Neuroprotective effects of total saponins from Rubus parvifolius L. on cerebral ischemia/reperfusion injury in rats

This study examines the neuroprotective effects and mechanisms of action of total saponins from Rubus parvifolius L. （TSRP） on focal cerebral ischemia and reperfusion injury in rats. Focal cerebral ischemia and reperfusion injury was performed in rats using the suture method. The results indicate that intragastric injection of TSRP, at 5, 10 and 20 mg/kg, could decrease neurological impairment, reduce cerebral infarct volume, diminish pathological changes, and significantly inhibit the apoptosis of neurons surrounding the ischemic area. In addition, TSRP upregulated the expression of the anti-apoptotic factor Bcl-2, at the protein and mRNA levels, and it downregulated the expression of the pro-apoptotic factor Bax, at the protein and mRNA levels. These findings indicate that TSRP protects against cerebral ischemia/reperfusion injury, and that it may do so by regulating the expression of Bcl-2 and Bax.

Total saponins in Rubus parvifolius L.induce lymphoma cells apoptosis through upregulated Bax/Fas and downregulated Bcl-2 in vivo and in vitro

Purpose:To investigate the effect of total saponins of Rubus parvifolius L.(TSRP)on lymphoma Raji cells and further discuss its mechanism.Methods:The model of nude mice bearing Raji cells was established,the volume,weight and inhibition rate of the transplanted tumor were analyzed and compared after different concentrations of TSRP treatment.Cell apoptosis and expression of Bcl-2,Bax,Fas proteins were detected by TUNEL and immunohistochemiscal method respectively.Effects of TSRP on cell proliferation were tested with MTT assay in vitro.Cell apoptosis and expression of Caspase-9,Caspase-3,Bcl-2,Bax and Fas proteins were tested with DAPI staining and Western blot.Results:TSRP significantly reduced the volume and tumor weight of Raji subcutaneous transplanted tumor and induced the apoptosis of Raji cells in vivo.The tumor inhibition rate of high-dose(100 mg/kg)TSRP is 90.84%.The TUNEL test results show that the fluorescence intensity of the tumor issue treated with TSRP is significantly improved.Compared with the control group,the fluorescence intensity of high-concentration TSRP is 82.43 ± 7.81,which is significantly different(P<0.001).The results of immunohistochemistry test showed that the Bcl-2 expression of Raji cell treated with TSRP is obviously reduced,and Bax expression is obviously increased.Meanwhile,compared with that of control group,Fas expression is obviously reduced.MTT assay showed that TSRP can significantly inhibit proliferation of Raji cells with dose dependence.The inhibition rate of 400 μg/mL TSRP is 53.46 ± 4.90%(P<0.001).DAPI staining results showed that TSRP can significantly induce cell apoptosis.According to Western blot results,it is found that TSRP can significantly inhibit activity of Bcl-2 and increase Bax expression,and TSRP can also inhibit Fas expression.Meanwhile,expression of Caspase-9 and Caspase-3 is also increased.Conclusion:TSRP could inhibit the proliferation of lymphoma via induction of apoptosis in a time and dose-dependent manner.Apoptotic signaling induced by TSRP was characterized by up-regulating Bax,Fas and Caspase-8 protein expression,and down-regulating of Bcl-2 protein expression.

Total Saponins of Rubus Parvifolius L.Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways

Objective： To investigate the anti-leukemia effect of total saponins of Rubus parvifo/ius L. （TSRP） on K562 cell xenografts in nude mice and the mechanisms of action. Methods： The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group（Am-c） and 3 TSRP groups （20, 40 and 100 mg/kg）. The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin （HE） and terminal dexynucleotidyl transferase （TdT）-mediated dUTP nick end labeling （TUNEL） staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 （STAT3）, eukaryotic initiation factor 4E （eIF4E） and B-cell lymphoma-2 （bcl-2） were determined with immunohistochemistry tests. Results： Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a significant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a significant decrease in tumor growth rate and tumor weight in comparison to the control group （all P〈0.05）. The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of elF4E and STAT3 were decreased obviously after the treatment of TSRP. Conclusion： TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

