竹叶活性组分的含量筛选及高效制备工艺研究
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摘要
竹子是禾本科,竹亚科多年生常绿植物,在我国有悠久的食用和药用历史。竹叶含有丰富的黄酮和多糖等生物活性组分,由于其具有优良的抗自由基、抗氧化、抗衰老、及保护心血管等方面的生物学功效,目前已受到国内外市场的广泛关注。为了提高竹叶活性组分的高效利用,本论文通过建立竹叶黄酮和多糖活性组分的含量检测方法,筛选高含量的品种;重点研究竹叶黄酮和多糖活性组分的高效提取、分离纯化及其高效制备技术,取得了如下结果:
选用了分光光度法和高效液相色谱法,对比研究竹叶中黄酮含量的差异性,确立了定量测定方法。NaNO2-Al(NO3)3-NaOH比色法较为常用,但是测定结果偏高,以荭草苷、牡荆苷为标准品,HPLC法测定结果更为可靠、准确。
对27个不同品种竹叶黄酮的含量进行了测定,得干竹叶黄酮的含量为0.50%～1.72%,即5.0～17.2mg/g,与文献[1]报道的范围基本相符。对不同品种竹叶多糖的含量进行了测定,得多糖含量在1.94%～5.69%之间,即19.4～56.9mg/g。
淡竹,绿竹,毛竹分布广、黄酮和多糖含量较高,成为工业上提取竹叶黄酮和多糖活性组分的最佳原料;水竹黄酮含量高,鹅毛竹多糖含量特别高,但是分布较少,不适宜为工业原料。对于不同年龄的竹叶,老叶片中多糖含量明显大于新生嫩叶片;而黄酮的含量则因竹种不同存在明显差异。
经单因素优选试验和正交试验,确定了竹叶黄酮的最佳提取工艺。水浴提取最佳条件为:温度50℃,提取时间2.0h,乙醇浓度60%(体积分数,下同),固液比1∶20(g∶mL),各因素对提取率的影响程度依次为:固液比>乙醇浓度>温度>时间;超声波提取最佳条件为:超声波作用时间30min,乙醇浓度60%,固液比1∶15(g∶mL),各因素对提取率的影响程度依次为:提取时间>乙醇浓度>固液比。
通过对几种树脂的比较,AB-8树脂的分离纯化效果最好;通过静态解吸试验,70%的乙醇效果好于90%的乙醇,乙醇的解吸效果要好于甲醇;树脂柱吸附过程中,吸附和解吸附都是渐变的曲线过程,用10%～90%乙醇解吸附剂梯度洗脱时,40%乙醇洗脱出最多的黄酮,解吸附量为47.0mg(47.1cm3的装柱体积)。
竹叶提取物经过组合膜处理后变得澄清透明,色泽鲜亮;在试验所用提取液体积为90L左右,提取液浓度为9.32g/L过陶瓷膜、7.08g/L过超滤膜时,膜使用状况良好,流速稳定,没有发生浓差极化现象;膜处理后的黄酮提取物,再用树脂和聚酰胺树脂处理,聚酰胺纯化效果明显好于AB-8,80%乙醇洗脱,黄酮含量能够达到40.35%;竹叶中试水提取后,经无机和有机膜处理得到多糖提取物,再经85%以上乙醇沉淀,多糖含量提高到40.20%。
Bamboo is perennial evergreen plant,belongs to Bambuaoidease Nees of the family poaceae.It has been used for food and medicines for a long time in China.There is a large body of evidence showing that bamboo leaves is rich in flavinoids and polysaccharides. It has many fine functions,such as anti-free radical,oxidation resistance and protecting vasular system etc.Nowadays,it has received broad attention from all over the world.In order to take full use of bamboo,the work had been done in the paper was focused on the method of determination and high content,especially the extration and purification for flavinoids and polysaccharides in bamboo leaves. The main results are as follows:
HPLC and UV-VIS spectrophotometry were applied to detect the content of flavonoids in the experiment.and the results of the two detection were compared.The method of quantitative detection were established. NaNO2-Al(NO3)3-NaOH UV-VIS spectrophotometry was been common used.But it's result was higher than true. The method of HPLC was reliable and precise by taking Orientin and Isoviextin as the index ingredient.
