Anti-tumor Effect of Paclitaxel Enhanced by Psoralen at the Cellular Level

[Objectives]To explore the effect of psoralen combined with paclitaxel on the apoptosis of MCF-7 cells.[Methods]The effects of different concentrations of psoralen,paclitaxel,or the combination of psoralen and paclitaxel on cell viability were detected using CCK-8 assay kit.Cell cycle distribution and apoptosis after 24 h of psoralen(0.16,0.32,0.64 mmol/L),paclitaxel(0.1μmol/L),combined action of psoralen(0.32 mmol/L)and paclitaxel(0.1μmol/L)were detected using flow cytometry.[Results]Lower concentration of psoralen(0.04-0.32 mmol/L)showed no significant inhibitory effect on cells.After combined with paclitaxel,the inhibitory effect on MCF-7 cell proliferation was significantly higher than that of the group treated alone.Compared with the paclitaxel group,the cell apoptosis rate in the drug combination group was significantly increased.Different low concentrations of psoralen can block the cell cycle of MCF-7 at G 0/G 1 phase,while paclitaxel can block the cell cycle at G 2/M phase.After combined action,the number of cells blocked at G 2/M phase decreased.[Conclusions]Overall,the combined effect of psoralen and paclitaxel can enhance anti-tumor ability by inhibiting cell proliferation,inducing apoptosis,and blocking cell cycle.

Effects of psoralens as anti-tumoral agents in breast cancer cells

This review examines the biological properties of coumarins, widely distributed at the highest levels in the fruit, followed by the roots, stems and leaves, by considering their beneficial effects in the prevention of some diseases and as anti-cancer agents. These compounds are well known photosensitizing drugs which have been used as pharmaceuticals for a broad number of therapeutic applications requiring cell division inhibitors. Despite this, even in the absence of ultraviolet rays they are active. The current paper mainly focuses on the effects of psoralens on human breast cancer as they are able to influence many aspects of cell behavior, such as cell growth, survival and apoptosis. In addition, analytical and pharmacological data have demonstrated that psoralens antagonize some metabolizing enzymes, affect estrogen receptor stability and counteract cell invasiveness as well as cancer drug resistance. The scientific findings summarized highlight the pleiotropic functions of phytochemical drugs, given that recently their target signals and how these are modified in thecells have been identified. The encouraging results in this field suggest that multiple modulating strategies based on coumarin drugs in combination with canonical chemotherapeutic agents or radiotherapy could be a useful approach to address the treatment of many types of cancer.

Comparative Study on Management of Vitiligo with Psoralen plus Steroid (Oxabet Formula) Alone VS Psoralen Formula plus Narrow Band of Ultraviolet B 311 nm in Khartoum Teaching Hospital of Dermatology and Venereology (KTHDV)

A comparative study and management has been conducted in (KTHDV). For some patients who attend the out patients clinic concern on treatment of the vitiligo with a new formula (Oxabet) alone VS Oxabet (oxpsolaren plus betamethasone) formula plus NB. UVB311 is during a period from (Jan 2011-Jan 2013). The study sample includes different age groups of both sexes. The study revealed that the formula alone gives good results. The localized vitiligo has a good response to the formula than generalized one. The early lesions have good responses than the old ones. The continuations of applying the treatment and the follow up of the patients enhance the efficacy of the treatment.

Qualitative Identification and Content Determination of Psoralen in Ficus Pandurata Hance

[Objectives]This study aimed to establish a qualitative identification and content determination method for psoralen in Ficus pandurata Hance and compare the psoralen contents in roots,stems and leaves of F.pandurate Hance and Ficus pandurata Hance var.holophylla Migo.[Methods]Thin-layer chromatography(TLC)was used for qualitative identification,and the content of psoralen was determined by high-performance liquid chromatography.The chromatographic conditions were as follows:column,Agilent ZORBAX Eclipse Plus C18(250 mm×4.6 mm,5μm);mobile phase,methanol-water(55∶45);flow rate,1 mL/min;detection wavelength,246 nm;column temperature,30℃;and injection volume,10μL.[Results]The TLC chromatogram of F.pandurate Hance showed clear spots and good separation.The concentration of psoralen detected in the range of 2.06-41.20μg/mL had a good linear relationship with the peak area(r=0.9999).The RSD values of the precision,stability and reproducibility tests were all less than 2%.The average recovery rate was 100.2%(RSD=1.13%,n=6).[Conclusions]The established method is simple,easy,accurate and reproducible.It can be used for quality control of F.pandurate Hance.

