烟73葡萄皮中七种花色苷单体的制备及分析应用
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摘要
花色苷属黄酮类物质,是一种对人体有益的天然色素,在食品中应用广泛。葡萄中天然花色苷主要存在于果皮上,经酿造过程赋予了葡萄酒亮丽的红色。Dp、Cy、Pt、Pn、Mv、Ma、Mc是欧亚种葡萄中七种含量最多的花色苷。
根据研究报道,目前已发现500多种花色苷,但现在市场上能购买到的花色苷标样较少,并且价格较贵。在葡萄酒花色苷研究中,定量大都是采用Mv一种标样对所有花色苷进行相对定量,无法做到绝对定量。花色苷标样的缺乏使花色苷颜色、口感、生理活性等方面的深入研究受到了限制。
本论文发明了一种从葡萄皮中分离制备七种高纯度花色苷单体的方法。先对赤霞珠葡萄皮进行浸提,得到花色苷的粗提取物,再用XAD-7HP大孔树脂对粗提取物进行纯化。将初步纯化后的花色苷提取物进行制备液相分离,得到花色苷的高纯单体,包括Ma和Mc两种没有商业标样的花色苷。主要结论有:
(1)葡萄皮中七种主要花色苷的提取
以HPLC为检测手段,分析考察了葡萄皮中的七种主要花色苷,Dp、Cy、Pt、Pn、Mv、Ma、Mc,建立了快速提取方法。实验以乙醇为提取溶剂,通过单因素试验和正交试验设计,研究了三种花色苷的最佳提取条件。实验表明,液料比4 mL/g,温度40℃,提取时间30 min,提取液乙醇中含0.1%盐酸(v/v),超声处理2 min,两次提取,可快速提取葡萄皮中的七种主要花色苷。
(2)葡萄皮花色苷的树脂纯化
以烟73葡萄为花色苷提取材料,采用HPLC的分析方法,对10种树脂的吸附/解析效果进行了比较。选用性能较优的XAD-7HP树脂进行纯化实验。纯化处理使花色苷在干物质中的含量由之前48.16 %提高到了94.93 %,色价由47.7提高到了291.6,回收率为85.7%。
(3)葡萄皮中七种花色苷单体的制备
使用waters制备液相(Preparative HPLC)自动纯化系统,在Xbridge Prep C18柱上分离得到了Dp、Cy、Pt、Pn、Mv、Ma、Mc七种花色苷的高纯单体,纯度分别为99.9%、99.9%、99.9%、99.9%、99.9%、91.7%和94.5%。
(4)花色苷单体稳定性的研究
以分离纯化得到的花色苷单体为研究对象,以HPLC为检测手段,考察了七种单体的光稳定性、热稳定性和pH稳定性。研究表明,花色苷单体对光照、温度、pH的稳定性并不相同,但变化趋势一致:随着光照强度、温度、pH的升高,花色苷的不稳定性明显加剧。Cy、Pt、Pn、和Mv的稳定性高于Dp、Ma和Mc。花色苷在避光、pH 3、4℃时一周之内保留率在95%以上,适宜花色苷的短期保存。
(5)张裕主产葡萄酒品种、产地分析
将分离得到的花色苷单体作为标样,对2010年张裕主产红葡萄酒进行了定性定量分析。研究证明Dp、Cy、Pt、Pn、Mv、Ma和Mc是赤霞珠、蛇龙珠、梅鹿辄、烟73四种酿酒葡萄中的主要花色苷。Mv是各品种中含量最高的花色苷,其次为Ma或Pn。七种花色苷的总量在品种和产地上都表现出了规律性变化。葡萄酒中七种花色苷的指纹图谱可以为葡萄酒的产地和品种鉴别提供依据。
Anthocyanins are widely distributed in the plant kingdom, responsible for red and blue pigmentation in fruits, berries, and petals. They are good antioxidant and have positive effect on human health. Structurally, anthocyanins are glycosides of anthocyanidins. Dp, Cy, Pt, Pn, Mv, Ma, and Mc are considered as the most important compounds for the quality and color of Vitis vinifera grapes and wine.
It is reported that there are more than 500 kinds of anthocyanins, but most of them are not commercial. The anthocyanin standards which can be bought are expensive. In many researches, anthocyanins were only semi-quantitified by Mv. Standard anthocyanins prove to be very useful for biochemical studies, because they provide quick identification of these compounds in the field of anthocyanins biosynthesis and food quality determination.
In the present work, the isolation method of seven anthocyanins monomers from raw grape extracts was introduced, including extraction, purification and isolation. Anthocyanins were extracted with ethanol, purified by XAD-7HP and then directly fractionated by Xbridge Prep C18 column on Preparative HPLC (auto-purity system, waters). This work prepared two kinds of purified acylated anthocyanins successfully, named Ma and Mc, whose commercial standards are not available up to now. The stability of the seven isolated anthocyanins to light, heat, and pH was also examined.
