RNAi沉默PLCε对Crocin抑制人膀胱癌BIU-87细胞增殖作用调控的研究

目的:观察RNAi沉默膀胱癌BIU-87细胞磷脂酶C-ε(Phospholipase C epsilon,PLCε)表达后对藏花素(Crocin)抑制人膀胱癌BIU-87细胞增殖作用的影响。方法:将携有siRNA基因的真核表达质粒PGenesil-PLCε转染BIU-87细胞后,分为5组:未转染组、pGenesil-NP阴性质粒组、pGenesil-PLCε质粒组、Crocin组、pGenesil-PLCε联合Crocin组。MTT法观察不同处理组对BIU-87细胞的生长抑制作用。RT-PCR法检测转染后对PLCεmRNA表达的影响及各处理组PCNA(Proliferating cell nuclear antigen)、CyclinD1 mRNA表达。Western-blot检测PCNA、CyclinD1蛋白表达。结果:RNAi可抑制BIU-87细胞78.06%的PLCεmRNA表达。pGenesil-PLCε联合Crocin组可增强Crocin对BIU-87细胞的生长抑制作用,48h抑制率达45.30%,且与pGenesil-PLCε质粒组和Crocin组相比较有显著性差异(P<0.01)。RT-PCR和Western-blot结果显示与RNAi沉默PLCε组比较,RNAi PLCε联合Crocin组明显下调了PCNA、Cyclin D1基因和蛋白的表达(P<0.05);较单独Crocin组比较,RNAi PLCε联合Crocin组显著降低了PCNA基因和蛋白的表达(P<0.05),降低了Cyclin D1蛋白的表达(P<0.05),Cyclin D1 mRNA表达变化两组间无显著性差异(P>0.05)。结论:沉默PLCε基因可增强Crocin对BIU-87细胞的抑制作用,其机制与进一步下调PCNA、CyclinD1的表达有关。

Effect of crocin carotenoid on BDNF and CREB gene expression in brain ventral tegmental area of morphine treated rats

Objective: To investigate the effect of crocin carotenoid on BNDF and CREB gene expression in the brain ventral tegmental area(VTA) and the serum level of BDNF in morphine-treated rats compared to control. Methods: In this study, 40 male Wistar rats(200-250 g) were used in 5 experimental groups: 1) non morphine treat rats(control); 2) non morphine-treated rats with 25 mg/kg crocin carotenoid(i.p., for 21 d); 3) morphine treated rats(10 mg/kg twice a day, s.c., 21 d); 4 and 5) morphine-treated rats with 12.5 and 25 mg/kg crocin carotenoid, respectively. By the end of research, BDNF and CREB expression was determined by real-time-PCR method. ELISA analysis was also applied for assessing the serum BDNF level. Results: The data indicated that morphine treatment could cause a significant decrease in BDNF and CREB gene expression(P<0.01 and P<0.001, respectively) in brain VTA as well as serum level of BDNF(P<0.01) in comparison to control group. Treatment with 25 mg/kg crocin carotenoid caused a significant enhancement in BDNF and CREF gene expression(P<0.01 and P<0.05, respectively) and serum level of BDNF(P<0.01) in morphine-treated rats in comparison to morphine-treated group. Conclusions: Regarding to obtained results, crocin carotenoid can inhibit unfavorable effects of morphine on the neural system to some extent through enhancing BDNF and CREB gene expression in brain VTA and serum level of BDNF.

Effects of Crocin on Nox2 Expression and ROS Level of Hypoxia/Reoxygenation-induced Injury of Cardiomyocytes

[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia/reperfusion injury.The cells were randomly divided into four groups:normal cell group(control group),crocin group),hypoxia/reoxygenation group(H/R group),hypoxia/reoxygenation+crocin group(H/R+crocin group).H/R+crocin group selected the concentration of crocin 1,10,and 100μmol/L,and determined the optimal concentration of crocin by detecting the cell proliferation ability.After the cells were pretreated using the optimal concentration of crocin,the levels of superoxide anion,cell proliferation,apoptosis and Nox2 levels in each group of cells were detected.[Results]Compared with the control group,the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced(P<0.05),while cell apoptosis and intracellular superoxide anion levels were significantly increased(P<0.01);the CMs pretreated with crocin can reduce the level of Nox2(P<0.01),increase the cell proliferation ability of CMs,reduce cell apoptosis,and accordingly reduce the level of superoxide anion in the cell(P<0.05).[Conclusions]Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury.

