灵芝孢子粉中脂溶性成分分析方法的建立

运用高效液相建立灵芝孢子粉中脂溶性成分的分析测定方法。通过色谱柱、洗脱条件、ELSD参数的优化,建立了12种脂溶性成分的测定方法,结果表明,该方法简单、准确、稳定,可以实现孢子粉中甘油三酯、脂肪酸、甾醇三类脂溶性成分的同时提取、分析;破壁孢子粉中脂溶性成分为301.49-397.37mg/g,孢子油产品中脂溶性成分的含量为626.00-713.07mg/g,远高于三萜含量;1,2-二油酸-3-棕榈酸甘油酯、甘油三油酸酯、1,2-二油酸-3-亚油酸甘油酯是主要的脂溶性成分,脂肪酸以不饱和脂肪酸亚油酸及油酸为主,甾醇中麦角甾醇含量最高。研究结果明确了灵芝孢子粉中脂溶性成分的物质基础,为深入研究其活性成分、全面评价孢子粉质量提供了依据。     

灵芝孢子粉中核苷类成分分析

本文利用高效液相色谱方法（HPLC）同时对灵芝孢子粉中的15种核苷类成分的含量进行测定。采用Ultimate AQ-C₁₈（4.6mm×250mm,5μm）色谱柱,以甲醇和水为流动相进行梯度洗脱,流速1.0mL/min,检测波长259nm,柱温30℃,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,适用于灵芝孢子粉中核苷类成分的测定分析。运用建立的方法对不同破壁时间、不同采收时期龙泉、奉化、大别山、黄山4个产区的灵芝孢子粉中的15种核苷类成分的含量进行测定。结果表明破壁处理对灵芝孢子粉中核苷类成分提取率的影响不大,不同产地的灵芝孢子粉中核苷类成分的组成和含量具有显著差异,且孢子粉中的核苷含量随着产粉时间的延长有所增加。各待测样品中均含有胞嘧啶、尿苷、腺嘌呤、鸟苷、腺苷等成分,其中尿苷、鸟苷、腺苷3种核苷的含量占总量的比例在待测样品中均达到70%以上,为灵芝孢子粉中的主要核苷类成分。     

破壁灵芝孢子粉的质量探讨

研究和探讨破壁灵芝孢子粉的质量标准,利用性状鉴别、显微鉴别、理化鉴别和主要成分含量测定的方法,初步掌握破壁孢子粉质量特性。薄层色谱鉴别可见清晰荧光斑点,其醇溶液加入硫酸后可见棕色环。总灰分测定显示:采自岫岩灵芝孢子粉和山东一等孢子粉灰分较低,山东三等孢子粉灰分含量较高。不同来源的样品总三萜类含量各不相同,高者达2.24%,低者1.26%。综合各项检测结果,初步确定了破壁灵芝孢子粉质量评价指标,方法稳定,可操作性强。     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

灵芝子实体中三萜类物质抗肿瘤活性的谱效关系

通过HPLC指纹图谱结合多元线性回归分析对不同产地灵芝子实体的功效性特征进行评价,为寻找灵芝中活性三萜提供理论依据。利用高效液相分析方法,结合样品对肿瘤细胞L1210的增殖抑制率,运用“中药色谱指纹图谱相似度评价系统2004A版”软件和多元线性回归分析11批不同品种灵芝子实体中的三萜活性成分。样品与标准指纹图谱的相似度均在0.9以上,共标定了12个共有物质峰,其中与抗L1210肿瘤细胞活性关系密切的三萜物质有灵芝酸C₂、灵芝酸G、灵芝烯酸B、灵芝烯酸A、灵芝酸K、灵芝酸A、灵芝酸F和灵芝醛A。     