(1) Optimal extraction of seven key anthocyanins from grape skin
The rapid extraction method of Dp, Cy, Pt, Pn, Mv, Ma, and Mc from the wine pomace was optimized by using high HPLC. The ANOVA of factors and orthogonal experimental design showed that it was a rapid approach to extract these main anthocyanins in following conditions: 4 liquid-solid ratio (mL/g), 0.1% hydrochloric acid (v/v) in ethanol, 40℃, 30 min and twice extraction, ultrasonic treatment for 2 min.
(2) Purification and stabilities of anthocyanins from grape skin
Purification and stability of anthocyanins from Yan73 grape were studied. HPLC was used to analyze the residual anthocyanins in the stability test. According to the experiment results, XAD-7HP exhibited the excellent characteristics for anthocyanins purification. The purity of grape anthocyanins was raised from 48.16 % to 94.93 %, the colour value was improved to 291.6 and the recovery rate of anthocyanins was 85.7%.
(3) Isolation of seven anthocyanins from Yan73 grape
Anthocyanin monomers, including Dp, Cy, Pt, Pn, Mv, Ma, and Mc were successfully isolated with Xbridge Prep C18 column on a preparative scale. Pigments Dp, Cy, Ma, and Mc were yielded in a one-step separation by preparative HPLC, with purities up to 99.9%, 99.9%, 91.7% and 94.5%, respectively. Pigments Pt, Pn, and Mv were further purified with another elution method and their purities were all improved up to 99.9 %.
(4) Stabilities study on the seven anthocyanin monomers
The stabilities of the seven anthocyanin monomers to light, heat, and pH were different, but the trend in changes was similar. The higher light intensity, temperature, and pH values, the more unstable the anthocyanins were. Among the seven anthocyanins, Dp, Ma and Mc were more sensitive to light heat and pH than Cy Pt, Pn, and Mv. When stored at pH 3, 4℃and in dark, the residual anthocyanins kept upon 95% in one week, so the situation was suitable for anthocyanins short storage.
(5) Varieties and viny regions analysis of ChangYu red wines
Dp, Cy, Pt, Pn, Mv, Ma, and Mc were the main anthocyanins of Cabernet Sauvignon wine, Cabernet Gernischt wine, Merlot wine and Yan wine. In four wines, the content of Mv is highest in Cabernet Sauvignon, followed by Ma or Pn. Anthocyanin fingerprints of red wine were used to categorize wine to different varieties and different viny regions. That helps to appraisal of red wines.
根据研究报道,目前已发现500多种花色苷,但现在市场上能购买到的花色苷标样较少,并且价格较贵。在葡萄酒花色苷研究中,定量大都是采用Mv一种标样对所有花色苷进行相对定量,无法做到绝对定量。花色苷标样的缺乏使花色苷颜色、口感、生理活性等方面的深入研究受到了限制。
本论文发明了一种从葡萄皮中分离制备七种高纯度花色苷单体的方法。先对赤霞珠葡萄皮进行浸提,得到花色苷的粗提取物,再用XAD-7HP大孔树脂对粗提取物进行纯化。将初步纯化后的花色苷提取物进行制备液相分离,得到花色苷的高纯单体,包括Ma和Mc两种没有商业标样的花色苷。主要结论有:
(1)葡萄皮中七种主要花色苷的提取
以HPLC为检测手段,分析考察了葡萄皮中的七种主要花色苷,Dp、Cy、Pt、Pn、Mv、Ma、Mc,建立了快速提取方法。实验以乙醇为提取溶剂,通过单因素试验和正交试验设计,研究了三种花色苷的最佳提取条件。实验表明,液料比4 mL/g,温度40℃,提取时间30 min,提取液乙醇中含0.1%盐酸(v/v),超声处理2 min,两次提取,可快速提取葡萄皮中的七种主要花色苷。
(2)葡萄皮花色苷的树脂纯化
以烟73葡萄为花色苷提取材料,采用HPLC的分析方法,对10种树脂的吸附/解析效果进行了比较。选用性能较优的XAD-7HP树脂进行纯化实验。纯化处理使花色苷在干物质中的含量由之前48.16 %提高到了94.93 %,色价由47.7提高到了291.6,回收率为85.7%。
(3)葡萄皮中七种花色苷单体的制备
使用waters制备液相(Preparative HPLC)自动纯化系统,在Xbridge Prep C18柱上分离得到了Dp、Cy、Pt、Pn、Mv、Ma、Mc七种花色苷的高纯单体,纯度分别为99.9%、99.9%、99.9%、99.9%、99.9%、91.7%和94.5%。
(4)花色苷单体稳定性的研究
以分离纯化得到的花色苷单体为研究对象,以HPLC为检测手段,考察了七种单体的光稳定性、热稳定性和pH稳定性。研究表明,花色苷单体对光照、温度、pH的稳定性并不相同,但变化趋势一致:随着光照强度、温度、pH的升高,花色苷的不稳定性明显加剧。Cy、Pt、Pn、和Mv的稳定性高于Dp、Ma和Mc。花色苷在避光、pH 3、4℃时一周之内保留率在95%以上,适宜花色苷的短期保存。
(5)张裕主产葡萄酒品种、产地分析
将分离得到的花色苷单体作为标样,对2010年张裕主产红葡萄酒进行了定性定量分析。研究证明Dp、Cy、Pt、Pn、Mv、Ma和Mc是赤霞珠、蛇龙珠、梅鹿辄、烟73四种酿酒葡萄中的主要花色苷。Mv是各品种中含量最高的花色苷,其次为Ma或Pn。七种花色苷的总量在品种和产地上都表现出了规律性变化。葡萄酒中七种花色苷的指纹图谱可以为葡萄酒的产地和品种鉴别提供依据。
