Nuclear factor-B (NF-B) and the signaling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts. NF-B regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and antiapoptotic responses. In our search for NF-B inhibitors from natural resources, we identified cardamomin, 2Ј,4Ј-dihydroxy-6Ј-methoxychalcone, as an inhibitor of NF-B activation from Alpinia conchigera Griff (Zingiberaceae). In present study, we demonstrated the effect of cardamomin on NF-B activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and LPS-induced mortality. This compound significantly inhibited the induced expression of NF-B reporter gene by LPS or tumor necrosis factor (TNF)-␣ in a dose-dependent manner. LPS-induced production of TNF-␣ and NO as well as expression of inducible nitric-oxide synthase and cyclooxygenase-2 was significantly suppressed by the treatment of cardamomin in RAW264.7 cells. Also, cardamomin inhibited not only LPS-induced degradation and phosphorylation of inhibitor B␣ (IB␣) but also activation of inhibitor B (IB) kinases and nuclear translocation of NF-B. Further analyses revealed that cardamomin did not directly inhibit IB kinases, but it significantly suppressed LPS-induced activation of Akt. Moreover, cardamomin suppressed transcriptional activity and phosphorylation of Ser536 of RelA/p65 subunit of NF-B. However, this compound did not inhibit LPS-induced activation of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun NH 2 -terminal kinase, but significantly impaired activation of p38 mitogen-activated protein kinase. We also demonstrated that pretreatment of cardamomin rescued C57BL/6 mice from LPS-induced mortality in conjunction with decreased serum level of TNF-␣. Together, cardamomin could be valuable candidate for the intervention of NF-B-dependent pathological condition such as inflammation.

Four ent-kaurane diterpenoids including two known, ent-7alpha,14beta-dihydroxykaur-16-en-15-one (1) and ent-18-acetoxy-7alpha-hydroxykaur-16-en-5-one (2), and two new, ent-1beta-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (3) and ent-18-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (4), were isolated from the leaves of Croton tonkinensis in a search for inhibitors of NF-kappaB activation and nitric oxide production. These ent-kauranoids inhibited LPS-induced NF-kappaB activation in murine macrophage RAW264.7 cells at IC50 values between 0.07 and 0.42 microM. Consistently, the ent-kauranoids markedly reduced LPS-induced NO production in a comparable concentration-dependent manner.

In recent years, eight tetracyclic ent-kaurane-type diterpenoids were isolated by us from the leaves of Croton tonkinensis GAGNEP. (Euphorbiaceae).1,2) In our continuing search for more bioactive compounds from this plant, six new diterpenoids 2-4, 6, 8, and 14 were isolated together with three known ent-kauranes ent-11a-acetoxy-7b,14a-dihydroxykaur-16-en-15-one (1), ent-kaur-16-en-15-one 18-oic acid (5), and ent-18-hydroxykaur-16-ene (7). The known compounds were identified by comparison of their spectroscopic data ( 1 H-, 13C-NMR) with published values. 3-5) Compounds 2-4 and 6, along with diterpenoids 9-13, which were previously isolated from C. tonkienesis, 1,2) were tested for their cytotoxicity in the brine shrimp lethality assay. This paper deals with the structure elucidation of the new compounds and the results of the brine shrimp lethality assay. Results and DiscussionCompound 2 C-NMR spectroscopic data of 2 with those of the diterpenoid 9, 1) 2 was suggested to belong to the entkaurane series. The acetyl group was assigned at C-7 (d C 73.6) on the basis of the heteronuclear multiple bond correlation (HMBC) cross-peaks ( (Table 1) and 13 C-NMR (Table 2) Six new ent-kaurane-type diterpenoids were isolated from the leaves of the endemic Vietnamese medicinal plant Croton tonkinensis GAGNEP. (Euphorbiaceae) together with three known ent-11a a-acetoxy-7b b,14a a-dihydroxykaur-16-en-15-one (1), ent-kaur-16-en-15-one 18-oic acid (5) and ent-18-hydroxykaur-16-ene (7). Their structures were determined by spectroscopic analyses to be ent-7b b-acetoxy-11a a-hydroxykaur-16-en-15-one (2), ent-18-acetoxy-11a a-hydroxykaur-16-en-15-one (3), ent-11a a-acetoxykaur-16-en-18-oic acid (4), ent-15a a,18-dihydroxykaur-16-ene (6), ent-11a a,18-diacetoxy-7b b-hydroxykaur-16-en-15-one (8), and ent-(16S)-1a a,14a a-diacetoxy-7b b-hydroxy-17-methoxykauran-15-one (14). ent-Kaurane-type diterpenoids from Croton tonkinensis 2-4, 6, and 9-13, were tested for toxicity in the brine shrimp lethality assay. Compounds 9, 10, and 12 demonstrated significant activity, compounds 2, 3, 6, and 11 showed weak activity, and compounds 4 and 13 were inactive.

