荔枝壳功能性成分制备与生理活性研究
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摘要
荔枝属无患子科,有“岭南果王”之称,为广东四大名果之一。我国是世界上栽培荔枝最早的国家,已知的种植品种有一百多个。2005年产量约165万吨,种植面积约60万公顷,分别占世界荔枝总产量的70.5%和荔枝栽培总面积的84.5%。荔枝壳重量占果实总重的16%左右,富含黄烷醇类和水溶性多糖等生理活性物质,具有很高的药用价值。而在荔枝加工和消费过程中,荔枝壳通常作为废弃物直接清理掉,造成了很大的资源浪费。因此,对这一资源进行深度开发,对于提高荔枝产品附加值、促进农业经济发展和保护环境具有重要意义。
本课题首先选取广东省常见的六个荔枝品种(槐枝、白蜡、三月红、妃子笑、糯米糍和桂味),测定其总黄酮含量、水溶性多糖含量和体外抗氧化性指标,比较结果得出,槐枝品种荔枝壳的总黄酮含量和水溶性多糖含量最高,分别为32.16 mg/g和5.51mg/g,各品种荔枝壳粗黄酮的抗氧化活性相当。因此,选取槐枝荔枝壳进行后续的分离纯化和生理活性研究。
在荔枝壳黄酮提取工艺试验中,分别探索了溶剂(水、60%乙醇溶液和乙酸乙酯)、盐酸、浸提时间、浸提温度、液料比、乙醇溶液浓度和浸提次数对提取效果的影响,并在单因素试验基础上,设计响应面分析方案,确定四个因素(浸提时间、浸提温度、液料比和乙醇溶液浓度),以总黄酮提取率为响应指标,建立该指标与四个因素间的数学模型,分析因素间的交互作用,并得出取得最高总黄酮提取率所需的工艺参数为:浸提时间3.9h,浸提温度78.2℃,液料比20.0mL/g,乙醇溶液浓度为80.0%。
在荔枝壳粗黄酮分离纯化过程中,首先采用有机溶剂(正己烷、乙酸乙酯、正丁醇和水)初步分级,测定各级分的总黄酮含量和抗氧化性,最终确定乙酸乙酯相进入下一步纯化工序。采用反相高效液相色谱柱纯化出三种物质,通过液质联用技术、核磁共振波谱技术和紫外可见光扫描技术鉴定为表儿茶素、原花青素B2和原花青素B4。
通过体外抗氧化试验、免疫细胞增殖试验和乳腺癌细胞增殖试验研究表明,这三种黄烷醇具有很强的自由基清除能力;乙酸乙酯相粗黄烷醇和表儿茶素的脾脏淋巴细胞增殖促进能力显著(P<0.05);表儿茶素和原花青素B2的乳腺癌细胞增殖抑制能力虽然低于对照品紫杉醇,但也具有显著效果(P<0.01)。
在荔枝壳水溶性多糖提取试验中,以水为提取溶剂,研究了浸提时间、浸提温度、液料比和浸提次数对提取效果的影响。并且在单因素试验基础上,设计响应面分析方案,确定三个因素(浸提时间、浸提温度和液料比),以水溶性多糖提取率为响应指标,建立响应指标与三个因素间的数学模型,分析因素间的交互关系,计算出取得最高水溶性多糖提取率所需的工艺参数为:浸提时间2.7h,浸提温度75.1℃,液料比13.8mL/g。
在水溶性多糖结构鉴定试验中,分别采用阴离子交换色谱和凝胶过滤色谱技术纯化出分子量均一多糖组分,通过气相色谱技术鉴定出该多糖由甘露糖(65.6%)、半乳糖(33.0%)和阿拉伯糖(1.4%)组成, 1→2糖苷键、1→3糖苷键和1→6糖苷键摩尔百分比约为8.7%:83.3%:8.0%。凝胶渗透色谱测定其分子量为14kDa,红外光谱分析表明甘露糖以β形式存在。比较不同纯化阶段的多糖样品自由基清除能力发现,随着纯化步骤的进行,多糖抗氧化性不断提高。
Litchi (Litchi Chinensis Sonn.), called fruit king of south China, is a world-wide famous fruit. China is the first country planting litchi in the world. There are more than one hundred of cultivars in China. In 2005, the yield and planting area in China are 1,650,000 tuns and 600,000 hectares, accounting for 70.5% of total yield and 84.5% of total area in the world, respectively. The pericarp tissues account for 16% of total weight of litchi fruit. They enrich in flavanols and water-soluble polysaccharides, and can be employed to produce healthful food and medicine. But, during the programme of direct consumption and agro-industrial process, litchi pericarp is eliminated as waste. Therefore, further development of this material means much for improving the by-product value of litchi fruit, facilitating the development of related agro-industry and enviroment protection.
