Two New C_(21) Steroids from Marsdenia tenacissima

Two new C21 steroids were isolated from the CHCl3 extract of the stem of Marsdenia tenacissima. On the basis of spectroscopic analysis and chemical methods, their structures were elucidated as 17β-tenacigenin B (2) and 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D- oleandropyranosyl-tenacigenin C (3). The structure of the known aglycon tenacigenin C was revised as 5α, 9α, 17β-pregnane-3β, 8β, 11α, 12β, 14β-pentanol-20-one. Compound 3 is the first reported glycoside of tenacigenin C.

Polyoxypregnanes from the stems of Marsdenia tenacissima

From the stems ofMarsdenia tenacissima two new polyoxypregnanes were isolated, their structures were elucidated by 1D, 2D-NMR as 11α,12β-di-O-tigloyl-tenacigenin B （1） and tenacigenoside E （2）.

A new C_(21) steroid glycoside from Marsdenia tenacissima

A new C21 steroid glycoside,11α-O-tigloyl-12-β-O-acetyl tenacigenin B 3-O-β-D-glucopyranosyl-（1→4）-β-D-glucopyr- anosyl-（1→4）-3-O-methyl-6-deoxy-β-D-allopyranosyl-（1→4）-β-D-oleandropyranoside（1）,named tenacissoside N was isolated from the stems of Marsdenia tenacissima.The structure of the glycoside was identified by HR-ESI-MS and NMR spectra.

Research progress on anti-tumor properties of Marsdenia tenacissima

Tongguanteng （Marsdenia tenacissima）, which is mainly distributed in the Yunnan and Guizhou provinces of China, wasfirst recorded in Diannanbencao by Lan Mao of the Ming dynasty of China. According to recent pharmacological studies,the chemical composition of Tongguanteng （Marsdenia tenacissima） is complex and contains C21 steroidal saponins,polysaccharides, alkaloids, and other molecules, which show anti-cancer effects on various tumor cell lines. It inhibitstumor cell proliferation and growth mainly by increasing the expression of apoptosis- and cell cycle-related proteins topromote apoptosis and arrest tumor cells in the G2/M or S phase. Downregulation of the expression of vascularendothelial growth factor-2/A and matrix metalloprotease-2/9 suppresses the formation of the tumor microvasculature,leading to tumor malnutrition, increased expression of interleukin-2, glutathione peroxidase, catalase, and superoxidedismutase, and decreased interleukin-10 and malondialdehyde expression, thereby enhancing immunity andantioxidation in the body. Additionally, inhibition of epidermal growth factor receptor, hepatocyte growth factor receptor,and tyrosine-protein kinase receptor activation enhances the anti-tumor efficacy of epidermal growth factorreceptor-tyrosine kinase inhibitors as well as inhibits P-glycoprotein and cytochrome P450 to increase the concentrationof anti-tumor drugs in tumor cells.

Marsdenia tenacissima extract induces G_0/G_1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase(MAPK) signaling pathway

Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.

Pregnane Glycosides from Stems of Marsdenia tenacissima

Objective To study the chemical constituents from the stems of Marsdenia tenacissima.Methods The chemical constituents were isolated by various column chromatography and their structures were identified by spectral and chemical analysis.Results Two pregnane glycosides were isolated from the stems of M.tenacissima and identified as 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O- tigloyltenacigenin B,named as tenacigenoside I(1)and 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-acetyltenacigenin B,named as tenacigenoside K(2).Conclusion Compound 1 is a new compound,1H-NMR and 13C-NMR data of compound 2 are reported for the first time.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis

Marsdenia tenacissima injection,a standard Marsdenia tenacissima extract(MTE),has been approved as an adjuvant therapeutic agent for various cancers.Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer(PCa)cells.However,the underlying mechanisms and active ingredients of MTE against PCa were not completely understood.This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells.In addition,MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7,Cyt c,and Bax.In vivo,DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size.TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE.Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets,and 709 PCa-associated targets were retrieved,from which 149 overlapped targets were screened out.Pathway enrichment analysis showed that the HIF-1,PI3K-AKT,and ErbB signaling pathways were closely related to tumor apoptosis.Western blot results confirmed that MTE increased the expression of p-AKT^(Ser473) and p-GSK3β^(Ser9),and decreased the expression of p-STAT3^(Tyr705) in vitro and in vivo.A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLCQTOF-MS/MS.Molecular docking analysis indicated that six compounds may interact with AKT,GSK3β,and STAT3.In conclusion,MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis,resulting in inhibition of PCa growth in vitro and in vivo.

