牡丹种皮黄酮提取分离与抗氧化及抗疲劳作用研究
摘要
牡丹种皮是牡丹籽油生产过程中的废弃物,占牡丹种子质量的三分之一,虽然生物量很大,但目前还没有有效的开发利用。笔者从牡丹种皮中提取黄酮类化合物,为其变废为宝、发展循环经济和提高牡丹籽油产品附加值发展新的研究方向。本文首先研究了使用匀浆提取和酶辅助匀浆提取技术提取牡丹种皮总黄酮的工艺,研究了工艺参数和提取率之间的关系,确定了酶辅助匀浆法提取牡丹种皮总黄酮的最佳工艺参数。优化了大孔树脂吸附分离牡丹种皮黄酮的工艺条件,采用多种方法研究了牡丹种皮黄酮的体外抗氧化活性,对小鼠体内抗氧化和抗疲劳作用的相关指标进行分析,主要研究内容如下：
1.以总黄酮和木犀草素提取率为考察指标,分析了酶辅助匀浆法与传统匀浆法两种提取工艺对牡丹种皮黄酮和木犀草素的提取效果。在最佳匀浆条件下,所得黄酮浓度最高可达49.82mg/go进行酶浓度和种类以及酶解孵育条件筛选后,得出果胶酶的效果好于纤维素酶和混合酶,果胶酶浓度最佳为0.05mg/mL,所得黄酮浓度最高可达74.66mg/g,相比于匀浆法所得黄酮含量高出33%。在最佳匀浆条件下,木犀草素含量最高可达0.75mg/g,晦辅助匀浆法所得木犀草素含量最高为3.20mg/g,是匀浆法所得木犀草素含量的四倍。所以酶辅助匀浆法能够高效提取牡丹种皮黄酮和木犀草素。
2.用大孔树脂对提取物进行了初步纯化,探讨了适宜的纯化条件。研究了7种树脂的静态和动态的吸附和解吸动力学,确定D101最佳吸附条件为上样流速控制为1mL/min,上样液浓度为4mg/mL,上样液的pH为6。而最佳的洗脱条件为：用浓度为80%的乙醇洗脱,洗脱速率控制在1.2mL/min。按照以上最优的牡丹种皮黄酮提取物大孔树脂纯化条件,总黄酮含量由纯化前的7.47%增加到纯化后的46.66%,木犀草素含量由纯化前的3.2‰增加到纯化后的2.08%,纯度都提高了6倍。
3.为了研究牡丹种皮黄酮的抗氧化作用,本研究通过体外抗氧化实验,测定了牡丹种皮黄酮和木犀草素的总还原能力和对生物体内常见的DPPH和ABTS·自由基的清除能力,以及Fe3+的还原能力,同时与2,6-二叔丁基-4-甲基苯酚(BHT)进行了比照。通过上述体外抗氧化能力评价体系的实验结果显示,牡丹种皮黄酮和木犀草素的总还原能力和对于DPPH和ABTS·自由基的清除能力和Fe3+的还原能力都强于BHT,木犀草素的抗氧化能力强于牡丹种皮黄酮。通过上述抗氧化能力分析体系,说明牡丹种皮黄酮和木犀草素都是一种很好的天然抗氧化剂,而且牡丹种皮黄酮的抗氧化能力与其所含有的木犀草素密切相关。
4.本文对牡丹种皮黄酮的体内抗氧化作用进行了系统研究。实验结果表明,牡丹种皮黄酮高剂量组、中等剂量组和维生素E组相对于空白组可以显著提高小鼠的血清、肝和肾组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)的活力,说明高剂量组和中等剂量组具有抵御膜脂质过氧化和清除血清、肝和肾组织中自由基的能力。并且高剂量组的肝组织中的丙二醛(MDA)含量、CAT和GSH-PX的活力与维生素E组差异不显著。说明牡丹种皮黄酮高剂量组在保护膜脂质过氧化和清除体内自由基能力与维生素E组相当,进一步表明牡丹种皮黄酮具有体内抗氧化活性。
5.本研究根据疲劳产生的机理,从宏观上,通过小鼠负重游泳力竭实验证明了牡丹种皮黄酮提取物高剂量组和中等剂量组与二十八烷醇组均能显著延长小鼠的负重游泳时间。相比于空白组,高剂量组负重游泳时间延长率为87.5%,中等剂量组延长率为52.5%,二十八烷醇组延长率为40%。从各种生化指标的检测来看,牡丹种皮黄酮提取物的高剂量组相比于二十八烷醇能明显降低小鼠运动后血清尿素氮(BUN)和血乳酸(BLA)含量,提高肝糖原(LG)和肌糖原(MG)储备。说明牡丹种皮黄酮可以使小鼠机体耐力增强,承受运动负荷能力加强,所以高剂量的牡丹种皮黄酮具有抗疲劳作用,其抗疲劳作用强于二十八烷醇。
The flavonoids of peony testa extracts has very high nutritional and medicinal value for a variety of nutrients and biological active substances.The extraction of flavonoids from peony testa,may shift the waste materialof value, improve the utilization value,and take on development of new direction.The author first studied the extraction technique of homogenate and enzyme assistant method to withdraw the flavonoid from peony testa, and studied the relationship between the parameters and the extraction rate,determined the optimum process parameters of extracting total flavonoids for enzyme assisted homogenate.Optimum conditions for the adsorption and desorption of macroporous resin, tested the in vitro antioxidant activity of flavonoids using a variety of methods, and carried on the related mechanism of the body antioxidant and research of the anti-fatigue.The main research contents are as follows:
1.Conditions for the extraction rate were studies under homogenate and enzyme assistant method, the resμLts for two above condition are compared.Under the optimum homogenate conditions, a flavonoids concentration couLd be over49.82mg/g.Screening enzyme concentration and types, as well as treatment conditions,the pectinase is better than cellμLase and mixed enzyme,optimum concentration is0.05mg/mL, the maximum flavonoids concentration74.66mg/g,33percent over the flavonoids content under homogenate. The content of luteolin under enzyme-assisted is relatively high, respectively3.2mg/g, was four times under simple homogenate that was0.75mg/g.
2.The macroporous resin purification of the extract were preliminarily discussed in suitable purification condition.The adsorption performance and desorption conditions of seven macroporous resin for peony testa extracts flavonoids was researched,the optimum parameters of adsorption and desorption were procured.The resμLts showed that D101was the optimum kind of macroporous resin among seven macroporous resins under the experimental condition.The optimum conditions of desorption by D101were as follows:the concentration was4mg/mL,the proper pH was6,the flow rate was1.0mL/min,80%ethanol was the eluent solvent,the eluent rate was1.2mL/min.The content of purity flavonoids and luteolin was46.66%and2.08%under the conditions mentioned above. The content of flavonoids and luteolin has been raised by6times
3.In order to study on antioxidation of peony testa flavonoids and luteolin, this research through the antioxidant experiment in vitro, the total reducing capacity of peony testa flavonoids, DPPH and ABTS free radical scavenging ability and the reduction ability of Fe3+were determined, and compared them with BHT.The assessment system of the in vitro antioxidant capacity of experimental resμLts show, the total reduction ability of peony testa flavonoids and luteolin, and for DPPH and ABTS free radical scavenging ability, and the reduction ability of Fe3+were stronger than BHT, although peony testa flavonoids was inferior to luteolin, but they were a good natural antioxidant. The antioxidation of peony testa flavonoids is closely related to the content of luteolin
4.The antioxidant effect in vivo of peony testa flavonoids were studied.Experimental resμLts show that high-dose group, middle-dose group and the vitamin E of the peony testa flavonoids compared with the the blank group can significantly improve the activity of SOD, CAT and GSH-PX in mice serum, liver and kidney tissue of.The high-dose group and mid-dose group has the ability to resist membrane lipid peroxidation and free radical scavenging activity in the serum, liver and kidney tissue.Differences in content of MDA and activity of SOD, CAT and GSH-PX in liver tissue was not significant compared with vitamin E, illustrated the ability of high dose group in the protection of membrane lipid peroxidation and scavenge free radical was equal to vitamin E group, further indicated that the peony testa flavonoids have antioxidant activity in vivo.
