柚核柠檬苦素类似物的提取纯化及稳定性研究
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摘要
柠檬苦素类似物是芸香料和楝科植物的主要次生代谢产物,是一类高度氧化的三萜类化合物。柑桔属包括柚类果实中含量丰富,尤其以种子中的含量最高;据文献报道,柠檬苦素类似物有抗癌、降低胆固醇、抗病毒等多种生物活性。本文主要研究了从湖南香柚种子中提取、分离纯化柠檬苦素类似物,对提取物进行定性鉴定,并对柠檬苦素稳定性的影响因素首次进行了初步探讨。主要研究结果如下:
1.用分光光度法建立了湖南香柚果实中的柠檬苦素类似物含量的测定方法,检测方法的线性关系良好,回归方程为:Y=0.0034X+0.0203,相关系数R~2=0.9979。对方法的稳定性、精密度和回收率分析表明该法处理简单、方便、快捷,检测结果准确、可靠。
湖南香柚果实不同部位的柠檬苦素类似物含量依次为:柚种子＞果皮＞囊衣＞果肉;果皮0.529%、果肉0.166%、囊衣0.274%、柚种子1.209%。
2.柠檬苦素类似物的提取工艺研究
采用浸提脱脂,确定石油醚(60-90℃)为柚种子的脱脂溶剂;正交试验表明,影响脱脂因素为温度＞时间＞脱脂剂量;柚种子脱脂的最佳组合是温度32℃,时间10h,脱脂剂量40mL/g。
索氏抽提脱脂柚种子,确定丙酮为抽提溶剂,采用Box-Behnken法设计试验,得到回归方程,对回归方程作方差分析、绘制和分析响应面及等高线图。回归方程为:
Y=-125.1910+2.6958X_1+0.2930X_2+7.4845X_3-0.0164X_1~2-0.00158X_2~2-0.6025X_3~2
由回归方程预测提取柠檬苦素类似物的最佳条件为:提取温度78.9℃、溶剂用量89.7mL、提取时间4.6h,此时的柠檬苦素类似物的理论提取量为:11.58mg/g。
对回归方程作方差分析,表明各因素与响应值之间的线性关系是显著的;对最佳预测条件作验证试验,柠檬苦素类似物的提取量达到11.52mg/g。
3.分离纯化柚种子的丙酮提取物,得到两种柠檬苦素类似物晶体,经化学特征反应、TLC、IR、NMR检测,晶体Ⅰ是柠檬苦素,晶体Ⅱ为诺米林;给出了柠檬苦素的~1H-NMR和~(13)C-NMR数据分析及图谱。
4.首次初步探讨柠檬苦素晶体及溶液稳定性的影响因素,试验表明温度、pH值、水分、光照条件对柠檬苦素的稳定性影响显著。
低温和常温下,柠檬苦素稳定性良好;45℃以上时,随温度和时间的增加,降解显著,稳定性差;pH4-7,柠檬苦素稳定,分解率小于1.8%;pH＜4、强酸环境中稳定性较差;碱性环境中,在pH＞10时,不稳定,当pH=12时,分解率达94.92%;随水分含量的增加,柠檬苦素的稳定性变差,当水分达6%时,放置21d后,柠檬苦素的分解率达61.56%;不同光源对柠檬苦素稳定性的影响程度依次为紫外光＞太阳光＞室内光＞阴暗处。试验证明:紫外光照射35d后,柠檬苦素的分解率达61%。
Limonoids are a prominent group of secondary metabolites found in the Rutaceae a nd Meliacea families,and also a group of highly oxygenated triterpenoid compounds.Citr us limonoids are abundant in the Citrus fruits,including Pummelo,particularly the conten t in the seed is the highest.Evaluation of the biological activity of citrus limonoids has indicated the potential of these compounds to improve human health as anticancer,cho lesterol-lowering,and antiviral age-nts.In this paper,extraction,isolation and purification of limonoids from Hunan Xiangy-ou seeds have been researched.The purifications have be en qualitative identified.And the effects of factors on stability of limonin were prelimina ry studied for the first time at the domestic.The result of main research as follows:
1.A spectrophotometric method for quantifying the total limonoids in Pummelo fruit was established and a regression equation was obtained as y=0.0034x+0.0203,the regression coefficient R~2=0.9979.Limonoids content in different Pummelo parts were measured by this method.The result is seeds＞pericarp＞pulp capsule＞pulp,1.209%,0.529%,0.274%and 0.166%respectively.
