Establishment and application of experimental model of human fetal hepatocytes: protective effects of silybin and polyporus umbelalus polysaccharides on human fetal hepatocytes

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GPP(composition of Ganoderma lucidum polysaccharides and Polyporus umbellatus polysaccharides) protects against DSS-induced murine colitis by enhancing immune function and regulating intestinal flora

Previous study have demonstrated that a compound composed of water-soluable Ganoderma lucidum polysaccharides(GLP)and Polyporus umbellatus polysaccharides(PUP)in a ratio of 3:1 named GPP enhances innate immune function in mice through enhancing the function of macrophage cells and activity of natural killer(NK)cells.Here in our research,we further investigated the effect of GPP on the diversity and composition of intestinal flora,and explored its effect on colitis model mice.The immunoregulatory verification experiments of GPP were conducted in both normal and DSS-induced mice model.Our research showed that GPP increased the diversity of intestinal microorganisms in mice with the extension of administration time.Daily GPP intake attenuated DSS-induced colon injury,protected the splenic lymphocyte proliferation ability,enhanced the serum hemolysin synthesis,and increased peripheral phagocytes and NK cell activity in model mice.Comparisons of the predominant gene pathways of the bacterial microbiota showed that DNA repair and recombination,base mismatch repair pathways was stronger in GPP-treatment group than in control group,indicating the possible molecular mechanisms of immune function regulation.Our study showed that GPP regulated immune function in both health and colitis model,and had a positive effect on maintaining intestinal flora homeostasis.

Optimization of Ultrasonic Extraction Process of Polysaccharides from Polyporus umbellatus

[ Objective] This study aimed to investigate the optimal conditions for ultrasonic extraction of polysaccharides from Polyporus umbeUatus, thus providing a reliable basis for the development and utilization of P. umbellatus. [ Method ] By single-factor experiment, the ultrasonic extraction process of polyperus pelysac- charides was optimized. [Result] The optimal conditions for ultrasonic extraction of polysaccharides from P. umbeUatus were： solid-liquid ratio 1：40 （g： ml）, ex- traction duration 60 rain, extraction temperature 70 ℃, ultrasonic power 100 W. [ Conclusion] Compared with conventional water extraction method, ultrasonic ex- traction could significantly improve the content of polyporus polysaccharides with shorter extraction duration, lower solid-liquid ratio and lower extraction tempera-

Preparation of Polyporus Albicans Teng Sulfate andIts Anti-coagulation Activity

More studies have indicated that polysaccharide sulfate has anti-coagulant activity.Now,heparin is the most popular anticoagulant used in clinic,however,its side effects have also caused highly concern.It is still under intensive investigations to synthesize effective safe polysaccharide sulfate as heparin substitute.We extracted water-soluble polysaccharide from fermented mycelium of edible polyporus albicans（Imaz.） teng,and got the water-soluble polyporus albicans teng sulfate（PATS） by modifying the water-solubility polyose with the method of chlorosulfonic acid-pyridine.The anti-coagulant assay of PATS in vitro towards normal human plasma indicates its remarkable anticoagulant activity,while the dose could be as low as 5 mg/L for anticoagulation.The anti-coagulant effect was equivalent to that of heparin about 150 U when the concentration of PATS was 10 mg/L.The study on anti-coagulation mechanism suggests that PATS got involved in the intrinsic pathway.The anti-coagulation activity of PATS was due to the inhibition of the coagulation factors IIa and Xa activities mediated by antithrombin Ⅲ（ATIII）.The anti-coagulation mechanism of PATS is absolutely identical to that of heparin.In conclusion,we suggest that PATS has the similar anti-coagulation characteristic to heparin,but with a better anti-coagulation effect.Meanwhile,derived from edible fungus-polysaccharide,PATS has more bio-safety advantage.Therefore,PATS has promising future to be developed and used as an ideal substitute for heparin in clinic.

