Kaoru Umehara

Physalin and neophysalins from Physalis alkekengi var. francheti and their differentiation inducing activity

A methanol extract of Physalis alkekengi var. francheti showed a potent cell differentiation inducing activity toward mouse myeloid leukemia cell line (M1 cells). From the extract, a new physalin and two new neophysalins, 25,27-dihydro-4,7-didehydro-7-deoxyneophysalin A and 4,7-didehydroneophysalin B, were isolated along with physalin A, L and isophysalin B. The structures of physalins and neophysalins were determined by means of NMR, UV, IR and mass spectra. Of these compounds, physalin A showed potent cell differentiation inducing activity.

Antiplasmodial Triterpenoids from Ekebergia Capensis.

From the stem bark of Ekebergia capensis, 10 new triterpenoid compounds, ekeberins A (1), B (2), C1 (3), C2 (4), C3 (5), D1 (6), D2 (7), D3 (8), D4 (9), and D5 (10), were isolated together with 17 known compounds. The structures of these new compounds were elucidated on the basis of the results of spectroscopic analysis, and the absolute configuration of compounds 6-10 were determined by partial synthesis from known compounds and using the Mosher ester method. Several of these compounds were screened in vitro against both chloroquine (CQ)-sensitive and -resistant Plasmodium falciparum isolates and were found to exhibit moderate antiplasmodial activity, with compounds 20 (7-deacetoxy-7-oxogedunin) and 27 (2-hydroxymethyl-2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene) showing IC50 values of 6 and 7 microM, respectively. Compound 27 at a dose of 500 mg/kg showed moderate parasitemia suppression of 52.9% against P. berghei NK 65 in a mouse model.

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.

Development of a novel method for determination of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase activity and its application to screening for acetyltransferase inhibitors: Inhibition by magnolol and honokiol from Magnoliae cortex

A method was developed for determining the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), a key enzyme in the biosynthesis of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The assay involves measurement of the radioactivity in the trichloroacetic acid (TCA)-precipitated complex of radioactive product and albumin after incubation of 1-alkyl-sn-glycero-3-phosphocholine and [3H]acetyl-CoA with rat spleen microsomes or membrane fractions of human polymorphonuclear leukocytes (PMNs). The radioactive product associated with the precipitate was identified as PAF using an ultrahigh-sensitivity TV camera system after extraction and separation by TLC. This TCA method was then used to screen the components of crude preparations that inhibited acetyltransferase activity. Major components from the cortex of Magnoliae (magnolol and honokiol), which have anti-inflammatory and anti-bacterial actions, inhibited the acetyltransferase activity in rat spleen microsomes (IC50, 150 and 150 microM, respectively) and membrane fractions of human PMNs (IC50, 70 and 60 microM, respectively). The inhibitory action of magnolol and honokiol was reversible, and similar to or higher than that of nordihydroguaiaretic acid. PAF production in human PMNs stimulated by the ionophore A23187 was also suppressed dose dependently by magnolol and honokiol. These activities may be relevant to the claimed therapeutic effects of the extract from Magnoliae cortex.

Matrix Metalloproteinase-2 Inhibitors from Clinopodium chinense var. parviflorum

From the aerial parts of Clinopodium chinense var. parviflorum, nine new phenylpropanoids, clinopodic acids A-I (2-10), were isolated together with a known phenylpropanoid, rosmarinic acid (1). The structures of these new compounds were elucidated on the basis of spectroscopic analysis. Clinopodic acid C (4) showed MMP-2 inhibitory activity (IC(50) 3.26 microM).

Umbelliferone released from hairy root cultures of Pharbitis nil treated with copper sulfate and its subsequent glucosylation

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a β-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.

Bioactive ergostanoids and a new polyhydroxyoctane from Lentinus polychrous mycelia and their inhibitory effects on E2-enhanced cell proliferation of T47D cells

From mycelia of Lentinus polychrous, a Thai local edible mushroom cultured under solid-state fermentation, a new compound, 6-methylheptane-1,2,3,4,5-pentaol (1), and five ergostanoids, namely (22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol (2), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (3), ergosta-4,6,8(14),22-tetraen-3-one (4), (3β,5α,8α,22E)-5,8-diepoxy-ergosta-6,22-dien-3-ol (5) and 5,8-epidioxy-(3β,5α,8α,22E)-ergosta-6,9(11),22-trien-3-ol (6), was isolated and characterised. The compounds were determined for their oestrogenic and anti-oestrogenic activities by using human breast cancer T47D cells. All compounds had no oestrogenic activity but exhibited suppressive effect on oestradiol-enhanced cell proliferation. Among these compounds, only 4 significantly competed with oestradiol in the binding to oestrogen receptors (ERs) with higher selectivity to ERα than ERβ. These results may suggest that most compounds suppressed this oestradiol-enhanced T47D proliferation via other mechanisms rather than ER binding.

The Quenching Effect of Flavonoids on 4-Methylumbelliferone, a Potential Pitfall in Fluorimetric Neuraminidase Inhibition Assays

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10−5), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

Polygalasaponins XLII–XLVI from roots of Polygala glomerata

Five new oleanane-type saponins, polygalasaponins XLII-XLVI, along with two known saponins were isolated from the roots of Polygala glomerata Lour. The structures of polygalasaponins XLII-XLVI were elucidated as 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl- (1-->2)-{4-O-[(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl- (1-->3)]-[4-O-(E)-p-methoxycinnamoyl]-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl- (1-->3)]-{4-O-[(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[6-O- acetyl-beta-D-glucopyranosyl-(1-->3)]-{4-O- [(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D- glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)- beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl- (1-->2)-[6-O-acetyl-beta-D-glucopyranosyl-(1-->3)]-{4-O-[(Z)-3, 4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, respectively, on the basis of spectroscopic and chemical evidence.

Identification of a new angiotensin-converting enzyme (ACE) inhibitor from Thai edible plants

Eight Thai edible plants were tested for their inhibitory activity against an angiotensin-converting enzyme (ACE) using an in vitro assay. The methanol extract of Apium graveolens exhibited significant ACE inhibitory activity with an IC50 value of 1.7 mg/ml, and was then subjected to an isolation procedure that resulted in identification of a pure active constituent, junipediol A 8-O-β-d-glucoside (1-β-d-glucosyloxy-2-(3-methoxy-4-hydroxyphenyl)-propane-1,3-diol) (1), which had good ACE inhibitory activity with an IC50 value of 76 μg/ml. Another eight known compounds, isofraxidin-β-d-glucoside (2), roseoside (3), apigenin-7-O-β-d-glucoside (4), luteolin-7-O-β-d-glucoside (5), icariside D2 (6), apiin (7), chrysoeriol-7-O-β-d-apiosylglucoside (8), and 11,21-dioxo-3 β,15 α,24-trihydroxyurs-12-ene-24-O-β-d-glucopyranoside (9) were also identified. Although each of these five constituents (2-6) isolated from the same fraction as 1 showed no activity at concentrations of 500 μM, together, when each was present at 300 μg/ml, they enhanced the inhibitory activity of 500 μM of 1 from 64% to 81%.