Enrichment of flavonoid aglycones in licorice extract enhanced anti-inflammatory potential,but its hypnotic effect was not altered / 中国药理学与毒理学杂志

OBJECTIVE Licorice is used throughout the world as a traditional herbal remedy. Ac-cording to Chinese traditional medicine licorice alone can be used to treat inflammation.Although there have been some studies investigated the anti-inflammatory ingredients of licorice, but for the potency of flavonoid glycoside and their aglycones on inflammation are not evaluated.This study was designed to assess the contributions of licorice flavonoid glycosides and their aglycons to its anti-inflammatory and hypnotic effects. METHODS For the flavonoid aglycone's enrichment, the extract of licorice (EL) was fermented in submerged culture of the edible fungus Grifola frondosa HB0071 mycelia which can produce β-glucosidase and catalyze the flavonoid glycosides to aglycones.EL and fermented extract of licorice (FEL) were used in this study. The anti-inflammation test was carried out in arachidonic acid (AA)-induced ear edema model and the hypnotic test was performed by using electroencephalogram (EEG)analysis method in normal freely moving SD rats.The chemicals constituents were analyzed by HPLC.RESULTS During fermentation,the falvonoid glycosides of licorice were hydrolyzed by the time process.Along with fermentation time,the concentration of the major flavonoid glycosides,liquiritin and isoliquiritin were decreased obviously, and simultaneously their aglycons, liquiritigenin and isoliquiriti-genin were remarkably increased in FEL.Moreover,the content of another major constituent glycyrrhi-zic acid and glycyrrhetinic acid were not changed after the fermentation. In AA-induced mice ear ede-ma test,after topical application,FEL(effective dose range:5-20 μg·ear-1)showed more potent inhibito-ry activity than EL(effective dose range:25-100 μg·ear-1).On the other hand,oral administration of EL and FEL exhibited the same hypnotic potency and both enhanced the total sleep time including rapid eye movement (REM) sleep and non-REM sleep time. CONCLUSION These results suggested that the enrichment of flavonoid aglycons such as liquiritigenin and isoliquiritigenin enhanced the anti-inflam-matory potency of licorice extract,and this potentiation has nothing to do with glycyrrhizic acid or glycyr-rhetinic acid.In addition,enrichment of flavonoid aglycones did not alter the hypnotic effect of licorice.

Structure and immunomodulatory activity of extracellular polysaccharide from Grifola frondosa / 生物工程学报

We aimed at analyzing the structure of extracellular polysaccharide A from Grifola frondosa (EXGFP-A) and testing its immunomodulatory activity. Structural analysis shows that EXGFP-A was a contained α-D-glucoside bond and pyranose ring. GC analysis reveals that EXGFP-A was mainly composed of rhamnose, arabinose, xylose, mannose, glucose, galactose, by the molar ratio of 0.28:0.31:0.30:0.06:7.98:0.61. The results of MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicates when EXGFP-A was at a concentration of 80 μg/mL and treatment time of 48 h, RAW264.7 cells proliferation index reached a maximum of 137.5%. Meanwhile, the AO staining showed that EXGFP-A activated RAW264.7 cells and improved the level of intracellular nucleic acid metabolism. In addition, in a certain range of concentration, EXGFP-A was able to increase the release of NO in RAW264.7 cells, and upregulate the mRNA expression of immunological factor TNF-α, IL-1β, IL-6, IL-12, IFN-γ and iNOS of RAW264.7 cells. Our results confirm that EXGFP-A had immunomodulatory activity. Our findings provided scientific basis for the structural analysis and application of Grifola frondosa polysaccharide.

