小柴胡汤及其药群配伍抗肝癌作用与机理的研究
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摘要
利用现代科学研究经典名方的物质基础、配伍规律及其生物效用机制,对阐明中医药理论内涵具有特别重要的意义。“经方”具有用药精专,法度严谨,疗效卓著的特点,为历代医家所推崇。因此,选择具有重要配伍意义和较高临床运用价值的经方作为研究切入点不仅具有逻辑上的合理性,也具有技术上的可操作性及研究上的示范性。
小柴胡汤配伍严谨,组合巧妙,具有扶正祛邪,和解少阳,疏肝利胆,通达表里阴阳,协调脏腑等多种功用,临床上被广泛用于肝病,疗效确切,间有用本方加减用于肝癌也有一定疗效。现代药理研究表明,本方对小鼠S180实体瘤有显著的抑制作用;方中多数药味具有一定的抗肿瘤及免疫调节作用。提示本方可能具有抗肝癌的作用。
本课题拟以小柴胡汤可能的抗肝癌效用为切入点,采用H22和S180小鼠移植性肿瘤模型探查小柴胡汤全方的抗肿瘤效用,在此基础上依据该方的中医配伍学理,通过拆方,比较观察了全方和不同药群配伍的抑瘤效用及免疫机制;同时考察了小柴胡汤水煎法和乙醇梯度法等不同提取条件下所获部位的抑瘤效用;最后运用体外血清药理学、细胞学和免疫组化等研究技术,探讨了小柴胡汤水提物的抗肝癌作用机理。
论文分为两大部分。上篇文献研究部分主要对小柴胡汤实验研究与治疗肝病的临床研究进展进行了综述。总体上看,小柴胡汤实验研究主要涉及化学、制剂、用法、药理等的研究。其中药理研究开展最多,涉及本方对肝胆、循环、胃肠、生殖和泌尿、免疫-神经-内分泌等多系统的影响,较多集中在本方的抗肿瘤、解热、抗炎、抗感染等作用方面。小柴胡汤临床运用非常广泛,其中用于肝病主要涉及急慢性肝炎、肝纤维化及肝硬化、肝癌、脂肪肝等病,以原方及其加减为主,间有配合西药以提高疗效和降低毒副作用。下篇实验研究为论文主体,共分五个部分。
研究一小柴胡汤药学研究
方法采用高效液相色谱法(HPLC)对小柴胡汤处方药材柴胡、黄芩、人参、炙甘草以及小柴胡汤中指标成分柴胡皂苷a、黄芩苷、人参皂苷及甘草酸进行含量测定,以考察各药材的质量以及小柴胡汤煎剂制备工艺的合理性。结果柴胡药材中柴胡皂苷a含量为3.3351 mg/g,小柴胡汤煎剂中柴胡皂苷a的含量为0.7171mg/g,小柴胡汤中柴胡皂苷a的转移率为53.75%;黄芩药材中黄芩苷含量为10.867%,小柴胡汤煎剂中黄芩苷的含量为33.717mg/g,小柴胡汤中黄芩苷的转移率为58.61%;人参药材人参皂苷Rg_1和人参皂苷Re总量为0.902%、人参皂苷Rb_1为0.592%,小柴胡汤中人参皂苷Rg_1和人参皂苷Re总量为0.278mg/g、人参皂苷Rb_1为0.2069mg/g,小柴胡汤中人参皂苷Rg_1、Re的转移率为9.41%,人参皂苷Rb_1的转移率为10.62%;炙甘草药材中甘草酸含量为1.73%,小柴胡汤中甘草酸的含量为0.135mg/g,小柴胡汤中甘草酸的转移率为25.95%。
结论拟用的实验用处方药材均符合《中国药典》2005版的规定,小柴胡汤煎剂制备中柴胡皂苷a和黄芩苷50%以上的转移率,提示其工艺合理。
研究二小柴胡汤抗肝癌作用的研究
方法①建立小鼠H22肝癌实体瘤模型和腹水瘤模型,分别灌服高、中、低浓度小柴胡汤煎剂(27、13.5、6.75g生药/kg),腹腔注射5-氟尿嘧啶(5-FU)(20 mg/kg)做阳性对照;实体瘤模型给药10d后检测各组的瘤重,体重,脾脏指数和胸腺指数,腹水瘤模型在模型组出现死亡停止给药,观察各组的生存时间。②建立小鼠S180肉瘤实体瘤模型,灌服小柴胡汤中剂量(13.5g生药/kg),连续10天后,测定各组瘤重。结果①小柴胡汤高、中、低三个剂量组H22小鼠肝癌实体瘤明显减小,其抑瘤率分别为49.66%,48.52%,36.91%,三个剂量组均能明显延长腹水瘤小鼠的生存时间,生命延长率分别为23.59%,36.33%,26.96%,最佳给药剂量为中剂量。②小柴胡汤中剂量对小鼠肉瘤S180实体瘤的抑瘤率为36.88%。与正常组相比,荷瘤小鼠脾脏指数升高(P＜0.001),胸腺指数和体重变化不大(P＞0.05);与模型组比较,小柴胡汤各剂量组脾脏指数和胸腺指数无显著性差异(P＞0.05);5-FU组胸腺指数极显著降低(P＜0.001)。小柴胡汤各剂量组的小鼠体重均无明显变化,5-Fu组小鼠体重下降明显(P＜0.001)。
结论小柴胡汤对小鼠H22肝癌实体瘤生长有明显抑制作用,并能延长小鼠H22肝癌腹水瘤的生存时间,以中剂量为最佳剂量,而小柴胡汤对小鼠肉瘤S180实体瘤生长有一定抑制作用,但抑瘤作用低于H22肝癌模型,提示小柴胡汤可能主要针对肝癌产生治疗作用,符合该方临床主要用于肝系疾病。
研究三小柴胡汤药群配伍与抗肝癌效用的关系
方法①建立小鼠H22肝癌模型,分别灌服小柴胡汤药群配伍煎剂(柴-芩、姜-夏、参-枣-草、柴-芩+姜-夏、柴-芩+参-枣-草、姜-夏+参-枣-草,全方),共七组,腹腔注射5-FU(20 mg·kg~(-1))为阳性对照组;给药10d后检测各组的瘤重,体重,脾脏和胸腺指数。②小鼠H22肝癌模型分别灌服小柴胡汤及药群参-枣-草,连续给药10d,测定各组的瘤重和体重,MTT法测定各组自然杀伤细胞(NK)和T淋巴细胞增殖的活性,ELISA法测定其白细胞介素-2(IL-2)的活性。结果参-枣-草组和全方组的抑瘤率为41.55%和43.65%。与正常组相比,H22荷瘤小鼠脾脏指数显著升高(P＜0.001)。与模型组比,各配伍组小鼠脾脏指数、胸腺指数均无明显差异;5-FU组胸腺指数极显著降低(P＜0.001)。小柴胡汤及各配伍组小鼠体重无明显差异;5-Fu组小鼠体重显著下降(P＜0.001)。与模型组相比,小柴胡汤组小鼠NK细胞、T淋巴细胞增殖及IL-2活性均明显升高(P＜0.01);参-枣-草组NK细胞和IL-2活性升高(P＜0.05);5-FU组NK细胞、T淋巴细胞增殖及IL-2活性均显著降低(P＜0.01)。
