基于抗氧化活性的厚朴有效组分的研究
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摘要
厚朴是中国传统中药,燥湿消痰,下气除满,用于湿滞伤中,脘痞吐泻,食积气滞,腹胀便秘,痰饮喘咳。近年来,很多研究都集中在厚朴中木脂素类化合物厚朴酚与和厚朴酚的生物活性,对厚朴中的微量元素、挥发油和多糖的关注较少,因此本论文对厚朴中的微量元素、挥发油、各相萃取物和多糖进行了研究。
采用电感耦合等离子体发射光谱法(ICP-AES)对厚朴水煎液中人体所需的6种微量元素Fe、Mn、Zn、Cu、Cr和Ni的形态进行分析。原药中Fe、Cu、Zn和Mn的含量都比较高,其含量特征为Fe>Mn>Zn>Cu>Cr>Ni。在初级形态中,Ni的提取率最高。在次级形态中,微量元素在颗粒物中的浓度远大于在可溶态中,可溶态中各元素的含量分布为Fe>Mn>Zn>Cr>Cu>Ni。在可溶态中,微量元素只有少量以有机态结合,大多数都以无机态结合。Fe在水煎液中含量最高,Mn和Ni在水煎液中稳定螯合态在可溶态中所占比例分别为99.85%和94.38%。
用水蒸气提取法提取厚朴中的挥发油,并用GC-MS对其化学成分进行研究。结果表明,其主要成分为β-桉叶醇、10-反式-γ-桉叶醇、2-异丙烯-4a,8-二甲基-1,2,3,4,4a,5,6,7-八氢萘、β-芹子烯和β-石竹烯。对厚朴挥发油进行活性研究,发现其挥发油具有-定的抗氧化和抗菌作用,而且抗菌研究发现厚朴挥发油对金黄色葡萄球菌具有较为明显的抗菌活性。
通过抗氧化、抗菌和抗肿瘤活性实验发现,厚朴各相萃取物表现出不同的抗氧化、抗菌和抗肿瘤活性,并测量了各相萃取物中总多酚和总单宁含量。从抗肿瘤活性最高的萃取物中分离得到的主要成分厚朴酚与和厚朴酚表现出对KB、HepG 2和MCF-7肿瘤细胞生长的抑制作用。
对厚朴多糖的研究首先通过响应面分析法考察了提取时间、提取温度和水料比等对多糖提取率的影响,得到了水提法的最佳提取工艺：提取温度是93.7℃；提取时间为3.25小时；水料比为26.2：1。再以脱色率、脱蛋白率和多糖保留率为指标,筛选出最适宜的大孔树脂对厚朴多糖进行脱色工艺研究。选用ADS-7大孔吸附树脂作为研究对象,通过正交试验研究大孔吸附树脂对多糖的脱色工艺,得到最佳工艺条件为：多糖浓度为0.2%；脱色温度为45℃；脱色时间为2小时。在此条件下,厚朴多糖的脱色率可达到最大,同时也可以去除大量的蛋白。
在提取工艺和脱色工艺的基础上,通过分级沉淀法得到了三种厚朴多糖部分。这三种多糖部分的分子量分布范围分别是5600-9400 Da,4700-5400 Da,3900-4200 Da。通过对三种多糖部分的活性研究发现,分子量最小的多糖部分表现出更高的抗氧化活性和对肿瘤细胞生长更强的抑制作用,并对其进行分离纯化,初步研究表明,得到了一个均一多糖,其分子量为1052。
Magnolia Officinalis, a traditional herb in Chinese medicine decoction, has been widely used to treat dissolving phlegm, eliminating dampness, gastrointestinal complaints, anxiety and nervous disturbance. In recent years, many studies have fouced on its components, magnolol and honokiol, however, more studies have comfirmed that microelement, essential oil, different solvent extractions and polysaccharides are the bioactive ingredients in plants, thus it is necessary to study about microelement, essential oil, different solvent extractions and polysaccharides of Magnolia Officinalis.
The microelements are associated with the human life, but also related to the effect of chinese medicine in decoction. The chemical speciation of six microelements were traced and analyzed in the aqua decoction of magnolia officinalis by inductively coupled plasma emission spectrometry (ICP-AES). The contents of Fe, Cu, Zn were higher than the other three microelements'in crude drug, and the order was Fe>Mn>Zn>Cu>Cr>Ni. The results of the primary form analysis showed that the extration rate of Ni was the highest. Furthermore, the sub-form distribution indicated that the concentrations of these trace elements in the particulate species were far greater than in the soluble species, the order of microelements in soluble species was Fe>Mn>Zn>Cu>Cr>Ni. Most of microelements were inorganic species. Particularly, the content of Fe was the highest in the aqua decoction. The stable species of Mn and Ni were 99.85% and 94.38%, respectively.
