滨蒿提取物中3种二咖啡酰奎宁酸的含量测定

目的建立滨蒿提取物中3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸的含量测定方法。方法采用HPLC法测定,色谱条件:Shim-pack VP-ODS色谱柱(250mm×4.6mm,5μm);流动相:乙腈-0.036mol·L~(-1)磷酸二元梯度洗脱;流速:1.0mL·min~(-1);柱温:30℃,检测波长:327nm。结果 3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸分别在0.010 3~0.309,0.009 7~0.291和0.010 5~0.315mg·mL~(-1)范围内与峰面积呈良好的线性关系(r=0.999 9);加样回收率分别为99.23%,101.30%和100.63%;RSD值分别为1.59%,0.83%和1.23%(n=6);样品溶液在32h内稳定。结论该方法操作简便,重复性好,可用于滨蒿提取物的质量控制。     

RP-HPLC法同时测定茵陈中5种化学成分的含量

目的建立同时测定茵陈中芦丁、金丝桃苷、异槲皮苷、4,5-O-二咖啡酰奎宁酸、异鼠李素-3-O-葡萄糖苷含量的方法,为茵陈药材的质量控制提供依据。方法采用RP-HPLC法。色谱柱:LunaC18柱(250 mm×4.6 mm,5μm);流动相:乙腈-体积分数0.04%磷酸水溶液(体积比17∶83);检测波长:345 nm。结果芦丁、金丝桃苷、异槲皮苷、4,5-O-二咖啡酰奎宁酸、异鼠李素-3-O-葡萄糖苷质量浓度分别在0.942~18.84 mg.L-1(r=0.9995)、1.190~23.79 mg.L-1(r=0.9995)、1.107~22.14 mg.L-1(r=0.9994)、56.78~1.136×103mg.L-1(r=0.9990)、0.621 9~12.44 mg.L-1(r=0.9998)内与峰面积呈良好的线性关系(n=5);方法回收率(n=9)分别为98.1%、100.7%、98.4%、100.2%、101.7%。结论该方法可用于茵陈药材的质量控制。     

RP-HPLC同时测定颜复康胶囊中绿原酸、3,5-O-双咖啡酰基奎宁酸、迷迭香酸和黄芩苷的含量

目的建立了同时测定颜复康胶囊中绿原酸、3,5-O-双咖啡酰基奎宁酸、迷迭香酸、黄芩苷含量的反相高效液相色谱法(reversed phase high performance liquid chromatography,RP-HPLC)。方法色谱柱:依利特C18柱(200 mm×4.6 mm,5μm),流动相:乙腈(A)-体积分数为0.05%的磷酸水溶液(B)(梯度洗脱),流速:1.0 mL·min-1,柱温:30℃,检测波长:330 nm。结果绿原酸、3,5-O-双咖啡酰基奎宁酸、迷迭香酸、黄芩苷的线性分别为5.12⁴0.96(r=0.999 6)、3.9340.96(r=0.999 6)、3.93³1.41(r=0.9997)、0.8931.41(r=0.9997)、0.89⁷.12(r=0.999 6)、10.307.12(r=0.999 6)、10.30⁸2.44 mg·L-1(r=0.999 1),4种成分的平均加样回收率为99.7%82.44 mg·L-1(r=0.999 1),4种成分的平均加样回收率为99.7%¹01.8%,RSD为2.8%101.8%,RSD为2.8%⁴.0%(n=6)。结论建立的含量测定方法可用于颜复康胶囊的质量控制。     

UFLC法测定四季抗病毒合剂中4种成分的质量浓度

目的测定四季抗病毒合剂中绿原酸、1,3-二咖啡酰奎宁酸、木犀草苷和3,5-二咖啡酰奎宁酸的质量浓度。方法采用超快速液相色谱(UFLC)法。色谱条件:色谱柱为Kromasil C18色谱柱(250mm×4.6mm,5μm);流动相为乙腈-3.0g·L~(-1)磷酸溶液梯度洗脱;流速为0.8mL·min~(-1);检测波长为348nm;柱温为35℃。结果绿原酸、1,3-二咖啡酰奎宁酸、木犀草苷和3,5-二咖啡酰奎宁酸的线性范围分别为24.00~960.00,22.00~880.00,3.00~120.00和24.00~960.00μg·mL~(-1),相关系数分别为0.999 3,0.999 2,0.999 6和0.999 3;平均回收率分别为98.62%(RSD=1.16%),98.90%(RSD=1.67%),98.37%(RSD=0.95%)和98.35%(RSD=0.91%);12批制剂中绿原酸、1,3-二咖啡酰奎宁酸、木犀草苷和3,5-二咖啡酰奎宁酸的平均质量浓度分别为142.91,191.39,16.89和140.20μg·mL~(-1),RSD值分别为0.97%,1.05%,1.16%和1.20%。结论文章测定的4种成分及建立的方法,可用于四季抗病毒合剂的质量评价。     

