桔梗、党参药效物质的提取分离及活性研究
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摘要
本课题是国家科技重大专项（重大新药创制）“中药药效物质基础及物质资源库研究关键技术”（课题编号：2009ZX09502-011）的研究内容之一，课题研究目标主要是围绕中药作用的药效物质基础和作用机理等重要科学问题，运用多种分离、鉴定即对中药基准药效物质分析的新技术和新方法，开展对传统中药材桔梗和党参的相关研究。
桔梗为桔梗科桔梗属植物桔梗Platycodon gradiflorum（Jacq.）A.DC.的干燥根。研究表明，桔梗具有抗炎镇痛、祛痰止咳、扩张血管、解热镇静、降血糖及增强人体免疫力等广泛的药理作用。本论文首先考察了桔梗主要活性物质桔梗皂苷的提取工艺，并对其进行了进一步的分离；其次研究了桔梗总皂苷（TP）、桔梗次皂苷（TPS）及桔梗皂苷D（PD）的平喘和抗肝纤维化活性。本论文首次采用了液-质联用技术对桔梗皂苷D进行了分析。首次研究了桔梗提取物对人体气管平滑肌细胞增殖的影响，对桔梗的平喘机制就体外细胞水平进行了探讨，实验结果提示了桔梗皂苷活性部位可以直接干预气道重构中气管平滑肌细胞的增殖、肥大，进而可能减少分泌炎性介质，为桔梗预防、治疗哮喘提供了新的理论依据。首次就TP、TPS和PD对肝损伤的保护作用在分子水平进行了深入对比，实验结果提示了桔梗皂苷类活性物质对四氯化碳诱导肝纤维化进程的保护机制可能是与其能够在体内阻止肝脏炎症、抗脂质过氧化反应和及时调节、清除自由基等因素有关。
党参为桔梗科党参属植物党参Codonopsis pilosula（Franch.）Nannf.的干燥根。其性味甘平、无毒，具有补中益气、活血化淤等功效，可用于治疗虚喘咳嗽，脾肺虚弱等症状。本论文考察了党参主要有效成分党参多糖的提取工艺，进一步从其精制多糖CPPw中分离并首次得到了党参多糖CPPw1。首次对比研究了CPPw和CPPw1在体内和体外对抗肿瘤和免疫功能调节的作用，实验结果提示了党参多糖所分离的各组分能够直接抑制肿瘤细胞活性，其中CPPw的抗肿瘤作用与调节相关免疫功能有关，包括刺激脾细胞增殖、增强巨噬细胞吞噬功能以及促使巨噬细胞活化，产生NO等。目前CPPw1的作用机制尚不明确，有待进一步的研究。
Campanulaceae plant includes70generas, and has about2000species, which accordingto fruit and anatomical characteristics, can be divided into five subfamilies, widelydistributes in the world. China has17generas and about150species, most of them belongsto Campanuloideae and distributes in the area of southwest. Campanuloideae mainly usedfor medicinal, such as traditional Chinese medicine Platycodonis Radix, CodonopsisPilosula, Adenophora stricta and Lobelia chinensis. Most plants of this subfamily containsaponins bioactive substances, such as Platycodin of Platycodon, Glehnia root saponin ofAdenophora, etc. All of them have effects on relieving a cough, reducing phlegm, moistinglungs and so on; in addition they also contain many kinds of Carbohydrate substances, suchas inulin, with the effects of invigorating the spleen, nourishing and fluiding. PlatycodonisRadix and Codonopsis Pilosula are nontoxic, their plants can also be edible.
Platycodonis Radix and Codonopsis Pilosula not only contain saponins,polysaccharides and other common pharmacologically active ingredient, also include avariety of amino acids, metal elements, vitamins and other active substances. Peopledeveloped a series of products, such as compound preparations, nutrition and health careproducts, which can use its medicinal value and also can play its nutrition and health careefficacy. With constantly in-depth study on pharmacological effects of Platycodonis Radixand Codonopsis Pilosula, new Chinese medicine can be further developed in clinicalapplications, new medicinal uses can be explored, the related health products research can bespeeded up, which makes resource advantage to play a greater role in the social andeconomic benefits.
