不同品种柚子皮精油成分及抗氧化与抑菌能力对比研究

柚子皮是一种富含精油的具有药用价值的生物质资源,但其精油成分与生物活性尚未深入研究。本文首先通过水蒸汽蒸馏法提取了12个不同品种的柚子皮精油,接着用气相色谱质谱联用仪(GC-MS)对精油成分与相对含量进行了测定,然后采用DPPH自由基清除法、滤纸片法对不同品种柚子皮精油的抗氧化和抑菌能力进行比较,最后对不同品种柚子皮精油GC-MS分析数据进行主成分分析和聚类分析,结合抗氧化和抑菌实验结果,着力于理清柚子皮精油成分与抗氧化、抑菌能力的关系。实验结果表明:柚子皮精油的提取率为0.22%~1.52%;GC-MS法从不同品种柚子皮精油中鉴定出D-柠檬烯、β-月桂烯、α-蒎烯、α-水芹烯、芳樟醇等35种化学成分;DPPH自由基清除实验表明柚子皮精油的抗氧化能力与精油浓度呈正相关性;滤纸片法抑菌实验表明柚子皮精油有一定的抑菌效果,且对特定的菌种抑菌效果更明显。主成分分析和聚类分析结果表明,抗氧化和抑菌效果显著的成分有D-柠檬烯、柠檬醛、芳樟醇,而石竹烯、香叶烯醇、α-水芹烯也具有一定的抗氧化和抑菌能力。

荷叶碱药理作用的研究进展

荷叶碱是来源于睡莲科植物莲干燥叶中的一种阿朴啡类生物碱,也是《中国药典》收载荷叶这味中药的质控成分。现代研究表明,荷叶碱具有降脂、降糖、抗肿瘤、抗脑缺血等多种药理作用,具有广阔的开发前景。但荷叶碱自身存在水溶性差、药物安全性数据缺失、生物利用度争议较大等成药性缺陷,极大地影响了荷叶碱的深入研究与开发。因此,如何从众多药理活性中获得启发,进而选择最适合荷叶碱产品开发的临床适应症,依据制剂或化学手段改善其溶解性和生物利用度,同时降低毒副作用的发生,是当前迫切需要解决的问题。该文在回顾荷叶碱的理化性质及药动学研究报道的基础上,系统总结了其药理活性及作用机制,以期为荷叶碱药理作用的深入研究及创新品种的开发提供参考。

Erzhi pills ameliorates cognitive dysfunction and alter proteomic profiles of hippocampus in ovariectomized Alzheimer disease model rats induced by D-galactose and Aβ1-40 injection

