竹节参药效物质及药材质量分析研究
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摘要
竹节参(Rhizoma Panax japonicus)系五加科人参属植物竹节参Panaxjaponicus C.A.Mey.的干燥呈竹鞭状根茎,是我国民间常用中草药,收载于《中国药典》(2005年版一部),具有滋补强壮、活血散瘀、消肿止痛等作用,常用于病后虚弱,劳嗽咯血,风湿肿痛等。现代药理研究表明,竹节参具有抗炎、免疫调节、抗肿瘤、抗心肌缺血等方面药理活性。
本论文通过对竹节参不同部位进行药理活性筛选,明确了竹节参主要药理活性部位为竹节参总皂苷。同时,对竹节参总皂苷进行提取、分离,并对其工艺进行了系统考察。对竹节参总皂苷的药理作用进行了较系统研究,并探讨了其作用机制,为竹节参药用价值深度开发奠定坚实基础。
采用二甲苯所致小鼠耳肿胀及弗氏完全佐剂所致大鼠足肿胀模型,研究竹节参抗炎活性。结果显示,竹节参总皂苷可显著抑制二甲苯所致的小鼠耳肿胀及大鼠足肿胀率,且竹节参总皂苷高剂量组的抑制作用优于阳性药白芍总苷组,说明竹节参总皂苷具有较显著的抗炎活性。
采用大鼠佐剂性关节炎(AA)及胶原诱导性关节炎模型,研究竹节参总皂苷对完全弗氏佐剂所致大鼠关节炎模型的作用,及对体外培养胶原诱导的大鼠关节炎滑膜细胞的抑制作用,研究结果显示,竹节参可显著抑制AA大鼠足肿胀,降低AA大鼠血清中TNF-α与IL-1β的表达水平,并与剂量存在着相关性;竹节参总皂苷可显著抑制模型大鼠滑膜细胞的增殖与诱导滑膜细胞凋亡,说明竹节参总皂苷对大鼠关节炎模型有一定的治疗作用,其主要作用机制与抑制滑膜细胞增殖,调节体内TNF-α与IL-1β等炎症因子的水平相关。
采用整体实验与细胞实验,研究竹节参总皂苷对S180肉瘤、腹水瘤模型小鼠的抑制作用,以及对HL60白血病细胞增殖与分化作用,结果显示,竹节参总皂苷对荷瘤小鼠的肿瘤生长有显著的抑制作用,不仅显著改善小鼠生存质量,还能提高其生存期。竹节参总皂苷对HL60白血病细胞增殖有较显著的抑制作用,并能诱导其向粒细胞分化。
采用异丙肾上腺素所致大鼠心肌缺血模型研究竹节参总皂苷对心肌缺血的保护作用,结果显示,竹节参总皂苷能抑制ISO所致心肌缺血大鼠血清中LDH和CK水平的升高,并显著降低血清中MDA含量,升高SOD含量,说明其抗心肌缺血的作用机制与提高机体清除氧自由基的能力和增强机体抗脂质过氧化的能力有关。
以药理活性筛选为指导,并以传统分离和泡沫分离两种不同方法分别对竹节参中总皂苷的提取和纯化工艺进行了系统的考察,优选出最佳提取工艺条件。对竹节参有效部位的化学成分进行了研究,利用大孔吸附树脂、硅胶柱层析以及制备高效液相等技术手段进行分离,根据化合物的光谱数据鉴定其结构。从中初步分离得到了3个化合物,鉴定其中2个,分别为:人参皂苷Rd(Ginsenoside Rd)和人参皂苷Re(Ginsenoside Re)。
药理筛选结果显示竹节参药理活性部位为竹节参总皂苷,根据文献记载其苷元主要为齐墩果酸。为了有效控制竹节参质量,以比色法测定竹节参中总皂苷的含量。测定结果表明,不同批次的竹节参中,总皂苷含量均较高,达到16%以上。以HPLC法测定竹节参中齐墩果酸的含量:采用氯仿-25%盐酸溶剂体系对竹节参皂苷提取液进行水解,甲醇-水-冰醋酸-三乙胺(90:10:0.04:0.02)为流动相。结果显示竹节参中游离型齐墩果酸很少,结合型齐墩果酸占总齐墩果酸的99%以上。
为了更好的控制竹节参的质量,采用HPLC方法,并采用UV、ELSD两种不同检测方法建立了竹节参的指纹图谱,结果显示该方法灵敏度高,重现性好,从而完善了2005版《中华人民共和国药典》收载该中药的质量标准,为有效控制竹节参药材的质量提供了科学依据。
Rhizoma Panax japonicus is the dried rhizome of Panax japonicus, belonging to Panax species of Araliaceae.As a traditional Chinese medicine,it is widely used as a tonics.Meanwhile,it possesses many pharmacological effects,such as antiinflammation,immunoiogicai regulation,anti-tumor, protecting myocardial ischemia,activating blood circulation,detumescence and analgesia,etc.Clinically,it is commonly used in asthenia,cough and hemoptysis,rheumatism.