Six well-known litchi cultivars (Huaizhi, Baila, Sanyuehong, Feizixiao, Nuomici and Guiwei) were chosed in this project. The total flavonoid content, water-soluble polysaccharide content and in vitro antioxidant activities were determined. The results showed Huaizhi litchi pericarp had the highest total flavonoid content and water-soluble polysaccharide content with the value of 32.16 mg/g and 5.51 mg/g, respectively. The antioxidant activities of crude flavonoids from these six cultivar were not significantly different. Thus, Huaizhi was selected for the following trials.
In the experiments of developing the extraction program of flavonoids from litchi pericarp tissues, the effects of solvent (water, 60% (v/v) of ethanol solution and ethyl acetate), hydrochloric acid, extraction time, extraction temperature, solvent-material ratio, ethanol solution concentration and extraction times on extraction program were studied. Response surface methodology was further taken basing on the single factor experiments. Extraction time, extraction temperature, solvent-material ratio and ethanol solution concentration were chosed in this programme. The mathematic model was built between the response, total flavonoids yield, and four factors. The interactions between every two factors were analysed. The optimal conditions for obtaining the highest total flavonoid yield were as follows: 3.9h of extraction time, 78.2℃of extraction temperature, 20.0 ml/g of solvent-material ratio and 80.0% of ethanol solution concentration.
In the experiments of isolation and purification of crude flavonoids, they were classified into four fractions depending on solubility by hexane, ethyl acetate, butanol and water. The ethyl acetate phase was chosen for further purification for its high total flavonoid content and antioxidant activity. Three compounds were isolated and collected by reverse-phase high performance liquid chromatography (HPLC). They were identified as epicatechin, proanthocyanidin B2 and proanthocyanidin B4 by HPLC-mass spectrometry, nuclear magnetic resonnance spectrometry and ultraviolet-visible light spectrophotometer.
In vitro trials of determination of antioxidant activities, immune cell proliferation and breast cancer cell proliferation were made. The results showed these three flavanols had strong radical-scavenging activities. The ethyl acetate fraction and epicatechin had significant capacities of promoting spleen lymphocyte proliferation. Epicatechin and proanthocyanidin B2 had significant capacities of inhibiting breast cancer cell proliferation, though lower than that of paclitaxel.
Four factors, extraction time, extraction temperature, water-material ratio and extraction times, were selected for exploring the water-soluble polysaccharide extraction process. Response surface methodology was taken to optimize the process conditions basing on single factor trials. Extraction time, extraction temperature and solvent-material ratio were set. The mathetical model of water-soluble polysaccharide yield as a function of extraction time, extraction temperature and solvent-material ratio was built. The interactions between factors of each other were analysed and the optimal conditions for obtaining highest water-soluble polysaccharide yield were computed as follows: 2.7 h of extraction time, 75.1℃of extraction temperature and 13.8 ml/g of solvent-material ratio.
