桦褐孔菌提取物抗氧化活性研究

以乙酸乙酯和甲醇为提取剂,采用索氏提取法,剩余残渣采用热水浸提,最终得到桦褐孔菌不同极性提取物,对其DPPH自由基、羟基自由基及超氧阴离子自由基清除活性作用进行了研究,确定桦褐孔菌的抗氧化能力,为深入研究和开发桦褐孔菌功能性食品奠定理论基础。实验结果表明桦褐孔菌具有较好的抗氧化活性,其中乙酸乙酯提取物的DPPH自由基清除率、羟基自由基清除率和超氧阴离子自由基清除率均高于其他两组分及BHT,桦褐孔菌提取物有望成为功能性食品组分中合成抗氧化剂的天然替代品。     

桦褐孔菌三萜化合物提取工艺的优选

为了提高桦褐孔菌三萜化合物的产量,对其三萜化合物的提取工艺进行了初步优选。试验以桦褐孔菌发酵菌丝体为材料,齐墩果酸为标准品,用香草醛比色法测桦褐孔菌三萜化合物含量。通过单因素实验考察了不同提取剂、提取剂用量、提取温度、提取时间对桦褐孔菌三萜化合物提取的影响。结果表明,提取的最佳工艺为：以异丙醇为提取溶剂,用量为7倍,最佳提取温度为80℃,提取时间2h,在此条件下三萜化合物占菌丝体干质量的9．03％。该工艺的三萜桦褐物的提取率高,可用于桦褐孔菌中三萜化合物的提取。     

桦褐孔菌多糖的提取工艺优化及抗氧化活性评价

目的：优化桦褐孔菌多糖的超声波法提取工艺,对比不同产地桦褐孔菌多糖的抗氧化活性。方法：采用正交试验法确定超声波法的最佳工艺流程,检测不同产地桦褐孔菌的多糖提取率和抗氧化活性。结果：超声波法的最佳提取条件为超声时间25min、料液比1∶40、超声温度50℃、提取3次,在此条件下多糖提取率高达8.61 g/100g,与水提醇沉法相比提高了17.3%,耗时缩短了3/4,大大提高了提取效率。抗氧化能力检测结果证明超声波法得到的桦褐孔菌多糖都具有抗氧化活性,并且抗氧化能力与多糖提取率之间的相关性极其显著。结论：通过优化提取条件,超声波法明显提高了多糖提取率,保持了桦褐孔菌多糖的抗氧化活性,是一种可行、实用和高效的真菌多糖提取方法。     

桦褐孔菌的化学成分及抗炎活性

本实验研究了桦褐孔菌的化学成分及其抗炎活性。方法是采用溶剂法和柱色谱法提取分离桦褐孔菌的化学成分,根据化合物的理化性质及核磁共振氢谱、碳谱、质谱对其化学结构进行鉴定,然后采用二甲苯致小鼠耳肿胀和二甲苯致小鼠腹膜炎模型对所得化合物的抗炎活性进行考察。最终从桦褐孔菌的乙醇提取物中分离得到3个羊毛脂烷型三萜类化合物,分别鉴定为羊毛甾醇(1)、inotodiol(2)和trametenolic acid(3)。化合物1、2、3在10 mg/kg均显著抑制二甲苯致小鼠耳肿胀,化合物1和2在10 mg/kg时显著降低血管通透性。由此得出化合物1～3均有抗炎活性,其中trametenolic acid(3)的抗炎活性最强。     

桦褐孔菌子实体多糖提取研究

目的:为了获得桦褐孔菌多糖最大量的多糖收率,研究了影响多糖提取的最佳条件.方法:对影响桦褐孔菌胞内水溶性多糖提取效果的6个因素进行了单一因素影响实验,并对多糖提取影响因子的3个因素进行正交试验,对多糖提取工艺参数进行了优化.结果:正交试验确定桦褐孔菌子实体多糖的最佳提取条件是:料水比为1∶40,温度80℃,提取1.5h,多糖收率达2.53％.结论:确立了桦褐孔菌子实体多糖提取工艺,建立了一套简便、高效的桦褐孔菌胞内多糖的提取方法.     

