Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteus

Aims:  The medicinal fungi Inonotus xeranticus and Phellinus linteus in the family Hymenochaetaceae have been used as traditional medicines for the treatment of various diseases. However, the compound responsible for the antioxidant activity is still unknown. Therefore, this study was conducted to characterize the antioxidant substances present in cultured broths made from these fungi.
Methods and Results:  Antioxidant fractions of the cultured broths obtained from I. xeranticus and P. linteus were analysed using reversed-phase HPLC, which revealed several peaks that exhibited a potent free radical scavenging activity. To identify these antioxidant peaks, an I. xeranticus strain was mass-cultured, and the cultured broth was separated using antioxidant activity-guided fractionation. Four major active substances were purified and identified as hispidin and its dimers, 3,14'-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan based on spectroscopic analyses. All compounds exhibited a significant scavenging activity against these radical species in a concentration-dependent manner.
Conclusions:  Antioxidant substances found in the cultured broths of the medicinal fungi I. xeranticus and P. linteus were identified as hispidin and its dimers, 3,14'-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan.
Significance and Impact of the Study:  Polyphenol antioxidants were isolated from the cultured broth of the medicinal fungi I. xeranticus and P. linteus and identified based on extensive spectroscopic analyses. These compounds exhibited a strong antioxidant activity.     

Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms

Twenty-six species of Phellinus sensu lato, reported as medicinal mushrooms, are enumerated in this review. The species’ names were checked and revised according to contemporary taxonomy and the latest version of the International Code of Botanical Nomenclature (Vienna Code); two misapplied names of Phellinus baumii Pilát and Phellinus himalayensis Y.C. Dai in previous reports are also discussed. Of the 20 types of medicinal functions, the most shared functions are antitumor and improving immunity, both of which may be viewed as the basal functions of Phellinus s. l. In addition, alleviating septic shock, anti-inflammatory, and antioxidation are also a routine functions mentioned often. The main medicinal metabolites, including several kinds of polysaccharides and polyphenols, are introduced. Different methods and conditions could purify various polysaccharides with difference in activity level even from the same species, while all polyphenols are hispidin and its derivatives in general. Three aspects of mechanism contribute to antitumor activities of polysaccharides: (1) promoting an immune response, (2) inducing cell apoptosis, and (3) inhibiting metastasis. Other general mechanisms of the metabolites in antioxidant activity, and in treating diabetes, as well as complications are summarized. We also elaborate on potential scientific strategies for obtaining the medicinal metabolites from Phellinus s. l., such as artificial cultivation, the discoveries of more species with medicinal functions, the utilization of species growing quickly, and the optimization of culture conditions and media supplements in fermentation.     

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.     

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.     

Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus

This study describes the extraction and characterization of a platelet aggregation inhibitory peptide from Inonotus obliquus. Ethanol extract from I. obliquus ASI 74006 mycelia showed the highest platelet aggregation inhibitory activity (81.2%). The maximum platelet aggregation inhibitory activity was found when the mycelia of I. obliquus ASI 74006 was extracted with ethanol at 80 degrees C for 12 h. The platelet aggregation inhibitor was purified by systematic solvent fractionation, ultrafiltration, Sephadex G-10 column chromatography, and reverse-phase HPLC. The purified platelet aggregation inhibitor is a novel tripeptide with a molecular mass of 365 Da, having a sequence of Trp-Gly-Cys. The purified platelet aggregation inhibitor also showed high platelet aggregation inhibitory activity in Institute of Cancer Research (ICR) mice.     

Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus

A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC₅₀ of 6 μM and also showed antibacterial activity against S. aureus and MRSA.     

