地榆多酚的组分分析及功能研究

【作者】 刘海英；

【导师】 仇农学；

【作者基本信息】 陕西师范大学 ， 植物学， 2009， 博士

【摘要】 植物多酚是一类广泛存在于植物界的多羟基的酚类物质,对活性氧等自由基具有很强的捕捉能力,能预防自由基引发的多种疾病及延缓衰老,其对人类健康的独特作用已经得到了普遍认可。在对86种不同植物原料植物多酚含量及抗氧化能力进行测定的基础上,发现地榆的总酚含量和聚黄烷醇多酚含量最高,同时具有很强的清除自由基能力。但是,目前有关地榆多酚化学组分及抗氧化等生物活性的研究尚少。因此,分析地榆多酚的化学组成、研究其抗氧化等生物活性及提取纯化工艺,对于充分利用地榆这一中草药资源和提高其产品的价值具有重要意义。现就具体研究工作及结果简述如下:1.确定了地榆中两类多酚化合物的含量测定方法,研究其稳定性、精密度和加样回收率。建立了香草醛-硫酸法测定聚黄烷醇多酚含量的标准曲线,方程为Y=1.7839X+0.0017,方程的R²=0.9989,在0-0.30 mg/mL浓度范围内,线性良好;建立了高效液相色谱（HPLC）法测定没食子酸多酚含量,没食子酸和鞣花酸的加样回收率分别为99.76%和99.68%,RSD分别为0.34%和0.36%,方法重现性好、准确可靠。并测得地榆中聚黄烷醇多酚含量为84.90mg/g,没食子酸酯多酚的含量为62.81mg/g,地榆多酚总含量为147.71mg/g。2.分离与检测了地榆多酚的主要组分,采用超高效液相色谱-电喷雾质谱（UPLC-ESI-MS）联用技术对地榆多酚组成成分进行初步分析。（1）通过UPLC分离、ESI-MS定性分析,从地榆提取物中检测到了聚黄烷醇多酚三个单体、五个二聚体、三个三聚体。没食子酸二酰基酚两个、三酰基酚三个、四酰基酚一个、五酰基酚十二个;（2）利用Sephadex LH-20凝胶层析法初步分离富集地榆多酚各组分,结合RP-UPLC-ESI-MS进一步分离检测,确认了地榆多酚主要组分包括没食子酸、单酯型黄烷醇二聚体、儿茶素、鞣花酸。其中,单酯型黄烷醇二聚体为首次发现。3.研究地榆多酚的最佳提取、纯化工艺（1）采用二次回归旋转设计响应曲面法对地榆多酚提取工艺进行优化,地榆多酚最佳提取技术参数为:超声提取时间30min、丙酮溶液浓度70%、料液比1:28;以此条件进行试验,得到提取量平均值为202.65mg/g,与回归方程理论计算的提取量值212.40mg/g相差4.59%。回归方程:Y=196.10932+8.11188X₁+5.73496X₂+7.39815X₃-4.92490X₁²-5.06702X₂²（2）大孔吸附树脂纯化地榆多酚工艺通过静态吸附和洗脱试验,选择AB-8树脂作为地榆多酚的纯化树脂材料,其对地榆多酚的吸附率和解析率分别为89.6%和92.6%;动态洗脱试验结果表明:乙醇对AB-8树脂所吸附的多酚具有很强的洗脱作用,回收率可达90.4%,洗脱峰出峰时间比较集中,且对称性良好,干燥后地榆多酚纯度为95.3%。4.探讨了地榆多酚的体外抗氧化活性、酪氨酸酶抑制作用以及保护皮肤细胞紫外光损伤的作用机理:（1）地榆多酚的体外抗氧化能力与其浓度在一定范围内呈正相关,1g地榆多酚相当于30.566 mmol FeSO₄的抗氧化能力。同时,地榆多酚对DPPH·、·OH、O₂^-·自由基半数清除浓度(IC₅₀)也较低,依次为18.86mg/L、1.258mg/mL、18.53mg/L,显示出较强的体外清除自由基能力。（2）地榆多酚保护UVA诱导真皮成纤维细胞光损伤地榆多酚处理UVA氧化损伤的人真皮成纤维细胞后,细胞的形态结构的保持相对完整、细胞增殖活力、细胞内自由基防御系统SOD、GSH-Px的酶活显著升高,同时降低了因脂质氧化而产生的丙二醛（MDA）生成量。地榆多酚保护皮肤细胞紫外线损伤的作用机理在于:一是地榆多酚具有较强的紫外吸收能力,可以减少进入细胞的紫外线量;二与地榆多酚的结构和化学性质密切相关,地榆多酚的多元酚羟基能与自由基反应生成较稳定的半醌式,捕获·OH、O₂^-·等自由基,终止自由基链式反应,抑制了不饱和脂肪酸的过氧化,减少了MDA的生成,维持了细胞和正常形态和功能。（3）地榆多酚对酪氨酸酶活性的影响紫外线照射皮肤的另一效应,在于激活皮肤表皮黑素细胞中的酪氨酸酶的活性,黑色素生成量相应增多,最终引起了皮肤色素沉着及皮肤癌变。通过酪氨酸酶活力测试,地榆多酚表现了较强的酪氨酸酶活抑制能力,其对酪氨酸酶活的半数抑制浓度(IC₅₀)较低,仅为为14.5mg/mL,显示了作为皮肤防晒化妆品美白成分的潜力。更多还原

