Enhanced phenolic antioxidants production in submerged cultures of Inonotus obliquus in a ground corn stover medium

The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. It is currently ascribed to a number of phenolic compounds as well as triterpenoids and polysaccharides responsible for significant biological and pharmacological properties. A study was conducted to determine the effects of inclusion of lignocellulosic material, in this case corn stover on production and antioxidant activity of extracellular (EPC) and intracellular phenolic compounds (IPC) by Inonotus obliquus in submerged fermentation. The corn stover medium contained 3% ground corn stover and 3.5% corn flour but the control medium contained 5% corn flour without corn stover. All of the other components were same in the two media. Decomposition rates of cellulose, hemicellulose, and lignin in the corn stover substrate were 20.9%, 17.9%, and 19.8% through 288 h of submerged cultivation. Lignocellulose decomposition in the corn stover-containing medium yielded significantly higher EPC (118.9/135.7 mg GAE (gallic acid equivalents)) and IPC (21.2/23.7 mg GAE) than in the control medium (34.7/42.5 mg GAE of EPC and 12.5/13.5 mg GAE of IPC) per liter of culture broth (EPC) and per gram of mycelia (IPC) in shake flask cultures/10 L fermenter runs. Both EPC and IPC from the corn stover medium showed a higher scavenging activity against hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those from the control medium during the later fermentation period. In dose-dependent experiments, EPC from the corn stover medium at 216 h demonstrated a significantly stronger free radical scavenger activity against DPPH and hydroxyl radicals, shown as much lower IC50 values, than that from the control medium and IPC from the two media.     

两株地理隔离的桦褐孔菌深层发酵产生抗氧化次生代谢产物的代谢组学分析

采用基于核磁共振代谢组学的方法研究了两株地理隔离的桦褐孔菌中国牡丹江菌株(IOM)和芬兰万塔菌株(IOV)在深层培养过程中主要代谢产物组成的差异。IOM和IOV两菌株在培养过程中积累菌丝体生物量、菌丝体酚类(TMP)以及三萜化合物(TMT)的过程相似,但IOM菌株产生较高水平的TMP。代谢产物组成动力学比较结果表明,IOM在第3天含有较高水平的硬毛素类似物、桦褐孔菌二糖和TMT,但在第5天均有所下降。导致这些差异的代谢产物为fuscoparianolD,21,24-羊毛甾-7,9(11),23-三烯-3β,22,25-三醇,羊毛甾-8,23-二烯-3β,22,25-三醇,23,24-环戊羊毛甾-3β,21,25-三醇-8-烯,桦褐孔菌二糖,phelligridinsC,D及H,甲基地花菌素A和C,地花菌素B,davallialactone及methyl davallialactone。这些代谢产物在IOV的提取物中均参与清除自由基,而在IOM的提取物中主要是酚类化合物参与自由基的清除。两菌株的这些差异反应了环境条件对桦褐孔菌生长及代谢的影响,即不仅影响其次生代谢产物的合成,还影响代谢产物在防止细胞氧化损伤过程中所起的作用。     

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.     

Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity

The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3 % compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident.     

Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus

Inonotus obliquus BELYU1102 was selected from 12 different strains of Inonotus as a producer of immuno-stimulating polysaccharide. After a batch fermentation of I. obliquus BELYU1102 was carried out in a 300 l pilot vessel, endo-polysaccharide and exo-polysaccharide were both obtained. The proliferation activity of endo-polysaccharide for splenic cells was much higher than the activity of exo-polysaccharide. The active endo-polysaccharide was produced primarily during the late stationary phase. Enhanced proliferation and polyclonal IgM antibody production were observed in B cells by purified water-soluble endo-polysaccharide. Nitrite production and expression of IL-1beta, IL-6, TNF-alpha, and iNOS in macrophages were also enhanced. However, the endo-polysaccharide did not affect the proliferation of T cells, the IL-2 expression of Th1 cells, or the IL-4 expression of Th2 cells. The endo-polysaccharide showed activities similar to lipopolysaccharide (LPS) for B cells and macrophages, but there was a large difference between the two polysaccharides because cellular activations induced by endo-polysaccharide were not affected by polymyxin B, a specific inhibitor of LPS. The endo-polysaccharide appeared to have other cellular binding sites with TLR-4 and did not show a direct toxicity against tumor cells. However, indirect anti-cancer effects via immuno-stimulation were observed. The mycelial endo-polysaccharide of I. obliquus is a candidate for use as an immune response modifier. Submerged mycelial cultures are advantageous for industrial production of polysaccharides.     

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。     

桦褐孔菌菌丝体及甾类化合物的发酵条件优化

对桦褐孔菌深层发酵培养基进行了筛选,以菌丝体及甾类化合物产量为目标对发酵条件进行了优化,确定最佳发酵条件为：30g/L葡萄糖,2.5g/L黄豆粉,2.5g/L蛋白胨,3g/L KH2PO4,0.8g/L MgSO4,0.8g/L CaSO4,初始pH4.0,接种量15%,装液量100mL/500mL,转速150r/min,28℃恒温培养。此条件下培养11d,菌丝体干重达12.52g/L,甾体类化合物的产量达112.44mg/L。     

桦褐孔菌子实体多糖的提取及体外降血糖活性

通过响应面设计得到桦褐孔菌子实体Inonotus obliquus多糖的优化提取条件,利用酶和细胞体外实验,探究桦褐孔菌子实体多糖的降血糖活性,确定活性多糖组分,并初步分析其降糖机理及单糖组成.优化条件下多糖得率为(2.79±0.03)％.粗多糖经乙醇分级沉淀分别获得IOP30、IOP60、IOP80组分.其中IOP3...     