Study on suitable distribution areas of Grifola umbellate in Sichuan province based on remote sensing and GIS / 中国中药杂志

Grifola umbellate is the important medicinal materials in China which has a very high medicinal value. This study analyzedthe suitable distribution areasof G. umbellate and provided scientific basis for determining G. umbellate planting regions and planning production distribution reasonably. The suitable distribution areas of G. umbellate in Sichuan province was researched based on TM, ETM+, and DEM data,the key ecological factors that affect the growth of G. umbellate were extracted, including elevation, slope, aspect, average annual temperature,average annual precipitation,forest information,soil information, following remote sensing and GIS techniques, combining field researchdata. The results showed that the G. umbellate resources in Sichuan province were mainly distributed in Pingwu, Beichuan, Licountry, Yanyuan, Xichang, Dechang, Yanbian, Miyi, Huidong, Panzhihua and so on, the suitability distribution areas is 276.214 4 km² approximately and accounting for more than 0.143 3% of the total area.According to the related document information and the field investigation, showed that the suitability distribution based on RS and GIS were corresponded with the actual distribution areas of G. umbellate.

Anti-hepatocellular carcinoma effects of Grifola frondosa extract and its underlying mechanisms / 中国药学杂志

OBJECTIVE: To investigate the anti-hepatocellular carcinoma effect and underlying mechanisms. METHODS: In PLC/PRF/5 and HepG2, after treatment with Grifola frondosa extract, MTT method, chemical method, JC-1 staining and Western Blot were applied to determine cell viability, caspase 3 activity, mitochondrial membrane potential, the expression of Bcl-2 and Bax, and the phosphorylation of Akt/GSK3β. The anti-tumor activity of Grifola frondosa extract was further confirmed in PLC/PRL/5-xengrafted mice model. RESULTS: Grifola frondosa extract significantly reduced cell viability, mitochondrial membrane potential, the expression of Bcl-2 and the phosphorylation of Akt/GSK3β, and enhanced LDH release, caspase 3 activity and the expression of Bax in both PLC/PRF/5 and HepG2 cells. 12-day Grifola frondosa extract treatment significantly inhibited the PLC/PRF/5-xenografted tumor growth without influence the body weight of mouse. CONCLUSION: All these data indicate that Grifola frondosa extract-mediated anti-hepatocellular carcinoma effects are related to its modulation of the activations of Akt/GSK3β and mitochondrial pathway.

Immunomodulatory activity of macromolecular polysaccharide isolated from Grifola frondosa / 中国天然药物

The present study was designed to evaluate the immune-modulating effects of the polysaccharide from Grifola frondosa (GFP) by using mouse peritoneal macrophage and cytoxan (CTX) induced immunosuppression models. Our results from the phagocytotic and mononuclear phagocytic system function assays showed that GFP-A (one component from GFP) stimulated the phagocytosis of the phagocytes. The splenocyte proliferation assay showed that GFP-A acted the effect combing ConA or LPS in splenocyte proliferation. The results showed that GFP-A increased indices of thymus and spleen, the levels of LDH and ACP in the spleen, the mRNA levels of IL-1β, IL-2, IL-6 and IFN-γ in splenocyte. And GFP-A also significantly increased the expression of CD4(+) and CD8(+) splenic T lymphocytes, which were suppressed by the CTX in peripheral blood. In conclusion, our results indicate that the GFP-A is involved in immunomodulatory effects leading to its modulatory effects on immunosuppression.

Grifola frondosa Extract Induced Acute Hepatic Injury.

Aim: To describe a case of acute hepatic injury related to the use of Grifola frondosa in a patient with colon cancer. Case Presentation: Patient is a 67 year old female with stage IV poorly differentiated adenocarcinoma of the colon, who presented with epigastric pain one month after resection of her primary tumor. A staging PET scan revealed metastasis to regional lymph nodes without solid organ involvement. Her home medications include longstanding amlodipine and losartan, and a recently started Grifola frondosa derivative. Her laboratory data was significant only for acute transaminitis (AST:967 U/L, ALT:768 U/L) without hyperbilirubinemia. Alcohol, acetaminophen, and a viral panel (EBV, CMV, hepatitis A/B/C) were all negative. A CT scan revealed heterogenous liver parenchyma without focal lesions. A subsequent liver biopsy demonstrated active portal inflammation with eosinophilic infiltration. Discussion: The etiologies of significant acute transaminitis include viral hepatitis, ischemic liver injury, acetaminophen toxicity and drug-induced liver injury (DILI). Viral and ischemic hepatitis and acetaminophen toxicity were excluded based on laboratory analysis and imaging studies. Liver biopsy findings demonstrating the characteristic eosinophilic infiltration of a drug reaction favored DILI as the etiology of transaminitis in this case. With a RUCAM score of 7 calculated based on history, clinical course, and objective data, DILI was concluded to be probably attributed to the patient’s recent use of the Grifola frondosa extract. Conclusion: A diagnosis of drug induced liver injury probably secondary to the use of Grifola frondosa extract was made after excluding all other causes of significant acute transaminitis.

