赖氨大黄酸通过激活JNK诱导宫颈癌HeLa细胞凋亡

目的研究赖氨大黄酸(RHL)对人宫颈癌HeLa细胞增殖、凋亡的影响,并探讨其作用机制。方法用MTT法检测细胞增殖;用流式细胞仪检测细胞凋亡,用Western blot检测凋亡相关蛋白及JNK蛋白与蛋白磷酸化水平。结果 RHL能有效抑制宫颈癌HeLa细胞增殖,并能诱导其凋亡,随药物浓度的增加,细胞凋亡率也逐渐升高;RHL能够激活caspase-3、caspase-7和PARP,并激活磷酸化的JNK表达,磷酸化的JNK表达增加在RHL的诱导凋亡中起主要作用。结论 RHL通过激活JNK-caspase-PARP信号通路抑制HeLa细胞增殖,并诱导其凋亡,赖氨大黄酸解决了大黄酸不溶于水的问题,有望成为临床肿瘤辅助化疗药物。     

早期大黄酸干预对db/db小鼠胰岛功能的影响

目的大黄酸(4,5-二羟基蒽醌-2-羧酸)是大黄的蒽醌衍生物之一。本研究旨在通过db/db小鼠这一先天性2型糖尿病动物模型,探讨大黄酸对血糖水平的影响以及对胰岛β细胞的作用。方法 30只4周龄db/db小鼠随机分为两组:对照组(1%纤维素钠,n=15),大黄酸组(120 mg/kg,n=15),给予连续鼻饲灌胃给药8周。投药结束后行经腹腔葡萄糖耐量试验(IPGTT)并测定相应胰岛素水平,其曲线下面积(AUC)分别代表糖耐量和胰岛素分泌水平,并通过计算IPGTT的0 min至30 min胰岛素曲线下面积以评估早期相胰岛素分泌功能。同时行胰岛素免疫组化染色,通过免疫组化染色计算β细胞含量,并用TUNEL法检测胰岛β细胞的凋亡。结果与对照组相比,大黄酸治疗组糖负荷后0,30,60和120 min的血糖水平明显下降,同时30,60和120 min的胰岛素水平明显升高,尤其是早期相胰岛素水平(AUCINS0-30)明显升高。同时大黄酸治疗组胰岛素染色明显增强,胰岛β细胞含量明显增高,凋亡细胞明显减少。结论早期大黄酸治疗可以明显改善db/db小鼠的葡萄糖耐量,恢复早期相胰岛素分泌,并能抑制胰岛β细胞的凋亡,保护胰岛功能。大黄酸的这一作用可能使之成为一种新的预防或治疗2型糖尿病的手段。     

何首乌不同炮制品中大黄酸的含量测定

目的：通过HPLC法比较何首乌不同炮制品中大黄酸的含量。方法：采用Shim-pack VP-ODS（150mm×4．6mm,5μm）色谱柱;流动相为甲醇：水（70：30,磷酸调至pH=3）;柱温：24℃;流速：1．0mL／min;检测波长：437nm。按上述色谱条件测定峰面积,以峰面积积分值对进样量进行线性回归。结果：回归方程Y=673755．99X-33059．11,r=0．9991,大黄酸线性范围为0．0132～0．132mg,r=0．9991。大黄酸在不同何首乌含量分别为生首乌8．231mg／g,黑豆制首乌5．166mg／g,酒制首乌4．977mg／g,蒸制首乌5．023mg／g。结论：何首乌的不同炮制品中大黄酸的含量不同,以生首乌中大黄酸的含量最多,酒制何首乌中大黄酸的含量最少。     

HPLC法测定痛风胶囊中青藤碱和大黄酸的含量

目的建立痛风胶囊中青藤碱、大黄酸含量的测定方法。方法采用HPLC法,青藤碱含量测定的色谱柱为Agilent Eclipse Plus C18(5μm,4.6 mm×250 mm),流动相为乙腈-0.08%(体积分数)三乙胺(体积比21∶79),流速为1.0 mL.min-1,检测波长为262 nm;大黄酸含量测定的色谱柱为Kromasil C18(4.6mm×250 mm,5μm),流动相为甲醇-0.05%(体积分数)H3PO4(体积比85∶15),流速为1.0 mL.min-1,检测波长为254 nm。结果青藤碱在0.115 6～3.855μg范围内与峰面积呈良好的线性关系(r=0.999 5),平均回收率为99.48%,RSD为1.93%;大黄酸在0.083 2～0.832μg范围内与峰面积呈良好的线性关系(r=0.999 5),平均回收率为98.62%,RSD为1.13%。结论本方法专属性强、准确、重复性好,可用于痛风胶囊的质量控制。     

