渐尖毛蕨的生物活性研究
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摘要
渐尖毛蕨Cyclosorus acuminatus(Houtt)Nakai是蕨类植物门真蕨亚门金星蕨科毛蕨属成员。药用为干燥根茎或全草,全年采收。药用名黑舒筋、牛肋巴、小叶凤凰尾巴草等。味微苦、涩,性平,中医认为其具有消炎,健脾,镇静,清热解毒等功效。渐尖毛蕨药材在民间尤其是江浙一带广泛用于治疗消化不良,小儿疳积,痢疾泄泻,热淋,烧烫伤,狂犬咬伤,咽喉肿痛,风湿痹痛等。本课题组在前期的研究中发现渐尖毛蕨富含黄酮类化合物,并分离和结构鉴定了7个新颖的二氢黄酮化合物,但鲜有对其生物活性以及物质基础的科学研究报道。为了系统性揭示传统中药渐尖毛蕨的生物活性,本次研究从传统中医理论和实际民间药用出发,结合现代医学理论,重点考察渐尖毛蕨的生物活性和其药理作用机制,为传统民间药物渐尖毛蕨的后续开发奠定基础。
本文的内容主要分为四个部分。即是：一：渐尖毛蕨的体外药理活性分析和主要潜在有效部位的富集；二：进一步利用在体模型明确渐尖毛蕨在代谢综合征、下焦疾病等中的生物活性；三：考察各部位的协同作用,确定其有效部位。利用细胞和动物模型综合考察渐尖毛蕨有效部位的生物活性；四：明确渐尖毛蕨有效部位的药理活性的具体作用机制。
本文利用大孔树脂对渐尖毛蕨甲醇总提取物进行初步分段得G,S,H段冻干粉,并从渐尖毛蕨的水提取物中制得精制粗多糖。然后通过7种体外自由基清除模型、铜离子诱导的LDL氧化模型、肝组织自发过氧化模型、Fe2+-H2O2诱导的线粒体脂质过氧化模型和Fe2+-Vc诱导的线粒体脂质过氧化等实验,发现其甲醇提取物的主要潜在有效部位为H段。明确H段的主要成分为总黄酮类化合物后,采用正交设计法优化渐尖毛蕨总黄酮的最佳提取纯化工艺。
为了进一步明确渐尖毛蕨在代谢综合征,下焦疾病等中的生物活性,本文利用降低正常昆明种小鼠胆固醇活性模型和用餐后一过性高血脂模型来考察渐尖毛蕨的降低血脂活性,利用正常昆明种小鼠口服血糖耐受模型考察渐尖毛蕨的降低糖活性,利用维甲酸诱导的骨钙流失模型考察渐尖毛蕨的抗骨质疏松活性,利用重铬酸钾诱导的急性肾毒性模型考察渐尖毛蕨的肾保护活性。从该次体内实验的初步活性筛选可以看出,传统药材渐尖毛蕨具有较强的降血糖,降血脂和肾保护潜力。而通过初步的各分段考察,发现渐尖毛蕨总黄酮,H段和精制粗多糖之中,总黄酮和H段具有较强的降血脂,降血糖和肾保护潜力。而尤以总黄酮的药理活性最佳。同时精制粗多糖也表现出一定的降血糖潜力,但其作用效果远弱于总黄酮部位,同时粗多糖也并未表现出降血脂和肾保护等多样性的药理活性。提示渐尖毛蕨降血脂,降血糖和肾保护潜力的主要物质基础为其总黄酮部位。
为了明确在降血糖这一生理活性中,精制粗多糖与总黄酮之间是否可能出现药理协同效应,本文首先利用链脲霉素-高脂饮食诱导的小鼠糖尿病模型进行了其降血糖作用研究。在实验中的给药组选择上,除了阳性药对照组外,本文设立了总黄酮组,精制粗多糖组,总黄酮-精制粗多糖=1：1组,实验结果显示出总黄酮-精制粗多糖=1：1的给药组并未表现出协同效应(明显优于总黄酮组的降血糖活性),因此我们明确了渐尖毛蕨的主要有效部位为其总黄酮,并且在后续的实验中我们将重点研究总黄酮的药理活性并明确其具体作用机理。
渐尖毛蕨显示了较强的降血脂,降血糖和肾保护潜力,提示其在代谢综合征和肾保护方面的药效。而研究显示体内代谢混乱导致血脂血糖水平异常,是血管性病变的最常见诱因之一。同时血胆固醇、甘油三酯和血糖水平的持续性升高往往将进一步诱导肾血管病变最终引发肾脏疾病,极大的危害了人类健康。于是本文随后考察渐尖毛蕨的药效部位对人脐静脉内皮细胞氧化损伤的保护作用,对3T3-L1前脂肪细胞生长分化的抑制作用,对长期高脂饮食诱导的大鼠高血脂和脂肪肝的保护作用,对高脂饮食-嘌呤霉素诱导的小鼠高血脂性肾病综合症的保护作用和对糖尿病肾病的保护作用及其具体的作用机制。
Cyclosorus acuminatus (Houtt) Nakai is a member of Cyclosonis of Thelypteridaceae of Ferns subphylum of Pteridophyta. The whole plant or the rhizomes could be used as medicine and full-year harvest. It also has the medicinal name as Heisujing, niuneiba, xiaoyefenghuangweibacao and so on. It tastes bitter, astringent and flat. Chinese medicine believes that it has the effect of anti-inflammatory, spleen, calm, clearing heat and detoxification. Cyclosorus acuminatus is widely used in folk, especially in Jiangsu and Zhejiang provinces, to treat indigestion, infantile malnutrition, dysentery, diarrhea, hot shower, burns, rabies bites, sore throat and wind dampness. In the previous