Cloning and expression analysis of PlERG24 gene in ergosterol biosynthesis of Phellinus linteus / 中草药

Objective: To clone the gene full length of ergosterol C14 reductase (ERG24) in Phellinus linteus and analyze its bioinformatics and expression pattern. Methods: The primers of PlERG24 were designed according to the transcription sequence of P. linteus, the cDNA full-length sequence of PlERG24 was obtained by PCR, its bioinformatics was analyzed by Ex PASy and other online analysis software, and its expression pattern in mycelia of P. linteus was analyzed by real-time fluorescence quantitative PCR. Results: The full-length cDNA of PlERG24 gene was 1 412 bp, which encoding a protein of 441 amino acids with a predicted molecular weight of 49 358.61 and isoelectric point of 5.28; ERG24 protein was a hydrophobic protein without signal peptide, which was presumably located in the plasma membrane with six phosphorylation sites. Phylogenetic tree analysis indicated that amino acid sequences of ERG24 in P. linteus were genetically closely related to ERG24 in Sanghuangporus baumii. The qRT-PCR results showed that gene expression of PlERG24 reached the highest level of 6.36 at 25d during the growth cycle of mycelia in P. linteus. Conclusion The full length of PlERG24 gene was obtained, which lays a foundation for further studies on gene function and genetic regulatory mechanism of ergosterol biosynthesis.

Antitumor Effect of Phellinus Linteus Polysaccharide on Sarcoma S180 Cells in vivo and in vitro / 中国药房

OBJECTIVE:To study the antitumor effect of phellinus linteus polysaccharide on sarcoma S180 cells in vivo and in vitro. METHODS:Sarcoma S180 cells in logarithmic growth period were selected,adding into 0(blank control),2,4,8 mg/mL phellinus linteus polysaccharide solution and respectively culturing for 12,24,36,48 h. The in vitro proliferation inhibition rate of cells was determined by MTT method;its apoptotic morphology was observed by fluorescence staining and cell apoptosis rate was detected by flow cytometry. S180 tumor-bearing mice models were established and randomly divided into control group,phellinus linteus polysaccharide high-dose,medium-dose,low-dose groups(400,200,100 mg/kg),10 in each group. Model mice were in-tragastrically administrated related medicined,once a day,for 12 d. Mice were executed after 24 h of last administration,tumor weight was determined,tumor inhibition rate was calculated. Immunohistochemistry was conducted to detect the tumor suppressor gene PTEN and oncogene C-myc protein expressions in tumor tissue. RESULTS:Compared with blank control group,phellinus linteus polysaccharide can increase the proliferation inhibition rate of S180 cells and induce the increase of apoptosis rate(P<0.05 or P<0.01),showing a concentration-time manner. Compared with control group,the tumor inhibition rates in phellinus linteus polysaccharide groups were obviously increased (P<0.01),PTEN protein expressions were strengthened (P<0.05 or P<0.01) and C-myc protein expressions were weakened (P<0.05). CONCLUSIONS:Phellinus linteus polysaccharide shows antitumor ef-fect in vivo and in vitro,which can up-regulate the PTEN,down-regulate C-myc protein expressions.

Chemical Constituents of the Culture Broth of Phellinus linteus and Their Antioxidant Activity

The medicinal fungus Phellinus linteus, in the family Hymenochaetaceae, has been used as a traditional medicine for the treatment of various diseases. In this study, the chemical constituents of the culture broth of P. linteus were investigated. P. linteus was cultured in potato dextrose broth medium, and the culture broth was extracted with ethyl acetate. The ethyl acetate-soluble portion was concentrated and subjected to ODS column chromatography, followed by Sephadex LH-20 column chromatography. Six compounds (1~6) were purified by preparative reversed-phase high-performance liquid chromatography. Spectroscopic methods identified their structures as caffeic acid (1), inotilone (2), 4-(3,4-dihydroxyphenyl)-3-buten-2-one (3), phellilane H (4), (2E,4E)-(+)-4'-hydroxy-gamma-ionylideneacetic acid (5), and (2E,4E)-gamma-ionylideneacetic acid (6). Compounds 1, 2, and 3 exhibited potent dose-dependent antioxidant activity.

