内蒙古克什克腾旗大型真菌资源利用研究

【作者】 刘坤；

【导师】 王俊丽；

【作者基本信息】 中央民族大学 ， 民族生态学， 2012， 博士

【摘要】 本研究采用文献调查和实地调查相结合的方法对内蒙古地区大型真菌资源进行了系统分析。文献报道大型真菌共计990种,隶属于2个门23个目72个科218个属。调查结果表明：内蒙古地区的大型真菌种类十分丰富,其中种类超过或等于20种的有13科,共有686种,占内蒙古大型真菌总数的69.29%；种类超过或等于10种的有24属,共有484种,占内蒙大型真菌全部种数的48.88%。内蒙古的食药用真菌共有531种,其中药用真菌316种,食用真菌373种,食药兼用菌158种。经对克什克腾旗的实地调查,共采集样品41份,根据宏观特征与显微结构特征鉴定出26种,隶属于子囊菌门2目2科2属2种,担子菌门7目14科20属24种。为了开发利用内蒙古地区的真菌资源,本研究对克什克腾旗地区桦褶孔菌(Lenzites betulina)、大白桩菇(Leucopaxillus giganteus)、粗柄蘑菇(Agaricus spissicaulis)、疣孢黄枝瑚菌(Ramaria flava)和毛咀地星(Geastrum fimbriatum)等五种食药性真菌的化学成分和生物活性进行了研究。利用各种分离技术手段和现代光谱技术,从五种大型真菌中共分离鉴定了49个化合物,其中有1个新化合物和1个新天然产物。具体结果如下：(1)从桦褶孔菌的乙醇提取物中分离鉴定了14个化合物,包括：甾体(7个)、对三联苯(3个)、吡喃酮(1个)、氨基酸(1个)、芳香化合物(1个)和糖醇(1个)。其中2-甲氧基-3,6-二苯基-4H-呲喃-4-酮是新化合物,2-苯基-3-甲氧基-[1H-2-苯并哌喃][4,3-e]p]苯醌是新天然产物。(2)从疣孢黄枝瑚菌的乙醇提取物中分离鉴定了11个化合物,分别是：甾体(3个过氧化物麦角甾、2个甾体苷类和1个甾醇)、芳香化合物(2个)、碱基(1个)、糖(1个)和糖醇(1个)。这11个化合物均为首次从该菌中分离得到。(3)从粗柄蘑菇的乙醇提取物中分离鉴定了10个化合物,其中包括4个甾体、2个香豆素、1个脑苷脂、1个糖醇、1个碱基和1个脂肪酸。目前尚无关于粗柄蘑菇的化学成分的研究报道。(4)从毛咀地星的乙醇提取物中分离鉴定了6个化合物,有2个甾体、2个糖、1个糖醇和1个氨基酸。目前尚无关于毛咀地星的化学成分的研究报道。(5)从大白桩菇乙醇提取物的乙酸乙酯层中分离鉴定了化合物8个,分别为：甾体(2个)、吡啶甲酸(1个)、生物碱(1个)、酸性成分(2个)和酚(1个)。除了生物碱clitocine,其余均为首次从该菌中分离得到的化合物。本研究对五种真菌乙醇提取物和3个对联三苯的抗氧化活性进行了分析,同时还对这些提取物进行了抗细菌、抗真菌的活性研究,采用MTT法研究了各提取物对MDA-MB-231、BGC-803和NCI-H520等肿瘤细胞的抑制活性。研究结果如下：(1)五种大型真菌乙醇提取物对DPPH自由基和羟自由基都有一定的清除活性,且清除能力有显著性差异(P<0.05),大小顺序为桦褶孔菌>疣孢黄枝瑚菌>大白桩菇>毛咀地星>粗柄蘑菇。其中桦褶孔菌抗氧化活性最好,对DPPH自由基和羟自由基清除能力IC5o分别为24.12±0.61μg/mL和24.58±0.19μg/mL。五种真菌的总酚含量分别为17.79±0.57μg/mL(桦褶孔菌)、12.95±0.41μg/mg (疣孢黄枝瑚菌)、11.66±0.56μg/mg(大白桩菇)、10.53±0.53μg/mg毛咀地星)和3.32±0.45μg/mg(粗柄蘑菇)。总酚含量与DPPH自由基和羟自由基清除能力之间均呈正相关关系,相关系数分别为0.856和0.892。在实验浓度的范围内,3种对联三苯化合物的DPPH自由基和羟自由基清除率与浓度都呈正相关,化合物2表现出了较高的羟自由基清除活性,其IC5o为37.69±0.8μg/mL。(2)通过抑菌圈法对五种大型真菌乙醇提取物进行了抗细菌活性测定,发现桦褶孔菌、疣孢黄枝瑚菌、大白桩菇和粗柄蘑菇提取物对金黄葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)和大肠杆菌(Escherichia coli)均有抑制作用。其中大白桩菇对这三种细菌的抑菌活性最好,抑菌圈直径均大于2㎝,最低抑菌浓度(MIC)均为6.25mg/mL;桦褶孔菌抑细菌活性次之,对上述细菌的抑菌圈直径分别为2.24cm、1.90cm和1.72cm, MIC分别为6.25mg/mL、12.50mg/mL和25.00mg/mL采用菌饼法对五种大型真菌乙醇提取物进行抗真菌活性测定,这些真菌提取物分别表现出了不同的抑制效果,提取物对燕麦镰刀菌(Gibberella zeae)的抑制率高于禾谷镰刀菌(Fusarium graminearum)和白斑小尾孢(Cercosporella albo-maculans)。其中大白桩菇的抑菌活性最好,对禾谷镰刀菌、燕麦镰刀菌和白斑小尾孢的抑制率分别为35.80%、67.23%和52.05%。(3)五种大型真菌乙醇提取物对MDA-MB-231、BGC-803和NCI-H520等肿瘤细胞都有一定的抑制活性,在试验浓度范围内,抑制活性与提取物浓度呈正相关。大白桩菇提取物的抑肿瘤效果最佳,在浓度为200μg/mL时对MDA-MB-231、BGC-803和NCI-H520细胞的抑制率分别达到77.35%、68.92%和65.53%,其IC50分别为46.26μg/mL、65.06μg/mL和84.76μg/mL;桦褶孔菌提取物的抑肿瘤效果次之,在浓度为200μg/mL时,对MDA-MB-231、BGC-803和NCI-H520细胞的抑制率分别达到83.15%、55.54%和57.50%,其IC5o分别为51.47μg/mL.130.19μg/mL和106.54μg/mL更多还原

