Extraction of Astragalus Polysaccharides and Ganoderma lucidum Mycelia Polysaccharides and Their in Vitro Antioxidative Effects

[Objectives]To explore the extraction and in vitro antioxidant effects of astragalus polysaccharides(APS)and Ganoderma lucidum mycelia polysaccharides(GLMPS).[Methods]By studying the polysaccharides of the herbal medicinal material Astragalus membranaceus and the fungal medicinal material Ganoderma lucidum mycelia,two polysaccharides were mixed according to different proportions and concentrations by using the principle of traditional Chinese medicine compound combination.The effect of polysaccharides on the scavenging ability of hydroxyl radical system was determined by salicylic acid method.[Results]When the compound ratios of GLMPS and APS were 1∶1,1∶4,1∶5,4∶1,and 5∶1,the scavenging effect of compound polysaccharides was better than that of single-component polysaccharides,and with the increase of concentration,the scavenging effect increased.When the ratio of GLMPS and APS was 5∶1,the hydroxyl radical scavenging rate of the compound polysaccharide reached 59.77%,which was 18.72%higher than that of single GLMPS and 28.58%higher than that of single APS.The scavenging effect of compound polysaccharide is closely related to the compound ratio and concentration.[Conclusions]APS and GLMPS can obtain better hydroxyl radical scavenging ability than single-component polysaccharides through compounding in appropriate proportions.In addition,within a suitable concentration range,as the concentration increases,the scavenging ability also increases.

Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a pre-liminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H202-induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H202 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis poly-saccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood flow after ischemia. Our findings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.

Angelica sinensis polysaccharides ameliorate 5-flourouracil-induced bone marrow stromal cell proliferation inhibition via regulating Wnt/β-catenin signaling

Chemotherapy may cause cellular oxidative stress to bone marrow.Oxidative damage of bone marrow hematopoietic microenvironment is closely related to chronic myelosuppression after chemotherapeutic treatment.Angelica sinensis polysaccharides(ASP)are major effective ingredients of traditional Chinese medicine Angelica with multi-target anti-oxidative stress features.In the current study,we investigated the protective roles and mechanisms of ASP on chemotherapy-induced bone marrow stromal cell(BMSC)damage.The human bone marrow stromal cell line HS-5 cells were divided into control group,5-FU group,5-FU+ASP group,and 5-FU+LiCl group to investigate the mechanism of ASP to alleviate 5-FU-induced BMSC proliferation inhibition.The results showed that 5-FU inhibits the growth of HS-5 cells in a time and dose-dependent manner;however,ASP partially counteracted the 5-FU-induced decrease in cell viability,whereas Wnt signaling inhibitor Dkk1 antagonized the effect of ASP on HS-5 cells.ASP reversed the decrease in total cytoplasmicβ-catenin,p-GSK-3β,and CyclinD1 following 5-FU treatment and modulated nuclear expression ofβ-catenin,Lef-1,and C-myc proteins.Furthermore,ASP also enhanced the antioxidant capacity of cells and reduced 5-FU-induced oxidative stress,attenuated FoxO1 expression,thus weakened its downstream apoptosis-related proteins and G0/G1 checkpoint-associated p27^(Kip1) expression to alleviate 5-FU-induced apoptosis and to promote cell cycle progression.All the results above suggest that the protective role of ASP in 5-FU-treated BMSCs proliferation for the chemotherapy may be related to its activating Wnt/β-catenin signaling and keeping homeostasis betweenβ-catenin and FoxO1 under oxidative stress.The study provides a potential therapeutic strategy for alleviating chemotherapeutic damage on BMSCs.