Effects of Grifola frondosa Polysaccharide on T cell Subsets and Th1/Th2 Subsets of Spleen Deficiency Mice / 中国中医药信息杂志

Objective To observe the effects of Grifola frondosa polysaccharide on T cell subsets and Th1/Th2 subsets of spleen deficiency mice, and investigate its immunoregulation mechanism. Methods Forty-eight KM mice were divided into 6 groups:normal group, spleen deficiency group, positive group (lentinan), three dosage groups of Grifola frondosa polysaccharide, 8 mice in each group. The mice were injected corresponding drug intraperitoneally for 10 days. Then mice in each group were detected CD3+, CD4+ and CD8+T cell by flow cytometry. IFN-γ and IL-4 levels were detected by ELISA. Results The high dose of Grifola frondosa polysaccharide could significantly increase the CD4+T lymphocytes percentage in peripheral blood (P<0.05). Medium and high dose of Grifola frondosa polysaccharide could significantly increase CD4+/CD8+T cell ratio (P<0.01) and CD3+T lymphocyte percentage (P <0.01) of spleen deficiency mice. Medium and high dose of grifola frondosa polysaccharide could significantly increase IFN-γ level in serum of spleen deficiency mice (P<0.01), and high dose of Grifola frondosa polysaccharide could decrease IL-4 level in serum of spleen deficiency mice (P<0.01). Conclusion Grifola frondosa polysaccharide can improve the decreased CD4+/CD8+T cell ratio which caused by spleen deficiency, and promote the transformation of Th1 to Th2, thus treating spleen deficiency syndrome.

Lignin degradation, ligninolytic enzymes activities and exopolysaccharide production by Grifola frondosa strains cultivated on oak sawdust

Fourteen strains of Grifola frondosa (Dicks.) S. F. Gray, originating from different regions (Asia, Europe and North America) were tested for lignin degradation, ligninolytic enzyme activities, protein accumulation and exopolysaccharide production during 55 days of cultivation on oak sawdust. Lignin degradation varied from 2.6 to7.1 percent of dry weight of the oak sawdust substrate among tested strains. The loss of dry matter in all screened fungi varied between 11.7 and 33.0 percent, and the amount of crude protein in the dry substrate varied between 0.94 to 2.55 percent. The strain, MBFBL 596, had the highest laccase activity (703.3 U/l), and the maximum peroxidase activity of 22.6 U/l was shown by the strain MBFBL 684. Several tested strains (MBFBL 21, 638 and 662) appeared to be good producers of exopolysaccharides (3.5, 3.5 and 3.2 mg/ml respectively).

Effect of Hericium erinaceum (Yamabusitake) and Grifola frondosa (Maitake) on the Suppression of EL4-Tumor Cell Growth / 日本補完代替医療学会誌

The objectives of this study were to investigate the effects of <i>Hericium erinaceum</i> (Yamabusitake) and <i>Grifola frondosa </i>(Maiteke) on the proliferation for EL4-tumor and immunoregulatory function by flow cytometory.<br> It was found that Yamabushitake and Maitake tend to inhibit the proliferation of EL4-tumor individually. In the flow cytometory analysis, Maitake-treatment showed the preserve effect against the depression effect by bearing EL4-tumor on cytotoxic T cell and NK-cell from spleen cell. This effect was shown more clear in the group of mixture Yamabusitake and Maitake.<br>

