何首乌有效成分二苯乙烯苷的降血脂作用与药物代谢动力学研究
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摘要
何首乌为蓼属植物何首乌(Polygonum multiflorum Thunb.)的干燥块根,具有多种药理作用。近年来报道,何首乌具有显著的降血脂和抗动脉粥样硬化等作用。2, 3, 5, 4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(简称二苯乙烯苷,TSG)为中药何首乌的主要水溶性成分,许多文献资料表明TSG具有抗氧化和清除自由基的特性,近年的研究亦证明,TSG对一些慢性病,如衰老、老年性痴呆、高血脂及动脉粥样硬化等,具有预防和治疗的作用。
本文通过比较TSG单体成分与含相同量TSG的何首乌总提物(有效部位)的降血脂作用,确定TSG为何首乌降血脂的药效物质基础,并较深入地研究了TSG在动物体内的吸收、分布、代谢、排泄等一系列过程,为TSG的开发利用奠定了坚实的基础。
七宝美髯丹是以何首乌为君药的复方制剂,含有多种有效成分,为了更好的控制药品质量,本文建立了RP-HPLC法同时测定二苯乙烯苷等七种组分的含量。
第一部分何首乌降血脂作用的药效物质基础研究
目的:比较研究何首乌有效部位(HSWAF)及有效成分TSG的降血脂作用,明确何首乌降血脂作用的药效物质基础。
方法:观察HSWAF和TSG对正常小鼠血脂及肝指数的影响;小鼠腹腔注射Triton致急性高脂血症模型,观察HSWAF和TSG对模型动物血脂、谷草转氨酶(GOT)和谷丙转氨酶(GPT)水平的影响;以大鼠食饵性高脂血症为模型,给予高脂饲料的同时给予HSWAF和TSG,连续28天,测定血脂、丙二醛(MDA)、一氧化氮(NO)含量和超氧化物歧化酶(SOD)活性,肝组织中总胆固醇(TC)和甘油三酯(TG)含量以及肝指数。
结果:对正常小鼠,TSG和HSWAF均有升高血清高密度脂蛋白胆固醇(HDL-C),降低血清低密度脂蛋白胆固醇(LDL-C)的作用。HSWAF和TSG对腹腔注射Triton致高脂血症小鼠的血清TC和TG均有降低作用,对HDL-C均有升高作用。HSWAF 60 mg/kg明显降低大鼠食饵性高脂血症模型的肝指数;HSWAF 30 mg/kg和60 mg/kg明显降低大鼠食饵性高脂血症模型的血清TC、TG、LDL-C、MDA含量、TC/HDL-C比值,明显升高血清HDL-C和NO含量以及SOD活性,TSG对以上各项指标的作用强度略低于HSWAF,但无显著性差异。
结论:何首乌中有效部位及有效成分TSG均具有调血脂、抗氧化,保护血管内皮的功能,TSG为发挥药效作用的主要物质,提示可用于防治高脂血症。
第二部分二苯乙烯苷在高血脂模型大鼠体内的药动学研究与组织分布
目的:研究TSG在不同高血脂病理模型动物体内的药动学及组织分布情况。
方法:1.分别取正常大鼠,食靡性高脂血症模型大鼠和Triton致高脂血症模型大鼠,按60 mg/kg的剂量灌胃给予TSG,于给药后不同时间经眼球后静脉丛取血,置肝素化试管中,以3 000 r/min离心10 min,分离血浆。以虎杖苷为内标,用3倍量甲醇沉淀蛋白,取一定量上清液,于50℃氮气流吹干,用少量甲醇溶解后,取上清液20μl注入液相色谱仪。C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-甲醇-0.1%冰醋酸(12∶10∶78),检测波长320 nm ,流速为1.0 ml/min。记录TSG与虎杖苷的峰面积,计算TSG的血药浓度。2.分别取正常大鼠,食靡性高脂血症模型大鼠和Triton致高脂血症模型大鼠,按60 mg/kg的剂量灌胃给予TSG,分别于给药后5、15、45 min断头处死,分别取出心,肝,脾,肺,肾,脑,胃,小肠等组织,用生理盐水制成1∶2的组织匀浆。组织匀浆加3倍量甲醇沉淀蛋白,离心,取20μl上清液注入液相色谱仪。C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-甲醇-0.1%冰醋酸(15∶18∶67);检测波长320 nm ,流速为1.0 ml/min。测定组织中TSG的含量。
结果:1.血浆中TSG在0.13~81.00μg/ml浓度范围内线性关系良好,高、中、低3种不同浓度血浆样品的方法回收率分别为99.7 %、98.9 %、98.3 %,日内与日间精密度RSD均<3.0 %。与正常大鼠相比,TSG在triton致高脂血症模型大鼠体内的Tmax有降低趋势,t1/2非常显著性缩短(p<0.01),Cmax、Ke、AUC0-t与AUC0-∞均非常显著性增加(p<0.01);脂肪乳致高脂血症模型大鼠与正常大鼠相比,AUC0-t与AUC0-∞显著性增加(p<0.05)外,其它参数均无显著性差异(p>0.05)。Triton致高脂血症模型大鼠与食靡性高脂血症模型大鼠相对正常大鼠的相对生物利用度分别为188 %和155 %。2.各组织中TSG浓度在0.65~162.00μg/ml范围内线性关系良好,高、中、低3种不同浓度肝脏匀浆样品的方法平均回收率分别为100.5 %、102.3 %、100.9 %,日内与日间精密度RSD均<3.0 %。与正常大鼠相比,在triton高脂血症模型大鼠体内,TSG除在肾脏中的分布有所减少外,其它均无明显变化;在食靡性高脂血症模型大鼠的心脏中明显减少,而在肝脏、脾脏和肾脏中均明显增加。
结论:TSG在高脂血症模型动物体内的药动学及组织分布发生了某些变化,提示在进行药物的体内过程研究时应充分考虑机体的生理状态,在可能的情况下尽量选择相关的病理模型进行研究,使实验结果更接近真实情况。
第三部分二苯乙烯苷的血浆蛋白结合率测定
目的:研究TSG的血浆蛋白结合情况,并探讨用浊点萃取法(CPE)测定TSG血浆蛋白结合率的方法。
方法:分别采用平衡透析法、超滤法和CPE法测定浓度为0.05、0.25、1.25 mg/ml的TSG与人血浆,牛血清白蛋白(BSA)和α1-酸性脂蛋白(α1-AGP)的结合情况,并将3种方法的测定结果进行比较。浊点萃取方法为:200μl样品,精密加入1.0 ml浓度为5 % (w/v)的Triton X-114溶液,涡旋混合,置35℃恒温振荡器中保温10 min,进行相分离,于3 000 r/min离心5 min,上层为水层(游离型药物),体积为850μl,下层为表面活性剂层(结合型药物),体积为350μl,用标准曲线法测定结合型药物的含量。
结果:在不同浓度(0.05、0.25、1.25 mg/ml)下,用平衡透析法、超滤法和CPE法测得的TSG与血浆蛋白的结合率(%)非常相似,分别为:55.8±4.1、53.4±4.4、51.9±5.0,79.6±3.3、74.2±3.3、72.5±2.6,89.8±1.8、85.0±3.6、87.4±1.3。用平衡透析法和超滤法测得的结果均表明,TSG与血清白蛋白的结合率基本不随浓度发生变化(约60 %),与α1-AGP的结合率则随药物浓度的增加而明显降低,而用CPE法测定的两种蛋白的结合率均在80 %~90 %,与上述两种方法的差别很大。
结论:CPE法可用于测定TSG的血浆蛋白结合率。TSG既能与血清白蛋白结合又能与α1-AGP结合,血浆中还可能存在其他能和TSG结合的蛋白质。
第四部分二苯乙烯苷在大鼠体内的吸收动力学研究
目的:建立同时测定胃灌注液及肠循环液中TSG及酚红浓度的HPLC/PDA法,并研究TSG在大鼠胃、肠的吸收特性。
