成分A008的化学分离及预防肥胖的活性研究
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摘要
荷叶为睡莲科植物莲(Nelumbo nucifera Gaertn.)的干燥叶,具有清热解暑、升发清阳、凉血止血之功效。荷叶中含有丰富的黄酮类、生物碱类、有机酸类及挥发油类成份,其中黄酮类、生物碱类是荷叶现在研究的主要有效部位,其降脂减肥活性倍受人们的关注。
目的:从荷叶中分离成分A008,并考察其对3T3-L1前脂肪细胞增殖与凋亡的影响及对营养型肥胖大鼠体重增长的预防作用。
方法:采用大孔吸附树脂法获取荷叶总提取物;回流提取法精制总提取物,在考察提取溶剂种类、提取时间、溶剂用量等单因素的基础上,采用正交设计法优化提取工艺条件;进一步采用硅胶柱层析法、重结晶方法分离纯化成分A008。
MTT法测定成分A008对3T3-L1前脂肪细胞增殖的影响,AnnexinⅤ-FITC/PI染色法观察成分A008对其凋亡的影响,流式细胞术定量检测CyclinD1细胞周期蛋白的表达。
建立营养性大鼠肥胖模型,同时灌胃给予成分A008,考察成分A008对营养性肥胖大鼠的体重和体脂指标的影响,同时检测大鼠主要脏器系数的大小,考察成分A008预防营养型肥胖大鼠的体重增加的活性,并对其进行毒性评价。
结果:本实验大孔吸附树脂法提取荷叶总提取物;得到的洗脱物用25倍量的甲醇回流提取2次,每次2h;得到的提取物用硅胶柱层析法进行分离,用石油醚:乙酸乙酯进行梯度洗脱,在石油醚:乙酸乙酯=8:1时分离得到成分A008;采用甲醇对该组分进行重结晶,纯度达到99.99%。
本实验发现成分A008明显抑制3T3-L1前脂肪细胞的增殖,且具有浓度依赖性和时间依赖性;同时成分A008能促进3T3-L1前脂肪细胞的凋亡,该作用亦具有浓度依赖性。且检测到成分A008上调CyclinD1蛋白的表达
成分A008高剂量组能够明显抑制营养型肥胖大鼠体重的增加,降低其Lee’s指数及脂肪重量,且对各主要脏器重量没有明显毒性作用,说明治疗剂量的成分A008能预防营养型肥胖大鼠体重的增加且对大鼠各主要脏器无明显的毒副作用。
结论:本实验在以往荷叶荷叶总提取物提取的基础上加以改进,缩短了实验周期,简化了实验过程,避免了多种毒性较大的有机溶剂的使用,研究了成分A008对3 T3-L1前脂肪细胞抑制增殖、促进凋亡的作用,和预防营养型肥胖大鼠体重增加的活性,为成分A008进一步的预防肥胖作用机理的研究和体内药物代谢的研究提供了可靠的实验依据。目前,研制安全、有效、质量可控、作用机理明确的预防和治疗肥胖、高脂血症的药物是药物学研究热点之一,能够简便、高效地进行生产是药物工业化生产的主要目标。故本实验又为开发安全、有效的预防和治疗肥胖的药物、促进民族药物的发展提供了可能。
Folium Nelumbinis is the dried leaf of Nelumbo nucifera Gaertn. It has the effect of clearing away summer-heat and heat evil, ascending the lucid yang and cooling blood to stop bleeding. The components in Folium Nelumbinis include alkaloids, flavonoids, organic acids and aetheroleas. Alkaloids and flavonoids are known as the main bioactive ingredients in it, which have the effect of anti-obesity.
Objective: We established the methods to extract componentA008 from Folium Nelumbinis, investigate the inhibition effects of it on proliferation and apoptosis of 3T3-L1 preadipocytes, and evaluate the prevention effect of componentA008 on the body weight increase of nutritional obese rats.
