Study on polysaccharide content and monosaccharide composition of Polyporus umbellatus from different production areas / 中国中药杂志

In order to provide scientific basics for exploitation and sufficient application of Polyporus umbellatus resources and study the monosaccharide composition of P. umbellatus polysaccharides,the anthrone-sulfuric acid method was applied to compare polysaccharide content of P. umbellatus from 17 producing areas. The monosaccharides were derived by 1-phenyl-3-methyl-5-pyrazolone( PMP) and the derivatives were identified by UPLC-MS/MS and the content of each monosaccharide component was determined simultaneously. The results demonstrated that there was a certain difference in total polysaccharide content of P. umbellatus from different regions,and the content of total P. umbellatus polysaccharide from Shaanxi province and Sichuan province( 1. 15% and 1. 90%) was relatively higher than that of others areas. Polysaccharides from P. umbellatus was mainly composed of eight monosaccharides,including glucose,glucuronic acid,galactose,ribose,xylose,arabinose,mannose and fucose. The contents of glucose( 17. 65 mg·g-1) was higher than others. The ribose was the lowest( 0. 13 mg·g-1). In addition,fructose,rhamnose and galacturonic acid were also detected in some samples. Furthermore,the results of cluster analysis( CA) and principal component analysis( PCA) indicated that totally 17 batches of P. umbellatus polysaccharide could be classified into three clusters,samples collected from Wuchang in Heilongjiang province were clustered into one group separately. The study can provide a basis for rational utilization of P. umbellatus resources,and also implies the sequence of monosaccharide linking and pharmacological activity of P. umbellatus polysaccharides.

Molecular identification of Armillaria gallica by PCR-RFLP analysis / 中国中药杂志

Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.

A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus / 中国中药杂志

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

Ligninolytic fungus Polyporus sp. S133 mediated metabolic degradation of fluorene

ABSTRACT This study aimed to investigate the impact of nonionic surfactants on the efficacy of fluorine degradation by Polyporus sp. S133 in a liquid culture. Fluorene was observed to be degraded in its entirety by Polyporus sp. S133 subsequent to a 23-day incubation period. The fastest cell growth rate was observed in the initial 7 days in the culture that was supplemented with Tween 80. The degradation process was primarily modulated by the activity of two ligninolytic enzymes, laccase and MnP. The highest laccase activity was stimulated by the addition of Tween 80 (2443 U/L) followed by mixed surfactant (1766 U/L) and Brij 35 (1655 U/L). UV-vis spectroscopy, TLC analysis and mass spectrum analysis of samples subsequent to the degradation process in the culture medium confirmed the biotransformation of fluorene. Two metabolites, 9-fluorenol (λmax 270, tR 8.0 min and m/z 254) and protocatechuic acid (λmax 260, tR 11.3 min and m/z 370), were identified in the treated medium.

Habitat suitability assessment of medicinal Polyporus umbellatusin China based on Maxent modeling / 中国中药杂志

Geographic distribution of Polyporus umbellatus was predicted by using distribution records. Based on 42 distribution records from 12 provinces and bioclimatic data (1950-2000), georaphic distribution of P. umbellatus was modeled using Maxent. The results showed thatthe Receiver Operating Characteristic (ROC) curve analysis method was used to assess the accuracy of MAXENT model and the area under ROC curve (AUC) value of MAXENT was 0. 960 which suggested that the result of assessment was dependable. The geographic distribution pattern of were divided into three distribution block based on distribution values of 0.5-0.8: small area of Heilongjiang, Jilin, Liaoning and Hebei province, the board area of Yunnan, Guizhou and Sichuan, the southeast area of Tibet and the most area of Shanxi and Shannxi, the southeast board area of Shannxi, Gansu and Ningxia. Jackknife Test showed that average precipitation in warm seasons had the greatest contribution to the distribution gain of P. umbellatus, followed by mean temperature of driest quarter and annual mean temperature. The object suggests the potential distribution areasof P. umbellatus which is useful for the habitat conservation and introduction of P. umbellatus.

Molecular cloning and characterization of four small GTPase genes from medicinal fungus Polyporus umbellatus / 药学学报

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.