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.     

一氧化氮对桦褐孔菌抗氧化酚类化合物积累的影响

本研究探讨了NO对深层培养的桦褐孔菌积累抗氧化酚类化合物的影响。在桦褐孔菌的培养基中分别加入0.01mmol/L、0.1mmol/L以及1mmol/L的NO供体硝普钠,并测定总酚的含量及其抗氧化活性。发酵产物的抗氧化活性以清除DPPH和羟自由基活力表示。加入0.1mmol/L的硝普钠可使桦褐孔菌胞内外酚类化合物分别达到最高水平67mg/g和677mg/L。不同培养时期产生的胞内外酚类化合物都具有抗氧化活性,尤其是添加0.1mmol/L硝普钠的菌丝体。因此NO可用于上调深层发酵培养的桦褐孔菌酚类化合物的积累。     

Melanin complex of the fungus Inonotus obliquus

The fungus Inonotus obliquus (Pers.) Pil. synthesised high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was showed that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated the melanogenesis. The production of melanin correlated with the synthesis of o- and p-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

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。     

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。     

深层培养的斜生褐孔菌酚类化合物对铅处理小鼠耐力的增强作用

本文研究了不同条件下深层培养的斜生褐孔菌Phaeoporus obliquus与野生桦褐孔菌在酚类化合物组成及其含量上的差异以及对铅处理小鼠常压缺氧和游泳耐力影响。野生桦褐孔菌主要含有水溶性黑色素、硬毛素衍生物,两者各占总酚类化合物的12.1%和42.2%。而深层培养的桦褐孔菌则主要积累黄酮类化合物,其中正常生理条件下积累量达到总酚的35.5%,加入H2O2后积累量达到总酚的92.5%,同时加入H2O2和熊果苷后则为总酚的76.9%。正常培养基中水溶性黑色素的积累量达到总酚的20.2%,而加入H2O2或同时加入H2O2和熊果苷后水溶性黑色素则很少合成。口服野生斜生褐孔菌或不同条件下深层培养的斜生褐孔菌酚类化合物都能使铅处理小鼠忍耐缺氧和游泳的时间延长12–15min。同时,口服不同来源的斜生褐孔菌酚类化合物可提升铅处理小鼠外周淋巴细胞的活力和总超氧化物歧化酶活性,并降低丙二醛的积累。因此,斜生褐孔菌酚类化合物对铅处理小鼠耐缺氧和游泳的提升作用在于抑制了脂质过氧化反应,增强了过氧化物歧化酶的表达,从而保护了铅处理小鼠的细胞和器官免于氧化胁迫的损伤。     

Comparison of the antioxidant activity of aqueous and ethanolic extracts from chaga (Inonotus obliquus)

Additional extraction of biologically active compounds from a chaga shred has been carried out after completion of the aqueous extraction. The extraction was carried out using ethanol. We have shown that the antioxidant activity of the ethanolic extracts and their components is higher than that of the aqueous extracts.     

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。     

Antioxidant activities of five polysaccharides from Inonotus obliquus

Five polysaccharides (IOP1b, IOP2a, IOP2c, IOP3a and IOP4) were isolated and purified from Inonotus obliquus by DEAE-Sepharose fast flow and SepharoseCL-6B column chromatography. Their chemical and physical characteristics were determined and antioxidant activities were investigated on the basis of hydroxyl radical assay, superoxide radical assay and ferric-reducing antioxidant power assay. The results showed that five polysaccharides exhibited antioxidant activities, and the higher content of uronic acid and proteinous substances, the stronger antioxidant activities of polysaccharides. Besides, molecular weights of polysaccharides also influence their antioxidant activities. IOP3a and IOP4 showed higher antioxidant properties than IOP1b, IOP2a and IOP2c.     

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.     

桦褐孔菌野生分离菌株的比较与栽培选育

通过菌丝形态比较、ISSR分析鉴定及人工驯化栽培选育,对1株野生分离菌株和4株保藏菌株进行比较。结果显示:5个菌株在菌丝形态和长势上差异显著,1号菌株(野生分离)菌丝洁白,长速最快;ISSR扩增图谱谱带差异明显,1号菌株与5号菌株在遗传相似水平85%时聚为1类,其他菌株各自聚为独立1类。驯化栽培试验结果表明:1号菌株出核较快,菌核大,产量高,较其他菌株产量差异显著。被试菌株为各自独立菌株,1号菌株经驯化适宜高产栽培,是重要珍稀野生种质资源。本研究可为野生桦褐孔菌菌株种质资源收集和为实现大规模栽培育种提供亲本资源。