study, our group found the plant is rich in flavonoids. We separated and elucidated the structures of7new flavanone compounds. In order to systematic reveal the biological activity of the plant, this study was taken to investigate the biological activity and pharmacological mechanism of Cyclosorus acuminatus based on the traditional Chinese medicine theory, practical folk using and modern medical theory.
The contents of this paper are divided into4parts. That is:A:The in vitro pharmacological analysis and the enrichment of the valid target. B:Investigation of the activity in metabolic syndrome and the lower-jiao syndrome using animal model. C:Investigate the synergism of the various parts and to determine the finally effective parts. D:Identify the pharmacological activity and its mechanism.
In this study, macroporous resin was used to get segmentation G, S and H from the total methanol extract of Cyclosorus acuminatus. The refined crude polysaccharide was also get. Finally the H was identified as the main valid target via7in vitro free radical scavenging model, Cu2+induced LDL oxidation model, the liver spontaneous peroxidation model, the Fe2+-H2O2and Fe2+-Vc induced peroxidation of mitochondrial. And then, the main compounds of H were identified as total flavonoids using chemical color test and the best extraction and purification measure was optimized using orthogonal design.
Then, the normal KM mice TC-lowing model and postprandial blood lipids model were used to examine the activity of lowing blood lipids. The OGTT model was to examine the activity of lowing blood glucose. The retinoic acid-induced bone calcium losing model was to examine the activity of anti-osteoporosis. The K2Cr2O7-induced acute renal toxicity model was to examine the activity of renal protection. The whole results suggested that Cyclosorus acuminatus has the activity of lowing blood lipids and glucose, and renal protection potential. The total flavonoids have the strongest pharmacological activity. The refined crude polysaccharide showed a certain potential for lowing blood glucose, but its effect is much weaker than the total flavonoids. And the crude polysaccharide did not show a diversity activity in lowing lipids and renal protection. At this stage, it can be concluded that the material base of Cyclosorus acuminatus is the total flavonoids.