灵芝子实体、菌丝体及孢子粉中多糖成分差异比较研究

为探讨灵芝子实体、菌丝体和孢子粉3种材料中多糖成分的差异,分别运用苯酚硫酸法进行多糖含量测定,运用离子色谱分析其酸水解后单糖组成,并运用HPLC分析各多糖图谱及经α-淀粉酶和β-1,3-葡聚糖酶处理后HPLC图谱的变化,结果发现,灵芝菌丝体中多糖含量最高,达到3.81%,孢子粉多糖含量为1.8%,灵芝子实体中多糖含量最低,仅为0.59%;水解后的单糖组成及摩尔比也有差异,子实体的单糖主要为葡萄糖和半乳糖,菌丝体和孢子粉的单糖主要为葡萄糖;HPLC图谱显示3种多糖出峰位置和分子量也不同,酶解效果表明多糖结构也相差较大。各样品多糖对小鼠巨噬细胞RAW264.7释放NO的产量的影响上,菌丝体与子实体多糖都表现出了很好的活性,而孢子粉多糖却呈现出较低活性。实验结果表明灵芝子实体、菌丝体和孢子粉3种材料的多糖成分差异大,在医药保健品使用中应区分使用。     

猪苓甾酮类化合物的HPLC含量测定及指纹图谱研究

收集中国12个省区35个猪苓样品,利用高效液相色谱技术,测定5个甾酮类化合物含量,获得指纹图谱,利用相似度评价对图谱数据进行分析。共有28个样品能获得定量分析结果,其中25个样品中猪苓酮A和B的相对含量大于70%;经相关分析,猪苓酮A和B的含量之间呈极显著正相关(r=0.955,P0.01)。陕西省样品平均总甾酮含量以及猪苓酮A和B相对含量均最高,分别为182.70μg/g(C.V=82.4%)和92.3%(C.V=3.1%)。35个样品之间指纹图谱相似度0.412–0.943,标定了8个共有峰,指认了其中的3个色谱峰。11个陕西省样品之间指纹图谱相似度0.812–0.989,标定了17个共有峰,指认了其中的9个色谱峰。以上结果说明,猪苓酮A和B是猪苓主要甾酮类成分,对猪苓药材的质量控制有重要意义;陕西省的猪苓质量好且稳定。     

灵芝子实体和孢子粉三萜含量的测定及体外抗肿瘤活性的评价

目的测定灵芝子实体和破壁灵芝孢子粉中的三萜含量,并评价其醇提物的体外抗肿瘤活性。方法采用高氯酸显色法测定灵芝子实体和破壁灵芝孢子粉中的三萜含量,采用磺酰罗丹明B蛋白染色法(sulforhodamine B,SRB)评价灵芝子实体和破壁灵芝孢子粉醇提物对宫颈癌He La细胞、结肠癌HCT-116细胞和乳腺癌MCF-7-ADM细胞的体外抗肿瘤活性。结果研究的灵芝子实体中三萜质量分数为0.92%,破壁灵芝孢子粉供试品中三萜质量分数实测值为2.95%,但是灵芝孢子粉中脂肪油对高氯酸显色法有极大的干扰,因此破壁灵芝孢子粉三萜的实测值可能远高于实际值。活性评价结果显示,灵芝子实体的醇提物在100μg/m L时,其对人宫颈癌He La细胞和乳腺癌MCF-7-ADM细胞表现出了较强的抑制作用,抑制率分别为94.54%和71.30%;对结肠癌HCT-116细胞表现出较弱的抑制作用,抑制率为20.68%。破壁灵芝孢子粉的醇提物在100μg/m L和10μg/m L时,其对宫颈癌He La细胞表现出了弱的抑制作用,对另外2种细胞系仅在100μg/m L时表现出较弱的抑制作用。结论灵芝子实体的醇提物含有三萜并具有较好的抗肿瘤活性,可以将其开发为辅助治疗癌症的药物或保健品。含有脂肪油的灵芝孢子粉直接采用高氯酸比色法测定会高估其三萜的含量,需要进一步开发准确、可行的测定方法。     

