广西姜黄挥发油两种提取方法的比较研究

以广西姜黄那坡县种植的姜黄为原料,以水蒸汽蒸馏法和以石油醚为溶剂的索氏提取法,分别提取挥发油,并用GC-MS分析法对这些以不同加工途径获取的姜黄挥发油进行成分分析,比较了不同的加工办法对挥发油的加工得率、主要成分和含量的影响。研究表明:广西那坡的姜黄挥发油主要成分为:α-姜黄烯、芳姜黄酮、(-)-姜烯、β-倍半水芹烯、β-姜黄酮、α-姜黄酮、4-(1,5-二甲基-4-己烯基)-2-环己烯酮、β-没药烯。     

索氏法提取姜黄挥发油及其成分的研究

本文以广西那坡县种植的姜黄为原料,分别以沸点为30-60℃,60-90℃的石油醚以及石油醚与乙醚的混合物作为溶剂,采用索氏提取法提取挥发油,并采用GC-MS分析法研究了这几种姜黄挥发油的主要成分,比较了不同的加工办法对挥发油的加工得率、主要成分、含量的影响。研究表明：用索氏提取法提取的广西姜黄挥发油其得率在7％以上,主要成分为：α-姜黄烯、（-）-姜烯、β-倍半水芹烯、芳姜黄酮、β-姜黄酮、α-姜黄酮、β-没药烯。     

广西几个姜黄品种姜黄油GC-MS分析

通过对广西地区栽培的三个姜黄品种:博白姜黄、大新姜黄、市场姜黄的姜黄油GC-MS分析,比较不同姜黄品种姜黄油成分的差异性,为筛选特定姜黄品种提供依据。不同姜黄品种其主要成分含量差异显著,主成份姜黄酮(含芳香-姜黄酮)含量依次为博白姜黄56.07%、大新姜黄47.79%、市场姜黄28.38%,市场姜黄的姜黄酮含量只有博白姜黄的一半。因此从姜黄油主要成份(含量>20%)姜黄酮含量来考察,以种植博白姜黄为最好。     

泰国野生银边姜黄的组培快速繁殖

利用泰国野生银边姜黄的萌动芽做外植体 ,对泰国野生银边姜黄的离体快速繁殖进行了研究 ,旨在为银边姜黄在中国的扩大栽培提供技术支持。筛选出了泰国野生银边姜黄萌动芽的恢复生长培养基 :MS +6 BA 0 .8mg.L 1+NAA 0 .1 5mg.L 1;芽增殖培养基 :MS +6 BA 4mg.L 1+KT 1 .5mg .L 1;生根培养基 :MS+NAA 0～ 0 .5mg.L 1。     

姜黄色素增溶的研究

本研究以脂溶性姜黄色素为材料,探讨其增溶后的水溶性、耐光性、耐加热性、安全性及增溶费用.结果表明,增溶后的姜黄色素不仅水溶性好,而且色泽鲜艳、着色力强,耐光性、耐加热性也较好;增溶后的姜黄色素安全无毒;增溶费用低.从而为工业化生产水溶性姜黄色素提供了理论依据.     

中药治疗脊髓损伤的研究现状

脊髓损伤是严重危害人类健康的疾病,随着社会的发展,其发病率日益升高,给社会、家庭、个人带来巨大负担.中医认为脊髓损伤与外力损伤督脉,致使气乱血逆,瘀阻经络,气血不能温煦濡养肢体有关:中药治则以活血化瘀,通络,补肾,益气为主.中药治疗脊髓损伤具有较好疗效,主要包括丹参、三七、人参、川芎等单味中药及其有效成分,中药复方如补阳还五汤、血府逐瘀汤、防己黄芪汤等.治疗脊髓损伤的临床验方较多,但临床实用成药却很少;采用挤压/撞击损伤动物等模型筛选和评价中药有效方剂,创制高效低毒治疗新药将是该领域今后研究的重要方向.     