Croton tonkinensis GAGNEP. (Euphorbiaceae), commonly known as Kho sam cho la in Vietnamese, is a small plant indigenous to Northern Vietnam. In Vietnamese traditional medicine the species is used as remedies for gastric and duodenal ulcers and many other diseases.1) Recently, the antiinflammatory and cancer chemopreventive activity of C. tonkinensis extracts were discovered through its ability to inhibit the activation of the transcription factor nuclear factor kappa B (NF-kB), and the activity is assumed to be correlated with the ent-kaurane diterpenoid constituents.2) The presence of kaurane diterpenoids in the Croton species is very uncommon, although so far the ent-kauranes have been found in many plants of the genus Rabdosia (Labitae).3) Therefore further phytochemical study on the ent-kaurane diterpenoids accumulated in C. tonkinensis is necessary. Other constituents were also isolated from this plant including phytosterols, long-chain alkyl alcohols, and flavonoid glucosides. 4)In the continuation of our study, this paper deals with the isolation and structural elucidation of the four new ent-kauranetype diterpenoids 1-4 (Fig. 1).Compound 1 was isolated as an amorphous powder and its elemental composition was determined to be C 24 H 34 O 6 by the [MϩNa] ϩ peak at m/z 441.6 in the positive-ion electrospray ionization mass spectrometry (ESI-MS) and [MϩNa] ϩ peak at m/z 441.2243 in the positive-ion high-resolution (HR)-FAB-MS. The IR spectrum indicated the presence of a hydroxy (3558 cm (Table 1) and 13 C-NMR (Table 2) signals of 1 were found to be similar to those of ent-1a-acetoxy-7b,14a-dihydroxykaur-16-en-15-one (5), 2) which was isolated from the same extract, except for the presence , and H-7 and H-5 and H-9 in the nuclear Overhauser enhancement and exchange spectroscopy (NOESY) spectrum of 1 (Fig. 2) the acetoxyl groups at C-1 and C-14 were assigned to occupy an ent-a-orientation, and the hydroxyl group at C-7 an ent-b-orientation. Thus the structure of 1 was determined to be ent-1a,14a-diacetoxy-7b-hydroxykaur-16-en-15-one (Table 1) and 13 C-NMR (Table 2) data of 2 with those of 1 revealed very close agreement, showing the presence of a 20-carbon skeleton of an ent-kaurane diterpenoid together with two secondary acetoxyl groups. The main differences -1a a,14a a-diacetoxy-7b b-hydroxykaur-16-en-15-one (1), ent-1a a,7b b-diacetoxy-14a a-hydroxykaur-16-en-15-one (2), ent-18-acetoxy-14a a-hydroxykaur-16-en-15-one (3), and ent-(16S)-18acetoxy-7b b-hydroxykauran-15-one (4) were isolated. Their structures were elucidated by spectroscopic analyses.

Leonurus heterophyllus SW. (family: Lamiaceae, syn. Labiatae) is used in Vietnamese traditional medicine for the treatment of menstruation and child delivery in gynaecology, high blood pressure, blood stasis, heart disorders, and dysentery.1) The rich distribution of bis-spirolabdane-type diterpenoids with variations of functionalities in the plants of the genus Leonurus was reported in the previous studies on chemical composition of L. persicus, 2-5) L. cardiaca, 6,7) and L. sibiricus. 8,9) Similarly, two prefuranic labdane-type diterpenoids were isolated from L. heterophyllus collected in Guangdong Province, China.10,11) However, a systematic chemical investigation on L. heterophyllus is necessary for reasons of chemotaxonomic interest. It is noteworthy that bis-spirocyclic labdane-type compounds are not confined to the Leonurus species, and so far have been found in the genera of Leonotis (Lamiaceae), 12) Marrubium (Lamiaceae), 12)Otostegia (Lamiaceae), 13) and Vitex (Verbenaceae). 14,15) In our present study, systematic extraction and isolation afforded twelve natural bis-spirolabdane-type diterpenoids (1-12), eight of which are new (3, 6-12), together with hispanone (13) and galeopsin (14). The known diterpenoids, leopersin B (1), 15-epileopersin B (2), leopersin C (4), 15-epileopersin C (5), 13 and 14 are identified on the basis of physical ([a] D ), and 1 H-and 13 C-NMR data. 2,3,16,17) Air-dried aerial parts of L. heterophyllus were extracted with MeOH by percolation at room temperature. Concentration under reduced pressure yielded an extract, which was divided into n-hexane-, ethyl acetate-and 1-BuOH-soluble parts by sequential solvent partitioning with H 2 O. Bis-spirocyclic compounds 1-12, and compounds 13 and 14 were obtained by systematic fractionation of n-hexane-soluble fraction first over silica gel, then over reversed-phase octadecyl silica (ODS) gel, followed by preparative HPLC. While compounds 3, 8, 13 and 14 were obtained in pure form, the other compounds were isolated as epimeric pairs 1/2, 4/5, 6/7, 9/10 and 11/12 at C-15 position. This phenomenon of the co-occurrence of C-15 oxygenated epimeric pairs has been frequently seen in many examples of bis-spirolabdanetype diterpenoids of the genus Leonurus. [2][3][4][5][6][7]9) Although the absolute configurations of 1-12 were not individually confirmed herein, they are probably of the same normal labdane-type diterpenoids on the basis of the co-occurrence with 13 and 14, since the formation of bis-spirotetrahydrofuranes involves only C-9 side chain. Additionally, compounds 1/2 and 4/5 were previously isolated from the same extraction fractions together with 4-b-hydroxymethylpregaleopsin, the absolute assignments of p-bromobenzoate derivative of which have been unambiguously determined by single-crystal X-ray crystallographic analysis.2) Therefore, the stereochemistry of 1-12 is suggested to be presented as in the normal series of labdane-type diterpenoids.Leoheteronone A (3) , including eight new, named leoheteronones A-E (3, 6, 8, 9, 11), 15-...