Although it has some research reports in the various aspects, but the depth and breadthof them still not enough: such as the reported studies on effective parts of the monomer are less; the mechanism of the active ingredients that have not yet completely be clear; the basicof their pharmacological activities are not clear enough, which is to be further in-depth studyin the future. In light of the present with their applications in China's pharmaceutical market,the thesis thinks that, it is not only necessary and possible in exploring the basic traditionalChinese medicine resources, developing their new uses, and also has a broad prospects inmarketing.
This thesis, after the single factor experiments, through four factors and three levels[L9(34)] of the orthogonal experiments, determined the extraction methods of total platycodinand crude Codonopsis Pilosula polysaccharide. The extraction process of total platycodin is:dried pieces of Platycodonis Radix extracted for2times with petroleum ether at60～90℃,each time lasts2h for removing the fat-soluble impurities, rotary evaporation and recovery ofpetroleum ether; after extraction of dry dregs with ethanol refluxing method, rotaryevaporation recovery of ethanol, then placed in a water bath, steaming to dry extract.Dissolving the obtained extract in distilled water, extracting with water saturated butanol,rotary evaporation recovery of butanol when positive butanol near colorless, oven drying theresidue at105℃. The optimal conditions of extraction was: At90℃, solid-liquid ratio is1:8,80%ethanol refluxing extracting for3times, each time lasts1h.The average rate of totalsaponins is2.66%.
The extraction process of crude Codonopsis pilosula polysaccharide is: dried pieces ofCodonopsis pilosula extracted3times with petroleum ether at60～90℃, each time lasts6hfor removing the fat-soluble impurities, rotary evaporation and recovery of petroleum ether;overnight the dry dregs with80%ethanol soak, and extracted for2times, each time lasts2hfor removing the alcohol-soluble impurities, rotary evaporation and recovery of ethanol; thenextract with water in the way of refluxing, rotary evaporation concentrating, adding5timesthe volume of different concentrations of ethanol overnight static at4℃, centrifugalcollection polysaccharide precipitation, drying in vacuum. The optimal conditions ofextraction was: At100℃, solid-liquid ratio was1:10, extracting for2times with the hotwater, each time lasts3h. The average rate of crude Codonopsis pilosula polysaccharide is23.38%.
In determining the extraction method based on, this thesis investigated the purificationmethods of platycodin D and CPPw1. The separation method of total platycodin is: wetpacking in column, column height is60cm, dry sample is24.7g, the sample in the silica gelcolumn is about3cm high, under atmospheric pressure with different ratio of ethyl acetate,methanol eluent gradient elution. Through the TLC view, combined the stream with the sameRf value, purified repeatedly in reverse phase silica gel column Lobar Rp-8with methanoland water as mobile phase, recrystalled with methanol. In addition, analysised the purifiedplatycodin D with TLC, HPLC-ELSD, LC-MS. According to the calculation of crude drugs,the yield rate of platycodin D is0.19%.
The separation method of crude Codonopsis pilosula polysaccharide is: firstly removeprotein by using sevag method to get the refined Codonopsis pilosula polysaccharide (CPP),then purified in the DEAE-cellulose column (2.6×50cm) and Sephadex G-75column(2.6×100cm), the yield rate of CPPw1was0.20%. After determining the polysaccharidecontent of the polysaccharide fractions by phenol sulfuric acid method, the purity ofpolysaccharide and molecular weight distribution by high performance size exclusionchromatography (HPSEC) method, we finally found that the CPPw1is a homogeneouspolysaccharide composition, its molecular weight is256000Da. For the further study ofpolysaccharide in nature, using gas chromatographic method for the determination of themonosaccharide composition of CPPw1, we finally found that CPPw1is composed ofrhamnose, arabia, fructose, glucose, and its molar percentage is:23:27:9:11.