Hepato-and reno-protective effects of thymoquinone,crocin,and carvacrol:A comprehensive review

Medicinal plants are rich in nutrients and phytochemicals which prevent and treat a wide range of ailments.Accumulating experimental studies exhibit that some bioactive ingredients extracted from medicinal plants have suitable therapeutic effects on hepatic and renal injuries.This review focuses on the hepato-and reno-protective effects of thymoquinone,crocin,and carvacrol.The relevant literature was retrieved from PubMed,Scopus,Web of Science,and Google Scholar databases from the beginning of 2015 until the end of November 2021.According to the scientific evidence,the considered phytochemicals in this review have been applied with useful therapeutic effects on hepatic and renal damage.These therapeutic effects were mainly mediated through the amelioration of oxidative stress,suppression of inflammatory responses,and inhibition of apoptosis.Intracellular signaling pathways linked to nuclear factor kappa B(NF-κB),adenosine monophosphate-activated protein kinase,c-jun N-terminal kinase,and extracellular signal-regulated kinase 1/2 and Toll-like receptors are the most important pathways targeted by these phytochemicals.Up-regulation of transcription factor Nrf2 and down-regulation of transforming growth factor-beta 1 by these natural compounds also contribute to the alleviation of hepatic and renal injuries.

Dietary crocin reverses melanoma metastasis

Crocus sativus and its bioactive constituent crocin are well known for anti-tumor potential in different models.However, the efficacy of crocin on in-vivo melanoma metastasis is not yet reported. In this study, melanoma metastatic model was developed by tail vein injection of B16 F-10 cells in to C57 BL/6 mice. Metastatic mice treated with two different doses of crocin（250 and 500 μg/kg of bodyweight） for 10 days and parameters such as lung metastasis inhibition, mean survival time, lung hydroxyproline, uronic acid and hexosamine levels were analyzed after 21 days of treatment. Then blood was collected and serum gamma glutamyl transpeptidase（γ-GGT）, sialic acid,tumor necrosis factor alpha（TNF-a）, interleukin 10（IL-10）, IL-6, IL-2, and TIMP-1 levels were measured. Further, a lung histological examination was done in crocin treated metastatic mice. Subsequently hallmark metastatic parameters such as matrix metalloproteinases（MMPs）, extracellular regulated kinase 2（ERK2）, vascular endothelial growth factor（VEGF）, and K-ras gene expression were investigated in the lungs of crocin treated metastatic mice.Further, in-vitro adhesion, invasion and migration of B16 F-10 cells were examined after 24 hours of crocin（5 and 10μg/mL） treatment. Administration of crocin to tumor bearing C57 BL/6 mice reduced the lung metastasis by 85%.Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and y-GGT in metastatic control were found to be significantly reduced in crocin treated mice. Crocin also inhibited expression of MMP-2, MMP-9, ERK-2,K-ras, and VEGF. Crocin reduced the ability of B16 F-10 cells invasion（P〈0.05）, migration（P〈0.05） and adhesion by upregulating E-cadherin expression. In conclusion, crocin elicited marked anti-metastatic potential by regulating the metastasis induced biomarkers.

Experimental Investigation of Inhibition Efficiency of Crocin for Chloride-Induced Corrosion of Aluminum Alloys

The use of Crocin, derived from the flowers of Crocus sativus, is investigated as corrosion inhibitor for the AA1050, AA5083, AA5754 and AA6082 aluminum alloys in chloride ions environment. Aluminum and aluminum alloys are subjected to corrosion in the aggressive environment of chlorides, so several green corrosion inhibitors, mostly of plant origin, with minimum impact on health and the environment have been examined. In this study, the inhibition efficiency of 1.25 mM Crocin in a 0.01 M NaCl corrosive solution was assessed via electrochemical corrosion techniques and gravimetric mass loss measurements of the aluminum alloys. The surface of the specimens was examined using Scanning Electron Microscopy, Energy Dispersive Spectroscopy, Stereomicroscopy and Glossiness measurements. Experimental results reveal the protective anticorrosive action of Crocin for all aluminum alloys in the sodium chloride medium.