结论小柴胡汤和方中参-枣-草配伍药群有抑制小鼠H22肝癌实体瘤生长和提高荷瘤宿主免疫功能作用,其抑瘤作用机制可能与增强荷瘤宿主免疫功能有关,而与5-氟尿嘧啶的直接抑瘤作用有所不同。结果提示该方中的扶正作用药群(人参、大枣和甘草)可能是全方抑瘤作用的核心药群。
研究四小柴胡汤不同提取方法抗肝癌作用的比较
方法建立小鼠H22肝癌模型,分别灌服四种不同提取方法获取的小柴胡汤提取物(13.5g生药/kg)共4组,设腹腔注射5-FU(20mg/kg)做阳性对照组。给药10d后检测各组的瘤重,体重,脾脏指数和胸腺指数。结果小柴胡汤水提组、梯度乙醇组、醇水组及系统溶剂组对H22小鼠肝癌实体瘤均有不同程度地抑制作用,各组抑瘤率分别为32.35%、43.97%、31.37%和25.77%。与模型组相比,各提取组的小鼠体重均无明显差异,小鼠的脾脏指数均明显增加(P＜0.01);5-Fu组胸腺指数显著降低(P＜0.001),体重明显下降(P＜0.001)。结论小柴胡汤采用水煎法、梯度乙醇提取法及醇水法获得的提取物对小鼠H22肝癌实体瘤生长均有明显抑制作用;较佳的梯度乙醇提取法的抑瘤作用与传统经典的水煎法无明显差异,提示传统提取方法具有合理性。
研究五小柴胡汤抗肝癌作用机理的研究
方法建立小鼠H22肝癌模型,分别灌服小柴胡汤煎剂13.5 g·kg~(-1),设腹腔注射5-FU(20 mg·kg~(-1))为阳性对照组,各组连续给药10d。分别检测各组的瘤重,体重;ELISA法检测各组小鼠血清TNF-α和VEGF含量;免疫组化方法检测肿瘤组织的微血管密度(MVD)和细胞凋亡率。制备小柴胡汤含药血清,MTT法观察含药血清对体外培养H22细胞的增殖抑制作用。结果小柴胡汤组对小鼠H22肝癌实体瘤的抑瘤率为43.28%。与模型组比较,小柴胡汤组荷瘤小鼠血清中TNF-α的浓度明显升高(P＜0.05),血清中VEGF的浓度明显降低(P＜0.05);5-FU组荷瘤TNF-α浓度显著降低(P＜0.05)。与模型组比较,5-FU组和小柴胡汤组肝癌细胞凋亡率均显著增加(P＜0.01),两组的凋亡率分别为11.18%和13.03%;小柴胡汤组和5-FU组小鼠肝癌组织中的微血管密度均明显减少(P＜0.05);与小柴胡汤组比较,5-FU组小鼠肝癌组织中的微血管密度也有增加,但无显著性差异。小柴胡汤各浓度梯度的含药血清对体外培养的H22细胞的生长没有明显抑制作用。
结论小柴胡汤具有一定的抑制H22小鼠肿瘤生长、抗新生血管生成及提高免疫功能的作用,小柴胡汤抗肝癌效用可能与这些作用有关。
本文在对古今小柴胡汤研究文献中发现小柴胡汤具有抗肿瘤作用及潜在的防治肝癌效用,进一步推测该方可能具有抗肝癌药理作用,并围绕方药配伍和提取工艺对其与抗肝癌作用的关系及作用机理进行了探察。本研究发现及证实小柴胡汤全方和方中参-枣-草药群具有抗肝癌作用,该方的全方配伍及其传统水提液在抗肝癌的综合效应方面仍具有优势,小柴胡汤的抗肿瘤作用可能主要是通过其整体作用条件下的免疫增强、抑制新生肿瘤血管生成及诱导肿瘤细胞坏死等作用来达到的。本研究为小柴胡汤用于肝癌的防治提供了一定的实验依据,为理解该方配伍及汤剂的合理性提供了一定的现代证据,为古方资源的多向开发利用提供了有价值的探索启示。
It is very significant to illuminate theory of traditional Chinese medicine for studying classical formula of material fundament,compatibility rules and action mechanism by modern science and technology.The classical formula has some fine features,which its medication perfect,compatibility compact,effect outstanding,and is held in esteem by many doctors.So to select classical formula of fine compatibility and higher clinic value as key point is not only reasonable but maneuverability and demonstration as well.Xiaochaihu decoction(XCHD) being compatibility compact and combination ingenious possessed many kinds of effect of strengthening healthy qi to eliminate pathogens,relieving shaoyang disorder,disperse the depressed liver-energy and cholagogue,clear and logical interior and exterior yin-yang, concordance entrails.It was used to treating liver disease and liver cancer in clinic.According to modern pharmaco-study indicated:XCHD could markedly inhibit S180 tumor of mice.
