HPLC法测定紫花前胡中紫花前胡苷的含量

目的:建立HPLC法测定中药紫花前胡中紫花前胡苷含量的方法,为其质量控制提供依据.方法:采用反相高效液相色谱法,Agilent Zorbax SB-C18色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇-水(40:60),流速为1mL·min-1,检测波长334 nm,柱温40℃.结果:紫花前胡苷在此色谱条件下获得良好分离,线性范围为25.0～300.0 ng(r2=0.9999),平均回收率为98.6%,RSD1.8%.结论:首次建立了两相系统HPLC法测定紫花前胡药材中紫花前胡苷含量的方法,该方法准确,可靠,对紫花前胡药材及其制剂的质量控制有较重要意义,已被2010版<中国药典>采纳.     

GC测定紫花前胡中紫花前胡苷元的含量

目的　建立GC测定紫花前胡根中紫花前胡苷元 (NDT)含量的方法。方法　采用SE - 30石英毛细柱(0 .2 2mm× 2 5m) ,液膜厚度 0 .2 5 μm ,FID检测器 ,以正二十五烷为内标物 ,并采用GC -MS联用技术 ,鉴定了样品中的NDT色谱峰和进行专属性考察。结果　NDT在 0 .0 89～ 1.0 6 2 μg之间有较好的线性关系 (r =0 .9999) ,NDT的加样回收率为 99.31%± 0 .33%。结论　该法简便、快速、准确、灵敏度高     

HPLC法测定白有胡中白花前胡丙素和紫花前胡中紫花前胡苷的含量

目的：建立高效液相色谱法测定白花前胡根中花前胡丙素（dl-praeruptorin A,Pd-Ia)和紫花前胡根中紫花前胡苷（modakenin,NDK)含量的方法。方法：色谱法Shim-Pack CLC-ODS,Pd-Ia的流动相为甲醇－水（79：21）和NDK的流动相为乙腈－甲醇－水（20：5：75）,流速为1．0mL.min^-1,紫外检测波长分别为323和334nm。结果：Pd-Ia及NDK的线性范围分别为0．08－0．8μg和0．01－0．10μg,相关系数均为0．9999,平均回收率分别为98．53％－99．27％和97．60％、99．87％,RSD小于2％。结论：方法准确,简便,快速,灵敏度高,对常用中药前胡的质量控制有较重要意义。     

HPLC法测定白花前胡中白花前胡丙素和紫花前胡中紫花前胡苷的含量

目的 :建立高效液相色谱法测定白花前胡根中白花前胡丙素 (dl-praeruptorinA ,Pd -Ia)和紫花前胡根中紫花前胡苷 (nodakenin ,NDK)含量的方法。方法 :色谱柱Shim -PackCLC -ODS ,Pd -Ia的流动相为甲醇 -水 (79∶2 1)和NDK的流动相为乙腈 -甲醇 -水 (2 0∶5∶75 ) ,流速为 1 0mL·min- 1 ,紫外检测波长分别为 32 3和 334nm。结果 :Pd -Ia及NDK的线性范围分别为 0 0 8～ 0 8μg和 0 0 1～ 0 10 μg ,相关系数均为 0 9999,平均回收率分别为 98 5 3%～ 99 2 7%和 97 6 0 %～99 87% ,RSD小于 2 %。结论 :方法准确 ,简便 ,快速 ,灵敏度高 ,对常用中药前胡的质量控制有较重要意义。     

紫花前胡化学成分的研究

目的研究中药紫花前胡［Peucedanum decursivum(Miq.)Maxim］根的化学成分。方法用硅胶柱色谱和制备型高效液相色谱进行分离纯化,用光谱分析和化学方法鉴定其结构。结果从紫花前胡根中分得7个线型吡喃香豆素类化合物,其结构分别鉴定为3′(S)-hydroxy-4′(R)-angeloyloxy-3′,4′-dihydroxanthyletin(1);3′(S)-acetoxy-4′(R)-hydroxy-3′,4′-dihydroxanthyletin(2);3′(S)-acetoxy-4′(R)-angeloyloxy-3′,4′-dihydroxanthyletin(3);Pd-C-IV(4);Pd-C-II(5);(+)-3′S-Decursinol(6);(+)-trans-Decursidinol(7)。结论化合物1和2为新化合物,分别命名为紫花前胡素D和紫花前胡素F;6和7为首次从该植物中分得。     