Monosaccharide analysis and fingerprinting identification of polysaccharides from Poria cocos and Polyporus umbellatus by HPLC combined with chemometrics methods

Objective:Poria cocos and Polyporus umbellatus are similar medicinal fungi in traditional Chinese medicines.A method for fingerprint analysis of monosaccharide composition of polysaccharides by HPLC combined with chemometrics methods has been developed for characterization and discrimination of them in this research.Methods:The polysaccharides were extracted by decocting in water,and then completely hydrolyzed with hydrochloride.Monosaccharides in the hydrolyzates were derivatized with 1-phenyl-3-methyl-5-pyrazolone(PMP)for HPLC analysis.More than 20 batches of P.cocos and P.umbellatus from different regions were analyzed.Results:The fingerprints of P.cocos showed five common characteristic peaks,which were identified by comparing with the reference substances.The five peaks corresponded to the derivatives of mannose,ribose,glucose,galactose,and fucose.At the same time,the fingerprints of P.umbellatus showed eight common characteristic peaks,of which seven were identified as the derivatives of mannose,ribose,rhamnose,glucose,galactose,xylose,and fucose.Moreover,the similarity of their fingerprints was respectively calculated by the Similarity Evaluation System for Chromatographic Fingerprint of TCM published by China Pharmacopoeia Committee(Version 2004 A).And the data were further processed by hierarchical cluster analysis(HCA)and principal component analysis(PCA).The similarity evaluation and HCA indicated that there were no significant difference in P.cocos or P.umbellatus samples from different geographical regions,but PCA was performed to characterize the difference in monosaccharide constituents between P.cocos and P.umbellatus,and linear discriminant analysis(LDA)showed the overall correct classification rate was 100%.Conclusion:The fingerprint analysis method of monosaccharide composition of water-soluble polysaccharides can distinguish P.cocos and P.umbellatus,and can be applied for the authentication or quality control for P.cocos and P.umbellatus.

猪苓利水渗湿的药效物质、药理作用机制及临床应用研究进展

猪苓是我国传统常用的利水渗湿中药,主要含有甾体类、多糖类等活性成分,常用于治疗泌尿系统疾病。本文对猪苓利水渗湿药效物质基础、药理作用机制及临床应用研究进展进行综述,归纳出麦角甾酮、过氧麦角甾酮、麦角甾-7,22-二烯-3-酮等甾体类化合物和猪苓多糖PPS1、PPS2、PPS3、GUMP-1-1、GUMP-1-2等可通过利尿、肾脏保护、抗炎、抗菌及免疫调节等药理作用发挥利水渗湿作用,猪苓散、猪苓汤、五苓散等中药复方制剂在治疗尿路感染或结石、肾性水肿、肾脏病变等方面效果显著,可为猪苓的进一步开发应用提供参考。

响应面优化猪苓菌核多糖超声辅助提取工艺及抗氧化活性分析

为提高猪苓菌核活性多糖提取效率,应用超声辅助技术提取猪苓菌核多糖,通过单因素实验结合响应面法优化考察提取时间、超声功率、提取温度和液料比对多糖得率的影响并确定最佳提取工艺,同时应用DPPH自由基清除能力和还原力测试评估抗氧化活性。结果表明,猪苓菌核多糖得率与提取温度和超声功率、提取温度和液料比、超声功率和液料比、液料比和提取时间之间的交互作用显著相关,最佳提取工艺为:提取温度72.0℃、提取功率300 W、提取时间65 min和液料比22 mL/g,最大得率为2.47%±0.03%。提取液经分级醇沉制备得到PUPF30、PUPF60和PUPF80,多糖含量分别为39.99%、58.60%、50.35%,蛋白含量分别为25.80%、14.42%、25.54%;测试浓度范围内,最大DPPH清除率分别为50.63%、83.95%、87.48%,最大总还原力(A700 nm)分别为0.828、1.433、1.525。经响应面优化的超声辅助提取工艺适合用于猪苓菌核活性多糖的有效提取制备,本研究为猪苓菌核多糖的进一步开发利用奠定了基础。

The Regulatory Effects of Polyporus Polysaccharide on the Nuclear Factor Kappa B Signal Pathway of Bladder Cancer Cells Stimulated by Bacillus Calmette-Guerin

Objective：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.Methods：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.Results：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（Ratio2.0）,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（P0.05）.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.Conclusions： 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.

