大叶藻中黄酮类化合物的研究
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摘要
研究报道,黄酮类化合物具有广泛的生物活性,包括抗氧化、抗突变、抗衰老、抗肿瘤、抗菌等,是茶及众多中草药如黄芩、银杏、沙棘等的活性成分。黄酮可以改善血液循环,降低胆固醇,改善心脑血管疾病的症状。本文主要介绍从大叶藻中提取分离纯化黄酮类化合物,并研究黄酮粗提物的抗氧化活性。
本文首先综述了大叶藻的生物门类、生活习性,黄酮类化合物的分类、生物活性、各种理化性质和显色反应等,并对国内外文献中黄酮类化合物的提取、纯化、分离和分析过程的研究进展进行了概述和比较。
首先测定了大叶藻中各种基本成分的含量,总糖含量:7.1%,粗脂肪含量:1.2%,蛋白质含量:5.21%。在已测定的16中氨基酸中,每100mL的大叶藻处理液中中氨基酸含量可达21.134g,其中必须氨基酸有6种,共7.470g,占氨基酸总量的35.346%,非必须氨基酸总量为13.664g,占必须氨基酸总含量的64.654%。在必须氨基酸中,以缬氨酸含量最高,占必须氨基酸含量的23.655%,蛋氨酸含量最低,占必须氨基酸含量的7.416%。在非必须氨基酸中,谷氨酸含量最高,占非必须氨基酸含量的23.259%,以组氨酸含量最低,占非必须氨基酸含量的2.518%。
其次进行了黄酮类化合物的浸提试验,试验中总黄酮含量分析采用酸性条件下氯化铝显色的可见分光光度法。乙醇作为浸提液,研究了传统工艺浸提法、超声波辅助浸提法、纤维素酶辅助浸提法和纤维素酶-超声波辅助浸提法,得到了这四种提取方法对黄酮的最佳浸提条件,并对其最大提取率进行比较,传统工艺提取法最佳提取工艺:提取温度55℃,提取时间3h,乙醇浓度为85%,提取料液比为1:30,最大提取率为1.84%;超声波辅助浸提法最佳提取工艺条件:超声时间15min,超声功率300W,乙醇浓度20%,料液比1:10,最大提取率为3.80%;酶解辅助浸提法最佳提取条件:加酶量为0.030g/g原料、酶解时间为25℃、酶解pH为4、酶解时间1h,最大提取率为5.93%;纤维素酶-超声波辅助浸提法最佳提取条件:超声时间15min,超声功率250W,纤维素酶量0.025g/g原料,酶解PH5,最大提取率为7.86%。结果表明,纤维素酶-超声波辅助浸提法的提取率最高。
浸提得到的粗黄酮提取物,进行硅胶层析柱的初步分离纯化。首先是使用极性由小到大的各种有机溶剂连续抽提(石油醚-乙酸乙酯-正丁醇-水),从而将亲脂性的成分和亲水性的成分分开。然后详细的对液液萃取纯化后的成分用硅胶板进行了展开剂的选择,发现氯仿:甲醇系统对该混合物具有较好的展开性能,再用硅胶柱层析对液液萃取纯化后的粗提物进行了分离纯化,最后采用气相色谱质谱联用技术对纯化物的结构进行检测分析。分析得到了七种黄酮化合物:(1)3,4,5,4'-四甲氧基查耳酮;(2)3,4-二甲氧基-8,9-二氧亚甲基紫檀素;(3)5-羟基-7,4'-二甲氧基黄酮;(4)5,7,5'-三羟基-4'-甲氧基二氢黄酮;(5)5,7-二羟基-4'-甲氧基黄酮醇;(6)4',5'-二羟基-7-甲氧基黄酮醇;(7)5,7,4'-三羟基黄酮。
在所发现的七种黄酮类物质中,5,7,4'-三羟基黄酮具有与毛地黄黄酮相似的结构,所不同之处是由于5,7,4'-三羟基黄酮在3'位置是一个氢而不是一个羟基。根据相似结构具有相似特性,可推测5,7,4'-三羟基黄酮也应该与有与毛地黄黄酮相似的特性,需要做进一方面的验证和检测。5,7-二羟基-4'-甲氧基黄酮醇具有与金雀花异黄素相似的结构特征,而金雀花异黄素具有雌性激素的作用,因此可推测5,7-二羟基-4'-甲氧基黄酮醇具有雌激素的作用,需进一步的验证和分析。其中的3,4,5,4'-四甲氧基查耳酮,3,4-二甲氧基-8,9-二氧亚甲基紫檀素和5-羟基-7,4'-二甲氧基黄酮这三种物质含甲氧基较多,据研究发现多聚甲氧基的脂溶性黄酮具有较强的抗肿瘤特性,这是由于聚甲氧基的黄酮类呈脂溶性,更易于接近并浸透到肿瘤细胞而起到作用。因此这三种黄酮对于开发新的抗肿瘤药物具有一定的实际利用价值。而其中5,7,5'-三羟基-4'-甲氧基二氢黄酮、5,7-二羟基-4'-甲氧基黄酮醇、5,7,4'-三羟基黄酮属于多羟基黄酮醇,而多羟基黄酮醇类化合物经生物学试验,证实可用于升高血浆高密度脂蛋白胆固醇(HDL-C),降低血浆低密度脂蛋白胆固醇(LDL-C)和血浆甘油三脂(TG)。这些黄酮类物质对于预防和治疗诸如外周血管疾病、冠心病、中风疾病、血管再狭窄等血管疾病具有一定的功效。