Even-molecular-weight water-soluble polysaccharides were obtained by using techniques of DEAE anion-exchanging chromatography and gel filtration chromatography. It comprised mannose (65.6%), galactose (33.0%) and arabinose (1.4%). The molar percentage ratio of (1→2)-glycosidic linkages, (1→3)-glycosidic linkages and (1→6)-glycosidic linkages was about 8.7%:83.3%:8.0%. The molecular weight was determined as 14 kDa by gel permeation chromatography. The infra-red spectra showed mannose existed asβtype. By comparison of antioxidant activities of water-soluble polysaccharides from each purification step, we found that the antioxidant activity kept increasing as purification went on.
本课题首先选取广东省常见的六个荔枝品种(槐枝、白蜡、三月红、妃子笑、糯米糍和桂味),测定其总黄酮含量、水溶性多糖含量和体外抗氧化性指标,比较结果得出,槐枝品种荔枝壳的总黄酮含量和水溶性多糖含量最高,分别为32.16 mg/g和5.51mg/g,各品种荔枝壳粗黄酮的抗氧化活性相当。因此,选取槐枝荔枝壳进行后续的分离纯化和生理活性研究。
在荔枝壳黄酮提取工艺试验中,分别探索了溶剂(水、60%乙醇溶液和乙酸乙酯)、盐酸、浸提时间、浸提温度、液料比、乙醇溶液浓度和浸提次数对提取效果的影响,并在单因素试验基础上,设计响应面分析方案,确定四个因素(浸提时间、浸提温度、液料比和乙醇溶液浓度),以总黄酮提取率为响应指标,建立该指标与四个因素间的数学模型,分析因素间的交互作用,并得出取得最高总黄酮提取率所需的工艺参数为:浸提时间3.9h,浸提温度78.2℃,液料比20.0mL/g,乙醇溶液浓度为80.0%。
在荔枝壳粗黄酮分离纯化过程中,首先采用有机溶剂(正己烷、乙酸乙酯、正丁醇和水)初步分级,测定各级分的总黄酮含量和抗氧化性,最终确定乙酸乙酯相进入下一步纯化工序。采用反相高效液相色谱柱纯化出三种物质,通过液质联用技术、核磁共振波谱技术和紫外可见光扫描技术鉴定为表儿茶素、原花青素B2和原花青素B4。
通过体外抗氧化试验、免疫细胞增殖试验和乳腺癌细胞增殖试验研究表明,这三种黄烷醇具有很强的自由基清除能力;乙酸乙酯相粗黄烷醇和表儿茶素的脾脏淋巴细胞增殖促进能力显著(P<0.05);表儿茶素和原花青素B2的乳腺癌细胞增殖抑制能力虽然低于对照品紫杉醇,但也具有显著效果(P<0.01)。
在荔枝壳水溶性多糖提取试验中,以水为提取溶剂,研究了浸提时间、浸提温度、液料比和浸提次数对提取效果的影响。并且在单因素试验基础上,设计响应面分析方案,确定三个因素(浸提时间、浸提温度和液料比),以水溶性多糖提取率为响应指标,建立响应指标与三个因素间的数学模型,分析因素间的交互关系,计算出取得最高水溶性多糖提取率所需的工艺参数为:浸提时间2.7h,浸提温度75.1℃,液料比13.8mL/g。
在水溶性多糖结构鉴定试验中,分别采用阴离子交换色谱和凝胶过滤色谱技术纯化出分子量均一多糖组分,通过气相色谱技术鉴定出该多糖由甘露糖(65.6%)、半乳糖(33.0%)和阿拉伯糖(1.4%)组成, 1→2糖苷键、1→3糖苷键和1→6糖苷键摩尔百分比约为8.7%:83.3%:8.0%。凝胶渗透色谱测定其分子量为14kDa,红外光谱分析表明甘露糖以β形式存在。比较不同纯化阶段的多糖样品自由基清除能力发现,随着纯化步骤的进行,多糖抗氧化性不断提高。
Litchi (Litchi Chinensis Sonn.), called fruit king of south China, is a world-wide famous fruit. China is the first country planting litchi in the world. There are more than one hundred of cultivars in China. In 2005, the yield and planting area in China are 1,650,000 tuns and 600,000 hectares, accounting for 70.5% of total yield and 84.5% of total area in the world, respectively. The pericarp tissues account for 16% of total weight of litchi fruit. They enrich in flavanols and water-soluble polysaccharides, and can be employed to produce healthful food and medicine. But, during the programme of direct consumption and agro-industrial process, litchi pericarp is eliminated as waste. Therefore, further development of this material means much for improving the by-product value of litchi fruit, facilitating the development of related agro-industry and enviroment protection.
