Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

茯神挥发性成分及其生物活性研究

为研究茯神挥发性成分含量、构成及其生物活性,本实验采用二氧化碳超临界的方法提取茯神低极性成分,以提取率作为响应值,在单因素试验的基础上采用响应曲面法考察提取压力、温度、二氧化碳流速对提取效果的影响,优化提取工艺。运用气相色谱-质谱联用仪(GC-MS)对其主要化学成分进行鉴定,并采用生长速率法测定了挥发油对5种真菌的生物活性。得到提取模型极值点,即提取压力27.26MPa、提取温度55.97℃、提取流速10.68L/min时,提取率达到最大,提取率预测值为1.66mg/g。通过NIST14质谱库检索,鉴定了其中17个主要化合物,运用峰面积归一法确定各个组分的含量,占挥发油总量的84.5%。抗菌实验表明茯神挥发油对采绒革盖菌菌株有低浓度促进高浓度抑制的活性。     

Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia

Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans. Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition.     

Triterpenes from the fungus Poria cocos and their inhibitory activity on nitric oxide production in mouse macrophages via blockade of activating protein-1 pathway

Two new triterpenes, 29-hydroxydehydrotumulosic acid (1) and 29-hydroxydehydropachymic acid (2), together with six known compounds, dehydropachymic acid (3), dehydrotumulosic acid (4), 29-hydroxypolyporenic acid C (5), polyporenic acid C (6), tumulosic acid (7), and pachymic acid (8), were isolated from the dried sclerotia of Poria cocos. In the in vitro bioassays, these isolated compounds reduced, in a dose-dependent manner, nitric oxide (NO) production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, with compounds 5 and 6, the IC(50) values of which were 16.8±2.7 and 18.2±3.3 μM, respectively, exhibiting the greatest inhibition activity. Further Western blot analysis conducted on cells pre-treated with compounds 5 and 6, and luciferase assays on activator protein 1-dependent gene expression revealed that the inhibited NO release was attributed to the reduced expression of iNOs (=inducible NO synthase) enzymes, which might be regulated via the blockade of activator protein-1 signaling pathway.     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

发酵茯苓菌丝体和天然茯苓多糖的研究

本文对发酵茯苓菌丝体和天然茯苓中多糖的提取分离进行了研究,并分别测定了二者总多糖的提取率及总糖的平均含量。发酵茯苓菌丝体和天然茯苓中总多糖的提取率分别为24.47%和82.93%;发酵茯苓菌丝体和天然茯苓总糖的平均含量分别为45.17%和87.24%。     

Multinucleate nature,and mating by use of isozyme analysis inPoria cocos

Double staining study of nuclei and cell walls inPoria cocos indicated that the hyphal cells were multinucleate and had no clamp connections. Isozyme analysis of alcohol dehydrogenase (ADH) in 52 natural isolates revealed that there were three types of banding patterns: type I, five bands; type II, one slow band; type III, one fast band. Regenerants expressing type-II or type-III ADH-isozyme pattern were obtained from type-I isolates via protoplast manipulation. When the type-II regenerants were mated with the type-III regenerants, hyphae of type-I phenotype appeared. These data indicated that these type-II and type-III regenerants derived from protoplasts of the type-I isolates were primary hyphae. These primary hyphal cells were also multinucleate. Inter-strain mating ofP. cocos was performed and confirmed by ADH-isozyme analysis. Confronting cultures of a type-III regenerant derived from protoplasts of a type-I isolate and a type-II regenerant derived from a type-II isolate resulted in type-I hyphae.     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodology

Polysaccharides production from Poria cocos was carried out using aqueous NaOH with the assistance of ultrasonic. Experimental design was used to investigate the effect of three parameters (extraction time, extraction concentration of NaOH, and ratio of aqueous NaOH to raw material) on polysaccharides yields. The ranges of the factors investigated were 1–3 min for extraction time (X₁), 0.5–1.0 mol/L for extraction concentration of NaOH (X₂), and 30–50 for ratio of aqueous NaOH to raw material (X₃). The statistical analysis of the experiment indicated that extraction concentration of NaOH had significant effect on P. cocos polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.9935 for P. cocos polysaccharides yield. The optimal condition for P. cocos polysaccharides yield within the experimental range of the variables studied was at 2.44 min, 0.789 mol/L, and 53.0. At this condition, the predicted yield of polysaccharides extracted was 82.3%.     

Expression of phenolics and defence-related enzymes in relation to red root rot disease of tea plants

Red root rot caused by Poria hypolateritia is a dreadful disease in tea plant due to sudden death of bushes. In response to fungal pathogen, variation in the defence-related enzymes was investigated. The infected tea root was undertaken to study about various defence-related and pathogen-related enzymes. The infected root, as a prime response to disease attack, was subjected to the analysis of phenolics, phenylalanine ammonia lyase, tyrosine ammonia lyase, peroxidase, polyphenol oxidase, catalase, chitinase, β-1,3-glucanase and protease were assayed. The results on assay of defence-related enzymes revealed that the activity was significantly higher in infected roots when compared with healthy roots. Phenolics were accumulated more in infected roots. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis further confirmed the presence of induced pathogenesis-related proteins in the infected root tissues. The activity of all enzymes was increased up to threefold amount when compared with normal ones. The accumulation of defence enzymes in plants revealed the virulence of root pathogen in stimulating induced systemic resistance of tea plants and phytopathogenicity causing pathogenesis. This study exemplify to recognise underlying processes in causing infection and to identify the existence of host–pathogen relationship.