茅莓总皂苷联合化疗药通过Fas途径协同诱导白血病HL-60细胞凋亡研究

目的 :探讨茅莓总皂苷(TSRP)抑制HL-60细胞及分别与化疗药高三尖杉酯碱(Hom)、阿糖胞苷(Ara-c)的协同作用,并探讨其协同作用的可能机制。方法 :MTT法检测不同浓度的TSRP与化疗药联用对HL-60细胞增殖影响;DAPI染色法观察一定浓度下TSRP联合化疗药诱导HL-60细胞凋亡的形态变化;Annexin V-FITC/PI检测不同浓度的TSRP与化疗药联用对HL-60细胞凋亡情况;RT-PCR检测TSRP联合化疗药干预HL-60白血病细胞后Fas和Caspase-3 mRNA表达。以上实验均用药物相互作用指数(CDI)评价药物协同作用。结果:MTT显示在一定浓度范围内TSRP和化疗药有显著的协同作用;DAPI染色法和Annexin V-FITC/PI法检测到一定浓度范围内TSRP联合化疗药后显示明显的凋亡,CDI均<1,具有协同作用;RT-PCR检测到在一定浓度范围内TSRP联合化疗较单用TSRP的FasmRNA表达明显增加和Caspase-3 mRNA表达明显减少,CDI均<1,具有协同作用。结论:TSRP分别和化疗药Hom或Ara-c联用对HL-60细胞有明显的协同增殖抑制和诱导凋亡作用,可能是通过Fas-Caspase途径增强凋亡效应,从而增加化疗药物的敏感性,具有良好的临床应用前景。

茅莓总皂苷诱导HL-60白血病细胞凋亡机制研究

目的:明确茅莓总皂苷(TSRP)在体外对HL-60细胞抑制增殖和诱导凋亡作用。方法:MTT法检测不同浓度TSRP对HL-60细胞的增殖抑制;使用DAPI和Annexin V-FITC/PI法观察TSRP诱导凋亡作用;RT-PCR法检测TSRP对HL-60细胞Bcl-2、Bax、Caspase-3、Caspase-9和Fas mRNA表达的影响。结果:MTT显示TSRP在浓度200、400、800 mg/L对HL-60细胞生长抑制呈剂量依赖性,浓度800mg/L时吸光度为(0.232±0.030),与空白对照组比较差异显著(P<0.01)。DAPI显示TSRP在一定浓度内干预HL-60细胞后出现明显凋亡现象;Annexin V-FITC/PI凋亡检测显示经不同浓度TSRP处理后,HL-60细胞的早期凋亡率明显增加;RT-PCR检测到在一定浓度范围内,TSRP显著抑制细胞Bcl-2和Fas mRNA表达,同时Bax、Caspase-9和Caspase-3 mRNA表达明显增加,以上均呈剂量依赖性。结论:在体外TSRP是通过Bcl-2和Fas途径诱导HL-60细胞凋亡,具有较好的临床应用前景。

Rubus Parvifolius L.Inhibited the Growth of Leukemia K562 Cells in Vitro and in Vivo

Objective：To determine the antiproliferative activity of Rubus parvifolius L.（RP）extract,its medicinal serum and RP total saponins（RPTS）against K562 cells in vitro and in vivo.Methods：Nude mice models bearing leukemia tumors were treated with different concentrations of RP extract.The size,weight and histopathological change of leukemic tumors were determined.Semi-solid agar culture and methylthiazolyl tetrazolium（MTT）assay were used to determine in vitro the inhibition of colony formation and proliferation of K562 cells respectively by different concentrations of RP medicinal serum and RPTS.Results：RP extract had a tumor inhibition rate of 84.8%when administered to mice at a dose of 1.0 g/day of crude RP root equivalent.Semi-solid agar culture of K562cells in the presence of 20%（v/v）of RP medicinal serum and 150 mg/L RPTS demonstrated a 50.8%and 100%inhibition of the colony forming unit（CFU）-K562,respectively.The same doses of RP medicinal serum and RPTS showed a proliferation inhibition of 31.4%and 86.3%,respectively against K562 cells in MTT assay.Conclusion：RP extract and RPTS show effective antiproliferative activity against myeloid leukemia cells in vitro and in vivo.

