大蓟炭止血药效物质基础及止血增效作用机制研究
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摘要
大蓟为菊科植物蓟Cirsium japonicum DC.的干燥地上部分或根,是一味临床常用的止血药,在全国范围内均有广泛应用。大蓟用于临床常常炒炭止血,研究表明大蓟炒炭后止血作用增强,但由于炒炭后止血成分柳穿鱼叶苷含量下降,无法合理解释其炮制增效的机理,为解决这一问题,我们在本篇论文中进行了以下实验研究。
目的：
(1)优选实验原药材,比较不同产地大蓟炭止血药效与化学成分差异。
(2)对大蓟炭的化学成分进行研究,阐明大蓟炒炭后的主要成分变化,揭示其转化机理。
(3)建立实验所需动物出血模型,研究大蓟炭止血作用机制,筛选大蓟炭止血活性部位；对柳穿鱼叶苷、柳穿鱼黄素的止血药效差异,及其作用机制进行研究；对柳穿鱼叶苷、柳穿鱼黄素大鼠小肠吸收情况进行研究,比较其吸收差异。对柳穿鱼黄素大鼠小肠代谢产物进行分析。
(4)研究不同地区大蓟炭柳穿鱼黄素含量；响应面法研究大蓟炭炮制工艺；研究大孔树脂提取、纯化柳穿鱼叶苷工艺条件；研究大蓟炒炭对其微量元素含量变化的影响。方法：
(1)采用小鼠断尾法和玻片法测定了不同产地大蓟炭的止血药效、利用HPLC对不同地区生大蓟、大蓟炭成分差异进行比较。
(2)采用系统提取分离法、结合正反相柱层析、大孔树脂吸附柱层析、Sephadex LH-20等分离手段,利用质谱、核磁共振分析方法对大蓟炭成分进行分离、纯化、鉴定。利用HPLC分析大蓟炒炭后变化的主要成分。
(3)利用中医热药(附子、干姜)、干酵母、西医抗凝药物肝素钠与华法林制作小鼠出血模型,采用断尾法、毛细管法评价出血模型,比较大蓟炭对不同方法制作的出血模型小鼠出血、凝血时间的影响；系统溶剂萃取法分离大蓟不同极性部位,结合小鼠断尾法、毛细管法研究大蓟炭止血活性部位；采用小鼠断尾法、毛细管法、大鼠血凝四项、大鼠血小板CD62P阳性表达率等方法评价比较柳穿鱼叶苷、柳穿鱼黄素的止血药效差异；采用大鼠小肠外翻肠囊法、大鼠在体原位灌注法研究柳穿鱼叶苷、柳穿鱼黄素吸收机制,利用HPLC比较分析其吸收代谢产物；体外酶水解,结合LC-MS分析柳穿鱼黄素大鼠小肠吸收、代谢产物。
(4)采用HPLC比较不同产地大蓟炭柳穿鱼黄素含量；根据中心组合试验设计原理(Box-Benhnken),采用响应面分析方法研究大蓟炭炮制工艺条件；结合HPLC法,对XAD-16吸附大孔树脂分离、纯化水相部位柳穿鱼叶苷的工艺进行研究；利用微波消解-ICP-OES接合的方法,研究大蓟炒炭对其微量元素变化的影响。结果：
(1)不同产地的大蓟炭确实存在较大的止血作用差异,江西樟树、湖南地区产大蓟炭的止血作用最强,河北产大蓟炭也表现出一定的止血药效,安徽产大蓟炭则没有止血作用。HPLC分析显示,各地大蓟成分差异较大,相同成分含量亦存在较大差异。
(2)从大蓟炭中共分离得到14个单体化合物,并分别进行了结构鉴定,14个化合物分别是柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、邻苯二甲酸二丁酯、3-羟基-4-甲基苯甲酸、5-羟基-7,4’.二甲氧基黄酮醇、3.羟基苯甲酸、芹菜素-7-O-β.D.葡萄糖醛酸苷、3-羟基.4.乙基苯甲酸、粗毛豚草素葡萄糖醛酸苷、5,7-二羟基-4’-甲氧基黄酮-7-0-β-D-葡萄糖苷、5-羟基.6,4’-二甲氧基黄酮.7.O-β-D-葡萄糖苷、芹菜素。其中,四个化合物为植物首发,分别是5-羟基-7,4’-二甲氧基黄酮醇、芹菜素.7-0.β-D-葡萄糖醛酸苷、粗毛豚草素葡萄糖醛酸苷、5-羟基.6,4’-二甲氧基黄酮.7-0.β-D-葡萄糖苷。
大蓟在制炭后,其蒙花苷、柳穿鱼叶苷的峰面积会明显减少,而出现的两个新成分峰,分别为刺槐素与柳穿鱼黄素,结合四个成分的化学结构,推断两个新成分是由生大蓟在制炭过程中,蒙花苷与柳穿鱼叶苷水解脱去双糖后生成的黄酮苷元。
(3)中药热药、肝素钠、华法林均能造成动物出血时间的延长(P<0.05),干酵母有延长小鼠出血、凝血时间趋势,但不具统计学意义P>0.05。对各种动物出血模型,大蓟炭均显示出良好的止血效果,其止血机制可能与其能凉血止血、拮抗肝素钠对凝血酶的抑制以及类维生素K等作用有关。
大蓟炭氯仿、乙酸乙酯、正丁醇提取部位均显示出较强止血药效。
柳穿鱼黄素、柳穿鱼叶苷对肝素钠制作的小鼠出血模型均有明显的促凝血、止血作用,具统计学意义(P<0.05)；柳穿鱼黄素中、高剂量的止血活性要优于柳穿鱼叶苷(P<0.05)；柳穿鱼黄素止血药效与剂量存在一定的相关性,而柳穿鱼叶苷止血药效与剂量相关性不明显。
大鼠血凝四项实验显示柳穿鱼黄素、柳穿鱼叶苷能显著升高大鼠FIB值(P<0.05),高剂量的柳穿鱼黄素对大鼠APTT值具有明显的减少趋势(P>0.05),但无统计学意义,提示柳穿鱼黄素、柳穿鱼叶苷止血活性可能是通过增加机体纤维蛋白原的含量,影响纤溶与抗凝系统而发挥作用；同时,柳穿鱼黄素可能具有增加内源性途径中凝血因子Ⅷ、Ⅸ、Ⅺ或Ⅻ活性的作用。
大鼠活化血小板CD62P实验表明,柳穿鱼叶苷、柳穿鱼黄素均具能显著增加大鼠血小板CD62P阳性表达率(P<0.05)；柳穿鱼黄素具有更强的活化大鼠血小板的作用,尤其是柳穿鱼黄素高剂量,其CD62P阳性表达率高于低剂量组的柳穿鱼叶苷、低剂量组的柳穿鱼黄素,甚至高于阳性药云南白药组(P均<0.05),显示出极强的血小板活化能力。实验结果还表明,柳穿鱼叶苷、柳穿鱼黄素活化大鼠血小板CD62P阳性表达率的作用具有明显的剂量正相关性。
大鼠小肠外翻肠囊实验证明柳穿鱼叶苷、柳穿鱼黄素在十二指肠、空肠、回肠,结肠段的吸收符合零级动力学特点,空肠与回肠是两药在体内主要吸收场所,其吸收情况好于十二指肠与结肠。柳穿鱼叶苷在整个小肠段的吸收率要明显大于其苷元柳穿鱼黄素,但由于柳穿鱼黄素在大鼠小肠内发生了明显的生物转化,该实验结果并不能真实反映两成分的吸收率。
大鼠单向肠灌流法表明柳穿鱼黄素更易被大鼠小肠吸收,其吸收速率常数要明显大于柳穿鱼叶苷(P<0.01)；柳穿鱼黄素的肠流出液HPLC图和血浆HPLC图中均可以发现多个未知代谢成分,柳穿鱼叶苷肠流出液HPLC图和血浆HPLC图则显示该成分主要以原型形式吸收。
体外酶水解结果显示,柳穿鱼黄素在小肠与血浆中发生了明显的葡萄糖醛酸化反应,LC-MS显示新产生的成分可能为柳穿鱼黄素葡萄糖苷、其甲基化产物与代谢产物。
(4)不同地区柳穿鱼黄素含量差异较大,部分地区(河北、安徽)大蓟炭并不含柳穿鱼黄素,樟树与浙江产大蓟炭中柳穿鱼黄素含量最高,达到0.22%