Extracting the main effective component from Chinese herbal medicine is thefoundation of a research. In the thesis we first using the organization block posted methodsuccessfully separated and cultured human ASMCs, by morphological observation andα-actin immunocytochemical detection, more than99%of cells are human ASMCs. HumanASMCs in vitro in the level of cells for the study of bronchial asthma has provided aneffective platform for the biological behavior, through the cultivation of human ASMCs canbe a means of its pathogenesis and prevention research.
The total platycodin (TP) extracted from Platycodonis Radix (0mg/L、10mg/L、50mg/L、100mg/L) with dexamethasone (DXM) as positive control (1mg/L) were diluted with serum-free DMEM, which is used to culture the human ASMCs. By using the MTTmethod and cells counting method for detecting, we found that small doses of celandineactive substances that could inhibit the proliferation of human ASMCs, and in a time andconcentration dependent.
Secondly, this thesis used the model on carbon tetrachloride induced liver injury in micewhich was based on the discussiong about the liver fibrosis in the etiology and mechanisms.After giving the drugs for1week, through the indexes determination of liver function (ALT,AST) and oxidation (MDA, SOD), inflammatory (IL-6), the experiments analysised theseverity of liver damage comprehensively by reflecting from different aspects for totalplatycodin (TP), total prosapogenins (TPS) and platycodin D (PD) against hepatic fibrosisand its mechanism. The experimental results compared with control group,6h afteradministration, showed that the ALT, AST, MDA and IL-6of the mice in the model groupincreased, SOD decreased significantly, which explains that the model description wassucceed; compared with the model group, the ecperiment showed that the TP can decreasethe levels of ALT, AST, MDA and IL-6, increased level of SOD, the TPS can markedlydecrease in ALT, MDA and IL-6levels, increased in SOD level, but the effect in decreasingthe AST level was not obvious; the PD decreased the levels of ALT and AST was inverselyproportional to the giving doses, its effect in increase of SOD level was also not obvious.
Finally, study comparatively about the antitumor and immune regulation function of theobtained Codonopsis pilosula polysaccharide CPPw and CPPw1in vivo and in vitro. Theexperimental results showed that, Codonopsis pilosula polysaccharide CPPw and CPPw1can well suppress tumor growth in vivo, in which CPPw can increase the immunity organsweight of mice spleen, thymus; spleen cells stimulated with ConA and LPS for combination,which could induce the proliferation of T and B lymphocyte; enhanced peritonealmacrophages phagocytosis, which was dose dependent, and stimulated the activation ofmacrophage for producing NO. The effects of CPPw1were not significant.
This thesis firstly investigated the extraction technology about platycodin which is themain active substance of Platycodonis Radix, then carried on its further separation; secondlystudies the antiasthmatic and anti hepatic fibrosis activity about total platycodin (TP), total prosapogenins (TPS) and platycodin D (PD). In this paper, platycodin D were analyzed bythe way of LC-MS for the first time. And also study on proliferation of human airwaysmooth muscle cells about the extract of Platycodonis Radix for the first time, discussed oncells level about its antiasthmatic mechanism in vitro, the results suggest that platycodinactive site can directly intervention in airway remodeling about proliferation andhypertrophy of airway smooth muscle cells, and may reduce the secretion of inflammatorymediators, which provides a new theoretical basis for Platycodonis Radix to prevent andtreat asthma. This thesis for the first time compared thoroughly on liver injury protectiveeffects at the molecular level about TP, TPS and PD, the results suggest that the mechanismof Platycodon saponins bioactive substances on protection of carbon tetrachloride-inducedliver fibrosis may be related to prevent liver inflammation, resisting lipid peroxidationreaction and timely regulation, scavenging of free radicals and other factors in vivo.
In addition, this thesis investigated the extraction technology about polysaccharidewhich is the main effective component of Codonopsis Pilosula, then isolated its refinedpolysaccharide CPPw and obtained the Codonopsis Pilosula polysaccharides CPPw1for thefirst time. This thesis compared CPPw and CPPw1for their antitumor and immuneregulation function in vivo and in vitro, the results suggest that the polysaccharidecomponents separated from Codonopsis pilosula can directly inhibit the activity of tumorcells, and the anti-tumor effect of CPPw is related to regulating immune function, includingstimulate proliferation of spleen cells, enhance the phagocytic function of macrophages andinduce macrophage activation for generating NO. The mechanism of CPPw1is still not clear,and it needs to be further studied.