Beneficial effects of saffron(Crocus sativus L.) in ocular pathologies, particularly neurodegenerative retinal diseases

Saffron(Crocus sativus L.)has been traditionally used in food preparation and as a medicinal plant.It currently has numerous therapeutic properties attributed to it,such as protection against ischemia,as well as anticonvulsant,antidepressant,anxiolytic,hypolipidemic,anti-atherogenic,anti-hypertensive,antidiabetic,and anti-cancer properties.In addition,saffron has remarkable beneficial properties,such as anti-apoptotic,anti-inflammatory and antioxidant activities,due to its main metabolites,among which crocin and crocetin stand out.Furthermore,increasing evidence underwrites the possible neuroprotective role of the main bioactive saffron constituents in neurodegenerative diseases,such as Parkinson’s and Alzheimer’s diseases,both in experimental models and in clinical studies in patients.Currently,saffron supplementation is being tested for ocular neurodegenerative pathologies,such as diabetic retinopathy,retinitis pigmentosa,age-related macular degeneration and glaucoma,among others,and shows beneficial effects.The present article provides a comprehensive and up to date report of the investigations on the beneficial effects of saffron extracts on the main neurodegenerative ocular pathologies and other ocular diseases.This review showed that saffron extracts could be considered promising therapeutic agents to help in the treatment of ocular neurodegenerative diseases.

Carnosine and crocin ameliorate oxidative stress in rats with rhabdomyolysis-induced acute kidney injury through upregulating HO-1 gene expression

We aimed to investigate whether carnosine(a natural dipeptide found in humans)and crocin(a natural product extracted from saffron and gardenia)can prevent the harmful effects of acute kidney injury(AKI)caused by glycerol-induced rhabdomyolysis with a focus on the role of the nuclear factor E2-related factor-2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.Thirty-six male Wistar rats were divided into 6 groups:3 of them received saline,carnosine,and crocin IP for 7 days and the other 3 groups received the same treatment in addition to a single IM injection of 50%glycerol(10 ml/kg,divided between the two limb muscles)on 8th day.All animals were sacrificed after 24 h of the IM injection.Blood samples,muscles,and kidneys were collected for further investigations.Rhabdomyolysis was evidenced by the histopathological alterations in muscle sections and increased levels of myoglobin in kidney tissue homogenate samples.Kidney injury was characterized by increased creatinine,kidney injury molecule-1,malondialdehyde,nitric oxide,and decreased catalase activity.The injury also increased inflammatory markers and histopathological alterations in kidney sections.All these effects were corrected by pretreatment with carnosine and crocin.Both agents could also elevate HO-1 gene expression.However,both agents failed to restore the declined Nrf2 expression in glycerol-treated groups.Carnosine and crocin can effectively prevent rhabdomyolysis-induced AKI in rats through augmenting gene expression of HO-1 and antioxidant system and suppressing the generation of reactive oxygen species,lipid peroxidation,and inflammatory response.

HPLC法测定竹叶青酒专用栀子中藏红花素-I的含量

利用HPLC法测定竹叶青酒专用栀子中藏红花素-I的含量。考察藏红花素-I线性关系及该方法的精密度、稳定性、重现性和回收率。结果表明,竹叶青酒专用栀子中藏红花素-I的平均含量为0.594%,RSD为1.63%。该方法具有较好的线性关系,精密度、稳定性、重现性和回收率都符合要求(RSD<3%);此法可以作为栀子特征成分检验的依据,为其质量评价提供前期探索;另外,此法可为竹叶青酒功效成分研究提供科学依据。

Crocus genome reveals the evolutionary origin of crocin biosynthesis

Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.

二十五味珊瑚丸中西红花苷Ⅰ&Ⅱ的含量测定研究

目的:建立高效液相色谱法测定二十五味珊瑚丸中西红花苷含量的方法。方法:采用HPLC法,色谱柱:Dionex C18柱(250mm×4.60mm,5um);流动相为甲醇-水(52:48),流速为0.8ml/min,检测波长为440nm,柱温为25℃;进样量10μl;用标准曲线法定量,测定二十五味珊瑚丸中西红花苷含量。结果:西红花苷Ⅰ在33μg～330μg范围内与峰面积呈良好的线性关系(r=0.9999),西红花苷Ⅱ在62μg～310μg范围内与峰面积呈良好的线性关系(r=0.9998),平均回收率为103.73%,RSD为1.68%(n=6)。结论:本方法操作简单、重现性好,可用于测定二十五味珊瑚丸中西红花苷Ⅰ&Ⅱ含量。

