Constituents of Indonesian medicinal plant Averrhoa bilimbi and their cytochrome P450 3A4 and 2D6 inhibitory activities.

As constituents of Averrhoa bilimbi leaves we identified three new compounds (1-3) together with 12 known ones (4-15); their inhibitory activities on cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) were examined. Among the isolated compounds, the mixture of 1 and 2, and compounds 4 and 9 showed strong inhibition on CYP3A4, but mild or no inhibition on CYP2D6. These compounds revealed the characteristics of 1) time- and concentration-dependent inhibition, 2) requirement of NADPH for the inhibition, 3) no protection by nucleophiles, and 4) suppression of the inhibition by competitive inhibitor. Thus, they are suggested to be mechanism-based inactivators of CYP3A4 and CYP2D6. The kinetic parameters for the inactivation (k(inact) and K(I)) were 0.19 min(-1) and 36.7 µM for the mixture of 1 and 2, 0.126 min(-1) and 10.5 µM for 4, and 0.29 min(-1) and 23.4 µM for 9.

Two new diphenylmethyl-substituted xanthones from Seicuridaca longepedunculata.

Two new diphenylmethyl-substituted xanthones, named muchimangins K (1) and L (2), have been isolated from the roots of Securidaca longepedunculata (Polygalaceae) collected in the Democratic Republic of Congo. Their structures were established by analyses of the spectral data, including 2D NMR spectra, to be 1 ,3,6,8-tetrahydroxy-2.5-dimethoxy-4-[ -(2,4,5-trimethoxyphenyl)-1-phenylmethyl]xanthone (1) and 1,3,6-trihydroxy-4,7-dimethoxy-2-[1-(2,4,5-trimethoxyphenyl)- 1-phenylmethyl]xanthone (2).

Muchimangins G-J, fully substituted xanthones with a diphenylmethyl substituent, from Securidaca longepedunculata.

Four highly oxygenated xanthones, muchimangins G-J (1-4), have been isolated from the roots of Securidaca longepedunculata collected in Democratic Republic of Congo. Their structures were elucidated by analyses of spectroscopic data to be fully substituted xanthones with a diphenylmethyl substituent at C-2.

New guaian-type sesquiterpene from Wikstroemia indica.

From a MeOH extract of powdered roots of Wikstroemia indica, we isolated a new guaian-type sesquiterpene (1) and two known guaian-type sesquiterpenes [oleodaphnal (2), 1alpha,7alpha,10alphaH-guaia-4,11-dien-3-one (3)], together with twelve known compounds: (+)-arctigenin, (+)-matairesinol, (+)-trachelogenin, (+)-nortrachelogenin, (+)-hinokinin, (+)-kusunokinin, 7-methoxycoumarin, 7-hydroxycoumarin (umbelliferone), daphnogitin, daphnoretin, salicifoliol, and (-)-pinoresinol. The structure of compound 1 was determined to be 4,10,11-guaiatrien-3-one-14-oic acid, by the analyses of spectral data.

Antiausterity activity of arctigenin enantiomers: importance of (2R,3R)-absolute configuration.

From a MeOH extract of powdered roots of Wikstroemia indica, six dibenzyl-gamma-butyrolactone-type lignans with (2S,3S)-absolute configuration [(+)-arctigenin (1), (+)-matairesinol (2), (+)-trachelogenin (3), (+)-nortrachelogenin (4), (+)-hinokinin (5), and (+)-kusunokinin (6)] were isolated, whereas three dibenzyl-gamma-butyrolactone-type lignans with (2R,3R)-absolute configuration [(-)-arctigenin (1*), (-)-matairesinol (2*), (-)-trachelogenin (3*)] were isolated from Trachelospermum asiaticum. The in vitro preferential cytotoxic activity of the nine compounds was evaluated against human pancreatic PANC-1 cancer cells in nutrient-deprived medium (NDM), but none of the six lignans (1-6) with (2S,3S)-absolute configuration showed preferential cytotoxicity. On the other hand, three lignans (1*-3*) with (2R,3R)-absolute configuration exhibited preferential cytotoxicity in a concentration-dependent manner with PC50 values of 0.54, 6.82, and 5.85 microM, respectively. Furthermore, the effect of (-)- and (+)-arctigenin was evaluated against the activation of Akt, which is a key process in the tolerance to nutrition starvation. Interestingly, only (-)-arctigenin (1*) strongly suppressed the activation of Akt. These results indicate that the (2R,3R)-absolute configuration of (-)-enantiomers should be required for the preferential cytotoxicity through the inhibition of Akt activation.

Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

Synthesis of long-chain fatty acid derivatives as a novel anti-Alzheimer's agent.

In order to develop new drugs for Alzheimer's disease, we prepared 17 fatty acid derivatives with different chain lengths and different numbers and positions of double bonds by using Wittig reaction and stereospecific hydrogenation of triple bonds as key reactions. Among them, (4Z,15Z)-octadecadienoic acid (10) and (23Z,34Z)-heptatriacontadienoic acid (16) showed the most potent neurite outgrowth activities on Aß(25-35)-treated rat cortical neurons, which activities were comparable to that of a positive control, NGF. Both fatty acids 10 and 16 possess two (Z)-double bonds at the n-3 and n-14 positions, which might be important for the neurite outgrowth activity.

Heptaoxygenated xanthones as anti-austerity agents from Securidaca longepedunculata.

In a course of our search for anticancer agent based on a novel anti-austerity strategy, we found that the CHCl3 extract of the roots of Securidaca longepedunculata (Polygalaceae), collected at Democratic Republic of Congo, killed PANC-1 human pancreatic cancer cells preferentially in nutrient-deprived medium (NDM). Phytochemical investigation on the CHCl3 extract led to the isolation of 28 compounds including five new polymethoxylated xanthones [1,6,8-trihydroxy-2,3,4,5-tetramethoxyxanthone (1), 1,6-dihydroxy-2,3,4,5,8-pentamethoxyxanthone (2), 8-hydroxy-1,4,5,6-tetramethoxy-2,3-methylenedioxyxanthone (3), 4,6,8-trihydroxy-1,2,3,5-tetramethoxyxanthone (4), 4,8-dihydroxy-1,2,3,5,6-pentamethoxyxanthone (5)] and a new benzyl benzoate [benzyl 3-hydroxy-2-methoxybenzoate (6)]. Among them, 1,6,8-trihydroxy-2,3,4,5-tetramethoxyxanthone (1) and 1,6-dihydroxy-2,3,4,5,8-pentamethoxyxanthone (2) displayed the potent preferential cytotoxicity with PC50 of 22.8 and 17.4 µM, respectively. They triggered apoptosis-like PANC-1 cell death in NDM with a glucose-sensitive mode.

Anti-austeric activity of phenolic constituents of seeds of Arctium lappa.

From seeds of Arctium lappa L. (Asteraceae) we obtained arctigenin (1), arctiin (2), chlorogenic acid (3), 4,5-dicaffeoylquinic acid (4), 3,5-dicaffeoylquinic acid (5), 3,4-dicaffeoylquinic acid (6), matairesinol (11), isolappaol A (12), lappaol F (14), and lappaol B (15), together with 1:1 mixtures of isolappaol C (7) and lappaol C (8), arctignan E (9) and arctignan D (10), and 12 and lappaol A (13), while 3,3',4'-tri-O-demethylarctigenin (16), 3,3'-di-O-demethyl-4'-dehydroxyarctigenin (17), and 3-O-demethylarctigenin (18) were obtained by anaerobic microbiological metabolism of 1. Then, we evaluated the in vitro preferential cytotoxic activity of these pure compounds and 1:1 mixtures, together with enterodiol (19) and enterolactone (20), against human pancreatic cancer PANC-1 cells in nutrient-deprived medium (NDM). Among them, 1 and 18 showed potent activity, with PC50 values of 1.75 and 4.38 microM, respectively, while 11, 15, and 17 showed mild activity with PC50 values of 31.1, 30.9, and 38.7 microM, respectively. By comparing their structures and PC50 values, the following structural moieties could be concluded to be important for the preferential cytotoxicity of 1: 1) the 3-hydroxy-4-methoxyphenyl group at the 2-position on the gamma-butyrolactone ring, 2) the less polar substituent at the 3-position on the gamma-butyrolactone ring, and 3) the gamma-butyrolactone ring.

Chemical constituents of Thai propolis.

Phytochemical investigation on the constituents of Thai propolis led the isolation of a new phenylallylflavanone, (7″S)-8-[1-(4'-hydroxy-3'-methoxyphenyl)prop-2-en-1-yl]-(2S)-pinocembrin (1) and (E)-cinnamyl-(E)-cinnamylidenate (2) from methanolic extract of Thai propolis. Their structures were determined on the basis of extensive NMR spectroscopic analysis. In addition to this, 19 compounds (3-21) belonging to flavonoids and phenolic esters were isolated and identified.