大蓟炭炮制工艺的最佳条件为投药量165g、炮制温度310℃、炮制时间13min。
大蓟炭提取液上样质量浓度为0.077g/mL时,溶液最佳上样量为28BV,吸附流速为3BV/h,除杂溶剂为水,除杂体积为19BV,解吸液为50%乙醇,解吸液用量为12BV,解吸附流速为2BV/h。经此工艺纯化的大蓟炭样品,其所含的柳穿鱼叶苷成分质量分数可达到68%以上。
大蓟炭中微量元素含量差异较大,其中Ca、K离子含量最高达到30mg/g以上,Fe、Mg、Mn的含量次之,Zn、Na、Cu的含量最少,含量低于0.1mg/g。大蓟炒炭后,各微量元素含量比生品大蓟均有不同程度升高；结论：
大蓟炒炭止血增效的炮制机理与大蓟炒炭后,柳穿鱼叶苷水解生成止血作用更强的柳穿鱼黄素存在关联,且两活性单体成分的止血药效差异可能与其体内截然不同的吸收、代谢途径有关,仍值得进一步研究。同时,中药止血往往并非单一成分引起,炒炭过程中生成炭素、鞣质、游离Ca离子浓度均能影响其止血药效,制炭过程应同步考虑这些影响因素。
论文主要创新点：
①大蓟品种来源较为混杂,本文首先对不同地区大蓟止血药效及成分差异做了研究,优选了药材品种,一定程度上对本课题的顺利实施提供了保障。
②对大蓟炭化学成分进行了提取、分离、共分离得到14个化合物,其中四个黄酮类化合物为大蓟中首发,并在此基础上,对大蓟炒炭变化的主要化学成分的转化机理进行了阐述。
③首次对大蓟炭柳穿鱼黄素、柳穿鱼叶苷的止血药效及其止血作用机制进行了较为全面的研究,证明了两成分的止血作用,发现了两成分之间的止血药效差异与止血作用机制。为揭示大蓟炒炭止血增效的炮制机理提供了数据支持。
④学术思想上,较为创新的提出了柳穿鱼黄素、柳穿鱼叶苷的止血药效差异与其体内吸收难易是否关联这么一个科学假设。并在对柳穿鱼黄素、柳穿鱼叶苷大鼠在体、离体肠吸收实验中发现,两成分吸收差异明显,并且在大鼠体内存在明显不同的生物代谢途径。进一步结合体外酶水解与LC-MS技术,我们首次对柳穿鱼黄素大鼠体内代谢产物进行了结构分析,推测了其可能的化学结构。这些研究都为大蓟炭柳穿鱼黄素药理活性进一步研究提供了理论依据。
Cirsium japonicum DC. is widely distributed all around china, its aerial parts or roots are wildely used as a clinical traditional Chinese hemostatic medcine. Studies show after been processed into Carbonized Cirsium japonicum DC., its hemostatic activity can be enhanced and its major hemostatic content pectolinarin decreases, which is apparently illogical. In order to explain this seemingly contradictory question, we made some researches in this thesis as follows.
Objective:
(1)Using hemostatic efficacy as indicator to optimize the selection of Cirsium japonicum DC. in different Povinces and compare the chemical constituents in Cirsium japonicum DC. from different places.
(2)Study the chemical constituents in Carbonized Cirsium japonicum DC.; To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed; Reveal the mechanism of those changes during carbonizing process.
(3)To establish experimental animal models of bleeding and study the, hemostatic mechanisms of Carbonized Cirsium japonicum DC.; To study the hemostatic fractions of Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin; To study the absorption mechanism and absorption differences of pectolinarin and pectolinaringenin; To study metabolites of pectolinarin and pectolinaringenin and reveal its biotransformation mechanisms in rats
(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places; To optimize the processing method of carbonizing; To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC.
Method:
(1)The tail tip transection and glass test methods were applied to compare the hemostatic activity differences of Cirsium japonicum DC. from different places; HPLC method was applied to compare chemical constituents differences of Cirsium japonicum DC. from different places.