2.The factors which affect the extraction efficiency of limonoids in pummelo seeds were studied thoroughly.Determining petroleum ether(60-90℃) as degreasing solvents of pummelo seeds using pickling extraction.Orthogonal test showed that the order of degreasing factors is temperature＞time＞degreasing solvents dose;The best degreasing conditions is temperature at 3℃C,time10h,degreasing solvents dose 40 mL/g.
Soxhlet extraction degreasing pummelo seeds,identified acetone as extraction of solvent using Box-Behnken design of experiments.The regression equation is: Y=-125.1910+2.6958X_1+0.2930X_2+7.4845X_3-0.0164X_1~2-0.00158X_2~2-0.6025X_3~2 Extraction forecast by the regression equation limonoids optimum conditions:temperature 78.9℃,89.7mL solvent,extraction time 4.6h,the limonoids from the theory is 11.58 mg/g.
The establishment of the regression equation for the analysis of variance showed that:all factors and response of the linear relationship between the notable,the best prediction of the condition for certification testing,limonoids extraction amounted to 11.52mg/g.
3.Isolation and Purification of pummelo seeds acetone extracts,obtained two limonoids crystal.The crystals were identified as limonin(crystalⅠ) and nomilin(crystaⅡ) by the characteristics of chemical reaction,TLC,IR,NMR detection.Given limonin ~1H-NMR and ~(13)C-NMR spectrum of graph and data analysis.
4.Factors affecting the stability of limonin were first preliminary studied at dome stic.Experiments show that the temperature,pH,moisture andlight conditions on the stab ility of limonin influence significantly.
At room temperature and low temperature the stability of limonin is good.At more than 45℃,with the increase in temperature and time,limonin is poor stability.pH＜4,In strong acid environment,limonin is instability;pH4-7,limonin is stability,decomposition rate less than 1.8%,In alkaline environment,pH＞10,limonin is very unstable.When pH =12,the decomposition rate is 94.92%.With the increase in water content,the stability of limonin worsen tested.When the water reached 6%,after 21 days,the decomposition rate of limonin 61.56%;Different li-ghts influence limonin stability.The results are UV＞sunlig ht＞indoor＞shadow.
1.用分光光度法建立了湖南香柚果实中的柠檬苦素类似物含量的测定方法,检测方法的线性关系良好,回归方程为:Y=0.0034X+0.0203,相关系数R~2=0.9979。对方法的稳定性、精密度和回收率分析表明该法处理简单、方便、快捷,检测结果准确、可靠。
湖南香柚果实不同部位的柠檬苦素类似物含量依次为:柚种子＞果皮＞囊衣＞果肉;果皮0.529%、果肉0.166%、囊衣0.274%、柚种子1.209%。
2.柠檬苦素类似物的提取工艺研究
采用浸提脱脂,确定石油醚(60-90℃)为柚种子的脱脂溶剂;正交试验表明,影响脱脂因素为温度＞时间＞脱脂剂量;柚种子脱脂的最佳组合是温度32℃,时间10h,脱脂剂量40mL/g。
索氏抽提脱脂柚种子,确定丙酮为抽提溶剂,采用Box-Behnken法设计试验,得到回归方程,对回归方程作方差分析、绘制和分析响应面及等高线图。回归方程为:
Y=-125.1910+2.6958X_1+0.2930X_2+7.4845X_3-0.0164X_1~2-0.00158X_2~2-0.6025X_3~2
由回归方程预测提取柠檬苦素类似物的最佳条件为:提取温度78.9℃、溶剂用量89.7mL、提取时间4.6h,此时的柠檬苦素类似物的理论提取量为:11.58mg/g。
对回归方程作方差分析,表明各因素与响应值之间的线性关系是显著的;对最佳预测条件作验证试验,柠檬苦素类似物的提取量达到11.52mg/g。
3.分离纯化柚种子的丙酮提取物,得到两种柠檬苦素类似物晶体,经化学特征反应、TLC、IR、NMR检测,晶体Ⅰ是柠檬苦素,晶体Ⅱ为诺米林;给出了柠檬苦素的~1H-NMR和~(13)C-NMR数据分析及图谱。
4.首次初步探讨柠檬苦素晶体及溶液稳定性的影响因素,试验表明温度、pH值、水分、光照条件对柠檬苦素的稳定性影响显著。
低温和常温下,柠檬苦素稳定性良好;45℃以上时,随温度和时间的增加,降解显著,稳定性差;pH4-7,柠檬苦素稳定,分解率小于1.8%;pH＜4、强酸环境中稳定性较差;碱性环境中,在pH＞10时,不稳定,当pH=12时,分解率达94.92%;随水分含量的增加,柠檬苦素的稳定性变差,当水分达6%时,放置21d后,柠檬苦素的分解率达61.56%;不同光源对柠檬苦素稳定性的影响程度依次为紫外光＞太阳光＞室内光＞阴暗处。试验证明:紫外光照射35d后,柠檬苦素的分解率达61%。
