葛根芩连汤配伍毒效及主要成份谱药动学变化的研究
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摘要
目的
通过观察葛根芩连汤拆方后含黄连各配伍组对葡萄糖-6磷酸脱氢酶(G6PD)缺陷的痢疾杆菌感染(BD)大鼠模型(G6PD-BD)红细胞渗透脆性的影响,探讨黄连配伍对降低红细胞脆性影响的原因,通过大鼠粪便状态的改变比较各配伍组的疗效;通过观察分别灌服葛根芩连汤和黄连大鼠肝脏和肾脏病理切片,考察葛根芩连汤配伍对肝肾毒性的影响;采用高效液相色谱质谱联用技术测定葛根、黄连及葛根芩连汤中主要成分犬血中浓度,计算其药动参数,探索配伍对主要成分体内过程的影响。综合分析、探讨葛根芩连汤的配伍规律。
方法
1.建立动物模型:先用痢疾杆菌感染大鼠,第二天用乙酰苯肼造成G6PD缺陷,建立实验性G6PD-BD大鼠模型。
2.隐血实验:用含黄连的葛根芩连汤各拆方组水煎液给G6PD-BD大鼠灌胃,每天两次,连续三天,每天观察粪便状态,第四天腹主动脉取血,测定其始溶血盐浓度。
3.病理实验:分别给正常大鼠和G6PD-BD大鼠灌胃高中低剂量的黄连和葛根芩连汤水煎液,每天两次,连续三天,第四天麻醉后取其肝脏和肾脏,做病理切片,观察病理结果。
4.药动学实验:建立葛根和黄连主要成分的高效液相色谱质谱检测方法,测定不同时间点主要成分血清中含量,WinNonlin药动学软件计算药动学参数。
按处方配比取葛根芩连汤各味药(葛根24g,黄芩9g,黄连9g,甘草6g)5倍处方量,分别加水2400ml,浸泡15min,先煎葛根20min,余药共煎30min,煎二次,趁热用120目滤布滤过,合并滤液,浓缩至360mL,备用。黄连、葛根水煎液同上处理。
分别给不同比格犬葛根、黄连单煎液和葛根芩连汤,不同时间点股静脉取血,血样离心(3000r/min)20min,取上清液,乙腈沉淀杂质,涡旋混合器上震荡5min,离心(3000r/min)20min,取上清液氮气挥干溶剂,甲醇定容1ml,0.22μm滤膜过滤,得样品溶液。采用高效液相色谱质谱联用仪器(HPLC-MS~n)进行分析,色谱柱Zorbax Eclipse XDB-C18(4.6×100mm,1.8um):流动相A0.02mol/L的醋酸铵水溶液,B为乙腈,梯度洗脱,对葛根主要成分进行含量测定;流动相A 0.2%三乙胺的水溶液(调pH值为6),B为乙腈,梯度洗脱,对黄连主要成分进行含量测定。
结果
1.G6PD-BD大鼠具有体青、体凉,便黑,立毛现象,大便量多、稀、出现白膜,结果显示造模成功。
2.隐血实验表明,单味黄连能使G6PD-BD大鼠红细胞渗透脆性增加,黄连配伍葛根、黄芩、炙甘草后均能降低红细胞渗透脆性,其中葛根芩连汤全方和黄连、黄芩、葛根配伍组能使红细胞渗透脆性恢复到正常水平。通过对大鼠粪便状态的观察发现给予葛根芩连汤、黄连葛根甘草、黄连葛根的大鼠第二天粪便恢复正常状态,给予其他配伍组的大鼠第三天才恢复正常状态。
3.分别给G6PD-BD大鼠和正常大鼠高、中、低剂量的单味黄连和葛根芩连汤全方,比较其肝脏肾脏病理切片,模型组给药后肾脏充血比较:高剂量黄连＞中剂量黄连=低剂量黄连,高剂量葛根芩连汤＞中剂量葛根芩连汤=低剂量葛根芩连汤;正常组给药后肾脏充血情况比较:高剂量黄连＞中剂量黄连＞低剂量黄连。模型组和正常组给药肝脏充血情况比较:高剂量黄连=中剂量黄连＞低剂量黄连;模型组给药肝脏水肿情况比较:高剂量黄连＞中剂量黄连≥低剂量黄连,高剂量葛根芩连汤＞中剂量葛根芩连汤=低剂量葛根芩连汤;正常组给药肝脏水肿情况比较:高剂量黄连＞中剂量黄连≥低剂量黄连,中剂量葛根芩连汤＞低剂量葛根芩连汤＞高剂量葛根芩连汤。
4.主要成分采用药动学WinNonlin软件处理后结果如下:
(1)葛根芩连汤与单味葛根中主要成分药动参数变化对比:
①葛根素:AUC比葛根大,CL比葛根慢,t_(1/2)较葛根快,Tmax较葛根慢,Cmax较葛根高;②大豆苷:AUC比葛根大,CL比葛根快,t_(1/2)较葛根慢,Tmax较葛根快,Cmax较葛根高;③大豆苷元:AUC较葛根大,CL比葛根慢,t_(1/2)较葛根快,Tmax和全方一致,Cmax较葛根高。
葛根及葛根芩连汤中没有标准品对照的成分(物质A),血中产生代谢产物(物质B);④物质A:AUC比葛根大,CL比葛根快,t_(1/2)较葛根快,Tmax较葛根快,Cmax较葛根高;⑤物质B:AUC比葛根小,CL比葛根慢,t_(1/2)较葛根快,Tmax较葛根快,Cmax较葛根高。
