乌饭树黑色素中功能性成分的研究
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摘要
乌饭树(Vaccinium Bracteatum Thunb)为杜鹃花科,越桔属。别名:羊尘饭子,十月乌,珍珠花,乌饭子等。通常为常绿或落叶灌木,很少为小乔木,高可达3.5米,其叶是重要的中药材。乌饭树树叶、花及果实中含有丰富的多酚类物质,尤以叶中为多。古代医学研究表明乌饭树的树叶、果实、花具有益精气、强筋骨、明目止泻等功效。而现代医学研究表明乌饭树具有抗氧化、防衰老、抗菌、抗癌防癌等功效。
本文主要研究了乌饭树树叶中黑色素的定量检测方法;黑色素的提取工艺以及大孔吸附树脂的筛选:黑色素中功能性成分——黄酮类化合物的纯化与结构分析;黑色素中黄酮类化合物的抗氧化、去自由基活性;黑色素对视网膜光损伤的保护作用;并且对黑色素功能与结构之间的关系进行了初步的探讨。
以总黄酮含量为指标,利用紫外扫描图谱技术对乌饭树树叶黑色素提取物进行分析,同时使用三种分光光度比色法和HPLC测定结果进行比较,分析分光光度比色法的原理,得出AlCl_3比色法(390nm)适用于本研究。该方法是一种操作简便、经济易行、结果较为准确的方法。
通过单因素以及正交试验,得到了乌饭树树叶中黑色素的最佳提取工艺:乙醇浓度60%(v/v),温度60℃,固液比1∶35,提取次数2次。在此条件下从乌饭树树叶中提取出总的黄酮类化合物可以达到34.71mg/g原料,提取率为97%。
通过对11种大孔吸附树脂的静态吸附研究,得出AB-8型大孔吸附树脂是一种比较理想的树脂,吸附量大,解吸率高,较适合于乌饭树树叶黑色素中黄酮类化合物的初步纯化。通过对AB-8型大孔吸附树脂的动态吸附研究,确定了乌饭树树叶黑色素中黄酮类化合物的初分条件:pH为5.05左右,溶液浓度为0.68～1.33mg/mL,吸附流速为3mL/min,此时树脂对其中的黄酮类化合物吸附量最大。通过对吸附后的树脂进行洗脱研究,发现采用95%的乙醇,与树脂的比值为7∶1(v/w)时进行洗脱,解吸率可以超过95%,黄酮类化合物的纯度从原先的14.50%达到50.32%。另外树脂在使用了三次以后必须进行再生处理。
利用大孔吸附树脂、聚酰胺、硅胶以及TSKgel Toyopearl HW-40色谱柱对乌饭树树叶黑色素中的功能性成分进行纯化分离研究,从中分离出7种黄酮类化合物单体,同时利用样品波长扫描图谱、HPLC-MS以及核磁共振技术对其中的5种化合物进行结构研究确定分别为:槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素,相对含量分别为:35.71%,2.26%,9.57%,1.72%和15.16%。
通过亚油酸体系、脂质过氧化体系对乌饭树树叶黑色素中槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素五种黄酮类化合物的抗氧化功能进行了研究。研究结果表明,乌饭树树叶黑色素具有一定的抗氧化功能。其中槲皮素、芹黄素、山奈酚和木犀草素的抗氧化效果要强于芦丁,而白杨黄素的抗氧化效果和芦丁则较接近。而且所有化合物的抗氧化功能都表现出很强的浓度依赖关系。抑制亚油酸氧化的能力从高到低为:槲皮素≈木犀草素>芹黄素>山奈酚≈Vc>白杨黄素>芦丁>BHT>粗提物。槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素的半抑制浓度分别为:6.188×10~(-4)、3.048×10~2、2.9127、4.0040、2.5450μg/mL。对脂质
Vaccinium Bracteatum Thunb, a resource of Chinese traditional medicine, belongs to the genus Rhododendron. It is grown massively in China but can be found worldwide.In this work, systematic studies were carried out on quantitative detection, extraction technology, isolation, purification, identification, antioxidant activity, free radical scavenging activity, and retina light damage prevention activity determination of Vaccinium Bracteatum Thunb leaf (VBTL) black pigment. The relationship was then discussed between the molecular structures and the physiological functions of VBTL black pigment.Based on the UV spectra of VBTL extracts and probe into the reaction mechanisms by three spectrophotometric methods and comparison of the relevant results with HPLC method, a relatively simpler, more accurate method, i.e., a colorimetric method (390nm) using AlCl_3 as the reagent was established for quantitative analysis of VBTL black pigment.A two step extraction process of VBTL black pigment was established, with each step using following conditions: extraction solvent 60%(v/v) ethanol, temperature 60℃, duration 1 hour, and material /ethanol ratio 1:35. By this optimized process 34.71mg flavones could be obtained from one gram VBTL, which accounts for 97% of total flavones.Eleven kinds of macroporous resins were screened with regard to absorption and desorption capacity for VBTL black pigment flavones. The AB-8 resin was determined to be the best one for previous purification of VBTL black pigment due to its relatively higher absorption and desorption capacity. Dynamic absorption capacity tests reveal that AB-8 presents the highest absorption capacity at pH 5.05, sample concentration 0.68~1.33mg/mL, and sample loading rate 3mL/min. When 