【作者】 徐福平；

【导师】 刘奕明；

【作者基本信息】 广州中医药大学 ， 中药学， 2011， 硕士

【摘要】 背景药对是中药复方配伍中最简单、最基本的用药形式,药对配伍的研究是探究复方配伍与吸收的关系重要切入点。本研究以经典药对知母/黄柏中知母有效成分芒果苷为研究对象,通过考察芒果苷在体内的吸收动力学特征和肠吸收特性,并与知母单煎液和知母/黄柏药对合煎液比较,探讨知母、黄柏配伍对芒果苷吸收动力学的影响,阐释知母/黄柏药对的配伍机制,为应用知母/黄柏药对的临床应用的物质基础研究提供工作基础和理论依据。目的1建立同时测定知母/黄柏药对中活性成分芒果苷、新芒果苷和盐酸小檗碱超高效液相色谱(UPLC)方法,考察知母/黄柏配伍及煎煮方法对3种成分煎出量的影响,并控制煎液批间组成成分的稳定性。2建立液相色谱-质谱联用(LC/MS/MS)法测定芒果苷在大鼠血浆中的浓度,考察知母配伍黄柏对有效成分芒果苷在大鼠体内药物动力学的影响。3采用大鼠在体肠循环灌流实验模型和缚管翻转肠囊模型研究知母/黄柏药对中有效成分芒果苷的肠吸收动力学特征。方法1芒果苷、新芒果苷和小檗碱含量测定方法学的建立：采用UPLC色谱系统,ACQUITY UPLC(?)BEH C1H色谱柱(2.1 mm×50 mm,1.7μm),流动相为0.1%磷酸(三乙胺调PH=3.0)-乙腈,梯度洗脱(乙腈：0 min为5%,5.5min为18%,8 min为42%),流速0.25 mL·min-1,柱温35℃,检测波长260 nm。2芒果苷血药浓度的测定及体内药物动力学研究：采用日本资生堂CAPCELL PAKC18柱(150×2.0mm,5μm);流动相为甲醇-乙腈-1%乙酸(40：3：57)；流速250 uL·min；柱温为室温；用电喷雾离子化和正离子多离子反应监测(MRM)方式检测芒果苷；SD大鼠随机分为6组,分别单次灌胃芒果苷单体17.5、35、70 mg·kg-1、知母单煎液、知母-黄柏(1：1)合煎液和知母-黄柏(1：3)合煎液,测定血浆中芒果苷浓度,用DAS软件根据非房室模型法计算药物动力学参数。3芒果苷在体肠吸收特性的研究：采用大鼠在体肠循环灌流模型,利用超高效液相色谱法(UPLC-TUV)和紫外分光光度法(UV)分别测定肠液中芒果苷和酚红的浓度,考察芒果苷单体和知母、知母/黄柏药对中芒果苷在大鼠肠道的吸收特征。4翻转肠囊法研究芒果苷在大鼠小肠内吸收的动力学特征：采用UPLC-TUV法测定芒果苷的浓度,通过缚管翻转肠囊实验研究芒果苷单体、知母水煎液和知母/黄柏水煎液中芒果苷的吸收动力学差异。结果1芒果苷、新芒果苷和小檗碱含量的测定：芒果苷、新芒果苷和小檗碱分别在3.44～220.00,3.75～240.00,9.76～625.00μg·mL-1内线性关系良好(r≥0.9999),平均回收率分别为(95.00±3.28)%,(96.45±1.26)%和(97.78±4.84)%。2芒果苷血药浓度的测定及体内药物动力学研究：芒果苷的线性范围为3.01-601.2 ng.mL-1；方法回收率在100.5%-104.0%；日内和日间精密度均<9.1%。芒果苷单体17.5.35.70mg.kg-1给大鼠灌胃后,Cmax和AUC增加但与剂量的增加不成比例且数值较低,表面芒果苷单体吸收较差。知母单煎液组芒果苷血药浓度比单体组明显升高,配伍黄柏后有所下降。大鼠单次灌胃芒果苷单体(35mg.kg-1).知母单煎液、知母-黄柏(1：1)和(1：3)合煎液后的主要药动学参数AUC0-t,分别为0.626、122.4、94.36、53.97μg.mL-1.h,Cmax分别为0.149、21.52、16.26、8.27μg·mL-1,Tmax分别为1.0、3.0、4.0、6.0 h,t1-22分别为3.41、1.46、1.65、1.77 h。3芒果苷在体肠吸收特性的研究：芒果苷浓度为2.0,5.0,10.0,20μg·mL-1时,吸收速率常数(Ka)分别是0.0541,0.0467,0.0491,0.0220 h-1,吸收百分率(P)分别是14.05%,13.14%,12.43%和5.82%；随PH升高,Ka和P依次增加；芒果苷在肠段内吸收存在差异,各肠段的吸收速率常数按结肠、十二指肠、回肠和空肠依次下降；知母和知母/黄柏水煎液组中,肠循环液中新芒果苷含量的下降和芒果苷含量的上升呈同步变化。4翻转肠囊法研究芒果苷在大鼠小肠内吸收的动力学特征：大鼠肠外翻试验中,芒果苷在空肠、回肠和结肠的表观渗透系数(Papp)分别是2.0350×10-1,5.1788×10-4,3.3825×10-1cm·min-1；知母水煎液组和知母-黄柏水煎液组中,其成分芒果苷在空肠的表观渗透系数(Papp)分别是18.363×10-4,4.1063×10-4cm·min-1,在回肠的表观渗透系数(Papp)分别是10.190×10-4,3.9650×10-4cm·min-1。结论1知母黄柏等量配伍时芒果苷或小檗碱的含量与知母或黄柏单煎相比基本相同或更高,两药合煎小檗碱的含量比煎后合并更高,等量配伍共煎更有助于有效成分的提取。本方法简便、快速、准确,可用于同时测定芒果苷、新芒果苷和小檗碱的含量。2该方法准确、灵敏、特异、简便,适用于鼠血浆芒果苷测定。芒果苷单体给药后药物吸收较快,但血药浓度水平较低。知母单独(知母单煎液)给药后,芒果苷血药浓度和AUC显著升高,达峰时间Tmax延长至3h,半衰期t1-22缩短,配伍不同比例黄柏后,t1-22没有显著性变化,但血药浓度和AUC明显下降,Tmax延长。3在体肠吸收实验中,芒果苷在2～10μg·mL-1,符合一级吸收动力学,主要表现为被动扩散机制,当浓度为20μg·mL-1时,芒果苷在大鼠小肠段的吸收存在高浓度饱和现象；在结肠、十二指肠吸收好于回肠,空肠吸收最差；在小肠内的吸收随着肠循环液pH值的增加而增加；知母水煎液组中,随着灌流时间的增加,肠液中芒果苷含量逐渐增多、新芒果苷含量逐渐减少。4翻转肠囊实验中,芒果苷在空肠内的吸收最差,在回肠内吸收显著好于空肠(P<0.05),总趋势为回肠>结肠>空肠。芒果苷单体组和知母/黄柏组给药中,其成分芒果苷在肠中的表观渗透系数无明显变化,知母组中芒果苷的表观渗透系数显著增加。更多还原

【Abstract】 BackgroundHerbs couples as the basic composition units of Chinese Herbs is the foundation and cut-point for the investigation of prescription compatibility and drug absorption. Zhimu-huangbai is a classic clearing heat drug compatibility. In this study, the absorption kinetics of mangiferin was investigated and compared with those of mangiferin in Zhimu decoction and Zhimu-huangbai decoction. The elucidation of the influence of Zhimu and Zhimu-huangbai on mangiferin absorption will provide theory guidance for revealing the material foundation of Zhimu-huangbai in the process of clinical application.Objective1 To establish a UPLC method for simultaneous determination of three compounds (mangiferin, neomangiferin, berberine) in Zhimu-huangbai decoction, and study the changes in contents of three compounds when Rhizoma