Analysis Of Chemical Components In Xinhui Citrus Seeds And The Inhibition Effect On The Growth Of Hepatocellular Carcinoma HepG2 Cells

Background: Xinhui citrus,also known as ‘Chachi',is mainly produced in xinhui District,Jiangmen City,Guangdong Province.Its dried and ripe peel is the famous genuine medicinal materials “Guangchenpi”.According to statistical analysis,there are more than 100000 tons of citrus fruits and nearly 2000 tons of seeds produced in xinhui in 2019.However,most of xinhui citrus seeds are discarded directly along with the pulp due to the utilization way of "reserve the skin rather than the pulp",which not only results in environmental pollution,but also causes serious waste of resources.Citrus seeds have been used as Chinese traditional medicine named Citri Reticulatae Seed(CRS)since ancient times.However,there are few researches on CRS and it has not been widely used in application.The results showed that the CRS had analgesic,anti-cancer and anti infection activities.Therefore,in this paper,the chemical components of xinhui citrus seeds were analyzed and identified systematically.Furthermore,the pharmacological effects of the chemical components on human hepatoma cells Hep G2 were preliminarily studied combining with relevant pharmacological experiments,which provided scientific basis for the rational development and utilization of xinhui citrus seeds resources.Aims:(1)The main chemical components of xinhui citrus seeds were analyzed and identified;(2)The main chemical components contents of xinhui citrus seeds were determined;(3)The main chemical components of xinhui citrus seeds were separated and purified;(4)The effect of the main chemical components of xinhui citrus seeds on human hepatoma cells Hep G2 was preliminarily studied.Methods:(1)The methanol ultrasonic extracts and supercritical CO2 fluid extracts of xinhui citrus seeds were analyzed and identified by ultra-high performance liquid chromatography combined with Q-Exactive Orbitrap tandem mass spectrometry(UHPLC-Q-Exactive Orbitrap-MS).The contents of limonin,hesperidin,nobiletin,tangeretin,3,5,6,7,8,3?,4?-heptamethoxyflavonoid,synephrine and gallic acid in xinhui citrus seeds were determined by High Performance Liquid Chromatography(HPLC);(2)The main chemical component was isolated from xinhui citrus seeds by silica gel column chromatography combined with recrystallization method.The molecular weight and structure were respectively evaluated by Electrospray Ionization Mass Spectrometry(ESI-MS),Proton Nuclear Magnetic Resonance(1H-NMR)and Carbon Nuclear Magnetic Resonance(13C-NMR);(3)Cell Counting Kit-8(CCK-8)assay,Colony formation assay and Hoechst assay were used to detect the cell viability,colony formation ability and apoptosis rate of Hep G2 cells treated with limonin.Results:(1)35 and 28 chemical constituents were identified from xinhui citrus seeds respectively by UHPLC-Q-Exactive Orbitrap-MS method in methanol extracts and supercritical CO2 fluid extracts.The mean contents of limonin,hesperidin,nobiletin,tangeretin,3,5,6,7,8,3?,4?-heptamethoxyflavonoid,synephrine and gallic acid in xinhui citrus seeds were 18.989,0.382,0.345,0.312,0.081,0.033 and 0.003 mg · g-1 respectively measured by HPLC;(2)The main chemical component was separated and prepared from xinhui citrus seeds by silica gel column chromatography combined with recrystallization,and identified as limonin by ESI-MS,1H-NMR and 1C-NMR;(3)The CCK-8 results showed that limonin inhibited the proliferation of Hep G2 cells in a both time-dependent and dose-dependent manner;(4)The colony formation ability of Hep G2 cells decreased with the increase of limonin concentration;(5)The results of Hoechst experiment showed that the apoptosis of Hep G2 cells increased with the increase of limonin concentration.Conclusion:(1)The chemical components of xinhui citrus seeds can be rapidly and effectively identified by UHPLC-Q-Exactive Orbitrap-MS,of which 20 flavonoids were first found in methanol extracts and 9 esters were first found in supercritical CO2 fluid extracts.In addition,the content determination results showed that limonin in xinhui citrus seeds is rich and can be further separated and prepared;(2)Silica gel column chromatography combined with recrystallization method can be used for the separation and purification of limonin monomer from xinhui citrus seeds;(3)Limonin inhibited the proliferation of Hep G2 cells in a both time-dependent and dose-dependent manner;(4)Limonin inhibits proliferation by inducing apoptosis of Hep G2 cells.