防风药效物质基础和相关成分药代动力学研究
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摘要
本研究以传统中药防风(Saposhnikovia divaricata(Turcz.)Schischk.)为研究对象,对其抗炎作用、质量控制方法、药效物质基础和药效活性物质的体内药代动力学过程进行了初步的探索,内容如下:
采用急性(角叉菜胶所致大鼠足肿胀)和慢性(大鼠佐剂性关节炎)炎症模型,系统考察了防风的抗炎作用。急性炎症模型采用大鼠右后足跖皮下注射1%角叉菜胶溶液0.1 ml致炎,以肿胀抑制率为指标,结果表明:防风低、中、高剂量给药组(1g/mL,2 g/mL和4 g/mL)均能显著性抑制角叉菜胶引起的大鼠急性足肿胀。慢性炎症模型采用大鼠右后足跖皮内注射Freund's完全佐剂0.1mL致炎,以右后足跖(佐剂注射侧)肿胀度为指标,观察其对原发病变的影响。结果表明,防风低、中、高剂量给药组大鼠足跖肿胀度均显著降低(P<0.01),证明防风低、中、高剂量提取物均能够预防Freund's完全佐剂引起的原发性足肿胀。于注射佐剂后第9 d开始给药,每日2次,连续灌胃给药18 d,以左后足跖(佐剂注射侧对侧)肿胀度为指标,观察其对继发病变的影响。结果表明,防风高剂量给药组能够明显降低由于继发病变引起的大鼠左后足跖肿胀,与模型组比较差异显著(P<0.01),而防风的中、低剂量给药组对足肿胀的抑制作用不明显,证实大剂量防风对大鼠佐剂性关节炎的继发病变具有的防治作用。
采用现代分离纯化技术对防风抗炎作用有效部位的活性成分进行研究,以大鼠佐剂性关节炎继发性足肿胀为指标,考察了防风醇提物、氯仿萃取层、乙酸乙酯萃取层和正丁醇萃取层的抗炎作用。结果表明,乙酸乙酯萃取层和正丁醇萃取层具有较好的抗炎活性,利用常压硅胶柱色谱、凝胶柱色谱、制备HPLC等分离、纯化手段,从药材醇提物的乙酸乙酯萃取层和正丁醇萃取层中共分得8个化合物并鉴定了其结构,分别是亥茅酚、5-O-甲基维斯阿米醇、升麻素、亥茅酚苷、5-O-甲基维斯阿米醇苷和升麻素苷、香柑内酯和蔗糖,活性单体对照品的获得为下一步研究提供了保证。
采用HPLC-DAD法测定了19批不同来源防风的体外高效液相色谱指纹图谱,选用Jiangshen BDS C_(18)柱(250 mm×4.6 mm,5μm),以甲醇-水系统为流动相进行梯度洗脱,以5-O-甲基维斯阿米醇苷峰为内参比峰,建立了防风体外高效液相色谱指纹图谱;共标示了16个共有峰,指认了其中的6个共有峰,分别为升麻素苷、升麻素、5-O-甲基维斯阿米醇苷、香柑内酯、5-O-甲基维斯阿米醇和亥茅酚苷。以夹角余弦为测度,建立了相似度评价标准,为防风质量控制提供了依据。
首次建立了防风体内高效液相色谱指纹图谱,对正常组大鼠和佐剂性关节炎模型组大鼠灌胃给予防风后的血浆和尿液进行研究。采用与体外指纹图谱相同的色谱条件,对20只大鼠灌胃后的0,0.5,2 h的血浆样品和0-12h的尿液样品进行全组分分析,结果从正常组大鼠含药血浆中检测出了19个色谱峰,与体外指纹图谱相比较,确定了6个色谱峰来源于药材,指认其中5个色谱峰,分别是升麻素苷、升麻素、5-O-甲基维斯阿米醇苷、5-O-甲基维斯阿米醇和亥茅酚苷,均为色原酮类成分。对模型组和正常组的指纹图谱进行比较,结果表明:佐剂性关节炎模型组大鼠含药血浆中的成分比正常组大鼠少,而尿液中的代谢产物比正常组大鼠复杂,提示炎症病理状态可能会影响防风在大鼠体内的代谢过程。
应用指纹图谱技术对大鼠含药血浆进行全组分分析,将入血成分信息与防风抗炎作用的药理信息相关联,采用数理统计学方法,确定防风抗炎作用的药效物质基础。对18只佐剂性关节炎模型大鼠灌胃给予防风后的含药血浆进行分析,建立防风在炎症大鼠体内的血浆指纹图谱,共标记了18个共有峰,得到18*18的化学信息矩阵;同时采集灌胃给予低、中、高剂量防风后炎症模型大鼠的药理信息,采用SPSS 13.0软件相关分析、回归分析等方法将大鼠血浆中的化学信息与药理信息相关联,证实升麻素苷和5-O-甲基维斯阿米醇苷为防风抗炎作用的主要药效物质基础。