本文采用大叶藻为提取原料一方面有效利用了大叶藻,另一方面为抗肿瘤抗癌及心脑血管疾病药物的开发开辟了新的空间。也为大叶藻中黄酮结构-功能方面的研究提供了依据。
文章最后进行了大叶藻黄酮化合物粗提物的抗氧化活性实验,主要是研究其对超氧阴离子的清除能力、对羟自由基的清除能力,以及对油脂的抗氧化酸败等,大叶藻黄酮对羟自由基、超氧阴离子有较强的清除作用。当添加浓度为10mg·mL-1时,对羟自由基的清除作用最大可达91.56%;当添加浓度为8mg·mL-1时,对超氧阴离子的清除作用最大可达90.43%,说明适宜的添加浓度,大叶藻黄酮可以完全清除了已产生的超氧阴离子还抑制了超氧阴离子的产生。大叶藻黄酮对猪油、花生油、亚油酸有极强的抗氧化作用,能够明显地抑制猪油、花生油POV值地升高。研究结果表明,大叶藻黄酮具有一定的清除自由基作用和抑制油脂氧化酸败的作用,大叶藻具有一定的开发利用前景。
As is known, flavonoids have extensive biological activity, including antioxidation, mutation, anti-aging, antitumor, anti-bacteria, which are active ingredients of tea and many other herbs. Flavonoids can improve blood circulation, reduce cholesterol, eliminate cardiovascular diseases' symptoms. This paper mainly study on the extract technology ,the structure and antioxidant activity of flavonoids from Zostera marina L.
The paper firstly summarized the biological categories and living habits of Zostera marina L.And the classification, biological activity , physical and chemical properties of flavonoids were also related ,lastly the extraction, purification, separation and analysis process of flavonoids in Zostera marina L. were reviewed and compared in this paper.
In the tests, first,by detection of the basic ingredients of Zostera marina L. ,we found that the carbohydrate is 7.1%,fat is 1.2%,water is 8.0%,protein is 5.21%. Amino acids up to 21.134g in each 100mL Zostera marina L. There is 7.470g six essential amino acids, which is 35.346% of total amino acids in 100g Zostera marina L.The unnessary maino acid is 13.664g.In the necessary amino acids , the content of Val is the highest,accounting for 23.655%,methionine is the lowest accounting for 7.416%.In the unnecessary acids , the content of Gul is the highest and histidine is the lowest,respectively accounting for 23.259% and 2.518%.