Effects of Angelica Polysaccharide on telomere length in mice with benzene-induced aplastic anemia

Objective:To investigate the changes in telomere length and the level of burst-forming units-erythrocyte(BFU-Es)and colony-forming unit-erythrocytes(CFU-Es)in mice with benzene-induced aplastic anemia(AA),and follow-up the therapeutic effects of Angelica Polysaccharide(AP).Methods:Male BALB/c mice(n=120)were randomly divided into three groups(1,2,3):normal control(n=24),AA control(n=48),and treated AA(n=48),respectively.Mice in Group 2 received benzene inhalation for 2.5 months and 1 ml distilled water p.o.per day for 2 weeks after the establishment of AA models.Similar procedure was applied to the mice in Group 3 and AP was given for 2 weeks after the establishment of AA models.Real-time polymerase chain reaction was used to survey relative telomere length measurement of the bone marrow cells.A BFU-Es and CFU-Es survey was done to follow-up the therapeutic effects of AP.Results:Compared with normal control,significant reductions of RBC,WBC,and platelet counts were found in peripheral blood of AA mice.After treatment with AP,counts of BFU-Es and CFU-Es were restored up to 66.8%and 77.25%,respectively,and length of telomere was restored up to 76.34%,of the normal levels.The telomere length in treated AA group was higher than the control AA group.Conclusion:The AP can protect the telomere length and differentiation of hemopoietic stem/progenitor cells,accelerate the recovery of BFU-Es and CFU-Es of AA mice,and then improve the bone marrow failure.

Mitochondrial membrane stabilization by Angelica sinensis polysaccharide in murine aplastic anemia

为了证实当归多糖可以通过调控线粒体膜稳定性干预鼠再障模型设计了该实验。ICR小鼠随机分为对照组、再障组和当归多糖治疗组。其中再障小鼠使用60Coγ照射和腹腔注射环磷酰胺和环孢素的方法造模。对照组小鼠采用铅屏蔽照射。对照组和再障组小鼠使用生理盐水腹腔注射,治疗组小鼠口服当归多糖两周。分别检测骨髓单个核细胞的线粒体超微结构和膜电位。检测COX、MDH在三组中的寒凉差异。结果显示,再障组中线粒体数量和膜电位较对照组均有显著下降,应用当归多糖干预后,有不同程度的回升。治疗组的线粒体裂解时间较再障组大幅延迟(P<0.05)。我们认为当归多糖可以提升再障骨髓单个核细胞的线粒体膜稳定性,并可能抑制线粒体通路的凋亡。

当归多糖APS-2a的结构分析及抗肿瘤作用研究

当归粗多糖经阴离子交换纤维素柱、凝胶柱色谱分离纯化,得白色絮状多糖APS-2a。采用高效尺寸排阻色谱、红外光谱、气相色谱和糖醛酸还原等方法进行了结构的初步分析;采用小鼠肉瘤S-180移植模型,对当归多糖APS-2a体内抑瘤率、脾脏指数、胸腺指数进行测定。结果表明APS-2a为均一组分,重均相对分子质量为7.4×105Da,由阿拉伯糖-半乳糖-鼠李糖-葡萄糖-半乳糖醛酸(38.2∶7.5∶2.6∶1.0∶4.9)构成,是首次从当归中分离出的新型酸性多糖。20、50 mg/kg的多糖APS-2a对肉瘤S-180荷瘤小鼠具有一定的抑瘤作用并明显促进荷瘤小鼠的胸腺指数和脾脏指数的增加。

APS作为重组(CHO细胞)乙型肝炎疫苗佐剂的安全性及免疫效果

目的 探讨黄芪多糖（APS）作为重组（CHO细胞）乙肝疫苗免疫佐剂的可行性。方法APS与重组（CHO细胞）乙肝病毒表面抗原（HBsAg）混合制成疫苗,进行了动物毒性试验、过敏试验及效力试验。结果 该佐剂毒性轻微,无过敏反应,同一剂量组动物血清抗-HBs阳转率高于Al（OH）3对照组。结论APS是一种很有潜力的疫苗佐剂。

