巴戟天抗衰老活性成分及作用机制研究
摘要
巴戟天是我国“四大南药”之一,在我国南方民间被广泛使用,具有补肾阳、壮筋骨、祛风湿等功效。其用途广泛,如治疗阳痿遗精、宫冷不孕、月经不调、风湿痹痛、少腹冷痛、筋骨痿软等。巴戟天主要有蒽醌类、环烯醚萜类、糖类等化合物,药理研究表明其具有调节免疫、抗衰老、抗肿瘤、抗抑郁、壮阳等作用。中医理论认为衰老首先是肾虚,其次是脾虚,再次是气血两虚,所以,补肾、健脾、益气是抗衰老的基本途径,巴戟天具有补肾阳、强筋骨的作用,是抗衰老作用具体体现。现代研究对巴戟天化学成分和药理作用进行了较系统研究,但巴戟天抗衰老作用的物质基础和机理仍不清楚,为阐明巴戟天抗衰老作用的活性成分及作用机制,本课题对巴戟天提取物进行进行抗衰老活性筛选,对活性部位的化学成分进行研究,并应用细胞模型对巴戟天的化学成分进行抗衰老活性筛选,综合评价其抗衰老作用,使巴戟天抗衰老作用得到现代意义的阐明,为巴戟天质量控制、指导合理用药提供科学依据。
本课题考察不同巴戟天提取物对小鼠游泳时间的影响,初步筛选活性部位,采用现代色谱技术对活性部位对其化学成分进行了分离纯化,用化学方法和波谱方法(UV,IR,1H-NMR,13C-NMR,2D-NMR, MS等)鉴定其结构,应用H2O2诱导的小鼠心肌细胞(H9c2(2-1))和人脐静脉内皮细胞(HUVECs)衰老模型对本课题组从巴戟天分离得到的化合物进行筛选,评价其抗衰老作用,并选取活性作用最强的单体化合物进行抗衰老作用机制的研究。用人脐静脉内皮细胞进行作用机制的研究,MTT法测定细胞存活率,以β-半乳糖苷酶活性(染色法)和端粒酶活性(TRAP-银染法)检测细胞衰老,用活性氧荧光试剂盒检测细胞内ROS的生成,实时定量PCR检测细胞内klotho mRNA的表达。
从巴戟天中共分离得到12个化合物,其中6个蒽醌、2个萜类、3个糖类,1个香豆素类化合物。它们分别为：巴戟醇(1),甲基异茜草素-1-甲醚(2),1-羟基-6-羟甲基蒽醌(3),2-醛基蒽醌(4),1-羟基-2-羟甲基蒽醌(5),1-羟基-7-羟甲基蒽醌(6),1,2-二羟基-3-甲基蒽醌(7),东莨菪内酯(8),rotungenic acid (9),蔗果三糖(10),1F-果吠喃糖基奈斯糖(11)、β-D-果吡喃糖-(1→2)-β-D-果呋喃糖(12),通过考察本课题组从巴戟天共分离得到的12个化合物的细胞抗衰老活性,筛选结果显示各单体化合物均能不同程度降低H2O2诱导的内皮细胞衰老,化合物2抗衰老作用最强,所以选用化合物2进行抗衰老作用机制研究。
MTT法结果显示化合物2浓度(3-30μM)正常内皮细胞存活率无明显影响,而100μM化合物2能显著降低内皮细胞的存活率；经H2O2(100μM)处理的HUVECs,β-半乳糖苷酶活性和端粒酶活性显著下降,不同浓度的化合物2(3、10和30 gM)或阳性对照药白藜芦醇(30μM)能显著抑制H2O2(100μM)诱导的端粒酶活性降低；H2O2(100μM)孵育HUVECs 48 h,可明显增强荧光强度(反映活性氧水平),预处理不同浓度的化合物2(3、10和30μM)或阳性对照药白藜芦醇(30μM)能显著抑制H2O2(100μM)诱导的活性氧水平增加；H2O2(100μM)处理HUVECs 48 h, klothomRNA表达无明显变化。预处理不同浓度的化合物2(3、10和30μM)能显著增加klotho mRNA表达,阳性对照药白藜芦醇(30μM)可轻微上调klotho mRNA表达。
本论文分离得到12个化合物,化合物1为一个新化合物,化合物12为首次从巴戟天属植物中分离得到,化合物3、4、6、9为首次从巴戟天分离得到,首次提供化合物3的核磁数据。抗衰老活性筛选结果表明巴戟天抗衰老作用是其各类成分综合作用的结果。抗衰老作用机制研究提示巴戟天单体抗衰老作用可能与抗氧化,上调抗衰老基因klotho表达有关。
Morinda officinalis How., one of the "top four south authentic traditional Chinese medicines", is a commonly used Chinese herb distributed in the south of China, and its roots are widely used for the treatment of sexual impotence, spermatorrhea, irregular menstruation and female infertility. Previous phytochemical researches have revealed that anthraquinones, iridoids, saccharides are the main constituents of this specie.The pharmacology activities of Immunomodulation, anti-aging, anticancer, antidepressant, Fertility and oestrogenicity had been reported in modern pharmacology. TCM theory holds that Senescence is the kidney deficiency the first, then the spleen deficiency, and the qi and blood deficiency the last, therefore, to tonify kidney, invigorate spleen and benefit vital energy are the basic means of anti-aging. Morinda officinalis How.has a concrete manifestation of anti-aging effects on the its replenishing the kidney and strengthening the bones and muscles activities. To investigate the anti-aging active constituents in this plant, the study were performed.Although the chemical composition and pharmacological activities of M. Officinalis How.had been studied systematically, its anti-aging composition and mechanism is still unclear, in order to clarify the anti-aging active composition and the mechanism of this effects,the chemical composition of M. officinalis How. was studied, cell models of the chemical constituents were used for screening the different parts of M. officinalis extracted and its anti-aging effects were evaluated Synthetically.The studies provide the antiaging material foundation for the exploitation and application of M. officinalis and its preparations.