In our continuation of studies on the plants growing in Vietnam, 1,2) we have examined extracts of the rhizomes of Alpinia pinnanensis T. L. WU et SENJEN (Zingiberaceae), which was recently found in Tam Dao, Vinh Phuc Province, northern Vietnam. This plant is usually distributed on mountainous slopes, under forest shade, at 800-1300 m above sea level.3) No previous chemical study on this plant has been reported. In this paper, we describe the isolation and structure determination of three new diarylheptanoids (1-3) (Fig. 1), two known chalcones, 2Ј,4Ј-dihydroxy-6Ј-methoxychalcone 4,5) and 4Ј,6Ј-dimethylchalconaringenin, 6) two known flavanones, alpinetin 7) and naringenin 5-O-methyl ether, 4) and a known diarylheptanoid, (3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene, 8) together with stigmasterol, 9) b-sitosterol, 9) and b-sitosterol 3-O-b-D-glucopyranoside, 9) from the rhizomes of A. pinnanensis growing in Vietnam. The structures of the new diarylheptanoids (1-3) were elucidated on the basis of detailed analyses of 1D-and 2D-NMR spectroscopic data, while the known compounds were determined by comparing their physical ([a] D ) and spectral properties with literature data. 4-9)The oven-dried and powdered rhizomes of A. pinnanensis were extracted with MeOH by percolation. Sequential solvent partition of the crude MeOH extract gave n-hexane-and CH 2 Cl 2 -soluble fractions, which were subjected to repeated open column chromatography over silica gel and repeated preparative reversed-phase (RP)-HPLC to give the abovementioned compounds.Compound 1 (Fig. 2), the hydroxyl group was affixed at C-3 (d C 71.6), while the chalcone part was attached to C-7 (d C 43.2), causing the downfield shift of H-7. This assignment was confirmed by heteronuclear multiple bond correlation (HMBC) (Fig. 2) Three new diarylheptanoids, called alpinnanins A-C (1-3), together with two known chalcones, 2,4-dihydroxy-6-methoxychalcone and 4,6-dimethylchalconaringenin, two known flavanones, alpinetin and naringenin 5 -O-methyl ether, a known diarylheptanoid, (3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene, stigmasterol and b b-sitosterol as a mixture, and b b-sitosterol 3-O-b b-D-glucopyranoside were isolated from the rhizomes of Alpinia pinnanensis T. L. WU et SENJEN (Zingiberaceae). Their structures were elucidated by spectroscopic analyses.