Anemarrhenae combined with different proportions of Cortex Phellodendri and the quality control method for decoction was developed.2 To develop a LC/MS/MS method for the determination of mangiferin in rat plasma for investigating the effects of Rhizoma Anemarrhenae combined with different proportions of Cortex Phellodendri on the pharmacokinetics of mangiferin in rats.3 In situ perfusion method and everted-gut technique in rats was utilized to study the absorption properties of the mangifeirn in rat intestine.Methods1 UPLC method for simultaneous determination of mangiferin, neomangif-erin and berberin:Three compounds were simultaneously determined by UPLC with WATERS ACQUITY UPLC(?)BEH C1N (2.1 mm×50 mm,1.7μm) column. The mobile phase consisted of 0.1% phosphoric acid (the PH was adjusted to 3.0 with triethylamine) and acetonitrile with gradient elution (acetonitrile:5% at 0 min,18% at 5.5 min,42% at 8 min) at the flow rate of 0.25 mL·min-1. The column temperature was set at 35℃. The determination wavelength was 260 nm.2 Determination of mangiferin in rat plasma by LC/MS/MS and its pharmacokinetic study:Chromatographic separation was carried out on a C1N column (150×2.0mm,5μm,CAPCELL) by isocratic elution with methanol-acetonitrile-l%acetic acid (40:3:57, V/V/V). Mangiferin were detected by the positive electrospray ionization-MS method under multiple reaction monitoring mode. SD rats were randomized into 6 groups and administered by single intragastric administration of mangiferin compound 17.5,35,70 mg·kg-1, Zhimu decoction, Zhimu-Huangbai 1:1 and 1:3 decoctions respectively. LC/MS/MS method was used for determination of mangiferin in rat plasma. The main pharmacokinetic parameters were calculated with DAS software by noncompartmental methods.3 Intestinal absorption properties of mangiferin:The intestine in rats was cannulated for in situ recirculation. UPLC and UV were respectively applied to measure the concentration of mangiferin in the flux and that of phenolsulfonphthalein in intestine perfusate. The absorption of mangiferin had been studied and compared with those of mangiferin in Zhimu and Zhimu-Huangbai in rat intestines.4 Study on the absorption of mangiferin in isolated rat small intestine utilizing everted-gut technique:Everted-gut technique was utilized to study the absorption properties of mangiferin which was compared with that in Anemarrhena Rhizoma and Zhimu-Huangbai. UPLC-UV method was applied to measure the concentration of mangiferin and neomangiferin in intestine perfusate.Results1 The contents determination of mangiferin, neomangiferin and berberin The linear ranges of mangiferin, neomangiferin and berberine were 3.44～220.00, 3.75～240.00,9.76～625.00μg·mL-1(r≥0.9999). The average recoveries (n=6) of the method were (95.00±3.28)%, (96.45±1.26)% and (97.78±4.84) %, respectively.2 Determination of mangiferin in rat plasma and its pharmacokinetics:The calibration curve of mangiferin in plasma were linear over the range of 3.01-600.2 ng·mL-1; the relative recovery was 100.5%～104.0%; The within-day and between-day precisions were less than 9.1%. After single intragastric administration of free mangiferin 17.5,35,70mg·kg-1, Cmax and AUC increased but non-proportional to the dose. After intragastric administration of mangiferin, Zhimu decoction, Zhimu-Huangbai (1:1) and (1:3) decoctions to rats (the dose of mangiferin was 35 mg·kg-1), the plasma concentration level of mangiferin in Zhimu decoction group was