(2)葛根芩连汤与单味黄连中主要成分药动参数对比:
①盐酸小檗碱:AUC比黄连小,CL比黄连高,t_(1/2)较黄连慢,Tmax比黄连快,Cmax较黄连低;②巴马汀:AUC比黄连小,CL比黄连高,t_(1/2)较黄连快,Tmax较黄连慢,Cmax较黄连低;③黄连碱:AUC较黄连小,CL比黄连高,t_(1/2)较黄连慢,Tmax较黄连快,Cmax比黄连低。
结论
1.黄连配伍葛根炙甘草、葛根、黄芩炙甘草、黄芩、炙甘草均能拮抗单味黄连对红细胞渗透脆性的影响;葛根芩连汤、葛根黄连黄芩三药配伍对G6PD-BD大鼠红细胞渗透脆性无影响;说明葛根芩连汤通过配伍能减低黄连对红细胞渗透脆性的影响。粪便状态的改变初步说明葛根芩连汤、黄连葛根甘草、黄连葛根配伍组的疗效优于单味黄连。
2.在机体正常状态下给单味黄连和葛根芩连汤对肾脏和肝脏的毒性作用要大于在病理状态下的,结果提示在治疗疾病的情况下灌服单味黄连或葛根芩连汤全方对肝肾是安全的;在黄连同等剂量下单味黄连和葛根芩连汤对肝肾切片结果对比提示,单味黄连的毒性大于葛根芩连汤全方;中高剂量的单味黄连和葛根芩连汤能对肝脏产生一定的毒性,对肾脏的毒性小于肝脏。
3.葛根芩连汤及单味葛根中的葛根素、大豆苷、大豆苷元和物质A药动参数结果显示:全方中各成分的AUC都比单味葛根大,Cmax都比单味葛根高,说明葛根配伍黄芩、黄连和甘草能提高葛根主要成分的入血量和血清中浓度,突出了葛根在葛根芩连汤中君药的地位。四种成分在葛根芩连汤和单味葛根的t_(1/2)和Tmax有快有慢,也有一致的,CL值有高有低,说明葛根中各成分体内吸收代谢是多样性的。
葛根芩连汤全方中的盐酸小檗碱、巴马汀和药根碱AUC比单味黄连药材小,说明黄连配伍后可以降低这三种成分的入血量;CL都比单味黄连高,说明黄连在配伍后这三种成分代谢加快。黄连单煎液中的盐酸小檗碱、巴马汀和药根碱的Cmax也较全方高,该结果提示全方配伍能抑制黄连各成分吸收入血,从而使其在肠道内发挥作用。黄连中这三种主要成分的Tmax和t_(1/2)在单味黄连和葛根芩连汤全方中各有不同,再一次提示了中药成分体内过程的复杂性和多样性。
综上所述,大鼠的隐血实验和病理切片结果显示:黄连的部分毒性在通过配伍后能降低;通过比格犬中黄连主要成分药动学研究结果进一步证实配伍后黄连毒性减低的原因。通过葛根及全方配伍葛根中主要成分药动学结果显示,配伍后可增加葛根主要成分的吸收,从而佐证了葛根为君药的配伍规律。
Objective:
To observe the different change of erythrocyte osmotic fragility(EOF) of rats with G6PD deficiency and bacterium dysenteriae infection(G6PD-BD) by method of intragastric administration of coptis root's different compatibility of Gegen Qinlian Decoction,and to investigate the effect of compatibility on reducing the EOF of coptis root,Compared the efficacy of the each combined group,through changes in the state of rat feces.By observing the biopsy results of Gegenqinlian Decoction and Coptis for rat model of liver,study the safety of Gegenqinlian decoction.Using HPLC-MS" and mass spectrometry to analyse content changes of main ingredients of puerarin,berberine and Gegenqinlian decoction in Beagle,to explore the drug in vivo pharmacokinetics of the law and elucidate the principle of the effectiveness enhanced and the toxicity reduced.