The content of flavonoids of 27 breeds of bamboo leaves were determined.It was concluded that the total flavonoids content of per gram dry bamboo leaves was between 5.0-13.3 milligram.The result was basically consilient with the reported result.The polysaccharides content of different breeds of bamboo leaves were determined.It was concluded that the polysaccharides content of per gram dry bamboo leaf was between 19.4-56.9 milligram.
The breeds of Ph.glauca McClue, Dendrocauamopsis oldhami.(munro)Keng f, Ph.edulis(Carr)A.etC.Riv contentted more flavonoids and polysaccharides.They are good materials of industry.Phyllostachys Heteroclata Oliver contentted high flavonoids,and Sh.chinensis Nakai contentted a lot of polysaccharides. But they were not grow on every hedge and not good materials of industry.The content of polysaccharides in insenescent leaves was higher than young leaves.The content of flavonoids didn't match this rule.
By good single factor selecting experiment and orthogonal design,the best technology for extracting bamboo leaves was established.The best waterbath extraction conditons was:60% ethanol solution,50℃,1:20 (w/v) of material and 1.5 hours per time.The order of affect actors was:solid-to-liquid ratio≥ethanol solution≥temperature≥time.The best ultrasonic extraction conditions was:60 % ethanol solution,1:15 (w/v) of material and 30 min per time.The order of affect actors was:time≥ethanol solution≥solid-to-liquid ratio.
By reviewing for several Maro-reticular and Polyamide resins,it was founded that the optimal Maro-reticular resin was AB-8 for the purification of flavonoids. Flavonoids could almost be develop out by using 70 % ethanol solution. Ethanol was better than methanol as eluant.
Ceramic membrane,ultra filtration and nanofiltration was adoptted to purify the extract of bamboo leaves.They can make the extract solution turning clarify and transparentted.When the volume of extract solution was about 90L,and concentration was 9.32g/L and 7.08g/L,It's function was fine for use. The flavonoids extract which had been treated by membrane can be purified to 15.8%-17.8% by resins. Polyamide had better effect than AB-8 in purifying flavonoids extract that had been treated by membrane.And it's result can be optimized to 40.35%. The polysaccharides extract which had been treated by membrane can be purified to 40.20% by high ethanol concentration.
选用了分光光度法和高效液相色谱法,对比研究竹叶中黄酮含量的差异性,确立了定量测定方法。NaNO2-Al(NO3)3-NaOH比色法较为常用,但是测定结果偏高,以荭草苷、牡荆苷为标准品,HPLC法测定结果更为可靠、准确。
对27个不同品种竹叶黄酮的含量进行了测定,得干竹叶黄酮的含量为0.50%～1.72%,即5.0～17.2mg/g,与文献[1]报道的范围基本相符。对不同品种竹叶多糖的含量进行了测定,得多糖含量在1.94%～5.69%之间,即19.4～56.9mg/g。
淡竹,绿竹,毛竹分布广、黄酮和多糖含量较高,成为工业上提取竹叶黄酮和多糖活性组分的最佳原料;水竹黄酮含量高,鹅毛竹多糖含量特别高,但是分布较少,不适宜为工业原料。对于不同年龄的竹叶,老叶片中多糖含量明显大于新生嫩叶片;而黄酮的含量则因竹种不同存在明显差异。
经单因素优选试验和正交试验,确定了竹叶黄酮的最佳提取工艺。水浴提取最佳条件为:温度50℃,提取时间2.0h,乙醇浓度60%(体积分数,下同),固液比1∶20(g∶mL),各因素对提取率的影响程度依次为:固液比>乙醇浓度>温度>时间;超声波提取最佳条件为:超声波作用时间30min,乙醇浓度60%,固液比1∶15(g∶mL),各因素对提取率的影响程度依次为:提取时间>乙醇浓度>固液比。
通过对几种树脂的比较,AB-8树脂的分离纯化效果最好;通过静态解吸试验,70%的乙醇效果好于90%的乙醇,乙醇的解吸效果要好于甲醇;树脂柱吸附过程中,吸附和解吸附都是渐变的曲线过程,用10%～90%乙醇解吸附剂梯度洗脱时,40%乙醇洗脱出最多的黄酮,解吸附量为47.0mg(47.1cm3的装柱体积)。
竹叶提取物经过组合膜处理后变得澄清透明,色泽鲜亮;在试验所用提取液体积为90L左右,提取液浓度为9.32g/L过陶瓷膜、7.08g/L过超滤膜时,膜使用状况良好,流速稳定,没有发生浓差极化现象;膜处理后的黄酮提取物,再用树脂和聚酰胺树脂处理,聚酰胺纯化效果明显好于AB-8,80%乙醇洗脱,黄酮含量能够达到40.35%;竹叶中试水提取后,经无机和有机膜处理得到多糖提取物,再经85%以上乙醇沉淀,多糖含量提高到40.20%。