Effect of Marsdenia tenacissima extract on G2/M cell cycle arrest by upregulating 14-3-3σ and downregulating c-myc in vitro and in vivo

Objective: Marsdenia tenacissima extract(MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc.Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE.Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc,and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo.Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.

基于JAK1/STAT3信号通路的通关藤对人骨肉瘤细胞增殖的影响

目的探讨通关藤注射液和通关藤提取液对人骨肉瘤细胞增殖、迁移、侵袭和凋亡的影响及潜在机制。方法体外培养人骨肉瘤细胞MNNG/HOS、Saos-2和人骨髓间充质干细胞(BMSC),分别予通关藤注射液或通关藤提取液(40、60、80 mg/mL)处理,CCK-8实验和平板克隆实验检测细胞增殖能力,划痕实验和Transwell实验检测细胞迁移和侵袭能力,Hoechst33342染色和Annexin-V/PI双染实验检测细胞凋亡情况,RT-qPCR检测细胞Bax、Bcl-2、胱天蛋白酶-3(Caspase-3)mRNA表达,Western blot检测细胞Bax、Bcl-2、Caspase-3、Janus激酶1(JAK1)、p-JAK1、信号转导和转录激活因子3(STAT3)、p-STAT3、基质金属蛋白酶(MMP)9蛋白表达。结果与对照组比较,不同浓度通关藤注射液和提取液均能显著降低MNNG/HOS和Saos-2细胞存活率,抑制细胞克隆形成、迁移和侵袭,诱导细胞凋亡(P<0.05,P<0.01),促进细胞BaxmRNA和蛋白表达,抑制Bcl-2mRNA和蛋白表达,上调Caspase-3mRNA和CleavedCaspase-3蛋白表达。通关藤注射液可显著下调Saos-2细胞p-JAK1、p-STAT3、MMP9蛋白表达(P<0.05,P<0.01)。结论通关藤注射液和提取液均可显著抑制人骨肉瘤细胞MNNG/HOS和Saos-2增殖、迁移和侵袭,诱导其凋亡,二者抗肿瘤作用无明显差异,其机制可能与抑制JAK1/STAT3信号通路激活有关。

通关藤多糖结构表征及生物活性研究

以通关藤为原料,采用超声辅助水提法提取通关藤多糖,研究其结构表征及体外抗氧化、抗肿瘤细胞增殖作用。采用紫外可见分光光度计、离子色谱仪和红外光谱仪对通关藤多糖的结构进行表征,通过自由基清除能力评价通关藤多糖体外抗氧化活性。利用四甲基偶氮唑盐(MTT)法测定不同浓度通关藤多糖分别作用于人肝癌细胞HepG224 h、48 h的细胞抑制率。结果显示通关藤多糖的多糖含量为72.83%,由Glc、Gal、Ara、Rha、Xyl、Man、ManA、GalA和GlcA组成,其摩尔比为58.5∶17.8∶17.1∶2.4∶1.2∶0.9∶0.9∶0.6∶0.5;通关藤多糖对DPPH自由基、ABTS自由基和羟自由基都具有一定的清除作用,且呈现浓度依赖性。通关藤多糖可抑制人肝癌细胞HepG2增殖,且呈浓度与时间依赖性。因此,通关藤多糖具有良好的体外抗氧化和抗肿瘤细胞增殖作用,具有潜在的开发应用价值。

