补骨脂中新补骨脂素的分离与鉴定

从补骨脂(Psotalea corylifolia Linn)中分得4个香豆系成分,其中一个(1)为新化合物,命名为新补骨脂素。另3个已知成分为异补骨脂素(Ⅱ),补骨脂素(Ⅲ),补骨脂定(Ⅳ)。     

补骨脂中呋喃香豆素类物质的积累规律及组织定位

应用高效液相色谱技术,分析了补骨脂(Psoralea corylifolia)不同器官、不同发育时期补骨脂素和异补骨脂素的含量变化规律。2种呋喃香豆素类物质在补骨脂的根、茎、叶、花、果实和种子中均有积累,其含量在果实中最高,根中最低,幼嫩茎叶高于成熟茎叶,成熟期果实高于幼嫩期果实。通过组织化学和荧光显微技术对呋喃香豆素类物质的定位研究,同时结合分泌腔的系数与2种呋喃香豆素的含量相关性分析,证明补骨脂中分泌腔是2种呋喃香豆素积累的主要场所。     

异补骨脂素加锌对大鼠成骨细胞增殖与分化的影响

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入异补骨脂素与锌,MTT法检测加药后不同时间成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量.结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在24和48 h时促体外大鼠成骨细胞增殖的作用更加明显;在48和72 h时能促进成骨细胞碱性磷酸酶活性(ALP),其中ALP的测定在72h的活性更为显著.与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对体外培养的成骨细胞的增殖与分化作用更加显著.     

补骨脂不同炮制饮片炮制前后化学成分定性定量分析

为探讨补骨脂不同炮制方法下炮制前后化学成分的变化,采用超高效液相色谱-飞行时间质谱联用技术(UHPLC-Q-TOF-MS/MS),对补骨脂生品、清炒品及盐炙品进行化学成分分析,运用超高效液相色谱(UHPLC)对补骨脂中9个成分进行含量测定,结合统计学方法分析炮制前后含量变化。在定性分析中,共鉴定指认38个化合物,建立了补骨脂MS数据库,发现了5个成分存在于生品与清炒饮片,而盐炙品中未发现;在定量分析中,发现补骨脂生品与炮制品(盐炙和清炒)化学成分有显著性差异,并筛选出4个差异标志物(补骨脂酚、补骨脂素、补骨脂宁和新补骨脂异黄酮)。从整体来看,补骨脂生品与炮制品(盐炙和清炒)在化学成分轮廓上存在一定差异。由含量变化推测,在补骨脂的炮制过程中,补骨脂酚和补骨脂素、补骨脂宁和新补骨脂异黄酮可能存在转化。本实验结果与建立的补骨脂MS数据库可为补骨脂炮制机理进一步研究提供数据支撑。     

补骨脂素对大鼠成骨细胞增殖与分化的影响

为探讨补骨脂素体外对大鼠成骨细胞增殖与分化的影响,用改良的组织块法分离培养新生大鼠颅骨成骨细胞,补骨脂素以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。补骨脂素浓度在1μmol/L 24 h,5~20μmol/L浓度范围内48 h,1、10、20μmol/L浓度范围内72 h促进成骨细胞增殖,在10~15μmol/L范围内48 h及72 h提高成骨细胞内碱性磷酸酶的活性。补骨脂素体外能促进成骨细胞的增殖与分化。     

补骨脂素加锌对大鼠成骨细胞增殖与分化影响的实验研究

为探讨补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞增殖、分化作用的影响及其量效关系,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入补骨脂素与锌,MTT法检测加药后不同时间成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性;用放射免疫法(RIA)测定细胞外骨钙素(BGP)的含量,用改良的Lowry法测蛋白含量。补骨脂素加锌较单独应用补骨脂素或硫酸锌在48 h和72 h时促体外大鼠成骨细胞增殖的作用更加明显;在24、48 h和72 h时能提高成骨细胞碱性磷酸酶(ALP)活性,其中在48 h时的效果更为显著;在24 h和72 h时能提高细胞外液中的骨钙素含量;补骨脂素浓度为1×10-9mol/L和锌浓度为1×10-5mol/L两者联合用药较为合适。与单独应用补骨脂素或者锌相比,补骨脂素与锌联合应用能够协同增效,对体外培养的大鼠成骨细胞的增殖与分化作用更加显著。     