Sesquiterpenes from the rhizomes of Curcuma heyneana.

Four new germacranes [heyneanones A-D (1-4)], three new guaianes [4,10-epizedoarondiol (5), 15-hydroxyprocurcumenol (6), 12-hydroxycurcumenol (7)], and two new spirolactones [curcumanolides C (8) and D (9)] were isolated from the rhizomes of Curcuma heyneana together with 13 known sesquiterpenes and two known labdane-type diterpenes. Among the isolated compounds, heyneanone A (1), heyneanone C (3), 4,10-epizedoarondiol (5), procurcumenol (16), aerugidiol (17), zerumin A (23), and (E)-15,16-bisnorlabda-8(17),11-dien-13-one (24) inhibited protein tyrosine phosphatase 1B (PTP1B) with IC(50) values of 42.5, 35.2, 35.1, 45.6, 35.7, 10.4, and 14.7 µM, respectively.

Synthesis and antitumor evaluation of arctigenin derivatives based on antiausterity strategy.

A series of new (-)-arctigenin derivatives with variably modified O-alkyl groups were synthesized and their preferential cytotoxicity was evaluated against human pancreatic cancer cell line PANC-1 under nutrient-deprived conditions. The results showed that monoethoxy derivative 4i (PC(50), 0.49 µM), diethoxy derivative 4h (PC(50), 0.66 µM), and triethoxy derivative 4m (PC(50), 0.78 µM) showed the preferential cytotoxicities under nutrient-deprived conditions, which were identical to or more potent than (-)-arctigenin (1) (PC(50), 0.80 µM). Among them, we selected the triethoxy derivative 4m and examined its in vivo antitumor activity using a mouse xenograft model. Triethoxy derivative 4m exhibited also in vivo antitumor activity with the potency identical to or slightly more than (-)-arctigenin (1). These results would suggest that a modification of (-)-arctigenin structure could lead to a new drug based on the antiausterity strategy.

Protein tyrosine phosphatase 1B inhibitory activity of Indonesian herbal medicines and constituents of Cinnamomum burmannii and Zingiber aromaticum.

We screened water and methanol extracts of 28 Indonesian medicinal plants for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Nine water extracts, i.e., Alstonia scholaris leaf, Blumea balsamifera, Cinnamomum burmannii, Cymbopogon nardus, Melaleuca leucadendra, Phyllanthus niruri, Piper nigrum, Syzygium aromaticum, and Sy. polyanthum, exhibited ≥70 % inhibition at 25 µg/mL, whereas 11 methanol extracts, i.e., Als. scholaris, Andrographis paniculata, B. balsamifera, Ci. burmannii, Curcuma heyneana, Glycyrrhiza glabra, M. leucadendra, Punica granatum, Rheum palmatum, Sy. polyanthum, and Z. aromaticum, exhibited ≥70 % inhibition at 25 µg/mL. Water extracts of B. balsamifera (IC50, 2.26 µg/mL) and M. leucadendra (IC50, 2.05 µg/mL), and methanol extracts of Ci. burmannii (IC50, 2.47 µg/mL), Pu. granatum (IC50, 2.40 µg/mL), and Sy. polyanthum (IC50, 1.03 µg/mL) exhibited strong inhibitory activity, which was comparable with that of the positive control, RK-682 (IC50, 2.05 µg/mL). The PTP1B inhibitory activity of the constituents of Ci. burmannii and Z. aromaticum was then evaluated. 5'-Hydroxy-5-hydroxymethyl-4″,5″-methylenedioxy-1,2,3,4-dibenzo-1,3,5-cycloheptatriene (2; IC50, 29.7 µM) and trans-cinnamaldehyde (5; IC50, 57.6 µM) were the active constituents of Ci. burmannii, while humulatrien-5-ol-8-one (21; IC50, 27.7 µM), kaempferol-3,4'-di-O-methyl ether (32; IC50, 17.5 µM), and (S)-6-gingerol (33; IC50, 28.1 µM) were those of Z. aromaticum. These results suggest that these medicinal plants may contribute to the treatment and/or prevention of type II diabetes and/or obesity through PTP1B inhibition.

Chemical constituents of Blumea balsamifera of Indonesia and their protein tyrosine phosphatase 1B inhibitory activity.

