【作者】 王露；

【导师】 王星敏；

【作者基本信息】 重庆工商大学 ， 环境工程， 2019， 硕士

【摘要】 作为“药食同源”物,桑叶药理价值极高,因生产经营或加工技术等原因,存在利用率低等问题,导致高附加值的成份流失,造成桑资源浪费。本文以废次桑叶为原料,将水热反应引入天然产物提取技术中,开展提制高附加值天然活性成份异槲皮苷等研究,变废为宝,实现桑资源增值开发的高值利用,符合国家国民健康与资源利用战略。为提高桑叶中异槲皮苷等活性成份的有效溶出,采用水热反应方法,以异槲皮苷溶浸效率为评价指标,探讨水热时间、料液比、水热温度和乙醇体积分数等因素对异槲皮苷溶出的影响,经响应曲面法优化获得水热辅助醇提取桑叶中异槲皮苷的适宜参数,并开展提取物体外抗氧化作用研究;运用SEM（电镜扫描）、BET（比表面积）和FTIR（红外光谱）等手段,分析表征水热反应前后桑叶植物组织的物相结构及组分变化,探究水热降解植物组织的性能。为进一步揭示异槲皮苷溶浸的制约因素及传质规律,通过计算传质系数k、活化能Ea、相对萃余率y及有效扩散系数Ds等动力学参数,开展异槲皮苷溶浸的动力学研究;分析异槲皮苷溶浸的扩散过程,建立异槲皮苷的内扩散模型,获得异槲皮苷扩散速率常数。为获得高纯度异槲皮苷单体,采用制备色谱方法,开展流动相组成及比列、流速和进样量等条件比选,获得异槲皮苷分离、纯化的适宜条件,并评价提取物经济收益。结论如下:（1）采用响应曲面法优化获得水热反应辅助醇法提取异槲皮苷的适宜参数为:以体积分数为57%的乙醇为提取剂,桑叶质量（g）与乙醇体积（v）之比为1:7,在水热温度154℃、水热解80min后,5g桑叶提取异槲皮苷3.64mg·g^-1,是乙醇提法的51倍。响应曲面法拟合所得异槲皮苷溶浸方程R²=0.9582,F=22.91,P<0.0001,表明模型显著性极高,可用;F值检验可知,影响桑叶中异槲皮苷含量的因素顺序分别为:水热时间>水热温度>料液比>乙醇体积分数,且水热时间和水热温度的交互作用对异槲皮苷溶浸量影响显著。桑叶提取液对DPPH的清除率、超氧阴离子清除率、脂质过氧化抑制率、还原能力的实验表明,桑叶提取液在较低浓度0.02mg·mL^-1表现出抗氧化能力,且在0.02mg·mL^-10.1mg·mL^-1范围内呈现正相关。（2）红外分光谱分析表明,水热反应有助于桑叶中C-O、C-H、>C=C<键断裂,促进桑叶木质素中苯基丙烷结构单元分解;扫描电镜结果显示经水热解后的桑叶组织间出现孔洞且孔隙较大;BET结果表明经水热解后的底物比表面积是未经水热反应的桑叶底物的1.8倍左右;表明水热反应能有效降解桑叶植物纤维组织,有利于天然活性成份溶出。粒子扩散模模型表明,异槲皮苷的扩散存在渗透、内部扩散和边界扩散三个过程,扩散速率常数k随着边界层厚度增加而变小,其边界层厚度常数C依次为C_Ⅰ<C_Ⅱ<C_Ⅲ。（3）Fick第二定律获得的桑叶异槲皮苷溶浸动力学模型表明,在温度为120℃¹60℃时,速率常数k为0.0157min^-10.0301min^-1,表观活化能Ea=27.22kJ·mol^-1,指前因子A为0.0164min^-1,表观活化能越小,有利于反应的进行;有效扩散系数Ds为0.52×10^-4mm²·min^-10.99×10^-4mm²·min^-1,且随温度升高而增大;萃余率y随着温度升高而减少,在154℃下萃余率最小为9.75%,此时异槲皮苷的溶浸率最为91.25%;通过热力学分析求出ΔGm在-4.3743kJ·mol^-1-1.2582kJ·mol^-1<0,ΔHm=34.907kJ·mol^-1>0,ΔSm=92.47J·mol^-1>0,表明水热辅助醇提取异槲皮苷过程是吸热熵增的过程。（4）采用制备色谱分离纯化获得异槲皮苷,分离纯化适宜条件为:V_乙腈:V_水=20:80,进样量10mL,流速20mL·min-1,异槲皮苷分离度为1.74,视为完全分离,纯度较高,同时可得到绿原酸、芦丁和紫云英苷,其分离度均大于1.5;经冷冻干燥后,制得异槲皮苷纯度为98.78%,含率为取粗含量的4.26%;同时制得绿原酸纯度为99.47%,含率为粗提物的6.74%,芦丁纯度为98%、含率为粗提物的1.36%,紫云英苷纯度为98%,含率为粗提物的0.35%,为桑叶资源多元产品开发提供单体或复合物原料支撑。更多还原