Physicochemical properties of medicinal fungus Polyporus umbellatus sclerotial exudate / 中国中药杂志

This study was conducted to investigate the physicochemical properties of Polyporus umbellatus sclerotial exudate. Morphological characteristics of the sclerotia and its exudate were observed during different stages of sclerotial formation. The pH of the exudate was detected at different time during cultivation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of P. umbellatus sclerotial exudate during cultivating time. Additionally, the protein content was measured by means of BCA protein assay. Furthermore, CAT content was detected using ultraviolet absorption method. That the protein content of the exudate and CAT specific activity rose gradually during the passage of the cultivating time indicated a high level of oxidative stress during P. umbellatus sclerotial exudate formation. The results showed that the pH of the exudate increased gradually and then dropped down during sclerotial formation. That the pH of the exudate maintained the acidity state during the cultivation indirectly indicated that acidic environment would help sclerotial formation. The exudate produced gradually and was absorbed by the sclerotia itself.

Isolation of high quality RNA from Polyporus umbellatus (Pers. ) Fries

Background: The dried sclerotium of medicinal fungus Polyporus umbellatus (Pers.) Fries has many pharmacological functions such as diuretic and anticancer activity, in which high-content polysaccharides may play an important role. However, RNA isolation is difficult in filamentous fungi and lacking in P. umbellatus. Results: Five methods for RNA extraction from five strains collected from four provinces were assessed for their ability to recover a high-quality RNA applicable for sequence-related amplification polymorphism (SRAP) PCR and GDP-D-mannose pyrophosphorylase (GMP) gene expression profiles. Both A260/A280 and A260/A230 ratios of the best Trizol Plus + RNAiso-mate for Plant Tissue method are around 2 with a yield of 1122.00 +/- 0.21 ng ul-1. The Trizol method also showed good quality with the yield 469.60 ng ul-1. The SRAP PCR amplified clear and polymorphic bands in all five cDNA samples transcribed from RNA by using primer Me4-Em4. GMP gene fragment (1251 bp) was successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. Conclusion: All these results showed that the total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.

The regulatory effects of polyporus polysaccharide on the nuclear factor kappa B signal pathway of bladder cancer cells stimulated by Bacillus Calmette-Guerin / 中国结合医学杂志

<p><b>OBJECTIVE</b>To detect the effects of Polyporus polysaccharide (PPS), Bacillus Calmette-Guerin (BCG), and their combination on the nuclear factor kappa B (NF-κB) signaling pathway associated-gene expression and investigate the molecular mechanisms of the toxic-reducing effect of PPS in coordination with BCG against bladder cancer.</p><p><b>METHODS</b>After T739 cells were treated with PPS, BCG and their combination, the changes in mRNA and protein expression of inhibitor of kappa B kinase beta (IKKβ), NF-κB subunit p65 (NF-κB p65), intracellular adhesion molecule 1 (ICAM1) and chemokine (C-c motif) ligand 2 (CCL2) in bladder cancer cell line T739 were determined by relative quantitative real-time PCR, Western blot, and flow cytometry (FCM). NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA, and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry.</p><p><b>RESULTS</b>Compared with the T739 control group, the mRNA expression of IKBKB (IKKβ), Rel A (NF-κB p65), ICAM1 and CCL2 in T739 cells treated with BCG were increased obviously (Ratio>2.0), as well as the expression of IKKβ, CCL2 and ICAM1 proteins. Meanwhile, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression in T739 cells treated with BCG were up-regulated significantly (P<0.05). Compared with the control, the increased expression in T739 cells were simultaneously down-regulated after PPS treatment, except for ICAM1 protein expression. With cells treated with a combination of BCG and PPS, the expression of genes associated with the NF-κB signaling pathway, such as IKBKB, ICAM1 and CCL2, were all down-regulated compared to the BCG group, as well as Rel A mRNA expression, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression.</p><p><b>CONCLUSIONS</b>PPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy.</p>

Fundamental study of degradation of dichlorodiphenyltrichloroethane in soil by laccase from white rot fungi