猪苓多糖对二乙基亚硝胺诱发肝细胞癌早期炎症改善作用

目的:研究猪苓多糖(Polyporus umbellatus polysaccharide,PUPs)对二乙基亚硝胺(Diethylnitrosamine,DENA)诱发肝细胞癌早期炎症的改善作用。方法:SD大鼠分为3组:对照组(n=15)腹腔注射生理盐水;DENA组(n=15)腹腔注射200 mg/kg body weight(BW);PUPs给药组(n=15)腹腔注射200 mg/kg·BW DENA后给予PUPs 300(mg/kg·BW)/d灌胃给药8周。用比色法检测PUPs对血清中肝功能酶的影响,用ELISA法检测肝组织甲胎蛋白(AFP)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-alpha(TNF-α)水平的影响。HE染色和免疫组化观测PUPs对肝组织病理和CYP2E1酶在肝组织表达的影响。以Griess法测定PUPs对肝组织中NO水平的影响。结果:PUPs能显著降低DENA诱导肝功能损伤大鼠血清中酶的水平,降低促进肝脏炎症反应的细胞因子水平。大鼠口服PUPs明显降低肝细胞癌标志物水平,明显改善肝脏病理状况和DNA的损伤。结论:PUPs对DENA诱发肝脏炎症具有改善作用,对DENA诱发肝细胞癌的发生和发展具有化学预防作用。

Inhibitive Effect of Umbellatus Polyporus Polysaccharide on Cachexic Manifestation induced by toxohorone-l in rats

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猪苓多糖与IL-2协同诱导PBMC杀伤肿瘤细胞活性的实验研究

研究了猪苓多糖（ＰＵＰＳ）与ＩＬ－２协同诱导人外周血单个核细胞（ＰＢＭＣ）成为具有较高杀伤肿瘤活性的细胞，其培养体系总杀伤单位明显高于ＬＡＫ细胞，其诱导细胞的杀伤活性可能与其分泌ＩＦＮ和ＴＮＦ有关，此研究为ＰＵＰＳ与ＩＬ－２协同诱导细胞的进一步研究和临床应用打下了理论基础。

人参多糖和猪苓多糖对大鼠肠道黏膜淋巴细胞功能的影响

目的观察人参多糖和猪苓多糖对大鼠肠道黏膜淋巴细胞功能的影响。方法分离SD大鼠外周血单个核细胞(PBMC)、派伊尔结淋巴细胞(PPL)、肠道上皮内淋巴细胞(IEL)和黏膜固有层淋巴细胞(LPL),分别与不同质量浓度的人参多糖和猪苓多糖共同培养,检测上清液中肿瘤坏死因子-α(TNF-α)和γ-干扰素(IFN-γ)水平变化。结果人参多糖和猪苓多糖均可以使PBMC和PPL培养上清液中TNF-α和IFN-γ水平升高;2mg/mL猪苓多糖可以使IEL培养上清液中TNF-α和IFN-γ水平升高;人参多糖和猪苓多糖可以使LPL培养上清液中TNF-α和IFN-γ水平降低。结论人参多糖和猪苓多糖对大鼠肠道黏膜淋巴细胞功能具有调节作用。