【Abstract】 The author adopts the use of research methods of literature search and review of papers regarding macro fungi in Inner Mongolia over the past twenty years for this dissertation. Accordingly, papers regarding990species of macro fungi were collected. The diversity catalogue of these990species was arranged according to Ainsworth&Bisby’s Dictionary of the Fungi (2008,10th), and they belong to2phylums,23orders,78families,218genus.The survey of the flora diversity study indicated that macro fungi species are vey plentiful in Inner Mongolia. Thirteen different families each have over20species represented, comprising a total of686species, or69.29%of the total macro fungi in Inner Mongolia. Another24families have over10species represented, totaling to484species, or48.88%of the total. Of these species,531have either edible or medicinal characteristics, or both. Three hundred sixteen species have medicinal properties,373species are edible, and158species are both edible and have medicinal functions.As a result of conducting field sampling in Hexigten Banner, a total of41specimens were collected. Twenty-six species were identified through studying their gross features and their structures under a microscope. Among these26species,2phylums were identified. The first phylum was Ascomycota, containing2orders,2families,2genuses and2species. The second phylum identified was Basidiomycota, which contained7orders,14 families,20genuses and24species.Because of the diversity of the species and their unique chemical structures, the higher fungi are an extremely important resource for new natural medicines. In order to fully exploit the fungal resources in Inner Mongolia region and search for new active compounds, we investigated the chemical constituents and bioactivities of the five higher fungi species of Lenzites betulina, Ramaria flava, Agaricus spissicaulis, Geastrum fimbriatum and Leucopaxillus giganteus in Inner Mongolia region.Forty-nine different compounds, including1new compound and1new natural compound, have been isolated by the use of chromatographic methods. The structures of these compounds were elucidated by both spectroscopic methods (MS,1D&2D-NMR, IR, UV) and chemical methods, and the results are as follows:(1) Fourteen compounds were isolated from the extract of Lenzites betulina including seven sterides, three p-terphenyls, one pyranone, one amino acid, one aromatic compound and one D-allitol.(2) Eleven compounds were isolated from the extract of Ramaria flava including six sterides, one glucose, one base, two aromatic compounds and one D-allitol. All of these were first reported from the genus Ramaria.(3) This was the first time to investigate the chemical constituents of the Agaricus spissicaulis fruiting bodies. Ten compounds were isolated from its extract including four sterides, one cerebroside, one base, two coumarin compounds, one aliphatic acid and one D-allitol. (4) It was the first time to investigate the chemical constituents of the fruiting body of Geastrum fimbriatum. Subsequently, six compounds were isolated from its extract including two sterides, two glucose, one amino acid and one D-allitol.