高效液相色谱法同时测定楤木不同部位4种成分的含量

目的:建立HPLC同时测定楤木不同部位绿原酸、咖啡酸、3,5-二咖啡酰奎宁酸及4,5-二咖啡酰奎宁酸含量的方法。方法:色谱柱:Dikma Kromasil C18柱(250 mm×4.6mm,5μm);流动相:乙腈-0.1%磷酸水溶液,梯度洗脱;测定波长:325 nm。结果:绿原酸、咖啡酸、3,5-二咖啡酰奎宁酸及4,5-二咖啡酰奎宁酸的线性范围分别为0.101～5.040μg(r=0.999 8),0.003～0.128μg(r=0.999 6),0.078～3.900(r=0.999 8),0.014～0.715(r=0.999 7)。平均回收率分别为99.10%(RSD为0.96%),98.26%(RSD为1.38%),99.25%(RSD为0.83%),98.53%(RSD为1.22%)。结论:该方法简便快速、具有良好的重复性和回收率,可作为楤木中这4种成分的定量分析方法。     

灯盏细辛中4种酚酸类有效成分的HPLC法测定

建立了HPLC法同时测定灯盏细辛中3,5-二咖啡酰基奎宁酸、1,5-二咖啡酰基奎宁酸、4,5-二咖啡酰基奎宁酸和飞蓬酯乙4种酚酸类有效成分的含量.采用C 18柱,流动相为乙腈-甲醇-0.5%甲酸溶液(梯度洗脱),流速1.0 ml/min,检测波长325nm.4种成分分别在0.1～2.5μg(r=0.999 8)、0.01～1μg(r=0.999 8)、0.1～2.5μg(r=0.999 6)和0.1～2.5μg(r=0.999 8)范围内线性关系良好,平均回收率分别为101.2%、102.3%、101.3%和101.8%.     

HPLC-DAD法同时测定清开灵注射液中7个酚酸类成分

目的:建立同时测定清开灵注射液中7个酚酸类成分(新绿原酸、绿原酸、隐绿原酸、咖啡酸、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸)含量的方法。方法:采用Agilent Zorbax SB-C18色谱柱(250 mm×4.6 mm,5μm),流动相为0.1%甲酸水溶液(A)-乙腈(B),梯度洗脱,流速为0.5 mL·min-1,检测波长327 nm,柱温30℃。结果:80min内7个待测组分分离度良好,在各自的检测范围内与峰面积呈良好的线性,其相关系数均大于0.9998,加样回收率为96.1%～103.9%。应用所建立的方法同时测定了清开灵注射液中新绿原酸、绿原酸、隐绿原酸、咖啡酸、4,5-二咖啡酰奎宁酸、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸共7个成分的含量,并对5个不同批次的注射液进行了含量测定,其结果有一定的差异。结论:本法可为清开灵注射液中酚酸类成分的质量控制提供参考。     

HPLC法测定地胆草中4,5-二咖啡酰奎宁酸的含量

目的：建立高效液相色谱法测定地胆草中4,5-二咖啡酰奎宁酸含量的方法。方法：色谱柱为AgilentSBC．。柱（250mm×4．6mm,5μm）,流动相为乙腈．0．1％磷酸溶液（17：83）;流速为1．0ml·min-1;检测波长为327nm;柱温为30℃,进样量10μl。结果：4,5-二咖啡酰奎宁酸在0．0235—2．3520txg范围内线性关系良好（r=0．9999）,平均加样回收率为98．75％,RSD为1．24％（n=6）。结论：所用方法简便、快速、准确,可作为测定地胆草中4,5-二咖啡酰奎宁酸含量的方法。     

HPLC测定楤木花中的3,5-二咖啡酰奎宁酸

目的 建立测定楤木花中3,5-二咖啡酰奎宁酸含量的方法.方法 采用HPLC法,色谱柱为Dikma Kromasil C18柱(250 mm x4.6mm,5μm),流动相为乙腈-0.1%磷酸溶液(23:77),检测波长为328nm.结果 3,5-二咖啡酰奎宁酸0.404～4.040μg与峰面积呈良好的线性关系(r=0.9999),平均加样回收率为98.74%,RSD=0.62%(n=9).结论 所用方法简便、快速、准确,可作为测定楤木花中3,5-二咖啡酰奎宁酸含量的方法.     