基于热分析-炮制动力学的焦栀子火力/火候的量化研究

目的:采用热分析技术明确栀子及其主要成分的热解温度范围,以化学动力学原理研究焦栀子炮制过程中京尼平苷及西红花苷I变化规律,建立京尼平苷、西红花苷I动力学方程,为焦栀子生产工艺参数优化提供参考。方法:采用热重-微商热重(TG-DTG)技术,对生栀子、京尼平苷和西红花苷I进行程序升温及恒温的热解特性研究及热分析动力学研究,确立生栀子热解主要温度范围;以该温度范围为参考,确立焦栀子不同炮制温度,利用HPLC测定不同炮制温度-时间焦栀子过程饮片京尼平苷及西红花苷I含量。结果:生栀子主要热解温度范围为229.7~329.4℃,所得京尼平苷动力学方程为ln K=9.5938-6968.3•T^(-1),西红花苷I动力学方程为ln K=5.4161-472.7•T^(-1)。符合一级动力学特征。结论:焦栀子最佳温度为260℃,京尼平苷、西红花苷I动力学方程的建立可为建立焦栀子火候量化标准提供参考。

西红花苷对培养的牛内皮细胞内钙的调节作用

目的 观察西红花苷对不同刺激因子5-羟色胺（5-HT）、组胺（His）、过氧化氢（H2O2）所致内皮细胞内钙升高的影响。方法 采用Fluo-3/AM荧光染色剂负载细胞,在激光共聚焦扫描显微镜上测定培养的单个牛内皮细胞内游离钙的浓度。结果 在含钙的Hank’s液中,5-经色胺、组胺、过氧化氢能明显升高静息状态的细胞内钙,增幅分别为280％、320％、285％。西红花苷1、10μmol/L对5-羟色胺、组胺、过氧化氢引起的钙增高有显著的抑制作用,并呈一定的剂量依赖性。0.1μmol/L西红花苷对细胞内钙升高无显著影响。结论 西红花苷抗动脉粥样硬化、高血压及炎症作用可能与其内皮细胞保护作用有关。

藏红花素Ⅰ的分离制备及抗氧化与稳定性研究

建立硅胶柱层析法分离制备栀子黄色素中藏红花素Ⅰ的方法,并对所得产品进行抗氧化性与稳定性研究。建立了乙酸乙酯-甲醇-水(体积比为100∶20∶15至100∶25∶15)的梯度洗脱体系,以及层析柱硅胶与栀子黄色素的质量比为350∶1的上样量比例,并制备出纯度为98.39%的藏红花素Ⅰ单体样品,其得率为15.58%,回收率为50.42%。抗氧化与稳定性研究结果表明,藏红花素Ⅰ具有优良的抗氧化活性;藏红花素Ⅰ在高温、强光照射、强酸强碱环境中均会产生损失。含藏红花素Ⅰ成分的产品应贮存于低温、避光、pH值为6的环境中,且与适量抗坏血酸联用可以提高其稳定性。

响应面优化西红花中西红花苷提取工艺的研究

建立了西红花药材中西红花苷-Ⅰ和西红花苷-Ⅱ的最优提取工艺。采用乙醇-水体系超声提取西红花苷(超声功率100W),在单因素实验基础上运用响应面法优化西红花苷-Ⅰ和西红花苷-Ⅱ提取工艺,最佳提取工艺参数为:乙醇浓度50%、提取时间30min、提取温度45℃。在此优化的提取工艺条件下,西红花苷-Ⅰ和西红花苷-Ⅱ得率分别为14.12%和4.98%。该提取工艺可用于西红花中西红花苷-Ⅰ和西红花苷-Ⅱ的提取。

10种含栀子中药制剂中栀子苷与西红花苷-Ⅰ的同时测定

目的采用HPLC法与一测多评法,同时测定10种含栀子中药制剂中栀子苷与西红花苷-Ⅰ的含有量。方法制剂分析采用Aglient 1200色谱柱;流动相为乙腈-0.1%磷酸水溶液,梯度洗脱;检测波长238 nm（0-20 min）、440nm（20-30 min）;柱温30℃。结果 HPLC图谱的峰形及分离度良好。以栀子苷为内标物,西红花苷-Ⅰ的相对校正因子为1.085（RSD=0.73%）,其重现性不受色谱柱影响。结论除清火栀麦片与小儿清热片外,其余8种含栀子中药制剂中栀子苷的含有量均符合药典标准。

