六神丸诱导HL-60细胞凋亡的实验研究

目的:从细胞凋亡角度探讨六神丸治疗白血病的机理,为临床应用提供依据。方法:分别制备六神丸乙醇提取剂和六神丸含药血清,用MTT法检测六神丸对HL-60细胞增殖的抑制作用,吉姆萨染色观察细胞凋亡的形态学变化,原位末端标记(POD)法检测细胞凋亡现象。结果:六神丸乙醇提取剂对HL-60细胞的增殖有明显的抑制作用,且呈现浓度依赖关系;中剂量组所得六神丸含药血清对HL-60细胞增殖的抑制作用优于低剂量组和高剂量组。吉姆萨染色显示六神丸乙醇提取剂和六神丸含药血清处理过的HL-60细胞均出现典型的凋亡形态。原位末端标记表明,六神丸乙醇提取剂诱导HL-60细胞凋亡在一定范围内呈现时间-浓度依赖关系;六神丸含药血清诱导HL-60细胞凋亡作用优于As_2O_3血清组和空白血清组(P<0.01)。结论:六神丸体外诱导HL-60细胞凋亡可能是六神丸治疗白血病的作用机制之一。     

表没食子儿茶素没食子酸酯诱导HL-60细胞程序性死亡

 目的：探讨儿茶素类化合物的抗癌作用机制。方法：以儿茶素的主要成分表没食子儿茶素没食子酸酯(EGCG)为实验材料，使用噻唑蓝(MTT)还原法，DNA凝胶电泳，透射电镜技术，观察了EGCG对人急性早幼粒白血病细胞(HL 60)程序性死亡的影响。结果：0.55×10^-3mol·L^-1EGCG作用5 h后，琼脂糖凝胶电泳下，HL 60细胞呈现典型的DNA条带，透射电镜观察到凋亡小体。结论：EGCG对HL-60细胞程序性死亡的诱导活性平行于它的细胞杀伤作用。     

A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines

The anticancer activity of eight crude extracts of Smilax china L. rhizome (SCR) against HeLa cells was assessed by MTT assay and clonogenic assay, the fraction rich in flavonoids had show good activity against HeLa cells. A bioassay-guided separation on this extract lead to the detection of kaempferol-7-O-beta-D-glucoside (KG), which belongs to flavonoid glycoside, displayed marked anticancer activity. We evaluated its in vitro cytotoxicity and antiproliferative effect in a panel of established cancer cell lines by MTT assay and clonogenic assay. KG induces A375 and HL60 cells apoptosis, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis. Fluorescent staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the A375 and HL60 cells. Flow cytometric analysis shown that A375 and HL60 cells treated with KG resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Quantitation of the hypodiploid cells shows a dose-dependent response to KG, and this result is in good accordance with that of the DNA fragmentation assay by agarose gel electrophoresis. Our results suggested that cell cycle arrest at G(1) phase and induce apoptosis as a mechanism by which KG exerts an antiproliferative effect.     

白花蛇舌草药材的HPLC指纹图谱研究

目的:建立白花蛇舌草药材HPLC指纹图谱,结合化学计量学手段,对多批次药材进行质量控制。方法:采用C18柱(250 mm×4.6 mm,5μm),以乙腈-水(各含0.1‰乙酸)为流动相梯度洗脱,流速0.8 mL/min,检测波长238 nm,柱温30℃;建立白花蛇舌草药材HPLC指纹图谱,通过比对化学分离对照品的保留时间,对主要特征峰进行化学指认;对22批药材进行相似度评价,并进行主成分分析和聚类分析。结果:建立了专属性、精密度、重现性和稳定性均较好的白花蛇舌草药材HPLC指纹图谱;11个特征峰得到明确化学指认;17批白花蛇舌草指纹图谱相似度值明显高于5批伪品;主成分分析结果显示,17批白花蛇舌草密集分布在一个区域,而5批伪品离散分布在此区域外;对22批药材进行聚类分析,17批白花蛇舌草聚为一类,5批伪品均未与其聚为一类;进一步运用聚类分析对17批白花蛇舌草进行质量评价,总共可聚为4类。结论:将HPLC指纹图谱与化学计量学手段相结合,可对白花蛇舌草进行真伪鉴别和质量评价,可为提高其整体质量控制方法提供参考。     