猪苓多糖对S180细胞培养上清免疫抑制作用影响的研究

目的 :研究猪苓多糖对肿瘤细胞系S180培养上清免疫抑制作用的影响。方法 :用MTT比色法检测有或无猪苓多糖作用的肿瘤细胞S180培养上清 (简称肿瘤上清 ) ,对ConA诱导的小鼠脾细胞增殖和IL 2产生 ,对小鼠脾细胞的杀伤活性 ,以及对CTLL 2细胞对IL 2反应性的影响。用流式细胞仪检测该培养上清对小鼠脾细胞表面IL 2Rα表达的影响。结果 :无猪苓多糖作用的肿瘤上清可强烈抑制上述 5项指标 ;而猪苓多糖作用的肿瘤上清则可明显上调上述方法中所测 5项指标。若先用含猪苓多糖培养液培养肿瘤细胞 2 4h或 48h ,而后洗去或不洗去猪苓多糖 ,再培养肿瘤细胞 2 4h ,所获肿瘤上清也可明显上调上述 5项指标。结论 :猪苓多糖可抵消肿瘤上清的免疫抑制作用 ,下调肿瘤细胞S180合成和

猪苓硒多糖连续合成工艺及抑菌性能研究

采用化学合成法,以猪苓多糖和亚硒酸钠为原料制备猪苓硒多糖。试验设计连续式合成工艺流程,并对硒多糖的连续式超声波辅助化学合成工艺条件进行研究;对所制备的硒多糖进行抑菌性能研究,并与猪苓多糖进行比较。结果表明,猪苓多糖、氯化钡、亚硒酸钠溶液浓度均为0.2mg/mL,采用连续式合成猪苓硒多糖的最佳工艺条件为多糖进料流量30 mL/min,亚硒酸钠溶液与猪苓多糖溶液流量配比1∶1,反应温度70℃、反应釜内硝酸的浓度0.008mL/mL,超声辅助合成频率为30kHz,其合成率达26.5mg/g;抑菌性能研究表明,猪苓硒多糖对大肠杆菌、金黄色葡萄球菌、啤酒酵母和黑曲霉均有抑制作用,且其抑菌能力明显优于猪苓多糖。

猪苓多糖超声提取工艺条件优化

采用超声提取方法研究了猪苓菌核多糖提取的优化工艺条件。结果表明,在超声功率为100W时,其最佳提取条件为60℃,料液比为1 g∶30 mL,pH值为7.0,提取时间为40 min,P.umbellatusLBZ多糖的量为20.3 mg/g。与常规的沸水浴提取法比较,超声提取能显著提高猪苓多糖含量、缩短提取时间、减小料液比和降低提取温度。

液体浅层培养对猪苓菌丝体生长及其多糖含量的影响

通过液体浅层培养方式培养猪苓菌丝体,在培养28d后,猪苓菌丝体的鲜重达到最高,而菌丝体干重在21d后增幅不大;培养28d后的培养液pH由接种前的3 9升高到5 0,菌丝体旺盛生长阶段的pH为4 0～4 6;菌丝体中总糖含量在培养14d后达到最大,浅层培养猪苓菌丝体生产猪苓多糖的适宜培养周期约为2周;对从猪苓菌丝体和猪苓菌核中提取的猪苓多糖进行红外吸收光谱比较分析,两者红外光谱图相似,表明多糖的主要组成成分相同,但含量上有差异。

中药用于血吸虫病抗独特型抗体疫苗佐剂的研究

目的 从中药中筛选血吸虫病抗独特型抗体疫苗的佐剂 ,并初步探讨其作用机制。方法 日本血吸虫抗独特型抗体 NP30主动免疫 BAL B/ c小鼠 ,分别联用虫草多糖、猪苓多糖、黄芪、甘草酸和人体免疫调理剂 (HIM) ,观察其对小鼠的保护作用 ,并检测小鼠血清特异性 Ig G、Ig G1和 Ig G2 a抗体水平。结果 黄芪和 HIM能明显提高 NP30对小鼠的保护性免疫作用 ,减虫率显著提高 ,从单用 NP30的 37.8%分别提高到 5 0 .6 %和 5 3.6 % ;但两者对血清特异性 Ig G、Ig G1和 Ig G2 a抗体均无明显升高作用。结论 黄芪和 HIM可明显提高血吸虫病抗独特型抗体疫苗 NP30的保护性免疫力 ,可作为 NP30的佐剂 ,其佐剂作用与所检测的抗体水平似不相关 ,推测可能与细胞免疫应答有关。

