富硒灵芝中一种新含硒蛋白的纯化、性质及其自由基清除活性研究

通过硫酸铵沉淀、离子交换层析和分子排阻层析等方法, 从富硒灵芝中获得了一种新的含硒蛋白, 命名为Se-GL-P, 并研究了此蛋白的性质、抗氧化活性与其硒含量间的关系. 结果表明, 此蛋白的分子量为36600, 分子中约含有19.8%的糖链, N端的氨基酸残基序列为DINGGGATLPQKLYLTPDVL, 属于DING蛋白家族. 硒含量为4.87 mg/g, 具有较高的羟自由基和超氧自由基清除活性. 研究发现, Se-GL-P的抗氧化活性的提高与其中硒含量的提高相关.     

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.     

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.     

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；     

灵芝发酵液中蛋白酶抑制剂GLPIA2的纯化及其特性

采用乙醇分级沉淀、凝胶色谱纯化、阴离子交换色谱分离等步骤从灵芝深层发酵液中提取得到蛋白酶抑制剂GLPIA1 与GLPIA2。其中GLPIA2仅在215 nm处有紫外吸收,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定为单一条带,相对分子质 量为15000。由其氨基酸组成分析谱图可看出,其酸性氨基酸含量较高,碱性氨基酸及芳香族氨基酸含量较低。GLPIA2抑 制剂的底物特异性研究表明,它对天冬氨酸族的胃蛋白酶和酵母蛋白酶A有相对较强的抑制作用。     

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。     

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.     

Purification and Characterization of PRL Protein Tyrosine Phosphatases

PRLs constitute a subfamily of protein tyrosine phosphatases(PTPs). In the present paper are reported the molecular cloning, expression, purification, and characterization of all the three members of the PRL enzymes in human and the only PRL in C. elegans. These enzymes were expressed as glutathione S-transferase (GST) fusion proteins in DE3pLysS E. coil cells, and the recombinant fusion proteins were purified on glutathione-Sepharose affinity columns. Having been cleaved with thrombin, GST-free enzymes were further purified on an S-100 Sepharose gel filtration column. The purified proteins show single polypeptide bands on SDS-polyacrylamide gel electrophoresis. With para-nitrophenyl phosphate(p-NPP) as a substrate, PRLs exhibit classical Michaelis-Menten kinetics with Vmax values two orders of magnitude smaller than those of classic PTPs. The responses of PRLs to ionic strength, metal ions and phosphatase inhibitors are similar to those of other characterized PTPs, but their optimal pH values are different. These data thus reveal distinct common biochemical properties of PRL subfamily PTPs as well.     

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。     

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.     

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.     

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.     

Purification and Characterization of a Proteolytic Enzyme from Fig Latex

Ficin is an important component of plants in Ficus family such as fig latex. It is of special significance in medicine and industry because it exhibits activity throughout a wide range of temperature and pH values. In this work, we purified a component of ficin from the latex homogeneity of Shandong fig trees, and the properties of the purified ficin were studied. The current findings revealed that heavy metal ions were able to inhibit ficin, while DTT, L-cysteine, and β-ME were found to promote ficin activity. It was also observed that the half life of ficin at 65 °C was longer than 1 h and the Michaelis constant（Km） for casein hydrolyzation was determined to be 1.56 mg/mL. Our study shows that this purified ficin is a cysteine protease.     

Deciphering the chemical composition of Ganoderma lucidum from different geographical origins by mass spectrometry molecular networking coupled with multivariate analysis

Ganoderma lucidum is a medicinal fungus that has been widely used in China and many Asian countries for thousands of years. This once rare macrofungus has now been artificially cultivated in a number of regions in China. However, detailed knowledge of its composition across different geographical origins is still lacking, as are analytical methods for comprehensive profiling of the diverse phytochemicals contained in G. lucidum. In this work, an on-demand strategy based on high-resolution MS and molecular networking is applied for natural product characterization, which led to the identification of 84 constituents in G. lucidum. Moreover, multivariate analysis, including hierarchical cluster analysis and orthogonal partial least squares-discriminant analysis, was used to analyze the (dis)similarity of the G. lucidum samples collected from the three main production areas (i.e., Jilin, Henan and Shandong Province). The results revealed a significant variation in the chemical composition of samples from different provinces. Marker constituents corresponding to the differentiation were then screened in terms of the variable importance in projection value, P-value and fold change. A total of 24 constituents were identified as geoherbalism markers, such as ganoderenic acid A for Henan, ganolucidic acid B for Jilin and ganodernoid D for Shandong. This proof-of-concept application demonstrates that combining MS molecular networking with meticulous multivariate analysis can provide a sensitive and comprehensive analytical approach for the quality assessment of traditional Chinese medicine ingredients. This study also suggests that the bioactivity and efficacy from different origins should be further evaluated considering the large difference in chemical compositions.     

Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum

Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at −80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase (Erk1/2, p38, and JNK) and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts.     

Purification and Characterization of Superoxide Dismutase （SOD） from Camellia Pollen

A superoxide dismutase（ SOD ） was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose（ DE52 ）, Sephadex G-100 and phenyl sepharose^TM 6 Fast Flow columns. Its specific activity could reach to 4034 U/mg protein and it was determined to be Cu/ Zn-SOD according to its different sensitivities to different inhibitors. The molecular weight of the SOD and its subunit were 69500 and 34700, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）, which implicates that the SOD in camellia pollen is a dimmer composed of two identical subunits. The isoelectric point of the enzyme was determined to be 4. 1 by isoelectric focusing electrophoresis and the N-terminal amino acid was identified to be Gly by the DNS-Cl method. Its α-Helix was also calculated to be approximately 21.8% according to the circular dichroism（CD） spectra.     

Studies on the Constituents of Ganoderma Lucidum

The methanolic extract of fruit bodies of cultivated Ganoderma lucidum was separated by silica gel column chromatography and preparative thin-layer chromatography to give ten compounds. On the basis of spectral analysis, chemical procedures and gas chromatography, d-mannitol (1), ergosta-7, 22-dien-3β-yl palmitate (2), ergosterol (3), ergosta-7, 22-dien-3β-ol (4), 5α-lanosta-7,9(11),24-trien-3β,26-diol (5), ergosterol peroxide (6), 24,25,26-trihydroxy-5α-lanosta-7,9(11)-dien-3-one (7), 5α-lanosta-7,9(11)-dien-3β,24,25,26-tetraol (8) and 8,9-epoxyergosta-5,22-dien-3β,15-diol (9) were identified. Among these compounds, 8,9-epoxyergosta-5,22-dien-3β,15-diol was first separated from Ganoderma lucidum.     

Platelet Aggregation Inhibitor from Ganoderma lucidum

Lucidenic acid A ( 1 ) and a new lactone, lucidenolactone ( 2 ) were isolated from the fruiting bodies of Ganoderma lucidum. Their structures were elucidated by spectroscopic method and single-crystal X-ray analysis. Lucidenolactone ( 2 ) showed significant antiplatelet aggregation activity. The NMR spectral data of lucidenic acid A were also reassigned.     

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。