This topic examined different M.officinalis extracts on the influence of mice swimming time and preliminary screened active site, the use of modern chromatographic techniques for the active site of its chemical constituents were isolated and purified by chemical and spectroscopic methods (UV, IR,1H-NMR,13C-NMR,2D-NMR, MS, etc.) to identify its structure.H2O2-induced cardiac cells in mice and human umbilical vein endothelial cell from aging model was used in this paper to screen compounds isolated from M. officinalis and evaluate their anti-aging effects, the most active single compounds were selected for anti-aging mechanism research and umbilical vein endothelial cells were employed for mechanism research. Cell viability was analyzed by using MTT assay, cell senescence was evaluated using galactosidase staining and telomerase activity detected by telomerase repeat amplification protocol(TRAP)-silver staining. The level of intracellular reactive oxygen species(ROS) was determined using fluorescent ROS detection kit and the level of klotho mRNA was detected by real time PCR.
As a result,twelve compounds have been isolated, including six anthraquinones, two terpenoids, three saccharides and one coumarin. their structures were identified as: officinaliso(1), rubiadin-1-methyl ether(2), 1-hydroxy-6-hydroxymethylanthraquinone(3), anthraquinone-2-aldehyde(4), digiferruginol(5), 1-hydroxy-7-hydroxymethylanthraquinone(6),1,2-dihydroxy-3-methylanthraquinone (7), scopoletin(8), rotungenic acid(9), kestose(10),1F-fructofuranosylnystose(11),β-D-fructopyranosyl-(1→2)-β-D-fructofuranose (12). Those compounds were able to reduce the H2O2-induced cell senescence at different level, anti-aging activity of the twenty-four compounds from our team screened showed different levels of the compounds were able to reduce the H2O2-induced endothelial cell senescence,compound 2 manifested the strongest anti-aging effects,so compound 2 was selected for anti-aging mechanism research.
MTT assay showed that with the concentrations of compound 2 (3-30μM) normal endothelial cells had no effect on survival,and 100μM compound 2 could significantly reduce the survival of endothelial cells by H2O2 (100μM) treatment with HUVECs,β-galactosidase activity and telomerase activity decreased significantly,different concentrations of compound 2 (3,10, and 30μM) or the positive control of resveratrol (30μM)could significantly inhibite the decreasing of telomerase activity in H2O2 (100μM)-induced; by H2O2 (100μM)-incubated treatment with HUVECs 48 h, the fluorescence intensity (reflecting the level of reactive oxygen species)was significantly enhanced, pretreatment with different concentrations of compound 2 (3,10, and 30 uM) or the positive control of resveratrol (30μM) could significantly inhibit the H2O2 (100μM)-induced elevation of intracellualr ROS level. By H2O2 (100μM) treatment with HUVECs 48 h, klotho mRNA expression did not change. Pretreatment with different concentrations of compound 2 (3,10, and 30μM) could significantly increase the expression of klotho mRNA, positive control of resveratrol (30μM) could upregulate the mRNA expression of klotho slightly.