【Abstract】 As a kind of polyphenol mixtures,polyphenols exist in a wide range of plant tissues. Polyphenols have great capacity of trapping and scavenging oxygen free radicals and reduce risk of some diseases（stroke,tumor and cancer,etc.）.Through comparing the content of polyphenols,Radix Sanguisorbae ranks the highest among 86 different plant materials.There are abundant plant resources of Radix Sanguisorbae which are widely distributed in many provinces of China.To make full use of Radix Sanguisorbae and its value-added processing,it is of great importance to extract and analyze the polyphenols from Radix Sanguisorbae.In this paper,the following contents of polyphenols from Radix Sanguisorbae were studied:1.In this study,the quantitative method for hydrolysable tannin and condensed tannin were investigated.Vanillin-H₂SO₄ assay was selected to determine the content of condensed tannin of Radix Sanguisorbae.In this paper,（+）-catechin was used as the standard substance,and there is a very good linearity when the concentration of（+）-catechin changing from zero to 0.30mg/mL.The standard curve equation is Y=1.7839Xq-0.0017 and the R² is 0.9989.The Vanillin-H₂SO₄ assay is suitable especially for the new plant resources which do not have the mature product of their standard substance for its sufficient precision and ruggedness.The content of hydrolysable tannin is quantitatively determined by HPLC assay and the standard curve equation of gallic acid and ellagic acid were established too.The RSD of precision and recovery of tis experiments are 0.34%and 0.36%resctively.The content of hydrolysable tannin and condensed tannin were messured as 84.90mg/g and 62.81mg/g respectively,so the total content of polyphenols is 147.71 mg/g in Radix Sanguisorbae.2.The qualitative analysis of oligomeric polyphenols in crude Radix Sanguisorbae extracts can be performed by reversed-phase high-performance liquid chromatograhy coupled with electrospray ionization mass spectrometry（RP-HPLC-ESI-MS）.Pseudomolecular ion and fragment information of ESI-MS made it possible to analyze the composition of oligmeric polyphenols directly without standards.By RP-HPLC-ESI-MS,Three monomers,five dimers,three trimers and one tetramer of condensed tannin were tentatively identified in crude Radix Sanguisorbae extract,at the same time,two monomers,two dimers,three trimers,one tetramer and twelve pentamer of hydrolysable tannin were tentatively identified too,but due to the large number of isomers and their similar ESI-MS characters, the isomers are not identified precisely except monomers.In fact,only oligomers with MW below 1000 can be well detected by RP-HPLC-MS,the higher polymers,with more isomers and more diversepolarity,can neither be separated nor be analyzed by RP-HPLC.3.Quadratic regression orthogonal rotary experiments for acetone extraction of polyphenols were used in this paper.And the results were analyzed by DPS.The results indicated that the 3 factors in the experiments-extraction time（X1）,acetone concentration（X2）,ratio between solvent and material（X3）-had strong influences to the extraction effects.The best extraction conditions are: extraction time（X1） is 30min,acetone concentration（X2） is 70%,ratio between solvent and material（X3） is 28:1.Under these conditions,the average extraction amount is 198.28mg/g,and it’s about 4.59%higher than the theoretical calculation value.The equation which showed the relationship between the extraction effect and the 3 factors is: Y=196.10932 + 8.11188X1 + 5.73496X2 + 7.39815Z3 - 4.92490 X1²-5.06702 X2²The results of static adsorption,desorption and the kinetics of static adsorption show that AB-8 macroporous absorbent resin is suitable for Raidix Sanguisorbae polyphenol.The absorbing and eluting rate of AB-8 resin was 89.6%and 92.6%respectively,which possessing higher absorbing and eluting capacities.Through dynamic desorption of polyphenols absorbed on the AB-8 resin,the results showed that absolute ethanol had a strong eluting effect and the polyphenols can be fully eluted by 4BV of absolute ethanol.The symmetry of elution peak was quite well and the recovery rate was 90.4%.After vacuum freeze-drying,the purity of polyphenols can reach 95.3%.4.The antioxidant capability,enzyme inhibition of tyrosinase and protection effects of human skin fibrolast of Radix Sanguisorbae polyphenols were observed in this paper.The reducing power increased with the growing concentration of polyphenols,and the Ferric Reducing/Antioxidant Power（FRAP） value was 30.566mmol FeSO₄/g Polyphenols of Radix Sanguisorbae.Polyphenols from Radix Sanguisorbae showed a high scavenging activity of 1,1- diphenyl-2-picryl-hydrazyl （DPPH·）,and the 50%inhibitory concentration(IC₅₀) was 18.86mg/L.For the scavenging activity for hydroxyl radical（·OH） and superoxide anion radical（O₂^-·）,the IC₅₀ was 1.258mg/mL, 18.53mg/L,respectivily.At the present of Radix Sanguisorbae polyphenols,activity of the cell multiplication,SOD and GSH-Px of fibrolast increased obviously,and the content of MDA decreased obviously.This activity of Radix Sanguisorbae polyphenols is dependent on it’s molecular structure,for the phenolic hudrooxyl group of polyphenols can capture free radicals,interrupt Chain reaction of unsaturated fatty acid and inhibit peroxidation of unsaturated fatty acid,that finally result in the content of MDA decreasing and the activity of fibrolast multiplication has been keeped.Ultraviolet irradiation can excite Langerhans cells to form more melanogenesis and cause to skin pigmentation.Through tyrosinase activity analysis,Polyphenols from Radix Sanguisorbae showed a high inhibition activity of tyrosinase enzyme,and the 50%inhibitory concentration(IC₅₀) was 14.5mg/mL,indicate that Radix Sanguisorbae polyphenols could be used as skin cosmetics active component.更多还原