【作者】 于治国；

【导师】 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 博士

【摘要】 本研究以具有活血化瘀、通经活络之功效的中药复方“脑得生”为研究对象，以药代动力学（PK）和药效动力学（PD）为研究手段，以血清药化学数据和活血化瘀的药理学表征为指标，研究中药复方的配伍规律、药效物质基础及作用机理，探索中药复方药效物质基础研究的新方法。采用现代分离提取技术，提取复方中各味药材的有效部位，以合理的处方和工艺制备脑得生注射液。在对主要有效成分人参皂苷Rg₁、Rb₁、红花黄色素A和葛根素的HPLC定量分析基础上，建立了脑得生注射液的HPLC色谱指纹图谱，确定35个共有色谱峰，并对其中的13个色谱峰进行了指认，建立了质量控制标准；同时进行了注射液中的多组分同时定量分析方法的研究，建立了人参皂苷Rb₁、Rg₁、红花黄色素A、阿魏酸和葛根素的HPLC-UV测定法和人参皂苷Rb₁、Rd、Re、Rg₁、Rg₂、Rh₁、三七皂苷R₁、红花黄色素A、葛根素和大豆苷元的HPLC-MS测定法。所建立的HPLC测定法符合定量分析要求，为脑得生注射液的质量稳定、可控提供了技术保证。建立了血浆中红花黄色素A、葛根素、3′-羟基葛根素、3′-甲氧基葛根素、葛根素芹菜糖苷、大豆苷、大豆苷元-7，4′-二葡萄糖苷的HPLC-UV测定法和人参皂苷Rb₁、Rd、Rg₁、三七皂苷R₁和大豆苷元的HPLC-MS测定法，并进行了药代动力学研究。研究结果显示：随给药剂量的增加，红花黄色素A药代动力学过程由二室模型转向一室模型、葛根素由近于二室模型转向完全二室模型；二者消除速度均有减缓，CL呈下降趋势；AUC与给药剂量呈现非剂量依赖性，在高剂量下，AUC增加显著。在8个葛根主要黄酮成分[3，-羟基葛根素（P3）、3′-甲氧基葛根素（P4）、葛根素芹菜糖苷（P5）、大豆苷（P6）、大豆苷元、大豆苷元-7，4′-二葡萄糖苷（P2）、未知物Ⅰ（U1）、未知物Ⅱ（U2）]和代谢物（M1）中，M1、U1、P4、P5的药代动力学过程符合二室模型；P2显示出明显的雌雄差异，雌性符合二室模型，雄性符合一室模型；P6、U2呈一室模型；P3表现为类似口服给药的一级吸收过程。M1的血浆浓度在3～8h出现第二个峰平台，P3在1h时出现吸收峰；大豆苷元、U1、P4、P5在体内的药动学特征与葛根素相近。人参皂苷Rb₁、Rd、Rg₁及三七皂苷R₁在大鼠体内的药代动力学过程均符合二室模型。人参皂苷Rg1和三七皂苷R1在大鼠体内均快速分布，消除速度较慢；人参皂苷Rb₁和Rd则分布和消除速度均较慢，在体内可存在蓄积现象。与给予单体人参皂苷Rb₁或三七总皂苷比较，给予脑得生后人参皂苷Rg₁和三七皂苷R₁的分布速度加快、消除延缓，消除半衰期显著延长。大鼠静脉注射脑得生后，人参皂苷Rb₁、Rd、Re、Rg₁、Rh₁、三七皂苷R₁、红花黄色素A、葛根素和大豆苷元迅速向组织分布。红花黄色素A主要分布于心脏，其分布量相当于血浆中的3.3倍、肝脏中的7倍、肾脏中的2.1倍。大豆苷元脂溶性较强，在大鼠的17个脏器中均有分布，在多数脏器中的分布量随时间延长而下降。但在1.5 h时主要分布于小肠和胃中，且分布量分别是10 min时的7.5倍和5.3倍，在结肠中也有较高的分布量，说明大豆苷元在大鼠体内存在较强的肝-肠循环。大豆苷元在大脑中有一定的分布（分布量相当于血浆中浓度），在1.5h时大脑和心脏中的分布量显著高于单独给予大豆苷元。人参皂苷Rb₁和Rd均存在肝-肠循环，人参皂苷Rd同时为Rb₁的肠道代谢产物，在注射10min时仅在肺、心、子宫、胃及小肠中可检测到；在1.5和4 h时可在多数脏器中检出；人参皂苷Rg₁注射10 min时主要分布于膀胱和肾，在心脏和大脑中有少量分布，在1.5h时仅在膀胱中检测到，4h后主要分布于肝脏；人参皂苷Rh₁同时为人参皂苷Rg₁的肠道代谢产物，在大鼠体内的分布范围亦较广、但滞留时间较短。在三七总皂苷中，Rb₁和Rd仅在心脏中有分布，其他成分在心脏和大脑中均有分布。脑得生具有活血化瘀、通经活络之功效，主要用于瘀血阻络所至的心脑血管病症的治疗，本研究主要对该方的活血化淤作用进行研究。研究结果显示：家兔给予脑得生注射液后，对家兔血液流变学、凝血因子活力和凝、出血时间均有显著性影响，且作用具有时间依赖性。在给药30min时红细胞的聚集性开始呈现下降趋势；1h时血液粘度开始下降；1.5h时红细胞聚集指数显著下降，血浆复钙时间和出血时间开始延长；2.5h时凝血时间开始延长；3h后红细胞压积显著下降，血液流变学开始部分恢复；血浆复钙和凝、出血时间的恢复在6h以后。经含药血清回注验证：脑得生在家兔体内的作用时间滞后约1～1.5h，且该时间滞后主要是由于脑得生中有效成分（包括具有相互协同或拮抗作用的成分）在家兔体内动力学过程（在靶标部位达到某种平衡）所致。红花黄色素可显著延长家兔的血浆复钙时间，三七皂苷有一定作用，葛根黄酮则呈现负相关；葛根黄酮和红花黄色素对降低红细胞压积有一定作用，三七皂苷则呈现负相关；三七皂苷对降低红细胞聚集性有显著作用，红花黄色素有一定作用；葛根黄酮对红细胞变形能力的提高有显著作用，红花黄色素有一定作用。脑得生对大鼠和家兔的血液流变学特性和凝血因子活力的影响趋势一致，对高切变率下的全血粘度、凝血时间和血浆复钙时间的作用有延长趋势。对家兔血液流变学特性的改变强于单独给予三七总皂苷，复方中的川芎、红花、葛根和山楂对三七改变血液流变学特性具有协同作用；脑得生灌胃给药有效剂量相当于注射给药剂量的4倍；但体内的效应受体具有饱和性，同一给药途径的药理效应与给药剂量之间无显著的线性量效关系。血清药化学与药代动力学（PK）和药效动力学（PD）相结合是中药复方药效物质基础研究方法的尝试。本研究采用线性模型拟合，以不同时间点的血浆药物浓度与药效学表征为指标，将脑得生中各指标成分在大鼠血浆中的动力学过程与其对血液流变学特性和凝血因子活力的改变趋势进行相关性分析，探索脑得生的药效物质基础。研究结果显示：三七中的三七皂苷R₁是抗凝血的主要药效物质，人参皂苷Rg₁、Rd和Rb₁亦具有较强的抗凝血（主要表现为抑制凝血因子活力）作用，大豆苷元和3′-羟基葛根素具有一定的作用；大豆苷元、红花黄色素A及其他葛根黄酮（葛根素芹菜糖苷、葛根素、3′-甲氧基葛根素、3′-羟基葛根素）主要具有降低血液粘度的作用，人参皂苷Rb₁和人参皂苷Rd具有一定的作用；人参皂苷Rg₁和三七皂苷R₁具有降低血液“浓”度的作用，与红细胞压积的下降具有相关性；葛根黄酮的代谢物M1与全血粘度、红细胞压积、红细胞聚集性的下降和血浆复钙时间的延长均具有一定的作用，而与凝血时间的延长具有负相关；人参皂苷Rb₁和Rd与红细胞聚集指数的下降存在相关性，而人参皂苷Rg₁和三七皂苷R₁与红细胞聚集指数的下降则存在负相关，但人参皂苷Rb₁和Rd的t_1／2β约为3d，连续多次给药后临床表现为主导作用，故而三七总皂苷与红细胞聚集指数的降低存在显著相关性。上述结果说明，在中药复方中各有效成分的药理作用存在相互协同和相互制约的现象，药效的终端表现是多成分、多途经、多靶点药理作用的综合表现。更多还原