(2)To investigate the chemical constituents of Carbonized Cirsium japonicum DC. by repeated silicagel column chromatography, macroporous resin adsorption chromatography, Sephadex LH-20methods, etal. The structural identification was performed by nuclear resonance spectrometry and mass spectrometry.
To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed by HPLC method; Reveal the mechanism of those changes during carbonizing process based on structures of those chemical constituents been changed.
(3)To establish animal models of hemorrhage in mice by giving an oral administration of tradional chinese herbs (Monkshood, rhizoma zingiberis, etal)、 warfarin or giving an iv of yeast or heparin sodium in mice tail; The tail tip transection and capillary tube methods were applied to study the hemostatic activity of different extracted parts from Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin by tail tip transection methods capillary tube method, blood coagulation funtion tests and platelet activity tests; To study the absorption mechanism of pectolinarin and pectolin-aringenin by everted rat intestinal Sac Method, single-pass perfusion and HPLC mthods; To study metabolites of pectolinaringenin by LC-MS and enzymatic hydrolysis methods in vitro.
(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places by HPLC method; To optimize the processing method of carbonizing by Response Surface Methodology (RSM); To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC. by microwave digestion and ICP-OES methods.
Results:
(1)Carbonized Cirsium japonicum DC from Zhangshu and Hunan show stronger hemostatic effect than Carbonized Cirsium japonicum DC from Hebei. The Cirsium japonicum DC. from Anhui show no hemostatic effect and even promote the bleeding. There were differences of hemostatic effect and compounds between Carbonized Cirsium japonicum DC. from different places.
(2)Fourteen compounds were isolated from Carbonized Cirsium japonicum DC. All their structures were identified as pectolinarin, pectolinaringenin, linarin, acacetin, dibutyl phthalate,3-hydroxy-4-methyl-benzoic acid,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one,3-hydroxy-benzoic acid, apigenin-7-O-β-D-glucuronide,4-ethyl-3-hydroxy-benzoic acid、hispidulin-7-O-glucuronide,5-hydroxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one,5-hydr oxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one, apigenin by NMR and mass spectrometry method. Among those compounds,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC.
After Cirsium japonicum DC. been carboinzed., the contents of linarin and pectolinarin decreased sharply and contents of pectolina-ringenin and acacetin increased, we surmised that linarin and pectolinarin were hydrolyzed into acacetin and pectolinaringenin during the Carbonized processing based on their chemical structures.