补骨脂素(Psoralen)的合成

补骨脂素是中药补骨脂的主要成分。临床用于治疗由于口服长效避孕药(甾体激素)引起的子宫出血。初步说明它具有类似止血灵(中药补骨脂制剂)的止血效果。作者介绍了由间苯二酚出发合成补骨脂素的方法。此方法与已报道的合成路线收率相似,但步骤简化了,成本降低了三分之一。

Determination of icariside,hyperoside and psoralen in food by liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was built to determine icarside,hyperoside and psoralen in food.The samples were extracted with 70%methanol,the solid and semi-solid hotpot seasoning samples were purified by solid phase extraction column,and then determined by HPLC-MS/MS.Acetonitrile and 0.1%formic acid solution were used as the mobile phase,and the gradient elution was adopted for analysis.As shown in the results,the analytes had good linearity in the range of 0.05−100 ng/mL,and the correlation coeffificients(R^(2))were greater than 0.999.In this method,the limits of quantitation(LOQ)of psoralen,icariside and hyperoside in liquid samples were 1.25,25.0 and 12.5μg/L respectively;while the LOQs of psoralen,icariside and hyperoside in solid samples and hotpot seasoning samples were 1.25,25.0 and 12.5μg/kg,respectively.The liquid beverage,solid beverage,health food(in the form of oral liquid,capsule,tablet),integrated alcoholic beverage and solid hotpot seasoning were selected as representative samples and used for method validation.The average spiked recoveries at 3 levels(LOQ,2 LOQ,10 LOQ)were in the range of 83.7%−115.0%,and the relative standard deviations were in range of 0.5%−9.4%(n=6).The method is rapid,accurate and sensitive,which is suitable for the simultaneous determination of icariside,hyperoside and psoralen in different food matrices.

五指毛桃不同部位补骨脂素合成酶基因的表达及其代谢产物含量

为探究五指毛桃(Ficus hirta Vahl)不同部位补骨脂素合成酶基因(psoralen synthetase,PS)表达与补骨脂素含量的关系,本研究分别采用实时荧光定量PCR法和高效液相色谱法测定来自广西和福建的五指毛桃根、茎、叶和果实PS基因的表达量及补骨脂素含量,分析不同五指毛桃种质不同部位PS基因表达及其代谢产物含量的差异。结果显示:PS基因在广西和福建五指毛桃的根、茎、叶和果实中均有表达,且其表达量具有组织特异性,在根中的相对表达量最高(侧根高于主根),叶中次之(全缘叶高于缺刻叶),茎和果实的表达量极低。使用高效液相色谱测得广西和福建的五指毛桃中补骨脂素含量存在差异,在2个五指毛桃种质的主根、侧根、缺刻叶、全缘叶、茎、果实中均有积累,侧根中的含量最高,分别为3.206、2.947 mg/g,主根次之(2.136、0.419 mg/g),缺刻叶中的含量分别为0.022、0.028 mg/g,全缘叶中的含量分别为0.069、0.035 mg/g,茎(0.003、0.003 mg/g)和果实(0.002、0.001 mg/g)中含量较低。五指毛桃PS基因表达与补骨脂素含量具有相关性,该研究结果为解析PS基因在五指毛桃补骨脂素合成中的作用提供参考。