茅莓总皂苷对局灶性脑缺血/再灌注大鼠的比较蛋白质组学研究

目的采用蛋白质组学技术研究茅莓总皂苷对脑缺血/再灌注大鼠脑组织差异蛋白的影响,进一步揭示茅莓总皂苷的脑保护作用及抗损伤修复机制。方法建立脑缺血/再灌注大鼠动物模型,分别提取蛋白质后进行2-DE电泳分离,应用相关软件比较分析,找出差异蛋白,酶切消化后进行肽指纹图谱分析,通过数据检索,初步鉴定差异蛋白质。结果与模型组比较,茅莓总皂苷组找到29个差异点,其中18个蛋白点表达上调,11个蛋白点表达下调,鉴定了其中7个蛋白。结论茅莓总皂苷对脑保护作用及抗损伤修复机制的机制是多个蛋白质共同参与的结果。

茅莓总皂苷和高三尖杉酯碱、阿糖胞苷体外协同诱导白血病细胞凋亡的机制研究

目的:探讨茅莓总皂苷(TSRP)抑制K562和人原代白血病细胞及分别与化疗药高三尖杉酯碱(Hom)、阿糖胞苷(Ara-c)的协同作用,并探讨其协同作用的可能机制。方法:MTT法和PHA-LCM液相-半固体二步培养法检测不同浓度的TSRP对K562和人原代白血病细胞增殖及其与化疗药联用后的影响;观察TSRP分别和Hom、Ara-c联用的抑制作用,用药物相互作用指数评价药物协同作用;DAPI染色法观察一定浓度下TSRP协同化疗药诱导K562细胞凋亡;Western blot检测促凋亡蛋白Cleaved caspase-3、Cleaved caspase-9和Cleaved PARP以及抗凋亡蛋白Survivin的表达。结果:TSRP能够抑制K562和人原代白血病细胞的增殖,呈浓度依赖性,在一定浓度范围内TSRP和化疗药有显著的协同作用;DAPI染色法观察TSRP和化疗药协同较单用化疗药出现明显凋亡现象,诱导细胞发生凋亡;Western blot检测TSRP联合Hom较单用Hom显著提高了K562细胞内PARP、Cleaved caspase-3和Cleaved caspase-9的活性;TSRP联合Ara-c较单用Ara-c显著提高了K562细胞内PARP和Cleaved caspase-3的活性,但是对Cleaved caspase-9的活性影响差别不大;TSRP联合Hom或Arac对Survivin的活性影响均未见明显差别。结论:TSRP分别和化疗药Hom或Ara-c联用对K562和人原代白血病细胞有明显的增殖抑制和诱导凋亡作用,且主要是通过线粒体途径增强凋亡效应,从而增加化疗药物的敏感性,具有良好的临床应用前景。

茅莓总皂苷对三种皮肤肿瘤的体外抗肿瘤作用

目的评价茅莓总皂苷对三种皮肤肿瘤的体外抗肿瘤药效作用。方法用A375黑色素瘤细胞、Hut-78人皮肤T细胞淋巴瘤细胞和A431人表皮癌细胞为癌细胞株,观察茅莓总皂苷的体外抗肿瘤作用。结果茅莓总皂苷对A375黑色素瘤细胞和Hut-78人皮肤T细胞淋巴瘤两种皮肤肿瘤细胞的增殖有明显的抑制作用,与对照组比较差异有显著性(P<0.05);对人表皮肤癌细胞无明显抑制作用。结论茅莓总皂苷对A375黑色素瘤细胞和Hut-78人皮肤T细胞淋巴瘤两种皮肤肿瘤细胞有明显的抗肿瘤活性。