Six well-known litchi cultivars (Huaizhi, Baila, Sanyuehong, Feizixiao, Nuomici and Guiwei) were chosed in this project. The total flavonoid content, water-soluble polysaccharide content and in vitro antioxidant activities were determined. The results showed Huaizhi litchi pericarp had the highest total flavonoid content and water-soluble polysaccharide content with the value of 32.16 mg/g and 5.51 mg/g, respectively. The antioxidant activities of crude flavonoids from these six cultivar were not significantly different. Thus, Huaizhi was selected for the following trials.
In the experiments of developing the extraction program of flavonoids from litchi pericarp tissues, the effects of solvent (water, 60% (v/v) of ethanol solution and ethyl acetate), hydrochloric acid, extraction time, extraction temperature, solvent-material ratio, ethanol solution concentration and extraction times on extraction program were studied. Response surface methodology was further taken basing on the single factor experiments. Extraction time, extraction temperature, solvent-material ratio and ethanol solution concentration were chosed in this programme. The mathematic model was built between the response, total flavonoids yield, and four factors. The interactions between every two factors were analysed. The optimal conditions for obtaining the highest total flavonoid yield were as follows: 3.9h of extraction time, 78.2℃of extraction temperature, 20.0 ml/g of solvent-material ratio and 80.0% of ethanol solution concentration.
In the experiments of isolation and purification of crude flavonoids, they were classified into four fractions depending on solubility by hexane, ethyl acetate, butanol and water. The ethyl acetate phase was chosen for further purification for its high total flavonoid content and antioxidant activity. Three compounds were isolated and collected by reverse-phase high performance liquid chromatography (HPLC). They were identified as epicatechin, proanthocyanidin B2 and proanthocyanidin B4 by HPLC-mass spectrometry, nuclear magnetic resonnance spectrometry and ultraviolet-visible light spectrophotometer.
In vitro trials of determination of antioxidant activities, immune cell proliferation and breast cancer cell proliferation were made. The results showed these three flavanols had strong radical-scavenging activities. The ethyl acetate fraction and epicatechin had significant capacities of promoting spleen lymphocyte proliferation. Epicatechin and proanthocyanidin B2 had significant capacities of inhibiting breast cancer cell proliferation, though lower than that of paclitaxel.
Four factors, extraction time, extraction temperature, water-material ratio and extraction times, were selected for exploring the water-soluble polysaccharide extraction process. Response surface methodology was taken to optimize the process conditions basing on single factor trials. Extraction time, extraction temperature and solvent-material ratio were set. The mathetical model of water-soluble polysaccharide yield as a function of extraction time, extraction temperature and solvent-material ratio was built. The interactions between factors of each other were analysed and the optimal conditions for obtaining highest water-soluble polysaccharide yield were computed as follows: 2.7 h of extraction time, 75.1℃of extraction temperature and 13.8 ml/g of solvent-material ratio.
Even-molecular-weight water-soluble polysaccharides were obtained by using techniques of DEAE anion-exchanging chromatography and gel filtration chromatography. It comprised mannose (65.6%), galactose (33.0%) and arabinose (1.4%). The molar percentage ratio of (1→2)-glycosidic linkages, (1→3)-glycosidic linkages and (1→6)-glycosidic linkages was about 8.7%:83.3%:8.0%. The molecular weight was determined as 14 kDa by gel permeation chromatography. The infra-red spectra showed mannose existed asβtype. By comparison of antioxidant activities of water-soluble polysaccharides from each purification step, we found that the antioxidant activity kept increasing as purification went on.
引文
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