Studies On The Bioactive Constituets Of Pholidota Yunnanesis Rolfe And Pithecellobium Clypearia Benth.

In this dissertation, the bioassay of the inhibitory effects on nitric oxide (NO) production in a murine macrophage-like cell line (RAW 267.4) activated by lipopolysaccharide (LPS) and interferon-γ(IFN-γ) was used to search NO inhibitors from herbs Pholidota yunnanensis Rolfe and Pithecellobium clypearia Benth from which 40, and 8 compounds were isolated respectively. The structures of 48 compounds were elucidated on the basis of chemical evidences and spectral analysis.The structures of 40 compounds isolated from Pholidota yunnanensis are trans-3,3'-dihydroxy-2',4',5-trimethoxystilbene (1), trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene (2), trans-3,3'-dihydroxy-2',5-dimethoxystilbene (3), trans-3-hydroxy-2',3',5-trimethoxystilbene (4), trans-3,3',5-trihydroxystilbene (5), trans-3,3',5-trihydroxy-2'-methoxystilbene (6), trans-2',3,3'-trihydroxy-5-methoxystilbene (7), cis-3,3'-dihydroxy-5-methoxystilbene (8), 3,3'-dihydroxy-5-methoxybibenzyl (batatasin-III) (9), 3,4'-dihydroxy-3',5-dimethoxybibenzyl (gigantol) (10), 3,3',5-trihydroxybibenzyl(11), 3,3',α-trihydroxy-5-methoxybibenzyl (12), n-nonadecanoic acid (13), 1,5-dihydroxy-2,7-dimethoxy-9,10-dihydrophenanthrene (eulophiol) (14), 4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (lusianthridin) (15), 2,4,7-trihydroxy-9,10-dihydrophenan threne (16), 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (coelonin) (17), 2,7-dihydroxy-6-methoxy-9,10-dihydro-5H-phenanthro-[4,5-bcd]pyran (imbricatin) (18), 6-[2'-(3',3"-dihydroxy-5'-methoxybibenzy)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (19), 6-[6'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (20), 1-[2'-(trans-3',3"-dihydroxy-5'-methoxystilbeny)]-4,7-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (21), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene (22), 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,6'-biphenanthrene (23),1 -[(9,10-dihydro-4-hydroxy-2-methoxy-7-phenanthrenyl)oxy]-4,7-dihydroxy-2-methoxy-9,10-dihydrophena nthrene (24), 7-O-[6'-(3",4'-dihydroxy-5'-methoxybibenzy)]-4-hydroxy-2-methoxy-9,10-dihydrophenanthrene (25), 4-hydroxy-3-methoxyphenyl)-3-acetoxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-ol (26), 2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-2,3,9,10-tetrahydro-phenanthro[2,3-b]furan-5-oI (27), 2-(4-hydroxy-3,5-dimethoxy phenyl)-3-hydroxymethyl-8-methoxy-2,3,10,11-tetrahydro-phenanthro[1,2-b]furan-5,6-diol (28), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-hydroxymethyl-1-methoxy-2,3,9,10-tetrhydro- phenanthro[2,3-b]furan-7-ol (29), 4,5-epoxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(3-hydroxy-5- methoxyphenyl)-7-methoxy-2,3,9,10-tetrhydro-phenanthro[2,3-b]furan-8-ol (30), 4'-hydroxy-3',7- dimethoxy-5-(3"-hydroxyphenethyl)flavan-3-ol (31), 4'-hydroxy-3',5',7-trimethoxy-5-(3"- hydroxystyryl)flavan-3-ol (32), (±)-pinoresinol (33), (±)-syringaresinol (34), (±)-lyoniresinol (35), (±)-isolariciresinol (36), (±)-5-methoxy-isolariciresinol (37), 4-(3-hydroxyphenyl)-2-butanone (38), 4-(3-hydroxy-2-methoxyphenyl)-2-butanone (39), (+)-(3R)-Lasiodiplodin (40), respectively. All the compounds include eight stilbenes (1—8), four bibenzyls (9—12), seventeen 9,10-dihydrophenanthrene derivatives (14—30), two flavans (31, 32), five lignans (33—37), three phenolic acid compounds (38—40) and one fatty acid (13). Compounds 1—4, 6—8, 19—21, 23—30 and 32 are new compounds, compounds 38 and 39 were isolated from plants for the first time, compounds 5, 