【Abstract】 Naodesheng, consisted of radix notoginseng, rhizoma chuanxiong, flos carthami, radix puerariae lobatae and fructus crataegi, is one of traditional Chinese medicine recipe for dredging the meridian passage and activating blood circulation to dissipate blood stasis In this dissertation, Naodesheng was systematically studied with Pharmacokinetics （PK） and pharmacodynamics （PD） methods to illuminate therapeutical basis, and to explore the rule of compatibility and the mechanism of effectiveness of the traditional Chinese recipe.By using modern techniques, active sites （active components groups） of crud drugs were isolated and prepared into Naodesheng injections. On the base of quantitative analytical methods for ginsenoside Rg₁, Rb₁, safflor yellow A and puerarin, the HPLC fingerprint chromatograph of Naodesheng injection was developed, in which 35 common peaks were standardized and 13 characteristic peaks were identified. The methods for simultaneous determination of multi-components in Naodesheng injection were developed, including HPLC-UV method for gensenoside Rg₁, Rb₁ safflor yellow A, ferulic acid and puerarin, and HPLC-MS method for gensenoside Rb₁, Rd, Re, Rg₁, Rg₂, Rh₁, notoginsenonide R₁, safflor yellow A, puerarin and daidzein.Simultaneous determination of multi-compounds in rat plasma were developed and were applied to pharmacokinetic research, including HPLC-UV method for safflor yellow A, puerarin, 3’-hydroxyl-puerarin （P3）, 3’-methoxyl-puerarin （P4）, puerarinapioside （P5）, daidzeine （P6）, daidzein-7,4’-diglucoside （P2） and HPLC-MS method for safflor yellow A, puerarin, daidzein, ginsenoside Rg₁, Rb₁, Rd, and notoginsenoside R₁. The results showed that by dosage increasing, the pharmacokinetic property of safflor yellow A alter from two-compartment model to one-compartment model while puerarin from approximate two-compartment model to complete two-compartment model; the rate of elimination and the CL of the both decrease when given a high dosage. In the eight of the main flavones, including 3’-hydroxyl-puerarin （P3）, 3’-methoxyl-puerarin （P4）, puerarinapioside （P5）, daidzeine （P6）, daidzein-7,4’-diglucoside （P2）, unknownⅠ（U2）, unknownⅡ（U2） and metabolite （M1）, four of them （M1, U1, P4, P5） showed a two-compartment model; P2 showed a significant difference between male and female, the female followed two-compartment model and the male follows one-compartment model; P6 and U2 consistented with one-compartment model; while P3 was consistent with oral first-order absorption process. The plasma concentration of M1 showed a second platform between the 3rd and the 8th hour and P3 showed a peak absorption at the 1st hour. Daidzein, U1, P4 and P5 show similar pharmacodynamic property with puerarin. Ginsenoside Rb₁, Rd, Rg₁ and notoginsenoside R₁ were consistent with