【作者】 周尽花；

【导师】 周春山；

【作者基本信息】 中南大学 ， 应用化学， 2007， 博士

【摘要】 从植物中分离提取有效活性成分一直是医药领域研究的重点。该文立足于实际,以柚果食用后的废弃物——柚皮和柚籽为原料,采用酶法提取、超滤和大孔吸附树脂纯化等多种现代分离技术,从柚皮和柚籽中共分离出香精油、生物类黄酮、果胶、水不溶性食用纤维和柠檬苦素类化合物等五种有效成分,并从中得到了三种高纯活性成分,同时,对各有效成分的性质进行了研究,开发了一条对柚果资源进行精深加工和综合利用的新工艺路线,并取得了一系列有意义的成果。采用压榨-水蒸气蒸馏法,以5%氯化钠溶液为盐析剂进行提取,香柚柚皮和蜜柚柚皮中香精油得率分别达0.935%和0.984%。经GC-MS和计算机联用技术,分别分离确定出香柚皮和蜜柚皮香精油中三十多种化学成分及其相对含量。从提取完香精油的柚皮中提取黄酮,黄酮一次提取率为72.57%,提取三次,其总提取率达83.09%;以C树脂吸附分离黄酮提取液,其动态吸附率为86.6%;以体积分数60%乙醇-水溶液（中性）洗脱,解吸率达87.5%;从提取完柚黄酮的柚皮中提取果胶,酶B辅助酸提取法具有较高的果胶提取率,提取三次,果胶的提取率可达为90.4%;大孔吸附树脂A对果胶提取液有较好的脱色效果;无机管式陶瓷膜超滤处理果胶提取液可以同时实现果胶提取液的浓缩、纯化及脱色;以硫酸铝钾为盐析剂的果胶沉淀得率达91.4%;以氯化铁为盐析剂,果胶沉淀得率为95.8%;直接醇沉法沉淀得率为94.7%;按酶B辅助酸法提取、无机管式陶瓷膜超滤处理并经喷雾干燥所得的果胶质量经湖南省质量监督检测所检测,其色泽为白色,灰分仅为0.97%,总半乳糖醛酸含量为76.1%,各项指标均较好地满足QB2484-2000的要求。从提取完果胶等有效成分的柚皮残渣中提取水不溶性食用纤维,水不溶性食用纤维的得率约46%左右;其脱色的适宜脱色剂为5%的脱色剂A溶液。以丙酮为提取剂,从柚籽中渗滤法提取柠檬苦素类化合物,脱脂原料与未脱脂原料的提取率分别为56.88%和41.37%。从柚果废弃物的提取物中经分离、纯化获得了三种活性单体:柚黄酮单体柚皮甙C₂₇H₃₂O₁₄·2H₂O;柠檬苦素类化合物经纯化后得两种单体:脱乙酰基诺米林C₂₆H₃₂O₈和柠檬苦素C₂₆H₃₀O₈。研究了柚香精油对家蝇及蚕豆蚜的生物活性、柠檬苦素类化合物粗品对粘虫及小菜蛾虫的生物活性;柚香精油显示出对家蝇及蚕豆蚜的生物活性可能是柠烯、香茅醛等活性成分共同作用的效果,柠檬苦素类化合物的生物活性可能与其含有呋喃环结构有关。建立了描述柚皮果胶提取液超滤过程膜污染的凝胶-衰减模型:当跨膜压力△P＜△P_opt时,超滤行为遵循凝胶极化模型,通量随跨膜压力的增加而上升,当达到稳定态时有:J=Kln（c_G/c_B）;当跨膜压力△P≥△P_opt,超滤行为遵循衰减模型,其衰减方程为:J=γ（△P）^δ(γ=5.208×10¹⁰,δ=-3.022),该模型首次合理地解释了膜通量与跨膜压力之间呈现钟形关系的原因,有助于指导、控制柚皮果胶提取液的超滤工艺。研究了柚皮果胶的黏流性能,温度对果胶溶液黏度的影响可用黏流方程η=η₀exp（Ea/RT）来描述,其平均黏流活化能为17.14kJ·mol^-1,果胶浓度对黏度的影响在一定程度上可用等温指数方程η=K₂exp（A₂C）来描述。由特性黏度理论,计算出了柚皮果胶的黏均分子量为7×10⁴-8×10⁴。采用传质理论解释了逆流萃取法可以提高果胶提取率的原因:其原因在于逆流萃取法可以保证较大的传质驱动力——浓度差。研究了等温条件下C树脂静态吸附柚黄酮的规律,其吸附规律符合Langmuir方程:C_e/q_e=C_e/q₀+1/Kq₀=0.01415C_e+0.10008。以量的形式确定了盐析剂的用量并初步探讨了三价铁盐与铝盐的盐析机理:盐析剂的用量可按0.1588 mol Mⁿ⁺/100g果胶计算;三价铁盐与铝盐盐析果胶的机理相似:果胶的羧基离子取代金属水合物中的羟基,与金属离子发生反应,生成果胶盐而产生沉淀实现分离。该工作以柚果废弃物为原料,开发出一系列的产品:香精油、柚黄酮类化合物、柠檬苦素类化合物、果胶及水不溶性食用纤维,真正做到资源的最大综合利用,为柚子综合开发利用提供了新方法和新思路,具有显著的经济效益和社会效益。本研究在天然药物的深加工开发、促进我国柚果产业的发展、加快中药现代化进程、帮助农民增收方面,必将具有深远的意义。更多还原

【Abstract】 Extracting and separating active components from plants has been an important and eye-catching area in medical and pharmaceutical fields. Citrus grandis Osbeck（Shaddock）is a good,useful fruit and is widely grown in China.However,most of the time,after eating the pulp,people discard shaddock peel and shaddock seeds.It is not only wasting resources but also polluting the environment.At the same time,shaddock growers can’t make enough money to keep their enthusiasm to grow shaddock continually.Based on the realties,in this dissertation,starting from the castoffs of shaddock-shaddock peel and shaddock seeds,five effective ingredients-shaddock essential oil,flavonoid,pectin,water insoluble dietary fiber and limonoids were extracted and separated out by modern extraction and separation technologies,such as enzyme-aided extraction,ultrafiltration and purification by macroporous adsorption resin.At the same time,three kinds of high-purity active compounds were obtained.Therefore,a new technical line for deep processing and comprehensive utilizing shaddock has been developed and a series of significant experimental results has been achieved.With salting-out agent 5%NaCl solution,shaddock essential oil was