药对知母/黄柏中知母有效成分芒果苷的吸收动力学研究
|
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。
Background
Herbs 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.
Objective
1 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.
Methods
1 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.
Results
1 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.
Conclusion
1 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.
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。
Background
Herbs 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.
Objective
1 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.
Methods
1 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.
Results
1 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.
Conclusion
1 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.
引文
[1]宋增锋,彭娟,马辰.LC-MS/MS测定大鼠大黄素血药浓度及脑组织含量[J].药物分析杂志,2009,29(6)：926-930.
[2]Kim NJ, Song WY, Yoo SD, et al. Pharmacokinetics of magnolin in rats [J]. Arch Pharm Res,2010,33(6):933-938.
[3]Han D, Chen C, Zhang C, et al. Determinnation of mangiferin in rat plasma by liquid-liquid extraction with UPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis,2010,51(1):260-263.
[4]谢华,马越鸣,王天明,等.桃核承气汤及单味大黄中大黄酸在家兔体内的药代动力学[J].中药药理与临床,2005,21(2)：1-3.
[5]刘昌孝.中药药代动力学研究的难点和热点[J]。药学学报,2005,40(5)：395-401。
[6]Mourad FH. Animal and human models for studying effects of drugs on intestinal fluid transport in vivo [J]. J Pharmacol Toxical Methods,2004, 50(1):3-12.
[7]曹颖,李永吉,吕邵娃.大鼠在体肠灌流模型在中药研究中的应用[J].中医药学报,2010,38(3)：134-136.
[8]Xie Y, Zeng X, Li G, et al. Assessment of intestinal absorption of total flavones of Hippophae rhamnoides L. in rat using in situ absorption models [J].Drug Dev Ind Pharm,2010,36(7):787-794.
[9]Zuo Z, Zhang L, Zhou L, et al. Intestinal absorption of hawthorn flavonoids-in vitro, in situ and in vivo correlations[J]. Life Sci,2006, 79(26):2455-2462.
[10]解江纯,刘志东,王晓玉,等.芒果苷大鼠在体肠吸收动力学研究[J].中南药学,2010,8(5)：340-343.
[11]廖正根,平其能,萧伟,等.桂枝茯苓胶囊中有效成分的大鼠在体肠吸收研究[J].中国天然药物,2005,3(5)：303-307.
[12]Kunes M, Svoboda Z, Kvetina J, et al. Intestinal single-pass in situ perfusion technique in rat:the influence of L-carnitine on absorption of 7-methoxytacrine[J]. Biomed Pap Med Fac Univ Palacky olomouc Czech Repub, 2005,149(2):433-435.
[13]Andlauer W, Kolb J, Siebert K, et al. Assessment of resveratrol bioavailability in the perfused small intestine of the rat [J]. Drugs Exp Clin Res,2000,26(2):47-55.
[14]Amidon GL, Sinko PJ, FleisherD. Estimating human oral fraction dose absorbed:a correlation using rat intestinal membrane permeability for passive and carri er-mediated compounds [J]. Pharm Res,1988,5(10):651-654.
[15]Cattoor K, Bracke M, Deforce D, et al. Transport of hop bitter acids across intestinal Caco-2 cell monolayers[J]. J Agric Chem,2010,14(58):4132-4140.
[16]Nait Chabane M, Al Ahmad A, Peluso J, et al. Quercetin and naringenin transport across human intestinal Caco-2 cells[J]. J Pharm Pharmacol,2009, 61(11):1473-1483.
[17]Lan K, He JL, Tian Y, et al. Intra-herb pharmacokinetics interaction between quercetin and isorhamentin[J]. Acta Pharmacol Slin,2008, 29(11):1376-1382.
[18]Nishimura N. Effects of Chinese herbal medicines on intestinal drug absorption [J]. Yakugaku Zasshi,2005,125(4):363-369.
[19]辛华雯,吴笑春,李罄,等.黄连素对P-糖蛋白底物在Caco-2细胞和L-MDR1细胞跨膜转运的影响[J].中国药理学通报,2007,23(6)：799-803.
[20]Lee YJ, Chung SJ, Shim CK. Limited role of P-glycoprotein in the intestinal absorption of cyclosporin A[J].Biol Pharm Bull,2005,28(4):760-763.
[21]谢海棠王广基赵小辰,等.Caco-2细胞对人参皂苷Rg3的摄取及代谢研究[J].中国临床药理学与治疗学,2004,9(3)：257-260.