紫花前胡苷在大鼠体内的药代动力学研究

目的研究中药狭叶羌活和宽叶羌活主要化学成分之一的紫花前胡苷(ND)在大鼠体内的药代动力学。方法♂SD大鼠(200～220)g随机分组,每组5只,单次尾静脉注射,给药量为80mg.kg-1,从眼眶静脉丛分时取血、处理。采用反相高效液相色谱法、恒速洗脱,应用外标法测定ND在SD系大鼠血浆中的浓度,应用3P87软件计算主要药代动力学参数。结果在所建立的方法学下,ND的保留时间为6.7min;标准曲线为Y=2.0×10-4X+0.4(r=0.9999),在0.2～80mg.L-1的血浆浓度范围内呈良好的线性关系;血浆中最低检测浓度为0.01mg.L-1(S/N=3),最低定量浓度为0.1mg.L-1(S/N=10);在1.0、10.0和40.0mg.L-1低、中、高3个浓度下的提取回收率为76.23～83.72;日内和日间RSD均小于5.60%(n=3)。在室温和冷冻—解冻试验中,ND具有良好的稳定性。按80mg.kg-1单剂量单次静脉给药后,ND在大鼠体内的药代动力学过程符合开放二房室模型,主要药代动力学参数t1/2α、t1/2β、AUC、Vc、Vp、Vss和CL分别为7.02min、219.27min、1384.34mg.min.L-1、0.92L.kg-1、6.04L.kg-1、6.96L.kg-1和0.06L.kg-1.min-1。结论所建立的方法快速、准确、简便,能够满足ND药代动力学研究要求。按80mg.kg-1单剂量单次静脉给药后,ND在大鼠体内分布广泛,消除速度中等。     

HPLC法筛查百花定喘制剂中前胡投料

目的： 通过测定白花前胡甲素、白花前胡乙素和紫花前胡苷的含量,建立筛查百花定喘制剂中投料为前胡还是紫花前胡的HPLC方法。方法：采用Symmentry-C18（4.6 mm×250 mm,5 μm）色谱柱,流动相：甲醇（A）-0.1%磷酸溶液（B）,梯度洗脱,检测波长为321 nm,流速为1.0 mL·min^-1。结果：5批药材的标示名称与试验判断结果不一致;47批百花定喘制剂的筛查结果表明,未见紫花前胡投料,前胡存在投料不足的情况。结论：该方法专属性良好,可作为定性筛查白花定喘制剂中投料前胡的方法。     

高效液相色谱-串联质谱法测定羌活中异欧前胡素和紫花前胡苷北大核心CSCD

目的:建立高效液相色谱-串联质谱法测定羌活中异欧前胡素和紫花前胡苷含量。方法:羌活样品超声萃取甲醇稀释后分析。色谱分离采用C18反相色谱柱(150 mm×2.1 mm,3.5μm),流动相为0.1%乙酸水溶液和乙腈,梯度洗脱。串联质谱在多反应监测模式下检测目标分析物,以保留时间和特征离子对(母离子和2个碎片离子)信息比较进行定性和定量分析。结果:异欧前胡素和紫花前胡苷的检出限分别为0.03 ng·mL-1和0.015 ng·mL-1,定量限分别为0.10 ng·mL-1和0.05ng·mL-1,异欧前胡素和紫花前胡苷平均回收率分别为93.6%和94.5%,RSD分别为2.4%和2.6%。结论:该方法经方法学验证,可用于羌活中异欧前胡素和紫花前胡苷的分析。     

紫花牡荆素对人宫颈癌细胞凋亡和侵袭迁移的影响

目的 观察紫花牡荆素(CAT)对人宫颈癌HeLa和SiHa细胞凋亡和侵袭转移的影响。方法 不同浓度CAT作用于HeLa和SiHa细胞不同时间后,采用MTT法观察药物对细胞活力的影响,流式细胞仪检测凋亡率,划痕试验观察细胞迁移率,基质胶法测定细胞粘附百分率,Transwell试验检测侵袭细胞数,Western blotting分析CAT对SiHa细胞抑制转移蛋白nm23-H1和促转移蛋白MTA1表达的影响。结果 CAT显著抑制HeLa和SiHa细胞活力,增加细胞凋亡率;降低细胞迁移百分率和黏附百分率;减少侵袭细胞数;明显增加SiHa细胞nm23-H1蛋白表达,降低MTA1蛋白表达。结论 CAT显著诱导人宫颈癌HeLa和SiHa细胞凋亡,抑制其侵袭迁移,提示其具有潜在的抗宫颈癌作用。     

Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specific features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer.     

