灵芝及灵芝孢子提取物的抗肿瘤及对DNA拓扑异构酶作用的研究

目的:观察灵芝子实体水提物及醇提物混合品(灵芝提取物)、灵芝孢子粉提取物(油)的体内外抗肿瘤作用,并测定它们对DNA拓扑异构酶Ⅰ、Ⅱ的作用。方法:台盼蓝计数法、MTT法观察体外对肿瘤细胞的抑制作用;应用小鼠移植瘤模型研究体内抗肿瘤作用,同时测定荷瘤小鼠的胸腺指数及脾指数;DNA超螺旋解旋法测定拓扑异构酶Ⅰ、Ⅱ活性。结果:灵芝提取物对K562、HL60、SGC-7901细胞作用24h的IC50值分别为0.44mg/ml、0.39mg/ml、0.90mg/ml,灵芝孢子油对K562、HL60、SGC-7901细胞作用24h的IC50值分别为1.13mg/ml、2.27mg/ml、6.29mg/ml;体内实验中,对S180、H22移植瘤株的抑制率灵芝提取物4g/kg分别为39.1%、44.6%,灵芝孢子油1.2g/kg分别为30.9%、44.9%;灵芝提取物1g/kg和2g/kg对S180的抑制率分别为15.6%和19.0%,对H22的抑制率分别为28.8%和38.5%;灵芝提取物对荷瘤小鼠的免疫功能有一定的上调影响,灵芝孢子油则无明显作用;两者对拓扑异构酶Ⅰ、Ⅱ的活性均有抑制作用。结论:灵芝提取物、灵芝孢子油均具有体内外抗肿瘤作用,二者的抗癌机制与抑制拓扑异构酶Ⅰ、Ⅱ的活性有关。

泰山赤灵芝中灵芝酸和灵芝酸A的测定

[目的]研究泰山赤灵芝中灵芝酸的检测方法并应用该方法测定灵芝酸A的含量。[方法]利用反相高效液相色谱和电喷雾质谱联用技术,通过研究已知的灵芝酸化合物的相对分子质量及结构特征,鉴定泰山赤灵芝中生物碱提取物中的灵芝酸类化合物,并通过灵芝酸A标准品的标准曲线测定其中灵芝酸A的含量。[结果]在泰山赤灵芝样本中检测到灵芝酸A、A1、B、C1、R、beta、delta、epsilon、gamma、zeta,灵芝酸A的含量为115.4μg/g。[结论]泰山赤灵芝含有较高的灵芝酸,液质联用仪对泰山赤灵芝天然产物结构的分析以及含量测定准确可靠。

灵芝

灵芝(JP19,图1),古称瑞草,又称“三秀”,属担子菌亚门,多孔菌科植物,菌盖呈肾形,上赤褐色,有云状环纹和漆样光泽,多生于山地枯根上。《神农本草经》将它列为上药,主治胸中结,益心气,增智慧。《本草纲目》和《花镜》等都有详尽记载。它具有调节免疫等药用价值,为“中华人民共和国法定中药材”。

Studies on Active Polysaccharides from Ganoderma lucidum vith Immune Activity

从云头状灵芝[Ganoderma Lucidum(Leyss.ex Fr.)Karst.]中分离得到三种灵芝多糖BN3A，BN3B及BN3C，它们均表现明显免疫调节作用。从灵芝多糖BN3B及BN3C中各分离得到四个多糖均一体，对其中主要成分BN3C1，BN3C3，BN3B1及BN3B3进行了物理及化学研究，它们的平均分子量依次为1.6×10^4，2.5×10^4，3.5×10^4及4.0×10^4。经完全酸水解、红外光谱测定、过碘酸氧化、甲酸生成、Smith降解等证明，BN3B1及BN3C1均为β－（1→6)(1→3）甙键相连的葡聚糖。BN3B3为阿拉伯半乳聚糖。BN3C3为由葡萄糖和阿拉伯糖组成的肽多糖。它们均为β－（1→6）（1→3）甙键相连。