This topic planed to select XCHD inhibiting liver cancer as elementary point.Used H22 and S180 tumor model of mice to affirm antitumor action of XCHD.On that basis,according its formula compatibility reason to comparison observe antitumor action of XCHD and its different herbal formulation of component.Then we found that XCHD and active drug group (shen-zao-cao) could markedly inhibit tumor and have immunologic enhancement.Meanwhile, we investigated the different extraction method to gain part which its inhibiting tumor action.At last,we approached antihepatocarcinoma effect mechanism of XCHD by extraorgan blood-serum pharmacology,cytology and immunohistochemical method.
This thesis was made of two parts.The first part mainly summarized XCHD study progress of experiment and liver disease clinic.As a whole,empirical study of XCHD refered to its chemistry,preparation,application and pharmaco-study:effect on liver and gall system, recirculating system,gastro intestinal system,genito-urinary system,immune system, neuro-endocrine system and antitumor,relieve fever,antiinflammatory,anti-infection action. Liver disease clinical research of XCHD refered to acu-chronic hepatitis,hepatic fibrosis and cirrhosis,liver cancer,adiposis hepatica,chole-stomach disease and depressive disorder etc.we found that XCHD adopted prescription,add-deduct and drug combination to promote effect and degrade toxic and side-effect.The second part was consisted of five researchs.
Research 1:Pharmaceutical research of Xiaochaihu Decoction.
Method:Used the HPLC to determine the index component of bupleurum chinense, Baical Skullcap Root,Radix Ginseng,Prepared Radix Glycyrrhizae.To determine saikoside-a, baicalin,panaxsaponin,glycyrrhizic acidcontent of XCHD by the HPLC which it can considerate preparation process of XCHD if it is reasonable.Result:Saikoside-a content of bupleurum,chinense is 3.3351mg/g,Saikoside-a content of XCHD is 0.7171mg/g.Baicalin content of Baical Skullcap Root is 10.867%,Baicalin content of XCHD is 33.717mg/g.; Panaxsaponin Rg_1 and panaxsaponin Re total contents of Radix Ginseng are 0.902%, Panaxsaponin Rb_1 content of Radix Ginseng is 0.592%,Panaxsaponin Rg_1 and panaxsaponin Re total contents of XCHD are 0.278mg/g,Panaxsaponin Rb_1 content of XCHD is 0.2069mg/g; Glycyrrhizin content of Prepared Radix Glycyrrhizae is 1.73%,Glycyrrhizin content of XCHD is 0.135 mg/g;Baicalin diversion of XCHD had exceeded 50%and arrived by 58.61%.
Conclusion:Saikoside-a and Baicalin diversions of XCHD had exceeded 50%.Decoction preparation process of XCHD is rather reasonable.And these results suggest that pharmacologic action of XCHD have intimately relationship with these component.
Research 2:Study on inhibiting liver cancer action to affirm of Xiaochaihu Decoction.
Method:Transplanted tumor H22 liver cancer to mice for modelling of solid tumor and ascites tumor,then filling on high,moderate,low dose of Xiaochaihu decoction drag(27g,13.5g, 6.75 g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,solid tumor model mice were observed cancer weight,body weight, spleen index and thymus gland index.Aseites tumor model mice were observed live span after model mice having died mice and stopped administer for studying dose-effect relationship.For furtthering to study on inhibiting liver cancer action to affirm of Xiaochaihu decoction. Transplanted tumor S180 tumor carneus to mice,then filling on moderatedose of Xiaochaihu decoction(13.5g·kg~(-1)) and detect cancer weight.Result:Growth of H22 mice liver cancer was markedly inhibited by high,moderate,low dose group,the inhibiting rates were by 49.66%, 48.52%,36.91%,optimal administration dose is moderate dose by analyzing.And three dose groups of XCHD could extend live time of mice which its were transplanted by ascites tumor, live span extended rates were by 23.59%,36.33%,26.96%.Moderate dose group of XCHD inhibiting rate was by 36.88%on S180 tumor carneus mice..To compare with normal group, Spleen index of H22 rumor-bearing mice was higher than that of blank control group(P＜0.01), thymus gland index and body weight had not obvious changed(P＞0.05),spleen index of three dose groups had evidently increased(P＜0.01),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).
