雾化吸入与静脉注射羟基喜树碱在兔体内的药动学比较

 目的比较雾化吸入与静脉注射羟基喜树碱在兔体内的药动学特点。方法采用高效液相色谱法测定不同时间点兔血浆中羟基喜树碱浓度,对雾化吸入和静脉注射给药后的血药浓度数据进行了药动学分析。结果雾化吸入给药后血浆中的含量显著降低。雾化吸入组与静脉给药组相比,MRT,t_1/2显著延长,AUC显著减小,其绝对生物利用度仅为(10.69±3.29)%。结论雾化吸入给予羟基喜树碱血药浓度显著降低,绝对生物利用度较低。改用雾化吸入途径给药治疗肺癌可以显著提高疗效而相对降低毒副作用。     

羟基喜树碱注射液与注射用羟基喜树碱含量与有关物质的比较

 目的考查国内不同企业羟基喜树碱制剂的产品质量。方法用高效液相色谱UV检测法和蒸发光散射检测法(ELSD)对来源于国内5个企业7个批次的羟基喜树碱制剂进行含量和有关物质的比较。结果羟基喜树碱注射液B厂和C厂的产品中杂质喜树碱的含量大于3％,注射用羟基喜树碱中仅D厂，杂质喜树碱含量大于3%;其它各厂的产品中，杂质喜树碱含量均小于1％。结论羟基喜树碱制剂质量的好坏、稳定与否与其制剂是粉针剂还是注射液没有直接的必然的联系；不管是哪一种制剂，只要生产企业能够提高质量意识，严把质量关，都能够得到高质量的产品。     

单用羟基喜树碱治疗肺癌骨转移疼痛18例观察

目的：观察单独静脉滴注羟基喜树碱对肺癌骨转移患者疼痛的治疗效果。方法：18例肺癌骨转移患者,持续静脉滴注羟基喜树碱(HCPT)10mg．10d为1周期．1疗程后观察患者疼痛情况及毒性反应。结果：18例患者中CR6例,部分缓解8例．稳定2例，进展2例，有效率77．8％。不良反应有白细胞下降、呕吐、腹泻等。结论：单用羟基喜树碱能较好地缓解肺癌骨转移患者骨性疼痛，且毒性可以耐受。     

羟基喜树碱对A549人肺腺癌细胞增殖的影响

目的观察羟基喜树碱对体外A549人肺腺癌细胞增殖的影响。方法采用细胞培养及MTT比色法检测羟基喜树碱对体外A549人肺腺癌细胞的抑制作用。结果低浓度羟基喜树碱作用24h对A549人肺腺癌细胞没有明显影响,当药物浓度达到50μg/mL时,抑制作用明显增强,抑制率达44.17%,药物浓度达到100μg/mL时抑制率达50.28%;随着药物作用时间的延长,抑制更明显,100μg/mL药物组作用48h抑制率达70.98%。结论羟基喜树碱具有抑制A549人肺腺癌细胞增殖的作用,抑制作用具有明显的浓度和时间依赖性。     

羟基喜树碱对人胚肺成纤维细胞HFLl生长的影响

目的观察羟基喜树碱（HCPT）对体外培养的人胚秭成纤维细胞（HFLI）的增殖抑制和促凋亡作用。,方法采川四甲块偶氮唑蓝法（MTT）检测不同浓度的HCVF对HFI．1细胞作用24h、48h、72h的增殖抑制效果,计算细胞生长抑制率和半数抑制量（IC50）,确定HCPT抑制HFL1细胞增殖的最佳给药浓度和最佳作用时间;姬姆萨染色观察细胞形念学变化;流式细胞仪分析细胞周期;琼脂糖凝胶电泳观察捌亡细胞的DNA梯度。对所得数据用SPSS15．0软件进行统计分析。结果HCPT对HFL1细胞增殖有明显的抑制作用,在0．008-32mg／L范围内呈时间和剂量依赖性;与对照组比较,各实验组24h、48h、72hA值及细胞生长抑制率差异有统计学意义（P〈0．05,P〈0．01）HCPT作用HFL1细胞48h时的IC50为0．466mg／L,故确定48h、0．5mg／L为最佳作用时间和最佳给药浓度;0．5mg／I．HCPT作用HFL1细胞24h后,Cu,／G．期细胞所占比例为（50．46±3．91）％;作用72h后,G0／G．期细胞所占比例为（88．19±2．13）％,怀对照组比较,其余各组各期细胞所占比例差异均有统计学意义（P〈0．05,P〈0．01）;姬姆萨染色显示,HFLI细胞形态发生明显变化;琼脂糖凝胶电泳可见明显的DNA梯度带形成。结论HCVF对体外培养的HLI细胞具有抑制增殖和诱导凋亡作用,可能是一种潜在的抗肺纤维化的药物。     

羟基喜树碱对H22肝癌模型小鼠药效学的研究

目的 考察羟基喜树碱(HCPT)对小鼠H22模型的抗肿瘤作用.方法 以小鼠H22肝癌模型为实验观察对象.观察HCPT对肿瘤的抑制效果、化疗前后的体重、食物摄取量及生长状况,同时观察HCPT对H22肝癌模型小鼠肝肾功能的保护作用.结果 阳性药对照组及HCPT高、中、低剂量化疗组对小鼠H22肝癌模型均有抑制作用(P<0.0...     

