六棱菊标准提取物和抗炎胶囊抗炎保肝作用研究
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摘要
六棱菊(L.alata)作为一种民间药用植物,具有清热解毒、抗菌消炎等功效,疗效甚著。该植物长期广泛应用于民间和中医实践中,且资源丰富。以“六棱菊”为主要成分的制剂在临床上治疗感冒、扁桃体炎、腮腺炎、急性呼吸道感染等取得了显著疗效,具有较高的研究和开发价值。目前国内对六棱菊的药理和临床研究主要停留在其粗提物上,缺乏药效物质基础相关研究,而国外有关这种植物的研究报道很少。总之,国内外尚无这种植物抗炎保肝作用系统的研究报道。本课题通过抗炎活性跟踪分离和标化这种植物的有效部位,并对其标准提取物抗炎保肝作用进行系统研究,以期为该植物的临床应用和深入开发打下基础。
“抗炎胶囊”是一种民间植物药的有效部位,“抗炎胶囊的抗炎保肝作用研究”为本研究室与制药企业的合作开发项目。本研究考察了“抗炎胶囊”的抗炎保肝作用,为该胶囊的临床应用提供了部分依据。
第一部分 六棱菊抗炎保肝作用研究
采用“二甲苯致小鼠耳肿胀模型”跟踪分离并比较六棱菊不同药用部位、不同容剂及不同工艺提取物的抗炎活性,结合急性毒性试验结果最终确定六棱菊的抗炎活性部位,并对该部位进行标化。急性毒性测定结果显示六棱菊提取物的LD_50大于7.5 g/kg。采用比色法测定六棱菊提取物的总酚含量为56.5 g GAE/100 g提取物。采用HPLC测定其主要成分为二咖啡酰奎尼酸,二咖啡酰奎尼酸在提取物中的含量为53%。采用植化方法从提取物中分离鉴定了3种二咖啡酰奎尼酸化合物(3,4-二咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸和4,5-二咖啡酰奎尼酸)。
1 六棱菊提取物抗炎作用及其机理研究
运用代表炎症发展3个阶段的8个动物模型和1个免疫性炎症模型考察了六棱菊提取物的抗炎作用。这些模型分别是:(1)代表炎症反应第一时相的二甲苯致小鼠耳肿胀模型、角叉菜胶致大鼠足肿胀模型、甲醛致大鼠足肿胀模型和醋酸致小鼠毛细血管通透性增高模型,主要用来评价药物对炎性物质引起血管通透性增加的抑制作用;(2)代表炎症反应第二时相的CMC-Na诱导大鼠皮下气囊白细
Laggera alata, as a medicinal plant, possesses potent anti-inflammatory, antibacterial, antifebrile and alexipharmic actions etc. It has been used in folk and Chinese medicine for a few hundred years. The plant is widely distributed in china. The preparations consisting of L alata have remarkable curative effect on the rheum, tonsillitis, parotitis and acute respiratory tract infection etc. It suggests that the plant has great potentials in research and development. The researches on the L alata was only carried out in its crude extract in china. And it is short of the studies in its active components. Furthermore, it is few on the reports of the plant in other countries. In general, no systematic bioactivity studies were carried out on the herb. To provide the pharmacological evidence of the medicine in the clinical use and further development, the anti-inflammatory activity fraction of the plant was isolated by bioactivity-guided and standardized, and its anti-inflammatory and hepatoprotective actions were systematic studied in the current thesis.Kangyan Capsule is the anti-inflammatory activity faction of one folk herb. "Studies of the anti-inflammatory and hepatoprotective actions of Kangyan Capsule" is a cooperation development project with the pharmaceutical company. The project validated the anti-inflammatory and hepatoprotective activities of Kangyan Capsule, thereby providing its part evidence in the clinical use.Part I The anti-inflammatory and hepatoprotective effects of L alataThe anti-inflammatory fraction of L. alata was isolated by bioactivity-guided using the xylene-induced ear oedema model and by toxicity-guided with acute toxicity test. Acute toxicity test revealed that L. alata extract (namely its anti-inflammatory fraction) up to an oral dose of 7.5 g/kg body weight was almost nontoxic in mice. The phenolic content of L. alata extract (56.5 g GAE/100 g extract) was determined by the
colorimetry of Folin Ciocalteu. HPLC assay indicated that dicaffeoylquinic acids are the main components of the extract and its content is 53%. Moreover, three dicaffeoylquinic acids from the extract such as 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were isolated and authenticated.1 The anti-inflammatory and its action mechanism of I. alata extractThe anti-inflammatory effect L. alata extract was evaluated by one immune inflammation model and eight other inflammation models that represent the three phage of inflammation development. These models are listed as follows: the xylene-induced ear oedema, carrageenan-induced paw oedema and acetic acid-induced vascular permeability models were used to evaluate the effect of this extract on vascular permeability increased of the first phase of inflammation;the effect of the extract on leukocyte migration of the second phase of inflammation was determined by the CMC-Na induced air pouch leukocyte migration and peritonitis tests in rats;the carrageenan induced air pouch granuloma and cotton pellet-induced granuloma in rats were employed to assess the effect of this extract on granuloma proliferation of the third phase of inflammation;the adjuvant-induced developing arthritis in rats, as a immune inflammation model, was utilized to evaluate the effect of the extract on extrinsic antigen- mediated immune injury. The results showed that L. alata extract at a dose of 100 mg/kg possessed pronounced inhibitory effect on all inflammation models tested. Furthermore, L. alata extract did not influence on the thymus index, adrenal index and spleen index of the animals in the test.The effect of L. alata extract on intrinsic anti-inflammatory system (the hypothalamus - pituitary - adrenal system) was assessed using xylene-induced ear oedema test in mice with adrenal gland removed and by measuring of the concentrations of cholesterol and vitamin C in the adrenal glands of rats. The results indicated that the extract had still potent inhibitory action on xylene-induced ear oedema test in mice with