首次建立了同时测定生物样品中升麻素苷和5-O-甲基维斯阿米醇苷含量的HPLC-MS分析方法,对防风抗炎作用2个主要的药效活性成分在大鼠体内的药代动力学行为进行了较为深入的研究。选用电喷雾电离源(ESI源),选择正离子监测模式(SIM),以葛根素为内标,乙腈作为蛋白沉淀试剂,采用甲醇-0.1%甲酸水系统进行梯度洗脱。升麻素苷在1.0～100ng/mL,5-O-甲基维斯阿米醇苷在1.5～150 ng/mL范围内线性关系良好。方法的日内精密度RSD≤4.8%,日间精密度RSD≤9.0%,准确度RE为-1.9%～3.9%。测定了大鼠单次灌胃给予防风后升麻素苷和5-O-甲基维斯阿米醇苷的血浆药物浓度-时间曲线,并计算了相应的药物动力学参数,升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为1.31和1.96 h,C_(max)分别为39.92和41.53 ng/mL,t_(max)分别为0.54 h和0.56 h,AUC_(0-t)分别为66.77和65.65 ng/L*h。
首次比较了多剂量灌胃给予防风后正常大鼠和佐剂性关节炎模型大鼠体内升麻素苷和5-O-甲基维斯阿米醇苷的药动学差异,结果表明,多剂量给药后,正常组大鼠体内升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为1.68 h和1.97 h,Cmax分别为58.53和55.01 ng/mL,tmax分别为0.47h和0.5h,AUCss分别为111.0和108.4ng/L*h。佐剂性关节炎模型组大鼠的体内药动学行为与正常组大鼠有所不同,模型组升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为3.35 h和2.99 h,C_(max)分别为60.23和61.34 ng/mL,t_(max)分别为0.33 h和0.20 h,AUCss分别为71.79和153.8 ng/L*h。与正常组大鼠相比较,佐剂性关节炎模型组大鼠的达峰时间变快,半衰期延长,提示炎症的病理状态会影响升麻素苷和5-O-甲基维斯阿米醇苷在大鼠体内的吸收和代谢过程。
首次建立了大鼠血浆中升麻素苷和5-O-甲基维斯阿米醇苷的UPLC/MS/MS分析方法,考察了大鼠灌胃和静脉给予单体化合物后体内升麻素苷和5-O-甲基维斯阿米醇苷的药动学行为。选用电喷雾电离源(ESI源),选择正离子多反应监测模式(MRM),以葛根素为内标,以加入0.1%HCl的甲醇作为蛋白沉淀试剂,采用甲醇-0.1%甲酸水系统进行梯度洗脱。升麻素苷和5-O-甲基维斯阿米醇苷在1.0～100 ng/mL范围内线性关系良好。方法的日内精密度RSD≤9.5%,日问精密度RSD≤7.5%,准确度RE为-2.6%～5.4%。测定了大鼠灌胃和静脉给予单体化合物后体内升麻素苷和5-O-甲基维斯阿米醇苷的血浆药物浓度-时间曲线,并计算了相应的药动学参数,升麻素苷灌胃和静脉给药的t_(1/2)分别为1.65 h和1.44 h,C_(max)分别为39.14和93.28 ng/mL,t_(max)分别为0.54h和0.05 h,,AUC_(0-t)分别为68.22和92.00 ng/L*h;5-O-甲基维斯阿米醇苷灌胃和静脉给药的t_(1/2)分别为1.91 h和1.73 h,C_(max)分别为48.41和89.85 ng/mL,t_(max)分别为0.30h和0.05 h,AUC_(0-t)分别为23.28和45.77 ng/L*h。
比较了两种给药途径下升麻素苷和5-O-甲基维斯阿米醇苷的药时曲线下面积,结果表明,升麻素苷和5-O-甲基维斯阿米醇苷在大鼠体内的绝对生物利用度均不到1%,提示单体口服给药治疗时需改善2种活性成分在体内的吸收。比较了灌胃给予单体和防风药材后升麻素苷和5-O-甲基维斯阿米醇苷药动学差异,结果表明,灌胃给予单体化合物后体内升麻素苷的药动学行为与给予药材时的药动学行为相似,但灌胃给予单体化合物后体内5-O-甲基维斯阿米醇苷的药动学行为与给予药材时的药动学行为相比有所改变,达峰时间提前,吸收量减少,而半衰期没有变化,提示防风中的某些化学成分可以促进5-O-甲基维斯阿米醇苷在大鼠体内的吸收。
Saposhnikovia divaricata,usually called Fangfeng,is one of the important member of traditional Chinese medicines(TCM).In this paper,anti-inflammatory activity,quality control method, therapeutic basis and pharmacokintics on the active constituents of Fangfeng were studied.