In this thesis,twelve compounds were obtained from M. Officinalis, compound 1 was a new compound, compound 12 was obtained from the genus of Morinda for the first time, compound 3、4、6、9 was isolated from this species for the first time,the NMR data of compound 3 is provided firstly in the literature. M.Officinalis's anti-aging activity screening effect is the comprehensive results of various components. The mechanism of anti-aging effect may be related to its effects of inhibiting oxidative function and upregulating the expression of klotho.
本课题考察不同巴戟天提取物对小鼠游泳时间的影响,初步筛选活性部位,采用现代色谱技术对活性部位对其化学成分进行了分离纯化,用化学方法和波谱方法(UV,IR,1H-NMR,13C-NMR,2D-NMR, MS等)鉴定其结构,应用H2O2诱导的小鼠心肌细胞(H9c2(2-1))和人脐静脉内皮细胞(HUVECs)衰老模型对本课题组从巴戟天分离得到的化合物进行筛选,评价其抗衰老作用,并选取活性作用最强的单体化合物进行抗衰老作用机制的研究。用人脐静脉内皮细胞进行作用机制的研究,MTT法测定细胞存活率,以β-半乳糖苷酶活性(染色法)和端粒酶活性(TRAP-银染法)检测细胞衰老,用活性氧荧光试剂盒检测细胞内ROS的生成,实时定量PCR检测细胞内klotho mRNA的表达。
从巴戟天中共分离得到12个化合物,其中6个蒽醌、2个萜类、3个糖类,1个香豆素类化合物。它们分别为：巴戟醇(1),甲基异茜草素-1-甲醚(2),1-羟基-6-羟甲基蒽醌(3),2-醛基蒽醌(4),1-羟基-2-羟甲基蒽醌(5),1-羟基-7-羟甲基蒽醌(6),1,2-二羟基-3-甲基蒽醌(7),东莨菪内酯(8),rotungenic acid (9),蔗果三糖(10),1F-果吠喃糖基奈斯糖(11)、β-D-果吡喃糖-(1→2)-β-D-果呋喃糖(12),通过考察本课题组从巴戟天共分离得到的12个化合物的细胞抗衰老活性,筛选结果显示各单体化合物均能不同程度降低H2O2诱导的内皮细胞衰老,化合物2抗衰老作用最强,所以选用化合物2进行抗衰老作用机制研究。
MTT法结果显示化合物2浓度(3-30μM)正常内皮细胞存活率无明显影响,而100μM化合物2能显著降低内皮细胞的存活率；经H2O2(100μM)处理的HUVECs,β-半乳糖苷酶活性和端粒酶活性显著下降,不同浓度的化合物2(3、10和30 gM)或阳性对照药白藜芦醇(30μM)能显著抑制H2O2(100μM)诱导的端粒酶活性降低；H2O2(100μM)孵育HUVECs 48 h,可明显增强荧光强度(反映活性氧水平),预处理不同浓度的化合物2(3、10和30μM)或阳性对照药白藜芦醇(30μM)能显著抑制H2O2(100μM)诱导的活性氧水平增加；H2O2(100μM)处理HUVECs 48 h, klothomRNA表达无明显变化。预处理不同浓度的化合物2(3、10和30μM)能显著增加klotho mRNA表达,阳性对照药白藜芦醇(30μM)可轻微上调klotho mRNA表达。
本论文分离得到12个化合物,化合物1为一个新化合物,化合物12为首次从巴戟天属植物中分离得到,化合物3、4、6、9为首次从巴戟天分离得到,首次提供化合物3的核磁数据。抗衰老活性筛选结果表明巴戟天抗衰老作用是其各类成分综合作用的结果。抗衰老作用机制研究提示巴戟天单体抗衰老作用可能与抗氧化,上调抗衰老基因klotho表达有关。
Morinda officinalis How., one of the "top four south authentic traditional Chinese medicines", is a commonly used Chinese herb distributed in the south of China, and its roots are widely used for the treatment of sexual impotence, spermatorrhea, irregular menstruation and female infertility. Previous phytochemical researches have revealed that anthraquinones, iridoids, saccharides are the main constituents of this specie.The pharmacology activities of Immunomodulation, anti-aging, anticancer, antidepressant, Fertility and oestrogenicity had been reported in modern pharmacology. TCM theory holds that Senescence is the kidney deficiency the first, then the spleen deficiency, and the qi and blood deficiency the last, therefore, to tonify kidney, invigorate spleen and benefit vital energy are the basic means of anti-aging. Morinda officinalis How.has a concrete manifestation of anti-aging effects on the its replenishing the kidney and strengthening the bones and muscles activities. To investigate the anti-aging active constituents in this plant, the study were performed.Although the chemical composition and pharmacological activities of M. Officinalis How.had been studied systematically, its anti-aging composition and mechanism is still unclear, in order to clarify the anti-aging active composition and the mechanism of this effects,the chemical composition of M. officinalis How. was studied, cell models of the chemical constituents were used for screening the different parts of M. officinalis extracted and its anti-aging effects were evaluated Synthetically.The studies provide the antiaging material foundation for the exploitation and application of M. officinalis and its preparations.