significantly higher than that in mangiferin compound group, and was decreased by combination of Huangbai. The main pharmacokinetic parameters of mangiferin in 4 formulations were as follows: AUG0-t were 0.626,122.4,94.36,53.97μg·mL-1·h; Cmax were 0.149,21.52,16.26, 8.27μg·mL-1; Tmax were 1.0,3.0,4.0,6.0h; t1-2Z were 3.41,1.46,1.65,1.77 h.3 Intestinal absorption properties of mangiferin:Mangiferin in different concentration of 2.0,5.0,10.0,20μg·mL-1 had different the absorption rate(Ka) and the absorption percentage (P), it was 0.0541,0.0467,0.0491,0.0220 h-1 and 14.05%,13.14%,12.43%,5.82% respectively. The absorption of mangiferin increased with the increasing of pH value; there was significant difference in the absorption of mangiferin in different intetines, Ka and P drcrease from colon, duodenum, ileum to jejunum in order. In the groups of Anemarrhena Rhizoma, the mangiferin content increased along with the duration of time, while the neomangiferin content was decreased at the same time.4 Study on absorption of mangiferin in isolated rat small intestine utilizing everted-gut technique:The apparent permeability coefficients(Papp) of mangiferin in jejunum, ileum and colon were 2.0350×10-1,5.1788×10-4 3.3825×10-4cm·min-1respectively; the Papp of mangiferin in jejunum was 18.363×10-1,4.1063×10-1cm·min-1, the Papp of mangifering in ilem was 10.190×10-4 3.9650×10-1cm·min-1, respectively in the Zhimu decoction group and Zhimu-huangbai decoction group.Conclusion1 The content of mangiferin and berberin in mixed decotion is approximate or higher than the single decoction of Rhizoma Anemarrhenae or Cortex Phellodendri. The extracted quantity of berberin in Zhimu-Huangbai (1:1) was significantly higher than that of single Cortex Phellodendri or Cortex Phellodendri. The mixed decotion is favorable for the extraction of mangiferin, neomangiferin and berberin.The method is convenient, rapid and has been successfully applied to detect mangiferin, neomangiferin and berberin.2 The LC/MS/MS method is accurate, sensitive, and specific, it is suitable for the measurement of mangiferin plasma concentration. In this study, mangiferin can be quickly absorbed in vivo but the concentration of mangiferin in plasma and AUC is extremely lower than that of Zhimu decotion groups. The Tmax was prolonged to 3h and t, t1-2Z also was shorten accordingly. Following combined with Huangbai, the absorption of mangiferin from Zhimu was decreased and prolonged with the proportion increasing of Huanbai, while t1-2Z has no obvious change.3 The absorption of mangiferin is a first-order process with the passive diffusion mechanism with concentration from 2.0 to 10μg·mL-1; when the concentration increase to 20μg·mL-1, the uptake of mangiferin does not increase; Mangiferin can be better absorbed in the colon, duodenum, ileum than jejunum; Ka and P of mangiferin increased with of the increasing of pH value; the mangiferin content in intestine perfusate increased along with the duration of time, while the neomangiferin content was decreased at the same time.4 The mangiferin can be absorbed from different intestinal segments using everted gut sac. The Papp among three intestinal segments were sequenced as follows:ilem> colon> jejunum. Compared with Zhimu group, the Papp of mangiferin significantly decreased in mangiferin group and Zhimu-huangbai group in jejunum or ileum and did not have significantly change between mangiferin group and Zhimu-huangbai group.更多还原