Effects of Pueraria candollei var mirifica (Airy Shaw and Suvat.) Niyomdham on Ovariectomy-Induced Cognitive Impairment and Oxidative Stress in the Mouse Brain.

The effects of the phytoestrogen-enriched plant Pueraria mirifica (PM) extract on ovari-ectomy (OVX)-induced cognitive impairment and hippocampal oxidative stress in mice were investigated. Daily treatment with PM and 17ß-estradiol (E2) significantly elevated cognitive behavior as evaluated by using the Y maze test, the novel object recognition test (NORT), and the Morris water maze test (MWM), attenuated atrophic changes in the uterus and decreased serum 17ß-estradiol levels. The treatments significantly ameliorated ovariectomy-induced oxidative stress in the hippocampus and serum by a decrease in malondialdehyde (MDA), an enhancement of superoxide dismutase, and catalase activity, including significantly down-regulated expression of IL-1ß, IL-6 and TNF-α proinflammatory cytokines, while up-regulating expression of PI3K. The present results suggest that PM extract suppresses oxidative brain damage and dysfunctions in the hippocampal antioxidant system, including the neuroinflammatory system in OVX animals, thereby preventing OVX-induced cognitive impairment. The present results indicate that PM exerts beneficial effects on cognitive deficits for which menopause/ovariectomy have been implicated as risk factors.

α-Keto tetrahydrofuran lignan glucosides from the Bangladeshi medicinal plant Terminalia citrina inhibit estradiol (E2) induced proliferation in cancer cells.

EtOAc extract from the leaves of Terminalia citrina collected in Bangladesh were separated, and seven previously undescribed α-keto tetrahydrofuran lignan glucosides (terminalosides Q to W) were isolated and characterized. NOESY analysis of 1H NMR spectra and ECD spectroscopic data analysis revealed the absolute stereochemistry of the tetrahydrofuran ring of the isolated constituents as being a (7S,8R,8'S)- configuration in terminalosides Q to U and a (7R,8R,8'S)- configuration in terminalosides V and W. All of the isolated compounds were evaluated for their estrogenic and anti-estrogenic properties using two types of estrogen-responsive human breast cancer cell lines (MCF-7 and T47D). Terminaloside R, which has a dioxymethylene group in its aromatic ring, inhibited 90% of estradiol-enhanced cell proliferation in T47D and MCF-7 cells at concentrations of 0.01 µM and 0.1 µM, respectively. On the other hand, terminaloside T, the analogous compound which has two oxymethyl groups in place of dioxymethylene, suppressed 90% of cell proliferation selectively in T47D cells at a concentration of 0.01 µM. However, terminaloside W, the 7R-stereoisomer of terminaloside R, only showed moderate activity.

Naphthalene glycosides in the Thai medicinal plant Diospyros mollis.

This study evaluated methanol extracts from the leaves and branches of the Thai medicinal plant Diospyros mollis (Ebenaceae). Seven triterpenes and 22 aromatic compounds, including five new compounds, were isolated, and their structures were determined. The new compounds had the following structures: diospyrol glycoside (makluoside A, 1), 8,8'-di-O-6-ß-D-apiofuranosyl-ß-D-glucopyranosyl-6,6'-dimethyl-2,3'-binaphthalene-1-ol-1',4'- dione (makluoside B, 2), and 3-methyl-1,8- naphthalenediol glycosides (makluosides C-E, 3-5). Makluoside B is the first example of a naphthoquinone glycoside that has both a 3-methyl-1,8-naphthalenediol unit and a 5-hydroxy-7-methyl-1,4-naphthoquinone unit. The hyaluronidase inhibitory activity of the isolates was evaluated, revealing that one of the triterpene derivatives possessed moderate inhibitory activity.

2-Alkylquinolone alkaloid biosynthesis in the medicinal plant Evodia rutaecarpa involves collaboration of two novel type III polyketide synthases.