extracted from fragrant shaddock peel and sweet shaddock peel squeezing-vapor distillation.The yield of essential oil from fragrant shaddock peel and sweet shaddock peel was 0.935%and 0.984%, respectively.At the same time,more than thirty kinds of compounds from Jiangyong fragrant shaddock and sweet shaddock essential oil were analyzed and identified and their relative contents were determined by GC-MS.The residue of shaddock peel after the extraction of essential oil was treated by ethanol-water solution to get flavonoid,One-time extraction yield of flavonoid was 72.57%while the total yield after three times extraction was 83.09%.Macroporous adsorption resin C was used to separate the flavonoid extracts with dynamic adsorption rate 86.6%and 60%ethanol-water（v/v）was used to wash the flavonoid from resin C with the desorption rate 87.5%. As for extracting pectin from the residue of shaddock peel after the extraction of flavonoid,enzyme B-aided extraction features higher pectin yield compared with other extraction methods.After three times extractions,the pectin yield can reach 90.4%.Good decoloration effects and low pectin loss can be achieved by macroporous adsorption resin A while concentration,purification and decoloration of pectin extracts can be achieved simultaneously by ultrafiltration with inorganic tubular ceramic membrane.The pectin precipitation yield was 91.4%for salting-out agent Aluminum potassium sulfate and 95.8%for salting-out agent Ferric chloride while the pectin precipitation yield of direct ethanol precipitation method was 94.7%.The quality of shaddock peel pectin produced by enzyme B-aided extraction,which followed by ultrafiltration with inorganic tubular ceramic membrane and then spray-drying can satisfy the demands of QB2484-2000 after having been tested by Hunan quality supervision and detection institute.Extraction of water insoluble dietary fiber from the residue of shaddock peel after the extraction of pectin was studied.The water insoluble dietary fiber from shaddock peel features good quality,high coarse fiber content and light color with yield 46%or so.The proper decoloring agent for insoluble dietary fiber is 5%decoloring agent A solution.With extraction agent acetone,limonoids were extracted from shaddock seeds by percolation method and the limonoids yield was 56.88%and 41.37%for degreased seeds and non-degreased seeds, respectively.Three high-purity effective ingredients were obtained from the extracts of shaddock castoffs after further separation and purification. They were naringin(C₂₇H₃₂O₁₄·2H₂O,from flavonoid),Deacetylnomilin (C₂₆H₃₂O₈,from limonoids)and Limonin(C₂₆H₃₀O₈,from limonoids).The biological activities of shaddock essential oil on Musca domestiea and Aphis fabae and biological cativities of limonoids on Mythimna separate and Plutellla xylostera were studied.The biological activities of shaddock essential oil on Musca domestica and Aphis fabae is likely to be involved with the co-effects of limonene,citronellal and other effective ingredients while the biological activities of limonoids may be involved with their