The acute and chronic inflammation model were established to systematically investigated the anti-inflammatory activity of Saposhnikovia divaricata.The acute inflammation model was established by subcutaneous injection of 0.1%carrageenin solution into the right foot of rats.The inhibition ratio of swelling was used as index.The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling caused by carrageenin.The chronic inflammation model was established by subcutaneous injection of 0.1 mL FCA(Freund's complete adjuvant) into the right foot of rats.The swelling of the right foot,which was injected,was used as index to observe the effect of Saposhnikovia divaricata on primary affection of adjuvant arthritis(AA).The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling(P<0.01) and prevent the primary affection of AA.Nine days after injection,Saposhnikovia divaricata was successively given for 18 days by oral administrtion and two times each day.The swelling of the left foot was usea as the index to observe the Saposhnikovia divaricata on secondary affection of AA.The results of the study show that the high dose of Saposhnikovia divaricata could significantly inhibit the swelling(P<0.01),but the inhibition was not significant by given the low and middle dose.The preventive and therapeutic effect on the secondary affection of the high dose of Saposhnikovia divaricata could be confirmed.
The active ingredients with anti-inflammatory effect was isolated from Saposhnikovia divaricata by various modern isolation and purification techniques.The secondary swelling of AA was used as index to investigate the anti-inflammatory activity of alcohol extract,chloroform extract, acetoacetate extract and n-butanol extract.The results of the study show that the acetoacetate extract and n-butanol extract possess the better anti-inflammatory activity.Eight constituents were isolated from the acetoacetate extract and n-butanol extract by silica column chromatogrphy,gel column chromatography and preparation HPLC techniques.They were identified to be sec-o-glucosylhamaudol,hamaudol,prim-o-glucosylcimifugin,cimifugin, 4'-O-D-glucosyl-5-O-methylvisamminol,5-O-methylvisamminol,bergapten and sucrose.The standards could guarantee the further study.
HPLC fingerprints of nineteen batches Saposhnikovia divaricata from different origin was developed on Jiangshen BDS C_(18) column,with methanol-water system as mobile phase. 4'-O-D-glucosyl-5-O-methylvisamminol was used as the reference.Sixteen common peaks of the fingerprints were marked,and six of them were identified to be sec-o-glucosylhamaudol,hamaudol, 4'-O-D-glucosyl-5-O-methylvisamminol,5-0-methylvisamminol,prim-o-glucosylcimifugin and cimifugin.The quality evaluation standard was established by determination the cosine between the standard chromatogram and samples
HPLC fingerprints of the plasma samples and urine samples of normal rats and AA rats was firstly developed after oral administratin of Saposhnikovia divaricata.With the same chromatographic condition,twenty samples of rats containing plasma samples obtained at 0,0.5,2 h after oral administratin of Saposhnikovia divaricata and urine samples obtained 0-12 h after oral administratin of Saposhnikovia divaricata were analyzed.Nineteen chromatographic peaks were detected from the plasma samples of the normal rats.Compared with the fingerprints in vitro,six peaks were identified to be from Saposhnikovia divaricata.And five peaks were chromones,which were sec-o-glucosylhamaudol,5-O-methylvisamminol,4'-O-D-glucosyl-5-O-methylvisamminol, cimifugin and prim-o-glucosylcimifugin.The HPLC fingerprints of normal rats and AA rats were compared,the results show that the constituents from Saposhnikovia divaricata of the model plasma samples are less than the normal plasma samples,but the model urine samples were complex than the normal urine samples.The results suggest that the inflammatory condition could influence the metabolic process in rats.
The total componet analysis of the plasma samples was carried out by fingerprint method.The chemical data and the pharmacologic data was combined to confirmed the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata by methematical statistics method.The plasma fingerprints of eighteen AA rats were analyzed to obtained the chemical data.Eighteen chromatographic peaks were marked and calculated,and 18*18 matrix was used as the chemical information.The pharmacologic data were obtained by determination of the swelling after oral administration of low,middle and high dose of Saposhnikovia divaricata.The chemical data of plasma and the pharmacologic data was combined by the software SPSS 13.0.Through the analysis by correlation and regression,prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were validated to be the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata.