白花蛇舌草水提取物对多药耐药白血病细胞HL-60/ADR的作用

目的:研究白花蛇舌草水提取物对多药耐药白血病细胞HL-60/ADR的抑制作用及机制。方法:采用MTT比色试验观察白花蛇舌草水提取物对多药耐药细胞HL-60/ADR的抑制作用。采用光镜、电镜技术观察细胞形态结构的改变,利用流式细胞术(FCM)检测细胞凋亡率。结果:①白花蛇舌草水提取物能明显抑制HL-60/ADR细胞的生长。②形态学改变,呈典型的凋亡特征。③流式细胞术证实,凋亡率也呈时间和剂量的依赖性。结论:白花蛇舌草水提取物对多药耐药细胞HL-60/ADR的生长具有极强的抑制作用,诱导其凋亡是其主要机制。     

Cytotoxic and apoptosis-inducing activities of limonoids from the seeds of Azadirachta indica (neem)

Thirty-five limonoids, including 15 of the azadiradione type (1-15), five of the gedunin type (16-20), four of the azadirachtin type (21-24), nine of the nimbin type (25-33), and two degraded limonoids (34, 35), isolated from Azadirachta indica seed extracts, were evaluated for their cytotoxic activities against five human cancer cell lines. Seven compounds (3, 6, 7, 16, 18, 28, and 29) exhibited cytotoxic activity against one or more cell lines. Among these compounds, 7-deacetyl-7-benzoylepoxyazadiradione (7), 7-deacetyl-7-benzoylgeduin (18), and 28-deoxonimbolide (28) exhibited potent cytotoxic activity against HL60 leukemia cells with IC(50) values in the range 2.7-3.1 μM. Compounds 7, 18, and 28 induced early apoptosis in HL60 cells, observed by flow cytometry. Western blot analysis showed that compounds 7, 18, and 28 activated caspases-3, -8, and -9 in HL60 cells. This suggested that compounds 7, 18, and 28 induced apoptotic cell death in HL60 cells via both the mitochondrial- and the death receptor-mediated pathways. Futhermore, compound 7 was shown to possess high selective cytotoxicity for leukemia cells since it exhibited only weak cytotoxicity against a normal lymphocyte cell line (RPMI 1788).     

白花蛇舌草及其伪品伞房花耳草的比较鉴别

文章对白花蛇舌草与伞房花耳草的来源、性状、显微方面进行了比较鉴别。发现二者来源不同,性状、显微各异。性状方面在花(果)的排列方式上,既有相似之处,又有差异,花轴的有无及花轴与茎的不同点是鉴别白花蛇舌草与伞房花耳草的关键之处;显微方面在茎横切面特征上,茎的形状具有明显的差异。     

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

Ethnopharmacological relevance

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.

Aim of the study

The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.

Materials and methods

Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.

Results

Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.

Conclusion

Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G₀/G₁) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.     

白花蛇舌草、半枝莲、蒲公英治疗肿瘤体外研究近况

白花蛇舌草、半枝莲、蒲公英等是中医临床防治肝癌清热解毒治法的常用药物。文章通过CNKI检索了这3味中药近10年来治疗肿瘤体外实验报道,对其体外给药制备、用药剂量、疗效等进行综述,发现白花蛇舌草、半枝莲研究较为集中,与对照药比较在肝癌等肿瘤细胞增殖抑制、凋亡诱导、侵袭抑制、抑制耐药性等方面具有一定的优势。     

β‐Eudesmol Induces JNK‐Dependent Apoptosis through the Mitochondrial Pathway in HL60 Cells

β‐eudesmol, a natural sesquiterpenol present in a variety of Chinese herbs, is known to inhibit the proliferation of human tumor cells. However, the molecular mechanisms of the effect of β‐eudesmol on human tumor cells are unknown. In the present study, we report the cytotoxic effect of β‐eudesmol on the human leukemia HL60 cells and its molecular mechanisms. The cytotoxic effect of β‐eudesmol on HL60 cells was associated with apoptosis, which was characterized by the presence of DNA fragmentation. β‐eudesmol‐induced apoptosis was accompanied by cleavage of caspase‐3, caspase‐9, and poly (ADP‐ribose) polymerase; downregulation of Bcl‐2 expression; release of cytochrome c from mitochondria; and decrease in mitochondrial membrane potential (MMP). Activation of c‐Jun N‐terminal kinases (JNK) mitogen‐activated protein kinases was observed in β‐eudesmol‐treated HL60 cells, and the inhibitor of JNK blocked the β‐eudesmol‐induced apoptosis, downregulation of Bcl‐2, and the loss of MMP. These data suggest that β‐eudesmol induces apoptosis in HL60 cells via the mitochondrial apoptotic pathway, which is controlled through JNK signaling. Copyright © 2012 John Wiley & Sons, Ltd.     