[22]Hellinger E, Bakk ML, Pocza P, et al. Drug penetration model of vinblastine-treated Caco-2 cultures [J].Eur J Pharm Sci,2010,41(1):96-106.
[23]Braun A, Hammerle S, Suda K, et al. Cell cultures as tools in biopharmacy[J]. Eur J Pharm Sci,2000, Suppl 2:S51-S60.
[24]Irvine JD, Takahashi L, Lockhart K, et al. MDCK (Madin-Darby canine kidney)cells:a tool for membrane permeability screening[J]. J Pharm Sci, 1999,88(1):28-33.
[25]De Souza J, Benet LZ, Huang Y. Comparison of bidirectional lamivudine and zidovudine transport using MDCK, MDCK-MDR1, and Caco-2 cell monolayers[J]. J Pharm Sci,2009,98(11):4413-4419.
[26]Gumbleton M, Audus KL. Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier[J]. J Pharm Sci,2001,90(11):1681-1698.
[27]Constable PA, Lawrenson JG, Dolman DE, et al. P-Glycoprotein expression in human retinal pigment epithelium cell lines[J]. Exp Eye Res,2006,83(1): 24-30.
[28]Troutman MD, Thakker DR. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium [J]. Pharm Res, 2003,20(8):1210-1224.
[29]Li XX, Zhou ZW,Zhou SF.Role of P-glycoprotein in the transport of tanshinone I,one active triterpenoid from Salvia miltiorrhiza[J] Drug Metab Lett,2008,2(3):223-230.
[30]陈艳明王宁生.冰片对P-糖蛋白的影响[J].中药新药与临床药理,2003,14(2)：96-99.
[31]Kamau SW, Kramermer SD, Gunthert M, et al. Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells[J]. In Vitro Cell Dev Biol Anim,2005, 41(7):207-216.
[32]Chan K, Liu ZQ, Jiang ZH, et al. The effects of sinomenine on intestinal absorption of paeoniflorin by the everted rat gut sac model. J Ethnopharmacol, 2006,103(3):425-432.
[33]崔生淼,赵春顺,何仲贵。大鼠肠管外翻模型对葛根素吸收机制的研究[J].时珍国医国药,2008,19(7)：1715-1716。
[34]张玉杰,杨洁,邹晓翠,等.黄连肉桂药对配伍对黄连总碱大鼠小肠吸收的影响[J].中国中药杂志,2007,32(15)：1521-1524。
[35]Le Ferrec E, Chesne C, Artusson P, et al. In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods[J]. Altern Lab Anim,2001,29(6): 649-668.
[36]Tran HT, Tran PH, Lee BJ. New findings on melatonin absorption and alterations by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal segments[J]. Int J Pharm,2009,378(1-2): 9-16.
[37]章巧萍.药对探析[J].中华中医药杂志,2005,20(4)：204-206.
[38]华树生.中西医结合治疗成人原发性肾病综合征48例[J].长春中医药大学学报,2010,26(3)：380-381.
[39]徐英,李凡成.清热止嚏汤治疗变应性鼻炎热证临床观察[J].湖南中医药大学学报,2009,29(4)：59-60.
[40]易博,孙赫,原源,等RP-HPLC法测定知母黄柏药对中新芒果苷、芒果苷和盐酸小檗碱[J].中草药,2007,38(6)856-858.
[41]黄小桃,宓穗卿,王宁生.姜酮大鼠在体肠吸收动力学研究[J].中药新药与临床药理,2009,20(5)：432-435.
[42]林文慧,朱春燕,陈卫,等.葛根黄酮在大鼠肠道的吸收动力学研究[J].中国中药杂志,2008,33(2)：164-168.
[43]韩旻,傅韶,方晓玲.三七皂苷中人参皂苷Rg1与Rb1口服吸收及其体内药代动力学的研究和比较[J].药学学报,2007,42(8)：849-853.
[44]Zhang J, Chen M, Ju W, et al. Liquid chromatograph/tandem mass spectro-metry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study[J]. J Pharm Biomed Anal,2010,51(3):685-690.
[45]孙万晶,张玉杰,姚南,等.黄连吴茱萸药对配伍变化对黄连生物碱煎出及其对大鼠小肠吸收的影响[J].中国中药杂志,2008,33(22)：2614-2616.