A accurate and sensitive HPLC-MS method was firstly developed for simultaneous determination of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in biological samples.And the pharmacokinetics of the two active constituents was systematically investigated.The ESI source and the selected ion monitoring with positive ion mode was chosed. The plasma or urine samples were prepared by protein precipitation.Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C_(18) column with a mobile phase consisted of methanol,water and 0.1%formic acid.Puerarin was added as the internal standard.The method was validated with the concentration range 1.0-100 ng/mL in rat plasma for prim-O-glucosylcimifugin,1.5-150 ng/mL in plasma for 4'-O-D-glucosyl-5-O-methylvisamminol.The lower limit of quantitation(LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL, respectively.The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0%as terms of relative standard deviation(R.S.D.).Accuracy determined at three quality control concentrations(2.0,25 and 75 ng/mL for prim-O-glucosylcimifugin;3.0,37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9%as terms of relative error(R.E.).The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Saposhnikovia divaricata to rats.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow: t_(1/2) wasl.31h and1.96 h,C_(max) was39.92 and 41.53 ng/mL,t_(max) was 0.54 and 0.56 h,AUCo-t was 66.77 and 65.65 ng/L*h,respectively.
The differences of pharmacokinetics between normal rats and AA rats were firstly studied after multidose oral administraion of Saposhnikovia divaricata.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow:in normal rats t_(1/2) was1.68 and 1.97 h,C_(max) was 58.53 and 55.01 ng/mL,t_(max) was 0.47 and 0.5h,AUC_(SS) was 111.0 and 108.4 ng/L*h,respectively;in the AA rats,t_(1/2) was 3.35 and 2.99 h,C_(max) was 60.23 and 61.34 ng/mL,t_(max) was 0.33 and 0.20 h,AUC_(SS) was 71.79 and 153.8 ng/L*h.Compared with the normal rats group,t_(max) becomes faster,t_(1/2) bocames longer in the AA rats group,which suggest that the inflammatory condition could influence the absorption and metabolic process in rats
A selective,sensitive and rapid UPLC/MS/MS method was firstly developed and validated for determination of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in rat plasma.With puerarin as internal standard,the plasma samples were precipitated by methanol adding with 0.1%HC1.The separation was performed on an ACQUITY UPLC~(TM) BEH C_(18) column (50 mm×2.1 mm,i.d.,1.7μm) with the mobile phase consisting of methanol-water-0.1%formic acid at a flow rate of 0.25 mL/min.The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring(MRM). Linear calibration curves were obtained in the concentration range of 0.5 - 100 ng/mL,with the lower limit of quantification of 0.5 ng/mL.The intra- and inter-day precision(R.S.D.) values were below 15%and accuracy(R.E.) was from -2.6%to 0.84%at all QC levels(2.0,25 and 75 ng/mL).The new method was fully validated and successfully applied to a pharmacokinetic study of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in 12 rats by oral administration and intravenous injection.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow:after oral administration and intravenous injection of prim-O-glucosylcimifugin,t_(1/2) was 1.65 h and 1.44 h, C_(max) was 39.14 and 93.28 ng/mL,t_(max) was 0.54 and 0.05 h,AUC_(0-t) was 68.22 and 92.00 ng/L*h, respectively;after oral administration and intravenous injection of 4'-O-D-glucosyl-5-O-methylvisamminol,t_(1/2) was 1.91 and 1.73 h,C_(max) was 48.41 and 89.85 ng/mL, t_(max) was 0.30h and 0.05 h,AUC_(0-t) was 23.28 and 45.77 ng/L*h.
Compared the AUC of prim-O-glucosylcimifiigin and 4'-O-D-glucosyl-5-O-methylvisamminol afer administration the monomer by two ways,the results show that the absolute bioavailability of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was no more than 1%, which suggests that the absorption should be improved when given the monomer for clinical cure. Compared the pharmacokinetic differences of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol between oral administration of the monomer and the herb, the results show that the pharmacokinetics of prim-O-glucosylcimifugin was similar between oral administration of the monomer and the herb,but the pharmacokinetics of prim-O-glucosylcimifugin was some changed,which shows that after oral administration of the monomer t_(max) became shorter, absorption dose became less,but t_(1/2) was the same.The results suggest that some ingredients of Saposhnikovia divaricata could promote the absorption of 4'-O-D-glucosyl-5-O-methylvisamminol.