Grifola frondosa water extract alleviates intestinal inflammation by suppressing TNF-alpha production and its signaling

TNF-alpha is a major cytokine involved in inflammatory bowel disease (IBD). In this study, water extract of Grifola frondosa (GFW) was evaluated for its protective effects against colon inflammation through the modulation of TNF-alpha action. In coculture of HT-29 human colon cancer cells with U937 human monocytic cells, TNF-alpha-induced monocyte adhesion to HT-29 cells was significantly suppressed by GFW (10, 50, 100 microg/ml). The reduced adhesion by GFW correlated with the suppressed expression of MCP-1 and IL-8, the major IBD-associated chemokines. In addition, treatment with GFW significantly suppressed TNF-alpha-induced reactive oxygen species production and NF-kappaB transcriptional activity in HT-29 cells. In differentiated U937 monocytic cells, LPS-induced TNF-alpha production, which is known to be mediated through NF-kappaB activation, was significantly suppressed by GFW. In an in vivo rat model of IBD, oral administration of GFW for 5 days (1 g/kg per day) significantly inhibited the trinitrobenzene sulfonic acid (TNBS)-induced weight loss, colon ulceration, myeloperoxidase activity, and TNF-alpha expression in the colon tissue. Moreover, the effect of GFW was similar to that of intra-peritoneal injection of 5-aminosalicylic acid (5-ASA), an active metabolite of sulfasalazine, commonly used drug for the treatment of IBD. The results suggest that GFW ameliorates colon inflammation by suppressing production of TNF-alpha as well as its signaling through NF-kappaB leading to the expression of inflammatory chemokines, MCP-1 and IL-8. Taken together, the results strongly suggest GFW is a valuable medicinal food for IBD treatment, and thus may be used as an alternative medicine for IBD.

Determinación de la actividad inmunomoduladora de dos extractos del hongo Grifola frondosa Dicks. Fr Gray, mediante ensayos in vitro / Termination of the immunomodulatory activity of two extracts of the fungus Grifola frondosa Dicks. Fr Gray, by in vitro tests

Fue evaluada la actividad de dos extractos del hongo comestible Grifola frondosa (Dicks; Fr) S.F. Gray, conocido comúnmente como maitake sobre el sistema inmune humano. Se estudió la actividad inmunomoduladora de los extractos acuoso y etanólico obtenidos del cuerpo fructífero del hongo sobre los linfocitos por medio de un ensayo linfoproliferativo y fueron realizados ensayos hematolíticos in vitro para probar su influencia sobre las vías clásicas y alterna del sistema de complemento. El ensayo de linfoproliferación fue realizado colocando linfocitos purificados expuestos a los extractos y cultivados para una evaluación final colorimétrica con XTT, una sal de tetrazolio que es reducida a un compuesto coloreado y soluble por la actividad enzimática mitocondrial presente en células vivas...

Morphological Characteristics of Hyphal Interaction between Grifola umbellata and its Companion Fungus

Morphological characteristics of hyphal interaction between Grifola umbellata (Pers. Ex Fr.) Pilat and its companion fungus which related to sclerotia formation from hyphae were investigated by external observations, light microscopy and transmission electron microscopy (TEM). External observations showed that a dense antagonism line was formed by both G. umbellata and companion fungus after their hyphae contacted each other in dual culture. Many hyphal strands emerged on the colony of G. umbellata and differentiated to sclerotia from where hyphal strands crossed. Light microscope observations revealed the process of antagonism line formation. Mature antagonism with structural differentiation, was composed of three main layers: the rind, the rind underlayer and the hypha layer. TEM observations showed that after colonies hyphal contact, a series of reactions always occurred in both G. umbellata and companion fungus. Cells in the center of antagonism line were dead. Cells of G. umbellata adjacent to the antagonism line were usually large and hollow, with unilateral thickened wall, whereas those of companion fungus were empty, with thin or thick wall. Both hyphal interaction at the antagonism line may be one of the main reasons for sclerotia of G. umbellata differentiation from hypha.