茅莓总皂苷的含量测定

目的:建立茅莓总皂苷的含量测定方法。方法:通过对样品采用不同溶剂及不同提取方法所得的提取液显色后,以分光光度法测定总皂苷的含量。结果:本法测定的线性范围为22-176μg(r=0．9997);平均回收率为102．1％,RSD=2．82％ (n=6);样品以70％乙醇回流提取所得的总皂苷含量最高。结论:该方法灵敏、可靠,显色后的样品比较稳定,可用于茅莓药材的质量控制及品质评价。

大孔吸附树脂富集纯化茅莓总皂苷工艺研究

目的 :研究大孔吸附树脂富集、纯化茅霉总皂苷的工艺条件及参数。方法 :以茅莓总皂苷的洗脱率和纯度为考察指标 ,研究大孔吸附树脂富集、纯化茅莓总皂苷的吸附性能和洗脱参数。结果 :茅莓提取液 1 9.3mL(0 .77mg·mL- 1 )上大孔吸附树脂柱 (干重 2 .68g ,1cm× 2 0cm) ,用蒸馏水 1 0 0mL、50 %乙醇 1 0 0mL依次洗脱 ,茅莓总皂苷富集于 50 %乙醇洗脱液部分 ,且除杂质效果好。结论 :通过大孔吸附树脂富集、纯化后 ,茅莓总皂苷的洗脱率在 79.2 %以上 ,总固物中茅莓总皂苷含量可达 55 .3 %。说明采用本法富集。

红梅消总皂苷分离纯化及体外抗凝血活性研究

目的优选红梅消总皂苷纯化工艺,并对其进行体外抗凝血实验研究。方法以红梅消总皂苷吸附率和转移率为指标,考察大孔吸附树脂型号、上样液质量浓度与上样量等因素,优化纯化工艺,再通过研究凝血酶原时间和抗凝血酶作用进行体外抗凝血实验。结果红梅消总皂苷最佳纯化工艺为采用D101型大孔吸附树脂,用2 BV(60 ml)的0.5 g/ml上样液(以原药材计),以3 BV/h的流速进行上样,静态吸附30 min,再用4 BV的50%乙醇以3 BV/h的流速洗脱。红梅消总皂苷对凝血酶原时间和抗凝血酶作用影响较大。结论优选工艺稳定可靠,可用于红梅消总皂苷纯化及抗凝血实验研究。

茅莓提取物及有效部位的药理作用研究进展

茅莓Rubus parvifolius为蔷薇科悬钩子属落叶小灌木,其提取物及有效部位具有抗肿瘤、保护神经、保护肝损伤等药理作用。抗肿瘤是其研究热点,在体内外实验中,茅莓提取物对人恶性黑色素瘤、白血病、肝癌均表现出明显的抗肿瘤作用,可显著抑制肿瘤细胞生长、侵袭和迁移,以及诱导细胞凋亡,其抗肿瘤的主要有效部位为茅莓总皂苷。茅莓提取物对脑缺血-再灌注损伤也有神经保护作用,作用机制为增加Bcl-2表达和降低Bax表达,主要有效部位为茅莓总皂苷。茅莓提取物的抗氧化能力与清除羟自由基、超氧负离子、有机自由基及抑制丙二醛等有关,主要有效部位为茅莓总黄酮。茅莓提取物对肝脏损伤也有保护作用,与清除自由基活性相关,主要有效部位为酚类化合物。茅莓提取物及有效部位的其他药理作用还有抗炎、抗骨吸收、抗菌、抗疲劳等。综述了茅莓提取物及有效部位的药理作用及研究现状,以期为茅莓的进一步研究及新药开发提供参考。

正交试验优选茅莓总皂苷的提取工艺

目的:优选茅莓总皂苷的提取工艺。方法:在单因素考察的基础上,以乙醇浓度、固液比、提取温度和提取时间为考察因素,以茅莓总皂苷提取率为指标,优选茅莓总皂苷的提取工艺。结果:最佳提取工艺为乙醇浓度80%,提取时间为3h,固液比为1:20,提取温度为80℃。在此条件下,茅莓总皂苷提取率达到1.994%。结论:该工艺稳定、可行,可为工业化生产提供理论依据。