药用真菌江西青霉多糖抗肿瘤机制的研究

目的探讨药用真菌江西青霉多糖抗肿瘤的可能作用机制.方法采用MTT法测定细胞毒性,流式细胞仪PI单染法测定细胞周期,Hoechst荧光染色法检测细胞凋亡的形态学特征,Annexin-V FITC/PI双标法和TUNEL法分析细胞早期和晚期凋亡的特征.结果江西青霉精制多糖组分MPPJ4和MPPJ5有微弱抑制肿瘤细胞增殖作用,并使其细胞周期明显阻滞于S期,且subG1期细胞所占比例显著上调.Hoechst染色可观察到细胞产生凋亡小体等典型的细胞凋亡形态学特征.细胞凋亡的早期和晚期特征提示,MPPJ5有更强诱导肿瘤细胞凋亡的能力,在0～72 h范围,时间越长,细胞凋亡检测阳性比例也越高,与阴性对照相比,呈显著性相关.结论江西青霉多糖抗肿瘤的效应机制与细胞凋亡通路及阻断细胞生长周期有关.     

蟾蜍灵诱导人白血病HL60细胞凋亡的初步研究

目的 :探讨中药蟾蜍灵诱导白血病HL60细胞凋亡作用。方法 :应用台盼蓝染色法测定HL6 0细胞的生长曲线 ,应用相差显微镜、电子显微镜和DNA琼脂糖凝胶电泳等技术分析细胞凋亡。结果 :蟾蜍灵与HL60细胞共育时 ,能明显抑制细胞生长 ,细胞呈典型的凋亡形态学改变 :细胞皱缩 ,染色质凝集 ,沿核膜内侧分布 ,呈新月形 ,可见由膜包被细胞内容物的凋亡小体的产生 ;细胞DNA裂解成 180～200bp及其倍数的片段 ,电泳得到特有的梯形条带。结论 :蟾蜍灵诱导细胞凋亡是其抗肿瘤的作用机制之一。     

白花蛇舌草HPLC指纹图谱的研究

目的建立江苏产白花蛇舌草HPLC指纹图谱。方法以芦丁为参照物,采用Kramasil C18(250mm×4.6 mm,5μm)色谱柱,以甲醇-水为流动相,梯度洗脱,流速1 mL/min,检测波长为254 nm。结果共标出白花蛇舌草药材10个共有峰。结论指纹图谱分析可作为科学、快速地鉴别不同产地白花蛇舌草药材的有效方法。     

Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC

Aim of the study

Oldenlandia diffusa is a traditional Chinese herbal remedy with known cytotoxic activity in vitro and in vivo. The aim of the study was to identify the most cytotoxic constituents of a water extract (a decoction is traditionally used as a treatment) by HPLC and activity-guided fractionation. The bioavailability of the decoction and certain fractions, and the mode of cell death induced by these mixtures, were also investigated.

Materials and methods

A decoction of O. diffusa was prepared and separated by HPLC into eleven fractions (F1-11) for testing on the growth of HL60 leukaemia cells; two of the most active fractions were also tested on Caco-2 colon cancer cells. Cell viability was measured by trypan blue exclusion, DNA content (Cyquant NF assay) and neutral red uptake. Evidence of apoptosis was gained from cells stained with the nuclear dye DAPI, and detection of cleaved poly (ADP-ribose) polymerase (PARP) by Western blot.

Results

Fraction 9 was found to be the most active fraction, and, along with the decoction, induced apoptosis. Cells stained with DAPI showed a decrease in cell size and nuclear fragmentation characteristic of apoptosis. Detection of cleaved PARP further confirmed induction of apoptosis by O. diffusa decoction and fraction 9. The bioavailability of O. diffusa was investigated by production of post-absorption samples using Caco-2 intestinal epithelial monolayers. Addition of post-absorption samples (taken from the basolateral side after 3 h incubation with the decoction on the apical side) inhibited the growth of HL60 cells, and suggested a degree of bioavailability. The constituents in fraction 9 were further separated by HPLC and eight major compounds were identified by LC-MS: two of these were ursolic acid (UA) and its enantiomer oleanolic acid (OA). Concentrations of UA and OA in the decoction were then calculated by reference to the area of the peaks of UA and OA found in F9. The post-absorption sample of F9 contained six of the eight constituents in the original pre-absorption fraction 9.

Conclusions

Taken together, the results suggest that certain constituents, possibly including ursolic/oleanolic acid, may be bioavailable and at sufficient concentration to induce apoptosis in cancer cells in vitro through a mechanism including the cleavage of PARP.     