HPLC-DAD法同时测定小儿清解颗粒中9种成分的含量

目的建立HPLC-DAD法同时测定小儿清解颗粒中9种成分(新绿原酸、绿原酸、隐绿原酸、獐芽菜苷、断氧化马钱素、连翘酯苷A、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸)的含量。方法采用Agilent Zorbax SB C18色谱柱(250 mm×4.6 mm,5μm),流动相为0.1%甲酸水溶液-乙腈,梯度洗脱,流速为1.0 m L·min-1,新绿原酸、绿原酸、隐绿原酸、连翘酯苷A、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸的检测波长为325 nm,獐芽菜苷、断氧化马钱素为240 nm,柱温30℃。结果 68 min分析时间内9个成分分离度良好,在各自的检测范围内浓度与峰面积呈良好的线性关系,r均>0.9995,加样回收率为95.7%~100.6%,RSD均≤2.1%。应用所建立的方法同时测定了2个厂家生产的小儿清解颗粒中各成分的含量,其结果有一定差异。结论该法简便可行,结果可靠,可为本制剂多指标成分的质量控制提供参考方法。     

二咖啡酰奎尼酸片健康人体安全性及耐受性研究

目的:评价健康志愿者单剂量及多剂量口服二咖啡酰奎尼酸片的安全性和耐受性。方法:按照GCP要求设计试验方案。单剂量给药58名受试者随机分至7个剂量组,每组4~10名,多剂量给药30名受试者随机分至3个剂量组,男女各半,从低剂量开始给药,单剂量给药结束后进行多剂量给药,多剂量给药10 d,观察指标为临床症状、生命体征和实验室检查指标等,采用描述性分析及SAS软件进行统计学处理。结果:在单剂量及多剂量给予二咖啡酰奎尼酸片耐受性试验中,各组受试者入选时各项指标均在正常范围,条件均衡,具有可比性。共报告9件被研究者判定为可能与研究药物有关的不良事件,且未经处理均消失。不良事件主要表现为窦性心动过缓、血白细胞降低,无自觉症状,均完成临床试验,其他均未见有临床意义的改变。结论:88名健康受试者中单剂量口服二咖啡酰奎尼酸片,最大剂量至1 200 mg;多剂量口服二咖啡酰奎尼酸片,最大剂量至500 mg,每12 h 1次,均安全且能很好耐受。     

Prevention of lipopolysaccharide-induced injury by 3,5-dicaffeoylquinic acid in endothelial cells

Aim： To investigate the effect of 3,5-dicaffeoylquinic acid （3,5-diCQA） on lipopolysaccharide （LPS）-induced injury in human dermal microvascular endothelial cells （HMEC-1）. Methods： The anti-oxidant effect was detected using the malondialdehyde （MDA） assay in a rat liver microsome model of lipid peroxidation. Cell viability was analyzed using the 3-（4,5-dimethylthiazol-2-yl）-2,5 diphenyltetrazolium bromide assay. Cell lipid peroxide injury was measured by lactate dehydrogenase （LDH） release. Apoptotic cells were detected by flow cytometry, and confirmed by DNA fragmentation analysis. Caspase-3 activity was measured using a specific assay kit. The level of intracellular reactive oxygen species （ROS） was determined by flow cytometry with a 2,7-dichlorodihydro-fluorescein diacetate fluorescence probe. Results： The exposure of microsomes to ascorbate-Fe^2＋ resulted in lipoperoxidation according to an increase in the level of MDA. MDA formation decreased in a dose-dependent manner on treatment with 5, 10, or 50 μmol/L 3,5-diCQA. Treatment with LPS for 16 h resulted in a 60% decrease in cell viability and an increase in LDH release from 47.6% to 61.5%. DNA laddering was observed by agarose gel electrophoresis. The level of apoptotic cells peaked at 27% after treatment with LPS for 12 h. Following treatment with LPS for 12 h, intracellular ROS and caspase-3 activity increased. Pretreatment with 3,5-diCQA at 5, 10, or 50 μmol/L for 1 h attenuated LPS-mediated endothelial cell injury. The anti-apoptotic action of 3,5-diCQA was partially dependent on its capacity for anti-oxidation and the suppression of caspase-3 activity. Conclusion： 3,5-diCQA displays anti-oxidative and anti-apoptotic activity in HMEC-1 due to scavenging of intracellular ROS induced by LPS, and the suppression of caspase-3 activity.     