当归多糖APS-3c的结构特征及体外抗肿瘤作用的研究

中国当归粗多糖经阴离子交换纤维素柱(DEAE-sephadex A-25)、凝胶柱(Sephacryl S-400及Sephadex G-100)色谱分离纯化,得淡黄色粉末状多糖APS-3c。采用高效尺寸排阻色谱、红外光谱、气相色谱和糖醛酸还原等方法进行了结构的初步分析,用四甲基偶氮唑蓝比色法观察APS-3c对多种肿瘤细胞的生长抑制作用。结果表明APS-3c为均一组分,分子量为1.4×104Da,由阿拉伯糖-鼠李糖-葡萄糖-半乳糖-甘露糖-木糖-半乳糖醛酸(6.7:6.5:6.3:4.7:1.6:1.0:13.2)构成,是首次从中国当归中分离出的新型酸性多糖。APS-3c对人白血病HL-60细胞、人结肠癌SW1116细胞的增殖有一定的抑制作用。

当归多糖APS-bⅡ的结构特征及体外抗肿瘤作用

从当归(Angelica sinensis(Oliv)Diels)中提取粗多糖,经阴离子交换纤维素柱和Sephadex G-100凝胶柱色谱分离纯化,得白色粉末状多糖APS-bII。采用高效尺寸排阻色谱、红外光谱、高效液相色谱等方法进行了结构的初步分析;采用四甲基偶氮唑蓝比色法观察APS-bII对多种肿瘤细胞的生长抑制作用。结果表明APS-bII为均一组分,分子量为1.3×104Da,由葡萄糖-半乳糖-木糖-阿拉伯糖(8.4:2.7:1.8:1.0)构成,是首次从当归中分离出的杂多糖。APS-bII对多种肿瘤细胞的增殖有抑制作用。

黄芪多糖(APS)拮抗铅镉联合致鸡脾脏细胞凋亡作用的研究

为了探讨黄芪多糖注射液对铅镉联合致鸡脾脏细胞凋亡的拮抗作用。采用TUNEL法、RT-PCR和DNAladder方法检测分别对对照组、铅镉组和黄芪多糖-铅镉组的鸡脾细胞凋亡率和脾淋巴细胞凋亡基因Bcl-2mRNA转录水平。结果表明:与对照组比较,试验组的细胞凋亡率明显升高,Bcl-2mRNA表达水平降低;试验组和对照组差异显著或差异极显著,并且黄芪多糖-铅镉组数据介于对照组和铅镉组之间。试验结果显示:黄芪多糖注射液对铅镉联合致鸡脏脾细胞凋亡具有明显的拮抗作用。

APS对感染CDV犬血常规及抗氧化能力的影响

[目的]探讨黄芪多糖(APS)对感染犬瘟热病毒(CDV)犬血常规及抗氧化能力的影响。[方法]选择45d健康、未接种犬瘟热疫苗的幼犬19只,随机分为5组,其中Ⅰ组为空白对照组,Ⅱ组为人工染毒阳性对照组,III组为APS治疗组,IV组为APS与常规治疗结合组,V组为常规治疗组。自人工染毒当日进行治疗,连续治疗5d。各组分别于人工感染前1d及感染后第3、6天于前臂头静脉采血,进行血常规、血清SOD活性和MDA含量测定。[结果]经CDV感染后,血液中RBC、WBC、GRA、Hb含量及SOD活性均降低,MDA含量增加,均显著低于对照组(P<0.05)。经治疗的各组中,APS和常规治疗对RBC、WBC、GRA、Hb含量及SOD活性均有不同程度的改善,但以APS和常规治疗相结合效果最明显,但与空白对照组仍存在显著差异(P<0.05)。[结论]APS和常规治疗均可提高人工感染CDV犬的抗氧化能力,但以中西药结合效果较好。