紫外分光光度法测定破壁灵芝孢子粉提取物总三萜含量

确定一种破壁灵芝孢子粉提取物总三萜紫外分光光度测定方法。采用紫外分光光度法,以熊果酸为对照品测定样品。溶液在波长548 nm处具有最大吸光度,熊果酸含量在14~54μg范围内呈良好的线性关系,r为0.999 4,平均回收率为92.94%。提示使用上述方法绘制标准曲线可计算出样品中三萜类化合物的含量。测定方法操作简便、检验快速、稳定性高、重复性好,可以用于灵芝孢子粉产品的质量控制。     

基于UPLC指纹图谱和多指标成分定量的不同产地白茅根标准汤剂质量评价研究CSCD

本研究通过超高效液相色谱(UPLC)指纹图谱结合多成分定量对不同产地的白茅根标准汤剂的质量进行评价。采用相似度计算、聚类分析(HCA)、主成分分析(PCA)和正交偏最小二乘法-判别分析(OPLS-DA)对指纹图谱数据进行分析。结果显示,18批白茅根标准汤剂指纹图谱共标识出9个共有指纹峰,指纹图谱相似度在0.920~0.997;HCA将18批样品分为3类,河北产区的样品自成一类,江苏与安徽、河南的样品在分类上存在交叉;PCA结果显示,除江苏外,同一产区白茅根标准汤剂指纹图谱共有峰的差异较小;OPLS-DA筛选出3个差异标志性色谱峰,分别为峰9(对羟基肉桂酸)、峰6和峰5(绿原酸)。对白茅根标准汤剂中绿原酸、新绿原酸、隐绿原酸和对羟基肉桂酸的含量进行测定,结果显示,4种酚酸的总含量:河北>安徽>江苏>河南,不同产区的样品4种酚酸的总含量差异明显;其中河南产区的样品绿原酸、新绿原酸和隐绿原酸的含量明显低于其他产区,安徽产区的样品对羟基肉桂酸的平均含量最高且比较稳定,江苏产区的样品对羟基肉桂酸和4种酚酸的总含量波动较大。所建立的分析方法稳定可靠,重复性好,为白茅根标准汤剂及其相关制剂的质量评价提供参考。     

Mould growth and conidiation in cereal grains as affected by water activity and temperature

The effects of different water activities ( a _w) and temperatures on growth of storage moulds ( Aspergillus candidus and Penicillium implicatum ) on maize and paddy rice grains were measured using ergosterol content and dilution plating. The results showed that the mode of fungal growth depends on a _w. In all cases, conidiation occurred more readily at low a _w, whereas mycelial growth is more marked at higher a _w. The xerotolerant, thermophilic characteristics of A. candidus were more clearly revealed by the ergosterol content, which reflected mycelial development, than by enumeration of spores formed. The ergosterol content proved to be a better index of technological quality of cereals (assessed by acidity) than the number of fungal propagules. A 10-fold increase in the number of spores corresponded to a doubling of the ergosterol content.     

Distribution of free and esterified ergosterols in the medicinal fungus <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The fruiting bodies, spores, and lipid from the spores of Ganoderma lucidum have been widely used for medicinal purpose in China. Ergosterol content may be a suitable marker for evaluating the quality of ganoderma spore and ganoderma spore lipid (GSL) products. A gradient reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of free and esterified ergosterols in G. lucidum. The contents of free and esterified ergosterols in the different parts (the stipe, pileus, tubes, and spores) of G. lucidum and GSL were determined. The results showed that total ergosterol levels in the stipe, pileus, tubes, and spores of G. lucidum were between 0.8 and 1.6 mg/g. The relative abundances of free to esterified ergosterol were different in the different parts of G. lucidum. The spores and the tubes, the hymenophore tissue that contains the spore-producing cells, have a considerably higher percentage of ergosteryl esters (41.9 and 39.7% of total ergosterol) in comparison with the pileus and stipe tissues (3.6 and 6.2%).     

Effect of plasterboard composition on Stachybotrys chartarum growth and biological activity of spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-alpha production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

Comparison of methods for estimating the biomass of three food-borne fungi with different growth patterns.