方法:采用大鼠在体胃、肠吸收模型,以HPLC/PDA法测定胃灌注液及肠循环液中药物的含量,色谱条件为:Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);柱温30℃;流动相为乙腈-甲醇-0.2 %磷酸(35∶15∶50),流速1 ml/min;检测波长320 nm(TSG)和430 nm(酚红);进样量20μl。
结果:TSG及酚红的线性关系良好,线性范围分别为3.5~140μg/ml和1~40μg/ml,日内、日间精密度(RSD)均小于3.1 %,方法回收率均在99.48 %~102.5 %之间。不同质量浓度(2.5、5、10 mg/ml)的TSG在大鼠胃部的每小时吸收百分率分别为72.7、67.7、56.6;不同质量浓度(30、60、120μg /ml)的TSG在肠道内的吸收速率常数分别为0.047 7、0.051 4、0.056 3,三者之间无显著性差异(P>0.05)。
结论:本文首次建立了HPLC/PDA法同时测定胃灌注液及肠循环液中TSG及酚红的浓度,该法操作简便,结果准确,灵敏度高。研究结果表明,TSG在肠道内吸收较差,主要吸收部位是胃,为延长药物在胃内的停留时间,改善生物利用度,适合制成胃漂浮片。
第五部分二苯乙烯苷在大鼠体内外的代谢研究
目的:研究TSG在大鼠体内外的代谢产物和代谢途径。
方法:采用肝微粒体温孵法,大肠杆菌培养法等对TSG进行体外代谢研究;灌胃给予一定量TSG后,对大鼠血液、胆汁、尿液、粪便、小肠内容物和胃内容物进行体内代谢产物的分析,通过制备液相获得主要代谢物的纯品,用1H-NMR、13C-NMR及MS手段进行结构确证,从而推测TSG在体内的转化过程。
结果:在大肠杆菌培养液中,胃内容物中只有原型药物;尿液中既无原型也无代谢物;肠内容物中除原型外,还有少量代谢物M2;血浆中除原型外,还有代谢物M1(已经证明为TSG的葡糖醛酸结合物);胆汁中除可检测到原型药物外,还有大量的代谢物M2和少量的代谢物M1;粪便中检测到的主要是原型药物;在肝微粒体酶的作用下,TSG很快代谢为M2。结构鉴定证明,M2与M1结合部位不同,为TSGC3-OH的葡糖醛酸结合物。
结论:TSG在肝脏代谢为葡糖醛酸结合物,并经胆汁排泄,在肠道内菌或酶的作用下水解为原型随粪便排出体外。
第六部分二苯乙烯苷在大鼠体内的排泄研究
目的:建立大鼠胆汁中TSG及其代谢物浓度的HPLC测定方法,明确TSG的代谢途径。
方法:胆汁样品或用重蒸水稀释后的胆汁样品200μl,加入10μl甲醇后直接离心测定TSG及其代谢物的浓度。Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm),柱温30℃;流动相为乙腈-0.1 %冰醋酸梯度洗脱(0~10 min,15∶85;15~23 min,25∶75;25~30 min,100∶0),流速1 ml/min;检测波长320 nm。测定大鼠灌胃给予60 mg/kg的TSG后胆汁中TSG及其代谢物的累积排泄率。
结果:TSG及其代谢物在胆汁中的线性范围分别为12.0~0.8μg/ml和110~0.9μg/ml,日内和日间RSD均小于7.0 %,准确度在±4.33 %之间。提取回收率均大于99.14 %。大鼠灌胃给予60 mg/kg的TSG后24 h内胆汁中TSG及其代谢物的累积排泄率分别为(0.084±0.04) %和(36.87±12.94) %。
结论:经方法学考察符合生物样品的测定要求,可应用于大鼠胆汁中TSG及其代谢物浓度的测定,TSG在大鼠体内主要以代谢物的形式由胆汁排泄。
第七部分二苯乙烯苷体内过程研究模式的建立及其应用
目的:建立一种简便,快速的研究药物体内吸收、分布、代谢及排泄等全过程的方法模式,并应用于TSG的体内过程研究。
方法:麻醉后的大鼠,进行胆管插管,采取原位胃吸收试验方法给予0.5 ml预热到37℃的含15 mg TSG的人工胃液,收集给药后20 min内的胆汁,取20 min后的胃内容物、胃粘膜、门静脉血、腹动脉血及尿液,采用酶解后的HPLC法分别测定各样品中游离TSG,TSG硫酸结合物,TSG葡糖醛酸结合物及TSG葡糖醛酸-硫酸结合物的含量。
结果:给药20 min后有(64±9.8) %的TSG被胃部吸收,其中(4±2) %分布在胃粘膜内,(1.1±0.5) %经胆汁排泄,1 %左右存在于血液循环中。与门静脉血相比,在动脉血中游离TSG比例减少,结合型比例增加。胆汁中主要为TSG葡糖醛酸结合物。
结论:TSG在胃部以游离形式被胃粘膜吸收,经肝门静脉进入肝脏,在肝脏内发生代谢作用,主要是与体内的葡萄糖醛酸和硫酸等内源性物质结合,最终经胆汁排出体外。
第八部分七宝美髯丹制剂中二苯乙烯苷等七种组分的同时测定
目的:采用HPLC/PDA法同时测定七宝美髯丹中二苯乙烯苷等七种组分的含量,以控制该制剂的质量。
方法:Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);流动相为乙腈和0.1 %冰醋酸进行梯度洗脱;检测波长:245 nm、320 nm、290 nm、350 nm;柱温30℃;流速1.0 ml/min。样品的提取方法为无水乙醇水浴回流2 h。
结果:芦丁、二苯乙烯苷、阿魏酸、补骨脂素、异补骨脂素、大黄素、大黄素甲醚七种组分的线性关系均良好(r>0.999),线性范围分别为:0.0215~0.536、0.0450~1.125、0.0052~0.131、0.0113~0.283、0.0137~0.342、0.0046~0.116、0.0018~0.044 mg/ml,平均回收率分别为:102.1 %、101.4 %、100.4 %、100.9 %、100.0 %、100.2 %、99.99 %,精密度和重复性均良好。不同厂家生产的七宝美髯丹质量有很大差别。
结论:该方法准确、快速、稳定,可用于七宝美髯丹的质量控制。
Radix polygoni multiflori is the dried root of polygonum plant Polygonum multiflorum Thunb.. It has many pharmacological actions. Recent studies showed that Radix polygoni multiflori had antihyperlipidemic and antiatherosclerotic effects. 2, 3, 5, 4′-Tetrahydroxystilbene-2-O-β-D-glycoside (stilbene glycoside, TSG) is one of the water soluble bioactive components in Radix polygoni multiflori. Many studies have documented the beneficial properties of TSG, including its strong antioxidant and free radical-scavenging. Possibly because of these properties, recent studies suggested that TSG could have preventive and therapeutic effect against some chronic diseases, such as apolexis, senile dementia (Alzheimer’s disease), hyperlipemia and atherosclerosis and so on.