[46]孙洋,陈婷,徐强.从药对的角度考察复方配伍规律[J].世界科学技术：中医药现代化,2004,6(1)：17-20.
[47]Pardo-Andreu GL, Delgado R, Nunez-Selles AJ, et al. Mangifera indica L.extract (Vimang) inhibits 2-deoxyribose damage induced by Fe (Ⅲ)plus ascorbate[J]. Phytother Res,2006,20(2):120-124.
[48]Rivera DG, Balmaseda IH, Leon AA, et al. Anti-allergic properties of Mangifera indica L. extract (Vimang) and contribution of its glucosylxan-thone mangiferin[J].J Pharm Pharmacol,2006,58(3):385-392.
[49]王志萍,邓家刚,谭珍媛,等.芒果苷片体外抑菌杀虫作用的实验研究[J].时珍国医国药,2009,20(9)：2167-2168.
[50]Ichiki H, Miura T, Kubo M, et al. New antidiabetic compounds, mangiferin and its glueoside[J]. Biol Pharm Bull,1998,21(12):1389-1390.
[51]Miura T, Iwamoto N, Kato M, et al. The suppressive effect of mangiferin with exercise on blood lipids in type 2 diabetes[J]. Biol Pharm Bull,2001,24(9):1091-1092.
[52]彭志刚,罗军,赖永榕,等.芒果苷对白血病K562细胞周期分布及细胞周期素A、细胞周期素B1表达的影响[J].中华中医药杂志,2007,22(8)：510-513.
[53]Rajendran P,Ekambaram G,Sakthisekaran D. Effect of mangiferin on benzo(a)pyrene induced lung carcinogenesis in experimental Swiss albino mice[J]. Nat Prod Res,2008,20(22):672-680.
[54]Lee B, Trung Trinh H, Bae EA, et al. Mangiferin inhibits passive cutaneous anaphylaxis reaction and pruritus in mice[J]. Planta Med,2009, 75(13):1415-1417.
[55]秦怀洲,王樑,赵振伟,等.芒果苷对小鼠免疫功能影响的初步研究[J].中国临床药理学与治疗学,2007,12(8)：931-934.
[56]Yoshikawa M, Ninomiya K, Shimoda H, et al. Hepotoprotective and antiox-idative properties of Salacia reticulata:preventive efects of phenolic constituents on CCl4-induced liver injury in mice[J]. Biol Pharm Bull,2002, 25(1):72-76.
[57]Gottlieb M, Leal-Campanario R, Campos-Esparza MR, et al. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia [J]. Neurobiology Dis,2006,23(2):374-386.
[58]邓家刚,郑作文,杨柯,等.芒果苷对内毒素致热家兔体温的影响[J].中国实验方剂学杂志,2006,12(2)：72-72.
[59]Perrucci S, Fichi G, Buggiani C, et al. Efficacy of mangiferin against cryptosporidium parvum in a neonatal mouse model[J]. Parasitol Res,2006, 99(2):184-188.
[60]Lai L, Lin LC, Lin JH, et al. Pharmacokinetic study of free mangiferin in rats by microdialysis coupled with microbore high-performance liquid chromatography and tandem mass spectrometry [J]. Journal of Chromatography A,2003,987:367-374.
[61]Wang H, Ye G, Tang YH, et al. High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urin[J]. Biomed Chromatogr,2006,20:1304-1308.
[62]李玉娟,毕开顺.大鼠一次性灌服酸枣仁汤剂提取物后芒果苷的药代动力学研究[J].药学学报,2005,40(2)：164-167.
[63]Gottlieb M, Leal C R, Camposesparza M R, et al. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia [J]. Neuro-biol Dis,2006,23(2):374-386.
[64]Li X, Cui X, Sun X, et,al. Mangiferin prevents diabetic nephropathy progression in streptozotocin-induced diabetic rats[J].Phytother Res,2010,24(6):893-899.
[65]Li B, Zhu W L, Che K X, et al. Advances in the study of berberine and its derivatives[J]. Acta Pharmaceutica Sinica,2008,43 (8):773-787.