补骨脂素对环磷酰胺抑制小鼠骨髓间充质干细胞成骨分化的恢复作用

背景:补骨脂素具有很强的抗骨质疏松活性,可能对化疗导致的骨质疏松具有恢复作用。目的:探讨补骨脂素对环磷酰胺抑制小鼠骨髓间充质干细胞成骨分化的恢复作用及机制。方法:分离培养C57BL/6小鼠骨髓间充质干细胞,以MTT法检测补骨脂素对骨髓间充质干细胞活力的影响,以成骨诱导结合碱性磷酸酶染色确定补骨脂素对环磷酰胺抑制骨髓间充质干细胞成骨分化恢复作用的最佳剂量。采用RT-qPCR法检测补骨脂素干预不同时间点成骨分化标志基因Runx2、ALP、Osteocalcin、骨保护素及Wnt/β-catenin信号通路相关基因Wnt1、Wnt4、Wnt10b、β-catenin、c-MYC的mRNA表达;采用Western blot法检测补骨脂素干预不同时间点成骨特异性转录因子Runx2及Wnt/β-catenin信号通路相关基因Activeβ-catenin、DKK1、c-MYC、Cyclin D1的蛋白表达。结果与结论:(1)不同浓度补骨脂素对骨髓间充质干细胞活力没有显著影响;200μmol/L补骨脂素干预后对环磷酰胺诱导骨髓间充质干细胞成骨分化抑制的恢复作用最佳;(2)补骨脂素可以逆转环磷酰胺条件培养基导致的骨髓间充质干细胞成骨标志基因Runx2、ALP、Osteocalcin、骨保护素mRNA表达和Runx2蛋白表达的降低;(3)补骨脂素可以逆转环磷酰胺条件培养基导致的骨髓间充质干细胞Wnt/β-catenin通路相关基因Wnt4、β-catenin、c-MYC mRNA表达和Activeβ-catenin、c-MYC、Cyclin D1蛋白表达的降低以及DKK1蛋白表达的升高;(4)结果表明,环磷酰胺能抑制小鼠骨髓间充质干细胞成骨分化,补骨脂素对其具有恢复作用,且200μmol/L补骨脂素干预效果最佳,而这种保护作用可能与补骨脂素激活Wnt4/β-catenin信号通路有关。

补骨脂素对人牙周膜干细胞增殖、成骨分化的促进作用及其机制

目的观察补骨脂素对人牙周膜干细胞(hPDLSCs)增殖、成骨分化的促进作用,并探讨其机制。方法将第三代hPDLSCs分为6组,5、10、25、50、100μmol/L补骨脂素组加入5、10、25、50、100μmol/L补骨脂素,对照组正常培养,采用CCK-8法检测细胞增殖能力,比色法检测碱性磷酸酶(ALP)活性,茜素红染色检测成骨诱导矿化结节。另取第三代hPDLSCs并分为两组,25μmol/L补骨脂素组加入25μmol/L补骨脂素,对照组正常培养,采用RT-PCR法检测转录因子2(Runx2)、骨桥蛋白(OPN)mRNA。结果与对照组比较,5、10、25、50、100μmol/L补骨脂素组细胞增殖能力和ALP活性增加,25μmol/L补骨脂素组细胞成骨矿化结节增加(P均<0.05);与5μmol/L补骨脂素组比较,25、50、100μmol/L补骨脂素组细胞增殖能力及10、25μmol/L补骨脂素组细胞ALP活性和25μmol/L补骨脂素组细胞成骨矿化结节增加(P均<0.05);与10μmol/L补骨脂素组比较,25、50、100μmol/L补骨脂素组细胞增殖能力增加(P均<0.05);与25μmol/L补骨脂素组比较,50、100μmol/L补骨脂素组细胞ALP活性降低(P均<0.05)。与对照组比较,25μmol/L补骨脂素组Runx2、OPN mRNA相对表达量增加(P均<0.05)。结论5~100μmol/L补骨脂素均能促进hPDLSCs的增殖和成骨分化,在5~25μmol/L呈剂量依赖性增强,在50~100μmol/L变化不明显;补骨脂素促进hPDLSCs的增殖和成骨分化的机制可能与调节Runx2信号通路有关。