天然喜树碱和10-羟基喜树碱柱层析分离工艺研究

目的:利用硅胶柱层的方法分离喜树碱和10-羟基喜树碱.方法:采用常规的硅胶柱层析梯度脱洗的方法,通过比较不同种类硅胶、不同洗脱液、不同硅胶量与上样品量之比,确定最佳的分离效果.结果:以甲醇:三氯甲烷=0.3:100为喜树碱的最佳洗脱液,甲醇:三氯甲烷=1.3:100为10-羟基喜树碱的最佳洗脱液,硅脱容量40g加样量1.0g为最佳的层析柱分离条件,以500～800目的柱层析硅胶的分离效果最好,可以得到纯度高的喜树碱和10-羟基喜树碱,适用于工业化大生产.结论:利用硅胶柱层析的方法,以极性渐增的一系列混合溶剂作为梯度洗脱剂,可以很好的分离喜树碱和10-羟基喜树碱.     

HPLC测定羟基喜树碱及其注射液的含量

 目的：采用高效液相色谱法测定羟基喜树碱及其注射液的含量。方法：用YWG-C₁₈色谱柱，甲醇-水（60∶40）为流动相，以倍他米松为内标，检测波长为246nm。结果：以峰面积计算，羟基喜树碱在1．8μg·ml^－1～9．0μg·ml^－1浓度范围内呈良好线性（r＝0．9998），平均回收率为100．7％，RSD为1．4％。结论：本法专属性强，操作方便，结果准确。     

氯化两面针碱和羟基喜树碱对斑马鱼胚胎发育的影响

目的:研究氯化两面针碱和羟基喜树碱对斑马鱼发育的影响。方法:氯化两面针碱(NC)分成5.00,3.15,2.00,1.58,1.12 mg.L-1等5个含药剂量组,各组的DMSO终浓度为0.01%,并设含0.01%DMSO的孵化液组及对照组(纯孵化液组)。羟基喜树碱(HCPT)分为16.00,10.00,6.50,4.00,2.50 mg.L-1 5个含药剂量组,各组的DMSO终浓度为0.01%。并设对照组(纯孵化液组)及含0.01%DMSO的孵化液组。在显微镜下将正常发育6 h(6 hpf)和48 h(48 hpf)的斑马鱼胚胎,随机分入以上各组中,每个实验组20颗胚胎。观察药物处理至72 hpf时,胚胎的死亡和畸形情况。结果:氯化两面针碱的6 hpf实验组中,半数致死浓度(LC50)为1.66 mg.L-1,半数致畸效应浓度(EC50)为1.33 mg.L-1;48 hpf实验组中,LC50为2.51 mg.L-1,EC50为2.02 mg.L-1。羟基喜树碱的6 hpf实验组中,LC50为4.21 mg.L-1,EC50为3.29 mg.L-1;48 hpf实验组中,LC50为7.70 mg.L-1,EC50为6.18 mg.L-1。斑马鱼胚胎的死亡率和畸形率随着氯化两面针碱和羟基喜树碱药物浓度的降低和给药时间的推迟而显著降低(P<0.01)。结论:6 hpf胚胎对氯化两面针碱和羟基喜树碱的药物敏感性均大于48 hpf;氯化两面针碱对斑马鱼胚胎的发育毒性大于羟基喜树碱。     

羟基喜树碱微透析回收率的体外研究

目的:建立羟基喜树碱微透析探针回收率的测定方法,考察影响回收率的因素,为体内微透析研究提供依据。方法:采用浓度差法(增量法、减量法)测定羟基喜树碱探针回收率,考察流速、浓度、温度对回收率的影响,并考察回收率的稳定性。结果:增量法和减量法测得的回收率一致;回收率与探针周围媒介浓度无关,稳定性良好,随温度升高而升高。结论:微透析技术可用于羟基喜树碱药动学研究,反透析法可用于羟基喜树碱体内微透析回收率的体内测定。     