Metheds: During the extraction of componentA008, the yield of Nuciferine was observed to be obviously influenced by the kind and volume of extraction solvents, time of extraction and times of it. Based on experiment concerning one single factor, an orthogonal experiment was carried out to ascertain optimal extraction conditions. Then, the silica gel column chromatography was used to separate componentA008 from Folium Nelumbinis, and we used the thin layer chromatography (TLC) to carry on the synchronous detection, used the methanol to recryst componentA008 we separated.
In this study we investigated the effects of componentA008 on 3T3-L1 preadipocytes. We determined the proliferation of 3T3-L1 preadipocytes by MTT assays, measured the apoptosis by AnnexinⅤ- FITC / PI staining and Flow Cytometry (FCM), and detected the protein expression of CyclinD1 by FCM.
We evaluated the prevention on the body weight increase of componentA008 on nutritional obese rats. We established the model of nutritional obese rats, and inspected the changes in body weight and fat indicators, and the toxic effect in rats.
Results: We used macroporous resins and 25times of methanol to extract for two times, every time for two hours; After that, we used the silica gel column chromatography to separate componentA008, eluted with petroleum ether and acetic ether, when petroleum ether: cetic ether=8:1, we got the componentA008 and identified the purity of it.
In this study we investigated the effects of componentA008 on 3T3-L1 preadipocytes. And we found componentA008 significantly inhibited the proliferation of 3T3-L1 preadipocytes and its inhibition of proliferation in a concentration-dependent and time-dependent manner. At the same time componentA008 also promoted the apoptosis of 3T3-L1 preadipocytes, this was also a concentration-dependent manner. And we found componentA008 promoted the protein expression of CyclinD1.
We found the componentA008 significantly inhibit the increase in body weight, reduced the lee’s index and fat weight. The major organ weight had no obvious abnormality, so we can say the therapeutic dose of componentA008 can inhibits obesity and has no toxic effects in rats.
Conclusion: At present, the development of safe, effective, quality control and a clear mechanism of the prevention of anti-obesity, anti-hyperlipidemia t is currently one of the hot spots. We established the separation and purification method of componentA008, studied the proliferation inhibition, apoptosis promotion on 3T3-L1 preadipocytes of it, and prevention of weight increase in vivo. This is very important for the further research of componentA008 in vivo and vitro. And it may promote the development of the national drugs.
目的:从荷叶中分离成分A008,并考察其对3T3-L1前脂肪细胞增殖与凋亡的影响及对营养型肥胖大鼠体重增长的预防作用。
方法:采用大孔吸附树脂法获取荷叶总提取物;回流提取法精制总提取物,在考察提取溶剂种类、提取时间、溶剂用量等单因素的基础上,采用正交设计法优化提取工艺条件;进一步采用硅胶柱层析法、重结晶方法分离纯化成分A008。
MTT法测定成分A008对3T3-L1前脂肪细胞增殖的影响,AnnexinⅤ-FITC/PI染色法观察成分A008对其凋亡的影响,流式细胞术定量检测CyclinD1细胞周期蛋白的表达。
建立营养性大鼠肥胖模型,同时灌胃给予成分A008,考察成分A008对营养性肥胖大鼠的体重和体脂指标的影响,同时检测大鼠主要脏器系数的大小,考察成分A008预防营养型肥胖大鼠的体重增加的活性,并对其进行毒性评价。
结果:本实验大孔吸附树脂法提取荷叶总提取物;得到的洗脱物用25倍量的甲醇回流提取2次,每次2h;得到的提取物用硅胶柱层析法进行分离,用石油醚:乙酸乙酯进行梯度洗脱,在石油醚:乙酸乙酯=8:1时分离得到成分A008;采用甲醇对该组分进行重结晶,纯度达到99.99%。
本实验发现成分A008明显抑制3T3-L1前脂肪细胞的增殖,且具有浓度依赖性和时间依赖性;同时成分A008能促进3T3-L1前脂肪细胞的凋亡,该作用亦具有浓度依赖性。且检测到成分A008上调CyclinD1蛋白的表达
成分A008高剂量组能够明显抑制营养型肥胖大鼠体重的增加,降低其Lee’s指数及脂肪重量,且对各主要脏器重量没有明显毒性作用,说明治疗剂量的成分A008能预防营养型肥胖大鼠体重的增加且对大鼠各主要脏器无明显的毒副作用。
结论:本实验在以往荷叶荷叶总提取物提取的基础上加以改进,缩短了实验周期,简化了实验过程,避免了多种毒性较大的有机溶剂的使用,研究了成分A008对3 T3-L1前脂肪细胞抑制增殖、促进凋亡的作用,和预防营养型肥胖大鼠体重增加的活性,为成分A008进一步的预防肥胖作用机理的研究和体内药物代谢的研究提供了可靠的实验依据。目前,研制安全、有效、质量可控、作用机理明确的预防和治疗肥胖、高脂血症的药物是药物学研究热点之一,能够简便、高效地进行生产是药物工业化生产的主要目标。故本实验又为开发安全、有效的预防和治疗肥胖的药物、促进民族药物的发展提供了可能。