the two-compartment model. While ginsenoside Rg₁ and notoginsenoside R₁ had quick distribution and slow elimination rate; ginsenoside Rb₁ and Rd showed slow rate in both distribution and elimination, which may cause aceumulation in vivo. Compared with single chemicals, ginsenoside Rg₁ and notoginsenoside R₁ in Naodesheng injection had quick distribution rate, slow elimination rate and longer elimination half life.After intravenous administration of Naodesheng injection, ginsenoside Rb₁, Rd, Rg₁, Rh₁, notoginsenoside R₁, safflor yellow A, puerarin and daidzein showed a quick distribution into tissue in rat. Safflor yellow A mainly exist in heart, about 3.3 times to serum, 7 times to liver and 2.1 times to kidney. Daidzein was fond in 17 tissues in rat. Although the concentration decreases, in the 1.5th hour daidzein mainly exist in small intestine and stomach with 7.5 times and 5.3 times concentrations compared with that in the 10th minutes respectively. Daidzein was also found in colon with a high concentration. All of this illuminated that daidzein goes through a strong enterohepatic circulation （EHC）. Daidzein exists in cerebrum with a similar concentration with that in plasma while the concentration in cerebrum and heart were much higher than given alonly daidzein. Both ginsenoside Rb₁ and Rd go through EHC and ginsenoside Rd, which can be found in lung, heart, uterus, stomach, small intestine at 10th minute after administration and can be also found in most tissues from the 1.5th to the 4th hour, is a metabolite of Rb₁ in intestine. Ginsenoside Rg₁ was found a lot in urinary bladder and kidney but a little in brain 10 minutes after administration, while can only be found in urinary bladder 1.5 hours later and in liver 4 hours later. Ginsenoside Rh₁, widely distributed in rat tissue but has a short residence time, is also a metabolite of Rg₁ in intestine. In the total notoginsenosides, Rb₁and Rd only exist in heart while others exist in both heart and cerebrum.Naodesheng, which has an effect in dredging the meridian passage and activating blood circulation to dissipate blood stasis, is mainly used to heal heart and cerebrum disease caused by blood stasis. This study mainly aims at its effect that dissipates blood stasis. The result of research on rabbit shows that Naodesheng injection has remarkable effects on rabbit’s hemorrheology and blood coagulation effect and the effects show a significant dependency on time. The erythroeyte aggregation decreased 30 minutes after administration; the plasma recalcification time （RT） and bleeding time （BT） extend 1.5 hours after injection. 