Phytochemistry (v.53, #4)

Index (v).

Xanthone 6-hydroxylase activity was detected in the microsomal fractions from two plant cell cultures. The enzyme from cultured cells of Centaurium erythraea (Gentianaceae) exhibited absolute specificity for 1,3,5-trihydroxyxanthone as substrate, whereas xanthone 6-hydroxylase from cell cultures of Hypericum androsaemum (Hypericaceae) preferred the isomeric 1,3,7-trihydroxyxanthone but used 1,3,5-trihydroxyxanthone also to a small extent. Both xanthones were regioselectively hydroxylated in position 6. The xanthone 6-hydroxylases are cytochrome P450 monooxygenases, as shown by their dependence on NADPH and molecular oxygen and their inhibition by carbon monoxide and typical P450 inhibitors. In both cell cultures, xanthone accumulation was preceded by an increase in xanthone 6-hydroxylase activity.
Keywords: Centaurium erythraea; Gentianaceae; Hypericum androsaemum; Hypericaceae; Cell cultures; Xanthone biosynthesis; Xanthone 6-hydroxylase; Cytochrome P450 monooxygenase;

L-Ascorbic acid and L-galactose are sources for oxalic acid and calcium oxalate in Pistia stratiotes by Sarah E. Keates; Nathan M. Tarlyn; Frank A. Loewus; Vincent R. Franceschi (433-440).
Axenic Pistia stratiotes L. plants were pulse-chase labeled with [ 14 C ]oxalic acid, L-[1- 14 C ]ascorbic acid, L-[6- 14 C ]ascorbic acid, D-[1- 14 C ]erythorbic acid, L-[1- 14 C ]galactose, or [1- 14 C ]glycolate. Specific radioactivities of L-ascorbic acid (AsA), free oxalic acid (OxA) and calcium oxalate (CaOx) in labeled plants were compared. Samples of leaf tissue were fixed for microautoradiography and examined by confocal microscopy. Results demonstrate a biosynthetic role for AsA as precursor of OxA and its crystalline deposition product, CaOx, in idioblast cells of P. stratiotes and support the recent discovery of Wheeler, Jones and Smirnoff (. The biosynthetic pathway of vitamin C in higher plants. Nature, 393, 365–369) that L-galactose is a key intermediate in the conversion of D-glucose to AsA in plants. D-[1- 14 C ]Erythorbic acid (a diastereomeric analog of AsA) is utilized also by P. stratiotes as a precursor of OxA and its calcium salt deposition product in idioblasts. Labeled OxA is rapidly incorporated into CaOx in idioblasts, but microautoradiography shows there is also significant incorporation of carbon from OxA into other components of growing cells, contrary to the dogma that OxA is a relatively stable end product of metabolism. Glycolate is a poor substrate for synthesis of OxA and CaOx formation, further establishing AsA as the immediate precursor in the synthesis of OxA used for calcium precipitation in crystal idioblasts.
Keywords: Pistia stratiotes; Araceae; L-ascorbic acid; Calcium oxalate; Crystal idioblast cells; D-erythorbic acid; L-galactose; Glycolate; Oxalic acid;

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid by Heather A. Walczak; John V. Dean (441-446).
Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutathione were used to study the vacuolar transport of phenylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoplast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 μM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 μM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and V max values for uptake were 142 μM and 5.95 nmol mg−1 min−1, respectively. Based on these results, phenylpropanoid-glutathione conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.
Keywords: Beta vulgaris; Chenopodiaceae; Red beet; Vacuolar transport; Glutathione S-conjugate (GS-X) pump; ATP-binding cassette (ABC) transporter; Peroxidase-mediated glutathione conjugation; Phenylpropanoids; trans-Cinnamic acid-glutathione;

Cell suspension cultures of Pinus radiata metabolize the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) (1) via hydroxylation of the pentyl side chain. Examination of the culture medium following dosing studies with 1 revealed that 79–85% of this bioactive compound had been metabolised after 144 h. At that time, 34–40% of the metabolized dose was recovered as a series of monohydroxylated isomers of 1, the principal metabolite being 5-(2-pyron-6-yl)pentan-5-ol (7).
Keywords: Pinus radiata; Cell suspension cultures; Biotransformation; Hydroxylation; Fungal metabolite; 6-n-pentyl-2H-pyran-2-one (6PAP);