95% ethanol was used as the eluent, the desorption rate reached 95%, and the purity of the flavones was increased from 14.5% to 50.32%. It was found that AB-8 should be regenerated after three times usage.The functional components in black pigment from VBTL were chromatographically purified with AB-8, polyamide, silica gel, and TSK gel Toyopearl HW-40. Five components, respectively coded B, C, D, E, and F, were thus purified. And by structural analyses with UV spectroscopy, HPLC-MS and NMR spectroscopy, these components were determined to be quercetin, chrysin, apigenin, kaempferol, and luteolinidin, respectively.The antioxidant properties of the five purified components were investigated by the ferric thiocyanate method and liposome phospholipid peroxidation method. Besides chrysin showing slightly higher antioxidant activities than rutin, all the other four components showed antioxidant activities significantly higher than rutin. The antioxidant activities of all five components increased significantly with the increase of their concentration. The antioxidant activities of samples tested follows the order: quercetin≈luteolinidin>apigenin>kaempferol≈Vc>chrysin>rutin>BHT> raw extracts. The half-inhibition concentration (IC_(50)) of the five samples against ferric thiocyanate
peroxidation were determined as following: 0.0006188, 304.8, 2.9127, 4.0040 and 2.5450 u g/mL respectively for quercetin, chrysin, apigenin, kaempferol, and luteolinidin. All the samples can inhibit the generation of conjugated diene hydroperoxide during liposome oxidation following the order in capacity: quercetins?luteolinidin>apigenin>kaempferol>Vc>chrysin??rutinR=5BHT> raw extracts. The half-inhibition concentration (IC50) of the five samples were respectively 0.03986, 3.4417^ 0.1730, 0.5184, 0.2004ug/mL.The superoxide anion [O2 ? ] and hydroxyl radical [ ? OH] scavenging capacities of these componnents were investigated respectively in the pyrogallol-lumino and o-phenanthroline hydrate-CuSO4-Ascorbic acid-H2O2 luminescence system. The IC50 of [O2 ? ] scavenging of five components were determined: 5.48, 17.44, 10.73, 12.74, and 5.91 V mol/L. The IC50 of [ ? OH] scavenging of the five components were also determined: 5.63, 22.45, 11.58, 16.61, and 5.99 u mol/L.The electroretinogram (ERG) indicated that VBTL black pigment could effectively prevent rabbit retina from being light damaged. The total superoxide (T-SOD) levels and the maleic dialdehyde (MDA) levels in the rabbit retina were measured. It was found that T-SOD decreased and MDA increased in the control group (rabbit retina light damaged), while T-SOD and MDA levels of the test group (rabbit retina light damaged, and VBTL black pigment treated) remained similar to that of natural rabbit retina. Based on the mechanism of retina light damaging and experimental observations as above, the retina protection effects of VBTL black pigment was probable to be ascribed to their antioxidant and free radical scavenging activities.The relationship between the molecular structure and the antioxidant and free radical scavenging functions of the components were discussed. The activities of these components were not only associated with the conjugate double bond system, but with the hydroxyl substitutes in their molecular structures.