2.5 hours after administration, the clotting time （CT） extended; the hemorheology partly recover 3 hours later while the other effects will stretch to the 6th hour. It was proved that the effect of Naodesheng on rabbit in vivo will lag for about 1 to 1.5 hours and the lag is mainly caused by dynamic process in vivo.In the formula of Naodesheng, safflor yellow （SY） significantly prolonged the recalcifieation time （RT）, notoginsenosides （NGs） had assist effect and puerariae flavones （PF） showed negative effect; PF and SY illustrated some effect on hematocrit （HCT） while NGs seems to be negative; effect of NGs on depressing erythroeyte aggregation index （EAI） was noticeable and SY had certain effect on it; PF had a significant effect on heightening erythrocyte deformability, SY had certain effect and NGs shows negative effect on it. The effects of Naodesheng on improving hemorheology index and activity of blood coagulation factor were similar on both rat and rabbit, the effects prolonged the clotting time （CT）, RT, and blood viscosity （BV） under high shear pressure seems to be more remarkable for rats than for rabbits. In the recipe, chuanxiong, carthami, pueraria and erataegi had a joint effect with nogoginseng on hemorheology compared with given single NGs to rabbits. Although oral dosage is about 4 times to injection, it showed no better effect because the aceeptor are saturable and the effect shows no dependency on dosage.The combination of serum pharmachemistry, pharmacokinetics and pharmacodynamics is a new way to illuminate the therapeutical basis of traditional Chinese medicine recipe. Correlation analysis was used to analyze the relationship between pharmacodynamics of the main ingredients and pharmacodynamic actions. It was proved that notoginsoside R₁ was the main anticoagulated blood substance, and ginsenoside Rg₁, Rd and Rb₁ had strong effects on anticoagulating blood while daidzein and 3’-hydroxyl-puerarin have some effect on it; daidzein, sail]or yellow A and other puerariae flavones mainly decrease BV while ginsenoside Rb₁ and Rd had certain effect; ginsenoside Rg₁ and notogensenoside R₁ could reduce the "concentration" of blood, and the effect may be connected with depression of the HCT. Metabolite （M1） of Puerariae flavone has a positive effect on reduce of BV, HCT and erythrocyte aggregation, and prolongation of RT, but it showed a negative dependency on the elongation of CT. Ginsenoside Rd, safflor yellow A, daidzein, P2, P3, P4, P5 and puerarin showed negative dependency on reduce of HCT. Gensenoside Rb₁、Rd、Rg₁ and P3 also showed negative dependency on the elongation of CT. All of above show that ingredients in traditional Chinese medicine recipe active cooperatively and restrict each other.Above all, under the direction of Chinese medical science and the practice, this study had conjoined traditional Chinese pharmacology, analytical chemistry, pharmacology and serum pharmaceutical chemistry to develop the quality control method, approach the rule of compatibility, find out the therapeutical basis and illuminate the maehanism of Naodesheng injection. It is a meaningful approach for the way of researching traditional Chinese medicine recipe.更多还原