5.According to the mechanism of the fatigue, from on macroscopic, through the exhaustive swimming experiment that the high dose group and middle dose group of peony testa extract can significantly prolong the swimming time of mice. Compared with control group, the exhaustive swimming time, high dose group increased87.5%, middle dose group increased52.5%, octacosanol increased40%. From the detection of various biochemical, the high dose group of peony testa flavonoids extract coμLd significantly reduce the serum urea nitrogen (BUN) and blood lactate acid (BLA) content and increase liver glycogen(LG) and muscle glycogen (MG) reserves after mouse movement.So the high dose of peony testa extract has anti-fatigue effect.
1.以总黄酮和木犀草素提取率为考察指标,分析了酶辅助匀浆法与传统匀浆法两种提取工艺对牡丹种皮黄酮和木犀草素的提取效果。在最佳匀浆条件下,所得黄酮浓度最高可达49.82mg/go进行酶浓度和种类以及酶解孵育条件筛选后,得出果胶酶的效果好于纤维素酶和混合酶,果胶酶浓度最佳为0.05mg/mL,所得黄酮浓度最高可达74.66mg/g,相比于匀浆法所得黄酮含量高出33%。在最佳匀浆条件下,木犀草素含量最高可达0.75mg/g,晦辅助匀浆法所得木犀草素含量最高为3.20mg/g,是匀浆法所得木犀草素含量的四倍。所以酶辅助匀浆法能够高效提取牡丹种皮黄酮和木犀草素。
2.用大孔树脂对提取物进行了初步纯化,探讨了适宜的纯化条件。研究了7种树脂的静态和动态的吸附和解吸动力学,确定D101最佳吸附条件为上样流速控制为1mL/min,上样液浓度为4mg/mL,上样液的pH为6。而最佳的洗脱条件为：用浓度为80%的乙醇洗脱,洗脱速率控制在1.2mL/min。按照以上最优的牡丹种皮黄酮提取物大孔树脂纯化条件,总黄酮含量由纯化前的7.47%增加到纯化后的46.66%,木犀草素含量由纯化前的3.2‰增加到纯化后的2.08%,纯度都提高了6倍。
3.为了研究牡丹种皮黄酮的抗氧化作用,本研究通过体外抗氧化实验,测定了牡丹种皮黄酮和木犀草素的总还原能力和对生物体内常见的DPPH和ABTS·自由基的清除能力,以及Fe3+的还原能力,同时与2,6-二叔丁基-4-甲基苯酚(BHT)进行了比照。通过上述体外抗氧化能力评价体系的实验结果显示,牡丹种皮黄酮和木犀草素的总还原能力和对于DPPH和ABTS·自由基的清除能力和Fe3+的还原能力都强于BHT,木犀草素的抗氧化能力强于牡丹种皮黄酮。通过上述抗氧化能力分析体系,说明牡丹种皮黄酮和木犀草素都是一种很好的天然抗氧化剂,而且牡丹种皮黄酮的抗氧化能力与其所含有的木犀草素密切相关。
4.本文对牡丹种皮黄酮的体内抗氧化作用进行了系统研究。实验结果表明,牡丹种皮黄酮高剂量组、中等剂量组和维生素E组相对于空白组可以显著提高小鼠的血清、肝和肾组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)的活力,说明高剂量组和中等剂量组具有抵御膜脂质过氧化和清除血清、肝和肾组织中自由基的能力。并且高剂量组的肝组织中的丙二醛(MDA)含量、CAT和GSH-PX的活力与维生素E组差异不显著。说明牡丹种皮黄酮高剂量组在保护膜脂质过氧化和清除体内自由基能力与维生素E组相当,进一步表明牡丹种皮黄酮具有体内抗氧化活性。
5.本研究根据疲劳产生的机理,从宏观上,通过小鼠负重游泳力竭实验证明了牡丹种皮黄酮提取物高剂量组和中等剂量组与二十八烷醇组均能显著延长小鼠的负重游泳时间。相比于空白组,高剂量组负重游泳时间延长率为87.5%,中等剂量组延长率为52.5%,二十八烷醇组延长率为40%。从各种生化指标的检测来看,牡丹种皮黄酮提取物的高剂量组相比于二十八烷醇能明显降低小鼠运动后血清尿素氮(BUN)和血乳酸(BLA)含量,提高肝糖原(LG)和肌糖原(MG)储备。说明牡丹种皮黄酮可以使小鼠机体耐力增强,承受运动负荷能力加强,所以高剂量的牡丹种皮黄酮具有抗疲劳作用,其抗疲劳作用强于二十八烷醇。
The flavonoids of peony testa extracts has very high nutritional and medicinal value for a variety of nutrients and biological active substances.The extraction of flavonoids from peony testa,may shift the waste materialof value, improve the utilization value,and take on development of new direction.The author first studied the extraction technique of homogenate and enzyme assistant method to withdraw the flavonoid from peony testa, and studied the relationship between the parameters and the extraction rate,determined the optimum process parameters of extracting total flavonoids for enzyme assisted homogenate.Optimum conditions for the adsorption and desorption of macroporous resin, tested the in vitro antioxidant activity of flavonoids using a variety of methods, and carried on the related mechanism of the body antioxidant and research of the anti-fatigue.The main research contents are as follows:
1.Conditions for the extraction rate were studies under homogenate and enzyme assistant method, the resμLts for two above condition are compared.Under the optimum homogenate conditions, a flavonoids concentration couLd be over49.82mg/g.Screening enzyme concentration and types, as well as treatment conditions,the pectinase is better than cellμLase and mixed enzyme,optimum concentration is0.05mg/mL, the maximum flavonoids concentration74.66mg/g,33percent over the flavonoids content under homogenate. The content of luteolin under enzyme-assisted is relatively high, respectively3.2mg/g, was four times under simple homogenate that was0.75mg/g.