This research describes application of laccase from white-rot fungi [polyporus] to remove dichlorodiphenyltrichloroethane in soil. The degradation kinetics of dichlorodiphenyltrichloroethane in soil was also investigated by laboratory batch experiments. The results showed that laccase from white-rot fungi can effectively degrade dichlorodiphenyltrichloroethane and the degradation of total dichlorodiphenyltrichloroethane [the sum of the four dichlorodiphenyltrichloroethane compounds in a sample] was pseudo-first-order kinetics. The residues of almost all the dichlorodiphenyltrichloroethane components and total dichlorodiphenyltrichloroethane in soils treated with laccase decreased rapidly during first 15 days and then kept at a stable level during next 10 days. The residues of total dichlorodiphenyltrichloroethane in soils with different dosages laccase decreased by about 21-32%, 29-45%, 35-51% and 36-51% after 5, 10, 15 and 25 days of incubation, respectively. The half-life of total dichlorodiphenyltrichloroethane in soils with different dosages laccase ranged from 24.75 to 41.75 days. The residues of total dichlorodiphenyltrichloroethane in three different types of soils decreased by 25-29%, 39-43%, 44-47% and 47-52% after 5, 10, 15 and 25 days of incubation with laccase, respectively. The half-life of total dichlorodiphenyltrichloroethane in different types of soil ranged from 24.71 to 27.68 days. The residues of total dichlorodiphenyltrichloroethane in soils with different pH levels decreased by 18-24%, 29-39%, 36-39% and 39-50% after 5, 10, 15 and 25 days of incubation with laccase, respectively. The half-life of total dichlorodiphenyltrichloroethane ranged from 25.63 to 36.42 days. Laccase can be an efficient and safe agent for remediation of dichlorodiphenyltrichloroethane-contaminated soil

Decolorisation of synthetic dyes and textile wastewater using Polyporus rubidus.

Effluent from textile industries were treated with enzyme from white rot fungi isolated from outskirts of Mumbai and identified as Polyporus rubidus in our laboratory. Decolorisation of 4 Reactive dyes commonly found in the effluents such as Reactive bue, Reactive orange, Ramazol black and Congo red was examined by treatment with enzyme from Polyporus rubidus. Treatment of effluent was done in a laboratory scale bioreactor constructed with laccase immobilized Na-alginate beads. Greater than 80% of dyes were degraded within 5 days under stationary incubation conditions. The enzyme had a maxmimum activity of 17.1U after 3 days and was found to be secreted extracellularly by Polyporus rubidus. In this study the Polyporus rubidus has been reported for the first time to have laccase activity offering a promising possibility to develop an easy and cost effective method for degradation of dangerous dyes.

Effect of companion fungus on hyphal growth and polysaccharide content of Polyporus umbellatus / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of companion fungus on hyphal growth and polysaccharide content of Polyporus umbellata.</p><p><b>METHOD</b>The mycelia and culture filtrate of companion fungus were added to the liquid culture system, and the biomass yield and polysaccharide of P. umbellatus were measured.</p><p><b>RESULT</b>Mycelia and appropriate unsterilized culture filtrate of companion fungus could enhance the biomass yield of P. umbellatus significantly, while sterilized culture filtrate of companion fungus could decrease the biomass yield of P. umbellatus significantly. Either mycelia or culture filtrate of companion fungus could increase the intracellular polysaccharide content of P. umbellatus significantly. At the same time, they also could decrease extracellular polysaccharide content of P. umbellatus evidently.</p><p><b>CONCLUSION</b>The mycelia and culture filtrate of companion fungus could be used in further fermentation of P. umbellatus.</p>

Progress on antineoplastic constituents derived from polypore fungi / 中国中药杂志

Polypore fungi is a cluster of important pharmacological fungi with significant antitumor activity. In recent years, the antineoplastic constituents from polypore fungi have been comprehensively studied. Through investigating the domestic and overseas studied paper, the antitumor active constituents derived from polypore fungi including high molecular weight compounds such as polysaccharides, glycopeptides, glycoproteins, lectins, and lipid soluble low molecular weight compounds such as terpenoids, steroids, phenolics, benzopyranones, were reviewed. In addition, the significance in the exploitation of new drug for antitumor by the application of polypore fungi was discussed at the end of this paper.

Improvement of Ergone Production from Mycelial Culture of Polyporus umbellatus

Ergone,a fungal metabolite derived from ergosterol,was previously isolated and identified from Polyporus umbellatus. Ergone is a major component of P. umbellatus known to have anti-aldosteronic diuretic effect and also displays cytotoxic activities. Most of mushroom's fruit bodies used for test contained less than 10 microg/g of ergone. But P. umbellatus have larger amount of ergone than any other mushrooms. In order to improve the ergone production from the submerged culture of P. umbellatus, several factors including medium composition,culture conditions (temperature and pH) and different combinations of co-cultivation with various mycelia were studied. Among various carbon sources examined,starch proved to be most effective for the production of mycelia. The optimum pH and temperature for a flask culture of P. umbellatus mycelia were found to be 4.5 and 25degrees C,respectively. Under the optimized culture conditions,both the ergone production (86.9 microg/g) and mycelial growth (3.5 g/l) increased when P. umbellatus was cultured with Armillariella mellea. When the optimized conditions were applied,both mycelium and ergone production were significantly enhanced.