Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus

The endo-polysaccharide extracted from mycelia of Inonotus obliquus (Pers.:Fr.) Pil. (Hymenochaetaceae) is a specific activator of B cells and macrophages. However, the in vivo anti-cancer effects and the chemical structure of the endo-polysaccharide are unknown. We purified the endo-polysaccharide, investigated its anti-cancer effects via in vitro and in vivo assays, and performed a structural characterization. The endo-polysaccharide was extracted from I. obliquus mycelia cultivated in a 300-l pilot fermenter, followed by hot water extraction and ethanol precipitation. Purification was achieved by DEAE-cellulose ion-exchange chromatography and gel-permeation chromatography. Chemical analysis revealed that the purified endo-polysaccharide is an alpha-linked fucoglucomannan with a molecular weight of approximately 1,000 kDa. The anti-cancer activities of the endo-polysaccharide against various types of tumor cells were determined. No direct toxicity against either cancer or normal cells was observed. Intraperitoneal administration of the endo-polysaccharide significantly prolonged the survival rate of B16F10-implanted mice, resulting in a 4.07-fold increase in the survival rate at a dose of 30 mg/kg/day. After 60 days of feeding, approximately 67% of the initial number of mice survived with no tumor incidence based on macroscopic examination. These results indicate that the anti-cancer effect of endo-polysaccharide is not directly tumorcidal but rather is immuno-stimulating.     

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。     

Accumulation of antioxidant phenolic constituents in submerged cultures of Inonotus obliquus

Phenolic compounds produced by sclerotia of Inonotus obliquus are the active constituents responsible for antioxidant activities. In this study, I. obliquus was grown in a continuously stirred tank reactor (CSTR) to explore how it accumulates phenolic compounds in different culture media and whether these compounds possess antioxidant activities. Phenolic compounds produced by I. obliquus in the control medium consisted of melanins, flavonoids, polyphenols and small phenolics. Their accumulation was affected by adding H(2)O(2) to the medium, where increased levels of total intracellular phenols (TIP) and melanins, but less total extracellular phenol (TEP) occurred. Simultaneous exposure to H(2)O(2) and arbutin resulted in a further increase in TIP production and reduced accumulation of TEP. Both TIP and TEP obtained at different culture ages and media were active in scavenging superoxide anion and DPPH radicals. Therefore, production of phenolic compounds by I. obliquus is enhanced by imposing oxidative stress, which might allow it to be exploited as a reliable source of pharmaceutically important phenolic compounds.     

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。     

Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called ‘Chaga’. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.     

Lipids produced by Epicoccum nigrum in submerged culture

1. The lipids of the red mycelium of the fungus Epicoccum nigrum Link were investigated. Four carotenoid pigments were isolated and identified as beta-carotene, gamma-carotene, rhodoxanthin and torularhodin. 2. For the first time rhodoxanthin was isolated as a fungal metabolite. 3. Linoleic acid was the major fatty acid in the total lipids. 4. Ergosterol was present in the lipids in the non-esterified form.     

Physicochemical properties of melanins produced by Inonotus obliquus("chagi") in the nature and the cultivated fungus

Physicochemical properties of pigments isolated from the naturally occurring sterile form of Inonotus obliquus (Fr.) Pil. known as Chagi and comprising the major constituent of the medicine befungin were compared with those of melanins synthesized by this fungus in the culture in order to develop a new medicine. Elemental and functional group analyses, as well as UV-visible, IR, and EPR spectra, and thermolysis studies revealed structural differences in these pigments and allowed for assignment of the naturally produced melanin to allomelanins, whereas that of cultivated fungus was assigned to eumelanins.     

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.     

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。     

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。     

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...     

Studies of the survival ofFusarium oxysporum conidia produced by submerged culture

Summary To study the survival of conidia ofFusarium oxysporum produced by submerged culture on malt extract, a harvesting process and different packaging and storage conditions have been tested. Conidia dried with talc and stored at +4°C preserve their viability after about 4.5 months.     

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.     

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.     

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.     

Hypoglycemic effect of a Lentinus edodes exo-polymer produced from a submerged mycelial culture

The hypoglycemic effect of an exo-polymer produced from a submerged mycelial culture of Lentinus edodes was investigated in streptozotocin-induced diabetic rats. The administration of the exo-polymer (200 mg/kg BW) reduced the plasma glucose level by as much as 21.5%, and increased plasma insulin by 22.1% as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively. Gel chromatography of the exo-polymer revealed a single peak which is likely to have been a glycoprotein with a molecular weight of 52 kDa and was found to contain 83.5% carbohydrate and 16.5% protein. The Sugar and amino acid compositions of the exo-polymer were analyzed in detail.