OBJECTIVE Erzhi pills is a clas⁃sic prescription of Chinese medicine originated from Fu Shou Jing Fang in Ming dynasty,with the effects of nourishing kidney-Yin and hemosta⁃sis,black hair,strengthening muscles and bones.As a classical prescription for nourishing kidney-Yin,Erzhi pills has been used to treat senile dementia in China for many years.Herein,our study aimed to investigate the protective effects of Erzhi pills in rat models of Alzheimer disease(AD)induced by ovariectomy as well as D-galactose and Aβ1-40 injection and to explore its potential mechanism.METHODS The model of AD rats was established by ovariectomy com⁃bined with D-galactose and Aβ1-40 injection.Ovariectomized rats were randomly divided into four groups:model group,estradiol valerate(0.80 mg·kg-1)group,Erzhi pills high(1.50 mg·kg-1)and low(0.75 mg·kg-1)doses group.In addition,rats of sham operation were selected as the sham operated group.Except for the sham oper⁃ated group,rats were injected intraperitoneally with D-galactose(100 mg·kg-1 per day,for 49 d)on the 8th day,then they were given intracerebro⁃ventricular injection of Aβ1-40(10μg per rat,1 g·L-1)on the 36th day,while the corresponding drugs were given by gastrointestinal administra⁃tion on the 22nd day.In our study,Morris water maze test was used to evaluate the learning and memory abilities,while ELISA kit was used to an⁃alyze serum estrogen level.The morphology of hippocampal neuron cells was observed by HE staining,and Nissl staining was utilized to ob⁃serve the Nissl body in cytoplasm.Then,the ex⁃pression of ERβpositive cells and hippocampal Aβ1-40 and p-Tau404 proteins was determined by immunohistochemistry.In order to further explore the molecular mechanism of Erzhi pills preven⁃tion and treatment of AD,proteomics was used to find potential targets and related pathways,Western blotting was used to verify the expres⁃sion of candidate differential protein.According to the results of proteomics in our experiment,Western blotting was used to detect the protein expression of PI3K,Akt,Bcl-2,Bcl-xl,Bad,14-3-3 and GSK3β.RESULTS The escape latency was significantly shortened,the number of crossing platform was increased,the neuron arrange⁃ment was more orderly and with less nuclear pycnosis in rats of Erzhi pills groups compared to the model group.In rats treated with Erzhi pills,the number of neurons,Nissl bodies,the estro⁃gen levels and ERβpositive cells were increased,while the number of Aβ1-40 and p-Tau404 positive cells was significantly decreased.Proteomics found that there were more than one hundred differentially expressed proteins of rats treated with Erzhi pills,which were involved in 48 signal⁃ing pathways.Among these proteins,five of them were involved in the PI3K/Akt signal path⁃way.As the down-stream protein of PI3K/Akt sig⁃naling pathway,the content of 14-3-3 protein was significantly increased.Western blotting analysis showed that the expression of p-GSK3β/GSK3βand Bad was decreased,while that of p-Akt/Akt,p-PI3K/PI3K,14-3-3,Bcl-xl and Bcl-2 was up-regulated in rats from the Erzhi pills groups compared with the model group.CON⁃CLUSION Erzhi pills can improve estrogen levels,alter proteomics expression of the hippocampus and activate PI3K/Akt pathway in AD rats,reduce Aβaggregation,inhibit the hyperphos⁃phorylation of Tau protein,maintain the morphol⁃ogy of hippocampal neurons and decrease the apoptosis of hippocampal neurons,thereby improving the learning and memory abilities of ovariectomized AD rats induced by D-galactose and Aβ1-40 injection.This study may provide an experimental basis for the clinical treatment of Erzhi pills.

α-Hederin inhibits growth of lung cancer A549 cells in vitro and in vivo by decreasing c-Myc and HIF-1α dependent glycolysis

OBJECTIVEα-Hederin is an effective component of the traditional Chinese medicine Pulsatilla chinensis,which has been reported to exert many pharmacological activities.However,the effect ofα-hederin on metabolism is still unclear.This study aimed to illuminate the role ofα-hederin in glucose metabolism in lung cancer cells and investigate the molecular mechanism ofα-hederin.METHODS CCK8 and colony formation assays were employed to assess the anti-proliferative effects induced byα-hederin.Glucose uptake,ATP generation,and reduced lactate production were measured using kits,and an A549 tumor xenograft mouse model of lung cancer was used to assess the in vivo antitumor effect ofα-hederin(5,10 mg·kg^-1).Glycolytic-related key enzymes were detected by Western blotting and immunohisto⁃chemical staining.RESULTS Cell proliferation was significantly inhibited byα-hederin in a dose-dependent manner and thatα-hederin inhibited glucose uptake and ATP generation and reduced lactate production.Furthermore,α-hederin remarkably inhibited hexokinase 2(HK2),glucose transporters 1(GLUT1),pyruvate kinase M2(PKM2),lactate dehydro⁃genase A(LDHA),monocarboxylate transporter(MCT4),c-Myc,and hypoxia inducible factor-1α(HIF-1α)protein expres⁃sion.Using inhibitors,we proved thatα-hederin inhibits glycolysis by inhibiting glycolytic regulators.Moreover,a tumor xenograft mouse model of lung cancer further confirmed thatα-hederin inhibits lung cancer growth via inhibiting glycolysis in vivo.CONCLUSIONα-Hederin inhibits the growth of non-small cell lung cancer A549 cells by inhibiting glycolysis.The mechanism of glycolysis inhibition includesα-hederin inhibiting the expression of the glycolytic regulatory factors HIF-1α and c-Myc.

