Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

Pyrenes and pyrendiones from Uvaria lucida.

A chemical investigation of the chloroform extract of the roots of Uvaria ludida Benth. (Annonaceae), an important African traditional medicine, led to the isolation of six new compounds; three pyrenes, 2-hydroxy-1,8-dimethoxypyrene (1), 8-methoxy-1,2-methylenedioxypyrene (2), and 7-hydroxy-8-methoxy-1,2-methylenedioxypyrene (3), two pyrenediones, 2-hydroxy-1,8-pyrenedione (4) and 2-methoxy-1,8-pyrenedione (5), and a sesquiterpene, (-)-10-oxo-isodauc-3-en-15-oic acid (6), together with eight known compounds (7-14). The structural elucidation by spectroscopic studies of the compounds isolated is described. While pyrenes did not exhibit strong cytotoxicity against human promyelocytic leukemia HL-60 cells, pyrenediones showed strong cytotoxicity. The IC(50) of 4 was 70 ng mL(-1), which was close to that of etoposide (IC(50) = 60 ng mL(-1)).

A tolerance gene for prenylated flavonoid encodes a 26S proteasome regulatory subunit in Sophora flavescens.

Yeast functional screening with a Sophora flavescens cDNA library was performed to identify the genes involved in the tolerant mechanism to the self-producing prenylated flavonoid sophoraflavanone G (SFG). One cDNA, which conferred SFG tolerance, encoded a regulatory particle triple-A ATPase 2 (SfRPT2), a member of the 26S proteasome subunit. The yeast transformant of SfRPT2 showed reduced SFG accumulation in the cells.

Chemical studies on the roots of Uvaria welwitschii.

A chemical investigation of the chloroform extract of the roots of Uvaria welwitschii (Annonaceae), an African traditional medicine taken for stomach ache, led to the isolation of eight new compounds, named welwitschins A-H (1-8), together with five known compounds (9-13). The structural elucidation by spectroscopic studies of the compounds isolated is described. All new compounds were flavonoids having a 2-hydroxy-3,4,6-trimethoxyphenyl moiety in the A-ring, and unsubstituted phenyl in the B-ring. Four of them (1-4) were monomeric flavonoids while the others (5-8) were dimeric flavonoids. The cytotoxicity of the isolated compounds against human promyelocytic leukemia HL-60 cells was investigated.

Biotransformation of phenolic tetrahydroprotoberberines in plant cell cultures followed by LC-NMR, LC-MS, and LC-CD.

A metabolic pathway of 2,3,10,11-oxygenated tetrahydroprotoberberines having the OH group on ring D was demonstrated. Metabolism of (13)C- or D(2)-labeled precursors was studied in cell cultures of Macleaya, Corydalis, and Nandina species. The structures of alkaloid metabolites obtained from feeding experiments were determined by application of combined LC-NMR, LC-MS/MS, and LC-CD techniques. (S)-Tetrahydropseudoprotoberberine (5) was stereospecifically O-methylated to the S-isomer (12) in cell cultures of three plant species. This S-isomer was further N-methylated to the (S)-alpha-N-methyl salt (15), which was oxidized to produce the pseudoprotopine-type alkaloid (10) in cell cultures of Macleaya and Corydalis species. These transformations were the same as those of 2,3,9,10-oxygenated protoberberines. The tetrahydropseudoprotoberberines (5, 6, and 12) were dehydrogenated to pseudoprotoberberines (13, 16, and 14), respectively. Both the R- and S-enantiomers of 5 were dehydrogenated in Macleaya cordata different from the case of 2,3,9,10-oxygenated protoberberines. Precursor 7, with OH groups at C-10 and C-11, was O-methylated at C-10 in M. cordata and C. ochotensis var. raddeana, which was distinct from O-methylation in N. domestica, in which 7 was O-methylated at both C-11 and C-10. Stereoselective O-demethylation [(S)-5 to (S)-18] occurred in N. domestica.

Antinociceptive effects of the extracts of Xylopia parviflora bark and its alkaloidal components in experimental animals.