Occurrence and characterization of oils rich in γ-linolenic acid by J.L. Guil-Guerrero; F. Gómez-Mercado; F. Garcı́a-Maroto; P. Campra-Madrid (451-456).
Nineteen species of the genus Echium (Fam. Boraginaceae) collected in Macaronesia were surveyed in a search for new sources of γ-linolenic acid (GLA, 18:3ω6). High amounts of this acid were found in all of them, ranging from 9.15% (E. plantagineum) to 26.31% (E. callithyrsum) of total seed fatty acids. The amounts of GLA related to total seed weight were also significant, ranging from 1.77% (E. sventenii) to 5.02% (E. nervosum). In addition, considerable amounts of stearidonic acid (SA, 18:4ω3) were detected, ranging from 3.03% (E. auberianum) to 12.94% (E. plantagineum) of total fatty acids. These data allow us to consider the members of the genus Echium from Macaronesia as one of the richest sources of γ-linolenic acid found so far in nature. The results obtained from multivariable data analysis and the taxonomic relationships among the species is discussed.
Keywords: Boraginaceae; Echium; γ-linolenic acid; Macaronesia; Stearidonic acid; Fatty acid; Seed oil; Multivariable data analysis;

Anti-HIV-1 phorbol esters from the seeds of Croton tiglium by Sahar El-Mekkawy; Meselhy R. Meselhy; Norio Nakamura; Masao Hattori; Takuya Kawahata; Toru Otake (457-464).
Five phorbol diesters, together with three known ones, were isolated from a MeOH extract of the seeds of Croton tiglium, and their structures were determined by spectroscopic methods and selective hydrolysis of acyl groups. These compounds were assessed for their abilities to inhibit an HIV-induced cytopathic effect (CPE) on MT-4 cells and to activate protein kinase C (PKC) associated with tumor-promoting action. 12-O-Acetylphorbol-13-decanoate and 12-O-decanoylphorbol-13-(2-methylbutyrate) effectively inhibited the cytopathic effect of HIV-1 [complete inhibitory concentration (IC100) values of 7.6 ng/ml and 7.81 μg/ml, and minimum cytotoxic concentration (CC0) value of 62.5 and 31.3 μg/ml, respectively]; however, 12-O-acetylphorbol-13-decanoate showed no activation of PKC at concentrations of 10 and 100 ng/ml. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was found to be not only the most potent inhibitor of HIV-1-induced CPE (IC100 value of 0.48 ng/ml), but also the most potent activator of PKC (100% activation at 10 ng/ml).
Keywords: 12-O-Acetylphorbol-13-decanoate; HIV-1; Croton tiglium; Phorbol esters; Protein kinase C;

Delevoyin C, a tetranortriterpenoid from Entandrophragma delevoyi by Dulcie A. Mulholland; Sianne L. Schwikkard; Peter Sandor; Jean M. Nuzillard (465-468).
The hexane extract of the bark of Entandrophragma delevoyi has yielded a novel tetranortriterpenoid, delevoyin C. This compound contains a cyclobutanyl ring incorporating C-19 and a cycloheptanyl ring C including C-30. Gedunin and 11β-Acetoxygedunin were isolated from the hexane extract of the wood.
Keywords: Entandrophragma delevoyi; Meliaceae; Tetranortriterpenoid; Delevoyin C; 11β-acetoxygedunin; Gedunin;

Flavonoid glycosides and saponins from Astragalus shikokianus by Shoji Yahara; Mareshige Kohjyouma; Hiroshi Kohoda (469-471).
A new flavonol glycoside, kaempferol 3-O-α-L-rhamnopyranosy1-(1 → 6)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, named astrasikokioside I, was isolated from aerial part of Astragalus shikokianus, together with two flavonol glycosides, kaempferol 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, robinin, and three triterpenoid glycosides, soyasaponin I, sophoraflavoside II and robinioside E.
Keywords: Astragalus shikokianus; Legminosae; Flavonol glycoside; Astrasikokioside I; Robinin; Clovin; Triterpenoid glycoside; Soyasaponin I; Sophoraflavoside; Robinioside E;

Sesquiterpene lactone glucosides from Sonchus asper by Amr M. Helal; Norio Nakamura; Hesham El-Askary; Masao Hattori (473-477).
From the methanolic extract of the roots of Sonchus asper, two new sesquiterpene glucosides, together with two known sesquiterpene glucosides and a known aglycone were isolated. Structures were identified as 11β,13-dihydrourospermal A, 15-O-β-D-glucopyranosyl-11β,13-dihydrourospermal A, 15-O-β-D-glucopyranosylurospermal A, 15-O-[6′-(p-hydroxyphenylacetyl)]-β-D-glucopyranosylurospermal A and 14-O-methylacetal-15-O-[6′-(p-hydroxyphenylacetyl)]-β-D-glucopyranosylurospermal A, by spectroscopic means.
Keywords: Sonchus asper; Asteraceae; Melampolides; Sesquiterpene glucosides; Urospermal A derivatives;