Antitumor Activity of the Ganoderma Lucidum Spore Alcohol Extract in Vitro

Several cancer cell lines(epithelioma cells or leukemia cells)from human being or mouse were first used to study the antitumor activity of the Ganoderma lucidum spore alcohol extract(GLSAE)in vitro by the MTT test A comparision was made between the sporodermbroken(SB)and sporoderm nonbroken(SN)GLSAE It was showed that both GLSAE SB and GLSAE SN could inhibit the proliferation of these cancer cells,but the activity of GLSAE SB was much higher than that of GLSAE SN These results suggested that Ganoderma lucidum spore could probably be used for tumor treatment

Preliminary Study on the Liquid Fermentation and Polysaccharide Production of Ganoderma

Strain of Ganoderma japonicum with higher mycelial growth rate and higher levels of polysaccharide production was selected from seven tested strains of Ganoderma. The effects of nitrogen source, carbon source and metal ions on the polysaccharide production of selected Ganoderma japonicum were studied. The results showed that the optimum nitrogen source and carbon source were 2% and 0.2% ,respectively. And the existence of 0.2% Fe^2＋ could improve the polysaccharide yield significantly. The result of fermentor enlargement test indicated that extracellular crude polysaccharide content per 100 mL fermentation liquor and mycelium content yielded highest amount of 181.7 mg and 151.0 mg,respectively,with relatively mild change of pH value. It was concluded that fermentation in fermentor was more suitable for the secretion of C, anoderma polysaccharide.

Effects of Lingzhi (Ganoderma lucidum) on Hemorrheology Parameters and Symptoms of Hypertensive Patients with Hyperlipidemia and Sequelae of Cerebral Thrombosis

The effects of Lingzhi(Ganoderma lucidum)on hemorrheology parameters and symptoms of hypertensive patients with hyperlipidemia,sequelae of cerebral thrombosis and coronary heart disease are studied.The Lingzhi extract was given to 33 patients(15 male,18 female,65±8 years old)in dosage of 110mg qid po for 2 weeks.The blood viscosity was im- proved after treatment.The results showed that whole blood viscosity(at high shear rate and low shear rate)and plasma viscosity were significantly decreased(P<0.01).Lingzhi showed the effect of reducing blood pressure simultaneously.Several symptoms were also improved. However,there were no significant changes in haematocrit and erythrocyte sedimentation rate (P>0.05).In a few cases there occurred adverse reactions of palpitation and restlessness.

Triterpenoids from the Fruiting Body of Ganoderma lucidum

A new triterpenoid, named ganolactone, was isolated from the CH 2Cl 2 soluble fraction of the fruiting body of Ganoderma lucidum. Its structure was deduced as 7β hydroxy 3,11,15 trioxo lanosta 8 en 24→20s lactone (1) on the basis of spectral analysis (UV, IR, MS. 1H NMR, 13 C NMR and 2D NMR) and was confirmed by X ray diffraction. In addition, three known compounds, ganoderiol A (2), ganoderiol B (3) and ganodermatriol (4), were obtained.

Determination of ganoderic acids in rat plasma after oral administration of total triterpenoids from Ganoderma lucidum

To investigate the absorption of total triterpenoids from Ganoderma lucidum in rats. HPLC-DAD and LC-MS methods were used to identify ganoderic acids in rat plasma after oral administration of total triterpenoids from G. lucidum by comparing their HPLC retention behaviors, UV absorption spectra, and mass spectra with authentic samples. Five ganoderic acids, ganoderic acid C2, C6, G, B and A were simultaneously detected in rat plasma. Ganoderic acids can be directly absorbed into circulation after oral administration of total triterpenoids from G. lucidum in rats.