姜黄标准汤剂UPLC指纹图谱的建立和4种成分定量分析研究CSCD

采用超高效液相色谱法(ultra-high performance liquid chromatography, UPLC)建立姜黄标准汤剂指纹图谱并同时测定4种有效成分的含量,为姜黄标准汤剂质量标准的制定提供依据。建立姜黄标准汤剂指纹图谱,采用质谱分析和对照品比对,对共有指纹峰进行指认,借助化学计量学方法对16批样品指纹图谱进一步处理和分析。同时,对姜黄标准汤剂中姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素和阿魏酸的含量进行测定。结果显示,姜黄标准汤剂具有10个共有峰,指认出其中8个成分;聚类分析(hierarchical cluster analysis, HCA)和主成分分析(principal component analysis, PCA)将16批样品归为两类,正交偏最小二乘法-判别式分析(orthogonal partial least squares-discriminant analysis, OPLS-DA)共找到6个差异性标志物。含量测定结果显示,云南大理的姜黄原料制备的标准汤剂姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素及阿魏酸的含量要高于四川宜宾和乐山的样品。该方法准确可靠,能够为姜黄标准汤剂的质量评价提供参考。     

姜黄根茎中姜黄素类成分含量的产地差异及其与环境因子的CCA分析

采用HPLC法对产自四川崇州和犍为、广东四会、广西玉林和博白及金秀、云南马关的姜黄(Curcuma longaL.)根茎中姜黄素类成分含量进行测定,并利用典范对应分析方法(CCA)研究了不同产地姜黄根茎中姜黄素类成分含量与地理-气候因子及根际土壤养分因子间的相关性。结果表明:不同产地根际土壤中有机质、全N、全P和全K含量分别为14.03～32.79、0.39～0.92、0.56～1.55和2.29～9.23 g.kg-1,根际土壤养分含量差异较明显;姜黄多生长在中性偏酸、水肥性能良好的土壤中。姜黄根茎中姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素含量及姜黄素类成分总含量的平均值分别为1.53%、0.42%、0.67%和2.61%;不同产地姜黄根茎中姜黄素类成分含量有显著差异,且同一产地采自不同采样点及不同采样时间的样品姜黄素类成分的含量也有一定差异。姜黄素类成分总含量以广西博白产姜黄根茎最高(4.29%)、广东四会产姜黄根茎最低(1.73%)。CCA分析结果表明:在经度、纬度、海拔、年均气温、极端最高温、极端最低温、年降水量、日照时数和无霜期等地理-气候因子中,年均气温和极端最低温与姜黄素类成分含量极显著正相关;而在pH值及有机质、全N、全P、全K含量等根际土壤养分因子中,有机质含量与姜黄素类成分含量极显著正相关。分析结果显示:影响姜黄根茎中姜黄素类成分含量的主要环境因子是年均气温、极端最低温和根际土壤的有机质含量。     

姜黄地上部分化学成分的研究

为了解姜黄(Curcuma longa L.)地上部分的化学成分,采用硅胶、葡聚糖凝胶柱色谱和高效液相色谱从姜黄地上部分分离得到14个化合物。通过波谱分析,分别鉴定为槲皮素3-O-α-L-鼠李糖苷(1)、山柰酚3-O-α-L-鼠李糖(1→2)-α-L-鼠李糖苷(2)、橙皮素7-O-α-L-鼠李糖(1→6)-β-D-葡萄糖苷(3)、1,7-二(4-羟基苯基)庚烷-4E,6E-二烯-3-酮(4)、1,7-二(4-羟基苯基)庚烷-1E,4E,6E-三烯-3-酮(5)、3-羟基-4-甲氧基肉桂酸(6)、对羟基苯甲醛(7)、香草醛(8)、4-羟基-3-甲氧基苯甲酸(9)、异香草酸(10)、4-(1-羟基-1-甲基乙基)苯甲酸(11)、R-6-羟基-6-甲基-3-(2-羟基异丙基)-2-烯环己酮(12)、6,9-二羟基-4,7-巨豆二烯-3-酮(13)和β-胡萝卜苷(14)。化合物1、2、3、12和13首次从该植物中分离得到。经HPLC比较分析,姜黄地上部分缺乏姜黄药材的主要功能成分姜黄素。     