桔梗为桔梗科桔梗属植物桔梗Platycodon gradiflorum（Jacq.）A.DC.的干燥根。研究表明，桔梗具有抗炎镇痛、祛痰止咳、扩张血管、解热镇静、降血糖及增强人体免疫力等广泛的药理作用。本论文首先考察了桔梗主要活性物质桔梗皂苷的提取工艺，并对其进行了进一步的分离；其次研究了桔梗总皂苷（TP）、桔梗次皂苷（TPS）及桔梗皂苷D（PD）的平喘和抗肝纤维化活性。本论文首次采用了液-质联用技术对桔梗皂苷D进行了分析。首次研究了桔梗提取物对人体气管平滑肌细胞增殖的影响，对桔梗的平喘机制就体外细胞水平进行了探讨，实验结果提示了桔梗皂苷活性部位可以直接干预气道重构中气管平滑肌细胞的增殖、肥大，进而可能减少分泌炎性介质，为桔梗预防、治疗哮喘提供了新的理论依据。首次就TP、TPS和PD对肝损伤的保护作用在分子水平进行了深入对比，实验结果提示了桔梗皂苷类活性物质对四氯化碳诱导肝纤维化进程的保护机制可能是与其能够在体内阻止肝脏炎症、抗脂质过氧化反应和及时调节、清除自由基等因素有关。
党参为桔梗科党参属植物党参Codonopsis pilosula（Franch.）Nannf.的干燥根。其性味甘平、无毒，具有补中益气、活血化淤等功效，可用于治疗虚喘咳嗽，脾肺虚弱等症状。本论文考察了党参主要有效成分党参多糖的提取工艺，进一步从其精制多糖CPPw中分离并首次得到了党参多糖CPPw1。首次对比研究了CPPw和CPPw1在体内和体外对抗肿瘤和免疫功能调节的作用，实验结果提示了党参多糖所分离的各组分能够直接抑制肿瘤细胞活性，其中CPPw的抗肿瘤作用与调节相关免疫功能有关，包括刺激脾细胞增殖、增强巨噬细胞吞噬功能以及促使巨噬细胞活化，产生NO等。目前CPPw1的作用机制尚不明确，有待进一步的研究。
Campanulaceae plant includes70generas, and has about2000species, which accordingto fruit and anatomical characteristics, can be divided into five subfamilies, widelydistributes in the world. China has17generas and about150species, most of them belongsto Campanuloideae and distributes in the area of southwest. Campanuloideae mainly usedfor medicinal, such as traditional Chinese medicine Platycodonis Radix, CodonopsisPilosula, Adenophora stricta and Lobelia chinensis. Most plants of this subfamily containsaponins bioactive substances, such as Platycodin of Platycodon, Glehnia root saponin ofAdenophora, etc. All of them have effects on relieving a cough, reducing phlegm, moistinglungs and so on; in addition they also contain many kinds of Carbohydrate substances, suchas inulin, with the effects of invigorating the spleen, nourishing and fluiding. PlatycodonisRadix and Codonopsis Pilosula are nontoxic, their plants can also be edible.
Platycodonis Radix and Codonopsis Pilosula not only contain saponins,polysaccharides and other common pharmacologically active ingredient, also include avariety of amino acids, metal elements, vitamins and other active substances. Peopledeveloped a series of products, such as compound preparations, nutrition and health careproducts, which can use its medicinal value and also can play its nutrition and health careefficacy. With constantly in-depth study on pharmacological effects of Platycodonis Radixand Codonopsis Pilosula, new Chinese medicine can be further developed in clinicalapplications, new medicinal uses can be explored, the related health products research can bespeeded up, which makes resource advantage to play a greater role in the social andeconomic benefits.