药用植物微生物组及其与药用植物次生代谢产物的关系

药用植物是中药的原料,是中药产业的源头,其生长发育受遗传和环境等诸多因素的影响。以往研究强调植物基因型及生态因子对药用植物产量和品质的影响。近几年,随着人类微生物组研究的推进,植物微生物组作为植物整体的重要组成部分在药用植物的生长发育、品质形成甚至药效等方面的作用也日益受到重视,有关植物微生物组的多样性,微生物组在植物生长发育中的作用已有较详细的综述,而有关药用植物微生物组及其与药用植物次生代谢产物间关系的综述较少。本文重点总结了自2010年以来药用植物微生物组的研究进展,包括药用植物微生物组物种组成、功能及其与药用植物次生代谢产物产生的关系等,并对其在药用植物提质增效及其生态种植中的潜在应用进行了展望。     

Hypoglycemic effect of crude exopolysaccharides produced by a medicinal mushroom Phellinus baumii in streptozotocin-induced diabetic rats

The antidiabetic effect of the crude exopolysaccharides (EPS) produced from submerged mycelial culture of Phellinus baumii in streptozotocin (STZ)-induced diabetic rats was investigated. The produced EPS consisted of two different heteropolysaccharides and two proteoglycans. The food intake of the diabetic control rats (STZ) was increased by 28.1%, whereas body weight gain was reduced by 44.1% as compared to the nondiabetic animals (NC). The plasma glucose level in the EPS-fed rats (EPS) was substantially reduced by 52.3% as compared to the diabetic rats (STZ), which is the highest hypoglycemic effect among mushroom-derived materials documented in literature. The activities of alanine aminotransferase (ALT) and asparate aminotransferase (AST) were significantly decreased by administration of P. baumii EPS, thereby exhibiting a remedial role in liver function. The significant increase in weights of liver, spleen, and kidney was observed in diabetic groups (both STZ and EPS) compared to NC. The results suggest that orally administrated P. baumii EPS exhibited considerable hypoglycemic effect in STZ-induced diabetic rats and that these EPS may be useful for the management of diabetes mellitus.     

Variation of antioxidants and secondary metabolites in nitrogen-deficient barley plants

Barley (Hordeum vulgare cv. Bojos) plants cultured in low nitrogen (N) containing Hoagland solution (20 mg/l) were exposed to N deficiency (−N) over 15 days. Plants revealed relatively high tolerance to total N deficit because shoot length was not altered and dry biomass was depleted by ca. 30% while root length increased by ca. 50% and dry biomass remained unaffected. Soluble proteins and free amino acids decreased more pronouncedly in the roots. Antioxidants (glutathione and ascorbic acid) decreased in the shoots but increased or were not affected in the roots. Ascorbate peroxidase and glutathione reductase activities were depleted in shoots and/or roots while guaiacol peroxidase activity was stimulated in the shoots. In accordance, fluorescence signal of reactive oxygen species (ROS) and nitric oxide was elevated in shoots but no extensive changes were observed in roots if +N and −N treatments are compared. At the level of phenolic metabolites, slight increase in soluble phenols and some phenolic acids and strong elevation of flavonoid homoorientin was found in the shoots but not in the roots. Fluorescence microscopy in terms of detection of phenols is also discussed. We also briefly discussed accuracy of quantification of some parameters owing to discrepancies in the literature. It is concluded that N deficiency induces increase in shoot phenolics but also elevates symptoms of oxidative stress while increase in root antioxidants probably contributes to ROS homeostasis aimed to maintain root development.     

Current findings,future trends,and unsolved problems in studies of medicinal mushrooms

The target of the present review is to draw attention to many critically important unsolved problems in the future development of medicinal mushroom science in the twenty-first century. Special attention is paid to mushroom polysaccharides. Many, if not all, higher Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, and cultured broth. The data on mushroom polysaccharides are summarized for approximately 700 species of higher Hetero- and Homobasidiomycetes. The chemical structure of polysaccharides and its connection to antitumor activity, including possible ways of chemical modification, experimental testing and clinical use of antitumor or immunostimulating polysaccharides, and possible mechanisms of their biological action, are discussed. Numerous bioactive polysaccharides or polysaccharide–protein complexes from medicinal mushrooms are described that appear to enhance innate and cell-mediated immune responses and exhibit antitumor activities in animals and humans. Stimulation of host immune defense systems by bioactive polymers from medicinal mushrooms has significant effects on the maturation, differentiation, and proliferation of many kinds of immune cells in the host. Many of these mushroom polymers were reported previously to have immunotherapeutic properties by facilitating growth inhibition and destruction of tumor cells. While the mechanism of their antitumor actions is still not completely understood, stimulation and modulation of key host immune responses by these mushroom polymers appears central. Particularly and most importantly for modern medicine are polysaccharides with antitumor and immunostimulating properties. Several of the mushroom polysaccharide compounds have proceeded through phases I, II, and III clinical trials and are used extensively and successfully in Asia to treat various cancers and other diseases. A total of 126 medicinal functions are thought to be produced by medicinal mushrooms and fungi including antitumor, immunomodulating, antioxidant, radical scavenging, cardiovascular, antihypercholesterolemia, antiviral, antibacterial, antiparasitic, antifungal, detoxification, hepatoprotective, and antidiabetic effects.     