The second, the extraction of flavonoids tests are conducted from Zostera marina L.,tests analysis the content of flavonoid mainly by adopting the AlCl3'colour-display spectrophotometry in acidic condition. Single factor experiments and orthogonal tests were performed to optimize the extraction technology with traditional ethanol process, ultrasonic wave assisted process, cellulose assisted process and cellulose and ultrasonic wave assisted process.The beat total flavonoids yield of above four kinds extraction technology is the cellulose and ultrasonic wave assisted extraction technology, with yield 7.86%.
After, the flavonoids were separated and purificated by silica gel column chromatography together with other separation methods,such as TLC、the specific color reaction of flavone、Plates and so on. The first is continuous extraction with a variety of polar organic solvents(petroleum ether - ethyl acetate - butanol - water), thus the lipophilic character compositions and hydrophilic ingredients were aparted.Then by the choice of developing solvent with silicon sheets, discovered chloroform: methanol: acetic acid system has good performance to the mixture, then using silica gel column chromatography purified the extracts, finally, gas chromatography - mass spectrometry (GC-MS) instrument was used for detecting the structure of flavonoids.They were:3,4,5,4 '- tetramethoxy chalcone (1);3,4 - dimethoxy -8,9 - Methylenedioxy pterocarpine(2);5 - hydroxy -7,4 '- dimethoxy flavones(3);5,7,5 '- trihydroxy -4' - methoxy dihydroflavonol(4);5,7 - dihydroxy -4 '- methoxy flavonones(5);4 ', 5'-dihydroxy -7-- methoxy flavononesl(6);5,7,4' - trihydroxy flavones(7).
In the seven flavonoids ,5,7,4 '- trihydroxy flavone and luteolin have the similar structure, the difference is due to 5,7,4 ' - trihydroxy flavonoids in the 3 'position is a hydrogen instead of a hydroxyl group. Similar characteristics based on similar structure, we can inferred 5,7,4 '- trihydroxy flavone should also have similar characteristics with luteolin, needs to be further verified.5,7 - dihydroxy -4 '- methoxy flavonols has a similar structure with broom genistein, while the broom genistein has the role of estrogen, it can be speculated that 5,7 - dihydroxy-4 '- methoxy flavonols has estrogen's effects, these need further verification and analysis. 3,4,5,4 '- tetramethoxy chalcone, 3,4 - dimethoxy -8,9 - Methylenedioxy red sandalwood pigment and 5 - hydroxy -7,4' - dimethoxy flavone may be have strong anti-tumor properties,which are poly-methoxy flavonoids.Due to methoxy-poly fat-soluble flavonoids were moreaccessible and easily soaked into the tumor cells and play a role. Therefore, the three flavonoids has some practical value for the development of new anticancer drugs.5,7,5 '- trihydroxy -4' - methoxy dihydroflavonol、5,7 - dihydroxy -4 '- methoxy flavonones and 5,7,4' - trihydroxy flavones are morehydroxy flavonols. Morehydroxy flavonol compounds were tested that can be decrease the density of high-density lipoprotein cholesterol (HDL-C), lipoprotein cholesterol (LDL-C) and plasma triglyceride(TG). Morehydroxy flavonol have a certain effects for the prevention and treatment of vascular diseases ,such as peripheral vascular disease, coronary heart disease, stroke disease, vascular restenosis and other diseases .The flavonoids from Zostera marina L. provide a wider space for the development and highly utilization of Zostera marina L..
Antioxidative activity of flavonoids from Zostera marina L..were investigated by examining the scavenging effects on hydroxyl radicals (·OH) and superoxide anion(O2-) and the inhibitions on auto-oxidation of lard, rape seed oil. The results showed that Flavonoids from Zostera marina L.had significant scavenging functions to free radical and superoxide anion, and the scavenging effect increased with the increase of the content of flavonoids from Zostera marina L..Flavonoids from Zostera marina L. can remarkably inhibit auto-oxidation of lard, rape seed oil,indicating its potential as a natural antioxidant.