Methods:
1.Establish animal model:First infect rat by dysenteriae,and establish experimental rat model of G6PD-BD defected on the second day
2.Occult blood test:Give the animal model the split group decoction containing Gegenqinlian by intragastric administration twice a day for three days.Then to observe the state of rat feces every day.The fourth day take abdominal aortic blood and measure starting Hemolyticcon centration.
3.Pathology experiment:Give normal and G6PD-BD rats with high,medium and low doses of berberine and Gegenqinlian decoction twice a day for three days.The fourth day take their liver and kidney postanesthesia,then make biopsy to Observe the results of pathological biopsys.
4.Pharmacokinetic experiment:Using WinNonlin pharmacoinetic software to establish suitable HPLC- MS~n conditions for puerarin and berberine to study the major components of serum at different times.
Take the Chinese Medical Herbs according to prescription Gegenqinlian decoction(Puerarin 24g,Baicalin 9g,Coptis 9g,licorice 6g),soak for 15min by adding water 2400ml.At first decoct Puerarin for 20min,and then decoct the remaining drugs twice.Filter them by 120 mesh while they are hot and combined.
Administration to different Puerarin,Huanglian Gegenqinlian decoction and take the femoral vein blood at different time points.Then centrifuge the blood sample for 20min at the 3000r/min rotation speed.Shift the supematant,then precipite impurity with acetonitrile.Shocks on the vortex mixer for 5min,centrifuge the sample for 20min at the 3000r/min rotation speed. Obtained supernatant dry the solvent by nitrogen.Dissolved with methanol and metered the volume,0.22μm membrane filter,getthe sample solutions.Analyze by HPLC-MS~n and high performance liquid instruments.Use the column of Zorbax Eclipse XDB-C18(4.6×100mm, 1.8um),Take 0.02mol/L ammonium acetate as solution A and acetonitrile as solution B.Adopt the solutions as mobile phase gradient elution to detect the content of the main components of puerarin.At the same time,take 0.2%triethylamine as solution A and acetonitrile as solution B. Adopt the solutions as mobile phase gradient elution to detect the content of the main components of huanglian.
Results:
1.G6PD-BD rats with body cooler,melena,air shaft.
2.Occult blood test shows that Gegenqinlian decoction of the whole prescription and Berberine, baicalin,puerarin group Osmotic fragility of red blood cells can return to normal levels.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian
3.Give G6PD-BD and normal rats with high,medium and low doses of Single Coptis Decoction and Gegenqinlian Huanglian Decoction,compared to the results of liver biopsy of the kidney.After administration,compared renal hyperaemia of the model group:High dose group of Huanglian is higher than medium and low doses,and the latter two is equivalent.As the same for Gegenqinlian decoction.Normal group comparison of renal hyperemia after administration:The degree of congestive is reduced successively.Comparison of liver congestive of model group and normal group:High and medium dose groups are serious than the low dose group of Huamglian.However the first two are equivalent.Compared edema of the liver of Huanglian after administration for model group.The degree of edema of the liver is reduced successively.For Gegenqinlian decoction,High dose group is serious than medium and low doses,and the latter two is equivalent.Compared edema of the liver of Huanglian after administration for normal group,The degree of edema of the liver is reduced successively for high,medium and low doses.However,for For Gegenqinlian decoction,it is reduced successively for medium,low and high doses.
4.Using WinNonlin phannacokinetic software to process the results of LC-MS~n
(1) Compared the change of phmarrnacokinetic parameters of Gegenqinlian decoction and singal Puerarin.①The change of main components of Radix Puerariae:Gegenqinlian decoction contrast to single Radix Puerariae.①Puerarin:The AUC of the former is bigger than the latter, CL is higher,t_(1/2) is slower,Tmax is slower and Cmax is higher than the latter.②Daidzein:The AUC of the fonner is bigger than the latter,CL is lower,t_(1/2) is faster,Tmax is slower and peak Cmax is higher than the latter.③lsoflavoues Aglycone:The AUC of the former is bigger than the latter,CL is lower,t_(1/2) is faster,Tmax is same and Cmax is higher than the latter.