2.The macroporous resin purification of the extract were preliminarily discussed in suitable purification condition.The adsorption performance and desorption conditions of seven macroporous resin for peony testa extracts flavonoids was researched,the optimum parameters of adsorption and desorption were procured.The resμLts showed that D101was the optimum kind of macroporous resin among seven macroporous resins under the experimental condition.The optimum conditions of desorption by D101were as follows:the concentration was4mg/mL,the proper pH was6,the flow rate was1.0mL/min,80%ethanol was the eluent solvent,the eluent rate was1.2mL/min.The content of purity flavonoids and luteolin was46.66%and2.08%under the conditions mentioned above. The content of flavonoids and luteolin has been raised by6times
3.In order to study on antioxidation of peony testa flavonoids and luteolin, this research through the antioxidant experiment in vitro, the total reducing capacity of peony testa flavonoids, DPPH and ABTS free radical scavenging ability and the reduction ability of Fe3+were determined, and compared them with BHT.The assessment system of the in vitro antioxidant capacity of experimental resμLts show, the total reduction ability of peony testa flavonoids and luteolin, and for DPPH and ABTS free radical scavenging ability, and the reduction ability of Fe3+were stronger than BHT, although peony testa flavonoids was inferior to luteolin, but they were a good natural antioxidant. The antioxidation of peony testa flavonoids is closely related to the content of luteolin
4.The antioxidant effect in vivo of peony testa flavonoids were studied.Experimental resμLts show that high-dose group, middle-dose group and the vitamin E of the peony testa flavonoids compared with the the blank group can significantly improve the activity of SOD, CAT and GSH-PX in mice serum, liver and kidney tissue of.The high-dose group and mid-dose group has the ability to resist membrane lipid peroxidation and free radical scavenging activity in the serum, liver and kidney tissue.Differences in content of MDA and activity of SOD, CAT and GSH-PX in liver tissue was not significant compared with vitamin E, illustrated the ability of high dose group in the protection of membrane lipid peroxidation and scavenge free radical was equal to vitamin E group, further indicated that the peony testa flavonoids have antioxidant activity in vivo.
5.According to the mechanism of the fatigue, from on macroscopic, through the exhaustive swimming experiment that the high dose group and middle dose group of peony testa extract can significantly prolong the swimming time of mice. Compared with control group, the exhaustive swimming time, high dose group increased87.5%, middle dose group increased52.5%, octacosanol increased40%. From the detection of various biochemical, the high dose group of peony testa flavonoids extract coμLd significantly reduce the serum urea nitrogen (BUN) and blood lactate acid (BLA) content and increase liver glycogen(LG) and muscle glycogen (MG) reserves after mouse movement.So the high dose of peony testa extract has anti-fatigue effect.
引文
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