Phellinus linteus Extract Exerts Anti-asthmatic Effects by Suppressing NF-kappaB and p38 MAPK Activity in an OVA-induced Mouse Model of Asthma

Phellinus linteus has been used as a traditional herbal medicine in Asian countries and is known to have anti-tumor, immunomodulatory, anti-inflammatory, and anti-allergic activities. However, the protective effects of P. linteus against experimental asthma have not been fully investigated. The objective of this study was to determine whether P. linteus ethanol extract (PLE) suppresses inflammatory response in an OVA-induced asthma model. As expected, the oral administration of PLE significantly inhibited eosinophilic airway inflammation and airway hyperresponsiveness in OVA-challenged BALB/c mice. Supporting these data, the augmentation of Th2 cytokines (IL-4, IL-5, and IL-13), eotaxin, and adhesion molecules in lung tissues and bronchoalveolar lavage fluid after OVA inhalation was markedly attenuated by PLE. Furthermore, PLE reduced OVA-induced activation of NF-kappaB and p38 MAPK in lung tissues. Therefore, our results suggest the potential of P. linteus as a therapeutic agent for asthma.

Neuraminidase Inhibitors from the Fermentation Broth of Phellinus linteus

During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 microM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.

Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice

Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). Methods: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. Results: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one-and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.

Quality Characteristics of Yanggaeng Supplemented with Sanghwang Mushroom (Phellinus linteus) Mycelia

The principal objective of this study was to examine the quality characteristics of yanggaeng supplemented with powder derived from Sanghwang mushroom (Phellinus linteus) mycelia. We analyzed the potential of utilizing Phellinus linteus mycelia as a functional food material by estimating total polyphenol and flavonoid contents, electron-donating abilities, as well as antioxidative activities of the water and ethanol extracts of Sanghwang mushroom mycelia. The total phenol and flavonoid contents of ethanol extracts from Phellinus linteus mycelia were 0.69 mg/ml and 0.16 mg/ml, respectively, while the contents from the water extract of Phellinus linteus mycelia were 0.66 mg/ml and 0.22 mg/ml, respectively. The electron-donating abilities of ethanol and water extracts from Phellinus linteus mycelia were 88.64 and 90.29%, respectively. The ABTS radical scavenging activities of ethanol and water extracts from Phellinus linteus mycelia were 89.74 and 71.35%, respectively. In terms of color values, as the level of powder increased, the value of L (lightness) decreased, whereas those of a (redness) and b (yellowness) increased. In regard to the mechanical properties of the samples, we noted significant differences in hardness, springiness, chewiness, and gumminess (P<0.05). The results of the sensory evaluation showed that the score from SHM (Sanghwang mushroom mycelia) with 4% powder was significantly higher than other samples in terms of sweetness, color, taste, texture and overall quality (P<0.05). Taken together, the recommended level of Phellinus linteus mycelia powder in yanggaeng is 4% for optimal sensory characteristics.

Neuraminidase Inhibitors from the Culture Broth of Phellinus linteus

During the search for neuraminidase inhibitors from medicinal fungi, we found that the culture broth of Phellinus linteus exhibited potent inhibitory activity. Solvent partition, Sephadex LH-20 column chromatography, and high-performance liquid chromatography (HPLC) were performed for purification of two active substances from the culture broth. According to 1H NMR measurements and comparison of HPLC retention times with those of authentic compounds, their chemical structures were identified as hispidin and hypholomine B. Compounds (hispidin) 1 and 2 (hypholomine B) inhibited neuraminidase, with IC50 values of 13.1 and 0.03 microM, respectively.