Anthocyanins are widely distributed in the plant kingdom, responsible for red and blue pigmentation in fruits, berries, and petals. They are good antioxidant and have positive effect on human health. Structurally, anthocyanins are glycosides of anthocyanidins. Dp, Cy, Pt, Pn, Mv, Ma, and Mc are considered as the most important compounds for the quality and color of Vitis vinifera grapes and wine.
It is reported that there are more than 500 kinds of anthocyanins, but most of them are not commercial. The anthocyanin standards which can be bought are expensive. In many researches, anthocyanins were only semi-quantitified by Mv. Standard anthocyanins prove to be very useful for biochemical studies, because they provide quick identification of these compounds in the field of anthocyanins biosynthesis and food quality determination.
In the present work, the isolation method of seven anthocyanins monomers from raw grape extracts was introduced, including extraction, purification and isolation. Anthocyanins were extracted with ethanol, purified by XAD-7HP and then directly fractionated by Xbridge Prep C18 column on Preparative HPLC (auto-purity system, waters). This work prepared two kinds of purified acylated anthocyanins successfully, named Ma and Mc, whose commercial standards are not available up to now. The stability of the seven isolated anthocyanins to light, heat, and pH was also examined.
(1) Optimal extraction of seven key anthocyanins from grape skin
The rapid extraction method of Dp, Cy, Pt, Pn, Mv, Ma, and Mc from the wine pomace was optimized by using high HPLC. The ANOVA of factors and orthogonal experimental design showed that it was a rapid approach to extract these main anthocyanins in following conditions: 4 liquid-solid ratio (mL/g), 0.1% hydrochloric acid (v/v) in ethanol, 40℃, 30 min and twice extraction, ultrasonic treatment for 2 min.
(2) Purification and stabilities of anthocyanins from grape skin
Purification and stability of anthocyanins from Yan73 grape were studied. HPLC was used to analyze the residual anthocyanins in the stability test. According to the experiment results, XAD-7HP exhibited the excellent characteristics for anthocyanins purification. The purity of grape anthocyanins was raised from 48.16 % to 94.93 %, the colour value was improved to 291.6 and the recovery rate of anthocyanins was 85.7%.
(3) Isolation of seven anthocyanins from Yan73 grape
Anthocyanin monomers, including Dp, Cy, Pt, Pn, Mv, Ma, and Mc were successfully isolated with Xbridge Prep C18 column on a preparative scale. Pigments Dp, Cy, Ma, and Mc were yielded in a one-step separation by preparative HPLC, with purities up to 99.9%, 99.9%, 91.7% and 94.5%, respectively. Pigments Pt, Pn, and Mv were further purified with another elution method and their purities were all improved up to 99.9 %.
(4) Stabilities study on the seven anthocyanin monomers
The stabilities of the seven anthocyanin monomers to light, heat, and pH were different, but the trend in changes was similar. The higher light intensity, temperature, and pH values, the more unstable the anthocyanins were. Among the seven anthocyanins, Dp, Ma and Mc were more sensitive to light heat and pH than Cy Pt, Pn, and Mv. When stored at pH 3, 4℃and in dark, the residual anthocyanins kept upon 95% in one week, so the situation was suitable for anthocyanins short storage.
(5) Varieties and viny regions analysis of ChangYu red wines
Dp, Cy, Pt, Pn, Mv, Ma, and Mc were the main anthocyanins of Cabernet Sauvignon wine, Cabernet Gernischt wine, Merlot wine and Yan wine. In four wines, the content of Mv is highest in Cabernet Sauvignon, followed by Ma or Pn. Anthocyanin fingerprints of red wine were used to categorize wine to different varieties and different viny regions. That helps to appraisal of red wines.
引文
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