Limonoids are a prominent group of secondary metabolites found in the Rutaceae a nd Meliacea families,and also a group of highly oxygenated triterpenoid compounds.Citr us limonoids are abundant in the Citrus fruits,including Pummelo,particularly the conten t in the seed is the highest.Evaluation of the biological activity of citrus limonoids has indicated the potential of these compounds to improve human health as anticancer,cho lesterol-lowering,and antiviral age-nts.In this paper,extraction,isolation and purification of limonoids from Hunan Xiangy-ou seeds have been researched.The purifications have be en qualitative identified.And the effects of factors on stability of limonin were prelimina ry studied for the first time at the domestic.The result of main research as follows:
1.A spectrophotometric method for quantifying the total limonoids in Pummelo fruit was established and a regression equation was obtained as y=0.0034x+0.0203,the regression coefficient R~2=0.9979.Limonoids content in different Pummelo parts were measured by this method.The result is seeds＞pericarp＞pulp capsule＞pulp,1.209%,0.529%,0.274%and 0.166%respectively.
2.The factors which affect the extraction efficiency of limonoids in pummelo seeds were studied thoroughly.Determining petroleum ether(60-90℃) as degreasing solvents of pummelo seeds using pickling extraction.Orthogonal test showed that the order of degreasing factors is temperature＞time＞degreasing solvents dose;The best degreasing conditions is temperature at 3℃C,time10h,degreasing solvents dose 40 mL/g.
Soxhlet extraction degreasing pummelo seeds,identified acetone as extraction of solvent using Box-Behnken design of experiments.The regression equation is: Y=-125.1910+2.6958X_1+0.2930X_2+7.4845X_3-0.0164X_1~2-0.00158X_2~2-0.6025X_3~2 Extraction forecast by the regression equation limonoids optimum conditions:temperature 78.9℃,89.7mL solvent,extraction time 4.6h,the limonoids from the theory is 11.58 mg/g.
The establishment of the regression equation for the analysis of variance showed that:all factors and response of the linear relationship between the notable,the best prediction of the condition for certification testing,limonoids extraction amounted to 11.52mg/g.
3.Isolation and Purification of pummelo seeds acetone extracts,obtained two limonoids crystal.The crystals were identified as limonin(crystalⅠ) and nomilin(crystaⅡ) by the characteristics of chemical reaction,TLC,IR,NMR detection.Given limonin ~1H-NMR and ~(13)C-NMR spectrum of graph and data analysis.
4.Factors affecting the stability of limonin were first preliminary studied at dome stic.Experiments show that the temperature,pH,moisture andlight conditions on the stab ility of limonin influence significantly.
At room temperature and low temperature the stability of limonin is good.At more than 45℃,with the increase in temperature and time,limonin is poor stability.pH＜4,In strong acid environment,limonin is instability;pH4-7,limonin is stability,decomposition rate less than 1.8%,In alkaline environment,pH＞10,limonin is very unstable.When pH =12,the decomposition rate is 94.92%.With the increase in water content,the stability of limonin worsen tested.When the water reached 6%,after 21 days,the decomposition rate of limonin 61.56%;Different li-ghts influence limonin stability.The results are UV＞sunlig ht＞indoor＞shadow.
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