In the present study, we attempted to elucidate the antinociceptive activity of Xylopia parviflora bark using the acetic acid-induced writhing test, hot plate test, and formalin test in mice. The MeOH extract (100 and 200 mg/kg, administered intraperitoneally (i.p.)) had an antinociceptive effect demonstrated by its inhibitory effects on writhing number induced by acetic acid. Three alkaloidal fractions exhibited significant antinociceptive effects in three animal models; the chloroform-soluble fraction, including secondary and tertiary alkaloids, exhibited the strongest effect. This result supported its use in folk medicine as an analgesic agent. We tested the main alkaloids of these fractions for their antinociceptive effects to clarify the active components. (+)-Corytuberine (6.3 and 12.5 mg/kg, i.p.) showed very strong activity, had a significant antinociceptive effect in the acetic acid-induced writhing test (with 49.4 and 98.9% reduction of writhes), in the hot plate test, and in the formalin test (with 55.4 and 90.6% inhibition during the first phase, and 73.9 and 99.9% during the second phase, respectively). (+)-Glaucine (12.5 and 25 mg/kg, i.p.) showed strong activity in three animal models, too. The activity of these compounds was also observed following oral administration in the acetic acid-induced writhing test.

Hydroxyespintanol and schefflerichalcone: two new compounds from Uvaria scheffleri.

A chemical investigation of the petroleum ether extract and chloroform extract of the root of Uvaria scheffleri Diels (Annonaceae) led to the isolation of two new compounds, named hydroxyespintanol (1) and schefflerichalcone (2), together with eight known compounds (3-10). The structural elucidation of compounds 1 and 2 by spectroscopic studies is described. The cytotoxicity of the isolated compounds against human promyelocytic leukemia HL-60 cells was studied. Among these, 2'-hydroxy-3',4',6'-trimethoxychalcone (5) exhibited cytotoxicity (IC(50) 12 microM), and espintanol (3), which was the main ingredient, also showed some cytotoxicity (IC(50) 44 microM).

A new acyclic diterpene acid and bioactive compounds from Knema glauca.

Investigation of the chemical constituents of the fruits of Knema glauca (Myristicaceae) yielded a new acyclic diterpene acid, named glaucaic acid 4, together with four acylphenols, including 1-(2,6-dihydroxyphenyl) tetradecan-1-one 1, malabaricone A 6, dodecanoylphloroglucinol 7 and 1-(2,4,6-trihydroxyphenyl)-9-phenylnonan-1-one 8, two lignans sesamin 2 and asarinin 3, and a flavan, myristinin D 5. In addition, myristinin A 9 and (+/-)-7,4'-dihydroxy-3'-methoxyflavan 10 were isolated from its leaves and stems, respectively. When tested against small-cell lung cancer (NCI-H187), epidermoid carcinoma (KB) and breast cancer (BC) cell lines, compounds 1, 6-8 and 10 displayed weak to moderate cytotoxicity. The acylphenols 6-8 displayed antituberculosis activity against the microbe Mycobacterium tuberculosis with MIC values of 25, 50 and 100 microg/mL, respectively, and antiviral activity against herpes simplex virus type 1, with 7 as the most active compound (IC(50) = 3.05 microg/mL). Malabaricone A 6 was also active against the malarial parasite Plasmodium falciparum with an IC(50) value of 2.78 microg/mL.

A new arylnaphthalene lignan from Knema furfuracea.

A new arylnaphthalene lignan, named furfuracin, was isolated from the leaves of Knema furfuracea (Myristicaceae), whereas 7 known compounds including (+)-trans-1,2-dihydrodehydroguaiaretic acid, fragransin A(2), biochanin A, gingkolic acid, anarcardic acid, 2-hydroxy-6-(12-phenyldodecyl)-benzoic acid and 2-hydroxy-6-(12-phenyldodecen-8'Z-yl)-benzoic acid were obtained from its stems. The structure of the new lignan was established by analysis of spectroscopic data (UV, IR, MS and NMR).

Enantiomeric separation of racemic 1-benzyl-N-methyltetrahydroisoquinolines on chiral columns and chiral purity determinations of the O-methylated metabolites in plant cell cultures by HPLC-CD on-line coupling in combination with HPLC-MS.

Effective enantiomeric separations of 1-benzyl-N-methyltetrahydroisoquinolines were achieved using commercially available Chiralcel OD-H and OJ-H columns. Online LC-CD analysis allowed for the establishment of a correlation between the absolute configuration of the separated enantiomers and their characteristic CD transitions. LC-MS combined with LC-CD analysis permitted chiral purity determinations of O-methylated metabolites of nine phenolic 1-benzyl-N-methyltetrahydroisoquinolines in cell cultures of Corydalis, Macleaya, and Nandina species.

Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC-MS/MS, LC-NMR, and LC-CD analyses.

The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were identified from N. domestica. O-Methylpallidine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were also characterized for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were identified in the callus of N. domestica.

Biotransformation of phenolic 1-benzyl-N-methyltetrahydroisoquinolines in plant cell cultures followed by LC/NMR, LC/MS, and LC/CD.

(+/-)-1-Benzyl- N-methyltetrahydroisoquinolines 7-10 and 11-14 with one and two hydroxy groups on the aromatic rings, respectively, were fed individually to cultured cells of Corydalis and Macleaya species, respectively. The structures of the metabolites were determined by using combinatorial techniques, including LC/NMR, LC/MS-MS, and LC/CD. The enantiomeric excesses of the metabolites were derived from LC/CD and LC/MS-MS analyses. In cell cultures of Corydalis and Macleaya species, laudanine (7), with a hydroxy group at C-3', can form the berberine bridge at C-2' and C-6' to produce S- and R-enantiomers of 2,3,9,10- and 2,3,10,11-oxygenated protoberberines (20 and 21), respectively, whereas reticuline (11) and protosinomenine (12), incoporating a hydroxy group at C-3', form the berberine bridge at C-2' to furnish the S-enantiomer of 2,3,9,10-oxygenated protoberberines (23 and 21), respectively.

Secondary and tertiary isoquinoline alkaloids from Xylopia parviflora.

From the secondary and tertiary alkaloidal fractions of the root and the bark of Xylopia parviflora (Annonaceae), the isoquinoline alkaloids, 10,11-dihydroxy-1,2-dimethoxynoraporphine and parvinine were isolated, along with 39 known alkaloids. Their structures were determined on the basis of analysis of spectroscopic data.

Cytotoxic C-benzylated chalcone and other constituents of Ellipeiopsis cherrevensis.

A new natural C-benzylated chalcone, 2',4'-dihydroxy-3'-(2-hydroxybenzyl)-6'-methoxychalcone (2), along with two other flavonoids, tiliroside and kaempferol 3-O-rutinoside, and an oxoaporphine alkaloid, lanuginosine were isolated from the aerial parts of Ellipeiopsis cherrevensis (Annonaceae). Two known polyoxygenated cyclohexene derivatives, ferrudiol and zeylenol, and a new analog, ellipeiopsol D, were also isolated. The chalcone 2 exhibited cytotoxic activity against human small-cell lung-cancer (NCI-H187), epidermoid carcinoma (KB) and breast cancer (BC) cell lines with IC50 values of 1.40, 5.31 and 13.92 microg/mL, respectively. This compound also showed antimalarial activity against Plasmodium falciparum with an IC50 value of 7.1 microg/mL as well as antimicrobacterial activity against Mycobacterium tuberculosis with a MIC of 25 mg/mL.

Potential cancer chemopreventive activity of simple isoquinolines, 1-benzylisoquinolines, and protoberberines.

Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of beta-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 x 10(3) mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than alpha-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC50 4.5-5.8 microM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.

Structural analyses of metabolites of phenolic 1-benzyltetrahydroisoquinolines in plant cell cultures by LC/NMR, LC/MS, and LC/CD.

The metabolism of the phenolic 1-benzyltetrahydroisoquinoline alkaloids was studied in cell cultures of Macleaya and Corydalis species. The crude alkaloid fraction obtained from feeding experiments was investigated by application of the combined LC/NMR and LC/APCI-MS (/MS) techniques. Several metabolites were detected, and their structures (6 and 8-14) were identified. Bioconversion of the phenolic 1-benzyltetrahydroisoquinoline (2) into the pseudoprotoberberine (8) was demonstrated for the first time. LC/APCI-MS and LC/CD experiments carried out on a chiral column permitted the deduction of the major enantiomeric form of the chiral metabolites. Thus, the combination of NMR, MS, and CD data permitted the structural elucidation and stereochemical analysis of the metabolites in the extract matrix solution, without isolation and sample purification prior to the coupling experiments.

Induction of apoptosis in human promyelocytic leukemia cell line HL-60 by C-benzylated dihydrochalcones, uvaretin, isouvaretin and diuvaretin.