Eudesmane derivatives from Tessaria integrifolia by Masateru Ono; Chikako Masuoka; Yusuke Odake; Yasuyuki Ito; Toshihiro Nohara (479-484).
Five eudesmane-type sesquiterpenoids were isolated from the methanol extract of the aerial part of Tessaria integrifolia Ruiz. et Pavon (Compositae). Their structures were elucidated on the basis of spectroscopic analysis as well as chemical evidence.
Keywords: Tessaria integriforia; Compositae; Sesquiterpene; Eudesmane; Glycoside; Integrifosides A–D; Integrifonol A; Anti-hyaluronidase activity;

Steroidal glycosides from the aerial part of Asclepias incarnata by Tsutomu Warashina; Tadataka Noro (485-498).
The aerial part of Asclepias incarnata afforded 34 pregnane glycosides. These were confirmed to have lineolon, isolineolon, ikemagenin, 12-O-nicotinoyllineolon, deacylmetaplexigenin, metaplexigenin, rostratamine, 12-O-acetyllineolon, 15β-hydroxylineolon and 15β-hydroxyisolineolon moieties as their aglycones, and 2,6-dideoxyhexopyranose, glucopyranose and allopyranose as the corresponding sugar constituents. Their structures were determined using both spectroscopic and chemical methods.
Keywords: Asclepias incarnata; Asclepiadaceae; Pregnane glycoside; 2,6-Dideoxyhexopyranose;

Iridoid glycosides from Lonicera quinquelocularis by S. Kumar; O.P. Sati; V.D. Semwal; M. Nautiyal; S. Sati; Y. Takeda (499-501).
A new iridoid glycoside 6′-O-β-apiofuranosylsweroside was isolated from the ethanolic extract of the roots of Lonicera quinquelocularis along with the known compounds loganin and sweroside.
Keywords: Lonicera quinquelocularis; Caprifoliaceae; Iridoid glycoside; Loganin; Sweroside; 6′-O-β-apiofuranosyl sweroside;

Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum by Motoo Tori; Tatsue Shimoji; Emi Shimura; Shigeru Takaoka; Katsuyuki Nakashima; Masakazu Sono; William A. Ayer (503-509).
The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as β-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam.
Keywords: Lycopodium lucidulum; Lycopodiaceae; Alkaloids; Lucidine A; Lucidine B; Oxolucidine A; Oxolucidine B; Lucidulinone;

A chalcone and a dihydrochalcone from Uvaria dulcis by Kan Chantrapromma; Yanisa Rat-A-pa; Chatchanok Karalai; Vitchu Lojanapiwatana; Vatcharee Seechamnanturakit (511-513).
2′,3′-Dihydroxy-4′,6′-dimethoxychalcone and the corresponding dihydrochalcone were isolated from the leaves of Uvaria dulcis and characterized by chemical and spectral methods.
Keywords: Uvaria dulcis; Annonaceae; Chalcone; Dihydrochalcone; Flavanone;

A biflavanone from Cycas beddomei by B. Jayaprakasam; A.G. Damu; D. Gunasekar; A. Blond; B. Bodo (515-517).
A new biflavanone, 7,7′′-di-O-methyltetrahydrohinokiflavone together with tetrahydrohinokiflavone were isolated from the stems of Cycas beddomei. The structures were established on the basis of spectral and chemical studies.
Keywords: Cycas beddomei; Cycadaceae; Stems; Biflavanones;

Gibberellins in seedlings and flowering trees of Prunus avium L. by Patrick S. Blake; Gordon Browning; Lynda J. Benjamin; Lewis N. Mander (519-528).
Extracts of acids from mature seeds, germinating seeds, first, second and third year seedlings as well as mature, flowering trees of sweet cherry (Prunus avium L. cv. Stella) were analysed by gas chromatography–mass spectrometry. The presence of the known gibberellins (GAs) GA1 (1), GA3 (4), GA5 (7), GA8 (11), GA19 (14), GA20 (12), GA29 (13), GA32 (5), GA85 (2), GA86 (3) and GA87 (6) was confirmed by comparison of their mass spectra and Kovats retention indices with those of standards or literature values. In addition, 16α,17-dihydrodihydroxy GA25 (16) was identified and its stereochemistry confirmed by rational synthesis. The 12α,13-dihydroxy GAs, GA32 (5), GA85 (2), GA86 (3) and GA87 (6), were detected in mature seeds, germinating seeds and young seedlings, but not in flowering plants. The 13-hydroxy GAs, GA1 (1) and GA3 (4), were present in germinating seeds and, in addition to these, GA5 (7), GA8 (11), GA19 (14), GA20 (12) and GA29 (13) were detected in seedlings and mature flowering plants. In germinating seeds and seedlings (while the plants were growing actively), concentrations of the 12α,13-dihydroxy GAs, measured by bioassay, declined and those of the 13-hydroxy GAs increased. The results are discussed with reference to the known and predicted effects of the GAs on the vegetative growth and flowering of P. avium plants.
Keywords: Prunus avium; Rosaceae; Sweet cherry; Gibberellins; Identification; Juvenility; Phase-change;