This thesis focused on the screening of active fractions,observation of several pharmacological effects,elucidating mechanism of principal effects, establishment of qualitiy evaluation methods for crude drugs and optimization of producing procedure of crude active products,which lay a solid foundation for the further development of the titled medicine.
Its anti-inflammatory actions were observed in vivo against mice ear oedama induced by dimethylbenzene and mice paw swelling induced by Freund's complete adjuvant.Results showed that TSPJ significantly inhibited dimethlybenzene-induced ear oedama and Freund's complete adjuvant-induced paw swelling in mice,and the inhibitory effect of TSPJ at a high dose was superior to that of total glucosides of paeony,i.e.TSPJ had significantly anti-inflammatory actions.
The curative effects of TSPJ towards adjuvant arthritis and collagen-induced arthritis were observed in adjuvant arthritis rats induced by complete Freunds adjuvant in vivo and inhibitory action of rats induced by collagen on arthritis synovial cell in intro.Results showed that TSPJ decreased Freund's complete adjuvant-induced paw swelling and the levels of TNF-a and IL-1βin serum of AA rats,which was dose-related;in addition,the significantly inhibitory effects of the samples in the proliferation of synovial cells and the apoptosis rate of rats.The results above inferred that the anti-inflammatory actions of TSPJ be related to the regulation of the the level of TNF-a and IL-1β.
The thesis involved in the observation of the effects of TSPJ on a solid tumor、an ascites tumor and proliferation and differentiation of human leukemic cells(HL-60) in vivo and in vitro on mice.The experiments showed that TSPJ markedly inhibited the growth of S-180 tumor,improved the life quality and prolonged the life-span of the mice.The results revealed that TSPJ markedly inhibited the growth of HL-60,and invoked differentiation of granular cells.
The protection of TSPJ on the heart muscle ischemia of rats were studied. TSPJ significantly decreased the level of cytokine LDH,CK,MDA in the serum of rats,and promoted the superoxide dismutase in MI rats.It was concluded that the effects above were paralleled with its capacities to remove active oxygen free radicals and inhibit lipid peroxidation.
Based on pharmacological activity screening,two extraction methods were adopted to separate and purify total saponins of Panax japonicus,i.e. traditional method and foam separation.The latter was a newly established procedure to separate saponins and related parameters were optimized.The chemical constituents of the titled plant were obtained by column chromatography and HPLC over macroporous adsorptive resins and silica gel. Three compounds were obtained,and two of them were identified as ginsenoside Rd and ginsenoside Re,respectively.
According to reports and our experimental results,ginsenosides accounted for most of the activities of Panax japonicus.In order to evaluate the quality of crude drugs,a photometric method was established to determine the content of TSPJ in Rhizoma Panax japonicus with vanillin-perchloric acid as chromogenic agent and detection wavelength at 552 nm.The results showed that the contents of TSPJ were more than 16%in different batches of Panax japonicus.A HPLC method was used to determine the content of oleanolic acid with methanol:water:glacial acetic acid:triethylamine= 90:10:0.04:0.02 as the mobile phase.The results suggested that the content of free oleanolic acid was much less that of combined oleanolic acid,with the latter being up to 99%.