不同干燥方法对猪苓中多糖及麦角甾醇含量的影响

以陕西省汉中市略阳县同一采收期的猪苓为研究对象,应用不同干燥方法制备样品,采用苯酚-硫酸法显色,紫外-可见分光光度法测定不同干燥方法猪苓样品的猪苓多糖含量;RP-HPLC法测定不同干燥方法制备的猪苓中麦角甾醇的含量差异;并优化猪苓药材的干燥加工方法。结果表明:紫外-分光光度法测得70℃2h,50℃烘干法的多糖含量最高(0.668 9%),阴干法多糖含量较低;HPLC法测得自然条件晒干及50℃烘干所得麦角甾醇含量较高,而高温干燥得到的麦角甾醇含量较低。说明不同干燥方法对猪苓多糖及麦角甾醇含量有显著影响,根据不同的药用途径应当选择不同的干燥加工方法。为了提高猪苓多糖的含量,应采用先高温干燥再低温烘干加工方法,为了提高麦角甾醇含量,应采用低温烘干加工方法。

酶解-超声法对猪苓多糖正交优选提取及抗氧化活性的初步实验研究

利用复合酶解法正交优化猪苓多糖的提取工艺,并附加超声波辅助处理。酶法-超声提取的多糖进行体外抗氧化活性的初步评价。结果表明对猪苓多糖的酶法提取产生影响的因素主要有:加酶比例、温度、p H值和时间,各因素对猪苓多糖提取效果的影响由大到小依次为:温度>p H值>酶用量>提取时间。最佳提取工艺为:纤维素酶∶果胶酶复合酶(比例:1∶1,以质量计为猪苓的1%),酶解温度50℃,p H=6.5,反应时间60 min,收率为4.31%。酶法提取后加超声提取超声处理30 min,多糖得率提高至5.48%。提取的猪苓多糖进行体外抗氧化活性的初步实验,表明其抗氧化能力与Vc大致相当。因此酶法-超声波辅助提取猪苓中的猪苓多糖的具有工艺简便易行、反应条件温和、绿色环保等优点,该法提取的多糖具有抗氧化活性能力。

猪苓多糖下调Colon26细胞肿瘤免疫抑制的体外研究

目的体外研究猪苓多糖(polyporus umbellatus polysacharide,PUP)对结直肠癌Colon26细胞肿瘤免疫抑制的影响。方法获取经PUP作用后的Colon26再培养上清,未经作用的同步培养上清作对照,检测PUP对Colon26肿瘤细胞所致NK杀伤和诱导转化(MTT法测定),以及IL-2Rα、CD3ε+ζ+和CD3ε-ζ+表达(流式细胞计数分析)5项免疫功能抑制的影响,定量ELISA法测定上清中TGF-β1、VEGF、IL-4、IL-6和IL-10五种免疫抑制分子含量,多元相关分析PUP下调Colon26免疫抑制与分泌免疫抑制分子的关系。结果对照上清中5种免疫抑制分子均可测到,TGF-β1含量最高,对5项免疫功能均有显著抑制。PUP作用后的第一次再培养上清,TGF-β1含量及对5项免疫功能的抑制均明显降低;与其相比,第二次再培养上清的5种免疫抑制分子含量及对NK杀伤、IL-2Rα和CD3ε-ζ+表达抑制均明显提高。TGF-β1与抑制诱导转化、NK杀伤及CD3ε+ζ+表达正相关。结论通过下调肿瘤细胞分泌免疫抑制分子而削弱肿瘤免疫抑制,可能是PUP抗瘤效应机制之一。