The hydro-distilled essential oil from Magnolia Officinalis was analyzed by GC/MS, and the major components wereβ-eudesmol,10-epi-γ-eudesmol,2-isopropenyl-4a,8-dimethyl-1,2, 3,4,4a,5,6,7-octahydronaphthalene,β-selinene, (3-caryophyllene, cadalene andβ-cadinene. 10-epi-γ-eudesmol,2-isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene and cadalene were found in magnolia species. The essential oil of Magnolia officinalis possessed antioxidant and antibateria activities. Moreover, the oil exhibited obvious activity against Staphylococcus aureus.
The antioxidant and antibateria experiments were carried out to evaluate different solvent extractions. Different solvent extractions exhibited different antioxidant, antibacteria and antitumor activities. And the Total polyphenols content and total tannin content were measured to find out the relationship between the TPC/TTC and antioxidant acitivities. Magnolol and honokiol were isolated from the extraction which showed the high antitumor activity.
The polysaccharides were the major bioactive ingredients of Magnolia Officinalis. In the study, we investigated the yield of polysaccharides from magnolia officinal under different extraction temperatures, extraction times and water/material ratios by using the response surface methodology (RSE). The optimal water extraction conditions for polysaccharides were as followes:extraction temperature of 93.7℃, extraction time of 3.25 h and a water/material ratio of 26.2:1. And macroporous adsorption resin of ADS-7 which had the best effect on decolorization of magnolia officinalis polysaccharides was chosen from 6 kinds of macroporous adsorption resins with decolorization rate, deproteinization rate and retention rate of polysaccharides as indices, and the optimum technique of decolorization was studied by orthogonal experimental method. The optimum conditions of decolorization were as follows:polysaccharides concentration was 0.2%, decoloring temperature was 45℃and decoloring time was 2 hours. In the optimum contiditons, the macroporous resin of ADS-7 could remove most pigments, and at the same time it could remove a lot of proteins.
And then, the bioactive activities of polysaccarides of Magnolia Officinalis were studied. The polysaccharides were extracted by hot water and divided into three fractions. The molecular weights of the three polysaccharides fractions ranged from 5.6 to 9.4 kDa,4.7 to 5.4 kDa and 3.9 to 4.2 kDa, respectively. The results of experiments about antioxidant acvities showed that compared with the polysaccharides fractions with molecular weight 5.6 to 9.4 kDa and 4.7 to 5.4 kDa, the lowest molecular weight polysaccharides fractions with 3.9 to 4.2 kDa exhibited the highest antioxidant activities and the strongest cytotoxic activity in MCF-7 cells. On the base of antioxidant activities, the homogeneous polysaccharide was isolated from the lowest molecular weight polysaccharides, its molecular weight was 1052.
采用电感耦合等离子体发射光谱法(ICP-AES)对厚朴水煎液中人体所需的6种微量元素Fe、Mn、Zn、Cu、Cr和Ni的形态进行分析。原药中Fe、Cu、Zn和Mn的含量都比较高,其含量特征为Fe>Mn>Zn>Cu>Cr>Ni。在初级形态中,Ni的提取率最高。在次级形态中,微量元素在颗粒物中的浓度远大于在可溶态中,可溶态中各元素的含量分布为Fe>Mn>Zn>Cr>Cu>Ni。在可溶态中,微量元素只有少量以有机态结合,大多数都以无机态结合。Fe在水煎液中含量最高,Mn和Ni在水煎液中稳定螯合态在可溶态中所占比例分别为99.85%和94.38%。
用水蒸气提取法提取厚朴中的挥发油,并用GC-MS对其化学成分进行研究。结果表明,其主要成分为β-桉叶醇、10-反式-γ-桉叶醇、2-异丙烯-4a,8-二甲基-1,2,3,4,4a,5,6,7-八氢萘、β-芹子烯和β-石竹烯。对厚朴挥发油进行活性研究,发现其挥发油具有-定的抗氧化和抗菌作用,而且抗菌研究发现厚朴挥发油对金黄色葡萄球菌具有较为明显的抗菌活性。
通过抗氧化、抗菌和抗肿瘤活性实验发现,厚朴各相萃取物表现出不同的抗氧化、抗菌和抗肿瘤活性,并测量了各相萃取物中总多酚和总单宁含量。从抗肿瘤活性最高的萃取物中分离得到的主要成分厚朴酚与和厚朴酚表现出对KB、HepG 2和MCF-7肿瘤细胞生长的抑制作用。
对厚朴多糖的研究首先通过响应面分析法考察了提取时间、提取温度和水料比等对多糖提取率的影响,得到了水提法的最佳提取工艺：提取温度是93.7℃；提取时间为3.25小时；水料比为26.2：1。再以脱色率、脱蛋白率和多糖保留率为指标,筛选出最适宜的大孔树脂对厚朴多糖进行脱色工艺研究。选用ADS-7大孔吸附树脂作为研究对象,通过正交试验研究大孔吸附树脂对多糖的脱色工艺,得到最佳工艺条件为：多糖浓度为0.2%；脱色温度为45℃；脱色时间为2小时。在此条件下,厚朴多糖的脱色率可达到最大,同时也可以去除大量的蛋白。
在提取工艺和脱色工艺的基础上,通过分级沉淀法得到了三种厚朴多糖部分。这三种多糖部分的分子量分布范围分别是5600-9400 Da,4700-5400 Da,3900-4200 Da。通过对三种多糖部分的活性研究发现,分子量最小的多糖部分表现出更高的抗氧化活性和对肿瘤细胞生长更强的抑制作用,并对其进行分离纯化,初步研究表明,得到了一个均一多糖,其分子量为1052。
Magnolia Officinalis, a traditional herb in Chinese medicine decoction, has been widely used to treat dissolving phlegm, eliminating dampness, gastrointestinal complaints, anxiety and nervous disturbance. In recent years, many studies have fouced on its components, magnolol and honokiol, however, more studies have comfirmed that microelement, essential oil, different solvent extractions and polysaccharides are the bioactive ingredients in plants, thus it is necessary to study about microelement, essential oil, different solvent extractions and polysaccharides of Magnolia Officinalis.