(3)The tradional chinese herbs (Monkshood、rhizoma zingiberis, etal)、warfarin and heparin sodium can all increase bleeding and coagulating time of mice, differences were significant (p<0.05), while the yeast can also increase bleeding and coagulating time of mice, but differences weren't significant (p>0.05). Carbonized Cirsium japonicum DC. can decrease bleeding and coagulating time of mice in all bleeding animal models.
Both chloroform, ethyl acetate and n-butanol extracted parts from Carbonized Cirsium japonicum DC. show hemostatic activity.
Both pectolinarin and pectolinaringenin can decrease the bleeding and coagulating time of mice of bleeding model induced by heparin sodium, differences were significant (p<0.05). The bleeding and coagulating time of mice in high and median dose group of pectolinaringenin were significantly shorter than pectolinarin group (p<0.05). the hemostatic activity of pectolinaringenin is dose related.
Both pectolinarin and pectolinaringenin can significantly increase Fib in blood of rats (p<0.05). High dose group of pectolinaringenin can decrease APTT in blood of rats, but there were no significant differences (p>0.05). the results show the realtionshiop between hemostatic effection of pectolinaringenin, pectolinarin and theirs'FIB promotion effection, it also show that pectolinarin may can enhance the activity of coagulation factor VIII、IX、 XI or XII.
Both pectolinarin and pectolinaringenin can significantly increase positive rate of CD-62P protein expression in rat platelet (p<0.05). The positive rate of CD-62P protein expression in high dose group of pectolinaringenin is dose related higher than Yunnan Baiyao group, low dose group of pectolinarin and pectolinaringenin, the differences were significant (p>0.05)
The result reveals that the absorption of pectolinarin and pectolinaringenin both increased over time. The absorption of both ingredients among jejunum and ileum parts were higher than other Small intestinal parts. The absorption rate of pectolinarin among entire small intestinal was much higher than the absorption rate of pectolinaringenin. The absorption approach of each sample is quiet different, Obviously some bioconversion take place in the process of pectolinarigenin adsorbtion, while the pectorinarin is aborbed in prototype, so the real absorbtion rate maynot been revealed in this experiment.
The absorption rate constant, the effective permeability coefficients (Peff), the absorption (Ma) of pectolinarin were significantly decreased than pectolinaringenin (P<0.01); We found a few unknown components in intestinal effluent and blood of pectolinaringenin group, while the pectorinarin is aborbed in prototype
By LC-MS and enzymatic hydrolysis methods in vitro, we inferred that metabolites of pectolinaringenin in intestinal effluent may be pectolinarin-genin-glucoside or its methylation products
(4)Pectolinarigenin in carbonized cirsium japonicum DC. from different places varied greatly. The content of pectolinarigenin in carbonized cirsium japonicum DC. from Zhangshu. Zhejiang is0.22%, which is the highest among all different places. The pectolinarigenin content in carbonized cirsium japonicum DC. from Anhui、Hebei is undetectable.
The best processing method of carbonizing Cirsium japonicum DC. is quantity165g processing temperature310℃、processing time13min.
The best macroporous adsorption resin method to purifying pectolinarin is loading quantity of sample28BV、adsorption velocity3BV/h、cleaning solvent water、cleaning solvent volume19BV、desorption solvent50%ethanol、desorption solvent volume12BV. desorption velocity2BV/h, after purification the purity of the pectolinarin can reach68%.
The content of trace elements in Carbonized Cirsium japonicum DC. varied greatly, content of Ca、K reach30mg/g, content of Fe、Mg、Mn is lower than Ca、K. content of Zn、Na、Cu reach only0. lmg/g. after Cirsium japonicum DC. been carbonized, the content of trace elements in carbonized Cirsium japonicum all increased at different extent.
New ideas of the reseacrh:
(1)Firstly, we made research on differences of hemostatic activity and chemical constituents in Cirsium japonicum DC. from different places in order to ensure the smooth implementation of our research.
(2)14components from Cirsium japonicum DC. were identified. Among them3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC. The chemical constituents been changed、 the mechanism of those changes during carbonizing process were firstly studied.
(3)The hemostatic activity difference and hemostatic mechanism between pectolinarin and pectolinaringenin was firstly studied, these results construct steady base to reveal the carbonizing processing mechanism of Cirsium japonicum DC.
(4)We formulate a scientific hypothesis which the hemostatic activity difference between pectolinarin and pectolinaringenin may connect with their different absorption rate in rat. we found in the following study that their absorption rate varied greatly and metabolic route is quiet different. The metabolites of pectolinaringenin were firstly studied by LC-MS and enzymatic hydrolysis methods in vitro. According to the results we try to infer the chemical structure of metabolites of pectolinaringenin.
Conclusion:
The pectolinarigenin has stronger hemostatic activity than pectolinarin, it can explain why Carbonized Cirsium japonicum DC. show stronger hemostatic activity while its hemostatic content pectolinarin decreased after been carbonized.. It also reveals that the stronger hemostatic effection of pectolinarigenin may connect with its different absorbtion approach from pectolinarin. Usually, the active ingredients in chinese medcine may not be one, so, other elements such as carbonaceous、tannins、Ca2+all should be considered, this is still worth studying.
