Determination And Purification Of Limonoids From Pomelo (Citrus Maxima) Fruit And Related Bioactivity Evaluation

In present study, the limonoids in different pomelo (Citrus maxima) were identified and quantified. A method system was established to purified the chief limonoids from pomelo fruits. The crude extracts and individual limonoids were analyzed for their effects on cellular glucose consumption promotion and tumor cell proliferation. The main results are as follows:1. Three dominant limonoids, limonin, nomilin and isoobacunone acid, were identified in pomelo fruit combining HPLC and LC-MS/MS. Their content varied depending on the varieties and tissues. These limonoids found to be distributed mainly in SM and seed, with a small amount detected in JS and albedo, while undetectable in flavedo. Limonin and nomilin were the chief limonoids in pomelo fruits. Seed of Qingtian has the highest amount of limonin, which was 5.45 ± 0.86 mg/g DW, segment membrane of Hongroupao has the highest amount of nomilin, which was 10.20 ± 1.87 mg/g DW. The content of isoobacunonic acid was much lower in all pomelo fruits compared to limonin and nomilin, with the highest amount of only 0.58 ± 0.05 mg/g DW in Qingtian seed.2. Efficient method system of the separation and purification of limonin, nomilin and isoobacunone acid were established by the combination of macroporous resin column chromatography, high-speed countercurrent chromatography (HSCCC) and semi-pre HPLC. After the first cleaning-up with HZ-816 resin, the purity of limonin increased from 0.29% in the crude extract to 19.80% and nomilin from 0.71% to 61.78% in the resin-refined sample, respectively. Further HSCCC purification resulted in a final purity of more than 95% for limonin, meanwhile, a mixture of nomilin and isoobacunone acid at a high purity was obtained. By subjecting the mixture into Semi-pre HPLC, nomilin and isoobacunone acid with a purity of more than 95% were obtained.3. Limonin promoted cellular glucose consumption and nomilin showed effect on inhibiting leukemia cell proliferation. Correspondingly, the glucose consumption promotion activity and cell proliferation inhibition activity of pomelo extracts was positively correlated with the content of limonin and nomilin in the extracts.