本文首先综述了大叶藻的生物门类、生活习性,黄酮类化合物的分类、生物活性、各种理化性质和显色反应等,并对国内外文献中黄酮类化合物的提取、纯化、分离和分析过程的研究进展进行了概述和比较。
首先测定了大叶藻中各种基本成分的含量,总糖含量:7.1%,粗脂肪含量:1.2%,蛋白质含量:5.21%。在已测定的16中氨基酸中,每100mL的大叶藻处理液中中氨基酸含量可达21.134g,其中必须氨基酸有6种,共7.470g,占氨基酸总量的35.346%,非必须氨基酸总量为13.664g,占必须氨基酸总含量的64.654%。在必须氨基酸中,以缬氨酸含量最高,占必须氨基酸含量的23.655%,蛋氨酸含量最低,占必须氨基酸含量的7.416%。在非必须氨基酸中,谷氨酸含量最高,占非必须氨基酸含量的23.259%,以组氨酸含量最低,占非必须氨基酸含量的2.518%。
其次进行了黄酮类化合物的浸提试验,试验中总黄酮含量分析采用酸性条件下氯化铝显色的可见分光光度法。乙醇作为浸提液,研究了传统工艺浸提法、超声波辅助浸提法、纤维素酶辅助浸提法和纤维素酶-超声波辅助浸提法,得到了这四种提取方法对黄酮的最佳浸提条件,并对其最大提取率进行比较,传统工艺提取法最佳提取工艺:提取温度55℃,提取时间3h,乙醇浓度为85%,提取料液比为1:30,最大提取率为1.84%;超声波辅助浸提法最佳提取工艺条件:超声时间15min,超声功率300W,乙醇浓度20%,料液比1:10,最大提取率为3.80%;酶解辅助浸提法最佳提取条件:加酶量为0.030g/g原料、酶解时间为25℃、酶解pH为4、酶解时间1h,最大提取率为5.93%;纤维素酶-超声波辅助浸提法最佳提取条件:超声时间15min,超声功率250W,纤维素酶量0.025g/g原料,酶解PH5,最大提取率为7.86%。结果表明,纤维素酶-超声波辅助浸提法的提取率最高。
浸提得到的粗黄酮提取物,进行硅胶层析柱的初步分离纯化。首先是使用极性由小到大的各种有机溶剂连续抽提(石油醚-乙酸乙酯-正丁醇-水),从而将亲脂性的成分和亲水性的成分分开。然后详细的对液液萃取纯化后的成分用硅胶板进行了展开剂的选择,发现氯仿:甲醇系统对该混合物具有较好的展开性能,再用硅胶柱层析对液液萃取纯化后的粗提物进行了分离纯化,最后采用气相色谱质谱联用技术对纯化物的结构进行检测分析。分析得到了七种黄酮化合物:(1)3,4,5,4'-四甲氧基查耳酮;(2)3,4-二甲氧基-8,9-二氧亚甲基紫檀素;(3)5-羟基-7,4'-二甲氧基黄酮;(4)5,7,5'-三羟基-4'-甲氧基二氢黄酮;(5)5,7-二羟基-4'-甲氧基黄酮醇;(6)4',5'-二羟基-7-甲氧基黄酮醇;(7)5,7,4'-三羟基黄酮。
在所发现的七种黄酮类物质中,5,7,4'-三羟基黄酮具有与毛地黄黄酮相似的结构,所不同之处是由于5,7,4'-三羟基黄酮在3'位置是一个氢而不是一个羟基。根据相似结构具有相似特性,可推测5,7,4'-三羟基黄酮也应该与有与毛地黄黄酮相似的特性,需要做进一方面的验证和检测。5,7-二羟基-4'-甲氧基黄酮醇具有与金雀花异黄素相似的结构特征,而金雀花异黄素具有雌性激素的作用,因此可推测5,7-二羟基-4'-甲氧基黄酮醇具有雌激素的作用,需进一步的验证和分析。其中的3,4,5,4'-四甲氧基查耳酮,3,4-二甲氧基-8,9-二氧亚甲基紫檀素和5-羟基-7,4'-二甲氧基黄酮这三种物质含甲氧基较多,据研究发现多聚甲氧基的脂溶性黄酮具有较强的抗肿瘤特性,这是由于聚甲氧基的黄酮类呈脂溶性,更易于接近并浸透到肿瘤细胞而起到作用。因此这三种黄酮对于开发新的抗肿瘤药物具有一定的实际利用价值。而其中5,7,5'-三羟基-4'-甲氧基二氢黄酮、5,7-二羟基-4'-甲氧基黄酮醇、5,7,4'-三羟基黄酮属于多羟基黄酮醇,而多羟基黄酮醇类化合物经生物学试验,证实可用于升高血浆高密度脂蛋白胆固醇(HDL-C),降低血浆低密度脂蛋白胆固醇(LDL-C)和血浆甘油三脂(TG)。这些黄酮类物质对于预防和治疗诸如外周血管疾病、冠心病、中风疾病、血管再狭窄等血管疾病具有一定的功效。
本文采用大叶藻为提取原料一方面有效利用了大叶藻,另一方面为抗肿瘤抗癌及心脑血管疾病药物的开发开辟了新的空间。也为大叶藻中黄酮结构-功能方面的研究提供了依据。
文章最后进行了大叶藻黄酮化合物粗提物的抗氧化活性实验,主要是研究其对超氧阴离子的清除能力、对羟自由基的清除能力,以及对油脂的抗氧化酸败等,大叶藻黄酮对羟自由基、超氧阴离子有较强的清除作用。当添加浓度为10mg·mL-1时,对羟自由基的清除作用最大可达91.56%;当添加浓度为8mg·mL-1时,对超氧阴离子的清除作用最大可达90.43%,说明适宜的添加浓度,大叶藻黄酮可以完全清除了已产生的超氧阴离子还抑制了超氧阴离子的产生。大叶藻黄酮对猪油、花生油、亚油酸有极强的抗氧化作用,能够明显地抑制猪油、花生油POV值地升高。研究结果表明,大叶藻黄酮具有一定的清除自由基作用和抑制油脂氧化酸败的作用,大叶藻具有一定的开发利用前景。
As is known, flavonoids have extensive biological activity, including antioxidation, mutation, anti-aging, antitumor, anti-bacteria, which are active ingredients of tea and many other herbs. Flavonoids can improve blood circulation, reduce cholesterol, eliminate cardiovascular diseases' symptoms. This paper mainly study on the extract technology ,the structure and antioxidant activity of flavonoids from Zostera marina L.