9—16, 22, 31and 33—37 were isolated from Pholidota genus for the first time, and compounds 17 and 18 were isolated from Pholidota yunnanensis for the fast time. The structures and bioactivities of dihydrophenanthrofurans were reported for the first time.The inhibitory effects of all the compounds on NO production were examined. The stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans showed strong inhibitory effects on NO production, and the primary structure-activity relationship was discussed. Furthermore, it was demonstrated that compound 2 exhibited inhibitory effects on NO production through inhibiting NO synthase (iNOS) mRNA expression.MTT method was also used to test the cytotoxic activity of the compounds on NCI-H460, SF-268, MCF-7, and HepG2 cell lines. Partial stilbenes showed selective cytotoxicity on HepG2 cell line, and compound 2, dimeric stilbenoids and dihydrophenanthrofurans exhibited cytotoxicity on all the cell lines. It was further indicated that compound 2 arrested the HepG2 cell cycle in G₂/M phase by up-regulating the protein level of Cyclin B1, and induced apoptosis by down-regulating Bcl2 protein. Compound 2 could cleave the protein PARP which is the target of caspase-3, suggesting the pathway of caspase-3 may induce the apoptosis on HepG2 cells.We also tested the DPPH free radical scavenging activity of all the compounds. Partial stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans were found to have strong antioxidant activity and primary structure-activity relationship.Those findings suggested that the anti-inflammation components are stilbenes, bibenzyls, 9,10-dihydrophenanthrene and dihydrophenanthrofurans, the anti-cancer components are stilbenes, dimeric stilbenoids and dihydrophenanthrofurans, and stilbenes, 9,10-dihydrophenanthrene derivatives and cyclolignans are the potent free radical scavenger. Stilbenes may be used as the selective iNOS inhibitors for the prevention and treatment of inflammatory and cancer.The structures of 8 compounds isolated from Pithecellobium clypearia Benth. are gallic acid (1), ethyl gallate (2), (-)-(2R,3R)-epigallocatechin (3), (-)-(2S)-5,7,3',4',5'-pentahydroxyflavan (4), (-)-(2R,3R)-epigallocatechin-7-gallate (5), (-)-(2S)-5,3',4',5'-tetrahydroxyflavan-7-gallate (6), quercitin-3-O-α-L-rhanmpyranoside (quercitrin) (7), myricitin-3-O-α-L-rhamnpyranoside (myricitrin) (8), respectively, which included two phenolic acid compounds (1, 2), four flavans (3-6), and two flavonol glycosides (7, 8). Compound 6 is a new compound, compound 4 was isolated from plants for the first time, and compounds 2, 5 and 8 were isolated from Pithecellobium genus for the first time.The inhibitory effects and their IC₅₀ values of 8 compounds on NO production in murine macrophage activated by LPS and IFN-γwere estimated, and compound 6 showed strong inhibitory effects and the inhibitory activity of compounds 5 and 8 were weak. All the compounds exhibited DPPH free radical and oxygen free radical scavenging effects. The inhibitory effects of all the compounds on T lymphocyte were assayed using mice splenocytes induced to proliferate by mitogens ConA. The results indicated that compound 5 had strong inhibitory effects on T lymphocyte. In summary, the polyphenolic compounds as the potent NO inhibitors, free radical scavengers and immunosuppressors play the important role in the anti-inflammatory effects of Pithecellobium clypearia. Our investigation on the bioactive constituents from Pithecellobium clypearia Benth. provided a reasonable ground for continuous study of this traditional Chinese medicine.