Conclusion:XCHD can markedly inhibit growth of H22 mice solid liver cancer and extend live span of H22 aseites tumor mice.The moderate dose of XCHD is the best dose by aggregate analysis.XCHD can also inhibit growth of S_(180) solid tumor cameus mice,but its action is not better than on H22 liver cancer.These results cue us that XCHD probably have therapeutic action to aim directly at liver cancer.
Research 3:Effect of Xiaochaihu Decoction and different herbal formulation of component on inhibiting H22 liver cancer in mice and enhancing the immune function.
Method:Transplanted tumor H22 liver cancer to mice,filling on Xiaochaihu decoction the different herbal formulations of its components(Chinese Thorowax Root- Baical Skullcap Root(A),Fresh Ginger-Pinellia Tuber(B),Ginseng- Chinese Date - Licorice Root(C),A+B, A+C,B+C and A+B+C.),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,we determined cancer weight,body weight,spleen index and thymus gland index for studying dose-effect relationship.Then Xiaochaihu decoction group (13.5 g·kg~(-1)) and Ginseng-Chinese Date-Licorice Root apozem group(5 g·kg~(-1)) were given the medicines for 10 days.To observing the weight of tumor,body weight and the NK cell activity, proliferation of the T lymphocytes by MTT and IL-2 level by ELISA.Result:In the study of compatibility role,only inhibiting rates of Ginseng-Chinese Date-Licorice Root apozem group and Xiaochaihu decoction group exceed 30%,the inhibiting rates were by 41.55%and 43.65%. To compare with normal group,spleen index of the different herbal formulation groups had evidently increased(P＜0.01),thymus gland index of the different herbal formulation groups had significant decreased(P＜0.05),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).Comparing the model group,the NK cell activity,proliferation of the T lymphocytes and IL-2 level of Xiaochaihu decoction were remarkable increased (P＜0.001),the NK cell activity and IL-2 level of Ginseng-Chinese Date-Licorice Root group were remarkable increased(P＜0.05),the NK cell activity and proliferation of the T lymphocytes and IL-2 level of 5-FU group were remarkable decreased(P＜0.01);Comparing the normal control group,body weight of 5-FU group had evidently decreased(P＜0.05).
Conclusion:Xiaochaihu decoction and Ginseng-Chinese Date-Licorice Root group can markedly inhibit growth of H22 mice solid liver cancer,and improve the immune function of tumor-bearing mice,its inhibiting effect probably closely related with improving rumor-bearing mice machine body immune function,there had a hint that compatibility Ginseng- Chinese Date -Licorice Root of strengthening body resistance was cores of inhibiting effect.
Research 4:Contradistinction of different extraction process of Xiaoehaihu Decoction on inhibiting H22 liver cancer in mice.
Method:Transplanted tumor H22 liver cancer to mice,filling on different extraction process of Xiaochaihu decoction drug(13.5 g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.Atter being administered for 10 days,we determined cancer weight,body weight,spleen index and thymus gland index for studying best extraction process.Result: Growth of H22 mice liver cancer was markedly inhibited by decoction process,gradient alcohol process,water-alcohol process group,the inhibiting rates were by 32.35%,43.97%and 31.37% optimal process is gradient alcohol process by analyzing.To compare with normal group,spleen index of the different extraction process groups had evidently increased(P＜0.01),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).Comparing the normal control group,body weight of 5-FU group had evidently decreased(P＜0.001).
Conclusion:Xiaochaihu decoction of decoction process,gradient alcohol process and water-alcohol process can markedly inhibit growth of H22 mice solid liver cancer.Gradient alcohol extraction process is the best process.But this process is too complex and its inhibiting rate is not far more than classical decoction process.All in all,classical decoction process has rather rationality.
Research 5:Study of mechanism of action of Xiaochaihu Decoction on inhibiting liver cancer
Method:Transplanted tumor H22 liver cancer to mice,filling on Xiaochaihu decoction drug(13.5g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,we determined cancer weight,body weight,detected TNF-αand VEGF of blood-serum by ELISA and detected Vascular Endothelial Growth Factor(MVD) and apoptosis rates of tumor tissue by immunohistochemical method.We prepared medicated blood-serum of XCHD which it was observed inhibitory action on H22 cells in culture fluid by MTT method.Result:Growth of H22 mice liver cancer was markedly inhibited by moderate dose group,the inhibiting rates were by 43.28%.To compare with analogue group,XCHD could increase markly TNF-αconcentration of mice blood-serum(P＜0.05) and degrade VEGF concentration of mice blood-serum(P＜0.05),but TNF-αconcentration of 5-FU group had evidently decreased(P＜0.05).XCHD could induced tumor apoptosis and markedly cut down MVD(P＜0.05).But medicated blood-serum of XCHD could not have any inhibitory action on H22 cells in culture fluid.
Conclusion:XCHD can inhibit tumor growed of H22 mice and new vascularization which it concern of elevating immune function,increasing TNF-αconcentration,degrading VEGF concentration and inducing tumor apoptosis.
This thesis had made system document research to find that XCHD had definite therapeutic on hepatopathy and liver cancer which is a key underlying pharmacologic action of XCHD.Our research results had also confirmed that XCHD had conclusive antitumor action.Compatibility study results indicated that XCHD inhibiting tumor action is the same as shen-zao-cao herb group.Mechanism of action research results hinted that XCHD inhibiting action were generated by immunological regulation,elevating activity of cell factor,inhibiting new tumor angiogenesis and inducing tumor cell necrosis.This research will provide,which modern study evidence for one formula more usage of traditional Chinese medicine,some study mode and experience for system studying of classical formula.Meanwhile,it will offer certain platform for developing and utilizating resources of classical formula.