The paper firstly summarized the biological categories and living habits of Zostera marina L.And the classification, biological activity , physical and chemical properties of flavonoids were also related ,lastly the extraction, purification, separation and analysis process of flavonoids in Zostera marina L. were reviewed and compared in this paper.
In the tests, first,by detection of the basic ingredients of Zostera marina L. ,we found that the carbohydrate is 7.1%,fat is 1.2%,water is 8.0%,protein is 5.21%. Amino acids up to 21.134g in each 100mL Zostera marina L. There is 7.470g six essential amino acids, which is 35.346% of total amino acids in 100g Zostera marina L.The unnessary maino acid is 13.664g.In the necessary amino acids , the content of Val is the highest,accounting for 23.655%,methionine is the lowest accounting for 7.416%.In the unnecessary acids , the content of Gul is the highest and histidine is the lowest,respectively accounting for 23.259% and 2.518%.
The second, the extraction of flavonoids tests are conducted from Zostera marina L.,tests analysis the content of flavonoid mainly by adopting the AlCl3'colour-display spectrophotometry in acidic condition. Single factor experiments and orthogonal tests were performed to optimize the extraction technology with traditional ethanol process, ultrasonic wave assisted process, cellulose assisted process and cellulose and ultrasonic wave assisted process.The beat total flavonoids yield of above four kinds extraction technology is the cellulose and ultrasonic wave assisted extraction technology, with yield 7.86%.
After, the flavonoids were separated and purificated by silica gel column chromatography together with other separation methods,such as TLC、the specific color reaction of flavone、Plates and so on. The first is continuous extraction with a variety of polar organic solvents(petroleum ether - ethyl acetate - butanol - water), thus the lipophilic character compositions and hydrophilic ingredients were aparted.Then by the choice of developing solvent with silicon sheets, discovered chloroform: methanol: acetic acid system has good performance to the mixture, then using silica gel column chromatography purified the extracts, finally, gas chromatography - mass spectrometry (GC-MS) instrument was used for detecting the structure of flavonoids.They were:3,4,5,4 '- tetramethoxy chalcone (1);3,4 - dimethoxy -8,9 - Methylenedioxy pterocarpine(2);5 - hydroxy -7,4 '- dimethoxy flavones(3);5,7,5 '- trihydroxy -4' - methoxy dihydroflavonol(4);5,7 - dihydroxy -4 '- methoxy flavonones(5);4 ', 5'-dihydroxy -7-- methoxy flavononesl(6);5,7,4' - trihydroxy flavones(7).