In this present study, the antihyperlipidemic effects of active fraction and TSG from Radix polygoni multiflori has been studied and compared. The study suggested that TSG was the active constitute of Radix polygoni multiflori for its antihyperlipidemic effects. Absorption, distribution, metabolism and excretion of TSG in rat were investigated. The results of this work will contribution to its development and utilization.
Qibaomeiran pill, a well-known compound preparation of traditional Chinese medicine, consists of seven herbs. Radix Polygoni Multiflori was the principal drug. In order to preferably control the quality of Qibaomeiran pill overall, a new RP-HPLC method was established for simultaneous determination of seven active components with photo diode array detection.
Part one Experimental studies on antihyperlipidemic effects of constitute from Radix polygoni multiflori
Objective: To investigate the antihyperlipidemic effects of active fraction (HSWAF) and active constitute stilbene glycoside (TSG) from Radix polygoni multiflori. To illuminate the therapeutical basis of Radix polygoni multiflori for its antihyperlipidemic effects
Methods: The effects of HSWAF and TSG on serum lipids and liver index were studied in normal mice. Using the concentrations of lipids, glutamic oxalacetic transaminase enzyme (GOT) and glutamate-pyruvate transaminase (GPT) in serum in the model mice with acute hyperlipidemia induced by intraperitoneal injection of Triton as markers, the influences of HSWAF and TSG were observed. The investigation was also employed in the hyperlipidemic model rats induced by feeding with the high-lipid diet accompanied by oral administration of HSWAF and TSG to the rats for 28 days, respectively. The effects of HSWAF and TSG were investigated by measurement of the concentrations of lipids, malondialdehyde (MDA), nitric oxide (NO), and the activity of superoxide dismutase (SOD) in serum in the hyperlipidemic rats. The contents of total cholesterol (TC) and triglyceride (TG) in liver as well as liver index were also determined.
Results: In the normal mice, HSWAF and TSG not only increased the level of serum high density lipoprotein cholesterol (HDL-C), also decreased the level of serum low density lipoprotein cholesterol (LDL-C). HSWAF and TSG could lower serum TC and TG, and increase serum HDL-C in the hyperlipidemic mice. Administration of HSWAF (60 mg/kg) significantly reduced the liver index in hyperlipidemic rats. Administration of HSWAF (30, 60 mg/kg) could remarkably decrease the levels of serum TC, TG, LDL-C, MDA as well as the ratio of TC/HDL-C, also remarkably increase serum HDL-C, NO and SOD. Compared with HSWAF, TSG had the somewhat lower effect but no significant difference.
Conclusion: HSWAF and TSG possess obvious lipid-regulating, antioxidative and protecting vascular endothelium effects. The main bioactive constitute in HSWAF was TSG. Such characteristics will be of significance to prevent and/ or treat hyperlipidemia.
Part two Studies on pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats
Objective: To study the characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats induced by different factor.
Methods: 1. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton, all of them were orally administrated TSG at a dose of 60 mg/kg, and blood samples were obtained from fossa orbitalis vein according to the specific schedule and collected in heparinized centrifuge tube, respectively. Plasma was obtained by centrifugation at approximately 3 000 r/min for 10 min. Polydatin was taked as the internal standard. Three-time volume of methanol was added and the content of tube was mixed to precipitate the plasma protein, and then centrifuged. The supernatant was evaporated to dryness under a stream of nitrogen in the thermostatically controlled water-bath maintained at 50℃. Thereafter, quantitative volume of methanol was added to it and vortexed for 45 s and then centrifuged at 12000r/min. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (12∶10∶78). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area ratio of TSG and Polydatin were used for predicting unknown concentrations from the regression equation. 2. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton were randomly assigned to three groups. After oral administration of 60 mg/kg water solution of TSG , Heart, liver, spleen, lung, kidney, brain, stomach and small intestine samples were obtained at 5, 15, and 45 min, respectively. Tissue samples were homogenized in saline solution (1∶2 w/v). Three-time volume of methanol was added to the tissue homogenate to precipitate the protein, and then centrifuged. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (15∶18∶67). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area of TSG was recorded to determine the content of TSG in each sample.
Results: The calibration curve in plasma was linear over the range of 0.13~81.00μg/ml and the RSD values of intra-day and inter-day were less than 3 %. The recoveries of TSG in three different concentrations (high, middle and low) were 99.7 %,98.9 % and 98.3 %, respectively. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the Tmax was lower, t1/2 was highly significant shorter (p<0.01), Cmax, Ke, AUC0-t and AUC0-∞were all highly significant increased (p<0.01); in the hyperlipidemic model rats induced by high-lipid diet, only AUC0-t and AUC0-∞had highly significant increase (p<0.05). Compared with the normal rats, the relative bioavailability of TSG in Triton model rats and high-lipid diet model rats were 188 % and 155 %, respectively. 2. The standard curve range were 0.65~162.00μg/ml in all tissue homogenate samples. The recoveries of TSG in three different concentrations (high, middle and low) were 100.5 %,102.3 % and 100.9 %, respectively, and the RSD values of intra-day and inter-day were less than 3 %. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the concentration of TSG in kidney was slightly decreased; in the hyperlipidemic model rats induced by high-lipid diet, the concentration of TSG in heart was highly significant decreased, while there were obviously increased in liver, spleen and kidney.
Conclusion: The characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats had taken place some change compared with the normal rats. The results suggested that the physiologic condition of animal must be consideration to during the investigation. Patho-model animal should be choosing under the possible conditions in order to draw the real conclusion.
Part three Drug-protein binding determination of TSG
Objective: To study the binding of TSG to plasma, albumin andα1-AGP and to prove the availability of cloud-point extraction (CPE) in the determination of protein binding ratio of TSG.
Methods: In the present investigation, the binding of TSG to plasma, albumin andα1-AGP (2.0, 10.0 or 50μg/ml) was investigated by three different methods- ultrafiltration, equilibrium dialysis and CPE, and compare the results obtained from the three methods. The process of CPE as follow: In a test tube 1.0 ml of a 5 % (w/v) aqueous solution of Triton X-114 is added to 0.2 ml of sample. The phase separation was observed at 35℃. The extraction mixture was kept at this temperature for 10 min. After centrifugation at 3 000 r/min for 5 min the upper dilute aqueous phase (850μl) with the free fraction of TSG was removed from the surfactant-rich lower phase (350μl). The quantitive surfactant-rich lower phase was transferred into a tube for determining the concentration of bound drug.
Results: The results to plasma obtained by CPE were in good agreement to these observed by ultrafiltration and equilibrium. The binding ratios(%) of TSG to plasma in three different concentrations (0.05, 0.25 and 1.25mg/ml) obtained by equilibrium, ultrafiltration and CPE were 55.8±4.1, 53.4±4.4, 51.9±5.0; 79.6±3.3, 74.2±3.3, 72.5±2.6; 89.8±1.8, 85.0±3.6, 87.4±1.3, respectively. The results obtained by ultrafiltration and equilibrium showed that binding albumin was constant (about 60 %) within concentration range studied, while the binding toα1-AGP decreased with increasing drug concentration, but the results obtained by CPE was very different form these.
Conclusion: CPE was a highly sensitive and selective method for the measurement to plasma protein binding of TSG. Both albumin andα1-AGP were important for TSG protein binding. It is possible that some other proteins could contribution to the binding of TSG to plasma.
Part four Studies on the absorption kinetics of TSG in rats
Objective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/PDA) method for simultaneous determination of TSG and phenolsulfonphthalein in the circulation solution and to study the absorption kinetics of TSG in stomach and intestine in rats with the in situ perfusion.