2-Alkylquinolone (2AQ) alkaloids are pharmaceutically and biologically important natural products produced by both bacteria and plants, with a wide range of biological effects, including antibacterial, cytotoxic, anticholinesterase, and quorum-sensing signaling activities. These diverse activities and 2AQ occurrence in vastly different phyla have raised much interest in the biosynthesis pathways leading to their production. Previous studies in plants have suggested that type III polyketide synthases (PKSs) might be involved in 2AQ biosynthesis, but this hypothesis is untested. To this end, we cloned two novel type III PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone synthase (AQS), from the 2AQ-producing medicinal plant, Evodia rutaecarpa (Rutaceae). Functional analyses revealed that collaboration of ADS and AQS produces 2AQ via condensations of N-methylanthraniloyl-CoA, a fatty acyl-CoA, with malonyl-CoA. We show that ADS efficiently catalyzes the decarboxylative condensation of malonyl-CoA with a fatty acyl-CoA to produce an alkyldiketide-CoA, whereas AQS specifically catalyzes the decarboxylative condensation of an alkyldiketide acid with N-methylanthraniloyl-CoA to generate the 2AQ scaffold via C-C/C-N bond formations. Remarkably, the ADS and AQS crystal structures at 1.80 and 2.20 Å resolutions, respectively, indicated that the unique active-site architecture with Trp-332 and Cys-191 and the novel CoA-binding tunnel with Tyr-215 principally control the substrate and product specificities of ADS and AQS, respectively. These results provide additional insights into the catalytic versatility of the type III PKSs and their functional and evolutionary implications for 2AQ biosynthesis in plants and bacteria.

Isolation of a new compound, 2-butanone 4-glucopyranoside 6'-O-gallate and other 8 compounds from the anti-inflammatory leave extracts of Memecylon edule Roxb.

This present study was designed to isolate the compounds of Memecylon edule. The chemical compounds were purified by chromatographic methods and their structures were established on the basis of spectroscopic analyses (UV, MS and NMR). The major isolated compounds were tested for anti-inflammatory activity. The methanolic extracts of M. edule leaves gave a new compound 2-butanone 4-glucopyranoside 6'-O-gallate (1) with eight known compounds, namely, 3,3'-di-O-methylellagic acid 4-O-ß-d-glucopyranoside (2), epigallocatechin-3-O-gallate (EGCG) (3), (2R, 3R)-dihydromyricetin-4'-ß-d-glucopyranoside (4), myricetin-3-O-α-l-rhamnopyranoside (5), benzyl-(6-O-α-L arabinofuranosyl) O-ß-d-glucopyranoside (6), benzyl-(6-O-α-l-rhanmopyranosyl) O-ß-d-glucopyranoside (7), 2-phenylethyl-(6-O-ß-d-apiofuranosyl)-O-ß-d-glucopyranoside (8) and methyl benzoate 2-(6-O-α-l-rhamnosyl)-O-ß-d-glucopyranoside (9). All compounds were isolated for the first time from this plant. The major compounds (2, 3 and 5) exhibited significant anti-inflammatory activity. In conclusion, M. edule was recognised to be a good source for phenolic compounds and these compounds may contribute to anti-inflammatory activity of the extract.

Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation.

Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L-P (1-5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity.

Furofuran Lignan Glucosides with Estrogen-Inhibitory Properties from the Bangladeshi Medicinal Plant Terminalia citrina.