茯苓多糖对B16黑色素瘤人工肺转移模型的影响

[目的]探讨茯苓多糖对B16黑色素瘤小鼠人工肺转移的影响,并对其作用机制进行初步研究。[方法]C57BL/6小鼠接种B16黑色素瘤,分为茯苓多糖高、低剂量组、顺铂组、模型组,茯苓多糖高、低剂量组分别给予每只小鼠茯苓多糖0.5 mg、0.33 mg,顺铂组给予顺铂0.04 mg,模型组给予生理盐水,接种瘤细胞后第2天开始尾静脉注射给药,隔天给药1次。观察小鼠一般状态,造模后21 d取肺,计数肺表面转移灶个数、HE染色检测肺微小转移灶个数,并检测外周血白细胞数量及脾质量、脾指数。[结果]顺铂能够抑制肺转移,同时降低小鼠体质量、外周血白细胞数量、脾质量和脾指数。而茯苓多糖高、低剂量组对B16荷瘤鼠的体质量无明显影响,茯苓多糖高剂量可明显降低肺表面转移灶个数,茯苓多糖高、低剂量可减少肺微小转移灶个数,高剂量组外周血白细胞数量明显降低,低剂量组与模型组无统计学差异,低剂量可增加B16荷瘤鼠的脾质量和脾指数,高剂量对B16荷瘤鼠的脾质量和脾指数无明显影响。茯苓多糖高、低剂量组脾质量和脾指数均高于顺铂组。[结论]茯苓多糖能够抑制尾静脉注射B16荷瘤小鼠肺转移,增加其脾质量和脾指数,无明显增加其外周血白细胞数量的作用,推测活化外周血白细胞,增强免疫功能可能参与茯苓多糖抑制肿瘤转移的机制。     

Citrus unshiu peel suppress the metastatic potential of murine melanoma B16F10 cells in vitro and in vivo

The peel of Citrus unshiu Marcow. fruits (CU) has long been used as a traditional medicine that has therapeutic effects against pathogenic diseases, including asthma, vomiting, dyspepsia, blood circulation disorders, and various types of cancer. In this study, we investigated the effect of CU peel on metastatic melanoma, a highly aggressive skin cancer, in B16F10 melanoma cells, and in B16F10 cells inoculated‐C57BL/6 mice. Our results show that ethanol extracts of CU (EECU) inhibited cell growth and increased the apoptotic cells in B16F10 cells. EECU also stimulated the induction of mitochondria‐mediated intrinsic pathway, with reduced mitochondrial membrane potential and increased generation of intracellular reactive oxygen species. Furthermore, EECU suppressed the migration, invasion, and colony formation of B16F10 cells. In addition, the oral administration of EECU reduced serum lactate dehydrogenase activity without weight loss, hepatotoxicity, nor nephrotoxicity in B16F10 cell‐inoculated mice. Moreover, EECU markedly suppressed lung hypertrophy, the number and expression of metastatic tumor nodules, and the expression of inflammatory tumor necrosis factor‐alpha in lung tissue. In conclusion, our findings suggest that the inhibitory effect of EECU on the metastasis of melanoma indicates that it may be regarded as a potential therapeutic herbal drug for melanoma.     

Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells

Ethnopharmacological relevance

Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine.

Aim of the study

The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells.

Materials and methods

Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in α-MSH-stimulated and unstimulated B16F10 cells.

Results

Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and α-MSH-stimulated melanin production in B16F10 cells.

Conclusions

Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry.     

Effect of Withania somnifera on B16F-10 melanoma induced metastasis in mice

Withania somnifera, a plant with known immunopotentiating activity and its bioactive fraction-Withanolide D were studied for their anti-metastatic activity using B16F-10 melanoma cells in C57BL/6 mice. Simultaneous administration of Withania extract (122 +/- 10 tumour nodules) and Withanolide (126 +/- 9 lung tumour nodules) could significantly (p < 0.001) inhibit the metastatic colony formation of the melanoma in lungs. 72.58% by extract and 69.84% by Withanolide treated, as compared to the untreated control animals also increased the survival days. Lung collagen hydroxyproline content was highly elevated in the control animals (23.5 +/- 0.9 micro g/mg protein), which was reduced by the simultaneous administration of both the extract (16.3 +/- 2.0 micro g/mg protein) and Withanolide (15.3 +/- 1.8 micro g/mg protein). The level of lung hexosamines (4.85 +/- 0.20 mg/100 mg tissue) and uronic acids (330.1 +/- 23.7 micro g/100 mg tissue) content was also elevated in the control animals. The elevated level of hexosamine was significantly reduced by the treatment with extract (1.92 +/- 0.05) and Withanolide (1.85 +/- 0.05). Similarly, the uronic acid content was also been reduced by the simultaneous administration of both Withania extract (194.2 +/- 17.4) and Withanolide (183.2 +/- 8.8). The control animals had 35.3 +/- 3.8 U/L gamma-glutamyl transpeptidase (gamma-GT), which was reduced by 50% by the treatment of extract and Withanolide to 17.5 +/- 4.0 U/L and 16.3 +/- 4.4 U/L respectively. There was a significant reduction in the levels of sialic acid in the serum of Withania extract (60.7 +/- 7.7) and Withanolide (67.16 +/- 5.8) treated animals compared to the higher level (102.2 +/- 8.7) in the control animals. Histopathological analysis of the lung tissues also correlated with these findings. Prophylactic administrations of both extract as well as Withanolide were ineffective in inhibiting the metastasis of B16F-10 melanoma cells.     