[66]Suryawanshi S,Asthana RK, Gupta RC. Simultaneous estimation of mangiferin and four secoiridoid glycosides in rat plasma using liquid chromatograhy tandem mass spectrometry and its application to pharmackinetic study of herbal preparation[J].J Chromatogr B,2007,858(1-2):211-219.[67]徐勤,刘布鸣,邓立东.芒果苷大鼠在体肠道吸收机制研究[J].中国药房,2009,20(21)： 1613-1615.[68]徐叔云,卞如濂,陈修,等。药理实验方法学[M].北京：人民卫生出版社,2003：70.[69]陈新民,李俊松,李文,等.五味子有效成分的大鼠在体单向灌流肠吸收[J].药学学报,2010,45(5)：652-658.[70]Sriangam P,Vidya SJ. Modulation of the P-glycoproein-mediated intestinal secretion of glibenclamide:in vitro and in vivio assessments[J]. J Yong Pharm,2010,2(4):379-383.[71]董宇,张英丰,杨庆,等.制吴茱萸提取物在肠外翻中的吸收研究与P-gp的关系[J].世界科学技术-中医药现代化
思路与方法：63-68.[72]阮丽萍,余伯阳,朱丹妮,等.吸收促进剂对苦参碱体外吸收的影响及小鼠体内肝脏保护作用[J].中国药科大学学报,2008,39(2)：116-121.[73]肖凤霞,邓少东,林励,等.基于翻转肠囊法的巴戟甲素吸收机制的实验研究[J].中药新药与临床药理,2010,21(6)：621-624.[74]Gilles Cornaire, John Woodley, Philippe Hermann, et al. Impact of excipients on the absorption of P-glycoprotein substrates in vitro and in vivo[J]. International Journal of Pharmaceutics,2004,278:119-131.[75]Barthe L, Woodley J, Houin G. Gastrointestinal absorption of drugs:methods and studys[J].Fundam Clin Pharmacol,1999,13(12):154.[76]Kilic F S,Batu O, Sirmagul B, et al. Intestinal absorption of digoxin and interaction with nimodipine in rats[J].Pol J Pharmacol,2004,56:137.[77]何盛江,栾立标.翻转肠囊法研究吸收促进剂对小肠吸收苦参碱的作用[J].药学进展,2004,28(3)：126.[78]郭竹君,崔志清,段洪泉,等.缚管翻转肠囊法研究委陵菜黄酮在大鼠小肠内的吸收行为[J].药学学报,2009,15(3)：348-350.[79]何卉,陈西敬,王广基。药物转运体在反式白藜芦醇肠道吸收中的作用[J].中国药科大学学报,2008,39(4)：324-328.
[2]Kim NJ, Song WY, Yoo SD, et al. Pharmacokinetics of magnolin in rats [J]. Arch Pharm Res,2010,33(6):933-938.
[3]Han D, Chen C, Zhang C, et al. Determinnation of mangiferin in rat plasma by liquid-liquid extraction with UPLC-MS/MS. Journal of Pharmaceutical and Biomedical Analysis,2010,51(1):260-263.
[4]谢华,马越鸣,王天明,等.桃核承气汤及单味大黄中大黄酸在家兔体内的药代动力学[J].中药药理与临床,2005,21(2)：1-3.
[5]刘昌孝.中药药代动力学研究的难点和热点[J]。药学学报,2005,40(5)：395-401。
[6]Mourad FH. Animal and human models for studying effects of drugs on intestinal fluid transport in vivo [J]. J Pharmacol Toxical Methods,2004, 50(1):3-12.
[7]曹颖,李永吉,吕邵娃.大鼠在体肠灌流模型在中药研究中的应用[J].中医药学报,2010,38(3)：134-136.
[8]Xie Y, Zeng X, Li G, et al. Assessment of intestinal absorption of total flavones of Hippophae rhamnoides L. in rat using in situ absorption models [J].Drug Dev Ind Pharm,2010,36(7):787-794.
[9]Zuo Z, Zhang L, Zhou L, et al. Intestinal absorption of hawthorn flavonoids-in vitro, in situ and in vivo correlations[J]. Life Sci,2006, 79(26):2455-2462.
[10]解江纯,刘志东,王晓玉,等.芒果苷大鼠在体肠吸收动力学研究[J].中南药学,2010,8(5)：340-343.
[11]廖正根,平其能,萧伟,等.桂枝茯苓胶囊中有效成分的大鼠在体肠吸收研究[J].中国天然药物,2005,3(5)：303-307.