In order to investigate the synergism of the various parts and to determine the finally effective parts, the STZ-high fat diet-induced diabetic mice were used to study the hypoglycemic effect. In addition to the positive control, the total flavonoids group, the refined crude polysaccharide group and the total flavonoids-refined crude polysaccharide=1:1group were set. The results showed that the total flavonoids-refined crude polysaccharide=1:1group did not exert a synergistic effect. So the total flavonoids were finally identified as the activity part.
Eventually, the protective effect of human umbilical vein endothelial cells against oxidative injury, the differentiation inhibition of3T3-L1preadipocytes, the protective activity in long-term high-fat diet-induced hyperlipemia mice and the renal protective effect in puromycin nephritic syndrome and diabetic nephropathy mice and the its specific mechanism were investigated.
本文的内容主要分为四个部分。即是：一：渐尖毛蕨的体外药理活性分析和主要潜在有效部位的富集；二：进一步利用在体模型明确渐尖毛蕨在代谢综合征、下焦疾病等中的生物活性；三：考察各部位的协同作用,确定其有效部位。利用细胞和动物模型综合考察渐尖毛蕨有效部位的生物活性；四：明确渐尖毛蕨有效部位的药理活性的具体作用机制。
本文利用大孔树脂对渐尖毛蕨甲醇总提取物进行初步分段得G,S,H段冻干粉,并从渐尖毛蕨的水提取物中制得精制粗多糖。然后通过7种体外自由基清除模型、铜离子诱导的LDL氧化模型、肝组织自发过氧化模型、Fe2+-H2O2诱导的线粒体脂质过氧化模型和Fe2+-Vc诱导的线粒体脂质过氧化等实验,发现其甲醇提取物的主要潜在有效部位为H段。明确H段的主要成分为总黄酮类化合物后,采用正交设计法优化渐尖毛蕨总黄酮的最佳提取纯化工艺。
为了进一步明确渐尖毛蕨在代谢综合征,下焦疾病等中的生物活性,本文利用降低正常昆明种小鼠胆固醇活性模型和用餐后一过性高血脂模型来考察渐尖毛蕨的降低血脂活性,利用正常昆明种小鼠口服血糖耐受模型考察渐尖毛蕨的降低糖活性,利用维甲酸诱导的骨钙流失模型考察渐尖毛蕨的抗骨质疏松活性,利用重铬酸钾诱导的急性肾毒性模型考察渐尖毛蕨的肾保护活性。从该次体内实验的初步活性筛选可以看出,传统药材渐尖毛蕨具有较强的降血糖,降血脂和肾保护潜力。而通过初步的各分段考察,发现渐尖毛蕨总黄酮,H段和精制粗多糖之中,总黄酮和H段具有较强的降血脂,降血糖和肾保护潜力。而尤以总黄酮的药理活性最佳。同时精制粗多糖也表现出一定的降血糖潜力,但其作用效果远弱于总黄酮部位,同时粗多糖也并未表现出降血脂和肾保护等多样性的药理活性。提示渐尖毛蕨降血脂,降血糖和肾保护潜力的主要物质基础为其总黄酮部位。
为了明确在降血糖这一生理活性中,精制粗多糖与总黄酮之间是否可能出现药理协同效应,本文首先利用链脲霉素-高脂饮食诱导的小鼠糖尿病模型进行了其降血糖作用研究。在实验中的给药组选择上,除了阳性药对照组外,本文设立了总黄酮组,精制粗多糖组,总黄酮-精制粗多糖=1：1组,实验结果显示出总黄酮-精制粗多糖=1：1的给药组并未表现出协同效应(明显优于总黄酮组的降血糖活性),因此我们明确了渐尖毛蕨的主要有效部位为其总黄酮,并且在后续的实验中我们将重点研究总黄酮的药理活性并明确其具体作用机理。
渐尖毛蕨显示了较强的降血脂,降血糖和肾保护潜力,提示其在代谢综合征和肾保护方面的药效。而研究显示体内代谢混乱导致血脂血糖水平异常,是血管性病变的最常见诱因之一。同时血胆固醇、甘油三酯和血糖水平的持续性升高往往将进一步诱导肾血管病变最终引发肾脏疾病,极大的危害了人类健康。于是本文随后考察渐尖毛蕨的药效部位对人脐静脉内皮细胞氧化损伤的保护作用,对3T3-L1前脂肪细胞生长分化的抑制作用,对长期高脂饮食诱导的大鼠高血脂和脂肪肝的保护作用,对高脂饮食-嘌呤霉素诱导的小鼠高血脂性肾病综合症的保护作用和对糖尿病肾病的保护作用及其具体的作用机制。
Cyclosorus acuminatus (Houtt) Nakai is a member of Cyclosonis of Thelypteridaceae of Ferns subphylum of Pteridophyta. The whole plant or the rhizomes could be used as medicine and full-year harvest. It also has the medicinal name as Heisujing, niuneiba, xiaoyefenghuangweibacao and so on. It tastes bitter, astringent and flat. Chinese medicine believes that it has the effect of anti-inflammatory, spleen, calm, clearing heat and detoxification. Cyclosorus acuminatus is widely used in folk, especially in Jiangsu and Zhejiang provinces, to treat indigestion, infantile malnutrition, dysentery, diarrhea, hot shower, burns, rabies bites, sore throat and wind dampness. In the previous study, our group found the plant is rich in flavonoids. We separated and elucidated the structures of7new flavanone compounds. In order to systematic reveal the biological activity of the plant, this study was taken to investigate the biological activity and pharmacological mechanism of Cyclosorus acuminatus based on the traditional Chinese medicine theory, practical folk using and modern medical theory.