一种新型补骨脂素衍生物BSP-1诱导B16细胞中黑色素合成的作用机制

8-甲氧基补骨脂素（8-MOP）和5-甲氧基补骨脂素（5-MOP）等补骨脂素类药物在临床上常用于治疗白癜风,但同时具有诸多副作用。因此,发掘作用更强、毒性更小的补骨脂素类化合物用以治疗白癜风成为研究热点。在我们的前期研究中,本课题组设计合成了一系列结构新颖的补骨脂素席夫碱衍生物,并评价了它们的抗白癜风活性。本论文选取了其中一个补骨脂素席夫碱衍生物（BSP-1）,研究了它对小鼠B16细胞中黑色素合成的作用及其信号通路。利用CCK 法、L-Dopa 氧化法、NaOH溶解法及Western印迹法分别分析BSP-1对细胞增殖、黑色素含量、酪氨酸酶(TYR)活性及相关蛋白表达的影响。结果显示,BSP-1能够促进B16细胞内黑色素生成和TYR活性,上调 TYR、TRP-1、TRP-2和MITF的蛋白表达,并呈浓度依赖性。机制研究发现,BSP-1通过提高Akt和GSK-3β的磷酸化水平,上调细胞核中β 联蛋白的含量,最终使得小眼相关转录因子（MITF）的蛋白表达增加。综上所述,本研究提示BSP-1可通过调节Wnt/β-联蛋白信号通路来促进B16细胞内的黑色素合成。     

甲氧补骨脂素对大鼠成骨细胞生物活性影响的实验研究

为探讨甲氧补骨脂素对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入甲氧补骨脂素,MTT法检测加药后不同时间细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量;用放射免疫法测定细胞内骨钙素含量。结果显示:与对照组相比,甲氧补骨脂素组在24 h和36 h时促进体外大鼠成骨细胞增殖的作用更明显;在24、48 h和72 h时均能提高成骨细胞碱性磷酸酶活性(ALP)和骨钙素(BGP)的分泌。甲氧补骨脂素对体外培养的大鼠成骨细胞的增殖与分化均有明显的促进作用。     

异补骨脂素加锌对大鼠成骨细胞相关细胞因子表达影响的实验研究

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞相关基因表达的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中加入异补骨脂素与锌,以雌激素为阳性对照,空白组为阴性对照。结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在48 h时促体外大鼠成骨细胞Ⅰ型胶原的表达;异补骨脂素加锌组可以明显上调大鼠成骨细胞TGF-β1的细胞信号转导因子Smad4 mRNA的表达(P0.01);能促进Runx2/Cbfa1 mRNA的表达(P0.05)。与空白对照组相比,异补骨脂素组,异补骨脂素加锌组均能增强Osterix mRNA的表达(P0.05)。与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对促进体外培养的成骨细胞相关转录因子的表达更加明显,探讨了异补骨脂素及其与锌配伍调节骨代谢的分子机制,为提高骨质疏松症的临床疗效以及抗骨质疏松新药的开发提供了实验依据。     

补骨脂素复配物对酪氨酸酶协同激活性能研究

从中药补骨脂中提取分离得到补骨脂素纯品。研究其与助剂的复配物对黑色素形成过程中主要限速酶酪氨酸酶的协同激活作用。结果表明,补骨脂素对酪氨酸酶有明显的激活作用,但是不随浓度的增加呈线性变化,与助剂复配的协同效应显著。     

Enhancement in furanocoumarin content and phenylalanine ammonia lyase activity in developing seedlings of Psoralea corylifolia L. in response to gamma irradiation of seeds

Gamma irradiation of seeds is known to be an important factor in stimulating biochemical and physiological processes. The aim of the present study was to investigate phenylpropanoids and associated enzymes responsible for the production of active metabolites. Furanocoumarin content was estimated in seeds of Psoralea corylifolia L. during two successive generations (G(1) and G(2)) where as phenylalanine ammonia lyase (PAL) activity was measured in leaves at different developmental stages of P. corylifolia L. raised from seeds irradiated with variable doses of gamma rays. Maximum accumulation of psoralen and isopsoralen was observed at 15 and 20 kGy doses during G(1) and G(2) generations, respectively. Psoralen proved to be the dominating metabolite in terms of its concentration, while isopsoralen was accumulated at relatively lower concentrations in successive generations. PAL activity was induced maximally following 15 and 20 kGy in G(1) plants and was preceded by psoralen and isopsoralen accumulation which peaked at the same dose rates in both generations. These effects were transmitted and prevalent in the next generation, that is, G(2) (indirectly irradiated). These long-term changes in plant metabolomics demonstrate genomic instability induced by gamma irradiation. However, no detrimental effects were seen at any irradiation dose in seeds. Furanocoumarin concentrations were also enhanced at 15 and 20 kGy. The present study further points out the persistence of changes in the biosynthesis of coumarin derivatives in the next generation. However, accumulation of these metabolites does not lead to any lethal effects.     