采用急性(角叉菜胶所致大鼠足肿胀)和慢性(大鼠佐剂性关节炎)炎症模型,系统考察了防风的抗炎作用。急性炎症模型采用大鼠右后足跖皮下注射1%角叉菜胶溶液0.1 ml致炎,以肿胀抑制率为指标,结果表明:防风低、中、高剂量给药组(1g/mL,2 g/mL和4 g/mL)均能显著性抑制角叉菜胶引起的大鼠急性足肿胀。慢性炎症模型采用大鼠右后足跖皮内注射Freund's完全佐剂0.1mL致炎,以右后足跖(佐剂注射侧)肿胀度为指标,观察其对原发病变的影响。结果表明,防风低、中、高剂量给药组大鼠足跖肿胀度均显著降低(P<0.01),证明防风低、中、高剂量提取物均能够预防Freund's完全佐剂引起的原发性足肿胀。于注射佐剂后第9 d开始给药,每日2次,连续灌胃给药18 d,以左后足跖(佐剂注射侧对侧)肿胀度为指标,观察其对继发病变的影响。结果表明,防风高剂量给药组能够明显降低由于继发病变引起的大鼠左后足跖肿胀,与模型组比较差异显著(P<0.01),而防风的中、低剂量给药组对足肿胀的抑制作用不明显,证实大剂量防风对大鼠佐剂性关节炎的继发病变具有的防治作用。
采用现代分离纯化技术对防风抗炎作用有效部位的活性成分进行研究,以大鼠佐剂性关节炎继发性足肿胀为指标,考察了防风醇提物、氯仿萃取层、乙酸乙酯萃取层和正丁醇萃取层的抗炎作用。结果表明,乙酸乙酯萃取层和正丁醇萃取层具有较好的抗炎活性,利用常压硅胶柱色谱、凝胶柱色谱、制备HPLC等分离、纯化手段,从药材醇提物的乙酸乙酯萃取层和正丁醇萃取层中共分得8个化合物并鉴定了其结构,分别是亥茅酚、5-O-甲基维斯阿米醇、升麻素、亥茅酚苷、5-O-甲基维斯阿米醇苷和升麻素苷、香柑内酯和蔗糖,活性单体对照品的获得为下一步研究提供了保证。
采用HPLC-DAD法测定了19批不同来源防风的体外高效液相色谱指纹图谱,选用Jiangshen BDS C_(18)柱(250 mm×4.6 mm,5μm),以甲醇-水系统为流动相进行梯度洗脱,以5-O-甲基维斯阿米醇苷峰为内参比峰,建立了防风体外高效液相色谱指纹图谱;共标示了16个共有峰,指认了其中的6个共有峰,分别为升麻素苷、升麻素、5-O-甲基维斯阿米醇苷、香柑内酯、5-O-甲基维斯阿米醇和亥茅酚苷。以夹角余弦为测度,建立了相似度评价标准,为防风质量控制提供了依据。
首次建立了防风体内高效液相色谱指纹图谱,对正常组大鼠和佐剂性关节炎模型组大鼠灌胃给予防风后的血浆和尿液进行研究。采用与体外指纹图谱相同的色谱条件,对20只大鼠灌胃后的0,0.5,2 h的血浆样品和0-12h的尿液样品进行全组分分析,结果从正常组大鼠含药血浆中检测出了19个色谱峰,与体外指纹图谱相比较,确定了6个色谱峰来源于药材,指认其中5个色谱峰,分别是升麻素苷、升麻素、5-O-甲基维斯阿米醇苷、5-O-甲基维斯阿米醇和亥茅酚苷,均为色原酮类成分。对模型组和正常组的指纹图谱进行比较,结果表明:佐剂性关节炎模型组大鼠含药血浆中的成分比正常组大鼠少,而尿液中的代谢产物比正常组大鼠复杂,提示炎症病理状态可能会影响防风在大鼠体内的代谢过程。
应用指纹图谱技术对大鼠含药血浆进行全组分分析,将入血成分信息与防风抗炎作用的药理信息相关联,采用数理统计学方法,确定防风抗炎作用的药效物质基础。对18只佐剂性关节炎模型大鼠灌胃给予防风后的含药血浆进行分析,建立防风在炎症大鼠体内的血浆指纹图谱,共标记了18个共有峰,得到18*18的化学信息矩阵;同时采集灌胃给予低、中、高剂量防风后炎症模型大鼠的药理信息,采用SPSS 13.0软件相关分析、回归分析等方法将大鼠血浆中的化学信息与药理信息相关联,证实升麻素苷和5-O-甲基维斯阿米醇苷为防风抗炎作用的主要药效物质基础。
首次建立了同时测定生物样品中升麻素苷和5-O-甲基维斯阿米醇苷含量的HPLC-MS分析方法,对防风抗炎作用2个主要的药效活性成分在大鼠体内的药代动力学行为进行了较为深入的研究。选用电喷雾电离源(ESI源),选择正离子监测模式(SIM),以葛根素为内标,乙腈作为蛋白沉淀试剂,采用甲醇-0.1%甲酸水系统进行梯度洗脱。升麻素苷在1.0～100ng/mL,5-O-甲基维斯阿米醇苷在1.5～150 ng/mL范围内线性关系良好。方法的日内精密度RSD≤4.8%,日间精密度RSD≤9.0%,准确度RE为-1.9%～3.9%。测定了大鼠单次灌胃给予防风后升麻素苷和5-O-甲基维斯阿米醇苷的血浆药物浓度-时间曲线,并计算了相应的药物动力学参数,升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为1.31和1.96 h,C_(max)分别为39.92和41.53 ng/mL,t_(max)分别为0.54 h和0.56 h,AUC_(0-t)分别为66.77和65.65 ng/L*h。
