Isolation, Identificantion And Anticancer Activities Of Bioactive Constituents From Juglans Mandshurica

Juglans mandshurica is one of rare species of trees for pharmacy resources.Its leaves, roots and seeds are widely used as detoxication,antitussive,bacteriostat,and anticancer ingredients according to the Chinese Pharmacopoeia.Recently,some pharmacological actions of Juglans mandshurica were discovered,such as anti-tumor effects,anti-oxidation, anti-bacteria and anti-inflammatory.Up to now,most researches focused on the seeds and roots of Juglans mandshurica.Few research reports are about the chemical constituents or biological activities of Juglans mandshurica leaves.We have undertaken an investigation on the anti-tumor constituents of the Juglans mandshurica leaves.A new quinone compound was isolated from it through bioassay-guided fractionation.We investigated the antitumor,activity of the compound on anti-proliferation and induction of apoptosis in tumor cells.In our search for new active ingredients from the Juglans mandshurica leaves,an ethyl acetate extract was found to possess anticancer activity against tumor cells.Subsequently,the fractionation and purification of the extract combined with cytotoxicity assay led to the isolation of a new quinone compound,p-hydroxymethoxybenzobijuglone(HMBBJ).The structure elucidation of the compound was established based on IR,HREIMS,and ¹H and ¹³C NMR spectroscopy,notably of the 2D NMR spectra.Then the anticancer activity in vitro of HMBBJ had been assayed for the first time.The studies found that HMBBJ obviously inhibited proliferation and induce apoptosis against tumor cells.(1)HMBBJ could inhibit tumor cells proliferation.To study the anti-proliferation of HMBBJ,MTT assay was employed to evaluate its effects on the viability of four cell lines including three human tumor ceils(Hela cervical carcinoma,MCF-7 breast adenocarcinoma, BGC823 gastric carcinoma cell)and one normal cell(3T3-L1 mouse preadipocytes).From the results of MTT assay,HMBBJ showed significant cytotoxic activity against three human tumor cells and less cytotoxic on the normal cell line.The IC₅₀values of HMBBJ were less than 17μM on three tumor cell lines.The inhibitory activity of HMBBJ to the four cell lines approached to Hydroxycampotothecin(HCPT)as positive control.Exposure of three human tumor cells to HMBBJ induced the leakage of lactate dehydrogenase(LDH),which showed that HMBBJ increased the leakage of LDH in a dose-dependent manner.These results indicated that HMBBJ could increase the membrane permeability of the treated cells.(2)HMBBJ could induce rumor cells apoptosis.Through the morphological observation of three human tumor cells treated with HMBBJ,several cellular changes indicative for apoptosis such as cell shrinkage,chromatin condensation,and apoptotic body formation were observed.Data from flow cytometry analysis showed that HMBBJ could induce three human tumor cells apoptosis and their cell cycle was arrested in the G2/M phase.The apoptosis in HMBBJ-induced three tumor cells was associated with loss of mitochondrial membrane potential,activation of caspase-3 and down-regulation of Bcl-2.Furthermore,HMBBJ not only increased the activation of caspases-8 and enhance calium ion concertration on HeLa cells and MCF-7 cells,but also increased the expression of Bax on HeLa cells.The results showed that HMBBJ could efficiently induce apoptosis through mitochondria dependent pathway and/or death receptor-related.(3)The inhibitory activity of HMBBJ against DNA Topo I was evaluated by agarose-gel electrophoresis experiments.The result manifested that HMBBJ exhibited potent inhibitory activities against Topo I at a serial concentrations(28.9-54.7μM).So HMBBJ may be a new inhibitor of DNA Topo I.In summary,the results suggested that HMBBJ may contribute partly to the tumor growth inhibitory activity of Juglans mandshurica leaves and was able to inhibit tumor cells proliferation and induce cells apoptosis through mitochondria dependent pathway,which provide forceful support for the notion that HMBBJ should be further explored as a possible chemotherapeutic drug in terms of its possible effectiveness in treatment of cancer.