Extracts from the leaves of the Bangladeshi medicinal plant Terminalia citrina were prepared, and 13 new furofuran lignan glucosides, terminalosides A-K (1-4, 6-12), 2-epiterminaloside D (5), and 6-epiterminaloside K (13), were characterized using various spectroscopic techniques. Twelve of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. Analysis of the NMR chemical shifts for the oxymethine signals in the furofuran ring suggested a pragmatic approach to determining the relative configuration of these compounds. The ECD and NOESY spectroscopic data obtained allowed for the deduction of the absolute configurations and conformations of the compounds. The isolates were tested for their estrogenic/antiestrogenic activity using the MCF-7 and T47D estrogen-responsive human breast cancer cell lines. Terminalosides B (2) and G (8) exhibited inhibitory effects for both cell lines, and estradiol-enhanced cell proliferation was suppressed by 90% at concentrations lower than 10 µM. Terminaloside E (6) showed inhibitory activity against the T47D cell line, whereas terminalosides C (3), F (7), and I (10) and 6-epiterminaloside K (13) displayed antiestrogenic activity against MCF-7 cells.

Phenolic constituents of the Bangladeshi medicinal plant Pothos scandens and their anti-estrogenic, hyaluronidase inhibition, and histamine release inhibitory activities.

Extracts from the stem and roots of the Bangladeshi medicinal plant Pothos scandens L. (Araceae) were isolated, and three hemiterpene glucoside aromatic esters, pothobanosides A (1), B (2), and C (3), and a phenylisobutanoid, pothobanol (4), along with 14 known compounds, were characterized. The isolates were tested for their estrogenic/anti-estrogenic activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D, and syringoyl derivatives (2, 3, and canthoside B) showed strong inhibitory activity against both cell lines. Their less oxygenated analogs (1, and markhamioside F) were almost inactive. The isolates were also evaluated for hyaluronidase and histamine release inhibitory activities, and pothobanoside A (1) showed significant hyaluronidase inhibitory activity among the isolated compounds, which was similar to that of the positive control rosmarinic acid. Because hyaluronidase produces an angiogenic response that has been implicated in tumor invasiveness and metastasis, 1 could be valuable as an anti-tumor compound with a different mechanism of action from related compounds (2, 3). Pothobanoside C (3) and pothobanol (4) were also found to inhibit histamine release to a similar degree to the positive control epigallocatechin 3-O-(3"-O-methyl)-gallate. The histamine release inhibitory potency of these isolates may support the traditional uses of this plant in folk medicine.

Effect of miroestrol on ovariectomy-induced cognitive impairment and lipid peroxidation in mouse brain.

Miroestrol (MR) is a phytoestrogen isolated from Pueraria candollei var. mirifica (KwaoKrueaKhao), a Thai medicinal plant used for rejuvenation. We examined the effects of MR on cognitive function, oxidative brain damage, and the expression of genes encoding brain-derived neurotrophic factor (BDNF) and cyclic AMP-responsive element-binding protein (CREB), factors implicated in neurogenesis and synaptic plasticity, in ovariectomized (OVX) mice. OVX decreased serum 17ß-estradiol level and uterine weight. OVX also impaired object recognition performance in the novel object recognition test and spatial cognitive performance in the Y-maze test and the water maze test. Daily treatment of MR dose-dependently attenuated OVX-induced cognitive dysfunction. Moreover, OVX mice had a significantly increased level of thiobarbituric acid-reactive substances, and down-regulated expression levels of BDNF and CREB mRNAs in the hippocampus and frontal cortex. MR treatment as well as hormone replacement therapy with 17ß-estradiol significantly reversed these neurochemical alterations caused by OVX. These results suggest that MR ameliorates cognitive deficits in OVX animals via attenuation of OVX-induced oxidative stress and down-regulation of BDNF and CREB mRNA transcription in the brain. Our findings raise the possibility that MR and Pueraria candollei var. mirifica, the plant of origin of MR, may have a beneficial effect on cognitive deficits like AD in which menopause/ovariectomy are implicated as risk factors.

Structure and antioxidant activity relationships of isoflavonoids from Dalbergia parviflora.