Antitumour effects of ursolic acid isolated from Oldenlandia diffusa

The cytotoxicity-guided fractionation of the MeOH extract of Oldenlandia diffusa (Rubiaceae) led to the isolation of ursolic acid (UA) as an active principle. Ursolic acid demonstrated a significant inhibition of the proliferation of cultured tumour cells, i.e. A549 (human lung), SK-OV-3 (ovary), SK-MEL-2 (skin), XF498 (brain), HCT-15 (colon), SNU-1 (stomach), L1210 (murine leukaemia) and B16-F₀ (murine melanoma). A marked increment of T/C (>200%) was also observed when UA was administered to mice bearing sarcoma-180 cells. The microscopic analyses (phase contrast microscope and TEM) of SNU-1 cell after continuous exposure to UA for 4 and 24 h showed typical morphological changes of the cell due to an apoptotic effect. The nucleosomal DNA of HL60 cells pretreated with UA was cleaved into several oligomeric fragments which was due to a typical apoptotic effect. However, the in vitro cytotoxic effect of UA on tumour cells was decreased in a dose dependent manner by the addition of nicotinamide, a poly-(ADP-ribose) polymerase inhibitor, or aurin tricarboxylic acid (ATA), an endonuclease inhibitor. These results suggested that the cytotoxicity of UA or the apoptotic effect of UA on tumour cells might be related to the activation of the endonucleolytic enzyme and subsequent activation of poly(ADP-ribose) polymerase in tumour cells and these could eventually lead to cell lysis. Copyright © 1998 John Wiley & Sons, Ltd.     

复方716合剂治疗上呼吸道感染的研究

复方７１６合剂由白花蛇舌草，乌蕨、地锦草、铁苋等中草药组成。为了观察７１６合剂治疗上呼吸道感染的疗效，我们将１５０例上呼吸道病毒感染阳性的患儿随机分为７１６治疗组（８９例）及西药对照组（６１例）进行临床观察，治疗组有效率达９２％，对照组有效率达６７％。实验研究亦证明７１６合剂对细胞的毒性很小，对鸡胚亦无明显毒性，并有显著抑制甲型流感病毒的作用。     

Antiproliferative and antimitotic effect, S phase accumulation and induction of apoptosis and necrosis after treatment of extract from Rhodiola rosea rhizomes on HL-60 cells

Rhodiola rosea is a medicinal plant having stimulating and adaptogenic properties, and some reports also indicate its anticancer and antimutagenic effect. However, the mechanism of its anticancer effect is unknown as there have been no cytological studies regarding cytostatics, cell cycle, induction of apoptosis or the mitotic activity of healthy and cancerous cells. In the present paper, those parameters were investigated using HL-60 cells, with flow cytometry and fluorescence microscopy. It has been found that the extract of Rhodiola rosea rhizomes inhibits division of HL-60 cells, which is preceded by an accumulation of cells at the prophase stage. This leads to induction of apoptosis and necrosis in HL-60 cells, and to marked reduction of their survival. The cells enter apoptosis from phase G2/M of the cell cycle. After treatment with the extract, no chromosome aberrations or micronuclei were observed, which indicates the mild action of the extract. The cytostatic and antiproliferative effect of the Rhodiola rosea rhizome extract, and its mild action, raises hope for its use in anticancer therapy by enhancing the effectiveness of cytostatics.     

薄层扫描法测定白花蛇舌草中齐墩果酸含量

目的：建立白花蛇舌草中齐墩果酸的含量测定方法。方法：薄层扫描法。结果：方法灵敏度高,重现性好,平均加样回收率为96．2％,RSD为1．94％。结论：该方法可用于白花蛇舌草的质量控制。     

8种中药煎剂对银屑病角质形成细胞C-erbB-1表达的抑制作用

目的:观察8种中草药(雷公藤、白花蛇舌草、黄柏、黄芩、茯苓、红花、板蓝根、丹参)煎剂对银屑病角质形成细胞内 C-erbB-1癌基因表达的抑制作用,探讨中草药治疗银屑病可能的作用机理。方法:8种中草药分别与体外培养的银屑病角质形成细胞和经银屑病患者外周血白细胞刺激的正常人角质形成细胞共同培养,采用细胞原位杂交的方法检测细胞中 C-erbB-1mRNA 的表达。结果:8种中草药对银屑病患者角质形成细胞内 C-erbB-1的表达有不同程度的抑制作用(P<0.001),抑制作用最强的为雷公藤;中药对经银屑病患者外周血白细胞刺激的正常人角质形成细胞内 C-erbB-1的表达也有不同程度的抑制作用(P<0.001),抑制作用最强的为茯苓。结论:这些中药可能通过抑制银屑病角质形成细胞 C-erbB-1的表达来抑制表皮细胞的增殖。     

Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria cocos

Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos , and their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evaluation of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549), melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145), stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC(50) values against one or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited potent cytotoxicities against six cell lines with IC(50) values of 1.2-5.5 μM. Poricotriol A induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF) from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell line (WI-38).