Morphological Characteristics of Pseudosclerotia of Grifola umbellata in In Vitro

The present study was carried out to investigate morphological characteristics of pseudosclerotia of Grifola umbellata formed by artificial cultures. Isolate G. umbellata DUM GUS-01 was obtained from sclerotium cultivated in field. The fungal isolate was cultured on PDYM broth, PDYMA(potato dextrose yeast malt agar) and oak sawdust media at 20degrees C under the dark condition. G. umbellata DUM GUS-01 showed a volumetric increment of fungal lumps rather than mycelial growth. Particularly, G. umbellata DUM GUS-01 produced a large amount of melanin pigments in all culture treatments. The color of the fungal mass has been changed into grey gradually, and then formed melanized rind-like structure on its superficial part. The fungal structures which were covered with melanized rind-like layer were named as pseudosclerotia of G. umbellata. The pseudosclerotia of G. umbellata DUM GUS-01 formed a new white mycelial mass, which was swollen out of the melanized rind structure for its volumetric increment. When the pseudosclerotia were sectioned, their structure was discriminated from two structures such as a melanized rind-like structure layer formed by aggregation of aged mycelia and a white mycelial mass with high density. As results of scanning electron microscopic examination, the pseudosclerotia of G. umbellata DUM GUS-01 which were formed in in vitro conditions were similar to the sclerotia of G. umbellata cultivated in natural conditions except for the crystals formed in medula layer of natural sclerotia. Although size, solidity of rind structure and mycelial compactness of pseudosclerotia were more poor than those of natural sclerotia, the morphological structure and growth pattern of pseudosclerotia were very similar to those of natural sclerotia. Therefore, it is probable to induce pseudosclerotia to sclerotia of G. umbellata in in vitro conditions. Consequently, it seems that the induced pseudosclerotia can be used as inoculum sources to substitute natural sclerotia in field cultivation.

Production of Polysaccharide by the Edible Mushroom, Grifola frondosa

The production of polysaccharide according to various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) in the edible mushroom Grifola frondosa was studied. The cap of the mature mushroom showed the highest amount of polysacchride. Mycelial growth and polysaccharide synthesis were optimal at pH 5 and 20degrees C. Polysaccharide synthesis was maximal after 12 days of cultivation, whereas maximum mycelial growth was shown after 18 days. Mannose, cellobiose and starch increased the level of polysaccharide as well as growth in submerged culture. Glucose and sucrose appeared to be good substrates for fruiting of Grifola frondosa.

A New Method for Cultivation of Sclerotium of Grifola umbellata

Sclerotia of Grifola umbellata were cultivated by two methods such as burying and root inoculation methods. The sclerotia of G. umbellata produced by the burying method were 6.0~6.8 x 3.4~4.6 x 1.8~1.9 cm (Width x Length x Thickness) in size and 17.3~19.6 g in weight, respectively. Their increase rate was 1.10~1.12 times. On the other hand, the sclerotia cultivated by the root inoculation method were 18.3~31.5 x 12.5~26.4 x 3.1~3.7 cm (Wx L x T) in size and 219.1~576.6 g in weight, respectively. Their growth increment was 11.18~39.77 times. The rhizomorphs of Armillaria mellea were developed with a high density under fallen leaves layer covering cultivation site, and distributed mainly between soil surface and soil depth of about 10 cm as well as colonized prominently on the inoculated wood logs. Fungal interaction between G. umbellata and A. mellea were observed mainly in the stage of white sclerotium of G. umbellata. The sclerotia of G. umbellata which were developed newly and harvested in the root inoculation method were twined with root hairs of host tree and rhizomorphs of A. mellea. The sclerotia of G. umbellata decomposing root hairs of host tree were confirmed through SEM examination. Physiochemical characteristics of soil in all cultivation sites had no significant differences. Soil pH were in the range of pH 3.98~4.40. Organic matters were the range of 17.97~23.86% and moisture contents of soil were 12.00~18.20%. Soil temperatures showed 12.9~13.8degrees C in November and 22.0~23.9degrees C in August, respectively. In conclusion, the root inoculation method seems to be a practical method for cultivating sclerotia of G. umbellata due to its many advantages such as simplicity of inoculation process, shortening of cultivation periods and facility of harvest.