adrenal gland removed and failed to influence on the concentrations of cholesterol and vitamin C in the adrenal glands and adrenal index. Thereby suggesting the anti-inflammatory mechanism of L. alata extract might not be related to the pituitary -adrenal system.The effect of L. alata extract on inflammatory mediators and other parameters was assessed by carrageenan-induced rat pleurisy model. The result showed that the extract possesses potent inhibitory effect on carrageenan-induced rat pleurisy and its action mechanisms are probably associated with the inhibition of prostaglandin and NO formation, the influence on the antioxidant systems, and the suppression of lysozyme release. The effect of L. alata extract on the cytokines was evaluated by the linoleic acid-LPS-induced acute lung damage test in rats. The results of immunohistochemistry indicated that the extract at doses of 50,100 and 200 mg/kg decreased the expression of ICAM-1 and inhibited the activation of of NF-kB p65 in the bronchial epithelium tissue of the rats with lung injury. Thus suggesting that interfering the NF-kB path and inhibiting the expression of ICAM-1 may be one of the anti-inflammatory mechanisms of I. alata extract.2 The hepatoprotective effect of I. alata extractThe hepatoprotections of L alata extract in vitro and in vivo were evaluated by four primary hepatocye injury models and six animal liver damage tests, respectively. The results showed that the extract at concentrations of 10-100 μg/ml significantly reduced cellular leakage of hepatocyte AST/ALT caused by H2O2, D-GalN, TAA and CCI4. Moreover, the extract afforded much stronger protection than the reference drug silybin. At doses of 50, 100, 200 mg/kg, L. alata extract have protection on D-GalN-, CCI4-, acetamidophenol-, BCG-LPS- and DHBV-induced liver damage. And it still improved liver histopathology damage in the different degree. It suggests that L. alata extract possesses hepatoprotection in vitro and in vivo.
Part II The anti-inflammatory and hepatoprotective effects of Kangyan CapsuleAcute toxicity, anti-inflammation and hepatoprotection of Kangyan Capsule were investigated. Acute toxicity test revealed that Kangyan Capsule up to an oral dose of 8.0 g/kg body weight was almost nontoxic in mice. The anti-inflammatory and hepatoprotective effects of Kangyan Capsule were described as follows.1 The anti-inflammatory effect of Kangyan CapsuleFour acute and chronic inflammatory models were employed to evaluate the anti-inflammatory effect of Kangyan Capsule. The results showed that the extract, at doses of 50, 100 and 200 mg/kg, inhibited significantly xylene-induced mouse ear oedema, carrageenan-induced rat paw oedema, acetic acid-induced mouse vascular permeability increased and cotton pellet-induced mouse granuloma. Thus suggesting the inhibitory effects of Kangyan Capsule on the different phases of inflammation development. These data provided the scientific evidences of Kangyan Capsule in the treatment of inflammatory disorders.2 The hepatoprotective effect of Kangyan Capsule in vitroThe hepatoprotective effect of Kangyan Capsule was evaluated with H2O2-, D-GalN-, TAA-, and CCLj-induced hepatocytes injury models in primary cultured neonatal rat hepatocytes. The cell viability was expressed with protection (%) and proliferation index. The results indicated that Kangyan Capsule possesses potent hepatoprotective activity on the hepatocyte injury models. Moreover, the extract afforded much stronger protection than the positive drug silybin. Thereby suggesting its activity against oxidative injury induced by the chemicals. These results provided the partial evidences of Kangyan Capsule as hepatoprotecive drug in clinic.In summary, this thesis validated the anti-inflammatory and hepatoprotective effects of the standardized extract from L. data, and studied the active components and
the primary anti-inflammatory mechanism of L. alata extract. These data provided the academic evidence for the clinical use and further development of the medicinal plant. Furthermore, the in vivo anti-inflammation and in vitro hepatoprotection of Kangyan Capsule were validated. However, the anti-inflammatory and hepatoprotective components and their action mechanisms of L. alata extract and Kangyan Capsule will desiderate to be further clarified.