Two HPLC methods,i.e.HPLC-UV and HPLC-ELSD,were established to obtain the HPLC fingerprint chromatograms of Panax japonicus. Experimental results showed that both methods exhibited high sensitivity and good reproducibility,which improved the quality standards of this medicine recorded on 2005 Edition of "People's Republic of China Pharmacopoeia".
本论文通过对竹节参不同部位进行药理活性筛选,明确了竹节参主要药理活性部位为竹节参总皂苷。同时,对竹节参总皂苷进行提取、分离,并对其工艺进行了系统考察。对竹节参总皂苷的药理作用进行了较系统研究,并探讨了其作用机制,为竹节参药用价值深度开发奠定坚实基础。
采用二甲苯所致小鼠耳肿胀及弗氏完全佐剂所致大鼠足肿胀模型,研究竹节参抗炎活性。结果显示,竹节参总皂苷可显著抑制二甲苯所致的小鼠耳肿胀及大鼠足肿胀率,且竹节参总皂苷高剂量组的抑制作用优于阳性药白芍总苷组,说明竹节参总皂苷具有较显著的抗炎活性。
采用大鼠佐剂性关节炎(AA)及胶原诱导性关节炎模型,研究竹节参总皂苷对完全弗氏佐剂所致大鼠关节炎模型的作用,及对体外培养胶原诱导的大鼠关节炎滑膜细胞的抑制作用,研究结果显示,竹节参可显著抑制AA大鼠足肿胀,降低AA大鼠血清中TNF-α与IL-1β的表达水平,并与剂量存在着相关性;竹节参总皂苷可显著抑制模型大鼠滑膜细胞的增殖与诱导滑膜细胞凋亡,说明竹节参总皂苷对大鼠关节炎模型有一定的治疗作用,其主要作用机制与抑制滑膜细胞增殖,调节体内TNF-α与IL-1β等炎症因子的水平相关。
采用整体实验与细胞实验,研究竹节参总皂苷对S180肉瘤、腹水瘤模型小鼠的抑制作用,以及对HL60白血病细胞增殖与分化作用,结果显示,竹节参总皂苷对荷瘤小鼠的肿瘤生长有显著的抑制作用,不仅显著改善小鼠生存质量,还能提高其生存期。竹节参总皂苷对HL60白血病细胞增殖有较显著的抑制作用,并能诱导其向粒细胞分化。
采用异丙肾上腺素所致大鼠心肌缺血模型研究竹节参总皂苷对心肌缺血的保护作用,结果显示,竹节参总皂苷能抑制ISO所致心肌缺血大鼠血清中LDH和CK水平的升高,并显著降低血清中MDA含量,升高SOD含量,说明其抗心肌缺血的作用机制与提高机体清除氧自由基的能力和增强机体抗脂质过氧化的能力有关。
以药理活性筛选为指导,并以传统分离和泡沫分离两种不同方法分别对竹节参中总皂苷的提取和纯化工艺进行了系统的考察,优选出最佳提取工艺条件。对竹节参有效部位的化学成分进行了研究,利用大孔吸附树脂、硅胶柱层析以及制备高效液相等技术手段进行分离,根据化合物的光谱数据鉴定其结构。从中初步分离得到了3个化合物,鉴定其中2个,分别为:人参皂苷Rd(Ginsenoside Rd)和人参皂苷Re(Ginsenoside Re)。
药理筛选结果显示竹节参药理活性部位为竹节参总皂苷,根据文献记载其苷元主要为齐墩果酸。为了有效控制竹节参质量,以比色法测定竹节参中总皂苷的含量。测定结果表明,不同批次的竹节参中,总皂苷含量均较高,达到16%以上。以HPLC法测定竹节参中齐墩果酸的含量:采用氯仿-25%盐酸溶剂体系对竹节参皂苷提取液进行水解,甲醇-水-冰醋酸-三乙胺(90:10:0.04:0.02)为流动相。结果显示竹节参中游离型齐墩果酸很少,结合型齐墩果酸占总齐墩果酸的99%以上。
为了更好的控制竹节参的质量,采用HPLC方法,并采用UV、ELSD两种不同检测方法建立了竹节参的指纹图谱,结果显示该方法灵敏度高,重现性好,从而完善了2005版《中华人民共和国药典》收载该中药的质量标准,为有效控制竹节参药材的质量提供了科学依据。
Rhizoma Panax japonicus is the dried rhizome of Panax japonicus, belonging to Panax species of Araliaceae.As a traditional Chinese medicine,it is widely used as a tonics.Meanwhile,it possesses many pharmacological effects,such as antiinflammation,immunoiogicai regulation,anti-tumor, protecting myocardial ischemia,activating blood circulation,detumescence and analgesia,etc.Clinically,it is commonly used in asthenia,cough and hemoptysis,rheumatism.