目的：
(1)优选实验原药材,比较不同产地大蓟炭止血药效与化学成分差异。
(2)对大蓟炭的化学成分进行研究,阐明大蓟炒炭后的主要成分变化,揭示其转化机理。
(3)建立实验所需动物出血模型,研究大蓟炭止血作用机制,筛选大蓟炭止血活性部位；对柳穿鱼叶苷、柳穿鱼黄素的止血药效差异,及其作用机制进行研究；对柳穿鱼叶苷、柳穿鱼黄素大鼠小肠吸收情况进行研究,比较其吸收差异。对柳穿鱼黄素大鼠小肠代谢产物进行分析。
(4)研究不同地区大蓟炭柳穿鱼黄素含量；响应面法研究大蓟炭炮制工艺；研究大孔树脂提取、纯化柳穿鱼叶苷工艺条件；研究大蓟炒炭对其微量元素含量变化的影响。方法：
(1)采用小鼠断尾法和玻片法测定了不同产地大蓟炭的止血药效、利用HPLC对不同地区生大蓟、大蓟炭成分差异进行比较。
(2)采用系统提取分离法、结合正反相柱层析、大孔树脂吸附柱层析、Sephadex LH-20等分离手段,利用质谱、核磁共振分析方法对大蓟炭成分进行分离、纯化、鉴定。利用HPLC分析大蓟炒炭后变化的主要成分。
(3)利用中医热药(附子、干姜)、干酵母、西医抗凝药物肝素钠与华法林制作小鼠出血模型,采用断尾法、毛细管法评价出血模型,比较大蓟炭对不同方法制作的出血模型小鼠出血、凝血时间的影响；系统溶剂萃取法分离大蓟不同极性部位,结合小鼠断尾法、毛细管法研究大蓟炭止血活性部位；采用小鼠断尾法、毛细管法、大鼠血凝四项、大鼠血小板CD62P阳性表达率等方法评价比较柳穿鱼叶苷、柳穿鱼黄素的止血药效差异；采用大鼠小肠外翻肠囊法、大鼠在体原位灌注法研究柳穿鱼叶苷、柳穿鱼黄素吸收机制,利用HPLC比较分析其吸收代谢产物；体外酶水解,结合LC-MS分析柳穿鱼黄素大鼠小肠吸收、代谢产物。
(4)采用HPLC比较不同产地大蓟炭柳穿鱼黄素含量；根据中心组合试验设计原理(Box-Benhnken),采用响应面分析方法研究大蓟炭炮制工艺条件；结合HPLC法,对XAD-16吸附大孔树脂分离、纯化水相部位柳穿鱼叶苷的工艺进行研究；利用微波消解-ICP-OES接合的方法,研究大蓟炒炭对其微量元素变化的影响。结果：
(1)不同产地的大蓟炭确实存在较大的止血作用差异,江西樟树、湖南地区产大蓟炭的止血作用最强,河北产大蓟炭也表现出一定的止血药效,安徽产大蓟炭则没有止血作用。HPLC分析显示,各地大蓟成分差异较大,相同成分含量亦存在较大差异。
(2)从大蓟炭中共分离得到14个单体化合物,并分别进行了结构鉴定,14个化合物分别是柳穿鱼叶苷、柳穿鱼黄素、蒙花苷、刺槐素、邻苯二甲酸二丁酯、3-羟基-4-甲基苯甲酸、5-羟基-7,4’.二甲氧基黄酮醇、3.羟基苯甲酸、芹菜素-7-O-β.D.葡萄糖醛酸苷、3-羟基.4.乙基苯甲酸、粗毛豚草素葡萄糖醛酸苷、5,7-二羟基-4’-甲氧基黄酮-7-0-β-D-葡萄糖苷、5-羟基.6,4’-二甲氧基黄酮.7.O-β-D-葡萄糖苷、芹菜素。其中,四个化合物为植物首发,分别是5-羟基-7,4’-二甲氧基黄酮醇、芹菜素.7-0.β-D-葡萄糖醛酸苷、粗毛豚草素葡萄糖醛酸苷、5-羟基.6,4’-二甲氧基黄酮.7-0.β-D-葡萄糖苷。
大蓟在制炭后,其蒙花苷、柳穿鱼叶苷的峰面积会明显减少,而出现的两个新成分峰,分别为刺槐素与柳穿鱼黄素,结合四个成分的化学结构,推断两个新成分是由生大蓟在制炭过程中,蒙花苷与柳穿鱼叶苷水解脱去双糖后生成的黄酮苷元。
(3)中药热药、肝素钠、华法林均能造成动物出血时间的延长(P<0.05),干酵母有延长小鼠出血、凝血时间趋势,但不具统计学意义P>0.05。对各种动物出血模型,大蓟炭均显示出良好的止血效果,其止血机制可能与其能凉血止血、拮抗肝素钠对凝血酶的抑制以及类维生素K等作用有关。
大蓟炭氯仿、乙酸乙酯、正丁醇提取部位均显示出较强止血药效。
柳穿鱼黄素、柳穿鱼叶苷对肝素钠制作的小鼠出血模型均有明显的促凝血、止血作用,具统计学意义(P<0.05)；柳穿鱼黄素中、高剂量的止血活性要优于柳穿鱼叶苷(P<0.05)；柳穿鱼黄素止血药效与剂量存在一定的相关性,而柳穿鱼叶苷止血药效与剂量相关性不明显。
大鼠血凝四项实验显示柳穿鱼黄素、柳穿鱼叶苷能显著升高大鼠FIB值(P<0.05),高剂量的柳穿鱼黄素对大鼠APTT值具有明显的减少趋势(P>0.05),但无统计学意义,提示柳穿鱼黄素、柳穿鱼叶苷止血活性可能是通过增加机体纤维蛋白原的含量,影响纤溶与抗凝系统而发挥作用；同时,柳穿鱼黄素可能具有增加内源性途径中凝血因子Ⅷ、Ⅸ、Ⅺ或Ⅻ活性的作用。