通关藤总皂苷通过IL-6/STAT3/NF-κB信号通路改善肝癌H22小鼠肿瘤炎症的研究

目的探讨通关藤总皂苷对H22荷瘤小鼠抗肿瘤炎症作用及机制。方法建立H22荷瘤小鼠模型,随机分为模型组,阳性组(阿霉素),联合给药组(阿霉素^(+)通关藤总皂苷中剂量),通关藤总皂苷低(62.5 mg/kg)、中(125 mg/kg)、高(250 mg/kg)剂量组,另设空白对照组。给药14 d。观察各组小鼠的行为学变化;剥离肿瘤组织进行称重,计算抑瘤率。采用HE染色、流式细胞术、酶联免疫吸附法(ELISA)、反转录实时定量聚合酶链式反应(RTPCR)和蛋白免疫印迹法(Western blot)对H22荷瘤小鼠的肝脏组织和肿瘤组织病理形态、外周血淋巴细胞比例、血清炎症因子、STAT3和核转录因子-κB(NF-κB)通路相关基因和蛋白等指标进行检测。结果通关藤总皂苷能够明显改善H22荷瘤小鼠的精神、行为等状态;各给药组抑瘤率明显,联合给药的抑瘤率高于通关藤总皂苷和阿霉素单独给药,差异具有统计学意义(P<0.01);模型组小鼠肝脏组织肝窦可见肿瘤细胞,且有炎症细胞浸润现象,各给药组情况得到不同程度改善。模型组小鼠肿瘤组织内肿瘤细胞生长密集,可见异型性和病理性核分裂现象,炎细胞浸润,各给药组肿瘤细胞出现大片坏死现象;相较于模型组,各给药组CD3^(+)、CD4^(+)T淋巴细胞百分率升高,CD8^(+)T淋巴细胞百分率降低,CD19^(+)、CD20^(+)B淋巴细胞和CD3^(-)CD16^(+)NK细胞百分率升高,差异具有统计学意义(P<0.01);通关藤总皂苷中、高剂量组血清中白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的浓度较模型组降低(P<0.01);各给药组中STAT3、MCL-1、XIAP、BCL-2、NF-κB mRNA的表达和STAT3、p-STAT3、MCL-1、XIAP、BCL-2、NF-κB蛋白的表达均较模型组降低(P<0.01)。结论通关藤总皂苷对H22荷瘤小鼠的肿瘤炎症有抑制作用,其机制可能与通关藤总皂苷下调STAT3和NF-κB信号通路中相关基因和蛋白的表达量及提高机体免疫功能有关。

通关藤水煎液中抗骨肉瘤细胞活性成分研究

目的:研究通关藤水煎液中抗骨肉瘤细胞活性成分。方法:采用大孔树脂柱层析、硅胶柱层析、制备型高效液相色谱(HPLC)、结合磁共振成像波谱(NMR)、高分辨率质谱(HR-MS)等方法,对通关藤水煎液的化学成分进行系统分离与结构鉴定;采用细胞计数检测(CCK-8)方法进行抗骨肉瘤细胞活性评价。结果:分离鉴定了33个化合物。其中,化合物1~8、化合物24~28均为首次从通关藤中分得;化合物6、化合物9、化合物21、化合物22、化合物25、化合物26、化合物29~31均能显著抑制骨肉瘤细胞MG63、143B的增殖。结论:通关藤水煎液中多个成分具有抑制骨肉瘤细胞活性。研究结果为通关藤的应用及进一步开发提供了科学依据。

通关藤总皂苷对LPS诱导的HepG2细胞炎症改善作用研究

目的探讨通关藤总皂苷(MaT)对脂多糖(LPS)诱导的HepG2细胞炎症模型的抑制作用。方法采用四氮唑盐(MTT)法、酶联免疫吸附法(ELISA)筛选出最适LPS造模浓度和诱导时间,建立LPS诱导的HepG2细胞炎症模型;MTT法、ELISA法筛选出MaT干预炎症HepG2细胞浓度范围。采用反转录实时定量聚合酶链式反应RT-PCR技术,检测不同浓度MaT干预后炎症信号通路STAT3和NF-κB通路相关基因的表达。结果以2μg/mL LPS与HepG2细胞在37℃,5%CO_(2)的条件下共培养24 h,建立LPS诱导的HepG2细胞炎症模型。相较于正常组,模型组白介素-6(IL-6)、白介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的分泌水平显著提高(P<0.01);MaT干预后,IL-6、IL-8、TNF-α浓度呈剂量依赖性下降,并且下调了炎症信号通路中STAT3、MCL-1、XIAP、BCL-2、NF-κB mRNA的表达。结论通关藤总皂苷对LPS诱导的HepG2细胞炎症模型具有抑制作用,其机制可能为通关藤总皂苷通过抑制STAT3/NF-κB信号通路相关基因表达,减少炎症因子的释放有关。