[12]Kunes M, Svoboda Z, Kvetina J, et al. Intestinal single-pass in situ perfusion technique in rat:the influence of L-carnitine on absorption of 7-methoxytacrine[J]. Biomed Pap Med Fac Univ Palacky olomouc Czech Repub, 2005,149(2):433-435.
[13]Andlauer W, Kolb J, Siebert K, et al. Assessment of resveratrol bioavailability in the perfused small intestine of the rat [J]. Drugs Exp Clin Res,2000,26(2):47-55.
[14]Amidon GL, Sinko PJ, FleisherD. Estimating human oral fraction dose absorbed:a correlation using rat intestinal membrane permeability for passive and carri er-mediated compounds [J]. Pharm Res,1988,5(10):651-654.
[15]Cattoor K, Bracke M, Deforce D, et al. Transport of hop bitter acids across intestinal Caco-2 cell monolayers[J]. J Agric Chem,2010,14(58):4132-4140.
[16]Nait Chabane M, Al Ahmad A, Peluso J, et al. Quercetin and naringenin transport across human intestinal Caco-2 cells[J]. J Pharm Pharmacol,2009, 61(11):1473-1483.
[17]Lan K, He JL, Tian Y, et al. Intra-herb pharmacokinetics interaction between quercetin and isorhamentin[J]. Acta Pharmacol Slin,2008, 29(11):1376-1382.
[18]Nishimura N. Effects of Chinese herbal medicines on intestinal drug absorption [J]. Yakugaku Zasshi,2005,125(4):363-369.
[19]辛华雯,吴笑春,李罄,等.黄连素对P-糖蛋白底物在Caco-2细胞和L-MDR1细胞跨膜转运的影响[J].中国药理学通报,2007,23(6)：799-803.
[20]Lee YJ, Chung SJ, Shim CK. Limited role of P-glycoprotein in the intestinal absorption of cyclosporin A[J].Biol Pharm Bull,2005,28(4):760-763.
[21]谢海棠王广基赵小辰,等.Caco-2细胞对人参皂苷Rg3的摄取及代谢研究[J].中国临床药理学与治疗学,2004,9(3)：257-260.
[22]Hellinger E, Bakk ML, Pocza P, et al. Drug penetration model of vinblastine-treated Caco-2 cultures [J].Eur J Pharm Sci,2010,41(1):96-106.
[23]Braun A, Hammerle S, Suda K, et al. Cell cultures as tools in biopharmacy[J]. Eur J Pharm Sci,2000, Suppl 2:S51-S60.
[24]Irvine JD, Takahashi L, Lockhart K, et al. MDCK (Madin-Darby canine kidney)cells:a tool for membrane permeability screening[J]. J Pharm Sci, 1999,88(1):28-33.
[25]De Souza J, Benet LZ, Huang Y. Comparison of bidirectional lamivudine and zidovudine transport using MDCK, MDCK-MDR1, and Caco-2 cell monolayers[J]. J Pharm Sci,2009,98(11):4413-4419.
[26]Gumbleton M, Audus KL. Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier[J]. J Pharm Sci,2001,90(11):1681-1698.
[27]Constable PA, Lawrenson JG, Dolman DE, et al. P-Glycoprotein expression in human retinal pigment epithelium cell lines[J]. Exp Eye Res,2006,83(1): 24-30.
[28]Troutman MD, Thakker DR. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium [J]. Pharm Res, 2003,20(8):1210-1224.
[29]Li XX, Zhou ZW,Zhou SF.Role of P-glycoprotein in the transport of tanshinone I,one active triterpenoid from Salvia miltiorrhiza[J] Drug Metab Lett,2008,2(3):223-230.
[30]陈艳明王宁生.冰片对P-糖蛋白的影响[J].中药新药与临床药理,2003,14(2)：96-99.
[31]Kamau SW, Kramermer SD, Gunthert M, et al. Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells[J]. In Vitro Cell Dev Biol Anim,2005, 41(7):207-216.
[32]Chan K, Liu ZQ, Jiang ZH, et al. The effects of sinomenine on intestinal absorption of paeoniflorin by the everted rat gut sac model. J Ethnopharmacol, 2006,103(3):425-432.
[33]崔生淼,赵春顺,何仲贵。大鼠肠管外翻模型对葛根素吸收机制的研究[J].时珍国医国药,2008,19(7)：1715-1716。
[34]张玉杰,杨洁,邹晓翠,等.黄连肉桂药对配伍对黄连总碱大鼠小肠吸收的影响[J].中国中药杂志,2007,32(15)：1521-1524。
[35]Le Ferrec E, Chesne C, Artusson P, et al. In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods[J]. Altern Lab Anim,2001,29(6): 649-668.