首次比较了多剂量灌胃给予防风后正常大鼠和佐剂性关节炎模型大鼠体内升麻素苷和5-O-甲基维斯阿米醇苷的药动学差异,结果表明,多剂量给药后,正常组大鼠体内升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为1.68 h和1.97 h,Cmax分别为58.53和55.01 ng/mL,tmax分别为0.47h和0.5h,AUCss分别为111.0和108.4ng/L*h。佐剂性关节炎模型组大鼠的体内药动学行为与正常组大鼠有所不同,模型组升麻素苷和5-O-甲基维斯阿米醇苷的t_(1/2)分别为3.35 h和2.99 h,C_(max)分别为60.23和61.34 ng/mL,t_(max)分别为0.33 h和0.20 h,AUCss分别为71.79和153.8 ng/L*h。与正常组大鼠相比较,佐剂性关节炎模型组大鼠的达峰时间变快,半衰期延长,提示炎症的病理状态会影响升麻素苷和5-O-甲基维斯阿米醇苷在大鼠体内的吸收和代谢过程。
首次建立了大鼠血浆中升麻素苷和5-O-甲基维斯阿米醇苷的UPLC/MS/MS分析方法,考察了大鼠灌胃和静脉给予单体化合物后体内升麻素苷和5-O-甲基维斯阿米醇苷的药动学行为。选用电喷雾电离源(ESI源),选择正离子多反应监测模式(MRM),以葛根素为内标,以加入0.1%HCl的甲醇作为蛋白沉淀试剂,采用甲醇-0.1%甲酸水系统进行梯度洗脱。升麻素苷和5-O-甲基维斯阿米醇苷在1.0～100 ng/mL范围内线性关系良好。方法的日内精密度RSD≤9.5%,日问精密度RSD≤7.5%,准确度RE为-2.6%～5.4%。测定了大鼠灌胃和静脉给予单体化合物后体内升麻素苷和5-O-甲基维斯阿米醇苷的血浆药物浓度-时间曲线,并计算了相应的药动学参数,升麻素苷灌胃和静脉给药的t_(1/2)分别为1.65 h和1.44 h,C_(max)分别为39.14和93.28 ng/mL,t_(max)分别为0.54h和0.05 h,,AUC_(0-t)分别为68.22和92.00 ng/L*h;5-O-甲基维斯阿米醇苷灌胃和静脉给药的t_(1/2)分别为1.91 h和1.73 h,C_(max)分别为48.41和89.85 ng/mL,t_(max)分别为0.30h和0.05 h,AUC_(0-t)分别为23.28和45.77 ng/L*h。
比较了两种给药途径下升麻素苷和5-O-甲基维斯阿米醇苷的药时曲线下面积,结果表明,升麻素苷和5-O-甲基维斯阿米醇苷在大鼠体内的绝对生物利用度均不到1%,提示单体口服给药治疗时需改善2种活性成分在体内的吸收。比较了灌胃给予单体和防风药材后升麻素苷和5-O-甲基维斯阿米醇苷药动学差异,结果表明,灌胃给予单体化合物后体内升麻素苷的药动学行为与给予药材时的药动学行为相似,但灌胃给予单体化合物后体内5-O-甲基维斯阿米醇苷的药动学行为与给予药材时的药动学行为相比有所改变,达峰时间提前,吸收量减少,而半衰期没有变化,提示防风中的某些化学成分可以促进5-O-甲基维斯阿米醇苷在大鼠体内的吸收。
Saposhnikovia divaricata,usually called Fangfeng,is one of the important member of traditional Chinese medicines(TCM).In this paper,anti-inflammatory activity,quality control method, therapeutic basis and pharmacokintics on the active constituents of Fangfeng were studied.