本文主要研究了乌饭树树叶中黑色素的定量检测方法;黑色素的提取工艺以及大孔吸附树脂的筛选:黑色素中功能性成分——黄酮类化合物的纯化与结构分析;黑色素中黄酮类化合物的抗氧化、去自由基活性;黑色素对视网膜光损伤的保护作用;并且对黑色素功能与结构之间的关系进行了初步的探讨。
以总黄酮含量为指标,利用紫外扫描图谱技术对乌饭树树叶黑色素提取物进行分析,同时使用三种分光光度比色法和HPLC测定结果进行比较,分析分光光度比色法的原理,得出AlCl_3比色法(390nm)适用于本研究。该方法是一种操作简便、经济易行、结果较为准确的方法。
通过单因素以及正交试验,得到了乌饭树树叶中黑色素的最佳提取工艺:乙醇浓度60%(v/v),温度60℃,固液比1∶35,提取次数2次。在此条件下从乌饭树树叶中提取出总的黄酮类化合物可以达到34.71mg/g原料,提取率为97%。
通过对11种大孔吸附树脂的静态吸附研究,得出AB-8型大孔吸附树脂是一种比较理想的树脂,吸附量大,解吸率高,较适合于乌饭树树叶黑色素中黄酮类化合物的初步纯化。通过对AB-8型大孔吸附树脂的动态吸附研究,确定了乌饭树树叶黑色素中黄酮类化合物的初分条件:pH为5.05左右,溶液浓度为0.68～1.33mg/mL,吸附流速为3mL/min,此时树脂对其中的黄酮类化合物吸附量最大。通过对吸附后的树脂进行洗脱研究,发现采用95%的乙醇,与树脂的比值为7∶1(v/w)时进行洗脱,解吸率可以超过95%,黄酮类化合物的纯度从原先的14.50%达到50.32%。另外树脂在使用了三次以后必须进行再生处理。
利用大孔吸附树脂、聚酰胺、硅胶以及TSKgel Toyopearl HW-40色谱柱对乌饭树树叶黑色素中的功能性成分进行纯化分离研究,从中分离出7种黄酮类化合物单体,同时利用样品波长扫描图谱、HPLC-MS以及核磁共振技术对其中的5种化合物进行结构研究确定分别为:槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素,相对含量分别为:35.71%,2.26%,9.57%,1.72%和15.16%。
通过亚油酸体系、脂质过氧化体系对乌饭树树叶黑色素中槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素五种黄酮类化合物的抗氧化功能进行了研究。研究结果表明,乌饭树树叶黑色素具有一定的抗氧化功能。其中槲皮素、芹黄素、山奈酚和木犀草素的抗氧化效果要强于芦丁,而白杨黄素的抗氧化效果和芦丁则较接近。而且所有化合物的抗氧化功能都表现出很强的浓度依赖关系。抑制亚油酸氧化的能力从高到低为:槲皮素≈木犀草素>芹黄素>山奈酚≈Vc>白杨黄素>芦丁>BHT>粗提物。槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素的半抑制浓度分别为:6.188×10~(-4)、3.048×10~2、2.9127、4.0040、2.5450μg/mL。对脂质
Vaccinium Bracteatum Thunb, a resource of Chinese traditional medicine, belongs to the genus Rhododendron. It is grown massively in China but can be found worldwide.In this work, systematic studies were carried out on quantitative detection, extraction technology, isolation, purification, identification, antioxidant activity, free radical scavenging activity, and retina light damage prevention activity determination of Vaccinium Bracteatum Thunb leaf (VBTL) black pigment. The relationship was then discussed between the molecular structures and the physiological functions of VBTL black pigment.Based on the UV spectra of VBTL extracts and probe into the reaction mechanisms by three spectrophotometric methods and comparison of the relevant results with HPLC method, a relatively simpler, more accurate method, i.e., a colorimetric method (390nm) using AlCl_3 as the reagent was established for quantitative analysis of VBTL black pigment.A two step extraction process of VBTL black pigment was established, with each step using following conditions: extraction solvent 60%(v/v) ethanol, temperature 60℃, duration 1 hour, and material /ethanol ratio 1:35. By this optimized process 34.71mg flavones could be obtained from one gram VBTL, which accounts for 97% of total flavones.Eleven kinds of macroporous resins were screened with regard to absorption and desorption capacity for VBTL black pigment flavones. The AB-8 resin was determined to be the best one for previous purification of VBTL black pigment due to its relatively higher absorption and desorption capacity. Dynamic absorption capacity tests reveal that AB-8 presents the highest absorption capacity at pH 5.05, sample concentration 0.68~1.33mg/mL, and sample loading rate 3mL/min. When 95% ethanol was used as the eluent, the desorption rate reached 95%, and the purity of the flavones was increased from 14.5% to 50.32%. It was found that AB-8 should