Chemical Constituents From Ardisia Crenata And Cajanus Cajan:Isolation, Identification, And Anticancer Effects

Malignant tumor or cancer is one of the major diseases that damage severely human health, and is the second leading cause of death in the world. Chemical medication is the major treatment method for cancer at present. Chemotherapeutic agents mainly contain synthetic and natural medicines. Many of synthetic drugs are of natural origin or derived from the structures of natural products. China is rich in medicinal plant resources, so it is a decided advantage on development of safe and effective anticancer drugs or lead compounds from medicinal herbs. The aim of this dissertation is to search natural anticancer leading compounds. It consists of two parts.Part One Ardisiacrispin A from Ardisia crenata:Isolation, Identification, Lot Manufacture, and Anticancer EffectBackground and ObjectiveAccording to our previous experiments and literatures, ardisiacrispin A, B from Ardisia crenata Sims., as well as their derivatives, exhibited significant anticancer activity They are worthy of our further and systemaic researches, including phytochemical study, in vitro and in vivo pharmacological studies for searching of anticancer leading compounds Method(1) Ardisiacrispin A and relavent constituents were isolated from the n-BuOH extract of Ardisia crenata by silica gel, MCI and ODS column chromatographies. The structures were elucidated on the basis of spectroscopic analysis, mainly on MS and NMR.(2) Effects of ardisiacrispin A on cancer cell lines were examined by MTT assay. The apoptosis was observed by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests.(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was also carried out.Result(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines and cell line ED-25, human liver vein endothelial cells, by MTT assay. The IC50values at48h were7.61μM,8.29μM,8.64μM,5.30μM,4.05μM and8.11μM, respectively. It was no significant difference between the IC50of cancer cells and that of ED-25(P>0.05). It indicated that the cytotoxicity of ardisiacrispin A was non-selective. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Exposed to8μM ardisiacrispin A for48h, partial HCT-15cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase. (3) Acute toxicity test showed ardisiacrispin A is a low-toxic compound. It's median lethal dose (LD50) was1.44g/kg. After4weeks of oral administration, the livers of the high-dose group rats were showed mild acidophilic change, cell shrinkage and loose connection. It suggested that ardisiacrispin A maight effect on liver.(4) The in vivo antitumor activity of ardisiacrispin A per os showed that relative tumor proliferation rate AT/AC of the human colon adenocarcinoma xenograft tumors in nude mice was more than42%. It was considered as no significant anticancer activity by the Division of Cancer Treatment, NCI, NIH.Conclusion(1) Three compounds, ardisiacrispin A (1), ardisicrenoiside B (2) and bergenin (3), were isolated from the n-BuOH extract of Ardisia crenata and their structures were identified by means of spectroscopic analysis. Forty-five grams of purified ardisiacrispin A was manufactured by means of repeated chromatography for in vivo studies.(2) Ardisiacrispin A dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines. The result of ardisiacrispin A against HCT-15cell lines in vitro was reported for the first time. Flow cytometric analysis displayed that ardisiacrispin A induced apoptosis of HCT-15cells and arrest of the cell cycle at S-phase.(3) Preliminary toxicology of ardisiacrispin A, when administered orally was carried out by acute and subacute toxicity tests. Acute toxicity test showed ardisiacrispin A is a low-toxic compound.(4) In vivo anticancer effect on human colon adenocarcinoma xenograft tumor in nude mice of ardisiacrispin A was carried out, and no significant anticancer activity for oral administration was observed. Part Two Studies on chemical constituents from the leaves of Cajanus cajan, and their anticancer effectBackground and ObjectiveAccording to our previous experiments and literatures, the stilbene-structural analogues of longistylin A, C from Cajanus cajan (L.) Millsp. exhibited significant anticancer effect. Meanwhile researches on stilbenes from Cajanus cajan were still insufficient. Therefore in order to discover new anticancer leading compounds with stibene strcture we carried out a systematic studies on isolation and structural identification of stilbenes from the leaves of Cajanus cajan, as well as their anticancer effect.Method(1) Stilbenes were isolated from the CHCl3extract of the leaves of Cajanus cajan by silica gel and Sephadex LH-20column chromatography. Their structures were elucidated on the basis of IR, MS and NMR spectrum, as well as elementary analysis.(2) Effects of the isolated and identified stilbenes on cell proliferation were examined in vitro by MTT assay. The apoptosis effect on cell line was evaluated by Wright-Giemsa's staining and Annexin V-FITC/PI dual staining. Cell cycle was also determined by FCM.Result(1) Seven compounds were isolated and identified as longistylin A (1), longistylin C (2), cajanstilbene H (3), cajanolactone A (4), pinostrobin (5), naringenin-4',7-dimethyl ether (6) and β-sitosterol (7). Among them cajanstilbene H (3) and cajanolactone A (4) were new compounds. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.(2) Stilbenes1-4exhibited significant anticancer effects in vitro. New compound cajanstilbene H dose-dependently reduced cell viability in NCI-H460, PC-3, MCF-7, Hela, HCT-15, KB-V1cell lines in a MTT assay test. The IC50values at48h were21.47μM,25.83μM,21.42μM,25.85μM,24.81μM, and22.29μM, respectively. After48h of exposure to42μM cajanstilbene H, partial NCI-H460cells presented characteristic morphological changes of apoptosis, including cell shrinkage, nuclear condensation. It suggested that apoptosis was possibly responsible for the cell death. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of NCI-H460cells, and arrest of the cell cycle at G0/G1phase.Conclusion(1) Two new stilbenes, cajanstilbene H and cajanolactone A, were found from the leaves of Cajanus cajan. The former was the first halogen-containing derivative of stilbene obtained from the genus Cajanus, and the latter was the first lactone-type of stilbene carboxylic acid isolated from genus Cajanus.(2) Cajanstilbene H showed significant in vitro anticancer effect on NCI-H460cells. Flow cytometric analysis displayed that cajanstilbene H induced apoptosis of the cells and arrest of cell cycle at G0/G1phase.