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG‐I) on melanoma cell growth and survival in vitro. We added okra RG‐I containing an almost pure RG‐I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2‐hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin‐3 (Gal‐3) protein. Incubation with okra RG‐I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N‐cadherin and α5 integrin subunit was reduced and that of the multifunctional carbohydrate‐binding protein, Gal‐3, at the cell membrane increased. These findings suggest that okra RG‐I induces apoptosis in melanoma cells by interacting with Gal‐3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur. Copyright © 2009 John Wiley & Sons, Ltd.     

Inhibition of melanogenesis in murine B16/F10 melanoma cells by Ligusticum sinensis Oliv

Ligusticum sinensis Oliv. (LSO) is an herbal drug commonly used as a topical treatment of epidermal hyperdepigmentation in Chinese medicine. However, the mechanism underlying the depigmentation by LSO is still unclear. The purpose of this study was to investigate the effects of LSO on the process of melanogenesis and its possible underlying mechanism. Suppressed DOPA oxidase activity of mushroom tyrosinase was first noted when incubated with aqueous extracts of LSO, demonstrating the direct inhibitory effect of LSO on mushroom tyrosinase. Further experiments were carried out in murine B16/F10 melanoma cells and the effects of LSO extract on melanin formation, tyrosinase activity and tyrosinase gene expression were tested. Under conditions without affecting the viability of murine B16/F10 melanoma cells, LSO extract significantly reduced the cellular melanin content in a dose-dependent manner. The DOPA oxidase activity of tyrosinase in B16/F10 cells was dose-dependently inhibited by LSO treatment, possibly mediated by the suppressed tyrosinase mRNA expression in LSO-treated B16/F10 cells. In conclusion, the inhibitory effect of LSO on melanogenesis is likely associated with decreased DOPA oxidase activity of tyrosinase that is most likely the result of the down-regulation of tyrosinase mRNA expression.     

Effects of phenolic compounds isolated from Rabdosia japonica on B16-F10 melanoma cells

Pedalitin isolated from the aerial part of Rabdosia japonica (Labiatae), exhibited cytotoxicity against the murine B16-F10 melanoma cell line with an IC(50) of 30 microm (9.5 microg/mL). As the cells were cultured with this flavone, melanin production was not suppressed, but rather enhanced. Quercetin isolated from the same source exhibited similar activities, but rutin showed neither activity.     

甘草查尔酮A对小鼠黑色素瘤B16F10细胞体外迁移能力的影响

目的:探讨甘草中黄酮类化合物甘草查尔酮A(licochalcone A,LCA)对小鼠黑色素瘤B16F10细胞株体外迁移能力的影响。方法:取对数生长期B16F10细胞按8 000个/孔接种于96孔板中,磺酰罗丹明B(SRB)法测定LCA(0,5,10,15,20μmol·L-1)作用24 h对细胞活力影响;取对数生长期B16F10细胞按1×105个/孔接种于24孔板中,划痕实验检测LCA(0,5,10,15μmol·L-1)作用24 h细胞体外迁移能力;取对数生长期B16F10细胞按5×105个/瓶接种于细胞培养瓶中,LCA(0,5,10,15μmol·L-1)作用24 h后,明胶酶谱法检测细胞培养上清基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)活性;ELISA检测MMP-2和MMP-9蛋白含量;实时荧光定量PCR(Q-PCR)和半定量PCR(RT-PCR)法检测MMP-2和MMP-9基因表达水平。结果:5~15μmol·L-1的LCA对小鼠黑色素瘤B16F10细胞作用24 h,对细胞活力没有显著的影响;划痕试验结果说明,LCA可剂量依赖性地降低B16F10细胞的体外迁移能力;明胶酶谱结果显示,LCA可明显抑制B16F10细胞分泌MMP-2和MMP-9的活性;ELISA试验表明,LCA可明显降低MMP-2和MMP-9蛋白表达;Q-PCR和RT-PCR结果显示,LCA可明显降低MMP-2和MMP-9的mRNA基因表达量。结论:甘草查尔酮A能抑制B16F10小鼠黑色素瘤细胞体外转移侵袭作用,其机制可能与抑制MMP-2和MMP-9表达有关。