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.

Isolation and Crystal Structure of Ent-kaurane Diterpenes from Rubus corchorifolius L. f.

In this study, ent-Kaurane-3β,16β,17-trioi （1） and ent-kaurane-2a,16β,17-triol （2）, were isolated from the leaves ofRubus corchorifolius L. f. Their structures were elucidated based on extensive spectroscopic analysis. NMR experiments identified the two compounds and X-ray crystallographic analysis confirmed their crystal structures. The crystal of 1 belongs to monoclinic with space group P21 and a = 10.7641（3）, b = 7.2789（3）, c = 11.7862（4） A, β = 95.275（3）°, V = 919.55（6） A3, Z = 2, C20H34O4, Mr = 322.49 g/mol, Dc = 1.230 g/m3, F（000） = 376, 2 = 1.54178 A,μ = 0.661 mm-1, R = 0.0319, and wR = 0.0811 for 10889 observed reflections （I〉 2σ（I））. The crystal of 2 is classified as orthorhombic with space group P212121 and a = 10.7641（3）, b = 12.72224（19）, c = 21.3929（3） A, V= 1748.94（4） A3, Z= 4, C20H3404, Mr = 322.47, De = 1.225 Mg/m3 F（000） = 712, λ = 1.54184 A,μ = 0.625 mm-1, R = 0.0303 and wR = 0.0759 for 10470 observed reflections （1〉 2σ（I））. Meanwhile, the compound revealed low inhibitory activities toward HepG2, MCF-7, and SCG-7901 cells.

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。

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.

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.

茅莓分散片的制备及溶出度测定

目的 优选茅梅分散片的制备工艺,并测定其溶出度。方法 在单因素试验的基础上,以微晶纤维素(MCC)、交联聚乙烯吡咯烷酮(PVPP)、低取代羟丙基纤维素(L-HPC)为影响因素,崩解时间为评价指标,采用中心复合设计-效应面法优化茅莓分散片的处方工艺。再以茅莓总皂苷为指标,参考中国药典方法测定溶出度。结果最优处方工艺为茅莓总皂苷浸膏粉用量为36.9%,MCC用量为42%,PVPP用量为13%,L-HPC用量为8%,微粉硅胶用量为0.1%,模型相关系数较高。所制分散片崩解时间小于3 min,在50 min时,累积溶出度达到88%以上。结论 优选工艺稳定可靠,可用于茅莓分散片的制备。

Structure analysis of ethyl ferulate from Rubus corchorifolius L.f.leaves and its inhibitory effects on HepG2 liver cancer cells

The tender leaves of Rubus corchorifolius L.f.(R.corchorifolius)have been made into herbal tea for relieving coughs in East and Southeast Asia from ancient times.Herein,we obtained an ethyl acetate extract from R.corchorifolius leaves and isolated a major bioactive compound within.Structural analysis of the isolated compound was performed by spectroscopic methods and the compound was identified as ethyl ferulate.Effect of ethyl ferulate on the growth and proliferation of human liver cancer HepG2 cells and normal human liver LO2 cells was determined by MTT assay.We found that ethyl ferulate significantly suppressed the cell proliferation of HepG2 cells(IC_(50)26.4μM),while it did not cause cytotoxicity on LO2 cells.In addition,flow cytometry analysis demonstrated that ethyl ferulate inhibited HepG2 cell growth via inducing cellular apoptosis.Specifically,ethyl ferulate reduced the electrical potential of the mitochondrial membrane and increased the concentration of calcium ions inside cells.Moreover,these changes were accompanied by increases in the expression of the pro-apoptotic proteins,such as cleaved caspase-3 and Bax.In contrast,cell morphology and growth of LO2 cells remained unaffected.In conclusion,for the first time,our observations demonstrated that ethyl ferulate possesses great potential as an effective and safe antitumor agent for cancer chemoprevention in humans.

茅莓化学成分的分离鉴定

目的研究茅莓RubusparvifoliusL的化学成分。方法采用柱色谱和制备型高效液相色谱进行分离纯化,通过理化方法和光谱分析鉴定化合物结构。结果从茅莓的乙醇提取物中分得4个三萜成分和1个二糖成分,分别鉴定为suavissimosideR1(1),nigaichigosideF1(2),camelliageninA(3),camelliageninC(4)和sucrose(5)。结论化合物3～5均为首次从该植物中分得。

茅莓化学成分研究

目的:研究茅莓的化学成分。方法:利用硅胶柱层析色谱进行分离纯化,通过波谱分析鉴定化合物结构。结果:从茅莓中分离得到了7个化合物,分别是:β-谷甾醇(Ⅰ)、月桂酸(Ⅱ)、邻硝基苯酚(Ⅲ)、β-胡萝卜苷(Ⅳ)、蔷薇酸(Ⅴ)、3β,16α2,2α,28-四羟基齐墩果-12-烯(Ⅵ)和(+)-儿茶素(Ⅶ)。结论:其中,化合物Ⅲ、Ⅶ为首次从该植物中分离得到。