藏红素在体保护大鼠脑缺血/再灌注损伤和可能的作用机制（英文）

通过中脑冠状动脉闭塞(MCAO)造成缺血/再灌注(I/R)损伤大鼠模型。藏红素预处理可以剂量依赖性地改善I/R损伤所造成的神经性机能障碍和降低脑梗死体积。免疫印迹分析显示藏红素能够上调MCAO大鼠大脑海马中Bcl-2的表达,下调Bax和Caspase-3的表达。由于氧化/硝化应激在I/R损伤中相当重要,建立了硝普钠(SNP)-诱导损伤的PC12细胞模型来模拟在I/R大脑中大量释放一氧化氮(NO)造成的神经毒性。研究结果与藏红素保护MCAO大鼠造成海马损伤一致,藏红素显著下调SNP-损伤PC12细胞所引起LDH和ROS水平的升高,并且剂量依赖性下调促凋亡蛋白Bax,Caspase-3和cytochrome c水平的升高,而上调抗凋亡蛋白Bcl-2蛋白的表达。所有证据表明,藏红素是一种可能通过调控凋亡蛋白活性及线粒体机能障碍机制有效保护I/R损伤的药物。

HPLC法同时测定水栀子中栀子苷和西红花苷-Ⅰ的含量

目的:建立水栀子中两种主要组分的双波长同时含量测定方法。方法:采用Cosmosil 5C18-AR-Ⅱ色谱柱(250mm×4.6mm,5μm);流动相为甲醇–0.1%乙酸溶液梯度洗脱;流速1.0m L/min;检测波长238nm(1)和440nm(2),测定水栀子中栀子苷(1)、西红花苷-Ⅰ(2)的含量。结果:两种组分色谱分离良好,栀子苷、西红花苷-Ⅰ的线性范围分别为50.16~501.6μg/m L(r=0.9999)、10.13~101.3μg/mL(r=0.9997),加样回收率分别为97.77%、97.39%。结论:该测定方法快速、准确,可为水栀子的质量分析方法提供参考。

栀子黄色素类单体对糖尿病小鼠降血糖作用

目的探讨栀子黄色素类单体对糖尿病小鼠降血糖的作用。方法采用四氧嘧啶制备糖尿病小鼠模型,测定栀子黄色素类单体对淀粉负荷和蔗糖负荷糖尿病小鼠血糖的影响。结果西红花苷Ⅰ100 mg/kg、西红花酸100 mg/kg和西红花苷Ⅲ 50 mg/kg可显著降低淀粉负荷糖尿病小鼠血糖;西红花苷Ⅰ100 mg/kg、西红花酸50 mg/kg、100 mg/kg和西红花苷Ⅲ 100 mg/kg可显著降低蔗糖负荷糖尿病小鼠血糖。结论栀子黄色素类单体西红花苷Ⅰ、西红花酸和西红花苷Ⅲ对淀粉负荷和蔗糖负荷糖尿病小鼠均有降血糖作用。

HPLC波长切换法同时测定白益镇惊丸中的京尼平龙胆二糖苷、栀子苷、西红花苷-Ⅰ、去氢茯苓酸和茯苓酸

目的:建立HPLC波长切换法同时测定白益镇惊丸中的京尼平龙胆二糖苷、栀子苷、西红花苷-Ⅰ、去氢茯苓酸和茯苓酸5种活性成分的含量。方法:以十八烷基硅烷键合硅胶为填充剂,乙腈(A)-0.5%磷酸溶液(B)为流动相,进行梯度洗脱(0~11 min,15.0%A;11~22 min,15.0%A→28.0%A;22~31 min,28.0%A→45.0%A;31~42 min,45.0%A→70.0%A;42~50 min,70.0%A→15.0%A),流速0.9 m L·min-1,柱温30℃,波长切换法检测(0~22 min,238 nm,京尼平龙胆二糖苷和栀子苷;22~31 min,440 nm,西红花苷-Ⅰ;31~50 min,210 nm,去氢茯苓酸和茯苓酸),进样量为10μL。结果:京尼平龙胆二糖苷、栀子苷、西红花苷-Ⅰ、去氢茯苓酸和茯苓酸5种成分的质量浓度分别在5.930~118.6、11.41~228.2、4.950~99.00、4.100~82.00、4.780~95.60μg·m L-1呈现良好的线性关系,相关系数分别为0.9999、0.9998、0.9994、0.9999、0.9997;平均加样回收率及相应的RSD分别为99.42%(0.96%)、98.54%(1.08%)、99.06%(1.28%)、97.76%(0.84%)、98.19%(1.59%);供试品溶液在室温下12 h内稳定。结论:本文建立的HPLC波长切换法同时测定白益镇惊丸中的5种成分,该方法简便、稳定、可靠,可作为白益镇惊丸全面可靠的质量控制方法。