枸杞多糖通过调控TLR9/AP-1信号通路对变应性鼻炎大鼠Th1/Th2细胞因子及鼻黏膜中嗜酸性粒细胞炎症的影响

目的探究枸杞多糖通过调控Toll样受体9/激活蛋白-1(TLR9/AP-1)信号通路对变应性鼻炎大鼠Th1/Th2细胞因子、鼻黏膜中嗜酸性粒细胞炎症的影响。方法选取60只SPF级SD雄性大鼠,随机分为正常(A)组,模型(B)组,糠酸莫米松(C)组,枸杞多糖(D)组,TLR9/AP-1信号通路抑制剂(E)组,TLR9/AP-1信号通路抑制剂联合枸杞多糖(F)组,每组各10只,对B、C、D、E、F组采用卵清蛋白致敏法建立变应性鼻炎模型,A组不建立该模型。建模成功后,对C组给予1喷/侧的糠酸莫米松喷鼻治疗,对D组灌胃100 mg/kg的枸杞多糖,对E组灌胃20 mg/kg AP-1抑制剂SP100030,对F组给予SP100030联合枸杞多糖,A、B组同期给予灌胃同体积生理盐水,对大鼠鼻部症状进行评分,HE染色法检测鼻黏膜组织病理形态及嗜酸性粒细胞并计数,ELISA法检测大鼠血清中Th1/Th2细胞因子水平,免疫印迹法检测鼻黏膜组织中TLR9/AP-1信号通路相关蛋白表达。结果与A组相比,B组鼻部症状评分、鼻黏膜中嗜酸性粒细胞数目、血清IL-4含量显著升高(P<0.05),血清中INF-γ含量显著降低(P<0.05);与B组比较,C、D两组鼻部症状评分、鼻黏膜中嗜酸性粒细胞数目、血清IL-4含量显著降低(P<0.05),血清中INF-γ含量显著升高(P<0.05),且D组比C组变化显著(P<0.05);A组大鼠鼻黏膜组织结构完整,B组鼻黏膜上皮结构紊乱且不完整,出血及嗜酸性粒细胞等炎性细胞浸润;与B组相比,C、D组病理状态明显改善,嗜酸性粒细胞明显减少;与A组比较,B组鼻黏膜组织中TLR9、AP-1蛋白表达显著升高(P<0.05),与B组比较,C、D组鼻黏膜组织中TLR9、AP-1蛋白表达显著降低(P<0.05),且D组比C组变化显著(P<0.05),而E组与D组相比无明显差异(P>0.05),F组比E组降低明显(P<0.05)。结论枸杞多糖可有效调控变应性鼻炎大鼠Th1/Th2细胞因子水平、减轻鼻黏膜中嗜酸性粒细胞炎症,其机制可能与抑制TLR9/AP-1信号通路有关。

Polysaccharide from Angelica Sinensis Suppresses Inflammation and Reverses Anemia in Complete Freund's Adjuvant-induced Rats

The antinlammatory and antianemic activities of Angelica sinensis polysaccharide(ASP)isolated from roots of Angelica sinensis(AS)was investigated in a complete Freund's adjuvant(CFA)-induced arthritic rat model.It was observed that serum iron(SI)and total iron binding capacity(TIBC)levels were elevated after 4-week oral administration of ASP.Red blood cell(RBC)count and hemoglobin(Hb)concentrations were ameliorated as well.Moreover,infammatory cytokines IL-6 and TNF-a were decreased strikingly in CFA-induced arthritic rats after treatment of ASP.Evidence also showed that ASP strongly inhibited hepcidin expression through the Janus kinase/signal transducers and activators of transcription(JAK2/STAT3)pathway.Furthermore,ASP exhibited reduced primary and secondary lesions in adjuvant arthritis,attenuating synovitis and inflammatory joint damage.Data presented in this article collectively indicated that ASP significantly decreased proinflammatory cytokines(TNF-a,IL-6),which might play a crucial role in the CFA-induced arthritic rats,and had a therapeutic effect on adjuvant arthritis in rats.Results of Western blot analysis indicated that ASP inhibited the activation of IL-6/JAK2/STAT3 signaling pathway in the CFA-induced arthritic rats.