小柴胡汤配伍严谨,组合巧妙,具有扶正祛邪,和解少阳,疏肝利胆,通达表里阴阳,协调脏腑等多种功用,临床上被广泛用于肝病,疗效确切,间有用本方加减用于肝癌也有一定疗效。现代药理研究表明,本方对小鼠S180实体瘤有显著的抑制作用;方中多数药味具有一定的抗肿瘤及免疫调节作用。提示本方可能具有抗肝癌的作用。
本课题拟以小柴胡汤可能的抗肝癌效用为切入点,采用H22和S180小鼠移植性肿瘤模型探查小柴胡汤全方的抗肿瘤效用,在此基础上依据该方的中医配伍学理,通过拆方,比较观察了全方和不同药群配伍的抑瘤效用及免疫机制;同时考察了小柴胡汤水煎法和乙醇梯度法等不同提取条件下所获部位的抑瘤效用;最后运用体外血清药理学、细胞学和免疫组化等研究技术,探讨了小柴胡汤水提物的抗肝癌作用机理。
论文分为两大部分。上篇文献研究部分主要对小柴胡汤实验研究与治疗肝病的临床研究进展进行了综述。总体上看,小柴胡汤实验研究主要涉及化学、制剂、用法、药理等的研究。其中药理研究开展最多,涉及本方对肝胆、循环、胃肠、生殖和泌尿、免疫-神经-内分泌等多系统的影响,较多集中在本方的抗肿瘤、解热、抗炎、抗感染等作用方面。小柴胡汤临床运用非常广泛,其中用于肝病主要涉及急慢性肝炎、肝纤维化及肝硬化、肝癌、脂肪肝等病,以原方及其加减为主,间有配合西药以提高疗效和降低毒副作用。下篇实验研究为论文主体,共分五个部分。
研究一小柴胡汤药学研究
方法采用高效液相色谱法(HPLC)对小柴胡汤处方药材柴胡、黄芩、人参、炙甘草以及小柴胡汤中指标成分柴胡皂苷a、黄芩苷、人参皂苷及甘草酸进行含量测定,以考察各药材的质量以及小柴胡汤煎剂制备工艺的合理性。结果柴胡药材中柴胡皂苷a含量为3.3351 mg/g,小柴胡汤煎剂中柴胡皂苷a的含量为0.7171mg/g,小柴胡汤中柴胡皂苷a的转移率为53.75%;黄芩药材中黄芩苷含量为10.867%,小柴胡汤煎剂中黄芩苷的含量为33.717mg/g,小柴胡汤中黄芩苷的转移率为58.61%;人参药材人参皂苷Rg_1和人参皂苷Re总量为0.902%、人参皂苷Rb_1为0.592%,小柴胡汤中人参皂苷Rg_1和人参皂苷Re总量为0.278mg/g、人参皂苷Rb_1为0.2069mg/g,小柴胡汤中人参皂苷Rg_1、Re的转移率为9.41%,人参皂苷Rb_1的转移率为10.62%;炙甘草药材中甘草酸含量为1.73%,小柴胡汤中甘草酸的含量为0.135mg/g,小柴胡汤中甘草酸的转移率为25.95%。
结论拟用的实验用处方药材均符合《中国药典》2005版的规定,小柴胡汤煎剂制备中柴胡皂苷a和黄芩苷50%以上的转移率,提示其工艺合理。
研究二小柴胡汤抗肝癌作用的研究
方法①建立小鼠H22肝癌实体瘤模型和腹水瘤模型,分别灌服高、中、低浓度小柴胡汤煎剂(27、13.5、6.75g生药/kg),腹腔注射5-氟尿嘧啶(5-FU)(20 mg/kg)做阳性对照;实体瘤模型给药10d后检测各组的瘤重,体重,脾脏指数和胸腺指数,腹水瘤模型在模型组出现死亡停止给药,观察各组的生存时间。②建立小鼠S180肉瘤实体瘤模型,灌服小柴胡汤中剂量(13.5g生药/kg),连续10天后,测定各组瘤重。结果①小柴胡汤高、中、低三个剂量组H22小鼠肝癌实体瘤明显减小,其抑瘤率分别为49.66%,48.52%,36.91%,三个剂量组均能明显延长腹水瘤小鼠的生存时间,生命延长率分别为23.59%,36.33%,26.96%,最佳给药剂量为中剂量。②小柴胡汤中剂量对小鼠肉瘤S180实体瘤的抑瘤率为36.88%。与正常组相比,荷瘤小鼠脾脏指数升高(P＜0.001),胸腺指数和体重变化不大(P＞0.05);与模型组比较,小柴胡汤各剂量组脾脏指数和胸腺指数无显著性差异(P＞0.05);5-FU组胸腺指数极显著降低(P＜0.001)。小柴胡汤各剂量组的小鼠体重均无明显变化,5-Fu组小鼠体重下降明显(P＜0.001)。
结论小柴胡汤对小鼠H22肝癌实体瘤生长有明显抑制作用,并能延长小鼠H22肝癌腹水瘤的生存时间,以中剂量为最佳剂量,而小柴胡汤对小鼠肉瘤S180实体瘤生长有一定抑制作用,但抑瘤作用低于H22肝癌模型,提示小柴胡汤可能主要针对肝癌产生治疗作用,符合该方临床主要用于肝系疾病。
研究三小柴胡汤药群配伍与抗肝癌效用的关系
方法①建立小鼠H22肝癌模型,分别灌服小柴胡汤药群配伍煎剂(柴-芩、姜-夏、参-枣-草、柴-芩+姜-夏、柴-芩+参-枣-草、姜-夏+参-枣-草,全方),共七组,腹腔注射5-FU(20 mg·kg~(-1))为阳性对照组;给药10d后检测各组的瘤重,体重,脾脏和胸腺指数。②小鼠H22肝癌模型分别灌服小柴胡汤及药群参-枣-草,连续给药10d,测定各组的瘤重和体重,MTT法测定各组自然杀伤细胞(NK)和T淋巴细胞增殖的活性,ELISA法测定其白细胞介素-2(IL-2)的活性。结果参-枣-草组和全方组的抑瘤率为41.55%和43.65%。与正常组相比,H22荷瘤小鼠脾脏指数显著升高(P＜0.001)。与模型组比,各配伍组小鼠脾脏指数、胸腺指数均无明显差异;5-FU组胸腺指数极显著降低(P＜0.001)。小柴胡汤及各配伍组小鼠体重无明显差异;5-Fu组小鼠体重显著下降(P＜0.001)。与模型组相比,小柴胡汤组小鼠NK细胞、T淋巴细胞增殖及IL-2活性均明显升高(P＜0.01);参-枣-草组NK细胞和IL-2活性升高(P＜0.05);5-FU组NK细胞、T淋巴细胞增殖及IL-2活性均显著降低(P＜0.01)。