补骨脂异黄酮通过miR-497-5p调控对瘢痕疙瘩成纤维细胞增殖和迁移的机制研究

目的:探讨补骨脂异黄酮通过调控miR-497-5p抑制瘢痕疙瘩成纤维细胞增殖和迁移的机制研究。方法:体外培养瘢痕疙瘩成纤维细胞,使用浓度为0、9、18、36μg/ml补骨脂异黄酮处理细胞,记为Con组、低剂量组、中剂量组、高剂量组;按照脂质体法miR-NC、miR-497-5p转染细胞,记为miR-NC组、miR-497-5p组;按照脂质体法anti-miR-NC、anti-miR-497-5p转染细胞后,使用36μg/ml补骨脂异黄酮处理细胞,记为高剂量组+anti-miR-NC组、高剂量组+anti-miR-497-5p组。CCK8、克隆实验检测细胞增殖;伤口愈合实验检测细胞迁移;Western blot实验检测Cyclin D1、MMP2、MMP9蛋白表达水平;qRT-PCR实验检测miR-497-5p表达水平。结果:补骨脂异黄酮呈剂量关系降低瘢痕疙瘩成纤维细胞活性、迁移率(P<0.05),减少克隆细胞数(P<0.05),降低Cyclin D1、MMP2、MMP9蛋白表达水平(P<0.05),增加miR-497-5p表达水平。与miR-NC组相比,miR-497-5p组细胞活性、迁移率降低(P<0.05),克隆细胞数减少(P<0.05),Cyclin D1、MMP2、MMP9蛋白表达水平降低(P<0.05)。抑制miR-497-5p可以逆转补骨脂异黄酮对瘢痕疙瘩成纤维细胞增殖、迁移的影响。结论:补骨脂异黄酮可能通过上调miR-497-5p抑制瘢痕疙瘩成纤维细胞增殖和迁移。

基于星点设计-效应面法联用正交设计优选抗骨质疏松颗粒的制剂工艺

目的用星点设计-效应面法(CCD-RSM)联用正交实验设计优选抗骨质疏松颗粒的制剂工艺。方法以溶媒用量、煎煮时间为考察因素,以有效成分补骨脂素、异补骨脂素总量和干膏收率的总评“归一值”(optical density,OD)为评价指标,用CCDRSM确定最佳提取工艺;以颗粒溶化性、吸湿率、水分、成型率、休止角为评价指标,用正交实验优选最佳成型工艺。结果最佳提取工艺为加水量12倍,提取2次,每次130 min;成型工艺为浸膏与辅料的比例为1∶2,可溶性淀粉与糊精的比例为1∶3,用体积分数为90%的乙醇制粒,75℃干燥,过16目筛整粒,即得。结论优选的制剂工艺条件稳定、可行,可为抗骨质疏松颗粒的实际生产提供依据。

HPLC法同时测定驻春胶囊中4个成分的含量

目的:建立以高效液相色谱法(HPLC)同时测定驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷、蛇床子素含量的方法。方法:采用HPLC法,以异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素作为指标成分,采用Agilent ZORBAX SB-C_(18)色谱柱(4.6 mm×250 mm,5μm);流动相为甲醇-水,梯度洗脱,体积流量为1.0 m·min^(-1);柱温为30℃,检测波长为254 nm,进样量为5μL。结果:异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素质量浓度分别在0.0059~0.1770 mg/mL、0.0049~0.1470 mg/mL、0.0031~0.093 mg/mL和0.0048~0.1440 mg/mL范围内与峰面积呈良好的线性关系,其相关系数分别为0.9996、0.9995、0.9997和0.9995,平均加样回收率分别为101.45%、100.95%、100.46%和101.17%;RSD分别为2.19%、1.83%、1.71%和2.01%。结论:该分析方法适用于驻春胶囊中异补骨脂素、补骨脂素、淫羊藿苷和蛇床子素的含量测定。

Formulation optimization, in situ intestinal absorption and permeability of psoralen and isopsoralen

Objective: To optimize a self-emulsifying drug delivery system(SEDDS) formulation for psoralen and isopsoralen(PSO and IPSO) isolated from Psoraleae Fructus.Methods: A D-optimal design was used to investigate the influence of oil percentage, surfactant percentage and cosurfactant percentage on several properties of SEDDS including particle size, polydispersity,equilibrium solubility, in situ intestine absorption rate and intestinal permeability. Furthermore, the desirability function approach was applied to obtain the optimal formulation for the system.Results: The oil percentage, surfactant percentage and cosurfactant percentage were optimized to be53.6%, 35.7% and 10.7%, respectively, which means the model is available.Conclusions: The D-optimal design is valuable to optimize the SEDDS formulation and understand formulation compositions’ functions on SEDDS properties.