桦褐孔菌的研究进展

概述了国内外桦褐孔菌的分类学、生态学、人工栽培及药用研究动态的最新进展和应用前景。     

正交实验法优化桦褐孔菌多酚类物质的提取工艺研究

以乙醇为提取溶剂,通过正交实验对影响桦褐孔菌多酚提取的乙醇浓度、提取时间、提取温度和料液比等因素进行探讨。结果显示,桦褐孔菌多酚的乙醇提取优化工艺参数为:乙醇的浓度为50%,料液比为1:10,温度为50℃,提取时间为45min,得到的多酚提取率可达到3.099%。用乙醇-水混合体系提取桦褐孔菌多酚是一种安全、经济、有效的方法。     

外源因子对桦褐孔菌发酵产桦褐孔菌醇的影响

食药用真菌因其丰富的天然活性物质成为具有开发潜力的药物来源。桦褐孔菌Inonotus obliquus作为一种珍稀的药用真菌,因其对糖尿病、消化系统疾病、心血管疾病、肝病和癌症等疾病有良好的治疗效果而受到广泛关注。桦褐孔菌醇（inotodiol）是桦褐孔菌特有的一种羊毛甾烷型三萜类化合物,具有多种抗癌活性。本文的主要目的是研究外源因子的添加对桦褐孔菌液态发酵产桦褐孔菌醇的影响,以及对桦褐孔菌醇合成途径中酶活的影响。结果表明：最佳外源因子是香叶醇,最佳添加浓度和添加时间分别为0.02%（V/V）和第144小时。发酵结束时（240h）桦褐孔菌醇的产量为27.89mg/L是对照组（9.23mg/L）的3.02倍。通过对比添加香叶醇后桦褐孔菌醇的产量变化以及合成途径中4种酶（法尼基焦磷酸合酶、角鲨烯合酶、角鲨烯环氧化酶和羊毛甾醇合酶）的活性变化,对香叶醇的作用机制进行了初步探究。研究结果表明添加香叶醇后,4种酶活性均较对照组有显著的提高,与此对应的桦褐孔菌醇产量也显著增加,说明这4种酶在桦褐孔菌醇合成途径中起到了积极的作用。     

桦褐孔菌提取物抗氧化与糖苷酶抑制活性研究

为探索桦褐孔菌(Inonotus obliquus (Fr.) Pila)生物活性与化学成分之间的相关性,本文用乙醇回流提取、梯度萃取得桦褐孔菌的石油醚、乙酸乙酯、正丁醇和水提取物,对其进行黄酮和多酚类化合物的含量测定,并从抗氧化和降血糖两个方面测定了总还原力、清除DPPH自由基、抑制肝脂质过氧化、抑制α-葡萄糖苷酶活性四种体外模型对不同提取物活性的影响.结果显示:乙酸乙酯和正丁醇提取物所含的黄酮和多酚类化合物较多(P<0.01)且抗氧化和降血糖活性较好.因此,桦褐孔菌抗氧化和降血糖活性的大小与其所含黄酮和多酚类化合物量密切相关,本研究结果为桦褐孔菌生物活性成分的深入研究奠定了良好的基础.     

桦褐孔菌资源分布及药理活性研究进展

对桦褐孔菌在我国的资源调查情况进行了汇总,在已报道桦褐孔菌综述的基础上,重点针对近5年来野生桦褐孔菌与桦褐孔菌发酵产物药理活性的研究成果进行了综述,并对桦褐孔菌的资源及其开发利用前景进行了展望。     

Analysis of antioxidant metabolites by solvent extraction from sclerotia of Inonotus obliquus (Chaga)

Introduction – The sclerotia of Inonotus obliquus (Chaga) are effective therapeutic agents to treat several human malignant tumours and other diseases without unacceptable toxic side‐effects. Objective – To investigate solvent effects on metabolic profiles and antioxidant activities of extracts of Chaga. Methodology – Chaga was extracted by petroleum ether, chloroform, ethyl acetate, acetone, ethanol and water. Solvent effects on metabolites in the extracts were assayed by NMR‐based metabolomic analysis. Antioxidant activities were indicated as capacities for scavenging superoxide anion, DPPH and hydroxyl radicals. Results – Petroleum ether and chloroform extracts contained primarily lanostane‐type triterpenoids (LT), whereas the extracts of ethyl acetate, acetone and ethanol were characterised by the predominant presence of hispidin analogues and LT, and water extracts by polysaccharides and phenolic compounds. The ethyl acetate, acetone, ethanol and water extracts revealed remarkable potential for scavenging the tested radicals, while those of petroleum ether and chloroform did not. Polyphenols are the major contributors for quenching the tested free radicals, while in LT only compounds 16 , 17 and 22 participated in scavenging hydroxyl radicals. Conclusion – Polyphenols in Chaga are the principles for quenching free radicals while polysaccharides and a few LT compounds contribute partially in scavenging DPPH and hydroxyl radicals, respectively. NMR‐based metabolomic analysis is a useful method by which to correlate ¹H‐NMR spectra of Chaga extracts with their antioxidant activities, and this allows the prediction of potentials for scavenging free radicals by ¹H‐NMR spectroscopy. Copyright © 2011 John Wiley & Sons, Ltd.     