【Abstract】 As"medicine and food homologue",mulberry leaves have extremely high pharmacological value.Because of the mode of production and operation,there are problems such as low utilization rate,resulting in the loss of high value-added components and the waste of mulberry resources.In this paper,the old mulberry leaves or wasted mulberry leaves are used as raw materials,andintroduced the hydrothermal reaction into the natural product extraction technology,carry out the extraction of high-purity and high value-added isoquercitrin and other active ingredients.To realize the value-added development and high-value utilization of mulberry resources.At the same time,turn abandoned mulberry leaves into valuable products,in line with the national resource depletion and"national health"strategy.In order to improve the effective dissolution of active ingredients such as isoquercitrin,using hydrothermal reaction method,evaluate the effects of hydrothermal time,solid-liquid ratio,hydrothermal temperature and ethanol volume fraction on the dissolution of isoquercitrin.Optimization of the parameters of hydrothermal-assisted alcohol extraction of isoquercitrin from mulberry leaves by response surface methodology.SEM,BET and FTIR were used to analyze the phase structure and composition changes of mulberry leafves tissue before and after hydrothermal reaction,explore the properties of hydrothermal degradation of plant tissues.In order to further reveal the restrictive factors and mass transfer laws of isoquercitrin leaching,the kinetic parameters such as mass transfer coefficient k,activation energy Ea,relative vacancy rate y and effective diffusion coefficient Ds were calculated,Study on the kinetics of isoquercitrin leaching using Fick’s second law.Analyzed the diffused process of isoquercitrin with different particle sizes,established an internal diffusion model of isoquercitrin,and obtained the diffusion rate constant of isoquercitrin.In order to obtain isoquercitrin with high-purity,the mobile phase composition,its ratio,flow rate and injection amount were compared to prepare conditions for separation and purification of isoquercitrin,and analyze the beneficial result.conclusion as below:（1）The suitable parameters for the extraction of isoquercitrin by hydrothermal reaction assisted alcohol method using response surface methodology are as follows:with a volume fraction of 57%ethanol as the extractant,the ratio of mulberry leaf mass（g）to ethanol volume（v）was 1:7,hydrothermal temperature is 154℃and hydrothermal time is 80 min,could extract the content of isoquercitrin was 3.6491mg·g^-1,It is about 51 times that of ethanol.The equation for the immersion of isoquercitrin was obtained by the response surface method,the value of R² is 0.9582,F is 22.91,P<0.0001,which indicates that the model is available.According to the F value,obtained the order of influence of the four factors is:hydrothermal time>hydrothermal temperature>liquid-to-liquid ratio>ethanol volume fraction,The interaction between hydrothermal time and hydrothermal temperature has a significant effect on the amount of isoquercitrin.The mulberry leaf extract showed the antioxidant capacity at a lower concentration of 0.02 mg·mL^-11 by the DPPH clearance rate,superoxide anion clearance rate,lipid peroxidation inhibition rate and reducing ability,a positive correlation was observed in the concentration range of 0.02 mg·mL^-11 to 0.1mg·mL^-1.（2）The results of FT-IR indicated that the hydrothermal reaction contributed to the C-O,C-H,C=C bond cleavage in mulberry leaves,and promoted the decomposition of phenylpropane structural unit in mulberry leaf lignin.The results of SEM showed that the pores between the mulberry leaves become larger and the pores appear.The results of BET showed that the specific surface area of the substrate after hydrothermal reaction is higher than that of the raw material,indicating that the hydrothermal reaction can effectively degrade the fiber tissue of mulberry leaves,which is beneficial to the dissolution of natural active ingredients.The diffusion model of particle shows that there are three processes,is permeation,internal diffusion and boundary diffusion in the diffusion of isoquercitrin,the thickness constant C of the boundary layer is C_I<C_II<C_III,the rate constant of diffusion k becomes smaller as the thickness of the boundary layer increases.（3）According to Fick’s second law,obtained the kinetic model of immersion of mulberry leaf isoquercitrin.When the temperature is between 120°C and 160°C,calculated the rate constant k is from 0.0157 min^-11 to 0.0301 min^-1,the apparent activation energy Ea is 27.22 kJ·mol^-1,and the pre-exponential factor A is 0.0164 min^-1,and the smaller the apparent activation energy is,which is beneficial to the reaction.The effective diffusion coefficient Ds is 0.52×10^-4mm²·min^-10.99×10^-4mm²·min^-1,and increased with increasing temperature.The raffinate rate decreased with increasing temperature,the minimum raffinate rate at 154℃is 9.75%,At this time,the leaching rate of isoquercitrin is 91.25%.The thermodynamic analysis determined thatΔGm was-4.3743kJ·mol^-1-1.2582kJ·mol^-1<0,ΔHm=34.907kJ·mol^-1>0,ΔSm=92.47J·mol^-1>0,indicating that the process of hydrothermal assisted alcohol extract isoquercitrin is a spontaneous process of endothermic entropy increase.（4）Purified by preparative chromatography to obtain isoquercitrin,the suitable conditions of separation and purification were:the ratio of the volume of acetonitrile to water is 20:80,the volume is 10mL,the flow rate is 20mL·min^-1.After lyophilization,the preparation rate of isoquercitrin was 26.36%,and the purity was 98.78%.At the same time,the preparation rate of chlorogenic acid was 30.37%,the purity was 99.47%,the preparation rate of rutin was 19.51%,the purity was 98%,the preparation rate of astragalin was 11.47%,and the purity was 98%.更多还原