Methods: The experiments were performed using the in situ perfusion method in rats. The concentrations of TSG and phenolsulfonphthalein were determined by HPLC/PDA with C18 column (250 mm×4.6 mm, 5μm) as an analytical column and a mixture of acetonitrile-methanol-0.2% phosphoric acid (v/v) (35∶15∶50) as a mobile phase at 1.0 ml/min of the flow rate. The column compartment was kept at the temperature of 30℃. The detection wavelenghs were chosen at 320 nm and 430 nm to record chromatograms of TSG and phenolsulfonphthalein, respectively.
Results: Calibration curves were generated over a concentration range of 3.5~140μg/ml for TSG and 1~40μg/ml for phenolsulfonphthalein, respectively. Intra- and inter-days variations were less than 3.1 %; Recoverys of TSG and phenolsulfonphthalein were among 99.48 % to 102.5 %. The hourly absorption percentages of TSG (2.5, 5 and 10 mg/ml) in stomach were 72.7 %, 67.7 % and 56.6 %, respectively. The absorption rate constants of TSG (30, 60 and 120μg/ml) in intestine were 0.047 7, 0.051 4 and 0.056 3, respectively and no significant difference (P>0.05) among them.
Conclusion: The measurement of TSG and phenolsulfonphthalein in the circulation solution was achieved by HPLC/PDA method. The method was sensitive, accurate, and simple. The results indicated that TSG was poor absorbed at intestine but well absorbed at stomach in rats. So TSG could be prepared as floating sustained-release tablet to prolong the retention time at stomach to improve bioavailability.
Part five Metabolism of TSG in vivo and in vitro
Objective: To study the metabolites and metabolic pathway of TSG in vivo and in vitro.
Methods: Culture solution of intestinal bacteria produced from rat feces and hepatic microsomal enzyme were used to research the metabolism of TSG in vitro. After taking orally TSG, blood, bile, urine, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC/PDA. Metabolites of TSG were purified by HPLC and identified by 1H-NMR, 13C-NMR and MS.
Results: Only TSG was present in both gastric content and culture solution of intestinal bacteria. TSG and its metabolites were not detected in the urine samples. In addition to free TSG, metabolite M2 was detected in the intestine content. TSG and metabolite M1 (TSG-glucuronide) were detected in the plasma sample. In the bile sample, slight TSG, metabolite M1 and considerable metabolite M2 were all detected. In the feces, free TSG was the major form of TSG. In the liver microsome incubation mixture, metabolite M2 was high in proportion. Metabolite M2 was C3-OH TSG-glucuronide identified by 1H-NMR, 13C-NMR and MS, which different from metabolite M1.
Conclusion: The results of this work demonstrated that TSG is metabolized to TSG-glucuronide mainly in rat liver, and be more easily excreted through bile. In the intestinal tract, TSG-glucuronides were hydrolyzed to TSG by bacteria or enzymes, and then out of body with defecation.
Part six Studies on the excretion of TSG in rats
Objective: To develop a HPLC method to assess the concentrations of TSG and its metabolite in rat bile and identify the metabolic pathway of TSG in rat.
Methods: After a single oral administration at a dose of 60 mg/kg, the bile of rat was collected. After methanol (10μl) was added into 200μl bile or diluted bile, and then centrifuged at a high speed for 10 min, the supernatant was injected into the liquid chromatographic system to determine the concentrations of TSG and its metabolite. Dikma Diamonsil C18 column (250 mm×4.6 mm, 5μm) was used. The mobile phase was composed of acetonitrile and 0.1 % (v/v) acetic acid solution (0~10 min, 15∶85; 15~23 min, 25∶75; 25~30 min, 100∶0). The flow rate was 1.0 ml/min and the column compartment was kept at the temperature of 30℃, 320 nm was chosen to record chromatograms.
Results: Calibration curves were generated over a concentration range of 12.0~0.8μg/ml for TSG and 110~0.9μg/ml for the metabolite, respectively. Intra- and inter- days variations were less than 7.0 %, and relative errors were ranged within±4.33 %. Extraction recoverys of TSG and metabolite were all above 99.14 %. Mean biliary accumulated excretion of TSG and metabolite for 24 h after administration were (0.084±0.04) % and (36.87±12.94) %, respectively.
Conclusion: This specific, sensitive and precise method is suitable for the determination of TSG and its metabolite in rat bile. Biliary excretion should be the main excretion route of TSG in the form of its metabolite. Part seven Development of the study model for physiological disposition of drug in rats and its application on TSG
Objective: To develop a simple study model to research the absorption, distribution, metabolism and excretion of drug in rat, and to make use of it on TSG.
Methods: The rats were anesthetized and kept alive under anesthesia throughout the experiments (In situ gastric absorption). At first, the biliary duct was cannulated. Bile was collected for up to 20 min after the administration. Second, the pylorus was ligated, and 15 mg of TSG in 0.5 ml artificial gastric juice at 37℃was injected into the stomach through the cardia by a syringe with plastic tubing. Immediately, the cardia was also ligated. At 20 min post-administration, blood was withdrawn with heparinized syringes from the portal vein and the celiac artery, respectively. Thereafter, the whole stomach was removed and immediately placed on ice (0℃). Urine was withdrawn from the bladder. Each sample was determined by HPLC after samples were prepared by the use of combined enzymatic hydrolysis to evaluate the content of free TSG, TSG-sulfate, TSG-glucuronide and TSG-diconjugate in each sample.
Results: After 15 mg of TSG was incubated in rat stomach in vitro for 20 min, (64±9.8) % of administered TSG seemed to be absorbed from the stomach, (4±2) % of the dose was stored in the gastric mucosa, and (1.1±0.5) % of the dose was excreted through bile, but none TSG was found in the urine. From the concentration of total TSG in the plasma, it could be also estimated that about 1 % of the dose was in the blood pool. Compared with the portal vein plasma, in the arteries plasma, the proportion of free TSG to total TSG was declined, while the proportion of conjugated TSG to total TSG was increased. In the bile, TSG-glucuronide was the major form of TSG.
Conclusion: TSG was absorbed from the stomach in the free form and transhepatic portal vein transported into the liver. In the liver, TSG was metabolized into conjugated TSG. TSG was excreted through bile mainly in the conjugated forms.
Part eight Simultaneous determination of seven components including stilbene glycoside in Qibaomeiran pill
Objective: To develop a high performance liquid chromatography coupled with photo diode array detection (HPLC/PDA) method for simultaneous determination of seven active components in Qibaomeiran pill.
Methods: The separation was performed by a Dikma Diamonsil C18 column using gradient acetonitrile-0.1 % glacial acetic acid as mobile phase. The detected wavelengths were set at 245, 320, 290 and 350 nm. The column temperature was 30℃. In order to extract the seven components completely, different extraction methods, solvents and extraction time were compared. The optimal method was refluxing extraction 2.0 hours with alcohol as solvent.
Results: The linear rangers of rutin, stilbene glucoside, ferulic acid, psoralen, isopsoralen, emodin and physcion were 0.0215~0.536, 0.0450~1.125, 0.0052~0.131, 0.0113~0.283, 0.0137~0.342, 0.0046~0.116 and 0.0018~0.044 mg/ml, respectively. Their average recoveries were 102.1 %, 101.4 %, 100.4 %, 100.9 %, 100.0 %, 100.2 % and 99.99 %, respectively. The results indicated that the precision and reproducibility were suitable. There is notable difference among the different manufacturers.
