Study On Limonoids And Oil From Lemon Seed

Lemon belongs to the family of Rutaceae,which widely distributed in subtropical regions.The fruit is rich in vitamin,sugar,citric acid,hesperidin and other elements,and used as upper class seasoning?such as beverage,cuisine,cosmetic and drug?.Lemon seeds are main by-product of the lemon processing industry,which contain a lot of oils,flavonoids and limonids.The action of discarding lemon seeds arbitrarily leads to resources loss and environmental pollution.This paper focuses on the study of limonoids from lemon seeds about extraction,purification,identification and in vitro antitumor activity.In addition,the composition of lemon seed oil obtained by ultrasonic and soxhlet extraction method were compared,which provide theoretical guidance and basis for the comprehensive utilization of lemon seeds.The specific experiments and results are as follows:1.In this paper,ultrasonic-assisted extraction of limonoids from skim lemon seed was optimized using response surface methodology?RSM?based on single factor experimental results.Furthermore,the extraction time,temperature and ultrasonic power were taken as independent variables,and the extraction yield was taken as the response value.Then,the antioxidant activity of the extract was determined by DPPH and superoxide radical scavenging assay.The optimized extraction conditions were as followings:extraction time of 56 min,temperature of56°C and ultrasonic power of 300 W.Under above conditions,the extraction yield reached 6.02±0.07%?n=3?.The IC₅₀ of the extract scavenging DPPH and superoxide radical are 0.22±0.05 mg/mL and0.51±0.03 mg/mL respectively.2.UPLC-TOF/MS and HPLC were used to determine the ingredient and content in the extract.And the extracts were separated and purified by silica gel column chromatography and recrystallization.In order to study the structure-activity relationship of limonins,limonin was modified and the structure was identified by NMR.UPLC-TOF/MS and HPLC-UV analyis showed that the major limonoids in the extract are limonin?71.54%?,nomolin?23.72%?and obakunone?4.74%?.Above compounds can be obtained by column chromatography,and the purity were 98.51%,97.83%and 97.37%respectively;limonin and nomilin can be separated by recrystallization with purity of 93.21%and 82.53%.NMR analysis result shown that the compound obtain by structural modification of limonin was limonin 7-methoxime.3.In the activity experiment,limonin,nomolin,obakunone and limonin 7-methoxime were used to investigate their cell growth inhibitory activity on MCF-7,HT-29 and LNCap cancer cells for 48 h,obakunone and limonin 7-methoxime was selected to be further study in LNCap to investigate its mechanism by cell cycle distribution and Westen Blot analysis.In CCK8 assay,limonin was precipitate easily at low concentration?50?M?and had no significant effect on cells.The IC₅₀ of nomolin on MCF-7 and LNCap were 485±0.04?M?P>0.05?and463±0.02?M?P<0.05?;the IC₅₀ of obakunone on HT-29 and LNCap were 662±0.02?M?P>0.05?and 236±0.03?M?P<0.05?;the IC₅₀ of limonin 7-methoxime on MCF-7,HT-29 and LNCap were 547±0.02?M?P>0.05?,366±0.03?M?P<0.05?and 249±0.04?M?P<0.05?respectively.According to the result of CCK8,the limonoids?especially obakunone and limonin 7-methoxime?possess stronger inhibition on LNCap cell,and the structure of limonin at the 7th position has a great influence on its solubility and anticancer activity.The result of cell cycle distribution analysed by flow cytometry indicate that obakunone coused greatly increase in G2/M and S stage,limonin 7-methoxime result in greatly increase in G2/M stage,which mean that both compounds play a role in cell cycle arrest.The result of protein expression analys shown that the level of P21 and Caspase 3 decreased,and the level of Active-caspase 3increased after treatment with obakunone and limonin 7-methoxime,which mean that both compounds play antiproliferative role by inducing cell apoptosis.4.GC-MS was applied to determine the ingredient of lemon seed oil obtained by ultrasonic-assisted and soxhlet extraction.The result shown that 20 and 18 components were identified from the sample of ultrasonic-assisted and soxhlet extraction respectively.And the main components of both samples were same,they are 9,12-Octadecadienoic acid,9-Octadecenoic acid,hexadecanoic acid,cis-Vaccenic acid and methyl stearate.