Psoralea corylifolia extracts stimulate cholinergic-like psoralen receptors of tadpole-tail melanophores,leading to skin darkening

The present work was carried out to determine the effects of lyophilized seed extracts of Psoralea corylifolia along with pure psoralen, its active ingredient on the isolated tail-piece melanophores of Bufo melanostictus, a type of disguised smooth muscle cells, which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. In the present study, it was found that lyophilized extract of P. corylifolia and its active ingredient psoralen induced powerful, dose-dependent, physiologically significant melanin dispersal effects in the isolated tail melanophores of B. melanostictus, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of P. corylifolia and its active ingredient psoralen were highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of P. corylifolia and psoralen are mediated by cholino-muscarinic or cholino-psoralen like receptors having similar properties that need to be studied further.     

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC-MS

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.     

Psoralen attenuates bleomycin‐induced pulmonary fibrosis in mice through inhibiting myofibroblast activation and collagen deposition

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) and chronic inflammation with limited therapeutic options. Psoralen, a major active component extracted from Psoralea corylifolia L. seed, has several biological effects. However, the role of psoralen in IPF is still unclear. Here, we hypothesized that psoralen played an essential role in IPF in the inhibition of fibroblast proliferation and inflammatory response. A murine model of IPF was established by injecting bleomycin (BLM) intratracheally, and psoralen was administered for 14 days from the 7^th to 21^st day after BLM injection. Our results demonstrated that psoralen treatment reduced body weight loss and improved the survival rate of mice with IPF. Histological and immunofluorescent examination showed that psoralen alleviated BLM‐induced lung parenchymal inflammatory and fibrotic alteration. Furthermore, psoralen inhibited proliferation and collagen synthesis of mouse fibroblasts and partially reversed BLM‐induced expression of α‐smooth muscle actin at both the tissue and cell level. Moreover, psoralen decreased the expression of transforming growth factor‐β1, interleukin‐1β, and tumor necrosis factor‐α in the lungs of BLM‐stimulated mice. Our results reveale for the first time that psoralen exerts therapeutic effects against IPF in a BLM‐induced murine model.     

Ethanolic extracts of Euphorbia and other ethnobotanical species as inhibitors of human tumour cell growth

Ethanolic extracts of 20 plant species, selected from the ethnobotanical literature, were analysed for their pharmacological potential as antineoplastic agents against the HEp-2 cell line. Psoralea corylifolia and E. grandidens were the most efficacious species eliciting IC50 values of 22 microg/ml and 57 microg/ml respectively. Psoralea corylifolia, additionally tested against lung carcinoma (A549) cells gave an IC50 value of 68 microg/ml. Such data would justify a search for active compounds from this species.     

Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco

Applied Microbiology and Biotechnology - Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by...     

Hypoxia-inducible factor-1 and nuclear factor-kappaB inhibitory meroterpene analogues of bakuchiol, a constituent of the seeds of Psoralea corylifolia

Two new meroterpenoids, 12,13-dihydro-12,13-dihydroxybakuchiol (2) and (12'S)-bisbakuchiol C (3), were isolated from the seeds of Psoralea corylifolia L. (Fabaceae). The structures of 2 and 3 were elucidated by spectroscopic and chemical methods. Six meroterpenoids isolated from P. corylifolia and three semi-synthetic analogues were evaluated for HIF-1 and NF-kappaB inhibition, and O-methyl and O-ethylbakuchiols (6 and 7) inhibited HIF-1 and NF-kappaB activation without significantly decreasing the viability of the AGS and HeLa cells, respectively.     

珊瑚菜不同部位香豆素含量的研究

应用高效液相色谱技术,对珊瑚菜（Glehnia littoralis Fr.Schmidt ex Miq.）的果实、叶和根以及采后去皮入药的根和根皮中的补骨脂素、欧前胡素和异欧前胡素3种香豆素的含量进行了测定。结果显示：（1）3种香豆素在果实、叶与根、根皮中均有积累,总含量在果实中最高,为0.6364mg·g-1,根中为0.0657mg·g-1,根皮中为0.0312mg·g-1,叶中为0.0151mg·g-1,采后去皮处理的根中最低,仅为0.0081mg·g-1。（2）珊瑚菜根经水烫去皮处理后香豆素含量急剧下降,与同期采收未去皮处理的根相比,去皮后根内香豆素总含量、补骨脂素、欧前胡素、异欧前胡素含量分别下降了87.7%、100%、82.76%、85.25%。（3）与未去皮的根相比,处理后的根皮中香豆素总量、补骨脂素、欧前胡素和异欧前胡素含量分别是未去皮处理的珊瑚菜根中的47.42%、31.37%、51.54%和53.28%。研究表明：从充分利用香豆素成分的角度出发,根入药时应带根皮使用,另外,叶和根皮不应丢弃,均可收集作为提取香豆素新的植物资源,果实中香豆素含量很高,亦可作为香豆素资源。