灵芝补气安神功效对动物焦虑行为影响的实验研究

目的:观察灵芝水煎液及其粗多糖对动物焦虑行为的抗焦虑作用及机制。方法:建立装置焦虑模型小鼠和心气虚焦虑装置大鼠模型,除正常对照组、焦虑模型对照组,大小鼠分别灌胃地西泮2.5 mg/kg、灵芝水煎液2、4、6 g/kg、灵芝粗多糖0.04、0.08、0.12 g/kg,末次给药后1 h,观察大、小鼠于高架十字迷宫(EPM)实验中开臂进入次数百分比(OE%)、开臂滞留时间百分比(OT%)及明暗箱(LDT)实验中穿梭次数;行为学实验结束后取大鼠血及大、小鼠脑组织,ELISA法测定小鼠脑组织中小鼠γ-氨基丁酸(GABA)、谷氨酸(GLU)、5-羟色胺(5-HT)的含量;试剂盒测定大鼠脑组织中GABA、GLU含量,γ-氨基丁酸转氨酶(GABA-T)、谷氨酸脱羧酶(GAD)、谷氨酰胺合成酶(GS)活力,血清超氧化物歧化酶(SOD)活力、丙二醛(MDA),血浆环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)、心钠素(ANP)含量;免疫组化法观察海马γ-氨基丁酸受体α1(GABAARα1)阳性表达。结果:随心气虚造模时间延长,各组大鼠体质量明显降低,19 d起,各给药组大鼠体质量降幅逐渐减小,22 d起各给药组大鼠体质量均大于心气虚焦虑模型组;灵芝水煎液及粗多糖均可显著升高大、小鼠的OE%、OT%、穿梭次数和脑组织中GABA含量,降低GLU含量及GLU/GABA的比值;降低小鼠脑中5-HT含量;降低大鼠脑中GABA-T活性;升高大鼠脑中GAD、GS活力,血清SOD活力、血浆cAMP含量,降低血清MDA、血浆cGMP、ANP含量,增强海马GABAARα1受体阳性表达;等质量灵芝饮片的水提取物、多糖提取物,表现出相似的抗焦虑效果。结论:灵芝水煎液及其粗多糖对动物焦虑行为有明显抗焦虑作用,抗焦虑主要活性部位可能为灵芝多糖。作用机制与通过调节GLU/GABA-Gln代谢环路中相关酶的活力,升高GABA含量和上调下游GABA/GABAARα1信号通路的表达,纠正兴奋-抑制性神经递质的失衡有关。

In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachlorideinduced liver injury

AIM： To investigate the possible mechanism of the protective effects of a bioactive fraction, Ganoderma lucidum proteoglycan （GLPG）isolated from Ganoderma luddum mycelia, against carbon tetrachloride-induced liver injury. METHODS： A liver injury model was induced by carbon tetrachloride. Cytotoxicity was measured by MTY assay. The activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined with an automatic multifunction-biochemical analyzer and the levels of superoxide dismutase （SOD） and TNF-α were determined following the instructions of SOD kit and TNF radioimmunoassay kit. Uver sections were stained with hematoxylin and eosin （H＆E） for histological evaluation and examined under light microscope. RESULTS： We found that GLPG can alleviate the L-02 liver cells injury induced by carbon tetrachloride （CCh） through the measurements of ALT and AST activities and the administration of GLPG to L-02 cells did not display any toxicity. Furthermore, histological analysis of mice liver injury induced by CCh with or without GLPG pretreatment indicated that GLPG can significantly suppress the toxicity induced by CCh in mice liver. We also found that GLPG reduced TNF-α level induced by CCh in the plasma of mice, whereas increased SOD activity in the rat serum. CONCLUSION： GLPG has hepatic protective activity against CCl4 induced injury both in vitro and in vivo. The possible antihepatotoxic mechanisms may be related to the suppression of TNF-α level and the free radical scavenging activity.

