桑黄(Phellinus linteus)水溶多糖提取技术研究

采用超声波+复合酶法预处理对Phellinuslinteus子实体水溶多糖进行提取,并对提取多糖成分差异进行初步分析。采用超声波技术预处理桑黄子实体,利用正交实验研究了料液比、时间、功率各因素对桑黄多糖得率的影响;在超声波优化结果基础上,进行纤维素酶+木瓜蛋白酶法处理,研究了纤维素酶用量、处理时间、木瓜蛋白酶用量、处理时间各因素对多糖得率的影响。研究表明:在料液比1:20、超声波(400W、20min)和纤维素酶(0.8%、pH5.5、45℃、60min)、木瓜蛋白酶(2%、pH5.5、50℃、120min)双重预处理条件下,再进行多糖提取,多糖得率最高。研究表明采用超声波+复合酶法预处理方法提取多糖,与传统的热水提取法相比,极大提高了提取效率,为Phellinuslinteus多糖的产业化打下了基础。

Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice

Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate(EtOAc)extract ol Phellinus linteus grown on germinated brown rice(PB).Methods:EtOAc extract of PB was partitioned with n-hexane,EtOAc,and water-saturated n-butanol.Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR,respectively.Cytotoxicity against HT-29 cells was tested by SRB assay.Results:The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line.Atractylenolide I,a eudesmane-type sesquiterpene lactone,a major anticancer substance of PB,was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC.This structure was elucidated by one-and two-dimensional NMR spectroscopic data.Atractylenolide I has not been reported in mushrooms or rice as of yet.The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells.Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.

基于rDNA ITS序列分析的桑黄真菌菌株分子鉴定

对6份桑黄真菌菌株的rDNA ITS区段进行克隆测序和序列特征比较分析,并与GenBank中获得的桑黄基源物种相关序列进行同源性比对,进一步将从GenBank检索获得的桑黄基源物种的ITS序列、复合外组ITS序列连同6份桑黄菌ITS序列一起作系统发育分析。结果表明:Sh-03、04、05、06与Phellinus linteus亲缘关系最近,Sh-01与Phellinus baumii亲缘关系最近,Sh-02与Phellinus baumii、Phellinus linteus、Phellinus igniarius的亲缘关系相差甚远,为一错误菌种。本实验的研究结果可以提供一种准确可靠且简单易行的桑黄真菌分子鉴定技术。

桑黄真菌分子鉴定及遗传多样性分析

药用真菌桑黄具有明显的抗肿瘤、抗氧化、增强免疫等药理活性,但研究者对其基源还没有达成共识,多种Phellinus属真菌被当作桑黄入药使用。采用rDNA ITS序列分析技术,对桑黄真菌进行分子鉴定及遗传多样性分析。通过rDNA ITS序列分析,成功鉴定出一份混淆样品(Phellinus spp-04),并将中国主要使用的桑黄真菌明确鉴定为P.baumii和P.linteus两种,未检测到P.igniarius的使用。依据rDNA ITS序列计算遗传距离并构建系统发育树,结果表明3种主要桑黄真菌存在明显的遗传分化,在系统发育树中明确聚为3个独立菌种类群。在3种桑黄真菌rDNA ITS序列中,存在颠换、转换及插入/缺失3种类型的变异位点,分别在P.linteus、P.baumii和P.igniarius中鉴定出9、9、8种rDNA ITS单倍型序列,不同单倍型菌种间遗传分歧度变化表现出明显的物种差异性。