基于网络药理学和实验验证探讨通关藤治疗乳腺癌作用机制

目的 基于网络药理学和实验验证探讨通关藤治疗乳腺癌的作用机制。方法 通过文献检索获得通关藤的活性成分,并利用SwissTargetPrediction数据库获得活性成分对应的靶点,通过GeneCards、GEPIA2、 OMIM、 PharmGKB、 TTD数据库获得乳腺癌的靶点,将药物靶点与疾病靶点取交集后,用Cytoscape3.9.0软件构建通关藤活性成分-乳腺癌-靶点网络,通过蛋白相互作用(PPI)网络筛选关键靶点,进行GO和KEGG通路富集分析,并筛选相关信号通路。对前10个关键靶点和主要活性成分进行分子对接验证。利用通关藤注射液体外干预人乳腺癌细胞株MDA-MB-231,CCK-8法和集落形成实验检测细胞增殖能力,Calcein-AM/PI染色和流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移能力,Western blot对PI3K-AKT信号通路进行验证。结果 从通关藤中筛选出11α-O-苯甲酰-12β-O-乙酰通关藤苷元B、咖啡酸、苦绳苷元A、山柰酚等37个活性成分和治疗乳腺癌的276个潜在作用靶点,PPI网络筛选出AKT1、EGFR、TNF、CTNNB1、IL-6等25个关键靶点,主要影响雌激素信号通路、ErbB信号通路、HIF-1信号通路和PI3K-AKT信号通路等。分子对接结果显示,通关藤的主要活性成分与核心靶点AKT1、ALB、CASP3、ESR1和TNF均有较好的结合活性。体外实验结果显示,通关藤注射液可抑制MDA-MB-231细胞增殖和迁移能力(P<0.01,P<0.001),诱导细胞凋亡(P<0.001),同时抑制PI3K-AKT信号通路激活(P<0.05,P<0.01)。结论 通关藤可能通过多靶点和多途径发挥抗乳腺癌作用,其机制可能与抑制PI3K-AKT信号通路有关。

基于网络药理学和体外实验探讨通关藤口服液抑制胃癌细胞增殖及联用厄洛替尼和阿帕替尼的增敏效应

目的利用网络药理学方法、分子对接及体外细胞实验探究通关藤(marsdenia tenacissima,MT)口服液抑制胃癌(gastric cancer,GC)增殖的潜在作用机制。方法通过搜索文献、检索TCMSP、Swiss Target Prediction数据库完成MT化学成分、靶点基因的收集;采用Gene Cards数据库获取疾病相关靶点;利用STRING平台和Cytoscape软件构建交集靶点的蛋白互作网络(protein-protein interaction,PPI);采用Metascape网络平台进行GO和KEGG信号通路分析;运用Auto Dock对部分活性成分与潜在靶点进行分子对接验证;采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测MT口服液对GC细胞增殖的抑制作用,分别与厄洛替尼和阿帕替尼联用的协同作用、流式细胞术检测MT口服液诱导GC细胞凋亡及对胃癌细胞周期的影响;采用RT-qPCR法检测MT口服液对GC细胞周期、凋亡相关基因表达的影响;采用Western blot法验证网络药理学和分子对接筛选的靶点。结果共得到MT主要活性成分17个,胃癌关键靶点1880个,主要信号通路167条。体外实验结果表明,40~200 mg/m L的MT口服液可有效抑制GC细胞增殖(P<0.01),与厄洛替尼和与阿帕替尼联用时,具有协同效应。40~160 mg/m L的MT口服液可诱导胃癌细胞凋亡(P<0.01),阻滞细胞周期G0/G1期(P<0.01)。RT-qPCR结果显示,通关藤口服液可以上调B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)相关X蛋白(Bcl-2-associated X protein,BAX)的表达量,下调Bcl-2、细胞周期蛋白依赖性激酶(cyclin dependent kinase,CDK)4、CDK6(P<0.05)的表达量。Western blot结果显示MT口服液,抑制磷酸化-表皮生长因子受体(phosphorylated-epidermal growth factor receptor,p-EGFR)、磷酸化-磷脂酰肌醇-3激酶(phosphorylated-phosphatidylinositol 3-kinase,p-PI3K)、磷酸化AKT丝氨酸/苏氨酸激酶(p-AKT)、磷酸化-雷帕霉素激酶的机制靶点(phosphorylated-mechanistic target of rapamycin kinase,p-mTOR)的表达水平,降低p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT1、p-mTOR/m TOR比值;同时上调BAX、肿瘤蛋白p53(tumor protein p53,P53)、细胞周期蛋白依赖性激酶抑制剂(cyclin dependent kinase inhibitor 1A,CDKN1A/P21),下调Bcl-2、CDK4、CDK6(P<0.05)水平。结论MT口服液可有效抑制胃癌细胞的增殖,阻滞细胞周期并诱导胃癌细胞凋亡,并提高小分子靶向药物厄洛替尼和阿帕替尼治疗胃癌的敏感性。其机制与经调控PI3K/AKT/m TOR信号通路上调BAX、P53、P21,下调Bcl-2、CDK4、CDK6的表达,并抑制EGFR信号通路有关,实验结果验证了网络药理学和分子对接的结果。