Metabolic profile of 1,5-dicaffeoylquinic acid in rats, an in vivo and in vitro study.

To explore the metabolism of 1,5-dicaffeoylquinic acid (1,5-DCQA) in rats, liquid chromatography-mass spectrometry in parallel to diode-array detection was used for the rapid detection/characterization of the metabolites formed in bile, urine, and plasma of rats following oral administration of 1,5-DCQA (160 mg/kg). The methylation and glucuronidation of 1,5-DCQA occurring in vitro using rat liver and small intestinal microsomes and cytosols were studied in comparison with those occurring in vivo, and the enzymes involved were also determined. In addition, the anti-HIV (human immunodeficiency virus) activity of three important metabolites was preliminarily evaluated in MT-4 cells infected with HIV-1. A total of 22 metabolites in vivo and in vitro were identified, including four isomeric O-mono-methylated metabolites (M8-M11), nine isomeric O-di-methylated metabolites (M3, M6, M22, and M12-M17), four isomeric O-mono-methyl-glucuronidated metabolites (M2 and M19-M21), four isomeric O-di-methyl-glucuronidated metabolites (M1, M4, M5, and M7), and one glucuronidated metabolite (M18). The O-methylation positions of three important metabolites (M8, M9, and M12) were determined (3'-, 3'-, and 3',3'-) by comparing with synthesized standards. The efficacy experiments showed that M8, M9, and M12 could inhibit HIV replication with IC(50) values of about 25, 25, and 46 microM, respectively. These results suggest that O-methylation and glucuronidation are two important metabolic pathways of 1,5-DCQA, that both rat liver and small intestine can catalyze such reactions by catechol-O-methyltransferase and UDP-glucuronosyltransferases, and that the HIV-1 inhibitory activity of M8, M9, and M12 is comparable to or slightly weaker than that of 1,5-DCQA.     

Effect of 1,5-dicaffeoylquinic acid on ethanol-induced hypertriglyceridemia. Short communication.

Male Wistar rats were administered acutely and chronically ethyl alcohol. The effect of 1,5-dicaffeoylquinic acid (cynarine) on ethanol induced hypertriglyceridemia and on hepatic concentration of triglyceride was investigated. In rats treated acutely with ethanol the serum and hepatic triglycerides showed significant rise by 216 and 111%, resp. The prolonged administration of ethanol caused insignificant increase of the serum triglyceride concentration by 40%, while its hepatic content was significantly increased by 100%. In rats receiving ethanol and cynarine simultaneously, distinct reduction of the serum and hepatic triglyceride concentration was observed.     

HPLC法测定心脑络通颗粒中野黄芩苷和4,5-二-O-咖啡酰奎宁酸

目的建立HPLC法同时测定心脑络通颗粒中野黄芩苷和4,5-二-O-咖啡酰奎宁酸的方法。方法色谱柱为岛津InterstilODS柱(250mm×4.6mm,5μm),流动相为乙腈-0.1%三氟乙酸(25:75),体积流量1.0mL/min,检测波长335nm,柱温37℃,进样量10μL。结果 2种成分均能达到基线分离,重现好,且呈现较好的线性关系;加样回收率分别为97.1%、99.8%,RSD值分别为0.66%、0.82%。结论该方法准确、灵敏、可靠,重复性好,可用于心脑络通颗粒的快速检测。     

Pharmacokinetics and metabolism of 1,5-dicaffeoylquinic acid in rats following a single intravenous administration