奇应内消巴布剂定性定量方法研究

目的:建立奇应内消巴布剂定性定量方法。方法:采用TLC法鉴别方中重楼、大黄、山柰;采用HPLC法测定大黄酸、大黄素、大黄酚含量。HPLC色谱条件:采用Agilent ZORBAX SB-C18(4.6 mm×250 mm,5μm)色谱柱,流动相为甲醇-0.2%磷酸水溶液(87∶13),流速1.0 mL.min-1,检测波长440 nm,柱温30℃。结果:定性鉴别薄层色谱特征明显;HPLC测定,大黄酸浓度在4.00～25.00μg.mL-1范围内线性关系良好(r=0.9994),平均回收率(n=5)为98.39%(RSD=2.0%);大黄素浓度在17.92～112.00μg.mL-1范围内线性关系良好(r=0.9998),平均回收率(n=5)为97.25%(RSD=1.5%);大黄酚浓度在33.92～212.00μg.mL-1范围内线性关系良好(r=0.9992),平均回收率(n=5)为96.55%(RSD=1.8%)。结论:该法可以准确地对奇应内消巴布剂进行定性、定量检测,有效地控制该制剂的质量。     

波长切换HPLC法同时测定元和正胃片中5种成分的含量

目的建立RP-HPLC法同时测定元和正胃片中龙胆苦苷、甘草苷、甘草酸、芦荟大黄素、大黄酸5种成分的含量。方法采用Diamonsil C18色谱柱(4.6 mm×200 mm,5μm)进行分离;流动相为乙腈(A)-体积分数0.05%磷酸溶液(B),梯度洗脱,流速1.0 mL·min-1;检测波长为0～35 min、276 nm,35～61 min、254 nm;柱温30℃。结果龙胆苦苷、甘草苷、甘草酸、芦荟大黄素、大黄酸质量浓度分别在29.4～294 mg·L-1(r=0.999 5,n=6)、4.35～43.5 mg·L-1(r=0.999 3,n=6)、8.25～82.5 mg·L-1(r=0.999 7,n=6)、1.27～12.7 mg·L-1(r=0.999 3,n=6)、0.68～6.8 mg·L-1(r=0.999 7,n=6)内与峰面积呈良好的线性关系,方法的平均回收率分别为99.5%(RSD=1.6%)、100.1%(RSD=1.4%)、99.4%(RSD=1.5%)、98.9%(RSD=1.1%)和99.4%(RSD=1.5%)。结论该方法简便、准确、重现性好、专属性强,可用于元和正胃片的质量控制。     

Reduced system exposures of total rhein and baicalin after combinatory oral administration of rhein,baicalin and berberine to beagle dogs and rats

Ethnopharmacological relevance

Rhein (Rh), baicalin (BG) and berberine (Be) are important coexisted constituents of San-Huang-Xie-Xin-Tang, which was widely used in traditional Chinese medicine for the treatment of gastritis, hypertension, gastric bleeding and peptic ulcers, etc.

Aim of the study

Based on the extensive phase II conjugation reactions of polyphenols (Rh and BG) in vivo, the aims of the present study were to investigate the effects of combination (Rh, BG and Be) on the system exposures of total Rh and BG involving the phase II conjugates metabolites and its possible mechanism.

Materials and methods

A 3×3 Latin square single heavy design was used to investigate the pharmacokinetics influence of total Rh and BG after combination of Be by treating plasma samples with β-glucuronidase/sulfatase both in beagle dogs and Wistar rats. In vitro and in situ experiment models including in situ rat intestinal perfusion, Caco-2 cell monolayer transport and small intestinal flora incubation system were used to discuss the possible mechanism.

Results

The results of pharmacokinetic interactions showed that combination significantly reduced the system exposures of total Rh and BG. Compared with Rh or BG alone, the mean area under concentration-time curves (AUC_0–t) of total Rh and BG reduced by 31% and 77% in beagle dog experiment. In Wistar rat experiment, the AUC_0–t of total Rh and BG reduced by 22% and 21%. Subsequently, the results of in situ rat intestinal perfusion and small intestinal flora incubation system tests revealed that combination may decrease the absorption and metabolism of BG. However, combination could not affect the transport profile of BG across the Caco-2 cell. Moreover, combination did not affect the absorption or metabolism profile of Rh in all three in situ/in vitro experiments.