Proceedings towards a natural classification of the worldwide taxa Phellinus s.l. and Inonotus s.l., and phylogenetic relationships of allied genera

The classification of Phellinus s.l., Inonotus s.l. and the phylogenetic relationships of allied genera were studied using nuc-lsu rDNA sequence data. The worldwide taxon sampling comprised 107 species, 99 of them belonging to the Hymenochaetales. The phylogenetic trees were discussed in relation to morphological and anatomical features of the fruit bodies. The Hymenochaetales formed no monophyletic group and several non-Hymenochaetales appeared as intermingled with the Hymenochaetales. Trichaptum abietinum and Oxyporus populinus showed no certain affinities within the Hymenochaetales, whereas Basidioradulum radula was closely related to Phellopilus nigrolimitatus, and Hyphodontia quercina and Schizopora paradoxa were related to Coltricia, Coltriciella and Pyrrhoderma adamantinum. Phellinus s.l. and Inonotus s.l. formed no monophyletic groups, and a subdivision in the following genera is accepted: Phellinus s.s., Inonotus s.s., Inocutis, Fomitiporella, Aurificaria, Phylloporia, Fulvifomes, Mensularia, Pseudoinonotus, Fomitiporia, Porodaedalea, Onnia, Fuscoporia, and Inonotopsis. Coltricia and Coltriciella were confirmed as seperate genera. The taxonomic status of Phellinidium and Pyrrhoderma remained uncertain. 16 new combinations are proposed.     

Quantitative response of cell growth and polysaccharide biosynthesis by the medicinal mushroom Phellinus linteus to NaCl in the medium

The effect of NaCl on cell growth and polysaccharide biosynthesis in the medicinal mushroom Phellinus linteus was studied. With the increase of NaCl concentration between 1 g/l and 7 g/l in the culture medium, the cell growth and intracellular polysaccharide (IPS) accumulation were decreased; extracellular polysaccharide (EPS) concentration was enhanced, with an increase of NaCl concentration from 1 g/l to 3 g/l. Under the optimum NaCl concentration of 3 g/l, the maximum EPS and IPS production reached 2.2±0.15 g/l and 53.6±2.45 mg/g DW on day 12, which improved 32.27% and decreased 16.89% compared to the control, respectively. Both EPS and IPS showed new polysaccharide components by fractionation with DEAE-cellulose ion exchange chromatography compared to the control. The results presented in this study are considered helpful for further investigation on the diversity of polysaccharide biosynthesis of this medicinal fungus under NaCl environments.     

pH control strategy in a shaken minibioreactor for polysaccharide production by medicinal mushroom Phellinus linteus and its anti-hyperlipemia activity

A process at various pH values ranging from 4.5 to 7.5 for production of mycelia and extracellular polysaccharide (EPS) by P. linteus fermentation in pH-controlled shaken bioreactor was investigated. A two-stage pH control strategy in which pH value was kept at 6.5 for the first 24 h, and then switched to 4.5 was developed successfully to enhance simultaneously the cell growth and EPS production. The maximum cell density and EPS production reached 15.13 ± 0.1 g/l on day 6 and 6.74 ± 0.1 g/l on day 4, respectively. The anti- hyperlipemia effect of EPS and intracellular polysaccharide (IPS) extracted from mycelia were observed that both EPS and IPS can obviously reduce the serum triglyceride (TG), the blood cholesterol (TC) and serum low density lipoprotein (LDL) level, and increase the high density lipoprotein (HDL) level of the hyperlipemia mice. Polysaccharides from submerged cultivation of medicinal fungus P. linteus have favorable potency to develop anti-hyperlipermia drugs.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Current analytical methods to study plant water extracts: the example of two mushrooms species, Inonotus hispidus and Sparassis crispa