To evaluate the effectiveness of steps taken to reduce the growth of molds in food and feed, methods that can accurately quantify the degree of fungal contamination of solid substrates are needed. In this study, the ergosterol assay has been evaluated by comparing the results of this assay with spore counts and hyphal length measurements made with a microscope and with CFU counts. Three fungi with different growth patterns during cultivation on a synthetic agar substrate were used in these experiments. For the nonsporulating Fusarium culmorum, there was good agreement between changes in hyphal length, CFU, and ergosterol content. Penicillium rugulosum and Rhizopus stolonifer produced many spores, and the production of spores coincided with large increases in CFU but not with increases in hyphal length or ergosterol content. Spores constituted between 3 and 5% of the total fungal mass. Changes in ergosterol level were closely related to changes in hyphal length. It was concluded that ergosterol level is a suitable marker for use in quantitatively monitoring fungal growth in solid substrates.     

Effect of Plasterboard Composition on Stachybotrys chartarum Growth and Biological Activity of Spores

The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied. S. chartarum was grown on 13 modified plasterboards under saturated humidity conditions. The biomass was estimated by measuring the ergosterol content of the S. chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores. The ergosterol content of S. chartarum correlated with the number of spores collected from plasterboards. The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core. Spores collected from all the plasterboards were toxic to the macrophages. The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity. The conventional additives used in the core had inhibitory effects on growth. Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF-α production in macrophages. In summary, this study shows that the growth of a strain of S. chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides. However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores.     

柘荣太子参HPLC指纹图谱的研究

建立太子参的 HPLC指纹图谱分析条件,为太子参药材内在质量评价积累数据.方法：应用RP-HPLC法;Cosmosil C18分析柱;乙腈-水二元梯度洗脱;流速为1.0 mL/in;检测波长203 nm;分析时间60 min.结果：建立太子参药材指纹图谱,特征共有峰有15个.结论：该方法准确可靠,重复性好,可用于太子参的HPLC的指纹图谱分析.     

云参化学成分研究——Ⅳ.云参、党参脂溶性成分和水溶性成分的薄层层析图谱比较

本文比较了云参和党参脂溶性成分和水溶性成分的薄层层析图谱。结果表明,云参、党参的脂溶性成分(乙醚、乙酸乙酯提取物)和水溶性成分(95％乙醇、水提取物、糖类成分)均基本相同,为云参代替党参使用提供了可靠的科学依据。     

紫外指纹图谱结合化学计量学对黄精基原植物的鉴别

运用紫外光谱技术结合化学计量学,建立快速鉴别不同基原黄精的方法。通过单因素实验确定黄精最佳提取溶剂、时间和用量,制备测试液,采用紫外光谱技术建立3种基原黄精的紫外指纹图谱,光谱数据转化后进行主成分(PCA)和系统聚类分析(HCA)。该方法重现性、精密度、稳定性较好,结果表明不同种类黄精紫外指纹图谱具有指纹特性,3种基原植物黄精紫外光谱图在210 nm、220 nm、280 nm附近差异明显;聚类分析和主成分分析三维投影图反映出不同种类黄精的化学成分积累具有差异,能较好地区分滇黄精(Polygonatumkingianum)、黄精(P.sibiricum)与多花黄精(P.cyrtonema)。紫外光谱结合化学计量学能快速鉴别不同种类黄精,可作为黄精的鉴别和质量控制新方法,为黄精临床应用、资源开发及黄精属植物分类提供辅助方法。     

Mycelial lipids as an aid in identifying rust fungi in culture-Cronartium fusiforme.

The sterol and fatty acid content of mycelium from germinating basidiospores of Cronartium fusiforme was determined. The mycelium contained stigmast-7-enol, fungisterol, and possibly stigmasta-5,7-dienol. No ergosterol was detected. The mycelium contained the expected fatty acids and low relative proportions of 9,10-epoxyoctadecanoic acid. The absence of ergosterol, and presence of the epoxy C18 acid and sterols typical of certain rust spores may be used for a relatively rapid confirmation of rust fungi in culture. Based on these chemical criteria, yeast-like cells isolated from the cultures of germinating basidiospores appear not to be C. fusiforme.