VEGF-C对宫颈癌凋亡影响的研究进展

血管内皮生长因子-C（VEGF-C）是一类很重要的淋巴管生成因子,其在宫颈癌淋巴管转移方面的研究较多,也证明了VEGF-C在宫颈癌发生发展、侵袭转移中起重要作用。然而,VEGF-C在宫颈癌中的作用机制并未完全明确,其对宫颈癌凋亡的影响值得继续探索。本文就VEGF-C在宫颈癌凋亡中的作用及其机制进行综述。     

白藜芦醇联合SB203580对宫颈癌细胞凋亡迁移的影响及机制

目的 探讨白藜芦醇联合SB203580对宫颈癌细胞凋亡迁移的影响及机制。方法 培养人宫颈癌HeLa细胞,CCK8实验检测不同浓度白藜芦醇作用于细胞48 h后对细胞增殖影响,挑选最佳作用浓度。将细胞分为四组,即对照组(A组):不做处理;SB203580组(B组),加10 μmol/L SB203580;白藜芦醇组(C组),加60 μmol/L白藜芦醇;白藜芦醇组+SB203580组(D组),加60 μmol/L白藜芦醇和10 μmol/L SB203580。流式细胞仪、Transwell小室检测四组细胞的凋亡率和迁移数,Western blot检测p38、p-p38、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase3)、B型白细胞/2型淋巴细胞样蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达。结果 白藜芦醇能显著抑制人宫颈癌HeLa细胞增殖,60 μmol/L白藜芦醇抑制了一半的细胞增殖(P＜0.001);B组(14.83±1.23)%细胞凋亡率显著低于A组(3.42±0.78)%,细胞迁移数(189.20±7.93)显著高于A组(135.40±7.23);C组(35.57±1.86)%和D组(23.45±1.45)%细胞凋亡率显著高于A组,C组(51.30±5.49)和D组(97.60±6.88)细胞迁移数显著低于A组;D组细胞凋亡率显著高于B组,低于C组,细胞迁移数显著低于B组,高于C组(P＜0.001);B组p-p38[(0.087±0.011比0.152±0.014)]、Cleaved-caspase3 [(0.162±0.011比0.093±0.009)]、Bax蛋白表达[(0.191±0.013)比(0.154±0.012)]显著低于A组,Bcl-2蛋白表达[(0.208±0.014)比(0.347±0.015)]显著高于A组;C组和D组p-p38[(0.282±0.016)、(0.201±0.015)]、Cleaved-caspase3[(0.315±0.013)、(0.223±0.012)]、Bax蛋白表达[(0.441±0.015)、(0.294±0.014)]显著高于A组,C组和D组Bcl-2蛋白表达[(0.071±0.010)、(0.156±0.012)]显著低于A组;D组p-p38、Cleaved-caspase3、Bax蛋白表达显著高于B组,低于C组,Bcl-2蛋白表达显著低于B组,高于C组(P＜0.001)。p38蛋白表达在各组之间差异无统计学意义(P＞0.05)。结论 白藜芦醇通过调控p38MAPK信号通路抑制人宫颈癌HeLa细胞增殖和迁移,并通过调节Cleaved-caspase3、Bax、Bcl-2促进细胞凋亡。     

Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in Leydig cells. This study investigated the effects of ZEA on rat Leydig cells. Results showed that ZEA at different doses significantly inhibited the growth of Leydig cells by inducing apoptosis. ZEA treatment upregulated Bax expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, followed by the cleavage of poly(ADP-ribose) polymerase (PARP), resulting in Leydig cell apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity.     

Nec-1对铝致神经细胞凋亡和自噬的作用

目的观察和探讨在铝致神经细胞死亡中,坏死抑制剂1(Nec-1)对凋亡和自噬的作用。方法用体外原代培养小鼠神经细胞的方法,制造铝损伤神经细胞模型,然后用细胞活力检测、荧光定量PCR(qRT-PCR)、蛋白印迹(Western-Blot)以及流式细胞术等方法从多角度对Nec-1的作用进行研究。结果①流式结果显示,在Al3+(2mmol/L)作用于神经细胞后,随着Nec-1剂量的增加,细胞的凋亡率呈下降趋势,且差异有统计学意义(P0.05);②qRT-PCR结果显示,以Al3+(2mmol/L)为对照,Nec-1(60和90μmol/L)可使神经细胞的凋亡和自噬相关基因的表达呈显著性下降(P0.05);③Western-Blot结果显示,同Al3+(2mmol/L)组比,Nec-1(60和90μmol/L)组凋亡相关蛋白caspase-3的表达下降(P0.01),Nec-1(30和60μmol/L)可使自噬相关蛋白LC3-Ⅱ表达下降(P0.05)。结论在本试验条件下,Nec-1可减少铝导致的神经细胞凋亡和自噬,起到保护神经细胞的作用。     

RASSFIA基因对胃癌细胞自噬的影响

目的探讨RASSFIA基因对胃癌细胞自噬的影响情况及其可能的机制。方法通过基因重组技术已构建了高表达RASSFIA基因的人胃癌SGC．7901细胞株,然后采用聚合酶链反应（PCR）技术检测SGC-7901细胞转染RASSFIA基因前后自噬BECLIN．1基因mRNA表达情况,并使用流式细胞仪检测SGC-7901细胞转染RASSFIA基因前后凋亡情况。结果胃癌SGC．7901细胞转染了RASSF1A基因后,凋亡增加,BELINE1表达下降。结论RASSFIA基因对胃癌细胞自噬有负调控作用。     