Sclerotial Development of Grifola umbellata

Sclerotial development of Grifola umbellata (Pers. : Fr.) Donk was investigated through microscopic examinations. The sclerotium of G. umbellata was bumpy and rugged, multi-branched, and dark-brown to black in color. The sclerotial development of G. umbellata was categorized into three stages such as sclerotial initial, development and maturation. Sclerotium development was initiated as the white fungal mass. The superficial part of white sclerotium changed into gray, light brown and then black as its development proceeded further. As a distinctive characteristic of this fungus, a large number of crystals were observed in the medulla layer of sclerotium during its maturation. For development of new sclerotium, G. umbellata formed a white sclerotial primordium on the matured sclerotium. Development of sclerotium in G. umbellata was intimately associated with rhizomorphs of Armillariella mellea and the developing sclerotia were often penetrated by rhizomorphs of A. mellea into medulla layer.

Effects of Grifola frondosa extracts on mouse immunity / 中国免疫学杂志

Objective :To investigate the effects of ethanol precipitate(ET-Pre) and RNA of Grifola frondosa on non-specific immunity in mouse.Methods:The common biological methods were used to examine the levels of cytokines and immunocyte activity. Results: The killing activity of the NK cells, the phagocytosis function of macrophages and the levels of TNF?/IL-1 in the animals treated with ET-Pre and RNA respectively were significantly higher than those in the control.The RNA was stronger than ET-Pre in increasing killing activity of NK cells and phagocytosis function of macrophages. Conclusion:Both ET-Pre and RNA extracts of Grifola frondosa may promote immunoactivity nonspecifi-cally and inhibit tumor cells indirectly.

THE PHYLOGENETIC RELATIONSHIPS OF GRIFOLA UMBELLATA AND ITS COMPANION FUNGUS: EVIDENCE FROM ITS SEQUENCE ANALYSIS / 微生物学通报

The sequences of 5.8S rDNA and the flanking internal transcribed spacers (ITS1 and ITS2) were sequenced from hypha, fruit body and sclerotia of Grifola umbellata and its companion fungus. Their ITS sequences similarity was 99.36%. The results suggested that G. umbellata was closely related to its companion fungus.

STUDIES ON PRODUCTION OF EXPOLYSACCHARIDES OF GRIFOLA FRONDOSA IN SACCHARKID SOLUTION / 微生物学通报

Effects of glucose concentration and pH\|control upon production of exopolysaccharides of Grifola frondosa was studied.The results showed that glucose at concentration of 5% was favorable to exopolysaccharides production,and the pellets benefited from the controlled start pH as well as the controlled final pH of 3 5～4 0 to produce exopolysaccharides.Attempt on the exopolysaccharides production with recycled pellets in the saccharoid solution was also made in this work.

PRODUCTION OF ACTIVE OXYGEN SPECIES AND CHANGES OF ENZYMES IN GRIFOLA UMBELLATA INDUCED BYARMILLARIA MELLEA ELICITOR / 微生物学通报

The changes of active oxygen species and some enzymes in Grifola umbellata induced by Armillaria mellea elicitor were studied.The results showed that active oxygen species appeared in both mycelia and sclerotia of G.umbellata after treated with A.mellea.There were two phases of active oxygen production upon addition of A.mellea elicitor.Phase I occured at 10 minute after addition of A.mellea elicitor.Phase Ⅱ occurred about 90 minute.The changes of some enzyme activity were also studied in this paper.Compared with control,the A.mellea elicitor could reduce the activity of superoxide dismutase and peroxidase.The catalase activity changed only little.The phenylanine ammonia lyase activity declined in the early stages and then increased in the late stages.