高效液相色谱测定温肾暖脾颗粒中补骨脂素和异补骨脂素含量

目的建立温肾暖脾颗粒补骨脂素、异补骨脂素的含量测定方法,为质量控制提供实验依据。方法以十八烷基硅烷键合硅胶为填充剂;流动相:甲醇-水(40:60);检测波长246 nm;结果峰面积和质量浓度之间线性关系良好,补骨脂素R^(2)=0.9992,异补骨脂素R^(2)=0.9993;精密度、稳定性均符合要求;补骨脂素平均回收率为98.25%,RSD为0.89%,异补骨脂素平均回收率为95.90%,RSD为1.18%。结论本试验研究并建立了高效液相色谱(high performance liquid chromatography,HPLC)补骨脂素、异补骨脂素的含量测定方法,可以作为温肾暖脾颗粒质量控制的依据。

补骨脂醇提物UPLC-Q-TOF-MS化学成分分析及促黑色素生成作用机制初探

分析补骨脂醇提物(Psoraleae Fructus alcohol extract,PFE)化学成分并探讨其促黑色素生成作用机制。采用超高效液相色谱-四级杆飞行时间质谱(UPLC-Q-TOF-MS)对PFE化学成分进行鉴定;观察PFE对斑马鱼黑色素生成的影响;通过实时荧光定量聚合酶链式反应(quantitative real-time PCR,qRT-PCR)考察PFE、补骨脂素和异补骨脂素对小鼠黑色素瘤细胞B16-F10中酪氨酸酶(tyrosinase,Tyr)、酪氨酸酶相关蛋白1(Tyr-related protein 1,Tyrp1)和相关蛋白2(Tyr-related protein 2,Tyrp2)mRNA表达的影响。结果在PFE中共鉴定47个化学成分,其中黄酮类27个,香豆素类10个,单萜酚类8个和其他类2个;发现PFE作用斑马鱼18 hpf(受精后小时,hours post fertilization)后,能明显增加其体内黑色素水平;qRT-PCR结果显示,与对照组相比,PFE能显著增加Tyr和Tyrp2 mRNA的表达,其他给药组均能不同程度地增加Tyr、Tyrp1和Tyrp2 mRNA的表达。

五指毛桃饮片的质量标准研究

目的:完善五指毛桃饮片的质量标准。方法:收集不同产地的五指毛桃饮片29批,对其进行外观性状、粉末鉴别和薄层鉴别,并测定水分、总灰分、酸不溶性灰分、水溶性浸出物的含量;采用HPLC法测定其补骨脂素、佛手柑内酯的含量;建立其HPLC特征图谱,以补骨脂素(4号峰)为参照,采用《中药色谱指纹图谱相似度评价系统(2012年版)》进行相似度评价,并确定共有峰。结果:五指毛桃饮片呈类圆形或不规则的厚片,表皮灰棕色至红褐色,皮部与木部易分离,质硬而脆,断面纤维性;粉末灰黄白色,可见石细胞、草酸钙方晶、淀粉粒、木栓细胞等;薄层鉴别结果发现,供试品和对照品在薄层板相应的位置显相同颜色的斑点;其水分、总灰分、酸不溶性灰分、水溶性浸出物、补骨脂素和佛手柑内酯的含量分别为5.67%~11.29%、1.90%~3.85%、0.06%~0.69%、5.71%~19.17%、0.01%~0.36%和0.01%~0.18%,平均值分别为8.02%、2.47%、0.23%、12.16%、0.17%和0.05%,拟定其水分、总灰分和酸不溶性灰分的含量分别不得超过12.0%、4.0%、0.7%,水溶性浸出物、补骨脂素和佛手柑内酯的含量分别不得低于6.0%、0.02%和0.01%;其特征图谱的相似度大于0.900,共确认了11个共有峰,指认4、5号峰分别为补骨脂素、佛手柑内酯。结论:所建标准可为科学评价五指毛桃饮片的质量提供参考。

Psoralen inhibits hepatitis B viral replication by down-regulating the host transcriptional machinery of viral promoters