Preparation and Identification of Angelica sinensis Polysaccharide-iron Complex

Angelica sinensis polysaccharide（ASP） was extracted from Angelica sinensis by boiling water. An Angelica sinensis polysaccharide-iron complex（APC） was prepared under the alkaline condition by adding a ferric chloride solution to the ASP solution. Then some identifiable properties of the complex were studied. The content of iron（ Ⅲ ） in the complex was determined with iodometry. The thermal property, the microscopic structure, the spectral characteristics, and N, C, H contents of the complex were examined by a variety of techniques including DSC, TEM, IR, NMR, and elemental analysis. The content of iron（ Ⅲ ） in the complex ranges from 10% to 40%. The DSC result shows that the melting point of the complex is about 450 ℃. The TEM result shows that the complex has an iron（ Ⅲ ） core（β-FeOOH core） linked by hydroxy and oxy bridges, with the polysaccharide chains attached to the surface of the core. The IR and NMR results also show that there is a β-FeOOH core in the complex. The elemental analysis shows that the contents of N, C, H in the complex are, respectively, lower than those of N, C, H in ASP. All our studies indicate that the APC consists of a β-FeOOH core surrounded by ASP.

Functional Metabolomics Reveals that Astragalus Polysaccharides Improve Lipids Metabolism through Microbial Metabolite 2-Hydroxybutyric Acid in Obese Mice

Polysaccharides are widely present in herbs with multiple activities,especially immunity regulation and metabolic benefits for metabolic disorders.However,the underlying mechanisms are not well under-stood.Functional metabolomics is increasingly used to investigate systemic effects on the host by iden-tifying metabolites with particular functions.This study explores the mechanisms underlying the metabolic benefits of Astragalus polysaccharides(APS)by adopting a functional metabolomics strategy.The effects of APS were determined in eight-week high-fat diet(HFD)-fed obese mice.Then,gas chromatography–time-of-flight mass spectrometry(GC–TOFMS)-based untargeted metabolomics was performed for an analysis of serum and liver tissues,and liquid chromatography–tandem mass spectrom-etry(LC–MS/MS)-based targeted metabolomics was performed.The potential functions of the metabo-lites were tested with in vitro and in vivo models of metabolic disorders.Our results first confirmed the metabolic benefits of APS in obese mice.Then,metabolomics analysis revealed that APS supplemen-tation reversed the HFD-induced metabolic changes,and identified 2-hydroxybutyric acid(2-HB)as a potential functional metabolite for APS activity that was significantly decreased by a HFD and reversed by APS.Further study indicated that 2-HB inhibited oleic acid(OA)-induced triglyceride(TG)accumula-tion.It was also found to stimulate the expression of proteins in lipid degradation in hepatocytes and TG lipolysis in 3T3-L1 cells.Moreover,it was found to reduce serum TG and regulate the proteins involved in lipid degradation in high-fat and high-sucrose(HFHS)-fed mice.In conclusion,our study demonstrates that the metabolic benefits of APS are at least partially due to 2-HB generation,which modulated lipid metabolism both in vitro and in vivo.Our results also highlight that functional metabolomics is practical for investigating the mechanism underlying the systemic benefits of plant polysaccharides.

Pharmacological Action of Adenophora Polysaccharides

Adenophora polysaccharides (AP), is an active principle extracted from the root of Adenophorae Potaninii Korsh originated in Gansu Province and isolated with boiling water(1). AP is isolated and purified from the crude drug by DEAE-cellulose and Sephadex G-200 column, with a white powder and mean molecular weight of 8. 3×104 , and [α]D20 of AP is +68(1). AP is only composed of glucose judging

Study on Extraction Technology for Polysaccharide from Blood-Supplementing Angelica Sinensis Decoction