The antioxidant activities of 24 isoflavonoids that were previously isolated as pure compounds from Dalbergia parviflora were evaluated using three different in vitro antioxidant-based assay systems: xanthine/xanthine oxidase (X/XO), ORAC, and DPPH. The isolates consisted of three subgroups, namely isoflavones, isoflavanones, and isoflavans, each of which appeared to have diversified substituents, and were thus ideal for the study of their structure-activity relationships (SARs). The SAR analysis was performed using the results obtained from both the inter-subgroup isoflavonoids with the same substitution pattern and the intra-subgroup compounds with different substitution patterns. The inter-subgroup comparison showed that the isoflavones exhibited the highest antioxidant activities based on all three assays. The intra-subgroup analysis showed that the additional presence of an OH group in Ring B at either R3' or R5' from the basic common structure of the R7-OH of Ring A and the R4'-OH (or -OMe) of Ring B greatly increased the antioxidant activities of all of the isoflavonoid subgroups and that other positions of OH and OMe substitutions exerted different effects on the activities depending on the subgroup and assay type. Therefore, based on the structural diversity of the isoflavonoids in D. parviflora, the present study provides the first clarification of the detailed antioxidant SARs of isoflavonoids.

Cytotoxic and immunomodulatory effects of polyhydroxyoctane isolated from Lentinus polychrous mycelia.

A polyhydroxyoctane, 6-methylheptane-1,2,3,4,5-pentaol (MHP), was first isolated from mycelia of the Thai edible mushroom Lentinus polychrous. MHP was evaluated for its cytotoxic and immunomodulatory effects in vitro. MHP was slightly cytotoxic to murine splenocytes but not to RAW264.7 cells or peripheral blood mononuclear cells. MHP decreased nitric oxide and intracellular O2 (-) production from lipopolysaccharide- and phorbol-12-myristate-13-acetate-activated RAW264.7 cells at levels of 78.98 ± 4.72 and 78.48 ± 2.41 % of controls, respectively. The mRNA expression of pro-inflammatory mediators, including iNOS, TNF-α, IL-1ß, IL-6, COX-1 and COX-2, were significantly suppressed by MHP. In addition, MHP significantly increased the proliferation of phytohemagglutinin and pokeweed mitogen-induced splenocytes. These results indicate that MHP is able to modulate inflammatory responses and the proliferation of both T- and B-lymphocyte cells, suggesting that MHP may be a good natural immunomodulator.

Induced production of mycotoxins in an endophytic fungus from the medicinal plant Datura stramonium L.

Epigenetic modifiers, including DNA methyltransferase (DNMT) or histone deacetylase (HDAC) inhibitors, are useful to induce the expression of otherwise dormant biosynthetic genes under standard laboratory conditions. We isolated several endophytic fungi from the medicinal plant Datura stramonium L., which produces pharmaceutically important tropane alkaloids, including scopolamine and hyoscyamine. Although none of the endophytic fungi produced the tropane alkaloids, supplementation of a DNMT inhibitor, 5-azacytidine, and/or a HDAC inhibitor, suberoylanilide hydroxamic acid, to the culture medium induced the production of mycotoxins, including alternariol, alternariol-5-O-methyl ether, 3'-hydroxyalternariol-5-O-methyl ether, altenusin, tenuazonic acid, and altertoxin II, by the endophytic fungus Alternaria sp. This is the first report of a mycotoxin-producing endophytic fungus from the medicinal plant D. stramonium L. This work demonstrates that treatments with epigenetic modifiers induce the production of mycotoxins, thus providing a useful tool to explore the biosynthetic potential of the microorganisms.

Flavonoid and stilbenoid production in callus cultures of Artocarpus lakoocha.