be regenerated after three times usage.The functional components in black pigment from VBTL were chromatographically purified with AB-8, polyamide, silica gel, and TSK gel Toyopearl HW-40. Five components, respectively coded B, C, D, E, and F, were thus purified. And by structural analyses with UV spectroscopy, HPLC-MS and NMR spectroscopy, these components were determined to be quercetin, chrysin, apigenin, kaempferol, and luteolinidin, respectively.The antioxidant properties of the five purified components were investigated by the ferric thiocyanate method and liposome phospholipid peroxidation method. Besides chrysin showing slightly higher antioxidant activities than rutin, all the other four components showed antioxidant activities significantly higher than rutin. The antioxidant activities of all five components increased significantly with the increase of their concentration. The antioxidant activities of samples tested follows the order: quercetin≈luteolinidin>apigenin>kaempferol≈Vc>chrysin>rutin>BHT> raw extracts. The half-inhibition concentration (IC_(50)) of the five samples against ferric thiocyanate
peroxidation were determined as following: 0.0006188, 304.8, 2.9127, 4.0040 and 2.5450 u g/mL respectively for quercetin, chrysin, apigenin, kaempferol, and luteolinidin. All the samples can inhibit the generation of conjugated diene hydroperoxide during liposome oxidation following the order in capacity: quercetins?luteolinidin>apigenin>kaempferol>Vc>chrysin??rutinR=5BHT> raw extracts. The half-inhibition concentration (IC50) of the five samples were respectively 0.03986, 3.4417^ 0.1730, 0.5184, 0.2004ug/mL.The superoxide anion [O2 ? ] and hydroxyl radical [ ? OH] scavenging capacities of these componnents were investigated respectively in the pyrogallol-lumino and o-phenanthroline hydrate-CuSO4-Ascorbic acid-H2O2 luminescence system. The IC50 of [O2 ? ] scavenging of five components were determined: 5.48, 17.44, 10.73, 12.74, and 5.91 V mol/L. The IC50 of [ ? OH] scavenging of the five components were also determined: 5.63, 22.45, 11.58, 16.61, and 5.99 u mol/L.The electroretinogram (ERG) indicated that VBTL black pigment could effectively prevent rabbit retina from being light damaged. The total superoxide (T-SOD) levels and the maleic dialdehyde (MDA) levels in the rabbit retina were measured. It was found that T-SOD decreased and MDA increased in the control group (rabbit retina light damaged), while T-SOD and MDA levels of the test group (rabbit retina light damaged, and VBTL black pigment treated) remained similar to that of natural rabbit retina. Based on the mechanism of retina light damaging and experimental observations as above, the retina protection effects of VBTL black pigment was probable to be ascribed to their antioxidant and free radical scavenging activities.The relationship between the molecular structure and the antioxidant and free radical scavenging functions of the components were discussed. The activities of these components were not only associated with the conjugate double bond system, but with the hydroxyl substitutes in their molecular structures.
引文
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