The hepatitis B virus(HBV) is a global public health challenge due to its highly contagious nature. It is estimated that almost 300 million people live with chronic HBV infection annually. Although nucleoside analogs markedly reduce the risk of liver disease progression, the analogs do not fully eradicate the virus. As such, new treatment options and drugs are urgently needed. Psoralen is a nourishing monomer of Chinese herb and is known to inhibit virus replication and inactivate viruses. In this study, we evaluated the potential of psoralen as an anti-HBV agent.Quantitative PCR and Southern blot analysis revealed that psoralen inhibited HBV replication in Hep G2.2.15 cells in a concentration-dependent manner. Moreover, psoralen was also active against the 3TC/ETV-dual-resistant HBV mutant. Further investigations revealed that psoralen suppressed both HBV RNA transcription and core protein expression. The transcription factor FOXO1, a known target for PGC1α co-activation, binds to HBV precore/core promoter enhancer II region and activates HBV RNA transcription. Co-immunoprecipitation showed that psoralen suppressed the expression of FOXO1, thereby decreasing the binding of FOXO1 co-activator PGC1αto the HBV promoter. Overall, our results demonstrate that psoralen suppresses HBV RNA transcription by downregulating the expression of FOXO1 resulting in a reduction of HBV replication.

基于转录组测序技术分析补骨脂素对HepG2细胞的作用机制

目的利用转录组测序(RNA-sequencing,RNA-seq)技术分析补骨脂素对肝癌HepG2细胞的作用机制。方法利用CCK-8法检测不同浓度的补骨脂素(0、62.5、125、250、500μM)作用HepG2细胞12 h和24 h后存活率的变化;流式细胞术检测250μM补骨脂素对HepG2细胞处理24 h后细胞周期的变化;RNA-seq测序获得对照组和补骨脂素组差异表达基因并进行GO富集分析和KEGG通路分析;实时荧光定量PCR技术验证差异基因表达水平。结果与对照组相比,补骨脂素作用HepG2细胞后,其存活率明显下降(P<0.01),细胞周期在G0/G1期比例提高(P<0.05);RNA-seq测序分析共有286个差异显著基因,其中包括130个上调基因和156个下调基因;GO功能富集和KEGG信号通路主要集中在细胞周期相关生物学过程及信号通路;qPCR结果显示HepG2细胞经补骨脂素处理后p 53和GADD 45B基因上调(P<0.05,P<0.01),CDK 2、RRM2和CCND 1基因下调(P<0.05,P<0.01)。结论补骨脂素对HepG2细胞产生毒性,阻滞细胞周期进程,其作用机制可能与调控细胞周期相关基因(p 53,CDK2,GADD45B,CCND1,RRM2)的表达有关。

补骨脂配方颗粒提取工艺研究

优选补骨脂配方颗粒最佳水提取工艺,以出膏率、补骨脂素和异补骨脂素总含量及转移率为考察指标,根据单因素考察结果,以煎煮时间、加水量和煎煮次数3个因素设计正交实验方案。结果提取次数和加水量对指标成分的提取率有显著影响,补骨脂配方颗粒的最佳提取工艺为提取3次,加水倍量分别为16/14/14倍,煎煮时间为1.5/1.5/1.5 h。该工艺条件可有效提高补骨脂中两个成分的转移率,可为补骨脂配方颗粒的生产工艺提供一定的参考依据。

Theoretical Study of Stability and Electronic Characteristics in Various Complexes of Psoralen as an Anticancer Drug in Gas Phase,Water and CCl_(4) Solutions

The present research employs density functional theory(DFT)computations to analyze the structure and energy of complexes formed by psoralen drug with alkali(Li^(+),Na^(+),K^(+))and alkaline earth(Be^(2+),Mg^(2+),Ca^(2+))metal cations.The computations are conducted on M06-2X/aug-cc-pVTZ level of theory in the gas phase and solution.The Atoms in Molecules(AIM)and natural bond orbital(NBO)analyses are applied to evaluating the characterization of bonds and the atomic charge distribution,respectively.The results show that the absolute values of binding energies decrease with going from the gas phase to the solution.Furthermore,the considered complexes in the water(as a polar solvent)are more stable than the CCl_(4)(as a non-polar solvent).The DFT based chemical reactivity indices,such as molecular orbital energies,chemical potential,hardness and softness are also investigated.The outcomes show that the considered complexes have high chemical stability and low reactivity from the gas phase to the solution.Finally,charge density distributions and chemical reactive sites of a typical complex explored in this study are obtained by molecular electrostatic potential surface.