灵芝多糖的结构特征分析

采用沸水回流法从灵芝子实体中提取多糖,经Sevage法除蛋白,乙醇沉淀,离心、透析、膜分离,浓缩、冻干后得灵芝多糖。经HIO4氧化、Smith降解及甲基化反应,并利用多糖及刚果红混合液在碱性溶液中的波长的红移变化,通过UV-VIS,IR,GC,GC-MS,NMR对灵芝水提多糖的结构特征及三螺旋体结构进行分析研究。结果表明:灵芝多糖含有三螺旋体构型,GC-MS分析灵芝多糖的主要单糖组分为葡萄糖,还有少量的半乳糖、甘露糖、木糖和艾杜糖,IR及1HNMR分析多糖为β-构型,HIO4氧化、Smith降解和甲基化分析表明:多糖主要为(1→3)糖苷键连接构型,并伴有少量的1→6位支链键连接的结构,灵芝多糖是由D-葡萄糖单元通过β-(1→3)糖苷键连接葡聚多糖,其主要构型特征为(1→3)β-D-线性连接的骨架结构。     

荧光光谱法测定灵芝多糖的含量

采用苯酚-硫酸法建立了灵芝多糖的荧光光谱测定方法。当葡萄糖浓度为20～100μg.mL-1时,λem=518nm处荧光强度与葡萄糖浓度有良好的线性关系,相关系数为0.9994,检出限为6.35μg.mL-1。用拟定的方法对9种灵芝样品进行了测定,其多糖含量在0.712%～2.548%之间,与《药典》法测定结果一致。加标平均回收率为99.82%,相对标准偏差为3.46%。该法结果稳定、重复性好,可用于天然产物中多糖含量的分析。     

重组灵芝免疫调节蛋白的纯化及其性质

采用超滤浓缩、强阴离子交换、疏水作用和凝胶色谱等方法, 对毕赤酵母表达的rGlip进行分离和纯化, 对离子交换色谱中rGlip与固相结合的最佳pH值进行了考察, 并对纯化产物的活性进行了鉴定. rGlip在215 nm处有强的紫外吸收, 经激光解析电离时间飞行质谱鉴定其相对分子量为12722, 经反相液相色谱鉴定纯度≥97%. 设计rGlip的疏水作用色谱, 有效地去除色素. 凝血实验结果表明, rGlip可以凝集绵羊血红细胞, 但对人血A, B, AB和O型等红细胞无凝集作用, 有类似凝集素的生物学活性.     

灵芝菌丝体多糖的化学组成和溶液性质

用磷酸盐缓冲液在80℃时从灵芝菌丝体中提取出多糖-蛋白质缀合物LM-A.成分分析表明,GLM-A主要由葡萄糖组成,蛋白质含量为13%.GLM-A在1∶1水稀释的饱和镉乙二胺溶液中30℃时的特性粘数[η]和均方根旋转半径〈S²〉^1/2的分子量依赖关系分别为[η]=5.1×10^-2M_w^0.06(cm³·g^-1)和〈S²〉^1/2=3.9×10^-2M_w^0.50(nm).按照Yamakawa-Fujii-Yoshizaki蠕虫状圆筒模型的粘度理论和Bohdanecky表达式,求得GLM-A的分子参数为:单位围长摩尔质量M_L=(530±10)nm^-1,持久长度q=(2.8±0.2)nm.链直径d=0.75nm.实验结果表明,GLM-A在该溶液中为无规线团构象.     

Chemotaxonomy of Triterpenoid Pattern of HPLC of Ganoderma lucidum and Ganoderma tsugae

Three strains of Ganoderma tsugae (CCRC36065, CCRC37034, CCRC37038) and three strains of Ganoderma lucidum (CCRC36021, CCRC37029, CCRC37033) were cultivated. Their triterpenoid patterns of the fruit body were analyzed by reverse phase HPLC using a gradient elution of acetonitrile/2% acetic acid (1/4 and 1/2). The triterpenoid patterns of G. tsugae and G. lucidum are different. But similar 2 and 3 dimensional patterns are obtained among three strains of G. tsugae. Different patterns are found among different strains of G. lucidum. Ganoderic acid A( 1 ), B( 2 ), C( 3 ) and D( 4 ) were isolated from the ethanol extract of G. tsugae.     