Although it has some research reports in the various aspects, but the depth and breadthof them still not enough: such as the reported studies on effective parts of the monomer are less; the mechanism of the active ingredients that have not yet completely be clear; the basicof their pharmacological activities are not clear enough, which is to be further in-depth studyin the future. In light of the present with their applications in China's pharmaceutical market,the thesis thinks that, it is not only necessary and possible in exploring the basic traditionalChinese medicine resources, developing their new uses, and also has a broad prospects inmarketing.
This thesis, after the single factor experiments, through four factors and three levels[L9(34)] of the orthogonal experiments, determined the extraction methods of total platycodinand crude Codonopsis Pilosula polysaccharide. The extraction process of total platycodin is:dried pieces of Platycodonis Radix extracted for2times with petroleum ether at60～90℃,each time lasts2h for removing the fat-soluble impurities, rotary evaporation and recovery ofpetroleum ether; after extraction of dry dregs with ethanol refluxing method, rotaryevaporation recovery of ethanol, then placed in a water bath, steaming to dry extract.Dissolving the obtained extract in distilled water, extracting with water saturated butanol,rotary evaporation recovery of butanol when positive butanol near colorless, oven drying theresidue at105℃. The optimal conditions of extraction was: At90℃, solid-liquid ratio is1:8,80%ethanol refluxing extracting for3times, each time lasts1h.The average rate of totalsaponins is2.66%.
The extraction process of crude Codonopsis pilosula polysaccharide is: dried pieces ofCodonopsis pilosula extracted3times with petroleum ether at60～90℃, each time lasts6hfor removing the fat-soluble impurities, rotary evaporation and recovery of petroleum ether;overnight the dry dregs with80%ethanol soak, and extracted for2times, each time lasts2hfor removing the alcohol-soluble impurities, rotary evaporation and recovery of ethanol; thenextract with water in the way of refluxing, rotary evaporation concentrating, adding5timesthe volume of different concentrations of ethanol overnight static at4℃, centrifugalcollection polysaccharide precipitation, drying in vacuum. The optimal conditions ofextraction was: At100℃, solid-liquid ratio was1:10, extracting for2times with the hotwater, each time lasts3h. The average rate of crude Codonopsis pilosula polysaccharide is23.38%.
In determining the extraction method based on, this thesis investigated the purificationmethods of platycodin D and CPPw1. The separation method of total platycodin is: wetpacking in column, column height is60cm, dry sample is24.7g, the sample in the silica gelcolumn is about3cm high, under atmospheric pressure with different ratio of ethyl acetate,methanol eluent gradient elution. Through the TLC view, combined the stream with the sameRf value, purified repeatedly in reverse phase silica gel column Lobar Rp-8with methanoland water as mobile phase, recrystalled with methanol. In addition, analysised the purifiedplatycodin D with TLC, HPLC-ELSD, LC-MS. According to the calculation of crude drugs,the yield rate of platycodin D is0.19%.
The separation method of crude Codonopsis pilosula polysaccharide is: firstly removeprotein by using sevag method to get the refined Codonopsis pilosula polysaccharide (CPP),then purified in the DEAE-cellulose column (2.6×50cm) and Sephadex G-75column(2.6×100cm), the yield rate of CPPw1was0.20%. After determining the polysaccharidecontent of the polysaccharide fractions by phenol sulfuric acid method, the purity ofpolysaccharide and molecular weight distribution by high performance size exclusionchromatography (HPSEC) method, we finally found that the CPPw1is a homogeneouspolysaccharide composition, its molecular weight is256000Da. For the further study ofpolysaccharide in nature, using gas chromatographic method for the determination of themonosaccharide composition of CPPw1, we finally found that CPPw1is composed ofrhamnose, arabia, fructose, glucose, and its molar percentage is:23:27:9:11.
Extracting the main effective component from Chinese herbal medicine is thefoundation of a research. In the thesis we first using the organization block posted methodsuccessfully separated and cultured human ASMCs, by morphological observation andα-actin immunocytochemical detection, more than99%of cells are human ASMCs. HumanASMCs in vitro in the level of cells for the study of bronchial asthma has provided aneffective platform for the biological behavior, through the cultivation of human ASMCs canbe a means of its pathogenesis and prevention research.