茅莓黄酮体外抗氧化活性研究

为了研究茅莓(Rubus parvifolius L.)不同部位(根、茎、叶)中总黄酮的体外抗氧化活性,试验分别考察了茅莓根、茎、叶中总黄酮对羟自由基(·OH)、超氧负离子(O2-)、有机自由基(DPPH·)的清除能力及对大鼠肝组织匀浆自发性脂质过氧化反应产物丙二醛(MDA)的抑制率。结果表明,茅莓各部位黄酮在一定浓度范围内,其清除羟自由基(·OH)、超氧负离子(O2-)、有机自由基(DPPH·)及抑制MDA的作用均呈量效关系。说明茅莓根、茎、叶中总黄酮均具有一定的体外抗氧化活性。

茅莓总黄酮提取工艺的优化

从茅莓(Rubus parvifolius L.)中提取总黄酮,采用吸光光度法测定提取液中总黄酮的浓度。在单因素试验的基础上采用L9(34)正交试验考察乙醇体积分数、料液比、超声处理时间及提取温度对总黄酮提取率的影响。结果表明,茅莓茎中总黄酮最佳提取工艺条件为乙醇体积分数30%、料液比1∶50(m∶V,g/mL)、提取温度80℃、超声处理时间90 min,此工艺条件下茅莓茎中总黄酮提取率为4.7 mg/g,此时叶中总黄酮的提取率高达12.2 mg/g,远高于根和茎中的。

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。

山莓中两个新二萜的分离与鉴定

为研究山莓(Rubus corchorifoliusL. f)的化学成分,应用硅胶柱色谱进行分离纯化,根据化合物的理化性质和波谱数据鉴定结构。本文分离鉴定了2个贝壳杉烷二萜化合物,其结构分别为对映-贝壳杉烷-3,α16,α17,19-四醇(1),对映-2-羰基-16α-羟基-贝壳杉烷-17-β-D-葡糖苷(2)。化合物1、2均为新化合物。

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。

茅莓根化学成分研究

目的:研究茅莓根的化学成分。方法:采用硅胶柱层析、葡聚糖凝胶柱和制备高效液相色谱等方法对茅莓根的化学成分进行分离纯化,并根据理化性质和波谱数据鉴定化合物结构。结果:从茅莓根的95%乙醇提取物中分离得到6个化合物,分别鉴定为:3β-乙酰氧基-11α,12α-环氧-齐墩果烷-28,13β-内酯(Ⅰ)、3-O-乙酰坡模酸(Ⅱ)、熊果酸(Ⅲ)、3β-乙酰氧基-12-烯-28-乌苏酸(Ⅳ)、蔷薇酸(Ⅴ)、β-谷甾醇(Ⅵ)。结论:其中,化合物Ⅰ、Ⅱ、Ⅳ为首次从该植物中分离得到。

茅莓的生药学研究

目的对药用植物茅莓进行形态组织学鉴别,为鉴定该药材提供参考依据。方法采用性状鉴定、显微鉴定及光谱鉴定。结果对茅莓根、茎、叶及粉末的显微特征进行了描述和测定,发现茅莓根和叶柄的横切面细胞内、叶肉组织内含有许多草酸钙簇晶,茎表皮、叶柄表皮及叶的上下表皮细胞均含有非腺毛。经光谱扫描发现其总皂苷提取液经显色后在可见区具有明显的吸收峰。结论形态组织学特征及光谱特征可用于茅莓的鉴定。

山莓叶挥发油成分分析及抑菌活性研究

目的:研究山莓叶挥发油的化学成分及抗菌活性。方法:用水蒸气蒸馏法提取山莓叶挥发油,利用GC-MS联用仪和Xcalibur软件对山莓叶挥发油的化学成分进行分析鉴定,并采用K-B滤纸片法对其抗菌活性进行研究。结果:鉴定其中44个化合物,占挥发油总量的88.52%;挥发油浓度为50%时具有很好的杀菌作用;挥发油的浓度为0.5%仍具有很好的抑菌作用。结论:该结果为山莓叶的应用提供了依据。

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。

山莓生态习性与叶的解剖结构观察

对湖南省广泛分布的山莓实地踏察和生态习性观察表明,山莓系荒地的一种先锋植物,耐贫瘠,适应性强,属阳性植物.在林缘、山谷阳坡生长,有阳叶、阴叶之分,是湖南省植被的一个广布种.叶的解剖结构为典型的双子叶植物二面叶,由表皮、栅栏组织、海绵组织和维管束组成.叶柄由表皮、基本组织和维管束组成.叶片显著特点为:第一,单细胞表皮毛的数量多于腺毛.腺毛头部大略为球形,多细胞柄部较长,由一行或两行细胞组成.第二,幼叶、阴叶栅栏组织呈“小丘”状,阳叶栅栏组织细胞体积较小,排列紧密,孔隙度小,海绵组织疏松,气下室空腔大.第三,主脉、叶柄的厚角组织和薄壁细胞内多数有晶簇,成含晶异细胞.