Conclution: The method was proved to be very accurate, quick and stable to the quality control for Qibaomeiran pill.
本文通过比较TSG单体成分与含相同量TSG的何首乌总提物(有效部位)的降血脂作用,确定TSG为何首乌降血脂的药效物质基础,并较深入地研究了TSG在动物体内的吸收、分布、代谢、排泄等一系列过程,为TSG的开发利用奠定了坚实的基础。
七宝美髯丹是以何首乌为君药的复方制剂,含有多种有效成分,为了更好的控制药品质量,本文建立了RP-HPLC法同时测定二苯乙烯苷等七种组分的含量。
第一部分何首乌降血脂作用的药效物质基础研究
目的:比较研究何首乌有效部位(HSWAF)及有效成分TSG的降血脂作用,明确何首乌降血脂作用的药效物质基础。
方法:观察HSWAF和TSG对正常小鼠血脂及肝指数的影响;小鼠腹腔注射Triton致急性高脂血症模型,观察HSWAF和TSG对模型动物血脂、谷草转氨酶(GOT)和谷丙转氨酶(GPT)水平的影响;以大鼠食饵性高脂血症为模型,给予高脂饲料的同时给予HSWAF和TSG,连续28天,测定血脂、丙二醛(MDA)、一氧化氮(NO)含量和超氧化物歧化酶(SOD)活性,肝组织中总胆固醇(TC)和甘油三酯(TG)含量以及肝指数。
结果:对正常小鼠,TSG和HSWAF均有升高血清高密度脂蛋白胆固醇(HDL-C),降低血清低密度脂蛋白胆固醇(LDL-C)的作用。HSWAF和TSG对腹腔注射Triton致高脂血症小鼠的血清TC和TG均有降低作用,对HDL-C均有升高作用。HSWAF 60 mg/kg明显降低大鼠食饵性高脂血症模型的肝指数;HSWAF 30 mg/kg和60 mg/kg明显降低大鼠食饵性高脂血症模型的血清TC、TG、LDL-C、MDA含量、TC/HDL-C比值,明显升高血清HDL-C和NO含量以及SOD活性,TSG对以上各项指标的作用强度略低于HSWAF,但无显著性差异。
结论:何首乌中有效部位及有效成分TSG均具有调血脂、抗氧化,保护血管内皮的功能,TSG为发挥药效作用的主要物质,提示可用于防治高脂血症。
第二部分二苯乙烯苷在高血脂模型大鼠体内的药动学研究与组织分布
目的:研究TSG在不同高血脂病理模型动物体内的药动学及组织分布情况。
方法:1.分别取正常大鼠,食靡性高脂血症模型大鼠和Triton致高脂血症模型大鼠,按60 mg/kg的剂量灌胃给予TSG,于给药后不同时间经眼球后静脉丛取血,置肝素化试管中,以3 000 r/min离心10 min,分离血浆。以虎杖苷为内标,用3倍量甲醇沉淀蛋白,取一定量上清液,于50℃氮气流吹干,用少量甲醇溶解后,取上清液20μl注入液相色谱仪。C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-甲醇-0.1%冰醋酸(12∶10∶78),检测波长320 nm ,流速为1.0 ml/min。记录TSG与虎杖苷的峰面积,计算TSG的血药浓度。2.分别取正常大鼠,食靡性高脂血症模型大鼠和Triton致高脂血症模型大鼠,按60 mg/kg的剂量灌胃给予TSG,分别于给药后5、15、45 min断头处死,分别取出心,肝,脾,肺,肾,脑,胃,小肠等组织,用生理盐水制成1∶2的组织匀浆。组织匀浆加3倍量甲醇沉淀蛋白,离心,取20μl上清液注入液相色谱仪。C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-甲醇-0.1%冰醋酸(15∶18∶67);检测波长320 nm ,流速为1.0 ml/min。测定组织中TSG的含量。
结果:1.血浆中TSG在0.13~81.00μg/ml浓度范围内线性关系良好,高、中、低3种不同浓度血浆样品的方法回收率分别为99.7 %、98.9 %、98.3 %,日内与日间精密度RSD均<3.0 %。与正常大鼠相比,TSG在triton致高脂血症模型大鼠体内的Tmax有降低趋势,t1/2非常显著性缩短(p<0.01),Cmax、Ke、AUC0-t与AUC0-∞均非常显著性增加(p<0.01);脂肪乳致高脂血症模型大鼠与正常大鼠相比,AUC0-t与AUC0-∞显著性增加(p<0.05)外,其它参数均无显著性差异(p>0.05)。Triton致高脂血症模型大鼠与食靡性高脂血症模型大鼠相对正常大鼠的相对生物利用度分别为188 %和155 %。2.各组织中TSG浓度在0.65~162.00μg/ml范围内线性关系良好,高、中、低3种不同浓度肝脏匀浆样品的方法平均回收率分别为100.5 %、102.3 %、100.9 %,日内与日间精密度RSD均<3.0 %。与正常大鼠相比,在triton高脂血症模型大鼠体内,TSG除在肾脏中的分布有所减少外,其它均无明显变化;在食靡性高脂血症模型大鼠的心脏中明显减少,而在肝脏、脾脏和肾脏中均明显增加。
结论:TSG在高脂血症模型动物体内的药动学及组织分布发生了某些变化,提示在进行药物的体内过程研究时应充分考虑机体的生理状态,在可能的情况下尽量选择相关的病理模型进行研究,使实验结果更接近真实情况。
第三部分二苯乙烯苷的血浆蛋白结合率测定
目的:研究TSG的血浆蛋白结合情况,并探讨用浊点萃取法(CPE)测定TSG血浆蛋白结合率的方法。
方法:分别采用平衡透析法、超滤法和CPE法测定浓度为0.05、0.25、1.25 mg/ml的TSG与人血浆,牛血清白蛋白(BSA)和α1-酸性脂蛋白(α1-AGP)的结合情况,并将3种方法的测定结果进行比较。浊点萃取方法为:200μl样品,精密加入1.0 ml浓度为5 % (w/v)的Triton X-114溶液,涡旋混合,置35℃恒温振荡器中保温10 min,进行相分离,于3 000 r/min离心5 min,上层为水层(游离型药物),体积为850μl,下层为表面活性剂层(结合型药物),体积为350μl,用标准曲线法测定结合型药物的含量。
结果:在不同浓度(0.05、0.25、1.25 mg/ml)下,用平衡透析法、超滤法和CPE法测得的TSG与血浆蛋白的结合率(%)非常相似,分别为:55.8±4.1、53.4±4.4、51.9±5.0,79.6±3.3、74.2±3.3、72.5±2.6,89.8±1.8、85.0±3.6、87.4±1.3。用平衡透析法和超滤法测得的结果均表明,TSG与血清白蛋白的结合率基本不随浓度发生变化(约60 %),与α1-AGP的结合率则随药物浓度的增加而明显降低,而用CPE法测定的两种蛋白的结合率均在80 %~90 %,与上述两种方法的差别很大。
结论:CPE法可用于测定TSG的血浆蛋白结合率。TSG既能与血清白蛋白结合又能与α1-AGP结合,血浆中还可能存在其他能和TSG结合的蛋白质。
第四部分二苯乙烯苷在大鼠体内的吸收动力学研究
目的:建立同时测定胃灌注液及肠循环液中TSG及酚红浓度的HPLC/PDA法,并研究TSG在大鼠胃、肠的吸收特性。
方法:采用大鼠在体胃、肠吸收模型,以HPLC/PDA法测定胃灌注液及肠循环液中药物的含量,色谱条件为:Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);柱温30℃;流动相为乙腈-甲醇-0.2 %磷酸(35∶15∶50),流速1 ml/min;检测波长320 nm(TSG)和430 nm(酚红);进样量20μl。
结果:TSG及酚红的线性关系良好,线性范围分别为3.5~140μg/ml和1~40μg/ml,日内、日间精密度(RSD)均小于3.1 %,方法回收率均在99.48 %~102.5 %之间。不同质量浓度(2.5、5、10 mg/ml)的TSG在大鼠胃部的每小时吸收百分率分别为72.7、67.7、56.6;不同质量浓度(30、60、120μg /ml)的TSG在肠道内的吸收速率常数分别为0.047 7、0.051 4、0.056 3,三者之间无显著性差异(P>0.05)。
结论:本文首次建立了HPLC/PDA法同时测定胃灌注液及肠循环液中TSG及酚红的浓度,该法操作简便,结果准确,灵敏度高。研究结果表明,TSG在肠道内吸收较差,主要吸收部位是胃,为延长药物在胃内的停留时间,改善生物利用度,适合制成胃漂浮片。