The acute and chronic inflammation model were established to systematically investigated the anti-inflammatory activity of Saposhnikovia divaricata.The acute inflammation model was established by subcutaneous injection of 0.1%carrageenin solution into the right foot of rats.The inhibition ratio of swelling was used as index.The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling caused by carrageenin.The chronic inflammation model was established by subcutaneous injection of 0.1 mL FCA(Freund's complete adjuvant) into the right foot of rats.The swelling of the right foot,which was injected,was used as index to observe the effect of Saposhnikovia divaricata on primary affection of adjuvant arthritis(AA).The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling(P<0.01) and prevent the primary affection of AA.Nine days after injection,Saposhnikovia divaricata was successively given for 18 days by oral administrtion and two times each day.The swelling of the left foot was usea as the index to observe the Saposhnikovia divaricata on secondary affection of AA.The results of the study show that the high dose of Saposhnikovia divaricata could significantly inhibit the swelling(P<0.01),but the inhibition was not significant by given the low and middle dose.The preventive and therapeutic effect on the secondary affection of the high dose of Saposhnikovia divaricata could be confirmed.
The active ingredients with anti-inflammatory effect was isolated from Saposhnikovia divaricata by various modern isolation and purification techniques.The secondary swelling of AA was used as index to investigate the anti-inflammatory activity of alcohol extract,chloroform extract, acetoacetate extract and n-butanol extract.The results of the study show that the acetoacetate extract and n-butanol extract possess the better anti-inflammatory activity.Eight constituents were isolated from the acetoacetate extract and n-butanol extract by silica column chromatogrphy,gel column chromatography and preparation HPLC techniques.They were identified to be sec-o-glucosylhamaudol,hamaudol,prim-o-glucosylcimifugin,cimifugin, 4'-O-D-glucosyl-5-O-methylvisamminol,5-O-methylvisamminol,bergapten and sucrose.The standards could guarantee the further study.
HPLC fingerprints of nineteen batches Saposhnikovia divaricata from different origin was developed on Jiangshen BDS C_(18) column,with methanol-water system as mobile phase. 4'-O-D-glucosyl-5-O-methylvisamminol was used as the reference.Sixteen common peaks of the fingerprints were marked,and six of them were identified to be sec-o-glucosylhamaudol,hamaudol, 4'-O-D-glucosyl-5-O-methylvisamminol,5-0-methylvisamminol,prim-o-glucosylcimifugin and cimifugin.The quality evaluation standard was established by determination the cosine between the standard chromatogram and samples
HPLC fingerprints of the plasma samples and urine samples of normal rats and AA rats was firstly developed after oral administratin of Saposhnikovia divaricata.With the same chromatographic condition,twenty samples of rats containing plasma samples obtained at 0,0.5,2 h after oral administratin of Saposhnikovia divaricata and urine samples obtained 0-12 h after oral administratin of Saposhnikovia divaricata were analyzed.Nineteen chromatographic peaks were detected from the plasma samples of the normal rats.Compared with the fingerprints in vitro,six peaks were identified to be from Saposhnikovia divaricata.And five peaks were chromones,which were sec-o-glucosylhamaudol,5-O-methylvisamminol,4'-O-D-glucosyl-5-O-methylvisamminol, cimifugin and prim-o-glucosylcimifugin.The HPLC fingerprints of normal rats and AA rats were compared,the results show that the constituents from Saposhnikovia divaricata of the model plasma samples are less than the normal plasma samples,but the model urine samples were complex than the normal urine samples.The results suggest that the inflammatory condition could influence the metabolic process in rats.
The total componet analysis of the plasma samples was carried out by fingerprint method.The chemical data and the pharmacologic data was combined to confirmed the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata by methematical statistics method.The plasma fingerprints of eighteen AA rats were analyzed to obtained the chemical data.Eighteen chromatographic peaks were marked and calculated,and 18*18 matrix was used as the chemical information.The pharmacologic data were obtained by determination of the swelling after oral administration of low,middle and high dose of Saposhnikovia divaricata.The chemical data of plasma and the pharmacologic data was combined by the software SPSS 13.0.Through the analysis by correlation and regression,prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were validated to be the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata.