The microelements are associated with the human life, but also related to the effect of chinese medicine in decoction. The chemical speciation of six microelements were traced and analyzed in the aqua decoction of magnolia officinalis by inductively coupled plasma emission spectrometry (ICP-AES). The contents of Fe, Cu, Zn were higher than the other three microelements'in crude drug, and the order was Fe>Mn>Zn>Cu>Cr>Ni. The results of the primary form analysis showed that the extration rate of Ni was the highest. Furthermore, the sub-form distribution indicated that the concentrations of these trace elements in the particulate species were far greater than in the soluble species, the order of microelements in soluble species was Fe>Mn>Zn>Cu>Cr>Ni. Most of microelements were inorganic species. Particularly, the content of Fe was the highest in the aqua decoction. The stable species of Mn and Ni were 99.85% and 94.38%, respectively.
The hydro-distilled essential oil from Magnolia Officinalis was analyzed by GC/MS, and the major components wereβ-eudesmol,10-epi-γ-eudesmol,2-isopropenyl-4a,8-dimethyl-1,2, 3,4,4a,5,6,7-octahydronaphthalene,β-selinene, (3-caryophyllene, cadalene andβ-cadinene. 10-epi-γ-eudesmol,2-isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene and cadalene were found in magnolia species. The essential oil of Magnolia officinalis possessed antioxidant and antibateria activities. Moreover, the oil exhibited obvious activity against Staphylococcus aureus.
The antioxidant and antibateria experiments were carried out to evaluate different solvent extractions. Different solvent extractions exhibited different antioxidant, antibacteria and antitumor activities. And the Total polyphenols content and total tannin content were measured to find out the relationship between the TPC/TTC and antioxidant acitivities. Magnolol and honokiol were isolated from the extraction which showed the high antitumor activity.
The polysaccharides were the major bioactive ingredients of Magnolia Officinalis. In the study, we investigated the yield of polysaccharides from magnolia officinal under different extraction temperatures, extraction times and water/material ratios by using the response surface methodology (RSE). The optimal water extraction conditions for polysaccharides were as followes:extraction temperature of 93.7℃, extraction time of 3.25 h and a water/material ratio of 26.2:1. And macroporous adsorption resin of ADS-7 which had the best effect on decolorization of magnolia officinalis polysaccharides was chosen from 6 kinds of macroporous adsorption resins with decolorization rate, deproteinization rate and retention rate of polysaccharides as indices, and the optimum technique of decolorization was studied by orthogonal experimental method. The optimum conditions of decolorization were as follows:polysaccharides concentration was 0.2%, decoloring temperature was 45℃and decoloring time was 2 hours. In the optimum contiditons, the macroporous resin of ADS-7 could remove most pigments, and at the same time it could remove a lot of proteins.
And then, the bioactive activities of polysaccarides of Magnolia Officinalis were studied. The polysaccharides were extracted by hot water and divided into three fractions. The molecular weights of the three polysaccharides fractions ranged from 5.6 to 9.4 kDa,4.7 to 5.4 kDa and 3.9 to 4.2 kDa, respectively. The results of experiments about antioxidant acvities showed that compared with the polysaccharides fractions with molecular weight 5.6 to 9.4 kDa and 4.7 to 5.4 kDa, the lowest molecular weight polysaccharides fractions with 3.9 to 4.2 kDa exhibited the highest antioxidant activities and the strongest cytotoxic activity in MCF-7 cells. On the base of antioxidant activities, the homogeneous polysaccharide was isolated from the lowest molecular weight polysaccharides, its molecular weight was 1052.
引文
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