第五部分二苯乙烯苷在大鼠体内外的代谢研究
目的:研究TSG在大鼠体内外的代谢产物和代谢途径。
方法:采用肝微粒体温孵法,大肠杆菌培养法等对TSG进行体外代谢研究;灌胃给予一定量TSG后,对大鼠血液、胆汁、尿液、粪便、小肠内容物和胃内容物进行体内代谢产物的分析,通过制备液相获得主要代谢物的纯品,用1H-NMR、13C-NMR及MS手段进行结构确证,从而推测TSG在体内的转化过程。
结果:在大肠杆菌培养液中,胃内容物中只有原型药物;尿液中既无原型也无代谢物;肠内容物中除原型外,还有少量代谢物M2;血浆中除原型外,还有代谢物M1(已经证明为TSG的葡糖醛酸结合物);胆汁中除可检测到原型药物外,还有大量的代谢物M2和少量的代谢物M1;粪便中检测到的主要是原型药物;在肝微粒体酶的作用下,TSG很快代谢为M2。结构鉴定证明,M2与M1结合部位不同,为TSGC3-OH的葡糖醛酸结合物。
结论:TSG在肝脏代谢为葡糖醛酸结合物,并经胆汁排泄,在肠道内菌或酶的作用下水解为原型随粪便排出体外。
第六部分二苯乙烯苷在大鼠体内的排泄研究
目的:建立大鼠胆汁中TSG及其代谢物浓度的HPLC测定方法,明确TSG的代谢途径。
方法:胆汁样品或用重蒸水稀释后的胆汁样品200μl,加入10μl甲醇后直接离心测定TSG及其代谢物的浓度。Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm),柱温30℃;流动相为乙腈-0.1 %冰醋酸梯度洗脱(0~10 min,15∶85;15~23 min,25∶75;25~30 min,100∶0),流速1 ml/min;检测波长320 nm。测定大鼠灌胃给予60 mg/kg的TSG后胆汁中TSG及其代谢物的累积排泄率。
结果:TSG及其代谢物在胆汁中的线性范围分别为12.0~0.8μg/ml和110~0.9μg/ml,日内和日间RSD均小于7.0 %,准确度在±4.33 %之间。提取回收率均大于99.14 %。大鼠灌胃给予60 mg/kg的TSG后24 h内胆汁中TSG及其代谢物的累积排泄率分别为(0.084±0.04) %和(36.87±12.94) %。
结论:经方法学考察符合生物样品的测定要求,可应用于大鼠胆汁中TSG及其代谢物浓度的测定,TSG在大鼠体内主要以代谢物的形式由胆汁排泄。
第七部分二苯乙烯苷体内过程研究模式的建立及其应用
目的:建立一种简便,快速的研究药物体内吸收、分布、代谢及排泄等全过程的方法模式,并应用于TSG的体内过程研究。
方法:麻醉后的大鼠,进行胆管插管,采取原位胃吸收试验方法给予0.5 ml预热到37℃的含15 mg TSG的人工胃液,收集给药后20 min内的胆汁,取20 min后的胃内容物、胃粘膜、门静脉血、腹动脉血及尿液,采用酶解后的HPLC法分别测定各样品中游离TSG,TSG硫酸结合物,TSG葡糖醛酸结合物及TSG葡糖醛酸-硫酸结合物的含量。
结果:给药20 min后有(64±9.8) %的TSG被胃部吸收,其中(4±2) %分布在胃粘膜内,(1.1±0.5) %经胆汁排泄,1 %左右存在于血液循环中。与门静脉血相比,在动脉血中游离TSG比例减少,结合型比例增加。胆汁中主要为TSG葡糖醛酸结合物。
结论:TSG在胃部以游离形式被胃粘膜吸收,经肝门静脉进入肝脏,在肝脏内发生代谢作用,主要是与体内的葡萄糖醛酸和硫酸等内源性物质结合,最终经胆汁排出体外。
第八部分七宝美髯丹制剂中二苯乙烯苷等七种组分的同时测定
目的:采用HPLC/PDA法同时测定七宝美髯丹中二苯乙烯苷等七种组分的含量,以控制该制剂的质量。
方法:Dikma Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);流动相为乙腈和0.1 %冰醋酸进行梯度洗脱;检测波长:245 nm、320 nm、290 nm、350 nm;柱温30℃;流速1.0 ml/min。样品的提取方法为无水乙醇水浴回流2 h。
结果:芦丁、二苯乙烯苷、阿魏酸、补骨脂素、异补骨脂素、大黄素、大黄素甲醚七种组分的线性关系均良好(r>0.999),线性范围分别为:0.0215~0.536、0.0450~1.125、0.0052~0.131、0.0113~0.283、0.0137~0.342、0.0046~0.116、0.0018~0.044 mg/ml,平均回收率分别为:102.1 %、101.4 %、100.4 %、100.9 %、100.0 %、100.2 %、99.99 %,精密度和重复性均良好。不同厂家生产的七宝美髯丹质量有很大差别。
结论:该方法准确、快速、稳定,可用于七宝美髯丹的质量控制。
Radix polygoni multiflori is the dried root of polygonum plant Polygonum multiflorum Thunb.. It has many pharmacological actions. Recent studies showed that Radix polygoni multiflori had antihyperlipidemic and antiatherosclerotic effects. 2, 3, 5, 4′-Tetrahydroxystilbene-2-O-β-D-glycoside (stilbene glycoside, TSG) is one of the water soluble bioactive components in Radix polygoni multiflori. Many studies have documented the beneficial properties of TSG, including its strong antioxidant and free radical-scavenging. Possibly because of these properties, recent studies suggested that TSG could have preventive and therapeutic effect against some chronic diseases, such as apolexis, senile dementia (Alzheimer’s disease), hyperlipemia and atherosclerosis and so on.
In this present study, the antihyperlipidemic effects of active fraction and TSG from Radix polygoni multiflori has been studied and compared. The study suggested that TSG was the active constitute of Radix polygoni multiflori for its antihyperlipidemic effects. Absorption, distribution, metabolism and excretion of TSG in rat were investigated. The results of this work will contribution to its development and utilization.
Qibaomeiran pill, a well-known compound preparation of traditional Chinese medicine, consists of seven herbs. Radix Polygoni Multiflori was the principal drug. In order to preferably control the quality of Qibaomeiran pill overall, a new RP-HPLC method was established for simultaneous determination of seven active components with photo diode array detection.