(5) Eight compounds were isolated from the extract of Lenzites betulina including two sterides, two acidic compounds, one hydroquinol, one alkaloid and one picolinic acid. All of these compounds, except the alkaloid, were first reported from this fungus.The antioxidant activities of the ethanol extracts of these five fungi and three p-terphenyls compounds were evaluated by DPPH radical scavenging activity and hydroxyl radical scavenging ability. Also, the antibacterial and antifungal activities of the extracts from these species were evaluated. Moreover, we also evaluated the in vitro cytotoxicity of these extracts against human tumor cells MDA-MB-231、BGC-803和NCI-H520, by the use of MTT assay. The results are as follows:(1) There were significant differences among the extracts of these five fungi (P<0.05) on DPPH and hydroxyl radical scavenging ability. The radical scavenging activity of these five fungi decreased in the order of Lenzites betulina, Ramaria flava, Leucopaxillus giganteus, Geastrum fimbriatum and Agaricus spissicaulis. Lenzites betulina displayed the best activity among these fungi in regard to DPPH and hydroxyl radical scavenging ability with the lowest IC50values (24.12±0.61μg/mL and24.58±0.19μg/mL respectively). The content of total phenol for these five fungi, in decreasing order, were17.79±0.57μg/mg (Lenzites betulina),12.95±0.41μg/mg (Ramaria, flava),11.66±0.56μg/mg (Leucopaxillus giganteus),10.53±0.53Hg/mg (Geastrum fimbriatum), and3.32±0.45μg/mg (Agaricus spissicaulis). The results indicated that the total phenols were positively correlated with DPPH and hydroxyl radical scavenging ability, displaying correlation coefficients of0.856and0.892, respectively. The three p-terphenyls compounds were positively correlated with DPPH and hydroxyl radical scavenging ability, and the compound2displayed higher hydroxyl radical scavenging activity (IC50=37.69±0.8μg/mL).(2) Using the inhibition zone method, we isolated the ethanol extracts from Lenzites betulina, Ramaria flava, Leucopaxillus giganteus and Agaricus spissicaulis. Lenzites betulina has an inhibitor effect on Staphylococcus aureus, Escherichia coli and Bacillus subtilis. Among these four fungi, Leucopaxillus giganteus displayed the best antibacterial activity. The inhibition zone diameters of this species were all greater than2cm, and the minimum inhibitory concentration of this species were all6.25mg/mL. The antibacterial activities of Lenzites betulina were better, and the inhibition zone diameters of this species were2.24cm,1.90cm and1.72cm respectively, with the MIC of6.25mg/mL,12.50mg/mL and25.00mg/mL respectively.Using the fungus cake method, antifungal activities of the extracts from these five species were evaluated. First, we found that the inhibition ratios of these five species on Gibberella zeae are higher than the inhibition ratios on Fusarium graminearum and Cercosporella albo-maculans. Second, it was discovered that Leucopaxillus giganteus has the best antifungi activities among the five fungi. At the concentration of1mg/mL, the inhibition ratios of Leucopaxillus giganteus on Gibberella zeae, Fusarium graminearum and Cercosporella albo-maculans were35.80%,67.23%,52.05%respectively.(3) The MTT test revealed that the ethanol extracts from these five mushrooms exhibited certain cytotoxicities against the human cancer cells identified as MDA-MB-231, BGC-803and NCI-H520. The IC50values of these five ethanol extracts also have positive correlation within this experiment scope of concentration. Of these five species, Leucopaxillus giganteus displayed the best cytotoxicities. At the concentration of200ug/mL, the inhibition ratios of Leucopaxillus giganteus against MDA-MB-231, BGC-803and NCI-H520cells were77.35%,68.92%,65.53%respectively, with corresponding IC50values of46.26μg/mL,65.06μg/mL and84.76μg/mL. At the concentration of200μg/mL, the inhibition ratios of Lenzites betulina against MDA-MB-231、BGC-803and NCI-H520cells were83.15%,55.54%and57.50%respectively, with corresponding IC50values of51.47μg/mL,130.19μg/mL and106.54μg/mL.更多还原