Isolation And Identification Of The Flower Of Citrus Maxima And Its Proliferation Inhibition Effect On 3T3-L1 Preadipocytes

Citrus maxima,a member of genus Citrus(Rutaceae),is evergreen tree fruit. Citrus maxima contains various pharmacological effects and nutritional value,which were recorded in Chinese medicine books and ancient prose. Its flower is rich in volatile oil,which have been applied in perfumes and cosmetics. At present,there are few studies on the flower of Citrus maxima,especially on its chemical composition and pharmacological effects. Reseach on its chemical components makes for the full use of the flower of Citrus maxima.The total extract was obtained by the extraction of heating the flower of Citrus maxima with 70% ethanol and then portioned with petroleum ether,chloroform,ethyl acetate,n-butanol and water,which yielded petroleum ether fraction,chloroform fraction,ethyl acetate fraction,n-butanol fraction,and water fraction,respectively. Different models,antioxidant,antitumor and the inhibition on 3T3-L1 cells were use to evaluate the activity of the total extract(70% ethanol) and five fractions extracted from the flower of Citrus maxima.Results showed that 70% ethanol extract and five fractions were exhibited poor antioxidant effect, showed better inhibition effect on tumor cells and 3T3-L1 cells. The main active ingredient is concentrated in the chloroform fraction after solvent extractions. Consequently,chloroform fraction was selected for further separation and purification.Chloroform fraction was separated and purified by various column chromatography(including silica gel column and Sephadex LH-20 gel column). Six compounds were obtained and their structure was identified by mass spectrometry and nuclear magnetic resonance(including1H-NMR、13C-NMR、DEPT-135、1H-1H COSY、HMBC、HMQC) as lup-20(29)-ene-3β-yl tetracosanoate, β- Sitosterol, 5-Hydroxy-6, 7, 3’,4’-tetramethoxyflavone, isolimonexic acid, 7’-[(6’, 7’-dihydroxy-3’,7’-dimethyl-2’-ocfenyl)oxy] coumarin, Limonexic acid, respectively.This paper studied the inhibition effect of 3T3-L1 preadipocyte proliferation induced by the compounds isolated from the flower of Citrus maxima. Among all compounds, Marmin could significantly inhibit the proliferation of 3T3-L1 cell,as well as change the morphologyof the cell and nucleus. Marmin showed antiproliferative activity on 3T3-L1 cells by induction of apoptosis,and significantly increase interacellular reactive oxygen species(ROS)level. Marmin could increase the ratio of Bax/ Bcl-2 and p53 at the m RNA level,but have little effect on the gene p21. Marimin could up-regulate the expression of p-JNK and down-regulate the expression of p-p65 and p-ERK1/2 at protein level. The suggested mechanisms of the pro-apoptosis activity of marmin were main through activation of JNK signaling pathway and inhibition of p-p65 and p-ERK1/2signaling pathway.Four chemical constituents(flavonoids,saponins,polysaccharides and volatile oil) were obtained from the flower of Citrus maxima by different extraction methods. Their contents were determined by standard curves of four reference substances,be 25.76%,35.28%,34.55%,and 2.61%,respectively. Flavonoids exhibited the best anti-proliferative activity on3T3-L1 cells lines among four chemical constituents. Flavonoids can induced early apoptosis cells,obviously increased intracellular ROS level and formed apoptotic bodies. Meanwhile,the relative m RNA expression of p21 and p53 gene and the ratio of bax/bcl-2 also increased significantly by flavonoids treatment. At the protein level, flavonoids can significantly up-regulate p-JNK protein expression and down-regulate p-p65 protein expression, but have little influence of p-ERK1/2 protein expression is not obvious.