Part one Experimental studies on antihyperlipidemic effects of constitute from Radix polygoni multiflori
Objective: To investigate the antihyperlipidemic effects of active fraction (HSWAF) and active constitute stilbene glycoside (TSG) from Radix polygoni multiflori. To illuminate the therapeutical basis of Radix polygoni multiflori for its antihyperlipidemic effects
Methods: The effects of HSWAF and TSG on serum lipids and liver index were studied in normal mice. Using the concentrations of lipids, glutamic oxalacetic transaminase enzyme (GOT) and glutamate-pyruvate transaminase (GPT) in serum in the model mice with acute hyperlipidemia induced by intraperitoneal injection of Triton as markers, the influences of HSWAF and TSG were observed. The investigation was also employed in the hyperlipidemic model rats induced by feeding with the high-lipid diet accompanied by oral administration of HSWAF and TSG to the rats for 28 days, respectively. The effects of HSWAF and TSG were investigated by measurement of the concentrations of lipids, malondialdehyde (MDA), nitric oxide (NO), and the activity of superoxide dismutase (SOD) in serum in the hyperlipidemic rats. The contents of total cholesterol (TC) and triglyceride (TG) in liver as well as liver index were also determined.
Results: In the normal mice, HSWAF and TSG not only increased the level of serum high density lipoprotein cholesterol (HDL-C), also decreased the level of serum low density lipoprotein cholesterol (LDL-C). HSWAF and TSG could lower serum TC and TG, and increase serum HDL-C in the hyperlipidemic mice. Administration of HSWAF (60 mg/kg) significantly reduced the liver index in hyperlipidemic rats. Administration of HSWAF (30, 60 mg/kg) could remarkably decrease the levels of serum TC, TG, LDL-C, MDA as well as the ratio of TC/HDL-C, also remarkably increase serum HDL-C, NO and SOD. Compared with HSWAF, TSG had the somewhat lower effect but no significant difference.
Conclusion: HSWAF and TSG possess obvious lipid-regulating, antioxidative and protecting vascular endothelium effects. The main bioactive constitute in HSWAF was TSG. Such characteristics will be of significance to prevent and/ or treat hyperlipidemia.
Part two Studies on pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats
Objective: To study the characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats induced by different factor.
Methods: 1. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton, all of them were orally administrated TSG at a dose of 60 mg/kg, and blood samples were obtained from fossa orbitalis vein according to the specific schedule and collected in heparinized centrifuge tube, respectively. Plasma was obtained by centrifugation at approximately 3 000 r/min for 10 min. Polydatin was taked as the internal standard. Three-time volume of methanol was added and the content of tube was mixed to precipitate the plasma protein, and then centrifuged. The supernatant was evaporated to dryness under a stream of nitrogen in the thermostatically controlled water-bath maintained at 50℃. Thereafter, quantitative volume of methanol was added to it and vortexed for 45 s and then centrifuged at 12000r/min. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (12∶10∶78). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area ratio of TSG and Polydatin were used for predicting unknown concentrations from the regression equation. 2. Normal rats, the hyperlipidemic model rats induced by feeding with the high-lipid diet and the hyperlipidemic model rats induced by intraperitoneal injection of Triton were randomly assigned to three groups. After oral administration of 60 mg/kg water solution of TSG , Heart, liver, spleen, lung, kidney, brain, stomach and small intestine samples were obtained at 5, 15, and 45 min, respectively. Tissue samples were homogenized in saline solution (1∶2 w/v). Three-time volume of methanol was added to the tissue homogenate to precipitate the protein, and then centrifuged. The supernatant (20μl) was injected into the HPLC system. The C18 column (250×4.6 mm, 5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile-methanol-0.1 % glacial acetic acid (15∶18∶67). The flow rate was 1 ml/min. The UV detector was set at 320 nm. The peak area of TSG was recorded to determine the content of TSG in each sample.
Results: The calibration curve in plasma was linear over the range of 0.13~81.00μg/ml and the RSD values of intra-day and inter-day were less than 3 %. The recoveries of TSG in three different concentrations (high, middle and low) were 99.7 %,98.9 % and 98.3 %, respectively. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the Tmax was lower, t1/2 was highly significant shorter (p<0.01), Cmax, Ke, AUC0-t and AUC0-∞were all highly significant increased (p<0.01); in the hyperlipidemic model rats induced by high-lipid diet, only AUC0-t and AUC0-∞had highly significant increase (p<0.05). Compared with the normal rats, the relative bioavailability of TSG in Triton model rats and high-lipid diet model rats were 188 % and 155 %, respectively. 2. The standard curve range were 0.65~162.00μg/ml in all tissue homogenate samples. The recoveries of TSG in three different concentrations (high, middle and low) were 100.5 %,102.3 % and 100.9 %, respectively, and the RSD values of intra-day and inter-day were less than 3 %. Compared with the normal rats, in the hyperlipidemic model rats induced by Triton, the concentration of TSG in kidney was slightly decreased; in the hyperlipidemic model rats induced by high-lipid diet, the concentration of TSG in heart was highly significant decreased, while there were obviously increased in liver, spleen and kidney.
Conclusion: The characteristics of pharmacokinetics and tissue distribution of TSG in the hyperlipidemia model rats had taken place some change compared with the normal rats. The results suggested that the physiologic condition of animal must be consideration to during the investigation. Patho-model animal should be choosing under the possible conditions in order to draw the real conclusion.
Part three Drug-protein binding determination of TSG
Objective: To study the binding of TSG to plasma, albumin andα1-AGP and to prove the availability of cloud-point extraction (CPE) in the determination of protein binding ratio of TSG.
Methods: In the present investigation, the binding of TSG to plasma, albumin andα1-AGP (2.0, 10.0 or 50μg/ml) was investigated by three different methods- ultrafiltration, equilibrium dialysis and CPE, and compare the results obtained from the three methods. The process of CPE as follow: In a test tube 1.0 ml of a 5 % (w/v) aqueous solution of Triton X-114 is added to 0.2 ml of sample. The phase separation was observed at 35℃. The extraction mixture was kept at this temperature for 10 min. After centrifugation at 3 000 r/min for 5 min the upper dilute aqueous phase (850μl) with the free fraction of TSG was removed from the surfactant-rich lower phase (350μl). The quantitive surfactant-rich lower phase was transferred into a tube for determining the concentration of bound drug.
Results: The results to plasma obtained by CPE were in good agreement to these observed by ultrafiltration and equilibrium. The binding ratios(%) of TSG to plasma in three different concentrations (0.05, 0.25 and 1.25mg/ml) obtained by equilibrium, ultrafiltration and CPE were 55.8±4.1, 53.4±4.4, 51.9±5.0; 79.6±3.3, 74.2±3.3, 72.5±2.6; 89.8±1.8, 85.0±3.6, 87.4±1.3, respectively. The results obtained by ultrafiltration and equilibrium showed that binding albumin was constant (about 60 %) within concentration range studied, while the binding toα1-AGP decreased with increasing drug concentration, but the results obtained by CPE was very different form these.
Conclusion: CPE was a highly sensitive and selective method for the measurement to plasma protein binding of TSG. Both albumin andα1-AGP were important for TSG protein binding. It is possible that some other proteins could contribution to the binding of TSG to plasma.
Part four Studies on the absorption kinetics of TSG in rats
Objective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/PDA) method for simultaneous determination of TSG and phenolsulfonphthalein in the circulation solution and to study the absorption kinetics of TSG in stomach and intestine in rats with the in situ perfusion.
