Component Analysis And Inhibitory Effect On Colon Cancer Cells Of The Citrus Aurantium L.var.amara Engl Flower Essential Oil

Citrus aurantium L.var.amara Engl is a variant of Citrus lime in the Rutaceae family.The Citrus aurantium L.var.amara Engl produces large amounts of flowers,these flowers rich in aromatic substances and active ingredients.The thining flowers and physiological fallen flowers cause a huge waste of resourses.Processing the Citrus aurantium L.var.amara Engl flowers into essential oil can use resources efficiently and increase their economic value.The differences of volatile components（VOCs）and content are important factors that affect the quality of essential oils,which are closely related to the methods and materials of extraction.In this study,essential oil samples A,B and C were extracted by over-water distillation,azeotropic distillation and ultrasonic-assisted azeotropic distillation from dried Citrus aurantium L.var.amara Engl flowers respectively.Then ultrasound-assisted azeotropic distillation was used to obtain essential oil samples D,E,F and G from the first,second,third and fourth-year Citrus aurantium L.var.amara Engl flowers,essential oil sample H are extracted by over-water distillation from Citrus aurantium L.var.amara Engl flowers.In order to meet the demand for different aroma components,the gas chromatography-ion mobility spectrometry（GC-IMS）and gas chromatography–mass spectrometry（GC-MS）were used to compare and analyze the VOCs of these eight kinds of essential oil samples,which provided advices for the selection of essential oil extraction methods and materials.The inhibitory effect on human colon cancer cell SW480 was determined to evaluate the anti-tumor activity of each essential oil sample,and explore the possible mechanism of the inhibitory effect of Citrus aurantium L.var.amara Engl flowers essential oil.1.The yield of dried flowers essential oils in over-water distillation,azeotropic distillation and ultrasonic-assisted azeotropic distillation are 0.38%,0.41%and 0.44%,respectively.Ultrasonic-assisted azeotropic distillation was selected to extract the flowers of different tree ages.The yield of essential oils from different tree ages flowers were 0.47%（first-year）,0.33%（second-year）,0.38%（third-year）,0.24%（fourth-year）,respectively.The yield of first-year Citrus aurantium L.var.amara Engl flowers essential oil was higher than others.The yield of Citrus aurantium L.var.amara Engl flowers essential oils by over-water distillation was 0.40%,which was higher than dried flowers essential oil.2.GC-IMS detected the VOCs of the dried flowers essential oils.The essential oil extracted by over-water distillation were less than others,but some of them,such as 1-butanol,ethanol,hexanal,citronellal,propyl acetate,ethyl propionate had a high concentration of fermented,fruity and floral fragrance.The concentration of components which were presented with the aroma of floral and fruity such asα-pinene,pentanal,methyl-5-hepten-2-one,methyl isobutyl ketone,3-methyl-butanal was highest in the essential oil extracted by azeotropic distillation.The components in essential oil extracted by ultrasound-assisted azeotropic distillation were similar to azeotropic distillation.The former had the most abundant types of ingredients and higher content of ethyl acetate and ethyl phenylacetate,which made it have a stronger fermented and honey aroma.The fourth-year flowers essential oil had the most abundant types and contents that were presented with the amora of fruity,green,peppermint,strong honey,freshly and almond,such as limonene,1-propanol,methyl-5-hepten-2-one,2-hexanone,ethyl phenylacetate,α-pinene,phenylacetalde-hyde.The first-year flowers essential oil was next to the fourth-year flower essential oil.The second-year and third-year flowers essential oils had fewer unique ingredients,and the second-year flower essential oil contained some irritating substance,which was not suitable to process essential oils.Fresh flower essential oil had abundant varieties of ingredients than dried flower essential oil,and the fragrance was fresher and sweeter.Fresh flower was preferred to be selected as the raw materials for essential oil.3.GC-MS detected eight samples of Citrus aurantium L.var.amara Engl flowers essential oil.74 chemical components were identified in total,including 25terpenes,23 alcohols,8 esters,6 aldehydes,4 ketones,4 alkanes,2 acids,1 aromatic hydrocarbon and 1 indole.Among them,β-pinene,β-myrcene,limonene,1,6-octadien-3-ol,3,7-dimethyl-,3-cyclohexen-1-ol,4-methyl-1-（1-methylethyl）-,2,6-octadien-1-ol,3,7-dimethyl-,acetate,（Z）-,2,6-octadien-1-ol,3,7-dimethyl-,acetate,（E）-,caryophyllene and 1,6,10-dodecatrien-3-ol,3,7,11-trimethyl-,（E）-were the common components in these eight essential oils.4.All of the Citrus aurantium L.var.amara Engl flowers essential oil samples could significantly inhibit the cell viability of SW480 cells.The order of the effect on SW480 cell viability inhibition were as follow:G>H>D>A>F>E>C>B.Among the different extraction methods,the essential oil extracted by azeotropic distillation had the best inhibitory effect.The fourth-year flowers essential oil had the best inhibitory effect among the different tree ages flowers essential oils.The essential oil extracted from fresh flowers had stronger inhibitory effect than extracted from dried flowers.5.The Citrus aurantium L.var.amara Engl flowers essential oil could increase content of ROS and Ca²⁺in human colon cancer cell SW480,which were concentration-dependently.It also could decrease the mitochondrial membrane potential,and arrest the cell cycle in the S phase.The m RNA and protein levels of Bcl-XL were down-regulated.Bid were up-regulated.The m RNA and protein expression of Cyto C and PARP were increased,which could activate the Caspase-9and Caspase-3 to increase their m RNA and protein expressions.These results showed that the Citrus aurantium L.var.amara Engl flowers essential oil resulted in the apoptosis of SW480 cells by regulating the mitochondrial signaling path way.According to the mentioned above,the Citrus aurantium L.var.amara Engl flowers essential oil was rich in aromatic substances and active ingredients.There were differences between samples extracted by different extraction methods and raw materials.It had strong inhibitory effect on SW480 cells and could induce apoptosis of SW480 cells through the mitochondrial signaling pathway.The Citrus aurantium L.var.amara Engl flowers essential oil had great potential to be a natural flavor and anti-tumor drug applied in the food,daily chemical and pharmaceutical industries.