Studies On Chemical Constituents And Antitumor Activities Of The Seeds Of Xylocarpus Granatum

The marine mangrove plant Xylocarpus granatum Koenig (Meliaceae) is commonly distributed along the shores of Indian Ocean and South-East Asia seacoast. The local Indian people have used it for a long time to treat fever diseases such as malaria and it is also used as a folk medicine in southeast Asia for the treatment of diarrhea, cholera and fever diseases such as malaria and also as an antifeedant. The genus Xylocarpus contains only three species in the world wild, they are Xylocarpus granatum, Xylocarpus moluccensis and Xylocarpus rumphii which are mainly distributed along the seacoast of southeastern Asia, Australia, and East Africa. There is only one species X. granatum distributed along the shores of Hainan Province in China. The laboratory chemical studies on the genus Xylocarpus is mainly focused on the seeds and the fruits of X. granatum and X. moluccensis.So far, over 100 compounds have been separated from this genus, including limonoids, monoterpenes, phenolic acids, alkaloids, flavanols, steroids and so on. Among them, limonoids are the major components of this plant. Limonoids belong to tetracyclic triterpenoids which are a series of highly oxygenated nortriterpenoids with their structural features either containing or being derived from a precursor with a furan ring at C17 skeleton. From 70's or 80's of last century, limonoids were widely studied and recognized as natural compounds which had potential biological activities. The pharmacological studies demonstrate limonoids have broad range of biological activities, such as antimicrobial, anthelminthic, anti-HIV, and anti-tumor. Animal experiments indicated that limonoids could inhibit the liver cancer, bowel cancer, mouth cancer and skin cancer, which were induced by chemical materials, and are nontoxic to animal model. This research mainly studied on the chemical composition of the seeds of X. granatum and their anti-cell proliferation activities on human cancer cells (HLE, KT and HeLa).Objective: The object of the study was the seeds of Xylocarpus granatum. Using extraction, separation and purification methods to establish effective methods for the isolatation of pure compounds. Using spectroscopic techniques including UV, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC, NOESY, MS and so on to determine the planar and stereoscopic configuration of the structures. To screen the antitumor activities of the compounds isolated from the plant on HLE, Hela, KT tumor cells to obtain new antitumor-activities precursors.Methods: Air-dried seeds of X. granatum (6.4 kg) were crashed and immersed in 30.0L of 95% ethanol for 3 days, repeat the procedure for 4 times. Filter and combine the filtrates, then evaporate the solvent under reduced pressure to obtain crude extract of X. granatum seeds (2598.5g). Suspend the crude extract in appropriate of water and extract with petroleum ether (60~90℃), dichloromethane and ethyl acetate, to obtain three extracta sicca groups (35.7g, 158.1g, 36.1g respectively). The three extract fractions were chromatographed on silica gel column, sephadex LH-20 , Pre-TLC, Pre-HPLC, crystallization and recrystallization respectively. The petroleum ether layer yielded compounds 1 and 2; the dichloromethane layer yielded compounds 1~25 ,and the ethyl acetate layer yielded compounds 1, 9 and 25.Use spectroscopic methods including UV, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC, NOESY and MS to characterize the structures of the isolated compounds.To obtain new antitumor-activities precursors, the MTT method is used to screen the antitumor activities of compounds isolated from the plant on HLE, Hela, KT tumor cells.Results: After systematically separation on extraction of the seeds of X. granatum, 25 compounds were obtained. These compounds were analysized on the basis of extensive spectral analysis and the structures of 21 compounds were determined, including 11 limonoids, 8 protolimonoids and 2 sterols. They wereβ-sitosterol (1), daucosterol (2), odoratone (3), xyloccensin K (4), piscidinol G (5), xylocarpin H (6), xylomexicanin A (7), 7-deacetyl-7-oxogedunin (8), xylogranatin C (9), xylogranatin D (10), 3β-detigloyoxy-2′- methylbutanoyloxy xylogranatin B (11) , hispidol B (15), spicatin (17), proceranolide (18), xylogranatin A (19), xylocarpin G (20), 3-hydroxydihydrocarapin (21), grandifoliolenone (22), sapelin E acetate (23), holstinone C (24), holstinone B (25).Antitumor activity studies upon seven compounds on HLE, Hela, KT cells indicated that three compounds had tumor growth inhibition activities in different degree. Conclusion: We studied the chemical components of X. granatum seeds on the extraction, separation, identification and antitumor activities preliminary screening systematically. Extracting with 95% alcohol at room temperature, adopting various kinds of chromatographic processes such as silica gel column, sephadex LH-20, Pre-TLC, Pre-HPLC, crystallization and recrystallization to isolate 25 compounds.Various of spectroscopic methods were used to elucidate the structures of 21 compounds, in which compound 21 is a new compound, compounds 22-25 are discovered for the first time on the genus. Antitumor activity study upon seven compounds on HLE, Hela, KT cells indicated that three compounds had the tumor growth inhibition activities in different degree, among which compound 9 indicated much strong inhibition activity to the three human tumor cells.