The analytical study of two hot-water extracts from the mushrooms Inonotus hispidus (Bull.) P. Karst and Sparassis crispa Wulf.:Fr was performed by NMR, HPLC-PAD-MS and GC-MS. The simultaneous use of different analytical techniques highlighted the diverse classes of natural products contained in these extracts. This study describes an attempt to adapt a useful phytochemical method to the direct investigation of plant water extracts, which represent the typical traditional manner for the administration of natural remedies. The heritage concerning plant processing procedures, known as traditional pharmaceutical knowledge, could play an important role in future research on medicinal species. This kind of study could be used as an update for current and future perspectives in this research field.     

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。     

药用植物华中五味子的种群遗传多样性及遗传结构

华中五味子（Schisandra sphenanthera）是著名的药用植物,具有悠久的药用历史和巨大的开发潜力。为了有效评估、利用和保护华中五味子资源,应用自主开发的9对SSR引物研究了华中五味子自然种群的遗传多样性与遗传结构。结果表明：在10个采样种群中,共检测到58个等位基因,平均预期杂合度H_E为0.528,平均观察杂合度H_O为0.519,较大的连续种群保持了较高的遗传多样性,而小种群的遗传多样性则相对较低;华中五味子总体表现为显著的杂合子缺失,内繁育系数F_IS为0.042;种群间总的遗传分化系数F_ST为0.108,两两种群间分化显著;贝叶斯聚类结果把10个采样种群按遗传组成分为江南组和江北组2组,长江所形成的特殊地理屏障对华中五味子江南和江北地区间较高的遗传分化造成了影响。     

分子标记在食（药）用真菌遗传多样性研究中的应用

食（药）用真菌在经济和生态方面都具有重要意义,其遗传多样性研究是资源可持续利用和生物保护学研究的基础,有利于食（药）用真菌种质资源的收集、保存、评价和利用,也有助于其分类学、系统学及进化等的研究。遗传多样性的研究方法很多,分子标记是目前最常用最有效的方法之一。综合分析了分子标记在食（药）用真菌遗传多样性研究中的应用,比较了各种标记的应用范围、优缺点,探讨了分子标记用于食（药）用真菌遗传多样性评价的前景及问题。     

Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice

The anti-diabetic activities of the exopolysaccharides (EPS) produced by submerged mycelial culture of two different mushrooms, Tremella fuciformis and Phellinus baumii, in ob/ob mice were investigated. All the animals were randomly divided into three groups with seven animals in each group: The control group received 0.9% NaCl solution; the diabetic groups were treated with EPS from T. fuciformis (Tf EPS) and P. baumii (Pb EPS) at the level of 200 mg/kg body weight using an oral zoned daily for 52 days. The plasma glucose levels in the EPS-fed mice were substantially reduced by about 52% (Tf EPS) and 32% (Pb EPS), respectively, as compared to control mice. The results of oral glucose tolerance test (OGTT) revealed that both EPS-fed groups significantly increased the glucose disposal after 52 days of EPS treatments. Furthermore, higher food efficiency ratios and reduced blood triglyceride levels were observed in the EPS-treated groups. Because peroxisome proliferator-activated receptor gamma (PPAR-γ) is indeed a key regulator of insulin action, we investigated the expression pattern of adipose tissue PPAR-γ messenger RNA (mRNA) and plasma levels of PPAR-γ. It was revealed that PPAR-γ was significantly activated in response to EPS treatments. The results suggested that both EPS exhibited considerable hypoglycemic effect and improved insulin sensitivity possibly through regulating PPAR-γ-mediated lipid metabolism. Our results indicated that two mushroom-derived EPS might be developed as potential oral hypoglycemic agents or functional foods for the management of non-insulin-dependent diabetes mellitus.