Influence of natural and synthetic compounds on cell surface expression of cell adhesion molecules, ICAM-1 and VCAM-1.

Various natural and synthetic compounds including alkaloids, terpenoids and phenolics were tested for inhibition of the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), both of which are crucial in the regulation of immune response and inflammation. Of 40 compounds tested, two compounds significantly downregulated the expression of VCAM-1 on murine endothelial cells (F-2) and ten compounds that of ICAM-1 on mouse myeloid leukemia cells (M1). Sanguinarine chloride (5) and isoliquiritigenin (13) were capable of lowering the levels of both ICAM-1 and VCAM-1. The structure-activity relationships study on chalcone and flavone derivatives related to 13 suggested that the inhibitory activity of the chalcone derivatives is attributable to the 4-hydroxy group as well as the possible coplanarity between the phenyl ring and the adjacent conjugated ketone.

H-NMR and mass spectral study of a D-enriched acetylenic norlignan, asparenyol, from cultured cells of Asparagus officinalis.

A suspension of cultured cells of Asparagus officinalis fed L-phenyl-D5-alanine-2,3,3-D3 (D5-phe) and DL-phenylalanine-3,3-D2 (D2-phe) yielded D-enriched asparenyol, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyllphenol (1). A H-NMR spectral study indicated that incorporation of deuterium atoms into the chains of the products was restricted to the C-9 position. The molecular ion regions of the MS of D-enriched metabolites, 1(D8-200), 1(D8-100) and 1(D8-50), obtained from feeding experiments using 200 mg, 100 mg and 50 mg of Ds-phe, respectively, coincided with the theoretical spectra. This confirmed that a single phenylalanine molecule supplies the nine carbon atoms in the -CH=CH-CH2-O-C6H4-O- moiety of 1. The biosynthetic sequence forming 1 as a norlignan class of metabolite is considered.

Biosynthesis of the acetylenic compounds in cultured cells of Asparagus officinalis from D- and 13C-labelled phenylalanines.

Studies on the biosynthetic pathway of acetylenic compounds, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]phenol and its related compounds, in cultured cells of Asparagus officinalis L. (Liliaceae) revealed that all of the 17 carbon atoms in their skeletons are supplied by phenylalanine. It is also concluded that p-substituted phenylacetylenic moieties (the C6-C2 unit) in these compounds are derived from C6-C3 shikimate pathway metabolites via phenylalanine. As a working hypothesis concerning the C6-O-C3 unit formation, a spirotetrahydrofuran type intermediate is predicted.

Biosynthesis of acetylenic compounds in cultured cells of Asparagus officinalis from [1-13C]- and [U-13C] Glucose.

The biosynthetic pathway of phenolics with an acetylenic group, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]phenol (1) and its related compounds, isolated from suspension-cultured cells of Asparagus officinalis L. (Liliaceae), was studied in feeding experiments using [1-13C]- and [U-13C] glucose. Analyses of the 13C-NMR spectra of the methyl ether and acetate of the 13C-labelled species of 1 indicated that the aromatic rings at both ends of the molecule are formed from shikimic acid. It was also assumed that the acetylenic carbons in the C5 chain could be incorporated together with its adjacent aromatic ring through any phenethyl class compound derived from phenylpropanoids, and the propenylene carbon through glycolysis metabolites with C3 carbon units.

Acetylenic compounds isolated from cultured cells of Asparagus officinalis.

Three new acetylenic compounds, compounds I, II and III were isolated from the cultured cells of Asparagus officinalis L. (Liliaceae) and their structures identified as 1-methoxy-4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]-benzene, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]phenol and 4-[5-(4-hydroxyphenoxy)-3-penten-1-ynyl]phenol, respectively, from chemical and spectral analysis.

Production of an anti-allergic triterpene bryonolic acid, by plant cell cultures.

Cell suspension cultures of Luffa cylindrica, Citrullus lanatus, and related cucurbitaceous plants accumulate large quantities of bryonolic acid (3 beta-hydroxy-D:C-friedoolean-8-en-29-oic acid) [1], an acidic, pentacyclic triterpene found exclusively in the roots of the intact plants. This compound could readily be isolated from cultured cells with CHCl3 and purified simply by recrystallization. Pharmacological tests using mice demonstrated that bryonolic acid or its derivative is active against at least three types of allergies and that its activity could be increased significantly by preparing synthetic derivatives, in particular a potassium salt of its succinate ester. The biosynthesis of bryonolic acid from mevalonic acid via isomultiflorenol has been elucidated by tracer and enzymological experiments using cultured cells of watermelon both in vitro and in vivo. Furthermore, cell fractionation and electron microscopic studies on subcellular structures of luffa cells suggested that minute vesicles originating from elongated, rough endoplasmic reticulum probably play an important role in the transport of bryonolic acid which largely accumulates in the cell wall of cultured cells. The results obtained from the present study indicate that plant cell culture would be useful not only as a biological system for elucidating biosynthetic mechanisms but also as a potential source of new pharmacologically active compounds.

Intracellular distribution of the hydrophobic triterpene, bryonolic acid, in cultured cells of Luffa cylindrica L.

Intracellular localization of bryonolic acid, an antiallergic pentacyclic triterpene, in cultured cells of Luffa cylindrica was investigated with reference to the sites of its biosynthesis and accumulation. The results of cell fractionation showed that bryonolic acid was mostly located in the cell wall fraction. The addition of FC-43 emulsion to the culture medium was found to cause the release of bryonolic acid from the cell wall into the medium without affecting cell growth and bryonolic acid production. Under this culture condition, (14)C-labeled sodium acetate administered to the cells was rapidly incorporated into bryonolic acid which was then excreted into the medium within 10 min after administration. Electron microscopic observations suggested that spherical vesicles (ca 0.1 µm in diameter) derived from the rough endoplasmic reticulum may be associated with the biosynthesis and excretion of this compound into the cell wall. Furthermore, the activity of 2,3-oxidosqualene cyclase, a key enzyme involved in the biosynthesis of bryonolic acid, was detected in the microsomal fraction containing the endoplasmic reticulum.

Anti-allergic effect of bryonolic acid from Luffa cylindrica cell suspension cultures.

The anti-allergic activity of bryonolic acid (1) isolated from the cultured cells of Luffa cylindrica L. (Cucurbitaceae) was compared with that of glycyrrhetinic acid (2), the aglycone of glycyrrhizin from licorice. Compound 1, when administered to rats intraperitoneally at a dose of 600 mg/kg, inhibited homologous passive cutaneous anaphylaxis more strongly than 2 at the same dose. Compound 1 also significantly inhibited delayed hypersensitivity in mice which could not be inhibited by 2. In contrast to 2, 1 showed not only little toxicity but no visible side effects on mice, without impairing the activity of the hepatic enzyme (4,5 beta-dihydrocortisone:NADP+ delta 4-oxidoreductase) involved in steroid catabolism.