合成蛋白FASN、SREBP1的表达显著下调,周期抑制蛋白p21和p27、脂滴分解蛋白ATGL及药物抵抗基因ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达水平显著上调;Gc1、Gc2、Gc3和5-FU作用48 h可呈浓度依赖性抑制2D和3D培养的HCT116细胞增殖,且Gc3的抑制作用显著强于Gc1和Gc2;在HCT116细胞3D培养模型中,Gc3可显著降低CDK2、CDK4、Bcl-xl、ATGL、LC3B的表达水平,升高p21、p27、Bax、Cleaved caspase-3、Cleaved PARP1的表达水平,过表达LC3B或ATGL均可减弱Gc3诱导的细胞毒性;Gc3还可显著抑制ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达,提高细胞内5-FU的浓度,并加强5-FU的抗肿瘤活性。结论 Gc3通过抑制细胞自噬和脂滴分解而诱导3D培养的HCT116细胞凋亡,并通过抑制ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达加强5-FU的抗肿瘤活性。     

灵芝破壁孢子粉总三萜提取工艺优化及体外抗肿瘤作用的研究

目的 优选出破壁灵芝孢子粉中总三萜的最佳提取工艺,并对其体外抗肿瘤活性进行评价。方法 正交试验法优化灵芝总三萜的提取工艺;MTT法测定体外抗肿瘤活性;流式细胞仪检测筛选出的细胞株的凋亡率。结果 最佳提取工艺条件为95%乙醇,液料比60：1,在85℃条件下,回流提取2 h,提取2次,总三萜的含量可达6.45%;灵芝总三萜对人结肠癌HCT116细胞、人肺癌A549细胞、人乳腺癌MDA-MB-231细胞的IC₅₀值分别为1.29,1.68,3.81 mg·mL^-1;不同浓度的灵芝总三萜（0.64,1.6,4.0,10 mg·mL^-1）作用于结肠癌HCT116 36 h能显著诱导结肠癌HCT116细胞发生凋亡。结论 该提取工艺提取效率较高,稳定、可行;灵芝总三萜在体外具有抗人结肠癌、人乳腺癌及人肺癌作用,并可显著诱导HCT116细胞产生凋亡作用。     

In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 microM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [(3)H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [(3)H]-digoxin polarized transport attained at 50 microM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [(3)H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 microg/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.     

松杉灵芝发酵产物对H22肝腹水瘤小鼠免疫功能影响及抗肿瘤作用

目的 研究松杉灵芝发酵产物对H22肝腹水瘤小鼠免疫系统的影响及抗肿瘤活性.方法 采用H22肝腹水瘤小鼠模型,设生理盐水空白组、阳性药对照组、发酵产物高、中、低剂量组,每天灌胃给药1次,连续15d,末次给药后检测脾脏T淋巴细胞和B淋巴细胞增值情况,用酶联免疫分析法检测血清中免疫球蛋白(IgG)和白细胞介素2(IL-2)的含量,MTT法检测H22肿瘤细胞的增殖情况.结果 与空白对照组相比,松杉灵芝发酵产物各组的白细胞介素2、胸腺指数、抑瘤率、B细胞反应均显著提高,茵丝中、低剂量组T细胞增殖显著增加,茵丝低剂量组、发酵液高剂量组和中剂量组免疫球蛋白均显著提高.结论 松杉灵芝发酵产物可显著提高小鼠的免疫功能,有效抑制H22肝腹水瘤细胞的增殖.     

苦瓜多糖抗肿瘤及免疫增强活性的研究

目的：研究苦瓜多糖的体内抗肿瘤及增强免疫作用,探讨可能作用机制。方法：通过建立小鼠S180肉瘤、H22肝癌肿瘤模型观察苦瓜多糖的抗肿瘤作用．此外应用MTT法观察苦瓜多糖对小鼠脾淋巴细胞增殖作用的影响．应用比色法观察苦瓜多糖对小鼠单核巨噬细胞RAW264．7吞噬功能的影响．运用Gfiess试剂检测苦瓜多糖促进小鼠单核巨噬细胞RAW264．7分泌No含量的变化。结果：苦瓜多糖高中低剂量组能明显抑制小鼠S180肉瘤、H22肝癌肿瘤的生长,同时能明显增加荷瘤小鼠的脾腺指数和胸腺指数。此外苦瓜多糖能显著刺激小鼠脾淋巴细胞增殖．明显提高小鼠单核巨噬细胞RAW264．7吞噬中性红的能力,明显促进小鼠单核巨噬细胞RAW264．7分泌No。结论：推测苦瓜多糖具有较强的免疫增强活性并可能通过刺激淋巴细胞、并增强巨噬细胞的活化来调节机体的免疫功能．从而抑制肿瘤细胞的生长。     

VRCTC-310 — A novel compound of purified animal toxins separates antitumor efficacy from neurotoxicity

Summary Two purified animal venom toxins, crotoxin and cardiotoxin, have been combined to produce a unique natural product (VRCTC-310) currently under investigation as an antitumor agent by the National Cancer Institute.In vitro, it has demonstrated cytotoxic disease specificity and a unique mechanism of action when submitted to COMPARE analysis.In vivo, tolerance was developed to the neurotoxic properties of crotoxin which allowed comparison of several schedules of fixed and escalating daily i.m. doses to mice bearing s.c. Lewis Lung carcinoma. An 83% inhibition of tumor growth was achieved using an escalating dose schedule starting at 1.8 mg/kg and reaching 6.3 mg/kg/day on day 20. Although some irritation around the sites of i.m. injection was noted, animal weight loss was negligible and there were no other signs of adverse toxicity. This natural product represents a new, membrane interactive anticancer agent which produces a unique spectrum of cytotoxicityin vitro and which has demonstrated interestingin vivo antitumor efficacy.