This topic examined different M.officinalis extracts on the influence of mice swimming time and preliminary screened active site, the use of modern chromatographic techniques for the active site of its chemical constituents were isolated and purified by chemical and spectroscopic methods (UV, IR,1H-NMR,13C-NMR,2D-NMR, MS, etc.) to identify its structure.H2O2-induced cardiac cells in mice and human umbilical vein endothelial cell from aging model was used in this paper to screen compounds isolated from M. officinalis and evaluate their anti-aging effects, the most active single compounds were selected for anti-aging mechanism research and umbilical vein endothelial cells were employed for mechanism research. Cell viability was analyzed by using MTT assay, cell senescence was evaluated using galactosidase staining and telomerase activity detected by telomerase repeat amplification protocol(TRAP)-silver staining. The level of intracellular reactive oxygen species(ROS) was determined using fluorescent ROS detection kit and the level of klotho mRNA was detected by real time PCR.
As a result,twelve compounds have been isolated, including six anthraquinones, two terpenoids, three saccharides and one coumarin. their structures were identified as: officinaliso(1), rubiadin-1-methyl ether(2), 1-hydroxy-6-hydroxymethylanthraquinone(3), anthraquinone-2-aldehyde(4), digiferruginol(5), 1-hydroxy-7-hydroxymethylanthraquinone(6),1,2-dihydroxy-3-methylanthraquinone (7), scopoletin(8), rotungenic acid(9), kestose(10),1F-fructofuranosylnystose(11),β-D-fructopyranosyl-(1→2)-β-D-fructofuranose (12). Those compounds were able to reduce the H2O2-induced cell senescence at different level, anti-aging activity of the twenty-four compounds from our team screened showed different levels of the compounds were able to reduce the H2O2-induced endothelial cell senescence,compound 2 manifested the strongest anti-aging effects,so compound 2 was selected for anti-aging mechanism research.
MTT assay showed that with the concentrations of compound 2 (3-30μM) normal endothelial cells had no effect on survival,and 100μM compound 2 could significantly reduce the survival of endothelial cells by H2O2 (100μM) treatment with HUVECs,β-galactosidase activity and telomerase activity decreased significantly,different concentrations of compound 2 (3,10, and 30μM) or the positive control of resveratrol (30μM)could significantly inhibite the decreasing of telomerase activity in H2O2 (100μM)-induced; by H2O2 (100μM)-incubated treatment with HUVECs 48 h, the fluorescence intensity (reflecting the level of reactive oxygen species)was significantly enhanced, pretreatment with different concentrations of compound 2 (3,10, and 30 uM) or the positive control of resveratrol (30μM) could significantly inhibit the H2O2 (100μM)-induced elevation of intracellualr ROS level. By H2O2 (100μM) treatment with HUVECs 48 h, klotho mRNA expression did not change. Pretreatment with different concentrations of compound 2 (3,10, and 30μM) could significantly increase the expression of klotho mRNA, positive control of resveratrol (30μM) could upregulate the mRNA expression of klotho slightly.
In this thesis,twelve compounds were obtained from M. Officinalis, compound 1 was a new compound, compound 12 was obtained from the genus of Morinda for the first time, compound 3、4、6、9 was isolated from this species for the first time,the NMR data of compound 3 is provided firstly in the literature. M.Officinalis's anti-aging activity screening effect is the comprehensive results of various components. The mechanism of anti-aging effect may be related to its effects of inhibiting oxidative function and upregulating the expression of klotho.
引文
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