In Vietnamese traditional medicine, the plant Leonurus heterophyllus SW. is known as Ich mau thao, Sung uy, or Choi den. The dried aerial parts of L. heterophyllus (Herba Leonuri) are applicable in the treatment of menstrual pain and child birth, as well as high blood pressure, blood stasis, heart disorders, and dysentery. The dried ripe fruits (Fructus Leonuri) are used to treat edema and as a diuretic.1) Several prefuranic and furanic labdane-type diterpenoids were isolated in previous studies on L. heterophyllus collected in Guangdong province, China.2,3) It is well known that the chemical composition of plants is geographically varied, and the reason for that may be related to the ability of each species to cope with its habitat environment. Therefore we carried out systematic extraction and isolation of the L. heterophyllus species grown in Vietnam, which led to the structure determination of nine labdane-type diterpenoids, including five new compounds, designated leoheteronins A-E (5-9), two phytosterols as a mixture of b-sitosterol and stigmasterol, and the flavone genkwanin (10). The structures of the known diterpenoids hispanone (1), leoheterin (2), hispanolone (3), and galeopsin (4), and 10 were determined by comparing their physical ([a] D ) and spectroscopic data with the literature values.3-6) Compounds 2-4 were among the previous isolates from L. heterophyllus, 1 was isolated from Ballota species (Lamiaceae) 4) but for the first time from a Leonurus species, and 10 is considered to be a chemotaxonomic marker of the genus Leonurus.7) This paper deals with the structure elucidation of the five new labdane-type diterpenoids 5-9. Results and DiscussionThe extraction of the dried aerial parts of L. heterophyllus with MeOH and sequential fractionation using solvents of increasing polarity gave n-hexane-, ethyl acetate-, and 1-BuOHsoluble fractions. Separation of the n-hexane-soluble fraction with silica (Si) gel open-column chromatography, octadecyl Si (ODS) gel open-column chromatography, and repeated preparative ODS HPLC led to the isolation of compounds 1-10 together with a mixture of b-sitosterol and stigmasterol.Leoheteronin A (5) C-16)], six methylene and one methine groups, and two quaternary carbons. Analysis of the data suggested the structure of 5 to be 15,16-epoxylabda-8,13-diene-7,15-dione, which was confirmed on comparison of the NMR data with those of the related compound leopersin G. (Table 1) and 13 C-NMR (Table 2) (Table 1) and 13 C-NMR (Table 2) spectroscopic data showed that 7 was closely related to 5, and the presence of an additional hydroxyl group was deduced from the mass unit of 7. Five new natural labdane-type diterpenoids (5-9), designated leoheteronins A-E, together with four known diterpenoids (1-4), two phytosterols as a mixture of b b-sitosterol and stigmasterol, and the flavone genkwanin (10) were isolated from the aerial parts of Leonurus heterophyllus SW. (Lamiaceae) collected in northern Vietnam. Compound 1 was isolated for the first time from a Leonurus species, an...

Amomum muricarpum ELMER (Zingiberaceae) is a medicinal plant 2-3 m tall which is distributed in dense forests in Guangdong and Guangxi provinces (China) at 300-1000 m above sea level.1) The plant is also distributed in the Philippines and recently was found in Vietnam by a group of botanic taxonomists of the Vietnam Academy of Science and Technology. The lack of phytochemical reports on A. muricarpum prompted us to investigate the species that grows in Vietnam. We succeeded in the isolation of the two new diarylheptanoids 1 and 2 together with the three known compounds 3-5, of which the isolation and structure determination are described in this paper.The extraction of the oven-dried rhizomes of A. muricarpum with MeOH and sequential fractionation using solvents of increasing polarities gave n-hexane-, CH 2 Cl 2 -, EtOAc-, and 1-BuOH-soluble fractions. The isolation of compounds 1-5 ( Fig. 1) . The locations of the carbonyl group at C-3 and methoxyl group at C-5 were unambiguously determined with the 1 H-1 H correlated spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) spectra of 1 (Fig. 2). The absolute stereochemistry of the methoxyl group was determined to be 5R on the basis of the negative sign of the optical rotation and positive Cotton effect associated with the carbonyl n→p* transition at 350 nm (De ϩ0.57) in the circular dichroism (CD) spectrum of 1 in comparison with those of a series of analogous compounds.2) Thus the structure of 1 was determined to be (5R)-5-methoxy-1,7-bis(3,4-dihydroxyphenyl)-3-heptanone, which was given the trivial name muricarpone A.Compound 2 was obtained as a syrup. Its molecular formula was determined to be C 19 H 22 O 5 based on the results of negative-ion HR-FAB-MS. The IR spectrum exhibited a hydroxyl absorption band at 3286 and a ketone band at 1696 cm Ϫ1. The 1 H-, 13 C-NMR (Table 1), and DEPT spectra of 2 disclosed the presence of seven carbons of the heptane chain including the isolated carbonyl group [d C 213.7 (s), 45.3 (t), 43.6 (t), 35.9 (t), 32.1 (t), 30.4 (t), 24.3 (t)] and two identical 3,4-dihydroxyphenyl moieties. The asymmetrical nature of the heptane chain was evident from the appearance of all seven carbons in the 13 C-NMR spectrum. The carbonyl group was located at C-3 by comparing the 13 C chemical shifts of 2 with those of yakuchinone A.3) This assignment was further confirmed in the HMBC experiment (Fig. 3). A and B, together with three known diarylheptanoids, 1,7-di-(3,4-dihydroxyphenyl)-4-hepten-3-one, 1-(3,4-dihydroxyphenyl)-7-(4؆-hydroxyphenyl)-4-hepten-3-one, and 1,7-bis(p-hydroxyphenyl)-4-hepten-3-one, were isolated from the rhizomes of Amomum muricarpum ELMER (Zingiberaceae). Their structures were determined using spectroscopic analyses.