“抗炎胶囊”是一种民间植物药的有效部位,“抗炎胶囊的抗炎保肝作用研究”为本研究室与制药企业的合作开发项目。本研究考察了“抗炎胶囊”的抗炎保肝作用,为该胶囊的临床应用提供了部分依据。
第一部分 六棱菊抗炎保肝作用研究
采用“二甲苯致小鼠耳肿胀模型”跟踪分离并比较六棱菊不同药用部位、不同容剂及不同工艺提取物的抗炎活性,结合急性毒性试验结果最终确定六棱菊的抗炎活性部位,并对该部位进行标化。急性毒性测定结果显示六棱菊提取物的LD_50大于7.5 g/kg。采用比色法测定六棱菊提取物的总酚含量为56.5 g GAE/100 g提取物。采用HPLC测定其主要成分为二咖啡酰奎尼酸,二咖啡酰奎尼酸在提取物中的含量为53%。采用植化方法从提取物中分离鉴定了3种二咖啡酰奎尼酸化合物(3,4-二咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸和4,5-二咖啡酰奎尼酸)。
1 六棱菊提取物抗炎作用及其机理研究
运用代表炎症发展3个阶段的8个动物模型和1个免疫性炎症模型考察了六棱菊提取物的抗炎作用。这些模型分别是:(1)代表炎症反应第一时相的二甲苯致小鼠耳肿胀模型、角叉菜胶致大鼠足肿胀模型、甲醛致大鼠足肿胀模型和醋酸致小鼠毛细血管通透性增高模型,主要用来评价药物对炎性物质引起血管通透性增加的抑制作用;(2)代表炎症反应第二时相的CMC-Na诱导大鼠皮下气囊白细
Laggera alata, as a medicinal plant, possesses potent anti-inflammatory, antibacterial, antifebrile and alexipharmic actions etc. It has been used in folk and Chinese medicine for a few hundred years. The plant is widely distributed in china. The preparations consisting of L alata have remarkable curative effect on the rheum, tonsillitis, parotitis and acute respiratory tract infection etc. It suggests that the plant has great potentials in research and development. The researches on the L alata was only carried out in its crude extract in china. And it is short of the studies in its active components. Furthermore, it is few on the reports of the plant in other countries. In general, no systematic bioactivity studies were carried out on the herb. To provide the pharmacological evidence of the medicine in the clinical use and further development, the anti-inflammatory activity fraction of the plant was isolated by bioactivity-guided and standardized, and its anti-inflammatory and hepatoprotective actions were systematic studied in the current thesis.Kangyan Capsule is the anti-inflammatory activity faction of one folk herb. "Studies of the anti-inflammatory and hepatoprotective actions of Kangyan Capsule" is a cooperation development project with the pharmaceutical company. The project validated the anti-inflammatory and hepatoprotective activities of Kangyan Capsule, thereby providing its part evidence in the clinical use.Part I The anti-inflammatory and hepatoprotective effects of L alataThe anti-inflammatory fraction of L. alata was isolated by bioactivity-guided using the xylene-induced ear oedema model and by toxicity-guided with acute toxicity test. Acute toxicity test revealed that L. alata extract (namely its anti-inflammatory fraction) up to an oral dose of 7.5 g/kg body weight was almost nontoxic in mice. The phenolic content of L. alata extract (56.5 g GAE/100 g extract) was determined by the
colorimetry of Folin Ciocalteu. HPLC assay indicated that dicaffeoylquinic acids are the main components of the extract and its content is 53%. Moreover, three dicaffeoylquinic acids from the extract such as 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were isolated and authenticated.1 The anti-inflammatory and its action mechanism of I. alata extractThe anti-inflammatory effect L. alata extract was evaluated by one immune inflammation model and eight other inflammation models that represent the three phage of inflammation development. These models are listed as follows: the xylene-induced ear oedema, carrageenan-induced paw oedema and acetic acid-induced vascular permeability models were used to evaluate the effect of this extract on vascular permeability increased of the first phase of inflammation;the effect of the extract on leukocyte migration of the second phase of inflammation was determined by the CMC-Na induced air pouch leukocyte migration and peritonitis tests in rats;the carrageenan induced air pouch granuloma and cotton pellet-induced granuloma in rats were employed to assess the effect of this extract on granuloma proliferation of the third phase of inflammation;the adjuvant-induced developing arthritis in rats, as a immune inflammation model, was utilized to evaluate the effect of the extract on extrinsic antigen- mediated immune injury. The results showed that L. alata extract at a dose of 100 mg/kg possessed pronounced inhibitory effect on all inflammation models tested. Furthermore, L. alata extract did not influence on the thymus index, adrenal index and spleen index of the animals in the test.The effect of L. alata extract on intrinsic anti-inflammatory system (the hypothalamus - pituitary - adrenal system) was assessed using xylene-induced ear oedema test in mice with adrenal gland removed and by measuring of the concentrations of cholesterol and vitamin C in the adrenal glands of rats. The results indicated that the extract had still potent inhibitory action on xylene-induced ear oedema test in mice with