Methods: The experiments were performed using the in situ perfusion method in rats. The concentrations of TSG and phenolsulfonphthalein were determined by HPLC/PDA with C18 column (250 mm×4.6 mm, 5μm) as an analytical column and a mixture of acetonitrile-methanol-0.2% phosphoric acid (v/v) (35∶15∶50) as a mobile phase at 1.0 ml/min of the flow rate. The column compartment was kept at the temperature of 30℃. The detection wavelenghs were chosen at 320 nm and 430 nm to record chromatograms of TSG and phenolsulfonphthalein, respectively.
Results: Calibration curves were generated over a concentration range of 3.5~140μg/ml for TSG and 1~40μg/ml for phenolsulfonphthalein, respectively. Intra- and inter-days variations were less than 3.1 %; Recoverys of TSG and phenolsulfonphthalein were among 99.48 % to 102.5 %. The hourly absorption percentages of TSG (2.5, 5 and 10 mg/ml) in stomach were 72.7 %, 67.7 % and 56.6 %, respectively. The absorption rate constants of TSG (30, 60 and 120μg/ml) in intestine were 0.047 7, 0.051 4 and 0.056 3, respectively and no significant difference (P>0.05) among them.
Conclusion: The measurement of TSG and phenolsulfonphthalein in the circulation solution was achieved by HPLC/PDA method. The method was sensitive, accurate, and simple. The results indicated that TSG was poor absorbed at intestine but well absorbed at stomach in rats. So TSG could be prepared as floating sustained-release tablet to prolong the retention time at stomach to improve bioavailability.
Part five Metabolism of TSG in vivo and in vitro
Objective: To study the metabolites and metabolic pathway of TSG in vivo and in vitro.
Methods: Culture solution of intestinal bacteria produced from rat feces and hepatic microsomal enzyme were used to research the metabolism of TSG in vitro. After taking orally TSG, blood, bile, urine, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC/PDA. Metabolites of TSG were purified by HPLC and identified by 1H-NMR, 13C-NMR and MS.
Results: Only TSG was present in both gastric content and culture solution of intestinal bacteria. TSG and its metabolites were not detected in the urine samples. In addition to free TSG, metabolite M2 was detected in the intestine content. TSG and metabolite M1 (TSG-glucuronide) were detected in the plasma sample. In the bile sample, slight TSG, metabolite M1 and considerable metabolite M2 were all detected. In the feces, free TSG was the major form of TSG. In the liver microsome incubation mixture, metabolite M2 was high in proportion. Metabolite M2 was C3-OH TSG-glucuronide identified by 1H-NMR, 13C-NMR and MS, which different from metabolite M1.
Conclusion: The results of this work demonstrated that TSG is metabolized to TSG-glucuronide mainly in rat liver, and be more easily excreted through bile. In the intestinal tract, TSG-glucuronides were hydrolyzed to TSG by bacteria or enzymes, and then out of body with defecation.
Part six Studies on the excretion of TSG in rats
Objective: To develop a HPLC method to assess the concentrations of TSG and its metabolite in rat bile and identify the metabolic pathway of TSG in rat.
Methods: After a single oral administration at a dose of 60 mg/kg, the bile of rat was collected. After methanol (10μl) was added into 200μl bile or diluted bile, and then centrifuged at a high speed for 10 min, the supernatant was injected into the liquid chromatographic system to determine the concentrations of TSG and its metabolite. Dikma Diamonsil C18 column (250 mm×4.6 mm, 5μm) was used. The mobile phase was composed of acetonitrile and 0.1 % (v/v) acetic acid solution (0~10 min, 15∶85; 15~23 min, 25∶75; 25~30 min, 100∶0). The flow rate was 1.0 ml/min and the column compartment was kept at the temperature of 30℃, 320 nm was chosen to record chromatograms.
Results: Calibration curves were generated over a concentration range of 12.0~0.8μg/ml for TSG and 110~0.9μg/ml for the metabolite, respectively. Intra- and inter- days variations were less than 7.0 %, and relative errors were ranged within±4.33 %. Extraction recoverys of TSG and metabolite were all above 99.14 %. Mean biliary accumulated excretion of TSG and metabolite for 24 h after administration were (0.084±0.04) % and (36.87±12.94) %, respectively.
Conclusion: This specific, sensitive and precise method is suitable for the determination of TSG and its metabolite in rat bile. Biliary excretion should be the main excretion route of TSG in the form of its metabolite. Part seven Development of the study model for physiological disposition of drug in rats and its application on TSG
Objective: To develop a simple study model to research the absorption, distribution, metabolism and excretion of drug in rat, and to make use of it on TSG.
Methods: The rats were anesthetized and kept alive under anesthesia throughout the experiments (In situ gastric absorption). At first, the biliary duct was cannulated. Bile was collected for up to 20 min after the administration. Second, the pylorus was ligated, and 15 mg of TSG in 0.5 ml artificial gastric juice at 37℃was injected into the stomach through the cardia by a syringe with plastic tubing. Immediately, the cardia was also ligated. At 20 min post-administration, blood was withdrawn with heparinized syringes from the portal vein and the celiac artery, respectively. Thereafter, the whole stomach was removed and immediately placed on ice (0℃). Urine was withdrawn from the bladder. Each sample was determined by HPLC after samples were prepared by the use of combined enzymatic hydrolysis to evaluate the content of free TSG, TSG-sulfate, TSG-glucuronide and TSG-diconjugate in each sample.
Results: After 15 mg of TSG was incubated in rat stomach in vitro for 20 min, (64±9.8) % of administered TSG seemed to be absorbed from the stomach, (4±2) % of the dose was stored in the gastric mucosa, and (1.1±0.5) % of the dose was excreted through bile, but none TSG was found in the urine. From the concentration of total TSG in the plasma, it could be also estimated that about 1 % of the dose was in the blood pool. Compared with the portal vein plasma, in the arteries plasma, the proportion of free TSG to total TSG was declined, while the proportion of conjugated TSG to total TSG was increased. In the bile, TSG-glucuronide was the major form of TSG.
Conclusion: TSG was absorbed from the stomach in the free form and transhepatic portal vein transported into the liver. In the liver, TSG was metabolized into conjugated TSG. TSG was excreted through bile mainly in the conjugated forms.
Part eight Simultaneous determination of seven components including stilbene glycoside in Qibaomeiran pill
Objective: To develop a high performance liquid chromatography coupled with photo diode array detection (HPLC/PDA) method for simultaneous determination of seven active components in Qibaomeiran pill.
Methods: The separation was performed by a Dikma Diamonsil C18 column using gradient acetonitrile-0.1 % glacial acetic acid as mobile phase. The detected wavelengths were set at 245, 320, 290 and 350 nm. The column temperature was 30℃. In order to extract the seven components completely, different extraction methods, solvents and extraction time were compared. The optimal method was refluxing extraction 2.0 hours with alcohol as solvent.
Results: The linear rangers of rutin, stilbene glucoside, ferulic acid, psoralen, isopsoralen, emodin and physcion were 0.0215~0.536, 0.0450~1.125, 0.0052~0.131, 0.0113~0.283, 0.0137~0.342, 0.0046~0.116 and 0.0018~0.044 mg/ml, respectively. Their average recoveries were 102.1 %, 101.4 %, 100.4 %, 100.9 %, 100.0 %, 100.2 % and 99.99 %, respectively. The results indicated that the precision and reproducibility were suitable. There is notable difference among the different manufacturers.
Conclution: The method was proved to be very accurate, quick and stable to the quality control for Qibaomeiran pill.
引文
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