大鼠活化血小板CD62P实验表明,柳穿鱼叶苷、柳穿鱼黄素均具能显著增加大鼠血小板CD62P阳性表达率(P<0.05)；柳穿鱼黄素具有更强的活化大鼠血小板的作用,尤其是柳穿鱼黄素高剂量,其CD62P阳性表达率高于低剂量组的柳穿鱼叶苷、低剂量组的柳穿鱼黄素,甚至高于阳性药云南白药组(P均<0.05),显示出极强的血小板活化能力。实验结果还表明,柳穿鱼叶苷、柳穿鱼黄素活化大鼠血小板CD62P阳性表达率的作用具有明显的剂量正相关性。
大鼠小肠外翻肠囊实验证明柳穿鱼叶苷、柳穿鱼黄素在十二指肠、空肠、回肠,结肠段的吸收符合零级动力学特点,空肠与回肠是两药在体内主要吸收场所,其吸收情况好于十二指肠与结肠。柳穿鱼叶苷在整个小肠段的吸收率要明显大于其苷元柳穿鱼黄素,但由于柳穿鱼黄素在大鼠小肠内发生了明显的生物转化,该实验结果并不能真实反映两成分的吸收率。
大鼠单向肠灌流法表明柳穿鱼黄素更易被大鼠小肠吸收,其吸收速率常数要明显大于柳穿鱼叶苷(P<0.01)；柳穿鱼黄素的肠流出液HPLC图和血浆HPLC图中均可以发现多个未知代谢成分,柳穿鱼叶苷肠流出液HPLC图和血浆HPLC图则显示该成分主要以原型形式吸收。
体外酶水解结果显示,柳穿鱼黄素在小肠与血浆中发生了明显的葡萄糖醛酸化反应,LC-MS显示新产生的成分可能为柳穿鱼黄素葡萄糖苷、其甲基化产物与代谢产物。
(4)不同地区柳穿鱼黄素含量差异较大,部分地区(河北、安徽)大蓟炭并不含柳穿鱼黄素,樟树与浙江产大蓟炭中柳穿鱼黄素含量最高,达到0.22%
大蓟炭炮制工艺的最佳条件为投药量165g、炮制温度310℃、炮制时间13min。
大蓟炭提取液上样质量浓度为0.077g/mL时,溶液最佳上样量为28BV,吸附流速为3BV/h,除杂溶剂为水,除杂体积为19BV,解吸液为50%乙醇,解吸液用量为12BV,解吸附流速为2BV/h。经此工艺纯化的大蓟炭样品,其所含的柳穿鱼叶苷成分质量分数可达到68%以上。
大蓟炭中微量元素含量差异较大,其中Ca、K离子含量最高达到30mg/g以上,Fe、Mg、Mn的含量次之,Zn、Na、Cu的含量最少,含量低于0.1mg/g。大蓟炒炭后,各微量元素含量比生品大蓟均有不同程度升高；结论：
大蓟炒炭止血增效的炮制机理与大蓟炒炭后,柳穿鱼叶苷水解生成止血作用更强的柳穿鱼黄素存在关联,且两活性单体成分的止血药效差异可能与其体内截然不同的吸收、代谢途径有关,仍值得进一步研究。同时,中药止血往往并非单一成分引起,炒炭过程中生成炭素、鞣质、游离Ca离子浓度均能影响其止血药效,制炭过程应同步考虑这些影响因素。
论文主要创新点：
①大蓟品种来源较为混杂,本文首先对不同地区大蓟止血药效及成分差异做了研究,优选了药材品种,一定程度上对本课题的顺利实施提供了保障。
②对大蓟炭化学成分进行了提取、分离、共分离得到14个化合物,其中四个黄酮类化合物为大蓟中首发,并在此基础上,对大蓟炒炭变化的主要化学成分的转化机理进行了阐述。
③首次对大蓟炭柳穿鱼黄素、柳穿鱼叶苷的止血药效及其止血作用机制进行了较为全面的研究,证明了两成分的止血作用,发现了两成分之间的止血药效差异与止血作用机制。为揭示大蓟炒炭止血增效的炮制机理提供了数据支持。
④学术思想上,较为创新的提出了柳穿鱼黄素、柳穿鱼叶苷的止血药效差异与其体内吸收难易是否关联这么一个科学假设。并在对柳穿鱼黄素、柳穿鱼叶苷大鼠在体、离体肠吸收实验中发现,两成分吸收差异明显,并且在大鼠体内存在明显不同的生物代谢途径。进一步结合体外酶水解与LC-MS技术,我们首次对柳穿鱼黄素大鼠体内代谢产物进行了结构分析,推测了其可能的化学结构。这些研究都为大蓟炭柳穿鱼黄素药理活性进一步研究提供了理论依据。
Cirsium japonicum DC. is widely distributed all around china, its aerial parts or roots are wildely used as a clinical traditional Chinese hemostatic medcine. Studies show after been processed into Carbonized Cirsium japonicum DC., its hemostatic activity can be enhanced and its major hemostatic content pectolinarin decreases, which is apparently illogical. In order to explain this seemingly contradictory question, we made some researches in this thesis as follows.