adrenal gland removed and failed to influence on the concentrations of cholesterol and vitamin C in the adrenal glands and adrenal index. Thereby suggesting the anti-inflammatory mechanism of L. alata extract might not be related to the pituitary -adrenal system.The effect of L. alata extract on inflammatory mediators and other parameters was assessed by carrageenan-induced rat pleurisy model. The result showed that the extract possesses potent inhibitory effect on carrageenan-induced rat pleurisy and its action mechanisms are probably associated with the inhibition of prostaglandin and NO formation, the influence on the antioxidant systems, and the suppression of lysozyme release. The effect of L. alata extract on the cytokines was evaluated by the linoleic acid-LPS-induced acute lung damage test in rats. The results of immunohistochemistry indicated that the extract at doses of 50,100 and 200 mg/kg decreased the expression of ICAM-1 and inhibited the activation of of NF-kB p65 in the bronchial epithelium tissue of the rats with lung injury. Thus suggesting that interfering the NF-kB path and inhibiting the expression of ICAM-1 may be one of the anti-inflammatory mechanisms of I. alata extract.2 The hepatoprotective effect of I. alata extractThe hepatoprotections of L alata extract in vitro and in vivo were evaluated by four primary hepatocye injury models and six animal liver damage tests, respectively. The results showed that the extract at concentrations of 10-100 μg/ml significantly reduced cellular leakage of hepatocyte AST/ALT caused by H2O2, D-GalN, TAA and CCI4. Moreover, the extract afforded much stronger protection than the reference drug silybin. At doses of 50, 100, 200 mg/kg, L. alata extract have protection on D-GalN-, CCI4-, acetamidophenol-, BCG-LPS- and DHBV-induced liver damage. And it still improved liver histopathology damage in the different degree. It suggests that L. alata extract possesses hepatoprotection in vitro and in vivo.
Part II The anti-inflammatory and hepatoprotective effects of Kangyan CapsuleAcute toxicity, anti-inflammation and hepatoprotection of Kangyan Capsule were investigated. Acute toxicity test revealed that Kangyan Capsule up to an oral dose of 8.0 g/kg body weight was almost nontoxic in mice. The anti-inflammatory and hepatoprotective effects of Kangyan Capsule were described as follows.1 The anti-inflammatory effect of Kangyan CapsuleFour acute and chronic inflammatory models were employed to evaluate the anti-inflammatory effect of Kangyan Capsule. The results showed that the extract, at doses of 50, 100 and 200 mg/kg, inhibited significantly xylene-induced mouse ear oedema, carrageenan-induced rat paw oedema, acetic acid-induced mouse vascular permeability increased and cotton pellet-induced mouse granuloma. Thus suggesting the inhibitory effects of Kangyan Capsule on the different phases of inflammation development. These data provided the scientific evidences of Kangyan Capsule in the treatment of inflammatory disorders.2 The hepatoprotective effect of Kangyan Capsule in vitroThe hepatoprotective effect of Kangyan Capsule was evaluated with H2O2-, D-GalN-, TAA-, and CCLj-induced hepatocytes injury models in primary cultured neonatal rat hepatocytes. The cell viability was expressed with protection (%) and proliferation index. The results indicated that Kangyan Capsule possesses potent hepatoprotective activity on the hepatocyte injury models. Moreover, the extract afforded much stronger protection than the positive drug silybin. Thereby suggesting its activity against oxidative injury induced by the chemicals. These results provided the partial evidences of Kangyan Capsule as hepatoprotecive drug in clinic.In summary, this thesis validated the anti-inflammatory and hepatoprotective effects of the standardized extract from L. data, and studied the active components and
the primary anti-inflammatory mechanism of L. alata extract. These data provided the academic evidence for the clinical use and further development of the medicinal plant. Furthermore, the in vivo anti-inflammation and in vitro hepatoprotection of Kangyan Capsule were validated. However, the anti-inflammatory and hepatoprotective components and their action mechanisms of L. alata extract and Kangyan Capsule will desiderate to be further clarified.
引文
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