姜黄属植物组培快繁技术的研究进展

姜科姜黄属植物是市场上重要的鲜切花和园林绿化品种，且多数物种具有很高的药用价值。但姜黄属目前主流的块茎分株繁殖法面临着诸如繁殖质量可控性差、速度慢等一系列问题，因而限制了其市场的规模发展。姜黄属植物的组织培养快繁技术能为其提供高效的繁殖途径，批量生产出优质可控的种苗，为满足市场需求提供重要的技术支撑。本研究总结出姜黄属组织培养过程中各阶段的技术现状，通过不同培养方法探讨了目前姜黄属植物组培技术存在的问题和未来的方向，希望为姜黄属植物组培快繁提供技术参考。     

Silencing MAP3K1 expression through RNA interference enhances paclitaxel-induced cell cycle arrest in human breast cancer cells

The objective of this study is to compare the expression level of MAP3K1 between normal mammary gland cells and breast cancer cells, and to analyze the effects of silencing MAP3K1 on breast cancer cells with paclitaxel treatment. Western blotting analysis was used to detect the expression level of MAP3K1 in MCF-7 and MCF-12F cells. The effect of gene silencing through different siRNAs was determined by realtime-PCR. MTT assay was used to test the cell proliferation. Cell cycle was detected by flow cytometry. MAP3K1 protein expression level in breast cancer cells was higher than that in normal mammary gland cells. MAP3K1 siRNA transfection significantly reduced the expression level of MAP3K1, and enhanced paclitaxel-induced cell proliferation inhibition and cell cycle arrest in breast cancer cells. Targeting MAP3K1 expression through small RNA interference can promote the therapeutic effects of paclitaxel in breast cancer.     

沉默DAXX基因表达促进人卵巢癌细胞凋亡和衰老

DAXX是Fas死亡结构域相关蛋白(Fas death-associated protein, DAXX),可与Fas死亡受体的死亡域结合.通过激活c-Jun NH2末端激酶通路,DAXX增强Fas介导的凋亡,同时也增强转录生长因子β依赖的凋亡.本研究通过免疫组化检测人正常卵巢组织和卵巢癌组织中DAXX蛋白表达,然后通过Tet-on诱导体系构建DAXX基因沉默的慢病毒,感染卵巢癌细胞后,加入嘌呤霉素筛选稳定表达的OV2008细胞,四环素诱导DAXX基因沉默,通过免疫印迹和定量PCR检测DAXX基因干扰效果,MTT检测细胞增殖,克隆形成实验检测克隆形成能力,免疫印迹和免疫荧光方法检测DNA损伤蛋白的变化,SA-β-gal检测细胞衰老.结果表明,在人正常卵巢组织中DAXX低表达,而在人卵巢癌组织中DAXX高表达,随着四环素浓度的增加DAXX的表达量降低,DAXX基因沉默抑制卵巢癌OV2008细胞的增殖和克隆形成. 免疫印迹结果显示,DAXX基因沉默可诱导DNA损伤相关蛋白p-H2AX和p-CHK2高表达.SA-β-gal检测结果表明,DAXX基因沉默可诱导OV2008细胞发生衰老,免疫印迹检测发现,p21和p27在 DAXX沉默的卵巢癌细胞中高表达.综上结果,DAXX沉默可以抑制卵巢癌细胞的增殖和克隆形成,同时也促进卵巢癌细胞发生DNA损伤和衰老.该研究为卵巢癌的基因治疗提供新的思路.     

Plant derived coumestrol phytochemical targets human skin carcinoma cells by inducing mitochondrial-mediated apoptosis,cell cycle arrest,inhibition of cell migration and invasion and modulation of m-TOR/PI3K/AKT signalling pathway

The current study was undertaken to investigate anticancer activity of coumestrol phytoestrogen against human skin cancer. MTT assay was performed for cell viability assessment and clonogenic assay for cell colony formation assessment. Apoptosis was analysed by Annexin V/FITC staining, AO/EB staining and western blotting assays. Effects on the m-TOR/PI3K/AKT signalling pathway were investigated by western blotting. Results indicated that coumestrol induced significant toxicity in human skin cancer cells in contrast to mouse skin cancer cells. The proliferation rate in normal skin cells remained almost intact. Annexin V-FITC and AO/EB staining assays indicated coumestrol induced cytotoxicity in skin cancer cells is mediated through apoptosis stimulation. The apoptosis in skin cancer cells was mediated through caspase-activation. Cell migration and invasion was inhibited by coumestrol in human skin cancer cells via inhibition of MMP-2 and MMP-9 expressions. Moreover, m-TOR/PI3K/AKT signalling pathway in SKEM-5 cells was blocked by coumestrol.     

Britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by targeting PD-L1 via abrogation of the crosstalk between Myc and HIF-1α in cancer

BackgroundProgrammed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects.PurposeIn this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells.MethodsIn vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model.ResultsBritannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model.ConclusionBritannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.     

Sanguinarine impedes metastasis and causes inversion of epithelial to mesenchymal transition in breast cancer

BackgroundA large number of breast cancer patients perishes due to metastasis instead of primary tumor, but molecular mechanisms contributing towards cancer metastasis remain poorly understood. Therefore, prompting development of novel treatment is inevitable. A vast variety of plant derived natural substance possesses several therapeutically active constituents, e.g. alkaloids, flavonoids, tannins, resins, terpenoids etc. that exhibit various pharmacological properties e.g. anti-inflammatory, anti-microbial and anti-cancer properties. Sanguinarine (SAN) alkaloid found its place among such naturally occurring substances that exerts several pharmacological activities, including anti-cancer effects.PurposeUntil now, role of SAN not only against epithelial-mesenchymal transition (EMT) but also against metastasis progression in breast cancer remains indistinct. Thus, aim of the present study was to investigate effects of SAN on EMT process and cancer metastasis in animal model.MethodsMTT assay was performed to assess SAN effects on proliferation in breast cancer. Scratch assay was performed to evaluate effects of SAN on migration in breast cancer. Colony formation assay was performed to determine effects of SAN on colonization characteristics of breast cancer. Western blotting was performed to measure EMT regulating protein expression as well as major pathway protein expression induced against TGF-β treatment in breast cancer. Tail vein method of injecting breast cancer cells in bulb/c mice was conducted to study metastasis progression and thereafter assessing effects of SAN against metastasis in mice.ResultsIn vivo results: MTT assay performed, demonstrated dose dependent inhibition of cell proliferation in breast cancer. Scratch assay results showed, SAN played a major role as migration inhibitor in estrogen receptor positive (ER+) breast cancer. Colony forming assay results demonstrated that SAN constrains ability of breast cancer to develop into well-defined colonies. Western blotting results for EMT regulating protein expression, after TGF-β treatment showed, SAN inhibited cadherin switch in ER+ breast cancer. Moreover, expression of pathway proteins involved in EMT process after TGF-β treatment i.e. Smad, PI3K/Akt and MAP kinase were significantly masked against SAN treatment.In vivo resultsThe appearance of metastatic nodules in lung tissues of mice model, helps to study the effects of SAN against metastasis in bulb/c mice. The obtained results have confirmed that SAN impeded lung metastasis. The macroscopic examination has confirmed metastasis inhibitory role of SAN in breast cancer. The Hematoxylin and eosin (H&E) staining results further advocate anti-metastatic characteristics of SAN, presented by fewer metastatic nodule and lesions appearance in SAN treated mice compared to untreated metastasis mice.ConclusionIn summary, SAN displayed prominent anti-metastatic effects in animal model and anti-proliferation effects together with significant inhibitory potential on EMT regulating protein expression against TGF-β treatment in ER+ breast cancer. So, overall findings of our study highlighted the pre-clinical significance of SAN in animal model therefore, further studies in humans as a part of clinical trial will be needed to establish pharmacokinetics and other effects of SAN, so that it can be a potential candidate for future treatment of metastatic breast cancer (MBC).     

Identification of TG100-115 as a new and potent TRPM7 kinase inhibitor,which suppresses breast cancer cell migration and invasion