Folium Nelumbinis is the dried leaf of Nelumbo nucifera Gaertn. It has the effect of clearing away summer-heat and heat evil, ascending the lucid yang and cooling blood to stop bleeding. The components in Folium Nelumbinis include alkaloids, flavonoids, organic acids and aetheroleas. Alkaloids and flavonoids are known as the main bioactive ingredients in it, which have the effect of anti-obesity.
Objective: We established the methods to extract componentA008 from Folium Nelumbinis, investigate the inhibition effects of it on proliferation and apoptosis of 3T3-L1 preadipocytes, and evaluate the prevention effect of componentA008 on the body weight increase of nutritional obese rats.
Metheds: During the extraction of componentA008, the yield of Nuciferine was observed to be obviously influenced by the kind and volume of extraction solvents, time of extraction and times of it. Based on experiment concerning one single factor, an orthogonal experiment was carried out to ascertain optimal extraction conditions. Then, the silica gel column chromatography was used to separate componentA008 from Folium Nelumbinis, and we used the thin layer chromatography (TLC) to carry on the synchronous detection, used the methanol to recryst componentA008 we separated.
In this study we investigated the effects of componentA008 on 3T3-L1 preadipocytes. We determined the proliferation of 3T3-L1 preadipocytes by MTT assays, measured the apoptosis by AnnexinⅤ- FITC / PI staining and Flow Cytometry (FCM), and detected the protein expression of CyclinD1 by FCM.
We evaluated the prevention on the body weight increase of componentA008 on nutritional obese rats. We established the model of nutritional obese rats, and inspected the changes in body weight and fat indicators, and the toxic effect in rats.
Results: We used macroporous resins and 25times of methanol to extract for two times, every time for two hours; After that, we used the silica gel column chromatography to separate componentA008, eluted with petroleum ether and acetic ether, when petroleum ether: cetic ether=8:1, we got the componentA008 and identified the purity of it.
In this study we investigated the effects of componentA008 on 3T3-L1 preadipocytes. And we found componentA008 significantly inhibited the proliferation of 3T3-L1 preadipocytes and its inhibition of proliferation in a concentration-dependent and time-dependent manner. At the same time componentA008 also promoted the apoptosis of 3T3-L1 preadipocytes, this was also a concentration-dependent manner. And we found componentA008 promoted the protein expression of CyclinD1.
We found the componentA008 significantly inhibit the increase in body weight, reduced the lee’s index and fat weight. The major organ weight had no obvious abnormality, so we can say the therapeutic dose of componentA008 can inhibits obesity and has no toxic effects in rats.
Conclusion: At present, the development of safe, effective, quality control and a clear mechanism of the prevention of anti-obesity, anti-hyperlipidemia t is currently one of the hot spots. We established the separation and purification method of componentA008, studied the proliferation inhibition, apoptosis promotion on 3T3-L1 preadipocytes of it, and prevention of weight increase in vivo. This is very important for the further research of componentA008 in vivo and vitro. And it may promote the development of the national drugs.
引文
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