1,5-Dicaffeoylquinic acid (1,5-DCQA) is a potentially important HIV-1 integrase inhibitor widely distributed in many plants. To characterize the pharmacokinetic and metabolic properties of 1,5-DCQA in rats following single intravenous administration (160 mg/kg), the plasma concentrations of 1,5-DCQA were measured by high-performance liquid chromatography (HPLC) and the metabolites formed in urine were identified by liquid chromatography-mass spectrometry (LC-MS) in parallel to diode-array detection (DAD). The results showed that the concentrations of 1,5-DCQA in plasma declined rapidly in a biphasic manner with a mean terminal half-life (t(1/2)) of 1.40 h. The mean clearance (CL) and the apparent volume of distribution (Vd(B)) of 1,5-DCQA were 0.44l/h/kg and 0.89l/kg, respectively. A total of 15 metabolites in rat urine were identified, including four isomeric O-mono-methylated (M1-M4), six isomeric O-di-methylated (M5-M10), one isomeric O-mono-methyl-glucuronidated (M11) and four isomeric O-di-methyl-glucuronidated (M12-M15) metabolites. The O-methylation positions of three important metabolites (M1, M2 and M5) were determined (3'-, 3'-, and 3',3'-) by comparing with synthesized standards. These results suggested that the disappearance of 1,5-DCQA from plasma was rapid, and that its quick urinary excretion and extensive metabolism, including methylation and glucuronidation, were two factors causing its rapid elimination from the circulation.     

液相色谱-电喷雾离子阱质谱法研究人尿中1,5-二咖啡酰奎宁酸的代谢产物

目的 研究健康受试者口服1,5-二咖啡酰奎宁酸后尿液中的代谢产物.方法 健康受试者每人口服1,5-二咖啡酰奎宁酸600 mg,收集0-24 h的尿样,经C_(18)小柱固相萃取纯化后,用液相色谱-电喷雾离子阱质谱联用技术对人尿中的代谢产物进行分析鉴定.结果 在人尿中发现了1,5-二咖啡酰奎宁酸的甲基化、葡萄糖醛酸化及甲基-葡萄糖醛酸化代谢产物共28个.其中,有2个代谢产物结构经标准品对照得到确证.结论 甲基化、葡萄糖醛酸化和异构化反应是1,5-二咖啡酰奎宁酸在人体内的3种重要代谢途径.     

灯盏细辛药效物质基础3,5-二咖啡酰奎宁酸对大鼠局灶性脑缺血-再灌注损伤的保护作用

目的 研究3,5-二咖啡酰奎宁酸对大鼠实验性局灶性脑缺血-再灌注损伤模型的保护作用.方法 采用线栓法阻塞大鼠脑中动脉( MCAO)制备局灶性脑缺血-再灌注损伤模型,观察3,5-二咖啡酰奎宁酸对模型大鼠状态、神经行为学的影响,以及对脑梗死比率及血清中丙二醛(MDA)、总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(G...     

一测多评法测定藏茴香中异绿原酸A、新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸C

目的 建立一测多评法（QAMS）测定藏茴香中6种咖啡酰奎宁酸类成分含量,并验证该方法在藏茴香质量评价中应用的可行性与适用性。方法 取藏茴香粉末（过三号筛）0.5 g,精密加入70%甲醇 20 mL ,超声处理（250 W、频率 53 kHz）30 min,制备供试品溶液;采用Phenomenex GeminiR C₁₈（250 mm×4.6 mm,5 μm）色谱柱,以乙腈-0.1%甲酸水溶液为流动相,梯度洗脱,检测波长为 330 nm,体积流量 1.0 mL·min^-1,柱温 30 ℃,进行专属性、供试品提取条件、检测波长选择、色谱条件、线性关系、精密度、重复性、稳定性、加样回收率方法学考察,建立异绿原酸A、新绿原酸、绿原酸、隐绿原酸、异绿原酸 B、异绿原酸 C成分含量检测的 HPLC法;以异绿原酸 A为内参成分,分别计算新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸C 5种成分的相对校正因子,分别采用3种不同色谱仪和3种色谱柱进行相对校正因子、相对保留时间耐用性考察,对藏茴香样品同时采用外标法与 QAMS 测定 6 种成分的质量分数,比较 2 种测定方法结果的差异。结果 建立的6种成分的HPLC检测方法的专属性、供试品提取条件、检测波长选择、色谱条件、线性关系、精密度、重复性、稳定性、加样回收率均符合要求;新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸C的相对校正因子平均值分别是 1.362、1.257、1.335、1.470、1.134,3种不同色谱仪和 3种色谱柱对相对校正因子、相对保留时间均无明显影响;QAMS与外标法2种方法测定3批藏茴香样品中6种成分得到的结果之间无显著差异。结论 建立的QAMS简便、准确、可靠,可用于藏茴香中6种咖啡酰奎宁酸类成分——异绿原酸A、新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸C的定量分析。