桦褐孔菌酚类化合物及其免疫促进作用

Phenolic compounds from field-grown Inonotus obliquus sclerotia (Chaga) consist mainly of hispidin analogs and melanins, and are thought to be the active constituents to treat several human diseases. In submerged cultures of the fungus, however, no information is currently available on the production of phenolic compounds and their corresponding pharmacological functions. In this study, phenolic compounds from Chaga and submerged cultures of the fungus were assayed for their composition and immune-stimulating effects. Phenolic compounds produced by I. obliquus in submerged cultures mostly consist of flavonoids, together with small amounts of hispidin analogs and melanins. This is quite contrary to the situation in Chaga, where flavonoids are determined as trace elements. Furthermore, phenolic compounds from Chaga show capacity about two-fold higher than those produced in submerged cultures in inhibiting cyclophosphamide-induced reduction of bodyweight, spleen index and viability of peripheral lymphocytes in test mice. Thus less production of hispidin analogs and melanins is likely to be responsible for less immune-stimulating effects in phenolic compounds from submerged cultures, and additional factors should be imposed during submerged cultures of I. obliquus to regulate biosynthesis of phenolic compounds directed to the composition similar to Chaga.     

New antioxidant polyphenols from the medicinal mushroom Inonotus obliquus

The fruiting body of Inonotus obliquus, a medicinal mushroom called chaga, has been used as a traditional medicine for cancer treatment. Although this mushroom has been known to exhibit potent antioxidant activity, the mechanisms responsible for this activity remain unknown. In our investigation for free radical scavengers from the methanolic extract of this mushroom, inonoblins A (1), B (2), and C (3) were isolated along with the known compounds, phelligridins D (4), E (5), and G (6). Their structures were established by extensive spectroscopic analyses. These compounds exhibited significant scavenging activity against the ABTS radical cation and DPPH radical, and showed moderate activity against the superoxide radical anion.     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

Folin-Ciocalteu比色法测定桦褐孔菌多酚的条件优化

以没食子酸为标准品,通过正交试验和单因素实验研究了Folin-Ciocalteu 比色法测定桦褐孔菌中多酚含量的适宜条件。结果表明,优化后的显色条件为Folin-Ciocalteu试剂用量0.3mL、10% Na2CO3溶液0.6mL,25℃时避光反应30min,于750nm处测定其吸光值。多酚质量浓度在0.0032－0.0256mg/mL范围内与吸光值有良好的线性关系。根据拟合的线性回归方程对桦褐孔菌多酚进行定量测定,野生桦褐孔菌和人工培养桦褐孔菌中多酚含量最高分别为（72.05±0.08）mg/g、（52.76±0.06）mg/g（n=6）。不同加标水平的加标回收率测定实验获得的平均回收率为98.95%,RSD为1.27%。该法测定桦褐孔菌多酚具有快速、准确,稳定性强,重现性好,精密度高的特点,可应用于实际样品的测定。     

桦褐孔菌乙酸乙酯提取物抗食管癌作用的研究

目的:探讨桦褐孔菌乙酸乙酯提取物对食管癌细胞EC-109、EC-9706增殖、凋亡和细胞周期等表型的影响。方法:用桦褐孔菌乙酸乙酯提取物处理食管癌细胞株EC-109、EC-9706,采用MTT法、流式细胞术、平板克隆实验分析桦褐孔菌乙酸乙酯提取物对食管癌细胞增殖、凋亡和细胞周期的影响。结果:桦褐孔菌乙酸乙酯提取物可显著抑制食管癌细胞的增殖,并降低肿瘤细胞的克隆形成能力。流式细胞术显示,桦褐孔菌提取物可将食管癌细胞周期阻滞在G1期,并促进肿瘤细胞的凋亡。结论:桦褐孔菌乙酸乙酯提取物可抑制人食管癌细胞株的增殖和克隆形成,阻滞细胞周期并诱导肿瘤细胞凋亡,具有一定的抗食管癌活性。