基于TLR4/NF-κB/NLRP3通路探讨二精丸减轻AD大鼠神经炎症的作用及机制

研究二精丸对D-半乳糖联合β淀粉样蛋白_(25-35)(Aβ_(25-35))致阿尔茨海默病(AD)大鼠神经炎症的改善作用及可能机制。SD大鼠随机分为假手术组、模型对照组、阳性药物组(多奈哌齐,1 mg·kg^(-1))、二精丸高剂量组(9 g·kg^(-1))、二精丸低剂量组(4.5 g·kg^(-1)),每组14只。D-半乳糖腹腔注射2周后,连续灌胃给药5周;D-半乳糖腹腔注射3周后,大鼠双侧海马注射Aβ25-35。灌胃给药4周后,新物体识别实验检测大鼠学习记忆能力。末次给药24 h后,取组织,免疫荧光法检测大鼠脑组织小胶质细胞活化情况;免疫组化检测大鼠海马CA1区Aβ1-42和磷酸化Tau404蛋白(phosphory protein Tau^(404),p-Tau404)阳性表达;酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法检测大鼠脑组织炎症因子白细胞介素1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor,TNF-α)、白细胞介素6(IL-6)水平;Western blot法检测大鼠脑组织Toll样受体4(Toll-like receptor,TLR4)/核因子κB(nuclear factor kappaB,NF-κB)/核苷酸结合寡聚化结构域样受体3(nucleotide-binding oligomerization domain-like receptors 3,NLRP3)通路相关蛋白表达。结果显示,与假手术组比较,模型对照组大鼠新物体识别指数明显下降,海马区Aβ_(1-42)沉积和p-Tau^(404)阳性蛋白显著增多,脑组织齿状回处小胶质细胞活化水平显著增高,海马组织IL-1β、TNF-α、IL-6水平明显升高;海马TLR4、p-NF-κB P65/NF-κB P65、p-IκBα/IκBα、NLRP3蛋白表达水平显著升高。与模型对照组比较,二精丸可提高大鼠新物体识别指数,减少海马区Aβ_(1-42)沉积和p-Tau^(404)阳性蛋白表达,抑制脑组织齿状回小胶质细胞活化;降低海马组织IL-1β、TNF-α、IL-6炎症因子水平;下调TLR4、p-NF-κB P65/NF-κB P65、p-IκBα/IκBα、NLRP3蛋白的表达。综上所述,二精丸可能通过改善小胶质细胞活化,降低神经炎症因子IL-1β、TNF-α、IL-6表达水平,抑制TLR4/NF-κB/NLRP3神经炎症通路,减少海马Aβ沉积和p-Tau表达,恢复海马形态结构,改善AD模型大鼠的学习记忆能力。

二精丸对去卵巢+D-半乳糖联合Aβ1-40所致肾阴虚AD大鼠海马蛋白质组学的影响

目的:探究二精丸对去卵巢+D-半乳糖联合β-淀粉样蛋白1-40(Aβ1-40)所致肾阴虚阿尔茨海默病(AD)大鼠生物学基础的影响。方法:大鼠去卵巢后随机分为5组,即模型组、多奈哌齐组、二精丸高、中、低剂量组,每组11只,另设11只为假手术组,术后1周,连续腹腔注射D-半乳糖7周,术后4周,单侧海马注射Aβ1-40。术后3周开始灌胃给药,模型组与假手术组灌胃生理盐水,二精丸高、中、低剂量组分别用对应浓度灌胃给药(9.0,4.5,2.25 g·kg^-1),多奈哌齐组灌胃1.0 mg·kg^-1多奈哌齐,给药体积为10 mL·kg^-1,每日1次,连续给药35 d。给药第31天,Y迷宫检测大鼠学习能力。末次给药后麻醉大鼠,分离大鼠海马,尼氏染色观察形态学。提取海马蛋白,Nanol-ESI液相-质谱联用系统检测蛋白,Protein Discovery软件鉴定蛋白,SIEVE软件对海马蛋白进行相对定量定性分析,PANTHER Classification System数据库对差异蛋白进行基因本体(GO)分析,String分析和京都基因与基因组百科全书(KEGG)富集信号通路。结果:与模型组大鼠比较,二精丸高、中剂量组大鼠Y迷宫正确交替率明显提高(P<0.05),海马CA1区神经元数量明显提高(P<0.01);二精丸高剂量组的差异蛋白(Ratio>1.5或Ratio<0.5)有胰岛素样生长因子-1受体(IGF-1R)等115个,中剂量组差异蛋白有突触素等94个,多奈哌齐组差异蛋白有乙酰辅酶A乙酰转移酶等87个。GO分析发现这些蛋白主要分为微管相关蛋白、热休克蛋白、能量代谢相关蛋白等与AD相关蛋白;KEGG分析发现上述差异蛋白共涉及Dopaminergic synaps等93条信号通路。结论:二精丸可能是通过磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)信号通路、胰岛素信号通路、酸活化蛋白激酶(AMPK)信号通路、雌激素信号通路,Dopaminergic synapse等多条通路相关蛋白的表达,从而达到防治肾阴虚AD的目的。