通关藤提取物联合化学治疗药物治疗肿瘤的系统评价再评价

目的为通关藤提取物联合化学治疗(简称化疗)药物治疗肿瘤提供科学、可靠的证据支持。方法采用计算机检索PubMed,The Cochrane Library,Web of Science,Embase及中国知网(CNKI)、万方(WanFang)、维普(VIP)、中国生物医学文献服务系统(CBM)、读秀数据库中自建库起至2023年3月13日的有关通关藤提取物联合化疗药物治疗肿瘤的相关文献,由2位研究者独立筛选出符合纳入标准的系统评价/Meta分析,采用多系统评价量表(AMSTAR 2)评价纳入文献的方法学质量,采用推荐分级的评估、制订与评价(GRADE)3.6系统工具对纳入文献的证据进行质量分级。结果共纳入12篇文献。AMSTAR 2量表评价结果显示,低质量1篇,极低质量11篇;GRADE分级结果显示,47项结局指标中,低质量证据9项,极低质量证据38项。与单用化疗药物比较,通关藤提取物联合化疗药物治疗肿瘤在提高临床有效率[OR=1.71,95%CI(1.53,1.91),P<0.00001]和疾病控制率[OR=1.89,95%CI(1.65,2.17),P<0.00001],改善卡氏(KPS)评分[OR=2.78,95%CI(2.39,3.22),P<0.00001],提高CD3+活性[MD=12.34,95%CI(11.63,13.06),P<0.00001]和CD4+活性[MD=9.14,95%CI(6.72,11.56),P<0.00001],降低神经毒性[OR=0.69,95%CI(0.54,0.86),P=0.001],降低白细胞减少[OR=0.46,95%CI(0.40,0.54),P<0.00001],改善恶心呕吐[OR=0.67,95%CI(0.55,0.80),P<0.0001],降低血小板减少[OR=0.52,95%CI(0.44,0.62),P<0.00001]方面的效果均显著更优。结论通关藤提取物联合化疗药物治疗肿瘤有助于提高临床有效率、疾病控制率、CD3+和CD4+活性,改善KPS评分和恶心呕吐,降低神经毒性、白细胞减少、血小板减少等,但因纳入文献的方法学质量与证据质量普遍较低,仍需更多高质量研究进一步证实。