Riccardin D induces cell death by activation of apoptosis and autophagy in osteosarcoma cells

Macrocyclic bisbibenzyls, characteristic components derived from liverworts, have various biological activities. Riccardin D (RD), a liverwort-derived naturally occurring macrocyclic bisbibenzyl, has been found to exert anticancer effects in multiple cancer cell types through apoptosis induction. However, the underlying mechanisms of such effects remain undefined. In addition, whether RD induces other forms of cell death such as autophagy is unknown. In this study, we found that the arrest of RD-caused U2OS (p53 wild) and Saos-2 (p53 null) cells in G1 phase was associated with the induction of p53 and p21^WAF1 in U2OS cells. RD-mediated cell cycle arrest was accompanied with apoptosis promotion as indicated by changes in nuclear morphology and expression of apoptosis-related proteins. Further studies revealed that the antiproliferation of RD was unaffected in the presence of p53 inhibitor but was partially reversed by a pan-inhibitor of caspases, suggesting that p53 was not required in RD-mediated apoptosis and that caspase-independent mechanisms were involved in RD-mediated cell death. Except for apoptosis, RD-induced autophagy occurred as evidenced by the accumulation of microtubule-associated protein-1 light chain-3B-II, formation of AVOs, punctate dots, and increased autophagic flux. Pharmacological blockade of autophagy activation markedly attenuated RD-mediated cell death. RD-induced cell death was significantly restored by the combination of autophagy and caspase inhibitors in osteosarcoma cells. Overall, our study revealed RD-induced caspase-dependent apoptosis and autophagy in cancer cells, as well as highlighted the importance of continued investigation on the use of RD as a potential anticancer candidate.     

Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells

Oxidative stress created by environmental toxicants activates several signaling pathways. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and up-regulated in response to intracellular reactive oxygen species (ROS). Recently, we reported that fipronil (FPN)-induced mitochondria-dependent apoptosis is mediated through ROS in human neuroblastoma SH-SY5Y cells. In this study, we explored the role of autophagy to prevent FPN neurotoxicity. We investigated the modulation of FPN-induced apoptosis according to autophagy regulation. FPN activated caspase-9 and caspase-3, and induced nuclear fragmentation and condensation, all of which indicate that FPN-induced cell death was due to apoptosis. In addition, we observed FPN-induced autophagic cell death by monitoring the expression of LC3-II and Beclin-1. Exposure to FPN in SH-SY5Y cells led to the production of ROS. Treatment with N-acetyl-cysteine (NAC) effectively blocked both apoptosis and autophagy. Interestingly, pretreatment with rapamycin, an autophagy inducer, significantly enhanced the viability of FPN-exposed cells; the enhancement of cell viability was partially due to alleviation of FPN-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment with 3-methyladenine (3MA) a specific inhibitor for autophagy, remarkably strengthened FPN toxicity and further induced activation of caspase-3 in these cells. Our studies suggest that FPN-induced cytotoxicity is modified by autophagy regulation and that rapamycin is neuroprotective against FPN-induced apoptosis through enhancing autophagy.     

Mulberry fruits extracts induce apoptosis and autophagy of liver cancer cell and prevent hepatocarcinogenesis in vivo

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. Many risks factors induce liver chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Mulberry fruits containing polyphenols to remove free radicals and mitigate inflammation has been reported to not only against gastric cancer, melanoma and leukemia but also prevent liver injury induced by alcohol or CCl₄ in previous researches. The aim of this study is to examine whether Mulberry could inhibit hepatocarcinogenesis. In animal experiment, diethylnitrosamine (DEN) was used to induce hepatic tumorgenesis. After injecting DEN, the rats treated with mulberry water extracts (MWE) had less and smaller tumor than others without MWE. Moreover, MWE reduced the serum ALT and AST, HCC marker, cleavage caspases, Ser-15-p53 and Ser46-p53 induced by DEN. Further, we observed that mulberry polyphenol extracts (MPE) inhibited the cell growth of HepG2 cell and Hep3B cell. By using flow cytometry and western blotting methods, MPE induced HepG2 cell apoptosis by increase subG1 cells and the elevated expression of caspase-3/8/9. Instead of apoptosis, MPE caused Hep3B cells autophagy by inhibiting Akt and mTOR phosphorylation. Comprehensively, mulberry extracts has a potential to be a health supplement to prevent hepatocarcinogenesis in the future.