A methanol extract of the leaves of Blumea balsamifera (L.) DC. (Asteraceae) afforded a new guaian-type sesquiterpene, epiblumeaene K (1), together with four known guaian-type sesquiterpenes (2-5), three known sesquiterpenes (6-8), and nine known flavonoids (9-17) by a combination of chromatography and preparative TLC techniques. Their structures were elucidated by extensive spectroscopic methods and comparison with the literature data. Among the isolated compounds, a known sesquiterpene, beta-caryophyllene 8R,9R-oxide (6), exhibited a significant PTP1B inhibitory activity in a dose-dependent manner, with an IC50 value of 25.8 microM (5.62 microg/mL).

Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC50 values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC50, 5.51 ± 0.04 µM).

Anti-austerity agents from Rhizoma et Radix Notopterygii (Qianghuo).

During a search for potent anticancer agents from natural products based on an anti-austerity strategy, we found that a CHCl3 extract of Rhizoma et Radix Notopterygii (Qianghuo), a Chinese crude drug, exhibited strong cytotoxicity against PANC-1 human pancreatic cancer cells, with a PC50 value of 17.5 µg/mL. Further fractionation and purification of this bioactive extract led to the isolation of 19 known compounds. The in vitro preferential cytotoxicity of the isolates was evaluated against two human pancreatic cancer cell lines, PANC-1 and PSN-1. Among the compounds isolated, ostruthin displayed the most potent activity against both PANC-1 (PC50, 7.2 µM) and PSN-1 (PC50, 7.8 µM) cells in nutrient-deprived medium (NDM) and may have induced necrotic nutrient-deprived PANC-1 cell death.

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara.

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.

Damnacanthal from the Congolese medicinal plant Garcinia huillensis has a potent preferential cytotoxicity against human pancreatic cancer PANC-1 cells.

Screening of eight Congolese medicinal plants showed that the CHCl(3) and MeOH extracts of Aframomum melegueta (PC(50) = 47.8 µg/mL and 13.8 µg/mL, respectively) and CHCl(3) extracts of Garcinia huillensis (PC(50) = 17.8 µg/mL) and Securidaca longepedunculata (PC(50) = 23.4 µg/mL) had preferential cytotoxicity against human pancreatic cancer PANC-1 cells under nutrient-deprived conditions. The active constituents of the CHCl(3) extract of G. huillensis were examined and 12 known anthraquinones were identified. Among them, damnacanthal (1) caused preferential necrotic cell death of PANC-1 and PSN-1 cells under nutrient-deprived and serum-sensitive conditions (PC(50) = 4.46 µm and 3.77 µm, respectively).

Flavanone glycosides from viscum coloratum and their inhibitory effects on osteoclast formation.

Two novel flavanone glycosides, homoeriodictyol 7-O-ß-D-[6-(3-hydroxybutanoyl)glucopyranoside] (viscumneoside IX; 1) and homoeriodictyol 7-O-ß-D-[6-(3-hydroxybutanoyl)glucopyranosyl](1→2)-ß-D-glucopyranoside (viscumneoside X; 2), together with four known flavanoids, 2-homoeriodictyol 7-O-ß-D-glucopyranoside (3), viscumneoside I (4), viscumneoside III (5), and 4',5-dihydroxy-3'-methoxy-7-(2-O-α-L-rhamnopyranosyl-ß-D-glucopyranosyloxy)flavanone (6) were isolated from stems and leaves of Viscum coloratum. Their structures were elucidated on the basis of their NMR spectra, HR-FAB-MS data, and acid hydrolysis. Inhibitory effects of the four compounds 1-4 on the formation of osteoclast-like multinucleated cells were investigated. As a result, all the four flavanoids showed significant inhibitory effects on the formation of osteoclast-like multinuclear cells even at a low concentration of 2 µg/ml. The activities of 1-4 at such a concentration exceeded or approximated to that of elcitonin, the positive control drug at a concentration of 2 U/ml, suggesting that they may be of interest for the development of new anti-osteoporosis drugs.

Pancreatic anticancer activity of a novel geranylgeranylated coumarin derivative.

A series of hydroxycoumarin derivatives has been synthesized and evaluated against human pancreatic PANC-1 cancer cells under nutrient-deprived conditions. Several compounds exhibited 100% preferential cytotoxicity at low micromolar concentrations under nutrition starvation, and showed no cytotoxicity under nutrient-rich conditions. In this study, a novel geranylgeranylated ether coumarin derivative 9 was found to exhibit the highest cytotoxic activity of 6.25 µM within 24h. The preferential anti-tumor activity exhibited by compound 9 against PANC-1 under low oxygen and nutrient environment illustrates its great potential as a promising lead structure for the development of novel agents to combat pancreatic cancer.