Conclusions

It was deduced that the possible mechanism of the reduction of the system exposures of total Rh and BG was related to that combination decreased the metabolism of BG to B or the phase II conjugates of Rh/BG excreted from liver/bile duct to their free aglycones in vivo by inhibiting intestinal flora. The potent effects of combination on the phase II conjugates of Rh and B in pharmacokinetics, shown in this paper, indicated that more attention should be paid to the phase II conjugates metabolites of these polyphenols (undergo extensive phase II conjugation reactions in vivo) when applied herbal products composed of these coexist compounds.     

大黄解热作用与降低血浆一氧化氮作用的PK-PD研究

药动学与药效学模型的结合是现代药物研究方法的一个热点,也是评价中药作用的一个重要手段.该文以内毒素复制大鼠发热炎症模型,灌胃大黄水煎液(1.54 g·kg-1)后不同时间点采血,测量体温及血浆NO浓度,同时采用高效液相色谱-荧光法(HPLC-FLD)进行大黄酸血药浓度测定.以Kinetica 5.0.11软件,分别对治疗组大黄酸平均血药浓度与治疗组体温及NO降低值进行PK-PD模型拟合,评价大黄作用特点及可能机制.实验结果表明大黄能够抑制LPS大鼠体温及血浆NO浓度的升高;最终PK-PD模型均以效应室联结的无滞后时间二房室-Sigmod Emax拟合较优;大黄酸在LPS大鼠体内的t1/2,Cmax,AUC与正常大鼠比较显著增大;大黄解热及降低血浆NO浓度的EC50分别为114.1,90.80 μg·L-1,两者较为接近;解热作用Emax约为造模后体温升高最大值的111.0％,对NO影响的Emax约为造模后血浆NO升高最大值的8.399％,两者有显著差异;两药效指标的药效动力学曲线均较为陡峭.大黄在正常及LPS大鼠体内的药物动力学过程存在差异;解热及降低血浆NO浓度的作用靶点可能处于同一部位;大黄解热作用机制除通过降低血浆NO浓度外,应具有其他微观作用机制;大黄解热和降低血浆NO浓度作用的量效关系范围较窄,效能较低.     

Stability-indicating spectrophotometric and spectrodensitometric methods for the determination of diacerein in the presence of its degradation product

Three sensitive, selective, and precise stability-indicating methods for the determination of the novel osteoarthritis drug, diacerein (DIA) in the presence of its alkaline degradation product (active metabolite, rhein) and in pharmaceutical formulation were developed and validated. The first method is a first derivative (D(1) ) spectrophotometric one, which allows the determination of DIA in the presence of its degradate at 322 nm (corresponding to zero crossing of the degradate) over a concentration range of 4-40 μg/mL with mean percentage recovery 100.21 ± 0.833. The second method is the first derivative of the ratio spectra (DD(1) ) by measuring the peak amplitude at 352 nm over the same concentration range as (D(1) ) spectrophotometric method, with mean percentage recovery 100.09 ± 0.912. The third method is a TLC-densitometric one, where DIA was separated from its degradate on silica gel plates using ethyl acetate:methanol:chloroform (8:1.5:0.5 v:v:v) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of DIA at 340 nm over a concentration range of 1-10 μg/spot, with mean percentage recovery 100.24 ± 1.412. The selectivity of the proposed methods was tested using laboratory-prepared mixtures. The proposed methods have been successfully applied to the analysis of DIA in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with reference method.     

大黄酸对Caco-2细胞多药耐药蛋白转运体表达的影响

目的:探讨大黄酸对人结肠腺癌上皮细胞(Caco-2)多药耐药蛋白mRNA表达的影响。方法:用大黄酸作用于Caco-2细胞,采用荧光定量PCR(real time PCR)法检测转运体MRP1-5 mRNA的表达量,数据分析采用2-ΔΔCT法。结果:高(5.00 mg.L-1)低(2.50 mg.L-1)质量浓度大黄酸均可以显著上调Caco-2细胞MRP3 mRNA表达(P<0.05或P<0.01);低浓度大黄酸可以显著下调Caco-2细胞MRP5 mRNA表达(P<0.05);大黄酸对MRP1,MRP2和MRP4 mRNA的表达没有显著影响。结论:大黄酸可能通过上调Caco-2细胞MRP3 mRNA的表达和下调MRP5 mRNA的表达而影响其口服药物的生物利用度。