A accurate and sensitive HPLC-MS method was firstly developed for simultaneous determination of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in biological samples.And the pharmacokinetics of the two active constituents was systematically investigated.The ESI source and the selected ion monitoring with positive ion mode was chosed. The plasma or urine samples were prepared by protein precipitation.Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C_(18) column with a mobile phase consisted of methanol,water and 0.1%formic acid.Puerarin was added as the internal standard.The method was validated with the concentration range 1.0-100 ng/mL in rat plasma for prim-O-glucosylcimifugin,1.5-150 ng/mL in plasma for 4'-O-D-glucosyl-5-O-methylvisamminol.The lower limit of quantitation(LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL, respectively.The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0%as terms of relative standard deviation(R.S.D.).Accuracy determined at three quality control concentrations(2.0,25 and 75 ng/mL for prim-O-glucosylcimifugin;3.0,37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9%as terms of relative error(R.E.).The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Saposhnikovia divaricata to rats.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow: t_(1/2) wasl.31h and1.96 h,C_(max) was39.92 and 41.53 ng/mL,t_(max) was 0.54 and 0.56 h,AUCo-t was 66.77 and 65.65 ng/L*h,respectively.
The differences of pharmacokinetics between normal rats and AA rats were firstly studied after multidose oral administraion of Saposhnikovia divaricata.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow:in normal rats t_(1/2) was1.68 and 1.97 h,C_(max) was 58.53 and 55.01 ng/mL,t_(max) was 0.47 and 0.5h,AUC_(SS) was 111.0 and 108.4 ng/L*h,respectively;in the AA rats,t_(1/2) was 3.35 and 2.99 h,C_(max) was 60.23 and 61.34 ng/mL,t_(max) was 0.33 and 0.20 h,AUC_(SS) was 71.79 and 153.8 ng/L*h.Compared with the normal rats group,t_(max) becomes faster,t_(1/2) bocames longer in the AA rats group,which suggest that the inflammatory condition could influence the absorption and metabolic process in rats
A selective,sensitive and rapid UPLC/MS/MS method was firstly developed and validated for determination of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in rat plasma.With puerarin as internal standard,the plasma samples were precipitated by methanol adding with 0.1%HC1.The separation was performed on an ACQUITY UPLC~(TM) BEH C_(18) column (50 mm×2.1 mm,i.d.,1.7μm) with the mobile phase consisting of methanol-water-0.1%formic acid at a flow rate of 0.25 mL/min.The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring(MRM). Linear calibration curves were obtained in the concentration range of 0.5 - 100 ng/mL,with the lower limit of quantification of 0.5 ng/mL.The intra- and inter-day precision(R.S.D.) values were below 15%and accuracy(R.E.) was from -2.6%to 0.84%at all QC levels(2.0,25 and 75 ng/mL).The new method was fully validated and successfully applied to a pharmacokinetic study of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol in 12 rats by oral administration and intravenous injection.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol were as follow:after oral administration and intravenous injection of prim-O-glucosylcimifugin,t_(1/2) was 1.65 h and 1.44 h, C_(max) was 39.14 and 93.28 ng/mL,t_(max) was 0.54 and 0.05 h,AUC_(0-t) was 68.22 and 92.00 ng/L*h, respectively;after oral administration and intravenous injection of 4'-O-D-glucosyl-5-O-methylvisamminol,t_(1/2) was 1.91 and 1.73 h,C_(max) was 48.41 and 89.85 ng/mL, t_(max) was 0.30h and 0.05 h,AUC_(0-t) was 23.28 and 45.77 ng/L*h.
Compared the AUC of prim-O-glucosylcimifiigin and 4'-O-D-glucosyl-5-O-methylvisamminol afer administration the monomer by two ways,the results show that the absolute bioavailability of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was no more than 1%, which suggests that the absorption should be improved when given the monomer for clinical cure. Compared the pharmacokinetic differences of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol between oral administration of the monomer and the herb, the results show that the pharmacokinetics of prim-O-glucosylcimifugin was similar between oral administration of the monomer and the herb,but the pharmacokinetics of prim-O-glucosylcimifugin was some changed,which shows that after oral administration of the monomer t_(max) became shorter, absorption dose became less,but t_(1/2) was the same.The results suggest that some ingredients of Saposhnikovia divaricata could promote the absorption of 4'-O-D-glucosyl-5-O-methylvisamminol.
引文
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