基于蛋白质组学探讨九蒸九晒地黄延缓去卵巢小鼠脑衰老的药效与机制

探究九蒸九晒地黄延缓去卵巢小鼠脑衰老的药效与机制。小鼠去卵巢之后随机分成模型组、戊酸雌二醇组(0.3 mg·kg^(-1))以及九蒸九晒地黄低、中、高剂量组(1.0、2.0、4.0 g·kg^(-1)),另设假手术组,每组15只。术后1周,开始灌胃给药连续15周。跳台实验和水迷宫实验检测小鼠行为学变化。HE染色和尼式染色观察小鼠脑组织形态学变化。免疫组化检测小鼠脑组织Aβ和ER_(β)的表达。试剂盒检测小鼠血清雌激素水平和脑组织胆碱酯酶及胆碱酯酶转移酶水平。提取海马蛋白,Nano-ESI-LC-MS联用系统检测蛋白,Protein Discovery软件鉴定蛋白,SIEVE软件进行蛋白相对定量定性分析,PANTHER Classification System数据库对差异蛋白进行GO分析和KEGG数据库信号通路富集。与假手术组比较,模型组学习记忆能力下降,跳台潜伏期显著缩短(P<0.05),Morris水迷宫实验中,逃避潜伏期显著延长(P<0.05),穿越平台次数和目标象限停留时间缩短;海马区神经细胞排列散乱,细胞间隙变大,Aβ阳性细胞表达显著增多(P<0.05),ER_(β)阳性细胞表达显著减少(P<0.05),雌激素水平明显下降(P<0.05),胆碱功能减退(P<0.05)。与模型组相比,九蒸九晒地黄组学习记忆能力改善,跳台潜伏期显著延长,雌激素水平明显升高(P<0.05),海马区神经细胞排列整齐,形态完整,Aβ阳性细胞数量减少,ER_(β)阳性细胞表达增多。蛋白质组学筛选差异蛋白得到146个,KEGG信号通路富集分析得到75条信号通路,其中参与dopaminergic synapse信号通路的蛋白最多,有13个蛋白参与。综上,九蒸九晒地黄可延缓去卵巢小鼠脑衰老进程,其机制可能为提高雌激素水平,介导dopaminergic synapse信号通路,提高胆碱功能。

基于应激颗粒的中药神经保护研究设想

应激颗粒是存在于胞质的一种无膜致密颗粒,通过包裹mRNA、转录起始因子、RNA结合蛋白等翻译起始阶段的重要元件,使细胞在遭受不利刺激时出现短暂的蛋白翻译阻滞,从而起到细胞保护作用。研究发现,应激颗粒广泛分布于海马和皮层的神经元细胞、星形胶质细胞、小胶质细胞和浦肯野细胞等神经细胞中,生物过程涉及蛋白异常聚集、突触的成熟和可塑性、免疫调节、抗氧化应激反应、抑制细胞凋亡等,在急性缺血性脑卒中和神经退行性疾病的发生发展中发挥着重要作用。尤其是其多样化的物质组成和广泛参与的生物过程,为中医证候生物学及“同病异治”“整体调节”等中医药传统理论和治法的研究提供了崭新的思路和视野。对应激颗粒的基本特征、神经保护及研究方法等进行系统的梳理,同时以急性缺血性脑卒中为范例,尝试将应激颗粒这一现代生物学现象纳入到中医药现代研究中,提出研究设想和思路,以期用科学的语言阐释传统中医药相关理论与治法,也为行业同仁的相关研究提供参考。