通关藤对胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平的影响

目的探讨通关藤对胃癌小鼠类固醇受体共激活因子(Steroid receptor coactivator,Src)/黏着斑激酶(Focaladhesion kinase,FAK)信号通路及Th1/Th2相关因子水平的影响。方法将SPF级成年雌雄各半C57BL/6小鼠60只,根据随机数字表法分为空白对照组、模型组(注射生理盐水)、环磷酰胺组(注射环磷酰胺)、通关藤低、中、高剂量组(分别注射0.1 ml、0.2 ml、0.4 ml通关藤注射液),每组各10只。干预1次/2 d,于干预第21天时处死所有小鼠,颈部取血。测量小鼠体质量以及胸腺、脾、肿瘤质量。HE染色观察小鼠瘤体病理形态变化。流式细胞术检测小鼠CD4^(+)、CD8^(+)以及CD4^(+)/CD8^(+)水平,ELISA法检测白细胞介素-2(Interleukin-2,IL-2)、白细胞介素-4(Interleukin-4,IL-4)、白细胞介素-10(Interleukin-10,IL-10)、干扰素-γ(Interferon-γ,INF-γ)水平,Western blot法检测Src、磷酸化Src(p-Src)、FAK、磷酸化FAK(p-FAK)蛋白表达水平。结果与模型组比较,通关藤各干预组脾指数、胸腺指数水平升高,且随通关藤剂量增加,脾指数、胸腺指数水平、抑瘤率均升高,差异有统计学意义(P<0.05)。环磷酰胺组脾指数和胸腺指数明显低于各通关藤剂量组,差异有统计学意义(P<0.05);环磷酰胺组和通关藤高剂量组抑瘤率明显高于通关藤低、中剂量组,差异有统计学意义(P<0.05)。HE染色结果显示,模型组肿瘤细胞排列整齐且密集,而环磷酰胺组和通关藤各剂量组肿瘤细胞密度有所减少,且分布不均,并呈不同程度的肿瘤细胞坏死灶。与模型组比较,通关藤各干预组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05);相较于环磷酰胺组,通关藤低、中、高剂量组IL-2、INF-γ水平升高,IL-4、IL-10水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,IL-2、INF-γ水平升高越明显,IL-4、IL-10水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组CD4^(+)、CD4^(+)/CD8^(+)水平升高,CD8^(+)水平降低,差异有统计学意义(P<0.05),且通关藤干预剂量越高,CD4^(+)、CD4^(+)/CD8^(+)水平升高越明显,CD8^(+)水平降低越明显(P<0.05)。与模型组比较,环磷酰胺组和通关藤各干预组p-Src/Src、p-FAK/FAK蛋白水平降低,差异有统计学意义(P<0.05);与环磷酰胺组比较,通关藤低、中、高剂量组p-Src/Src、p-FAK/FAK蛋白水平升高,差异有统计学意义(P<0.05),且通关藤干预剂量越高,p-Src/Src、p-FAK/FAK蛋白水平降低越明显。结论通关藤可调节胃癌小鼠Src/FAK信号通路及Th1/Th2相关因子水平,从而发挥抑癌作用,且剂量越高,效果越显著。

通关藤的适宜采收期研究

测定不同生长期通关藤(Marsdenia tenacissima)的茎粗和茎长,采用香草醛-高氯酸法显色,在462 nm处测定通关藤中总皂苷的含量,研究通关藤的最适宜采收期。结果表明,通关藤总皂苷含量在两年生时达到最高,以后缓慢下降;产量在三年生时增长最快,之后增长缓慢。通关藤的最佳采收期要兼顾其产量和有效成分总皂苷的含量,两者都在相对较高的时候才是最佳采收期,确定其最佳采收期为两年半生至三年生的藤茎。

通光藤的化学成分研究

目的 :研究中药通光藤Marsdeniatenacissima的化学成分。方法 :利用溶剂提取和硅胶、SephadexLH 2 0等色谱方法对云南昆明产通光藤的醋酸乙酯可溶部位进行分离纯化 ,通过化学和光谱方法鉴定结构。结果 :共分离得到 8个化合物 ,已鉴定出其中 5个化合物 :大叶牛奶菜苷丁 (Ⅰ ) ,大叶牛奶菜苷乙 (Ⅱ ) ,牛奶菜醇 (Ⅲ ) ,二氢牛奶菜醇 (Ⅳ ) ,喙柱牛奶菜苷乙 (Ⅴ )。结论 :这 5个化合物均为首次从该植物中分离得到 ;通光藤的醋酸乙酯可溶部位含有较多的甾体酯苷类化合物。

通光藤中两个新C_(21)甾体苷类成分

目的 研究中药通光藤的化学成分。方法 用现代色谱技术分离化合物 ,用化学方法及现代光谱技术(IR ,UV ,MS ,1HNMR ,13 CNMR和各种 2D NMR技术 )对所得化合物的结构进行鉴定。结果 从云南昆明产通光藤茎中分离得到 2个化合物 ,分别鉴定为通光藤苷J(tenacissosidesJ,I)和通光藤苷K(tenacissosidesK ,II)。