There is not standard reference substance(material A)in Ggenqinlian decoction and Puerarin,but there is blood metabolites(material B)④Material A:The AUC of the former is bigger than the latter,CL is faster,t_(1/2) is faster,Tmax is faster and Cmax is higher than the latter.⑤Material B:The AUC of the former is smaller than the latter,CL rate is slower,t_(1/2) is faster, Tmax is faster and Cmax is higher than the latter.
(2) Compared the change of phmarmacokinetic parameters of Gegenqinlian decoction and singal Coptis.①Berberine Hydrochloride:The AUC of the foriner is smaller than the latter,CL is faster,t_(1/2) is slower,Tmax is faster and Cmax is slower than the latter.②Palmatine:The AUC of the fonner is smaller than the latter,CL is higher,t_(1/2) is faster,Tmax is slower and Cmax is lower than the latter.③Coptisine:TheAUC of the former is smaller than the latter,CL rate is higher,t_(1/2) is slower,Tmax is faster and Cmax is lower than the latter.
Conclusions:
1.The effect of EOF were lower when G6PD-BD rats ate CR plus KR and PRG,CR plus KR、CR plus BSR and PRG、CR plus BSR or CR plus PRG than ate CR.The GegenQinlian Decoction or three traditional Chinese medicine including CR、KR and BSR can relive the effect of CR on EOF.Based on the above descriptions that Gegenqinlian decoction can reduce EOFtoxicity after combination.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian.
2.Under normal conditions of the body and administration the Huanglian Gegenqinlian decoction produce the side effects of kidney and liver than under the pathological state,from the impact of the study about the kidneys and liver show that in the case of treatment of disease Huanglian gavage or Gegenqinlian decoction to the liver and kidney is safe.The results of iver and kidney slices suggest that the toxicity of single is higher than the whole Gegenqinlian decoction.The high dose of single Huanglian and Gegenqinlian decoction have certain side effects for the liver and the side effects for Kidney is lower.
3.Measure the four components of Gegenqinlian decoction and single Radix Puerariae.The AUC of the AUC of the former is bigger than the latter and peak concentration is higher than the latter.It showed that Baicalin,Huanglian and licorice can increase the main ingredients of Puerarin into the blood and serum containing an effective concentration.Meanwhile,Highlighted the drug-jun status of Puerarin in Gegenqinlian decoction.The elimination half-life,the clearance rate and peak time of the four components of Gegenqinlian Decoction and single Puerarin is different.It showed that absorption and metabolism of Puerarin in the body is diverse.
The AUCs of berberine,jatrorrhizine and tetrahydropahnatine of the whole Gegenqinlian decoction are smaller than the single herbs.Indicate that Huanglian after combination can reduce the entry of these three components into blood.CL become high Descript that the metabolism of Huanglian step up after compatibility.Cmax of the whole decoction is higher than single component of berberine,jatrorrhizine and tetrahydropalmatine.It shows the whole decoction can restrain the absorption of Huanglian to blood.Coptiscan produce a marked effect in intestinal tract. Tmax and t_(1/2) of the three components are different in the single and whole decoction.It Prompts that study the role of traditional Chinese medicine having the complexity and diversity in vivo once again.
In the total,the results of the occult blood of experimental rats and pathology described that the part toxicity of berberine and berberine can be reduced after combining.Through the experiment of Beagle Pharmacokinetics to explanate the reasons for reducing the toxicity of Huanglian.The results of pharmacokinetic of the main components of puerarin and -wide side showed the puerarin can increase the absorption of the major components,whatever support the combined law of puerarin as King drug.