结论小柴胡汤和方中参-枣-草配伍药群有抑制小鼠H22肝癌实体瘤生长和提高荷瘤宿主免疫功能作用,其抑瘤作用机制可能与增强荷瘤宿主免疫功能有关,而与5-氟尿嘧啶的直接抑瘤作用有所不同。结果提示该方中的扶正作用药群(人参、大枣和甘草)可能是全方抑瘤作用的核心药群。
研究四小柴胡汤不同提取方法抗肝癌作用的比较
方法建立小鼠H22肝癌模型,分别灌服四种不同提取方法获取的小柴胡汤提取物(13.5g生药/kg)共4组,设腹腔注射5-FU(20mg/kg)做阳性对照组。给药10d后检测各组的瘤重,体重,脾脏指数和胸腺指数。结果小柴胡汤水提组、梯度乙醇组、醇水组及系统溶剂组对H22小鼠肝癌实体瘤均有不同程度地抑制作用,各组抑瘤率分别为32.35%、43.97%、31.37%和25.77%。与模型组相比,各提取组的小鼠体重均无明显差异,小鼠的脾脏指数均明显增加(P＜0.01);5-Fu组胸腺指数显著降低(P＜0.001),体重明显下降(P＜0.001)。结论小柴胡汤采用水煎法、梯度乙醇提取法及醇水法获得的提取物对小鼠H22肝癌实体瘤生长均有明显抑制作用;较佳的梯度乙醇提取法的抑瘤作用与传统经典的水煎法无明显差异,提示传统提取方法具有合理性。
研究五小柴胡汤抗肝癌作用机理的研究
方法建立小鼠H22肝癌模型,分别灌服小柴胡汤煎剂13.5 g·kg~(-1),设腹腔注射5-FU(20 mg·kg~(-1))为阳性对照组,各组连续给药10d。分别检测各组的瘤重,体重;ELISA法检测各组小鼠血清TNF-α和VEGF含量;免疫组化方法检测肿瘤组织的微血管密度(MVD)和细胞凋亡率。制备小柴胡汤含药血清,MTT法观察含药血清对体外培养H22细胞的增殖抑制作用。结果小柴胡汤组对小鼠H22肝癌实体瘤的抑瘤率为43.28%。与模型组比较,小柴胡汤组荷瘤小鼠血清中TNF-α的浓度明显升高(P＜0.05),血清中VEGF的浓度明显降低(P＜0.05);5-FU组荷瘤TNF-α浓度显著降低(P＜0.05)。与模型组比较,5-FU组和小柴胡汤组肝癌细胞凋亡率均显著增加(P＜0.01),两组的凋亡率分别为11.18%和13.03%;小柴胡汤组和5-FU组小鼠肝癌组织中的微血管密度均明显减少(P＜0.05);与小柴胡汤组比较,5-FU组小鼠肝癌组织中的微血管密度也有增加,但无显著性差异。小柴胡汤各浓度梯度的含药血清对体外培养的H22细胞的生长没有明显抑制作用。
结论小柴胡汤具有一定的抑制H22小鼠肿瘤生长、抗新生血管生成及提高免疫功能的作用,小柴胡汤抗肝癌效用可能与这些作用有关。
本文在对古今小柴胡汤研究文献中发现小柴胡汤具有抗肿瘤作用及潜在的防治肝癌效用,进一步推测该方可能具有抗肝癌药理作用,并围绕方药配伍和提取工艺对其与抗肝癌作用的关系及作用机理进行了探察。本研究发现及证实小柴胡汤全方和方中参-枣-草药群具有抗肝癌作用,该方的全方配伍及其传统水提液在抗肝癌的综合效应方面仍具有优势,小柴胡汤的抗肿瘤作用可能主要是通过其整体作用条件下的免疫增强、抑制新生肿瘤血管生成及诱导肿瘤细胞坏死等作用来达到的。本研究为小柴胡汤用于肝癌的防治提供了一定的实验依据,为理解该方配伍及汤剂的合理性提供了一定的现代证据,为古方资源的多向开发利用提供了有价值的探索启示。
It is very significant to illuminate theory of traditional Chinese medicine for studying classical formula of material fundament,compatibility rules and action mechanism by modern science and technology.The classical formula has some fine features,which its medication perfect,compatibility compact,effect outstanding,and is held in esteem by many doctors.So to select classical formula of fine compatibility and higher clinic value as key point is not only reasonable but maneuverability and demonstration as well.Xiaochaihu decoction(XCHD) being compatibility compact and combination ingenious possessed many kinds of effect of strengthening healthy qi to eliminate pathogens,relieving shaoyang disorder,disperse the depressed liver-energy and cholagogue,clear and logical interior and exterior yin-yang, concordance entrails.It was used to treating liver disease and liver cancer in clinic.According to modern pharmaco-study indicated:XCHD could markedly inhibit S180 tumor of mice.