The total platycodin (TP) extracted from Platycodonis Radix (0mg/L、10mg/L、50mg/L、100mg/L) with dexamethasone (DXM) as positive control (1mg/L) were diluted with serum-free DMEM, which is used to culture the human ASMCs. By using the MTTmethod and cells counting method for detecting, we found that small doses of celandineactive substances that could inhibit the proliferation of human ASMCs, and in a time andconcentration dependent.
Secondly, this thesis used the model on carbon tetrachloride induced liver injury in micewhich was based on the discussiong about the liver fibrosis in the etiology and mechanisms.After giving the drugs for1week, through the indexes determination of liver function (ALT,AST) and oxidation (MDA, SOD), inflammatory (IL-6), the experiments analysised theseverity of liver damage comprehensively by reflecting from different aspects for totalplatycodin (TP), total prosapogenins (TPS) and platycodin D (PD) against hepatic fibrosisand its mechanism. The experimental results compared with control group,6h afteradministration, showed that the ALT, AST, MDA and IL-6of the mice in the model groupincreased, SOD decreased significantly, which explains that the model description wassucceed; compared with the model group, the ecperiment showed that the TP can decreasethe levels of ALT, AST, MDA and IL-6, increased level of SOD, the TPS can markedlydecrease in ALT, MDA and IL-6levels, increased in SOD level, but the effect in decreasingthe AST level was not obvious; the PD decreased the levels of ALT and AST was inverselyproportional to the giving doses, its effect in increase of SOD level was also not obvious.
Finally, study comparatively about the antitumor and immune regulation function of theobtained Codonopsis pilosula polysaccharide CPPw and CPPw1in vivo and in vitro. Theexperimental results showed that, Codonopsis pilosula polysaccharide CPPw and CPPw1can well suppress tumor growth in vivo, in which CPPw can increase the immunity organsweight of mice spleen, thymus; spleen cells stimulated with ConA and LPS for combination,which could induce the proliferation of T and B lymphocyte; enhanced peritonealmacrophages phagocytosis, which was dose dependent, and stimulated the activation ofmacrophage for producing NO. The effects of CPPw1were not significant.
This thesis firstly investigated the extraction technology about platycodin which is themain active substance of Platycodonis Radix, then carried on its further separation; secondlystudies the antiasthmatic and anti hepatic fibrosis activity about total platycodin (TP), total prosapogenins (TPS) and platycodin D (PD). In this paper, platycodin D were analyzed bythe way of LC-MS for the first time. And also study on proliferation of human airwaysmooth muscle cells about the extract of Platycodonis Radix for the first time, discussed oncells level about its antiasthmatic mechanism in vitro, the results suggest that platycodinactive site can directly intervention in airway remodeling about proliferation andhypertrophy of airway smooth muscle cells, and may reduce the secretion of inflammatorymediators, which provides a new theoretical basis for Platycodonis Radix to prevent andtreat asthma. This thesis for the first time compared thoroughly on liver injury protectiveeffects at the molecular level about TP, TPS and PD, the results suggest that the mechanismof Platycodon saponins bioactive substances on protection of carbon tetrachloride-inducedliver fibrosis may be related to prevent liver inflammation, resisting lipid peroxidationreaction and timely regulation, scavenging of free radicals and other factors in vivo.
In addition, this thesis investigated the extraction technology about polysaccharidewhich is the main effective component of Codonopsis Pilosula, then isolated its refinedpolysaccharide CPPw and obtained the Codonopsis Pilosula polysaccharides CPPw1for thefirst time. This thesis compared CPPw and CPPw1for their antitumor and immuneregulation function in vivo and in vitro, the results suggest that the polysaccharidecomponents separated from Codonopsis pilosula can directly inhibit the activity of tumorcells, and the anti-tumor effect of CPPw is related to regulating immune function, includingstimulate proliferation of spleen cells, enhance the phagocytic function of macrophages andinduce macrophage activation for generating NO. The mechanism of CPPw1is still not clear,and it needs to be further studied.
引文
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