BackgroundTransient receptor potential melastatin 7 (TRPM7) regulates breast cancer cell proliferation, migration, invasion and metastasis in its ion channel- and kinase domain-dependent manner. The pharmacological effects of TRPM7 ion channel inhibitors on breast cancer cells have been studied, but little is known about the effects of TRPM7 kinase domain inhibitors due to lack of potent TRPM7 kinase inhibitors.MethodsScreening was performed by using TRPM7 kinase assay. Effects of TG100-115 on breast cancer cell proliferation, migration, invasion, myosin IIA phosphorylation, and TRPM7 ion channel activity were assessed by using MTT, wound healing, transwell assay, Western blotting, and patch clamping, respectively.ResultsWe found that CREB peptide is a potent substrate for the TR-FRET based TRPM7 kinase assay. Using this method, we discovered a new and potent TRPM7 kinase inhibitor, TG100-115. TG100-115 inhibited TRPM7 kinase activity in an ATP competitive fashion with over 70-fold stronger activity than that of rottlerin, known as a TRPM7 kinase inhibitor. TG100-115 has little effect on proliferation of MDA-MB-231 cells, but significantly decreases cell migration and invasion. Moreover, TG100-115 inhibits TRPM7 kinase regulated phosphorylation of the myosin IIA heavy chain and phosphorylation of focal adhesion kinase. TG100-115 also suppressed TRPM7 ion channel activity.ConclusionsTG100-115 can be used as a potent TRPM7 kinase inhibitor and a potent inhibitor of breast cancer cell migration.General significanceTG100-115 could be a useful tool for studying the pharmacological effects of TRPM7 kinase activity aimed at providing insight into new therapeutic approaches to the treatment of breast cancer.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

Circular RNA circPIP5K1A promotes non-small cell lung cancer proliferation and metastasis through miR-600/HIF-1α regulation

Circular RNAs (circRNAs) have an important function in human diseases, especially in cancer. circRNA hsa_circ_0014130 (circPIP5K1A), a particularly abundant circRNA, participates in the tumorigenesis of non-small cell lung cancer (NSCLC), although the underlying regulatory mechanism remains unclear. Here, we investigated the circPIP5K1A role in NSCLC. Expression of circPIP5K1A in NSCLC cell lines was explored with quantitative real-time PCR. The effect of circPIP5K1A on NSCLC was evaluated with circPIP5K1A silencing, miR-600 mimic transfection, and hypoxia-inducible factor (HIF)-1α overexpression, followed by assessment of cell proliferation, metastasis, and tumorigenesis in nude mice. The subcellular localization of circPIP5K1A was evaluated via fluorescence in situ hybridization (FISH), and correlation between circPIP5K1A, miR-600, and HIF-1α was assessed by luciferase assay. The data demonstrated that circPIP5K1A expression was increased in NSCLC cells. FISH showed that circPIP5K1A localized to the cytoplasm. The circPIP5K1A knockdown suppressed NSCLC cell metastasis and proliferation by promoting expression of miR-600. Overexpression of miR-600 inhibited HIF-1α-mediated metastasis and proliferation of NSCLC cell by downregulating the endothelial mesenchymal transition-related proteins, Snail and vimentin, and upregulating E-cadherin. In vivo experiments illustrated that circPIP5K1A silence suppressed tumor growth and pulmonary metastasis. The circPIP5K1A may function as an miR-600 sponge to facilitate NSCLC proliferation and metastasis by promoting HIF-1α. A bifluorescein reporter experiment confirmed that miR-600 was the circPIP5K1A target, and miR-600 interacted with the 3′ untranslated region of HIF-1α. These results show that circPIP5K1A acted as a tumor promoter through a novel circPIP5K1A/miR-600/HIF-1α axis, which provides candidate markers and therapeutic targets for NSCLC.     

雌激素快速激活MAPK途径促进前列腺间质细胞的增殖

为研究雌激素促进前列腺间质细胞增殖的机理,使用雌二醇（E2）或BSA雌二醇（BSA-E2）处理前列腺间质细胞系wpmy-1,用MTT法及细胞计数法检测细胞增殖情况;用实时RT-PCR以及Western印迹方法检测细胞增殖核抗原（PCNA）的表达水平;用抗磷酸化ERK1/2抗体检测细胞内MAPK途径的激活情况.结果显示,2种形式的雌二醇均能促进前列腺间质细胞系wpmy-1的增殖,并且显著上调增殖相关核抗原PCNA的表达水平;2种形式的雌激素均能快速激活wpmy-1细胞内的MAPK信号通路;MAPK途径的特异性抑制剂PD98059能够显著降低雌激素对细胞增殖以及PCNA表达水平的上调作用.结果表明,雌激素能够通过膜受体快速激活前列腺间质细胞中的MAPK信号通路,进而促进前列腺间质细胞的增殖.