Single- and repeated-dose toxicities of aloe fermentation products in rats / 한국실험동물학회지

In this study, aloe fermentation products were derived from mycelia from 3 mushrooms: Ganoderma lucidum (AG), Hericium erinaceum (AH), and Phellinus linteus (AP). Levels of aloin A and B increased with fermentation time. The highest levels were measured on the fifth day of fermentation. beta-Glucan levels decreased with fermentation time. The safety of aloe fermentation products were examined in male and female Sprague-Dawley rats. Rats were orally administered the three aloe fermentation products at dose levels of 1, 2 or 5 g/kg for single-dose toxicity test and 0.5, 1, or 2 g/kg for repeated-dose toxicity test. There were no significant differences in body weight gain between vehicle control and AG-, AH- or AP-treated rats. Also, significant changes in daily feed intake and water consumption were not observed. In hematological analysis, none of the parameters were affected by aloe fermentation products with mushroom mycelia. This suggests that there are no negative effects on homeostasis and immunity. In blood biochemistry analysis, none of the markers were affected by feeding rats with AG, AH or AP. Similarly, there were no significant effects on markers for liver, kidney, skeletal and heart muscle functions. No remarkable lesions were observed in these organs at histopathology. Since there were no adverse effects of AG, AH and AP in single- or repeated-dose toxicity tests, even at higher doses than normal, we conclude that the aloe fermentation products with mushroom mycelia possess long-term safety and could be candidates as multifunctional nutrients for the improvement of intestinal function and immunity.

Extraction and Characteristics of Anti-obesity Lipase Inhibitor from Phellinus linteus

To develop a potent anti-obesity lipase inhibitor from mushroom, the lipase inhibitory activities of various mushroom extracts were determined. Methanol extracts from Phellinus linteus fruiting body exhibited the highest lipase inhibitory activity (72.8%). The inhibitor was maximally extracted by treatment of a P. linteus fruiting body with 80% methanol at 40degrees C for 24 hr. After partial purification by systematic solvent extraction, the inhibitor was stable in the range of 40~80degrees C and pH 2.0~9.0. In addition to lipase inhibitory activity, the inhibitor showed 59.4% of superoxide dismutase-like activity and 56.3% of acetylcholinesterase inhibitory activity.

Optimal Extraction Conditions of Anti-obesity Lipase Inhibitor from Phellinus linteus and Nutritional Characteristics of the Extracts

In an effort to develop novel mushroom-derived anti-obesity nutraceuticals, water and ethanol extracts containing the lipaseinhibitory compound from Phellinus linteus were prepared, and their nutritional components were determined. The optimal conditions for the extraction of P. linteus lipase inhibitor involved the treatment of the fruiting bodies with distilled water at 80degrees C for 72 hr and 80% ethanol at 100degrees C for 60 hr, respectively. The distilled water extract and ethanol extract contained 10.9% and 6.11% of crude protein, and 0.96% and 15.86% of crude fat, respectively. Additionally, the distilled water extract contained a large quantity of minerals, including 239.5 mg of K, 39.3 mg of Mg, and 39.3 mg of Na. The free amino acid content of the distilled water extracts was also higher than that of the ethanol extracts, and in particular, the distilled water extracts contained 5,139 mg of asparagine, 3,891 mg of tryptophan, 2,598 mg of alanine, and 2,066 mg of serine in 100 g of the distilled water extracts. 100 g of the distilled water and ethanol extracts were found to contain 12.31 g and 8.16 g of malic acid, respectively.

Anti-tumor Effect of Phellinus Linteus and Coriolus Versicolor Capsule on S180 sarcoma and H22 hepatoma in mice / 中国实验方剂学杂志

Objective: To observe the anti-tumor effect of Phellinus Linteus and Coriolus Versicolor Capsules (PLCVC) in S180 sarcoma and H22 hepatoma animal models in mice. Methods: The sarcoma S1180 and hepatoma H22 models were established in mice. After 12 days of treatment, the animals were killed, and the subcutaneous sarcoma were separated and weighted. The levels of vascular endothelial growth factor(VEGF), CD4 and CD8 of S180 tumor tissue were investigated by immunohistochemical method. KM mice were intraperitoneal injected with H22 hepatoma cells, and treated with different experimental drugs. The survival time was observed and recorded, and life-prolongation rate was calculated. Result: PLCVC could inhibit the growth of S180 and H22 tumor, and inhibit the expression of VEGF, improve the expression of CD4 and CD8. The survival time of the mice treated by PLCVC were significantly longer than the untreated group. Conclusion: PLCVC can inhibit the growth of tumour, the mechanism is partially related to inhibiting angiogenesis and improving the immunological function.