Objective:
(1)Using hemostatic efficacy as indicator to optimize the selection of Cirsium japonicum DC. in different Povinces and compare the chemical constituents in Cirsium japonicum DC. from different places.
(2)Study the chemical constituents in Carbonized Cirsium japonicum DC.; To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed; Reveal the mechanism of those changes during carbonizing process.
(3)To establish experimental animal models of bleeding and study the, hemostatic mechanisms of Carbonized Cirsium japonicum DC.; To study the hemostatic fractions of Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin; To study the absorption mechanism and absorption differences of pectolinarin and pectolinaringenin; To study metabolites of pectolinarin and pectolinaringenin and reveal its biotransformation mechanisms in rats
(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places; To optimize the processing method of carbonizing; To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC.
Method:
(1)The tail tip transection and glass test methods were applied to compare the hemostatic activity differences of Cirsium japonicum DC. from different places; HPLC method was applied to compare chemical constituents differences of Cirsium japonicum DC. from different places.
(2)To investigate the chemical constituents of Carbonized Cirsium japonicum DC. by repeated silicagel column chromatography, macroporous resin adsorption chromatography, Sephadex LH-20methods, etal. The structural identification was performed by nuclear resonance spectrometry and mass spectrometry.
To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed by HPLC method; Reveal the mechanism of those changes during carbonizing process based on structures of those chemical constituents been changed.
(3)To establish animal models of hemorrhage in mice by giving an oral administration of tradional chinese herbs (Monkshood, rhizoma zingiberis, etal)、 warfarin or giving an iv of yeast or heparin sodium in mice tail; The tail tip transection and capillary tube methods were applied to study the hemostatic activity of different extracted parts from Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin by tail tip transection methods capillary tube method, blood coagulation funtion tests and platelet activity tests; To study the absorption mechanism of pectolinarin and pectolin-aringenin by everted rat intestinal Sac Method, single-pass perfusion and HPLC mthods; To study metabolites of pectolinaringenin by LC-MS and enzymatic hydrolysis methods in vitro.
(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places by HPLC method; To optimize the processing method of carbonizing by Response Surface Methodology (RSM); To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC. by microwave digestion and ICP-OES methods.
Results:
(1)Carbonized Cirsium japonicum DC from Zhangshu and Hunan show stronger hemostatic effect than Carbonized Cirsium japonicum DC from Hebei. The Cirsium japonicum DC. from Anhui show no hemostatic effect and even promote the bleeding. There were differences of hemostatic effect and compounds between Carbonized Cirsium japonicum DC. from different places.
(2)Fourteen compounds were isolated from Carbonized Cirsium japonicum DC. All their structures were identified as pectolinarin, pectolinaringenin, linarin, acacetin, dibutyl phthalate,3-hydroxy-4-methyl-benzoic acid,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one,3-hydroxy-benzoic acid, apigenin-7-O-β-D-glucuronide,4-ethyl-3-hydroxy-benzoic acid、hispidulin-7-O-glucuronide,5-hydroxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one,5-hydr oxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one, apigenin by NMR and mass spectrometry method. Among those compounds,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC.
After Cirsium japonicum DC. been carboinzed., the contents of linarin and pectolinarin decreased sharply and contents of pectolina-ringenin and acacetin increased, we surmised that linarin and pectolinarin were hydrolyzed into acacetin and pectolinaringenin during the Carbonized processing based on their chemical structures.
(3)The tradional chinese herbs (Monkshood、rhizoma zingiberis, etal)、warfarin and heparin sodium can all increase bleeding and coagulating time of mice, differences were significant (p<0.05), while the yeast can also increase bleeding and coagulating time of mice, but differences weren't significant (p>0.05). Carbonized Cirsium japonicum DC. can decrease bleeding and coagulating time of mice in all bleeding animal models.