In the seven flavonoids ,5,7,4 '- trihydroxy flavone and luteolin have the similar structure, the difference is due to 5,7,4 ' - trihydroxy flavonoids in the 3 'position is a hydrogen instead of a hydroxyl group. Similar characteristics based on similar structure, we can inferred 5,7,4 '- trihydroxy flavone should also have similar characteristics with luteolin, needs to be further verified.5,7 - dihydroxy -4 '- methoxy flavonols has a similar structure with broom genistein, while the broom genistein has the role of estrogen, it can be speculated that 5,7 - dihydroxy-4 '- methoxy flavonols has estrogen's effects, these need further verification and analysis. 3,4,5,4 '- tetramethoxy chalcone, 3,4 - dimethoxy -8,9 - Methylenedioxy red sandalwood pigment and 5 - hydroxy -7,4' - dimethoxy flavone may be have strong anti-tumor properties,which are poly-methoxy flavonoids.Due to methoxy-poly fat-soluble flavonoids were moreaccessible and easily soaked into the tumor cells and play a role. Therefore, the three flavonoids has some practical value for the development of new anticancer drugs.5,7,5 '- trihydroxy -4' - methoxy dihydroflavonol、5,7 - dihydroxy -4 '- methoxy flavonones and 5,7,4' - trihydroxy flavones are morehydroxy flavonols. Morehydroxy flavonol compounds were tested that can be decrease the density of high-density lipoprotein cholesterol (HDL-C), lipoprotein cholesterol (LDL-C) and plasma triglyceride(TG). Morehydroxy flavonol have a certain effects for the prevention and treatment of vascular diseases ,such as peripheral vascular disease, coronary heart disease, stroke disease, vascular restenosis and other diseases .The flavonoids from Zostera marina L. provide a wider space for the development and highly utilization of Zostera marina L..
Antioxidative activity of flavonoids from Zostera marina L..were investigated by examining the scavenging effects on hydroxyl radicals (·OH) and superoxide anion(O2-) and the inhibitions on auto-oxidation of lard, rape seed oil. The results showed that Flavonoids from Zostera marina L.had significant scavenging functions to free radical and superoxide anion, and the scavenging effect increased with the increase of the content of flavonoids from Zostera marina L..Flavonoids from Zostera marina L. can remarkably inhibit auto-oxidation of lard, rape seed oil,indicating its potential as a natural antioxidant.
引文
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[1]孙艳梅,徐雅琴,杨林.天然物质类黄酮的抗氧化活性的研究[J].中国油脂,2003,28(3):54-56
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[2]彭胜,海草矮大叶藻生物量与热值的动态研究,硕士论文集,广西大学,2007年6月
[3] Arber A.Water plants:a study of aquatic angiosperms.Cambridge Press,1920
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[7]刘平,玉米须黄酮的提取分离及生物活性研究,硕士论文,陕西师范大学,2006年4月
[8]陈从瑾,黄可瀛等,植物中黄酮类化合物的提取方法研究概况,生物质化学工程,2007,3 (41):42-45
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[14]纪大泽.碱水冷浸提取芦丁[J].四川大学学报:自然科学版,1991,28(2):265-267
[15]霍丹群,张文,李奇琳,等.超声波法与热提取法提取山楂总黄酮的比较研究[J].中成药,2004,26(12):1063-1064
[16]王延峰,李延清,郝永红,等.超声波法提取液银杏叶黄酮的研究[J].食品科学,2002,23(8):166-167
[17]郭孝武.超声提取杜仲叶中黄酮类物质工艺研究[J].陕西师范大学学报:自然科学版,2005,33(4):59-61
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