Antitumor Effects and Immunomodulating Activities of Phellinus linteus Extract in a CT-26 Cell-Injected Colon Cancer Mouse Model

The antitumor effects of Phellinus linteus extract (Keumsa Linteusan) were investigated in a CT-26 cell-injected colon cancer mouse model. When administered orally (250~1,000 mg/kg body weight), Keumsa Linteusan significantly inhibited the growth of solid colon cancer. The highest dose was highly effective, reducing tumor formation by 26% compared with the control group. The anticomplementary activity of Keumsa Linteusan increased in a dose-dependent manner. Lysosomal enzyme activity of macrophages was increased by 2-fold (100 microg/ml) compared with the control group. Keumsa Linteusan can be regarded as a potent enhancer of the innate immune response, and can be considered as a very promising candidate for antitumor action.

Anti-cancer Activities of Ginseng Extract Fermented with Phellinus linteus

In the present study, the anti-cancer effects of ginseng fermented with Phellinus linteus (GFPL) extract were examined through in vitro and in vivo assays. GFPL was produced by co-cultivating ginseng and Phellinus linteus together. Ginsenoside Rg3, Rh1 and Rh2 are important mediators of anti-angiogenesis and their levels in GFPL were enriched 24, 19 and 16 times, respectively, more than that of ginseng itself through the fermentation. GFPL exhibited distinct anti-cancer effects, including growth inhibition of the human lung carcinoma cell line A549, and promotion of immune activation by stimulating nitric oxide (NO) production in Raw 264.7 cells. Further evidence supporting anti-cancer effects of GFPL was its significant prolongment of the survival of B16F10 cancer cell-implanted mice. These results suggest that the GFPL may be a candidate for cancer prevention and treatment through immune activation and anti-angiogenic effects by enriching Rg3, Rh1 and Rh2.

Cultural Characteristics and Log-Mediated Cultivation of the Medicinal Mushroom, Phellinus linteus

The optimal conditions for mycelial growth of Phellinus linteus ATCC 26710 were determined to be a log length of 20 cm, temperature of 30degrees C and pH of 6.0. Mycelial growth was excellent on the mushroom complete medium, and was optimal when sucrose, mannose and glucose were supplied as carbon sources. Potassium nitrate and sodium nitrate as nitrogen sources supported good mycelial growth. To evaluate P. linteus mycelial colonization on logs, sterilized short log inoculation, drilling inoculation and log-end sandwich inoculation techniques were used. Only sterilized short log inoculation produced good mycelial colonization. Initial mycelial growth and full mycelial colonization were best on 20 cm logs having 42% moisture content. The initial mycelial growth of P. linteus was accelerated over 12 hr of sterilization. Basidiocarp formation was optimal using a burying method of logs after 5~6 months, and fruiting body formation was superior in cultivation house conditions of 31~35degrees C and in excess of 96% relative humidity.

Submerged Culture of Phellinus linteus for Mass Production of Polysaccharides

In order to increase the mycelial production of Phellinus linteus, which exhibits potent anticancer activity, some ingredients of the medium used to culture P. linteus were investigated. The optimal medium composition for the production of Phellinus linteus was determined to be as follows: fructose, 40 g/l; yeast extract, 20 g/l; K2HPO4, 0.46 g/l; KH2PO4, 1.00 g/l; MgSO4.7H2O, 0.50 g/l; FeCl2.62O, 0.01 g/l; MnCl2.4H2O, 0.036 g/l; ZnCl2, 0.03 g/l; and CuSO4.7H2O, 0.005 g/l. The optimal culture conditions were determined to be as follows: temperature, 28degrees C; initial pH, 5.5; aeration, 0.6 vvm; and agitation, 100 rpm, respectively. Under optimal composition and conditions, the maximum mycelial biomass achieved in a 5 l jar fermentor was 29.9 g/l.