Ameliorative effect of Ganoderma lucidum on carbon tetrachloride-induced liver fibrosis in rats

AIM： To investigate the effects of Reishi mushroom, Ganoderma lucidum extract （GLE）, on liver fibrosis induced by carbon tetrachloride （CCl4） in rats. METHODS： Rat hepatic fibrosis was induced by CCl4. Forty Wistar rats were divided randomly into 4 groups： control, CCl4, and two GLE groups. Except for rats in control group, all rats were administered orally with CCl4 （20%, 0.2 mL/100 g body weight） twice a week for 8 weeks. Rats in GLE groups were treated daily with GLE （1 600 or 600 mg/kg） via gastrogavage throughout the whole experimental period. Liver function parameters, such as ALT, AST, albumin, and albumin/globulin （A/G） ratio, spleen weight and hepatic amounts of protein, malondiladehyde （MDA） and hydroxyproline （HP） were determined. Histochemical staining of Sirius red was performed. Expression of transforming growth factor β1 （TGF-β1）, methionine adenosyltransferase （MAT1） 1A and MAT2A mRNA were detected by using RT-PCR. RESULTS： CCl4 caused liver fibrosis, featuring increase in plasma transaminases, hepatic MDA and HP contents, and spleen weight; and decrease in plasma albumin, A/G ratio and hepatic protein level. Compared with CCl4 group, GLE （600, 1 600 mg/kg） treatment significantly increased plasma albumin level and A/G ratio （P〈0.05） and reduced the hepatic HP content （P〈0.01）. GLE （1 600 mg/kg） treatment markedly decreased the activities of transaminases （P〈 0.05）, spleen weight （P〈 0.05） and hepatic MDA content （P〈0.05）; but increased hepatic protein level （P〈0.05）. Liver histology in the GLE （1 600 mg/kg）-treated rats was also improved （P〈0.01）. RT-PCR analysis showed that GLE treatment decreased the expression of TGF-β1 （P〈 0.05-0.001） and changed the expression of MAT1A （P〈0.05-0.01） and MAT2A （P〈 0.05-0.001）. CONCLUSION： Oral administration of GLE significantly reduces CCl4-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular necrosis by its free-radical scavenging ability.

Hydrodynamic Shear Stress Affects Cell Growth and Metabolite Production by Medicinal Mushroom Ganoderma lucidum

By investigating the shear effect on submerged cultivation of a traditional Chinese medicinal herb Ganoderma lucidum, a relatively high cell concentration of 13.8 g·L-1 by dry mass was obtained in bioreactor at an impeller tip speed (ITS) of 0.51m·s-1. At an ITS of 0.51,1.02 and 1.53m·s-1, a maximal production titer of intracellular polysaccharide was 2.64, 2.20 and 2.28g·L-1 and that of ganoderic acid was 306, 299 and 273g·L-1, respectively. Under these ITSs, the maximal mean projected area of dispersed hyphae was 3.70, 2.54 and 2.13×104μm2, and that of pellets was 0.91, 0.67 and 0.55mm2, respectively. The information obtained is useful for efficient submerged cultivation of mushrooms on a large scale.

Fermentation Production of Ganoderma lucidum by Bacillus subtilis Ameliorated Ceftriaxone-induced Intestinal Dysbiosis and Improved Intestinal Mucosal Barrier Function in Mice

Objective This study aimed to develop a type of Ganoderma lucidum(G.lucidum)-probiotic fermentation broth that can effectively improve the intestinal mucosal barrier function of mice with ceftriaxone-induced intestinal dysbiosis.Methods By means of absorbance of optical density(OD)value and phenol-concentrated sulfuric acid measurement of polysaccharide content,the probiotic species can grow on the medium of G.lucidum were screened out,and the concentration of the medium of G.lucidum was determined,and the fermentation broth was prepared for subsequent experiments.Thirty-two SPF grade male BALB/c mice were randomly divided into four groups(eight mice in each group),namely control group(CON),intestinal mucosal barrier damage model group(CS),fermentation broth intervention group(FT)and G.lucidum medium intervention group(GL),respectively.The intestinal dysregulation model was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(twice a day for seven consecutive days).From day 8,the FT group and GL group were gavage with 0.2 mL fermentation broth and G.lucidum medium,respectively.On day 15,all mice were sacrificed.To draw the weight curve and measure the cecal index;pathological examination of colon tissues with HE staining;serum levels of LPS,IL-10,TNF and IL-6 were detected by ELISA.Flow cytometry was used to analyze the changes of CD3+T cells,CD4+T cells,CD8+T cells and macrophages in spleen.16S rRNA sequencing was performed to detect the intestinal microbiota structure of mice.Results Bacillus subtilis can decompose and utilize G.lucidum fruiting body medium,and the suitable concentration of G.lucidum fruiting body medium is 33.2 mg/mL.The effect of Bacillus subtilis-G.lucidum fermentation broth on the damage of intestinal mucosal barrier caused by ceftriaxone sodium was reduced,the body weight of mice recovered and colon swelling improved,colon histopathological injury was alleviated,inflammatory cell infiltration was alleviated,serum IL-10 increased significantly,LPS、TNF-αand IL-6 decreased significantly compared with model group,and the proportion of T cells and intestinal dysbiosis was improved.Conclusions The experimental results suggest that Bacillus subtilis-G.lucidum fermentation broth can effectively improve the intestinal barrier function damage,immune dysfunction and intestinal dysbiosis caused by antibiotic overdose,and has a certain regulatory effect on intestinal mucosal barrier function.