furan nucleus structure.A gel-attenuation model was set up to describe ultrafiltration membrane pollution behavior of pectin extracts.When transmembrane pressureΔP is belowΔP_opt,the ultrafiltration of pectin extracts follows gel-polarization model,flux increases with the transmemberane and the equation J=Kln（c_G/c_B）is tenable when the stable state is achieved. However,whenΔP is aboveΔP_opt,the ultrafiltration of pectin extracts follows attenuation model with the attenuation equation J=γ（ΔP）^δ(γ=5.208×10¹⁰,δ=-3.022).With this gel-attenuation model,the bell-shape relations between transmembrane pressure and membrane flux can be explained properly for the first time.The model will be helpful to guide and control the ultrafiltration of shaddock peel pectin extracts.The flow properties of shaddock peel pectin solutions were also studied.Models describing the concentration dependence and temperature dependence of viscosity of shaddock peel pectin solution were set up.The effects of temperature on viscosity of pectin solution can be described by the viscous-flow equationη=η₀exp（Ea/RT）and the average viscous-flow activation energy is 17.14kJ·mol^-1while the isothermal exponential equationη=K₂exp（A₂C）can be used to describe the effects of concentration on viscosity of pectin solution.At the same time,intrinsic viscosities of Shaddock peel pectin were determined and thus the viscosity-average molecular weights of pectin were calculated as 7×10⁴--8×10⁴ according to the intrinsic viscosity theory.The mass transfer theory was used to explain the increasing of pectin yield when pectin was extracted by countercurrent extraction-countercurrent extraction guarantees higher driving force for mass transfer,i.e.,higher concentration difference.The regularity of static adsorption of flavonoid by macroporous resin C at isothermal conditions was studied.The experimental results showed that the adsorption can be described by Langmuir equation: C_e/q_e=C_e/q₀+1/Kq₀=0.01415C_e+0.1000.The usage of salting-out agents was determined quantitatively and the salting-out mechanism of Fe³⁺and Al³⁺were studied.The results showed that the usage of salting-out agents can be calculated according to 0.1588 mol Mⁿ⁺per 100 g pectin.Fe³⁺and Al³⁺have similar salting-out mechanism-the carboxyls of pectin substitute the hydroxyls of metal hydrates,react with the metal cations and form pectin salt which precipitate from the solutions and thus separation is achieved.In conclusion,the shaddock resource is fully and comprehensively utilized:the new technical line starts from the castoffs of shaddock-shaddock peel and shaddock seeds and presents a series of products,i.e., essential oil,flavonoid,limonoids,pectin and water insoluble dietary fiber.Therefore,the study in this dissertation has offered a new way of comprehensively utilizing shaddock resources and will feature remarkable economic and social benefits and profound significance in deep processing of natural product,helping farmers to increase their incomes,promoting the shaddock growing and processing industry and modernizing Chinese medicine.更多还原