Uvaretin, isouvaretin and diuvaretin, cytotoxic C-benzylated dihydrochalcones isolated from Uvaria acuminata, displayed growth inhibitory effects against human promyelocytic leukemia HL-60 cells. We examined the mechanism of the cytotoxicity. From the morphological study by staining with Hoechst 33258, the cells treated with C-benzylated dihydrochalcones exhibited significant chromosomal condensation and nuclear degradation. The cell cycle analysis showed the accumulation of cells in the G1 phase and the appearance of a sub-G1 peak. These results suggest apoptotic cell death. Further, from the detection of DNA fragmentation and the activation of caspase-3, the biological hallmarks of apoptosis, these C-benzylated dihydrochalcones appeared to induce apoptosis against HL-60 cells. The cytotoxicity of uvaretin and diuvaretin was stronger than that of isouvaretin, which suggest that the 5'-substitution of the 2-hydroxybenzyl group increased the cytotoxicity.

Effects of ellagic acid and 2-(2,3,6-trihydroxy-4-carboxyphenyl)ellagic acid on sorbitol accumulation in vitro and in vivo.

Caesalpinia ferrea MART. (Leguminosae) called as Juca is one of the medicinal plants in Brazil used for diabetes. From the fruits of this plant, ellagic acid (EA) and 2-(2,3,6-trihydroxy-4-carboxyphenyl)ellagic acid (TEA) have been recently isolated as aldose reductase (AR) inhibitors. In this study, we examined to prove the inhibitory activity against AR of EA and TEA in vitro, and EA in vivo by measurement of the accumulation of sorbitol, which is the product of glucose reduction catalyzed by AR. TEA was not examined in vivo because of its shortage of yield from the fruits. EA and TEA significantly and dose-dependently inhibited sorbitol accumulation in erythrocytes, lens and sciatic nerve under incubating with glucose in vitro. EA at a dose of 75 mg/kg/d showed the most potent inhibition of sorbitol accumulation in erythrocytes, lens and sciatic nerve at 50, 75 and 100 mg/kg/d in vivo. These results suggest that the inhibitory activity of EA against AR causes to inhibit sorbitol accumulation by in vitro and in vivo experiments. EA is distributed in fruits and vegetables, so that taking them might be able to relieve diabetic complications.

Quaternary isoquinoline alkaloids from Xylopia parviflora.

From the quaternary alkaloidal fraction of the bark and the root of Xylopia parviflora (Annonaceae), four isoquinoline alkaloids, xylopinidine, dehydrocoreximine, N, N-dimethylanomurine and N-methylphoebine were isolated along with the known compounds, pycnarrhine, lotusine, 6,7-dimethoxy-2-methyl-isoquinolinium salt, 1,2-dehydroreticuline, (-)-phellodendrine, (+)-tembetarine, (-)-litcubine, (+)-magnoflorine, tetradehydroreticuline, (-)-oblongine, (+)-menisperine, (+)-N-methylcorydine, stepharanine, (+)-xanthoplanine, dehydrodiscretine, jatrorrhizine and palmatine. 3,4-Dihydro-6,7-dimethoxy-2-methyl-isoquinolinium and N-methylpurpuerine were isolated as natural products for the first time. Their structures were determined on the basis of spectroscopic evidence.

G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Viticis Fructus (Vitex rotundifolia Linne fil.).

Flavonoids are distributed in many plants. We studied the antitumor effects of flavonoids isolated from Viticis Fructus, casticin, artemetin, quercetagetin and 5,3'-dihydroxy-6,7,4' -trimethoxyflavanone. Casticin inhibited the growth of KB cells markedly (IC(50)=0.23 microM), compared with the other flavonoids tested (IC(50)=15.3-18.6 microM). In contrast, casticin did not inhibit the proliferation of A431 cells similar to normal cell lines, 3T3 Swiss Albino and TIG-103. Flow cytometric analyses revealed that the exposure of KB cells to casticin led to significant arrest at G2-M. In immunostaining of KB cells, casticin disrupted mitotic spindles. These results suggest that G2-M arrest by casticin may be relevant to its antimitotic activity, although the mechanism of selective growth inhibition has been unknown. Further examinations are required to confirm that casticin is an antitumor drug for specific cancers with low toxicity.