Anti-tumor and immunodulational effect of Phellinus linteus / 吉林大学学报(医学版)

Objective To study the anti-tumor activity of Phellinus linteus(PL) and its immunomodulational effects.Methods Solid tumor was induced by injecting HepA,MFC,S_(180) cells(3?10~6cells/animal) subcutaneously to the right anterior limb in mice.The tumor weight,thymus weight and spleen weight were measured after mice were executed.The effects of PL on lymphocyte proliferation,mitogen-induced proliferatoin of ICR mouse splenocytes were measured by MTT.Meanwhile,lymphocytes were prepared as described above.SRBC(10%,0.2 mL) was injected to abdominal cavity to elevate the level of antibody production by B cells.Spleen cells suspension(1?10~7?mL~(-1)) was used as the effect cells and L_(929) cells was used as the target cells.L_(929) cells engulfing neutral red determined the cytotoxic activity of NK.Simultaneously,phagocytosis of mice was detected using the method that macrophages engulfed neutral red.The effect of PL on the production of antibody was studied,the lymphocytes from mice were prepared as described above.Results The high-dose(2.0 g?kg~(-1)) and mid-dose(1.0 g?kg~(-1)) of PL could significantly inhibit the growth of HepA,MFC and S_(180)tumor,the inhibitory rates were 38.19% and 37.15%,32.46% and 30.91%,36.0% and 34.16%,respectively(P

The Culture Conditions for the Mycelial Growth of Phellinus spp

Phellinus genus belonged to Hymenochaetaceae of Basidiomycetes and has been well known as one of the most popular medicinal mushrooms due to high antitumor activity. This study was carried out to obtain the basic information for mycelial culture conditions of Phellinus linteus, P. baumii, and P. gilvus. According to colony diameter and mycelial density, the media for suitable mycelial growth of them were shown in MEA, glucose peptone, and MCM. The optimum temperature for mycelial growth was 30degrees C. Carbon and nitrogen sources were mannose and malt extract, respectively. The optimum C/N ratio was 10 : 1 to 5 : 1 with 2% glucose concentration, vitamin was thiamine-HCl, organic acid was succinic acid, and mineral salt was MgSO4.7H2O.

Antitumor and Antioxidant Activities of the Extracts from Fruiting Body of Phellinus linteus

Fruiting bodies of Phellinus linteus were extracted by hot water and alkali methods. Sugar contents of PL-H (hot water extract) and PL-A (alkali water extract) were 81.1%, 37.4% and protein contents were 6.2%, 21.8%, respectively. Amino acid pattern showed that two extracts contained large amount of aspartic acid and alanine. Two extracts showed characteristic IR absorption pattern for glycosidic bond at 890 cm(-1). PL-H was divided two fractions by gel filtration chromatography and the molecular weights of each fraction were estimated to be about 10 kD and 225 kD, respectively and also PL-A was estimated 10 kD. Two extracts showed strong antitumor, immunomodulating and antioxidant activities, and were compared with commercialized glycopeptide anticancer drugs.

Mycelial Extract of Phellinus linteus Induces Cell Death in A549 Lung Cancer Cells and Elevation of Nitric Oxide in Raw 264.7 Macrophage Cells

In the present study, in order to investigate the anti-proliferative phenomenon of PLME, the effects of mycelial extract of Phellinus linteus (PLME) on the growth of human lung carcinoma cell line A549 was examined. We studied on the effects of PLME on the release of nitric oxide (NO) in mouse macrophage Raw 264.7 cells. Treatment of PLME to A549 cells resulted in the growth inhibition, morphological change and induction of apoptotic cell death in a dose-dependent manner as measured by MTT assay. We found that PLME stimulated a dose-dependent increase in NO production. These findings suggest that PLME enhances the anti-tumoral activity of macrophage and may be a potential therapeutic agent for the control of human lung carcinoma cells.