Callus cultures of Artocarpus lakoocha Roxb., established from seedling explants and maintained on woody plant medium containing 1mg/l 2,4-dichlorophenoxyacetic acid and 1mg/l benzyladenine, were studied for their chemical constituents and biosynthetic potential of secondary metabolites. Four prenylflavones and prenylated stilbenes, along with nine known polyphenolic compounds, were isolated and elucidated for their structures through extensive analysis of their NMR and MS data. Among the 13 isolates, it appeared that seven of them are prenylated derivatives of 5,7,2',4'-tetrahydroxyflavones, and four are prenylated derivatives of 2,4,3',5'-tetrahydroxystilbene (oxyresveratrol), suggesting that the biosynthetic pathways of these two polyphenolic groups and their prenylating enzymes are highly expressed in A. lakoocha callus cultures. A study on the growth-product relationship of the callus cultures showed that the secondary metabolites were all formed simultaneously during the rapid growth phase of the culture cycle, with various prenylflavones, and a prenylated stilbene as major constituents. In assays for DPPH free radical scavenging activity and tyrosinase inhibitory potential, the stilbenoids appeared to possess moderate effects, whereas the flavonoids showed only weak activity.

Estrogenic and anti-estrogenic compounds from the Thai medicinal plant, Smilax corbularia (Smilacaceae).

From the rhizomes of Smilax corbularia Kunth. (Smilacaceae), 11 compounds, (2R,3R)-2″-acetyl astilbin, (2R,3R)-3″-acetyl astilbin, (2R,3R)-4″-acetyl astilbin, (2R,3R)-3″-acetyl engeletin, (2R,3S)-4″-acetyl isoastilbin, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10R)-2H,8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10S)-2H, 8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxy-phenyl)-5-[(1E)-2-(3,4-dihydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, and 5,7,3',4'-tetrahydroxy-3-phenylcoumarin along with 34 known compounds were isolated and characterized as 19 flavonoids, 14 catechin derivatives, 6 stilbene derivatives, and 6 miscellaneous substances. All isolates had their estrogenic and anti-estrogenic activities determined using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. The major constituents were recognized as flavanonol rhamnosides by the suppressive effect on estradiol induced cell proliferation at a concentration of 1µM. Meanwhile, flavanonol rhamnoside acetates demonstrated estrogenic activity in both MCF-7 and T47D cells at a concentration of 100µM, and they enhanced the effects of co-treated E2 on T47D cell proliferation at concentrations of more than 0.1µM.

QSAR study of natural estrogen-like isoflavonoids and diphenolics from Thai medicinal plants.

Two species of Thai medicinal plants, Dalbergia parviflora R. (Leguminosae) and Belamcanda chinensis L. (Iridaceae), used traditionally for the regulation of menstrual disorders, have been found to contain a large number of potential estrogen-like compounds. A set of some 55 isolated isoflavonoids and diphenolics showed a wide range of estrogen activity as determined in breast cancer MCF-7 and T47D cell proliferation assays. This set of compounds was studied by means of computational techniques including quantitative structure-activity relationships (QSAR) and molecular modeling. It was found that the estrogenic potencies of the studied compounds depend mainly upon the presence/absence of hydroxyl groups attached to 3' and 5' positions of B ring of the isoflavone scaffold and the inter-atomic distance between the hydroxyl groups attached to the outer terminal positions 7 of A ring and 4' of B ring. In a QSAR model employing ligand-receptor interaction energy descriptors, the LigScore scoring function of Cerius(2) virtual screening module, which describes the receptor affinities of simultaneous binding to estrogenic receptors α and ß (ER(α) and ER(ß)), led to the best correlation between the observed estrogenic activities and computed descriptors. Consideration of independent binding to ER(α) and ER(ß) did not result in statistically significant QSAR models. It was thus concluded that simultaneous and possibly competitive interaction of the compounds with the ER(α) and ER(ß) receptors, in which the presence of hydroxyl groups at the abovementioned positions of the isoflavonoids and diphenolics molecular scaffold plays a dominant role, may determine the estrogenic potency of the considered phytochemicals.

[Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata].

A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Antiestrogenic constituents of the Thai medicinal plants Capparis flavicans and Vitex glabrata.