Purpose:Optimize water-alcohol technology for extracting polysaccharide from blood-supplementing angelica sinensis decoction to improve the extraction rate.Method:Draw the standard curve of glucose reference substance and build the regression equation to calculate the polysaccharide content.Investigate the effect of water addition times,extraction duration,extraction times,ethanol concentration for ethanol precipitation and times of ethanol precipitation on the extraction rate of polysaccharide.Result Add 8 times the medicinal material quality of water,and perform 2 extractions for 120min each time;it shows that conducting 2 ethanol precipitations with 80%ethanol concentration results in the maximum polysaccharide content,indicating the best extraction condition.Conclusion:The experiment establishes an easy and convenient water-alcohol method for extracting polysaccharide from blood-supplementing angelica sinensis decoction,and lays a research foundation on pharmacodynamics of polysaccharide in blood-supplementing angelica sinensis decoction.

当归多糖对低氧环境中骨髓间充质干细胞成骨分化潜能的影响

目的探讨当归多糖对低氧环境中骨髓间充质干细胞(BMSC)成骨分化潜能的影响。方法将BMSC随机分为空白组、低氧组、当归多糖组。使用OriCellTM成人BMSC成骨诱导分化完全培养基对3组细胞进行培养,其中当归多糖组的培养基含有50μg/mL的当归多糖。对空白组BMSC进行常规培养(21%氧浓度),将低氧组与当归多糖组BMSC置于6%氧浓度环境中进行培养。培养14 d后,观察3组的BMSC形态学变化和钙盐沉积情况,检测3组BMSC的成骨分化相关蛋白(骨桥蛋白和骨钙素)表达水平,以及成骨分化关键转录因子RUNT相关转录因子2(RUNX2)表达情况。结果与空白组相比,低氧组BMSC的骨钙素、骨桥蛋白、RUNX2的表达水平均降低(P<0.05),高密度钙结节及钙盐沉积减少。与低氧组相比,当归多糖组BMSC骨钙素、骨桥蛋白、RUNX2的表达升高(P<0.05),高密度钙结节及钙盐沉积增多。结论低氧环境可以抑制BMSC的成骨分化潜能,而当归多糖能够有效维持低氧环境中BMSC成骨分化潜能的稳定。

当归多糖组分AP-3诱生小鼠脾细胞IL-2和IFN-γ的作用

目的研究当归多糖组分AP-3对细胞因子IL-2和IFN—γ的诱生作用，以探讨其免疫调节的特点。方法流式细胞术测定培养的脾细胞中Cd4^＋细胞比例；酶联免疫法测定培养上清液中IL-2和IFN-γ的浓度；RT—PCR法测定IL-2和IFN-γ mRNA的转录水平。结果AP-3在0．6～2μmol·L^-1，能显著提高培养脾细胞CD^4＋细胞的百分率；在2～6μmol·L^-1，AP-3时间、剂量依赖性地增加培养的细胞上清液中IL-2的浓度和细胞内IL-2 mRNA的转录水平，而对于IFN-γ和IFN-7 mRNA，则先升高后降低，并呈现剂量依赖性。结论当归多糖能够促进IL-2和IFN-γ的分泌，激活Th1细胞，从而发挥免疫调节作用。

当归多糖组分AP-3对不同淋巴细胞亚群的作用

目的观察当归多糖组分AP-3对不同淋巴细胞亚群的作用.方法免疫磁珠法制备T、B细胞亚群;四甲基偶氮唑蓝(MTT)法检测细胞增殖;流式细胞术观察CD4+T细胞亚群比例的变化.结果在30～300 μg/ml范围内,AP-3能显著促进小鼠脾淋巴细胞、巨噬细胞、混合淋巴细胞和T淋巴细胞的增殖反应,而对B细胞增殖有一定的抑制作用;显著增加培养的脾细胞中CD4+T细胞亚群的比例.结论当归多糖具有独特的免疫调节特性,活化巨噬细胞、T细胞以及辅助性T细胞(Th细胞),但对B细胞有一定的抑制作用.