[36]Tran HT, Tran PH, Lee BJ. New findings on melatonin absorption and alterations by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal segments[J]. Int J Pharm,2009,378(1-2): 9-16.
[37]章巧萍.药对探析[J].中华中医药杂志,2005,20(4)：204-206.
[38]华树生.中西医结合治疗成人原发性肾病综合征48例[J].长春中医药大学学报,2010,26(3)：380-381.
[39]徐英,李凡成.清热止嚏汤治疗变应性鼻炎热证临床观察[J].湖南中医药大学学报,2009,29(4)：59-60.
[40]易博,孙赫,原源,等RP-HPLC法测定知母黄柏药对中新芒果苷、芒果苷和盐酸小檗碱[J].中草药,2007,38(6)856-858.
[41]黄小桃,宓穗卿,王宁生.姜酮大鼠在体肠吸收动力学研究[J].中药新药与临床药理,2009,20(5)：432-435.
[42]林文慧,朱春燕,陈卫,等.葛根黄酮在大鼠肠道的吸收动力学研究[J].中国中药杂志,2008,33(2)：164-168.
[43]韩旻,傅韶,方晓玲.三七皂苷中人参皂苷Rg1与Rb1口服吸收及其体内药代动力学的研究和比较[J].药学学报,2007,42(8)：849-853.
[44]Zhang J, Chen M, Ju W, et al. Liquid chromatograph/tandem mass spectro-metry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study[J]. J Pharm Biomed Anal,2010,51(3):685-690.
[45]孙万晶,张玉杰,姚南,等.黄连吴茱萸药对配伍变化对黄连生物碱煎出及其对大鼠小肠吸收的影响[J].中国中药杂志,2008,33(22)：2614-2616.
[46]孙洋,陈婷,徐强.从药对的角度考察复方配伍规律[J].世界科学技术：中医药现代化,2004,6(1)：17-20.
[47]Pardo-Andreu GL, Delgado R, Nunez-Selles AJ, et al. Mangifera indica L.extract (Vimang) inhibits 2-deoxyribose damage induced by Fe (Ⅲ)plus ascorbate[J]. Phytother Res,2006,20(2):120-124.
[48]Rivera DG, Balmaseda IH, Leon AA, et al. Anti-allergic properties of Mangifera indica L. extract (Vimang) and contribution of its glucosylxan-thone mangiferin[J].J Pharm Pharmacol,2006,58(3):385-392.
[49]王志萍,邓家刚,谭珍媛,等.芒果苷片体外抑菌杀虫作用的实验研究[J].时珍国医国药,2009,20(9)：2167-2168.
[50]Ichiki H, Miura T, Kubo M, et al. New antidiabetic compounds, mangiferin and its glueoside[J]. Biol Pharm Bull,1998,21(12):1389-1390.
[51]Miura T, Iwamoto N, Kato M, et al. The suppressive effect of mangiferin with exercise on blood lipids in type 2 diabetes[J]. Biol Pharm Bull,2001,24(9):1091-1092.
[52]彭志刚,罗军,赖永榕,等.芒果苷对白血病K562细胞周期分布及细胞周期素A、细胞周期素B1表达的影响[J].中华中医药杂志,2007,22(8)：510-513.
[53]Rajendran P,Ekambaram G,Sakthisekaran D. Effect of mangiferin on benzo(a)pyrene induced lung carcinogenesis in experimental Swiss albino mice[J]. Nat Prod Res,2008,20(22):672-680.
[54]Lee B, Trung Trinh H, Bae EA, et al. Mangiferin inhibits passive cutaneous anaphylaxis reaction and pruritus in mice[J]. Planta Med,2009, 75(13):1415-1417.
[55]秦怀洲,王樑,赵振伟,等.芒果苷对小鼠免疫功能影响的初步研究[J].中国临床药理学与治疗学,2007,12(8)：931-934.
[56]Yoshikawa M, Ninomiya K, Shimoda H, et al. Hepotoprotective and antiox-idative properties of Salacia reticulata:preventive efects of phenolic constituents on CCl4-induced liver injury in mice[J]. Biol Pharm Bull,2002, 25(1):72-76.