This topic planed to select XCHD inhibiting liver cancer as elementary point.Used H22 and S180 tumor model of mice to affirm antitumor action of XCHD.On that basis,according its formula compatibility reason to comparison observe antitumor action of XCHD and its different herbal formulation of component.Then we found that XCHD and active drug group (shen-zao-cao) could markedly inhibit tumor and have immunologic enhancement.Meanwhile, we investigated the different extraction method to gain part which its inhibiting tumor action.At last,we approached antihepatocarcinoma effect mechanism of XCHD by extraorgan blood-serum pharmacology,cytology and immunohistochemical method.
This thesis was made of two parts.The first part mainly summarized XCHD study progress of experiment and liver disease clinic.As a whole,empirical study of XCHD refered to its chemistry,preparation,application and pharmaco-study:effect on liver and gall system, recirculating system,gastro intestinal system,genito-urinary system,immune system, neuro-endocrine system and antitumor,relieve fever,antiinflammatory,anti-infection action. Liver disease clinical research of XCHD refered to acu-chronic hepatitis,hepatic fibrosis and cirrhosis,liver cancer,adiposis hepatica,chole-stomach disease and depressive disorder etc.we found that XCHD adopted prescription,add-deduct and drug combination to promote effect and degrade toxic and side-effect.The second part was consisted of five researchs.
Research 1:Pharmaceutical research of Xiaochaihu Decoction.
Method:Used the HPLC to determine the index component of bupleurum chinense, Baical Skullcap Root,Radix Ginseng,Prepared Radix Glycyrrhizae.To determine saikoside-a, baicalin,panaxsaponin,glycyrrhizic acidcontent of XCHD by the HPLC which it can considerate preparation process of XCHD if it is reasonable.Result:Saikoside-a content of bupleurum,chinense is 3.3351mg/g,Saikoside-a content of XCHD is 0.7171mg/g.Baicalin content of Baical Skullcap Root is 10.867%,Baicalin content of XCHD is 33.717mg/g.; Panaxsaponin Rg_1 and panaxsaponin Re total contents of Radix Ginseng are 0.902%, Panaxsaponin Rb_1 content of Radix Ginseng is 0.592%,Panaxsaponin Rg_1 and panaxsaponin Re total contents of XCHD are 0.278mg/g,Panaxsaponin Rb_1 content of XCHD is 0.2069mg/g; Glycyrrhizin content of Prepared Radix Glycyrrhizae is 1.73%,Glycyrrhizin content of XCHD is 0.135 mg/g;Baicalin diversion of XCHD had exceeded 50%and arrived by 58.61%.
Conclusion:Saikoside-a and Baicalin diversions of XCHD had exceeded 50%.Decoction preparation process of XCHD is rather reasonable.And these results suggest that pharmacologic action of XCHD have intimately relationship with these component.
Research 2:Study on inhibiting liver cancer action to affirm of Xiaochaihu Decoction.
Method:Transplanted tumor H22 liver cancer to mice for modelling of solid tumor and ascites tumor,then filling on high,moderate,low dose of Xiaochaihu decoction drag(27g,13.5g, 6.75 g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,solid tumor model mice were observed cancer weight,body weight, spleen index and thymus gland index.Aseites tumor model mice were observed live span after model mice having died mice and stopped administer for studying dose-effect relationship.For furtthering to study on inhibiting liver cancer action to affirm of Xiaochaihu decoction. Transplanted tumor S180 tumor carneus to mice,then filling on moderatedose of Xiaochaihu decoction(13.5g·kg~(-1)) and detect cancer weight.Result:Growth of H22 mice liver cancer was markedly inhibited by high,moderate,low dose group,the inhibiting rates were by 49.66%, 48.52%,36.91%,optimal administration dose is moderate dose by analyzing.And three dose groups of XCHD could extend live time of mice which its were transplanted by ascites tumor, live span extended rates were by 23.59%,36.33%,26.96%.Moderate dose group of XCHD inhibiting rate was by 36.88%on S180 tumor carneus mice..To compare with normal group, Spleen index of H22 rumor-bearing mice was higher than that of blank control group(P＜0.01), thymus gland index and body weight had not obvious changed(P＞0.05),spleen index of three dose groups had evidently increased(P＜0.01),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).
Conclusion:XCHD can markedly inhibit growth of H22 mice solid liver cancer and extend live span of H22 aseites tumor mice.The moderate dose of XCHD is the best dose by aggregate analysis.XCHD can also inhibit growth of S_(180) solid tumor cameus mice,but its action is not better than on H22 liver cancer.These results cue us that XCHD probably have therapeutic action to aim directly at liver cancer.