Both chloroform, ethyl acetate and n-butanol extracted parts from Carbonized Cirsium japonicum DC. show hemostatic activity.
Both pectolinarin and pectolinaringenin can decrease the bleeding and coagulating time of mice of bleeding model induced by heparin sodium, differences were significant (p<0.05). The bleeding and coagulating time of mice in high and median dose group of pectolinaringenin were significantly shorter than pectolinarin group (p<0.05). the hemostatic activity of pectolinaringenin is dose related.
Both pectolinarin and pectolinaringenin can significantly increase Fib in blood of rats (p<0.05). High dose group of pectolinaringenin can decrease APTT in blood of rats, but there were no significant differences (p>0.05). the results show the realtionshiop between hemostatic effection of pectolinaringenin, pectolinarin and theirs'FIB promotion effection, it also show that pectolinarin may can enhance the activity of coagulation factor VIII、IX、 XI or XII.
Both pectolinarin and pectolinaringenin can significantly increase positive rate of CD-62P protein expression in rat platelet (p<0.05). The positive rate of CD-62P protein expression in high dose group of pectolinaringenin is dose related higher than Yunnan Baiyao group, low dose group of pectolinarin and pectolinaringenin, the differences were significant (p>0.05)
The result reveals that the absorption of pectolinarin and pectolinaringenin both increased over time. The absorption of both ingredients among jejunum and ileum parts were higher than other Small intestinal parts. The absorption rate of pectolinarin among entire small intestinal was much higher than the absorption rate of pectolinaringenin. The absorption approach of each sample is quiet different, Obviously some bioconversion take place in the process of pectolinarigenin adsorbtion, while the pectorinarin is aborbed in prototype, so the real absorbtion rate maynot been revealed in this experiment.
The absorption rate constant, the effective permeability coefficients (Peff), the absorption (Ma) of pectolinarin were significantly decreased than pectolinaringenin (P<0.01); We found a few unknown components in intestinal effluent and blood of pectolinaringenin group, while the pectorinarin is aborbed in prototype
By LC-MS and enzymatic hydrolysis methods in vitro, we inferred that metabolites of pectolinaringenin in intestinal effluent may be pectolinarin-genin-glucoside or its methylation products
(4)Pectolinarigenin in carbonized cirsium japonicum DC. from different places varied greatly. The content of pectolinarigenin in carbonized cirsium japonicum DC. from Zhangshu. Zhejiang is0.22%, which is the highest among all different places. The pectolinarigenin content in carbonized cirsium japonicum DC. from Anhui、Hebei is undetectable.
The best processing method of carbonizing Cirsium japonicum DC. is quantity165g processing temperature310℃、processing time13min.
The best macroporous adsorption resin method to purifying pectolinarin is loading quantity of sample28BV、adsorption velocity3BV/h、cleaning solvent water、cleaning solvent volume19BV、desorption solvent50%ethanol、desorption solvent volume12BV. desorption velocity2BV/h, after purification the purity of the pectolinarin can reach68%.
The content of trace elements in Carbonized Cirsium japonicum DC. varied greatly, content of Ca、K reach30mg/g, content of Fe、Mg、Mn is lower than Ca、K. content of Zn、Na、Cu reach only0. lmg/g. after Cirsium japonicum DC. been carbonized, the content of trace elements in carbonized Cirsium japonicum all increased at different extent.
New ideas of the reseacrh:
(1)Firstly, we made research on differences of hemostatic activity and chemical constituents in Cirsium japonicum DC. from different places in order to ensure the smooth implementation of our research.
(2)14components from Cirsium japonicum DC. were identified. Among them3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC. The chemical constituents been changed、 the mechanism of those changes during carbonizing process were firstly studied.
(3)The hemostatic activity difference and hemostatic mechanism between pectolinarin and pectolinaringenin was firstly studied, these results construct steady base to reveal the carbonizing processing mechanism of Cirsium japonicum DC.
(4)We formulate a scientific hypothesis which the hemostatic activity difference between pectolinarin and pectolinaringenin may connect with their different absorption rate in rat. we found in the following study that their absorption rate varied greatly and metabolic route is quiet different. The metabolites of pectolinaringenin were firstly studied by LC-MS and enzymatic hydrolysis methods in vitro. According to the results we try to infer the chemical structure of metabolites of pectolinaringenin.
Conclusion:
The pectolinarigenin has stronger hemostatic activity than pectolinarin, it can explain why Carbonized Cirsium japonicum DC. show stronger hemostatic activity while its hemostatic content pectolinarin decreased after been carbonized.. It also reveals that the stronger hemostatic effection of pectolinarigenin may connect with its different absorbtion approach from pectolinarin. Usually, the active ingredients in chinese medcine may not be one, so, other elements such as carbonaceous、tannins、Ca2+all should be considered, this is still worth studying.
引文
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