Antiestrogenic compounds were investigated from Thai indigenous plants for galactogogues since estrogen is reported to suppress lactation in breastfeeding women. The aerial parts of the Thai medicinal plant Capparis flavicans, which has traditionally been used to promote lactation, gave the new compound capparoside A (1), along with 28 known compounds. The leaves of Vitex glabrata belong to the same genus as the chaste tree (Vitex agnus-castus), which is used traditionally to support lactation, and afforded the new compounds khainaoside A (14), khainaoside B (15), and khainaoside C (16), together with six known compounds. The isolates were tested for their biological activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. Syringaresinol (3) and principin (6), from C. flavicans, and khainaoside A (14) showed the most potent inhibitory effects on estrogen-enhanced cell proliferation among all compounds isolated. These results suggest that the lactation-promoting properties of C. flavicans might be related to the inhibitory effect on excess estrogen of women who experience insufficient breastfeeding and highlight the possibility of using V. glabrata leaves for their antiestrogenic properties.

Flavonoids from the heartwood of the Thai medicinal plant Dalbergia parviflora and their effects on estrogenic-responsive human breast cancer cells.

From the heartwood of Dalbergia parviflora, eight new compounds, khrinones A (1), B (2), C (3), D (4), and E (5), isodarparvinol B (6), dalparvin (7), and (3S)-sativanone (22), along with 32 known compounds, have been isolated and characterized as 17 isoflavones, nine isoflavanones, five flavanones, six isoflavans, and three miscellaneous substances. Isolates were evaluated for their cell proliferation stimulatory activity against the MCF-7 and T47D human breast cancer cell lines, and their luciferase inductive effects using luciferase transiently transfected MCF-7/luc and T47D/luc cells were also determined. Isoflavones such as genistein (10), biochanin A (11), tectorigenin (12), and 2'-methoxyformononetin (13) stimulated the proliferation of both cells, and concentrations of lower than 1 muM of these compounds showed equivalent activity to 10 pM of estradiol (E2). The new isoflavanone (22) also showed activity against both cell types, although it was weaker than that of the corresponding isoflavone (2'-methoxyformononetin, 13). Two optically active isoflavanones (22 and 24: (3S)-violanone) stimulated the proliferation of both cell lines at lower concentrations than three racemates (21: vestitone, 23: 7,3'-dihydroxy-4'-methoxyisoflavanone, and 25: 3-O-methylviolanone). Bowdichione (20), an isoflavone with a quinone structure in its B-ring, showed activity against only one cell line associated with MCF-7 in these assays.

Novel type III polyketide synthases from Aloe arborescens.

Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85-96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl-CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild-type PKS obtained from A. arborescens. Interestingly, the aloesone-forming activity was maximum at 50 degrees C, and the novel heptaketide pyrone was non-enzymatically converted to aloesone. In PKS3, the active-site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site-directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone.

Isolation and pharmacological characterization of fatty acids from saw palmetto extract.

Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary-tract symptoms secondary to benign prostatic hyperplasia. The mechanisms of pharmacological effects of SPE include the inhibition of 5alpha-reductase, anti-androgenic effects, anti-proliferative effects, and anti-inflammatory effects. Previously, we showed that SPE bound actively to alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine calcium channel (1,4-DHP) receptors in the prostate and bladder of rats, whereas its active constituents have not been fully clarified. The present investigation is aimed to identify the main active components contained in hexane and diethyl ether extracts of SPE with the use of column chromatography and preparative HPLC. Based on the binding activity with alpha(1)-adrenergic, muscarinic, and 1,4-DHP receptors, both isolated oleic and lauric acids were deduced to be active components. Authentic samples of oleic and lauric acids also exhibited similar binding activities to these receptors as the fatty acids isolated from SPE, consistent with our findings. In addition, oleic and lauric acids inhibited 5alpha-reductase, possibly leading to therapeutic effects against benign prostatic hyperplasia and related lower urinary-tract symptoms.