通过观察葛根芩连汤拆方后含黄连各配伍组对葡萄糖-6磷酸脱氢酶(G6PD)缺陷的痢疾杆菌感染(BD)大鼠模型(G6PD-BD)红细胞渗透脆性的影响,探讨黄连配伍对降低红细胞脆性影响的原因,通过大鼠粪便状态的改变比较各配伍组的疗效;通过观察分别灌服葛根芩连汤和黄连大鼠肝脏和肾脏病理切片,考察葛根芩连汤配伍对肝肾毒性的影响;采用高效液相色谱质谱联用技术测定葛根、黄连及葛根芩连汤中主要成分犬血中浓度,计算其药动参数,探索配伍对主要成分体内过程的影响。综合分析、探讨葛根芩连汤的配伍规律。
方法
1.建立动物模型:先用痢疾杆菌感染大鼠,第二天用乙酰苯肼造成G6PD缺陷,建立实验性G6PD-BD大鼠模型。
2.隐血实验:用含黄连的葛根芩连汤各拆方组水煎液给G6PD-BD大鼠灌胃,每天两次,连续三天,每天观察粪便状态,第四天腹主动脉取血,测定其始溶血盐浓度。
3.病理实验:分别给正常大鼠和G6PD-BD大鼠灌胃高中低剂量的黄连和葛根芩连汤水煎液,每天两次,连续三天,第四天麻醉后取其肝脏和肾脏,做病理切片,观察病理结果。
4.药动学实验:建立葛根和黄连主要成分的高效液相色谱质谱检测方法,测定不同时间点主要成分血清中含量,WinNonlin药动学软件计算药动学参数。
按处方配比取葛根芩连汤各味药(葛根24g,黄芩9g,黄连9g,甘草6g)5倍处方量,分别加水2400ml,浸泡15min,先煎葛根20min,余药共煎30min,煎二次,趁热用120目滤布滤过,合并滤液,浓缩至360mL,备用。黄连、葛根水煎液同上处理。
分别给不同比格犬葛根、黄连单煎液和葛根芩连汤,不同时间点股静脉取血,血样离心(3000r/min)20min,取上清液,乙腈沉淀杂质,涡旋混合器上震荡5min,离心(3000r/min)20min,取上清液氮气挥干溶剂,甲醇定容1ml,0.22μm滤膜过滤,得样品溶液。采用高效液相色谱质谱联用仪器(HPLC-MS~n)进行分析,色谱柱Zorbax Eclipse XDB-C18(4.6×100mm,1.8um):流动相A0.02mol/L的醋酸铵水溶液,B为乙腈,梯度洗脱,对葛根主要成分进行含量测定;流动相A 0.2%三乙胺的水溶液(调pH值为6),B为乙腈,梯度洗脱,对黄连主要成分进行含量测定。
结果
1.G6PD-BD大鼠具有体青、体凉,便黑,立毛现象,大便量多、稀、出现白膜,结果显示造模成功。
2.隐血实验表明,单味黄连能使G6PD-BD大鼠红细胞渗透脆性增加,黄连配伍葛根、黄芩、炙甘草后均能降低红细胞渗透脆性,其中葛根芩连汤全方和黄连、黄芩、葛根配伍组能使红细胞渗透脆性恢复到正常水平。通过对大鼠粪便状态的观察发现给予葛根芩连汤、黄连葛根甘草、黄连葛根的大鼠第二天粪便恢复正常状态,给予其他配伍组的大鼠第三天才恢复正常状态。
3.分别给G6PD-BD大鼠和正常大鼠高、中、低剂量的单味黄连和葛根芩连汤全方,比较其肝脏肾脏病理切片,模型组给药后肾脏充血比较:高剂量黄连＞中剂量黄连=低剂量黄连,高剂量葛根芩连汤＞中剂量葛根芩连汤=低剂量葛根芩连汤;正常组给药后肾脏充血情况比较:高剂量黄连＞中剂量黄连＞低剂量黄连。模型组和正常组给药肝脏充血情况比较:高剂量黄连=中剂量黄连＞低剂量黄连;模型组给药肝脏水肿情况比较:高剂量黄连＞中剂量黄连≥低剂量黄连,高剂量葛根芩连汤＞中剂量葛根芩连汤=低剂量葛根芩连汤;正常组给药肝脏水肿情况比较:高剂量黄连＞中剂量黄连≥低剂量黄连,中剂量葛根芩连汤＞低剂量葛根芩连汤＞高剂量葛根芩连汤。
4.主要成分采用药动学WinNonlin软件处理后结果如下:
(1)葛根芩连汤与单味葛根中主要成分药动参数变化对比:
①葛根素:AUC比葛根大,CL比葛根慢,t_(1/2)较葛根快,Tmax较葛根慢,Cmax较葛根高;②大豆苷:AUC比葛根大,CL比葛根快,t_(1/2)较葛根慢,Tmax较葛根快,Cmax较葛根高;③大豆苷元:AUC较葛根大,CL比葛根慢,t_(1/2)较葛根快,Tmax和全方一致,Cmax较葛根高。
葛根及葛根芩连汤中没有标准品对照的成分(物质A),血中产生代谢产物(物质B);④物质A:AUC比葛根大,CL比葛根快,t_(1/2)较葛根快,Tmax较葛根快,Cmax较葛根高;⑤物质B:AUC比葛根小,CL比葛根慢,t_(1/2)较葛根快,Tmax较葛根快,Cmax较葛根高。
(2)葛根芩连汤与单味黄连中主要成分药动参数对比:
①盐酸小檗碱:AUC比黄连小,CL比黄连高,t_(1/2)较黄连慢,Tmax比黄连快,Cmax较黄连低;②巴马汀:AUC比黄连小,CL比黄连高,t_(1/2)较黄连快,Tmax较黄连慢,Cmax较黄连低;③黄连碱:AUC较黄连小,CL比黄连高,t_(1/2)较黄连慢,Tmax较黄连快,Cmax比黄连低。
结论
1.黄连配伍葛根炙甘草、葛根、黄芩炙甘草、黄芩、炙甘草均能拮抗单味黄连对红细胞渗透脆性的影响;葛根芩连汤、葛根黄连黄芩三药配伍对G6PD-BD大鼠红细胞渗透脆性无影响;说明葛根芩连汤通过配伍能减低黄连对红细胞渗透脆性的影响。粪便状态的改变初步说明葛根芩连汤、黄连葛根甘草、黄连葛根配伍组的疗效优于单味黄连。
2.在机体正常状态下给单味黄连和葛根芩连汤对肾脏和肝脏的毒性作用要大于在病理状态下的,结果提示在治疗疾病的情况下灌服单味黄连或葛根芩连汤全方对肝肾是安全的;在黄连同等剂量下单味黄连和葛根芩连汤对肝肾切片结果对比提示,单味黄连的毒性大于葛根芩连汤全方;中高剂量的单味黄连和葛根芩连汤能对肝脏产生一定的毒性,对肾脏的毒性小于肝脏。
3.葛根芩连汤及单味葛根中的葛根素、大豆苷、大豆苷元和物质A药动参数结果显示:全方中各成分的AUC都比单味葛根大,Cmax都比单味葛根高,说明葛根配伍黄芩、黄连和甘草能提高葛根主要成分的入血量和血清中浓度,突出了葛根在葛根芩连汤中君药的地位。四种成分在葛根芩连汤和单味葛根的t_(1/2)和Tmax有快有慢,也有一致的,CL值有高有低,说明葛根中各成分体内吸收代谢是多样性的。
葛根芩连汤全方中的盐酸小檗碱、巴马汀和药根碱AUC比单味黄连药材小,说明黄连配伍后可以降低这三种成分的入血量;CL都比单味黄连高,说明黄连在配伍后这三种成分代谢加快。黄连单煎液中的盐酸小檗碱、巴马汀和药根碱的Cmax也较全方高,该结果提示全方配伍能抑制黄连各成分吸收入血,从而使其在肠道内发挥作用。黄连中这三种主要成分的Tmax和t_(1/2)在单味黄连和葛根芩连汤全方中各有不同,再一次提示了中药成分体内过程的复杂性和多样性。
综上所述,大鼠的隐血实验和病理切片结果显示:黄连的部分毒性在通过配伍后能降低;通过比格犬中黄连主要成分药动学研究结果进一步证实配伍后黄连毒性减低的原因。通过葛根及全方配伍葛根中主要成分药动学结果显示,配伍后可增加葛根主要成分的吸收,从而佐证了葛根为君药的配伍规律。
Objective:
To observe the different change of erythrocyte osmotic fragility(EOF) of rats with G6PD deficiency and bacterium dysenteriae infection(G6PD-BD) by method of intragastric administration of coptis root's different compatibility of Gegen Qinlian Decoction,and to investigate the effect of compatibility on reducing the EOF of coptis root,Compared the efficacy of the each combined group,through changes in the state of rat feces.By observing the biopsy results of Gegenqinlian Decoction and Coptis for rat model of liver,study the safety of Gegenqinlian decoction.Using HPLC-MS" and mass spectrometry to analyse content changes of main ingredients of puerarin,berberine and Gegenqinlian decoction in Beagle,to explore the drug in vivo pharmacokinetics of the law and elucidate the principle of the effectiveness enhanced and the toxicity reduced.