超临界流体萃取-高效液相色谱离线联用分析灵芝中三萜类化合物

在系统考察压力、温度和时间对萃取率影响的基础上,利用超临界流体萃取技术提取了灵芝子实体中的三萜类化合物。其最佳萃取条件为:压力15 MPa,温度35℃,动态萃取时间120 m in,CO2流量1 mL/m in,背压阀温度50℃。此外,还建立了高效液相色谱梯度洗脱分离三萜类化合物的方法。通过比较超临界流体提取物和甲醇提取物的色谱图,发现两者具有相似的峰形,说明超临界流体能够达到与甲醇相近的萃取效果,可以取代甲醇作为新一代的绿色萃取溶剂。     

灵芝富碘实验及碘的测定

对灵芝菌种ＴＷ富碘能力进行了研究，结果表明：灵芝ＴＷ富碘能力较强，添加１０００×１０－６～８０００×１０－６范围内的碘化钾，在２５℃、１３０ｒ／ｍｉｎ条件下培养，生长良好．富集碘的范围为３．０５×１０－３～７．７５×１０－３．     

灵芝多糖的结构分析

从灵芝菌丝和子实体热水提取液中分离和纯化得到两种多糖，凝胶过滤导析测得分了量分别为３．７×１０＾４和４．２×１０＾４。气相色谱分析表明菌丝体多糖含Ｄ－葡萄糖，Ｄ－半乳糖，Ｄ－甘露糖，Ｄ－木糖，Ｌ－岩藻糖，Ｌ－鼠李糖，其摩尔比为５．３５：２．６７：１．００：１．１９：０．３８：０．３７；子实体多糖含Ｄ－葡萄糖，Ｄ－半乳糖，Ｄ－甘露糖，Ｄ－木糖，Ｌ－阿拉伯糖，Ｌ－鼠李糖，其摩尔比为５．８２：２．２３；     

灵芝富碘实验中多糖含量变化的探讨

阐述了ＴＷ灵芝在液体深层培养中，进行富集碘实验过程菌丝体多糖含量的变化．实验表明，在ＴＷ灵芝液体培养中添加１０００×１０－６～８０００×１０－６浓度的碘化钾，菌丝体多糖含量为０．９２％～１．５８％．     

灵芝及虫草菌丝体中微量元素含量的分析

利用原子吸收光谱法和分光光度法对人工培育的灵芝,灵芝孢子粉,虫草菌丝体中具有重要生理意义的元素锌,铁,锰,镁,锗进行了检测,比较了灵芝子实体,灵芝孢子粉,虫草菌丝体中５种元素的含量,探讨了５种元素的药理作用。     

Purification and Characterization of Cyclic AMP-Binding Protein from Ganoderma lucidum

Cyclic AMP-binding protein was purified 30 fold from the mycelia of Ganoderma lucidum by the methods of ammonium sulfate precipitation, DEAE-cellulose, phospho-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of the purified protein is 34.5 kDa and 17 kDa by Sephacryl S-100 gel filtration and SDS-ployacrylamide gel electrophoresis, respectively. From these results it is suggested that the protein has a homometric dimmer structure. The pI of the purified protein is pH 8.2 by native isoelectric focusing gel. The half-life of the protein activity in 10% glycerol at 4 ℃ is 7 d in crude extract, but its half-life is only 3 d under purifying conditions. The optimal conditions of the protein activity are at 1 ℃ and pH 7.5. Its activity is increased 6 times by 1 mmol/L Zn^2 and is slightly inhibited by cGMP,Cu^2 and Mn^2 .     

各种栽培因子下培养的灵芝无机元素含量比较测定

研究表明,不同产地、段木材料和采收期所产出的灵芝,其无机元素含量有明显差异,为筛选最佳的灵芝野外生长条件提供了可靠依据。     

HPLC method for the determination and pharmacokinetic studies of four triterpenoids in rat plasma after oral administration of Ganoderma lucidum extract

Four major triterpenoids (ganoderic acids C(2), B, K and H) in rat plasma after oral administration of G. lucidum extract were analyzed quantitatively by high-performance liquid chromatography (HPLC). Plasma samples taken from rats were acidified with hydrochloric acid and extracted with dichloromethane-ethyl acetate (90:10). The chromatographic separation was achieved on an Agilent Zorbax SB-C(18) column (250 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 1.0 mL/min. The four triterpenoids and internal standard (hydrocortisone) were detected at a wavelength 252 nm. All calibration curves showed good linearity (r(2) > 0.99) within test ranges. The relative deviation of this method was less than 10% for intra- and inter-day assays, and the accuracy ranged from 89 to 108%. The extract recovery for the four triterpenoids and internal standard ranged from 95 to 67%, and the QC samples were found to be stable according to the results of the stability study. This is the first report on determination of the major triterpenoids in rat plasma after oral administration of G. lucidum extract and the results provided a firm basis for clarifying the pharmacological effect of G. lucidum and evaluating the clinical applications of this medicinal fungus.     