Bamboo is perennial evergreen plant,belongs to Bambuaoidease Nees of the family poaceae.It has been used for food and medicines for a long time in China.There is a large body of evidence showing that bamboo leaves is rich in flavinoids and polysaccharides. It has many fine functions,such as anti-free radical,oxidation resistance and protecting vasular system etc.Nowadays,it has received broad attention from all over the world.In order to take full use of bamboo,the work had been done in the paper was focused on the method of determination and high content,especially the extration and purification for flavinoids and polysaccharides in bamboo leaves. The main results are as follows:
HPLC and UV-VIS spectrophotometry were applied to detect the content of flavonoids in the experiment.and the results of the two detection were compared.The method of quantitative detection were established. NaNO2-Al(NO3)3-NaOH UV-VIS spectrophotometry was been common used.But it's result was higher than true. The method of HPLC was reliable and precise by taking Orientin and Isoviextin as the index ingredient.
The content of flavonoids of 27 breeds of bamboo leaves were determined.It was concluded that the total flavonoids content of per gram dry bamboo leaves was between 5.0-13.3 milligram.The result was basically consilient with the reported result.The polysaccharides content of different breeds of bamboo leaves were determined.It was concluded that the polysaccharides content of per gram dry bamboo leaf was between 19.4-56.9 milligram.
The breeds of Ph.glauca McClue, Dendrocauamopsis oldhami.(munro)Keng f, Ph.edulis(Carr)A.etC.Riv contentted more flavonoids and polysaccharides.They are good materials of industry.Phyllostachys Heteroclata Oliver contentted high flavonoids,and Sh.chinensis Nakai contentted a lot of polysaccharides. But they were not grow on every hedge and not good materials of industry.The content of polysaccharides in insenescent leaves was higher than young leaves.The content of flavonoids didn't match this rule.
By good single factor selecting experiment and orthogonal design,the best technology for extracting bamboo leaves was established.The best waterbath extraction conditons was:60% ethanol solution,50℃,1:20 (w/v) of material and 1.5 hours per time.The order of affect actors was:solid-to-liquid ratio≥ethanol solution≥temperature≥time.The best ultrasonic extraction conditions was:60 % ethanol solution,1:15 (w/v) of material and 30 min per time.The order of affect actors was:time≥ethanol solution≥solid-to-liquid ratio.
By reviewing for several Maro-reticular and Polyamide resins,it was founded that the optimal Maro-reticular resin was AB-8 for the purification of flavonoids. Flavonoids could almost be develop out by using 70 % ethanol solution. Ethanol was better than methanol as eluant.
Ceramic membrane,ultra filtration and nanofiltration was adoptted to purify the extract of bamboo leaves.They can make the extract solution turning clarify and transparentted.When the volume of extract solution was about 90L,and concentration was 9.32g/L and 7.08g/L,It's function was fine for use. The flavonoids extract which had been treated by membrane can be purified to 15.8%-17.8% by resins. Polyamide had better effect than AB-8 in purifying flavonoids extract that had been treated by membrane.And it's result can be optimized to 40.35%. The polysaccharides extract which had been treated by membrane can be purified to 40.20% by high ethanol concentration.
引文
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