Research 3:Effect of Xiaochaihu Decoction and different herbal formulation of component on inhibiting H22 liver cancer in mice and enhancing the immune function.
Method:Transplanted tumor H22 liver cancer to mice,filling on Xiaochaihu decoction the different herbal formulations of its components(Chinese Thorowax Root- Baical Skullcap Root(A),Fresh Ginger-Pinellia Tuber(B),Ginseng- Chinese Date - Licorice Root(C),A+B, A+C,B+C and A+B+C.),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,we determined cancer weight,body weight,spleen index and thymus gland index for studying dose-effect relationship.Then Xiaochaihu decoction group (13.5 g·kg~(-1)) and Ginseng-Chinese Date-Licorice Root apozem group(5 g·kg~(-1)) were given the medicines for 10 days.To observing the weight of tumor,body weight and the NK cell activity, proliferation of the T lymphocytes by MTT and IL-2 level by ELISA.Result:In the study of compatibility role,only inhibiting rates of Ginseng-Chinese Date-Licorice Root apozem group and Xiaochaihu decoction group exceed 30%,the inhibiting rates were by 41.55%and 43.65%. To compare with normal group,spleen index of the different herbal formulation groups had evidently increased(P＜0.01),thymus gland index of the different herbal formulation groups had significant decreased(P＜0.05),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).Comparing the model group,the NK cell activity,proliferation of the T lymphocytes and IL-2 level of Xiaochaihu decoction were remarkable increased (P＜0.001),the NK cell activity and IL-2 level of Ginseng-Chinese Date-Licorice Root group were remarkable increased(P＜0.05),the NK cell activity and proliferation of the T lymphocytes and IL-2 level of 5-FU group were remarkable decreased(P＜0.01);Comparing the normal control group,body weight of 5-FU group had evidently decreased(P＜0.05).
Conclusion:Xiaochaihu decoction and Ginseng-Chinese Date-Licorice Root group can markedly inhibit growth of H22 mice solid liver cancer,and improve the immune function of tumor-bearing mice,its inhibiting effect probably closely related with improving rumor-bearing mice machine body immune function,there had a hint that compatibility Ginseng- Chinese Date -Licorice Root of strengthening body resistance was cores of inhibiting effect.
Research 4:Contradistinction of different extraction process of Xiaoehaihu Decoction on inhibiting H22 liver cancer in mice.
Method:Transplanted tumor H22 liver cancer to mice,filling on different extraction process of Xiaochaihu decoction drug(13.5 g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.Atter being administered for 10 days,we determined cancer weight,body weight,spleen index and thymus gland index for studying best extraction process.Result: Growth of H22 mice liver cancer was markedly inhibited by decoction process,gradient alcohol process,water-alcohol process group,the inhibiting rates were by 32.35%,43.97%and 31.37% optimal process is gradient alcohol process by analyzing.To compare with normal group,spleen index of the different extraction process groups had evidently increased(P＜0.01),thymus gland index and body weight of 5-FU group had evidently decreased(P＜0.001).Comparing the normal control group,body weight of 5-FU group had evidently decreased(P＜0.001).
Conclusion:Xiaochaihu decoction of decoction process,gradient alcohol process and water-alcohol process can markedly inhibit growth of H22 mice solid liver cancer.Gradient alcohol extraction process is the best process.But this process is too complex and its inhibiting rate is not far more than classical decoction process.All in all,classical decoction process has rather rationality.
Research 5:Study of mechanism of action of Xiaochaihu Decoction on inhibiting liver cancer
Method:Transplanted tumor H22 liver cancer to mice,filling on Xiaochaihu decoction drug(13.5g·kg~(-1)),5-FU intraperitoneal injection(20 mg·kg~(-1)) as positive control.After being administered for 10 days,we determined cancer weight,body weight,detected TNF-αand VEGF of blood-serum by ELISA and detected Vascular Endothelial Growth Factor(MVD) and apoptosis rates of tumor tissue by immunohistochemical method.We prepared medicated blood-serum of XCHD which it was observed inhibitory action on H22 cells in culture fluid by MTT method.Result:Growth of H22 mice liver cancer was markedly inhibited by moderate dose group,the inhibiting rates were by 43.28%.To compare with analogue group,XCHD could increase markly TNF-αconcentration of mice blood-serum(P＜0.05) and degrade VEGF concentration of mice blood-serum(P＜0.05),but TNF-αconcentration of 5-FU group had evidently decreased(P＜0.05).XCHD could induced tumor apoptosis and markedly cut down MVD(P＜0.05).But medicated blood-serum of XCHD could not have any inhibitory action on H22 cells in culture fluid.
Conclusion:XCHD can inhibit tumor growed of H22 mice and new vascularization which it concern of elevating immune function,increasing TNF-αconcentration,degrading VEGF concentration and inducing tumor apoptosis.
This thesis had made system document research to find that XCHD had definite therapeutic on hepatopathy and liver cancer which is a key underlying pharmacologic action of XCHD.Our research results had also confirmed that XCHD had conclusive antitumor action.Compatibility study results indicated that XCHD inhibiting tumor action is the same as shen-zao-cao herb group.Mechanism of action research results hinted that XCHD inhibiting action were generated by immunological regulation,elevating activity of cell factor,inhibiting new tumor angiogenesis and inducing tumor cell necrosis.This research will provide,which modern study evidence for one formula more usage of traditional Chinese medicine,some study mode and experience for system studying of classical formula.Meanwhile,it will offer certain platform for developing and utilizating resources of classical formula.
引文
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