Autotoxicity of Endogenous Organic Acid Stress in Two Ganoderma lucidum Cultivars

Ganoderma lucidum has been used as a rare medical mushroom for centuries in China, due to its health-promoting properties. Successive cropping obstacles are common in the cultivation of G. lucidum, although the remaining nutrients in the germ substrate are sufficient for a second fruiting. Here, we aimed to study the metabolite profile of G. lucidum via nontargeted metabonomic technology. Metabonomic data revealed that organic acids played an important role in the cropping obstacles of G. lucidum, which is accordance with the pH decrease in the germ substrate. A Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis indicated that most differential acids participated in the metabolic pathways. Five acids were all significantly upregulated by two MS with high energy (MS^E) modes in two cultivars, among which 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid is also involved in purine metabolism regulation and microbial metabolism in diverse environments. Taken together, this work illustrated the organic acid stress generated by G. lucidum, which formed the autotoxicity feedback, and resulted in cropping obstacles. Determining the cause of the cropping obstacles in G. lucidum will promote the utilization rate of fungus substrate to realize the sustainable use of this resource.     

Lanostanoids Isolated from Ganoderma lucidum Mycelium Cultured by Submerged Fermentation

Three new lanostane triterpene acids, 3‐O‐acetylganoderic acid B ( 1 ), 8β,9α‐dihydroganoderic acid C ( 3 ), and 3‐O‐acetylganoderic acid K ( 4 ), as well as two new lanostane triterpene acid ethyl esters, ethyl 3‐O‐acetylganoderate B ( 2 ) and ethyl ganoderate J ( 5 ), were isolated and characterized from Ganoderma lucidum mycelia which was cultured by submerged fermentation method. Their structures were elucidated on the basis of spectroscopic methods. In addition, the identification of two known lanostane triterpene acid methyl esters, methyl O‐acetyl ganoderate C and methyl 3,7,11,15,23‐pentaoxo‐lanost‐8‐en‐26‐oate were identified by comparison of the NMR data with those reported in the literature.     

Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors

In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.     

Studies on the Constituents from the Bark of Bauhinia Purpurea

In a previous paper, were ported a new stigmasteroyl glucoside derivative together with four known compounds. In addition to the above compounds, the isolation and identification of twenty‐four compounds from the same source are described here. Their structures were deter mined on the basis of spectral evidence as well as comparison with authentic samples. Those compounds included four waxes, four triterpenes, eight steroids, two glyceride, three flavonoids, and three phenylpropanoids. Some new compounds were found in mixtures of three alkyl ω‐hydroxyalkanoate, glyceride, and phenylpropanoids.     

Improved Method of Extraction and High-Performance Liquid Chromatographic Separation of Ganoderic Acids from Ganoderma Lucidum

A rapid and efficient method is described for the extraction of ganoderic acids from Ganodemia lucidum by means of a small Sep-Pak C₁₈ cartridge. The application of high-performance liquid chromatography to determine the chemical compositions of strains of Ganoderma lucidum is also reported.     

傅立叶变换离子回旋共振质谱法对赤灵芝的化学成分鉴定和指纹图谱研究

建立了基于傅立叶变换离子回旋共振超高分辨质谱(FTICR-MS)的赤灵芝化学成分鉴定和指纹图谱分析方法,并应用于不同产地赤灵芝样品的来源区分。样品采用50%甲醇进行回流提取后,以流动注射的进样方式进行直接质谱分析。以ESI离子源在负离子模式下进行检测,质荷比扫描范围为100～1 000 Da。采用精确分子量测定和碰撞诱导解离实验进行化学成分鉴定,通过与文献进行比对,共鉴定出63种化学成分(1种萜烯醛、3种糖、4种三萜醇、6种有机酸和49种三萜酸类成分)。采用聚类分析和主成分分析(PCA)对所获得的质谱指纹图谱进行统计学分析,在95%的置信区间下,多个批次不同产地的赤灵芝样品得到了较好的来源区分。研究结果表明该方法可实现赤灵芝的直接、快速、高效分析和指纹图谱研究,在中药分析领域有着广阔的应用前景。     

Peroxidase-Like Platinum Clusters Synthesized by Ganoderma lucidum Polysaccharide for Sensitively Colorimetric Detection of Dopamine

The sensitive and selective detection of dopamine (DA) is very important for the early diagnosis of DA-related diseases. In this study, we reported the colorimetric detection of DA using Ganoderma lucidum polysaccharide (GLP) stabilized platinum nanoclusters (Pt_n-GLP NCs). When Pt₆₀₀-GLP NCs was added, 3,3’,5,5’-tetramethylbenzidine (TMB) was rapidly catalyzed and oxidized to blue oxTMB, indicating the peroxidase-like activity of Pt₆₀₀-GLP NCs. The catalytic reaction on the substrate TMB followed the Michaelis-Menton kinetics with the ping-pong mechanism. The mechanism of the colorimetric reaction was mainly due to the formation of hydroxyl radical (•OH). Furthermore, the catalytic reaction of Pt₆₀₀-GLP NCs was used in the colorimetric detection of DA. The linear range for DA was 1–100 μM and the detection limit was 0.66 μM. The sensitive detection of DA using Pt-GLP NCs with peroxidase-like activity offers a simple and practical method that may have great potential applications in the biotechnology field.