Methods:
1.Establish animal model:First infect rat by dysenteriae,and establish experimental rat model of G6PD-BD defected on the second day
2.Occult blood test:Give the animal model the split group decoction containing Gegenqinlian by intragastric administration twice a day for three days.Then to observe the state of rat feces every day.The fourth day take abdominal aortic blood and measure starting Hemolyticcon centration.
3.Pathology experiment:Give normal and G6PD-BD rats with high,medium and low doses of berberine and Gegenqinlian decoction twice a day for three days.The fourth day take their liver and kidney postanesthesia,then make biopsy to Observe the results of pathological biopsys.
4.Pharmacokinetic experiment:Using WinNonlin pharmacoinetic software to establish suitable HPLC- MS~n conditions for puerarin and berberine to study the major components of serum at different times.
Take the Chinese Medical Herbs according to prescription Gegenqinlian decoction(Puerarin 24g,Baicalin 9g,Coptis 9g,licorice 6g),soak for 15min by adding water 2400ml.At first decoct Puerarin for 20min,and then decoct the remaining drugs twice.Filter them by 120 mesh while they are hot and combined.
Administration to different Puerarin,Huanglian Gegenqinlian decoction and take the femoral vein blood at different time points.Then centrifuge the blood sample for 20min at the 3000r/min rotation speed.Shift the supematant,then precipite impurity with acetonitrile.Shocks on the vortex mixer for 5min,centrifuge the sample for 20min at the 3000r/min rotation speed. Obtained supernatant dry the solvent by nitrogen.Dissolved with methanol and metered the volume,0.22μm membrane filter,getthe sample solutions.Analyze by HPLC-MS~n and high performance liquid instruments.Use the column of Zorbax Eclipse XDB-C18(4.6×100mm, 1.8um),Take 0.02mol/L ammonium acetate as solution A and acetonitrile as solution B.Adopt the solutions as mobile phase gradient elution to detect the content of the main components of puerarin.At the same time,take 0.2%triethylamine as solution A and acetonitrile as solution B. Adopt the solutions as mobile phase gradient elution to detect the content of the main components of huanglian.
Results:
1.G6PD-BD rats with body cooler,melena,air shaft.
2.Occult blood test shows that Gegenqinlian decoction of the whole prescription and Berberine, baicalin,puerarin group Osmotic fragility of red blood cells can return to normal levels.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian
3.Give G6PD-BD and normal rats with high,medium and low doses of Single Coptis Decoction and Gegenqinlian Huanglian Decoction,compared to the results of liver biopsy of the kidney.After administration,compared renal hyperaemia of the model group:High dose group of Huanglian is higher than medium and low doses,and the latter two is equivalent.As the same for Gegenqinlian decoction.Normal group comparison of renal hyperemia after administration:The degree of congestive is reduced successively.Comparison of liver congestive of model group and normal group:High and medium dose groups are serious than the low dose group of Huamglian.However the first two are equivalent.Compared edema of the liver of Huanglian after administration for model group.The degree of edema of the liver is reduced successively.For Gegenqinlian decoction,High dose group is serious than medium and low doses,and the latter two is equivalent.Compared edema of the liver of Huanglian after administration for normal group,The degree of edema of the liver is reduced successively for high,medium and low doses.However,for For Gegenqinlian decoction,it is reduced successively for medium,low and high doses.
4.Using WinNonlin phannacokinetic software to process the results of LC-MS~n
(1) Compared the change of phmarrnacokinetic parameters of Gegenqinlian decoction and singal Puerarin.①The change of main components of Radix Puerariae:Gegenqinlian decoction contrast to single Radix Puerariae.①Puerarin:The AUC of the former is bigger than the latter, CL is higher,t_(1/2) is slower,Tmax is slower and Cmax is higher than the latter.②Daidzein:The AUC of the fonner is bigger than the latter,CL is lower,t_(1/2) is faster,Tmax is slower and peak Cmax is higher than the latter.③lsoflavoues Aglycone:The AUC of the former is bigger than the latter,CL is lower,t_(1/2) is faster,Tmax is same and Cmax is higher than the latter.
There is not standard reference substance(material A)in Ggenqinlian decoction and Puerarin,but there is blood metabolites(material B)④Material A:The AUC of the former is bigger than the latter,CL is faster,t_(1/2) is faster,Tmax is faster and Cmax is higher than the latter.⑤Material B:The AUC of the former is smaller than the latter,CL rate is slower,t_(1/2) is faster, Tmax is faster and Cmax is higher than the latter.