Optimization of enzyme assisted extraction of polysaccharides from Ganoderma lucidum

In the present work,an enzyme assisted extraction method is used to isolate crude polysaccharides from Ganoderma lucidum. The isolating effect was optimized with orthographic graph statistic method with three levels and four independent variables. Complex enzyme,extraction temperature,extraction time and extraction pH were combined to obtain the best possible combination to get maximum amount of extract and crude polysaccharides yield. The optimum extraction conditions were:complex enzyme amount of 3 %(w/v),extraction temperature at 45 ℃,extraction time of 3 h and extraction pH at 7. Under these conditions,the experimental amount of extract is 8.9 % and the yield of crude polysaccharides is 1.1 %,which are in close agreement with the value predicted by the model.

Study on Key Techniques for High-quality and High-yield Cultivation of Ganoderma lucidum Spore Powder

Comparative test of 4 Ganoderma lucidum varieties from different sources showed that the mycelium of Chizhi 1 grew fast with thick and dense hyphae,round and solid cap,and high spore powder yield.Chizhi 1 was proved to be an excellent variety because of its strong resistance and high spore powder yield.Different cultivation materials were chosen and combined to form 3 cultivation formulations.The results showed that Formulation(3),in which basswood was soaked in nutrient solution for 24 h,presented fast mycelial growth and high spore powder output,and therefore was proved to be a high-yield formulation of Ganoderma lucidum spore powder.

Utilization of Fungi for the Biological Control of Insect Pests and Ganoderma Disease in the Indonesian Oil Palm Industry

Biological control is usually the first choice of control and prevention method for integrated pest management （IPM） strategies and has now been widely implemented by Indonesian oil palm plantations. Entomopathogenic fungus, i.e., Metarhizium anisopliae, Cordyceps militaris and Beauveria bassiana have been demonstrated to control renowned pests of oil palm. Metarhizium has been used to control Oryctes larvae and the mortality has ranged from 91.67% to 100% in laboratory and 7.4% to 88.75% in the field. Metarhizium has been applied in combination with a termite baiting system （TBS） to control termites in the field for preventive and curative action as well. In many oil palm plantations in Indonesia, Cordyceps has been used to reduce the field moth population ofSetothosea asigna. Application of Cordyceps within the oil palm circle was able to infect S. asigna pupae up to 80%. Meanwhile, Beauveria in an effervescent formulation was demonstrated to have better efficacy on Darna trima larvae. A significant finding on the biological control of basal stem rot disease （Ganoderma） was the isolation of Trichoderma sp. and Gliocladium sp.. The efficacy was conducted with promising result and techniques on the application of Triehoderma have been developed, i.e., hole-in-hole system, surgery and a mounding method. However, as the roots developed, Trichoderma was no longer able to protect the palm from Ganoderma. In spite of that, the use of Trichoderma still prolonged the life ofoil palms by up to 2-3 years. Another fungi belonging to vesicular arbuscular mychorrhiza （VAM） has been developed to control Ganoderma. The efficacy in the nursery showed promising results and the Ganoderma incidence remained low compared to the untreated control. Large scale field trials are ongoing. Challenges on the implementation of biological control in oil palm plantations are because of application and availability of biopesticides/natural enemies. Therefore, advances in research on the formulation of biological control agents are still needed.