[57]Gottlieb M, Leal-Campanario R, Campos-Esparza MR, et al. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia [J]. Neurobiology Dis,2006,23(2):374-386.
[58]邓家刚,郑作文,杨柯,等.芒果苷对内毒素致热家兔体温的影响[J].中国实验方剂学杂志,2006,12(2)：72-72.
[59]Perrucci S, Fichi G, Buggiani C, et al. Efficacy of mangiferin against cryptosporidium parvum in a neonatal mouse model[J]. Parasitol Res,2006, 99(2):184-188.
[60]Lai L, Lin LC, Lin JH, et al. Pharmacokinetic study of free mangiferin in rats by microdialysis coupled with microbore high-performance liquid chromatography and tandem mass spectrometry [J]. Journal of Chromatography A,2003,987:367-374.
[61]Wang H, Ye G, Tang YH, et al. High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urin[J]. Biomed Chromatogr,2006,20:1304-1308.
[62]李玉娟,毕开顺.大鼠一次性灌服酸枣仁汤剂提取物后芒果苷的药代动力学研究[J].药学学报,2005,40(2)：164-167.
[63]Gottlieb M, Leal C R, Camposesparza M R, et al. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia [J]. Neuro-biol Dis,2006,23(2):374-386.
[64]Li X, Cui X, Sun X, et,al. Mangiferin prevents diabetic nephropathy progression in streptozotocin-induced diabetic rats[J].Phytother Res,2010,24(6):893-899.
[65]Li B, Zhu W L, Che K X, et al. Advances in the study of berberine and its derivatives[J]. Acta Pharmaceutica Sinica,2008,43 (8):773-787.
[66]Suryawanshi S,Asthana RK, Gupta RC. Simultaneous estimation of mangiferin and four secoiridoid glycosides in rat plasma using liquid chromatograhy tandem mass spectrometry and its application to pharmackinetic study of herbal preparation[J].J Chromatogr B,2007,858(1-2):211-219.[67]徐勤,刘布鸣,邓立东.芒果苷大鼠在体肠道吸收机制研究[J].中国药房,2009,20(21)： 1613-1615.[68]徐叔云,卞如濂,陈修,等。药理实验方法学[M].北京：人民卫生出版社,2003：70.[69]陈新民,李俊松,李文,等.五味子有效成分的大鼠在体单向灌流肠吸收[J].药学学报,2010,45(5)：652-658.[70]Sriangam P,Vidya SJ. Modulation of the P-glycoproein-mediated intestinal secretion of glibenclamide:in vitro and in vivio assessments[J]. J Yong Pharm,2010,2(4):379-383.[71]董宇,张英丰,杨庆,等.制吴茱萸提取物在肠外翻中的吸收研究与P-gp的关系[J].世界科学技术-中医药现代化
思路与方法：63-68.[72]阮丽萍,余伯阳,朱丹妮,等.吸收促进剂对苦参碱体外吸收的影响及小鼠体内肝脏保护作用[J].中国药科大学学报,2008,39(2)：116-121.[73]肖凤霞,邓少东,林励,等.基于翻转肠囊法的巴戟甲素吸收机制的实验研究[J].中药新药与临床药理,2010,21(6)：621-624.[74]Gilles Cornaire, John Woodley, Philippe Hermann, et al. Impact of excipients on the absorption of P-glycoprotein substrates in vitro and in vivo[J]. International Journal of Pharmaceutics,2004,278:119-131.[75]Barthe L, Woodley J, Houin G. Gastrointestinal absorption of drugs:methods and studys[J].Fundam Clin Pharmacol,1999,13(12):154.[76]Kilic F S,Batu O, Sirmagul B, et al. Intestinal absorption of digoxin and interaction with nimodipine in rats[J].Pol J Pharmacol,2004,56:137.[77]何盛江,栾立标.翻转肠囊法研究吸收促进剂对小肠吸收苦参碱的作用[J].药学进展,2004,28(3)：126.[78]郭竹君,崔志清,段洪泉,等.缚管翻转肠囊法研究委陵菜黄酮在大鼠小肠内的吸收行为[J].药学学报,2009,15(3)：348-350.[79]何卉,陈西敬,王广基。药物转运体在反式白藜芦醇肠道吸收中的作用[J].中国药科大学学报,2008,39(4)：324-328.