(2) Compared the change of phmarmacokinetic parameters of Gegenqinlian decoction and singal Coptis.①Berberine Hydrochloride:The AUC of the foriner is smaller than the latter,CL is faster,t_(1/2) is slower,Tmax is faster and Cmax is slower than the latter.②Palmatine:The AUC of the fonner is smaller than the latter,CL is higher,t_(1/2) is faster,Tmax is slower and Cmax is lower than the latter.③Coptisine:TheAUC of the former is smaller than the latter,CL rate is higher,t_(1/2) is slower,Tmax is faster and Cmax is lower than the latter.
Conclusions:
1.The effect of EOF were lower when G6PD-BD rats ate CR plus KR and PRG,CR plus KR、CR plus BSR and PRG、CR plus BSR or CR plus PRG than ate CR.The GegenQinlian Decoction or three traditional Chinese medicine including CR、KR and BSR can relive the effect of CR on EOF.Based on the above descriptions that Gegenqinlian decoction can reduce EOFtoxicity after combination.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian.
2.Under normal conditions of the body and administration the Huanglian Gegenqinlian decoction produce the side effects of kidney and liver than under the pathological state,from the impact of the study about the kidneys and liver show that in the case of treatment of disease Huanglian gavage or Gegenqinlian decoction to the liver and kidney is safe.The results of iver and kidney slices suggest that the toxicity of single is higher than the whole Gegenqinlian decoction.The high dose of single Huanglian and Gegenqinlian decoction have certain side effects for the liver and the side effects for Kidney is lower.
3.Measure the four components of Gegenqinlian decoction and single Radix Puerariae.The AUC of the AUC of the former is bigger than the latter and peak concentration is higher than the latter.It showed that Baicalin,Huanglian and licorice can increase the main ingredients of Puerarin into the blood and serum containing an effective concentration.Meanwhile,Highlighted the drug-jun status of Puerarin in Gegenqinlian decoction.The elimination half-life,the clearance rate and peak time of the four components of Gegenqinlian Decoction and single Puerarin is different.It showed that absorption and metabolism of Puerarin in the body is diverse.
The AUCs of berberine,jatrorrhizine and tetrahydropahnatine of the whole Gegenqinlian decoction are smaller than the single herbs.Indicate that Huanglian after combination can reduce the entry of these three components into blood.CL become high Descript that the metabolism of Huanglian step up after compatibility.Cmax of the whole decoction is higher than single component of berberine,jatrorrhizine and tetrahydropalmatine.It shows the whole decoction can restrain the absorption of Huanglian to blood.Coptiscan produce a marked effect in intestinal tract. Tmax and t_(1/2) of the three components are different in the single and whole decoction.It Prompts that study the role of traditional Chinese medicine having the complexity and diversity in vivo once again.
In the total,the results of the occult blood of experimental rats and pathology described that the part toxicity of berberine and berberine can be reduced after combining.Through the experiment of Beagle Pharmacokinetics to explanate the reasons for reducing the toxicity of Huanglian.The results of pharmacokinetic of the main components of puerarin and -wide side showed the puerarin can increase the absorption of the major components,whatever support the combined law of puerarin as King drug.
引文
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[2]鲁佳慧,江滨,曾元儿,中药配伍的研究进展.时珍国医国药,2003,14(7)419-420:
[3]杨守业.高晓山.黄连“诱发和加重”新生儿黄疸的研究概况.中国中药杂志,1996:21(3):187
[4]曾美怡,李敏民.国外有关盐酸小檗碱毒性反应的报道.中药药理与临床,1995,6(3):47-48
[5]De Smet,P.A.G.M.et al.Adverse effect of herbal drugs.1992,(1) Springer/Verlag:97-104
[6]Shanbhag,S.M.et al.Jap.J Pharmacol.1997,20:483-487
[7]Tyler,V.E.The new honest herbal,George F.Stickley,Philadephia,Adverse effects of herbal drugs.1992,(1)28-29
[8]林娜,高晓山,李建荣,等.黄连和盐酸小檗碱对实验性G6PD缺陷大鼠红细胞渗透脆性的影响.中国中药杂志,1998,23(9):562/565
[9]李建荣,高晓山,林娜,等.黄连及黄连、黄芩配伍对大鼠离体血红细胞渗透脆性的影响.中国实验方剂学杂志,1999,5(2):31-33
[10]林娜,高小山,李建荣,等.黄连配伍黄芩、甘草对G6PD缺陷大鼠红细胞渗透脆性的影响.中国中药杂志,1998,23(10):630-632
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