Comparison of the antitumor activity of polysaccharides extracted by boiling water and enzyme assistance from Ganoderma lucidum

Polysaccharides are the most important pharmacologically active constituents of Ganoderma lucidum. In this work, polysaccharides were extracted from Ganoderma lucidum with boiling water method and enzyme assisted method. The human liver hepatocellular carcinoma cell line HepG2 was used to compare the an- titumor effect of the two kinds of extraction with 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） test. Both of these two kinds of Ganoderma lucidum polysaccharides reduced cell viability of cancer cell HepG2 in a dose and time-dependent manner. At low concentrations, there was no significant difference in the effectiveness of L 1 and L2; while at concentrations over 0.8 ug/mL, the difference in the effectiveness of L2 in comparison to L1 became significant. At the concentrations of 3.2 ug/mL, the cancer cells were almost killed in 2 d.

灵芝孢子粉、破壁灵芝孢子粉对Lewis肺癌小鼠肿瘤生长和VEGF表达的比较研究

目的:比较灵芝孢子粉、破壁灵芝孢子粉对Lewis肺癌荷瘤小鼠肿瘤生长及血管内皮生长因子(VEGF)表达的影响。方法:采用C_(57)BL/6小鼠左前肢腋下皮下接种建立Lewis肺癌荷瘤小鼠模型,灵芝孢子粉、破壁灵芝孢子粉各设3、2、1g/kg三个剂量组,连续灌胃给药18天。给药期间观察小鼠体重,记录肿瘤体积变化;末次给药后处死,取肿瘤、胸腺和脾脏,称重并计算抑瘤率、胸腺指数和脾指数;HE染色观察肿瘤组织病理状况;Tunel法检测肿瘤组织细胞凋亡情况;免疫组化法检测肿瘤组织VEGF表达。结果:灵芝孢子粉与破壁灵芝孢子粉均有抑制Lewis肺癌荷瘤小鼠肿瘤的作用,但破壁灵芝孢子粉(3、2g/kg)在提高荷瘤小鼠体重、脾指数、抑制肿瘤生长方面的效果优于灵芝孢子粉;破壁灵芝孢子粉能诱导肿瘤细胞凋亡,降低VEGF蛋白表达。结论:破壁灵芝孢子粉抑制Lewis肺癌荷瘤小鼠肿瘤生长的效果优于灵芝孢子粉,其机制可能与诱导肿瘤细胞凋亡、降低VEGF表达有关。

灵芝多糖对D-氨基半乳糖所致小鼠急性肝损伤的保护作用

目的:研究灵芝多糖对D-氨基半乳糖(Gal N)所致小鼠急性肝损伤的保护作用以及对肝脏抗氧化活力的影响。方法:将60只小鼠随机分为正常组、模型组、灵芝多糖50 mg/kg剂量组、灵芝多糖100 mg/kg剂量组、灵芝多糖200 mg/kg剂量组及联苯双脂100 mg/kg剂量(阳性对照)组。每日灌胃给药1次,连续7 d。试验末期,腹腔注射Gal N(800 mg/kg)建立小鼠急性肝损伤模型,6h后摘眼球取血及肝脏,比色法检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)、总胆红素(TBIL),以及肝脏过氧化脂质(LPO)、丙二醛(MDA)、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性。结果:100、200 mg/kg灵芝多糖及100 mg/kg联苯双脂显著降低Gal N诱导的急性肝损伤小鼠血清ALT和AST活性及TBIL含量,提高血清ALB水平,显著降低肝组织LPO和MDA含量,升高GSH水平和SOD,CAT,GSH-Px活性。结论:灵芝多糖对Gal N所致小鼠急性肝损伤具有保护作用,其机制可能与抗氧化作用有关。