This thesis focused on the screening of active fractions,observation of several pharmacological effects,elucidating mechanism of principal effects, establishment of qualitiy evaluation methods for crude drugs and optimization of producing procedure of crude active products,which lay a solid foundation for the further development of the titled medicine.
Its anti-inflammatory actions were observed in vivo against mice ear oedama induced by dimethylbenzene and mice paw swelling induced by Freund's complete adjuvant.Results showed that TSPJ significantly inhibited dimethlybenzene-induced ear oedama and Freund's complete adjuvant-induced paw swelling in mice,and the inhibitory effect of TSPJ at a high dose was superior to that of total glucosides of paeony,i.e.TSPJ had significantly anti-inflammatory actions.
The curative effects of TSPJ towards adjuvant arthritis and collagen-induced arthritis were observed in adjuvant arthritis rats induced by complete Freunds adjuvant in vivo and inhibitory action of rats induced by collagen on arthritis synovial cell in intro.Results showed that TSPJ decreased Freund's complete adjuvant-induced paw swelling and the levels of TNF-a and IL-1βin serum of AA rats,which was dose-related;in addition,the significantly inhibitory effects of the samples in the proliferation of synovial cells and the apoptosis rate of rats.The results above inferred that the anti-inflammatory actions of TSPJ be related to the regulation of the the level of TNF-a and IL-1β.
The thesis involved in the observation of the effects of TSPJ on a solid tumor、an ascites tumor and proliferation and differentiation of human leukemic cells(HL-60) in vivo and in vitro on mice.The experiments showed that TSPJ markedly inhibited the growth of S-180 tumor,improved the life quality and prolonged the life-span of the mice.The results revealed that TSPJ markedly inhibited the growth of HL-60,and invoked differentiation of granular cells.
The protection of TSPJ on the heart muscle ischemia of rats were studied. TSPJ significantly decreased the level of cytokine LDH,CK,MDA in the serum of rats,and promoted the superoxide dismutase in MI rats.It was concluded that the effects above were paralleled with its capacities to remove active oxygen free radicals and inhibit lipid peroxidation.
Based on pharmacological activity screening,two extraction methods were adopted to separate and purify total saponins of Panax japonicus,i.e. traditional method and foam separation.The latter was a newly established procedure to separate saponins and related parameters were optimized.The chemical constituents of the titled plant were obtained by column chromatography and HPLC over macroporous adsorptive resins and silica gel. Three compounds were obtained,and two of them were identified as ginsenoside Rd and ginsenoside Re,respectively.
According to reports and our experimental results,ginsenosides accounted for most of the activities of Panax japonicus.In order to evaluate the quality of crude drugs,a photometric method was established to determine the content of TSPJ in Rhizoma Panax japonicus with vanillin-perchloric acid as chromogenic agent and detection wavelength at 552 nm.The results showed that the contents of TSPJ were more than 16%in different batches of Panax japonicus.A HPLC method was used to determine the content of oleanolic acid with methanol:water:glacial acetic acid:triethylamine= 90:10:0.04:0.02 as the mobile phase.The results suggested that the content of free oleanolic acid was much less that of combined oleanolic acid,with the latter being up to 99%.
Two HPLC methods,i.e.HPLC-UV and HPLC-ELSD,were established to obtain the HPLC fingerprint chromatograms of Panax japonicus. Experimental results showed that both methods exhibited high sensitivity and good reproducibility,which improved the quality standards of this medicine recorded on 2005 Edition of "People's Republic of China Pharmacopoeia".
引文
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