The contents of this paper are divided into4parts. That is:A:The in vitro pharmacological analysis and the enrichment of the valid target. B:Investigation of the activity in metabolic syndrome and the lower-jiao syndrome using animal model. C:Investigate the synergism of the various parts and to determine the finally effective parts. D:Identify the pharmacological activity and its mechanism.
In this study, macroporous resin was used to get segmentation G, S and H from the total methanol extract of Cyclosorus acuminatus. The refined crude polysaccharide was also get. Finally the H was identified as the main valid target via7in vitro free radical scavenging model, Cu2+induced LDL oxidation model, the liver spontaneous peroxidation model, the Fe2+-H2O2and Fe2+-Vc induced peroxidation of mitochondrial. And then, the main compounds of H were identified as total flavonoids using chemical color test and the best extraction and purification measure was optimized using orthogonal design.
Then, the normal KM mice TC-lowing model and postprandial blood lipids model were used to examine the activity of lowing blood lipids. The OGTT model was to examine the activity of lowing blood glucose. The retinoic acid-induced bone calcium losing model was to examine the activity of anti-osteoporosis. The K2Cr2O7-induced acute renal toxicity model was to examine the activity of renal protection. The whole results suggested that Cyclosorus acuminatus has the activity of lowing blood lipids and glucose, and renal protection potential. The total flavonoids have the strongest pharmacological activity. The refined crude polysaccharide showed a certain potential for lowing blood glucose, but its effect is much weaker than the total flavonoids. And the crude polysaccharide did not show a diversity activity in lowing lipids and renal protection. At this stage, it can be concluded that the material base of Cyclosorus acuminatus is the total flavonoids.
In order to investigate the synergism of the various parts and to determine the finally effective parts, the STZ-high fat diet-induced diabetic mice were used to study the hypoglycemic effect. In addition to the positive control, the total flavonoids group, the refined crude polysaccharide group and the total flavonoids-refined crude polysaccharide=1:1group were set. The results showed that the total flavonoids-refined crude polysaccharide=1:1group did not exert a synergistic effect. So the total flavonoids were finally identified as the activity part.
Eventually, the protective effect of human umbilical vein endothelial cells against oxidative injury, the differentiation inhibition of3T3-L1preadipocytes, the protective activity in long-term high-fat diet-induced hyperlipemia mice and the renal protective effect in puromycin nephritic syndrome and diabetic nephropathy mice and the its specific mechanism were investigated.
引文
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