万州尤力克柠檬果皮精油的提取工艺及其抗氧化、抑菌、抗肿瘤活性研究

Study on extraction process of essential oil from Wanzhou Eureka lemon peel and its antioxidant,antibacterial,and antitumor activities

为研究柠檬精油提取的最佳工艺条件及抗氧化、抑菌、抗肿瘤等生物活性,以尤力克柠檬皮为原料,通过单因素实验和响应面实验优化柠檬精油提取条件并采用气质联用技术对精油成分进行分析。以抗坏血酸为对照,进行抗氧化活性测定,运用滤纸片扩散法测定柠檬精油抑菌活性,通过噻唑兰(methyl thiazolyl blue tetrazolium bromide,MTT)比色法及蛋白免疫印迹法(western blotting,WB)分析柠檬精油的抗肿瘤活性。结果表明,柠檬精油提取的最佳工艺为:液料比为12∶1(mL∶g)、超声时间29 min、蒸馏时间133 min、氯化钠添加量5.20 g/L,在此条件下提取率可达2.72%,精油中含有102种化合物,其中烯类物质相对含量最高,达62.89%。柠檬精油对DPPH自由基具有较好的清除能力,当精油质量浓度为343 mg/mL时,清除率可高达92.68%。抑菌活性试验表明,柠檬精油对4种受试菌均有抑制作用,其中对维氏气单胞菌抑制效果最佳。通过MTT试验并结合半数抑制浓度(IC50)发现,柠檬精油表现出显著的抗癌活性,其中对结直肠癌细胞增殖活力的影响尤为明显,对结直肠癌细胞周期和自噬相关蛋白进行WB检测发现,柠檬精油通过周期阻滞及促进自噬抑制结直肠癌细胞增殖。该研究可为万州区柠檬深加工产业以及废渣利用的发展提供理论依据,并且为精油活性成分深度开发提供理论参考。

To study the optimal extraction conditions of lemon essential oil and its antioxidant,antibacterial,anti-tumor,and other biological activities,the extraction conditions of lemon essential oil(LEO)were optimized by complete randomized design and response surface methodology and the components of the essential oil were analyzed by gas chromatography-mass spectrometer.The antibacterial activity of LEO was determined by methyl thiazolyl blue tetrazolium bromide(MTT)and western blotting(WB).Results showed that the optimal extraction process conditions of LEO were as follows:Liquid-solid ratio of 12∶1,ultrasonic time of 29 min,distillation time of 133 min,and sodium chloride of 5.20 g/L.Under these conditions,the extraction yield of LEO could reach 2.72%,there were 102 compounds in essential oil,among which the relative content of alkenes was the highest(62.89%).LEO had a good scavenging ability on DPPH radical,and the scavenging rate was up to 92.68%when the concentration of LEO was 343 mg/mL.The antibacterial activity test showed that LEO had inhibitory effects on four kinds of tested bacteria,and the inhibitory effect was the best against Aeromonas vectori.It was found by the MTT test and median inhibitory concentration(IC50)that LEO showed significant antitumor activity.Among them,the effect on the proliferation of colorectal cancer cells was particularly obvious.WB detection of colorectal cancer cell cycle and autophagy-related proteins showed that LEO inhibited the proliferation of colorectal cancer cells by blocking the cell cycle and promoting autophagy.This study provides a theoretical basis for the development of the lemon deep processing industry and waste residue utilization in the Wanzhou district and provides a theoretical reference for the deep development of active ingredients.