Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MSn.

The prenylated phloroglucinol hyperforin, thought to be an essential component for the anti-depressant activity of St. John's Wort (Hypericum perforatum), is unstable. The facile oxidative degradation of hyperforin poses serious problems for standardisation, and may also dramatically affect the pharmacological activity of the extracts. Hyperforin was dissolved in hexane and stored at room temperature for 3 days and yielded various closely related degradation products which, although difficult to isolate on the preparative scale, have been analysed by on-flow and stop-flow HPLC-NMR and HPLC-MS/MS. From on-line spectroscopic data, and with the aid of complementary in-mixture standard NMR two-dimensional correlation experiments, the different oxidised forms of hyperforin were found to be phloroglucinol derivatives in which a hydroxy-dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric form) and the prenyl chain at C-8 of the core nucleus of hyperforin. The strategy followed for the on-line identification of these constituents is discussed.

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae).

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 microg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 microg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter.

Furostanol saponins from Tribulus terrestris.

An HPLC-ELSD-ESI-MS method has been developed for the analysis of the steroidal saponins in the aerial parts of Tribulus terrestris. Protodioscin, a new saponin (5,6-dihydroprotodioscin, neoprotodioscin) and their respective sulfates were detected. The structure of the new compound was elucidated on the basis of NMR and ESI-MS spectral analysis.

A simple HPLC-UV method for the assay of ginkgolic acids in Ginkgo biloba extracts.

Extracts from the leaves of Ginkgo biloba are among the most widely used phytotherapeutics. Some alkylphenols (ginkgolic acids, cardanols and cardols) have been described as potentially hazardous constituents in Ginkgo extracts. Accordingly, a requirement for a maximum concentration of ginkgolic acids has been proposed in the UE and US pharmacopoeias Ginkgo monographs establishing a limit value of 5 ppm. A novel HPLC-UV method, developed by the use of HPLC-APCI-MS HPLC-DAD techniques and allowing the identification of ginkgolic acids and related phenols, is described. The new analytical method, not requiring enrichment procedures, can be used for the quantification of ginkgolic acids in Ginkgo extracts.

Identification of Ruscus steroidal saponins by HPLC-MS analysis.

A novel HPLC-UV method has been developed for the fingerprint analysis of the steroidal saponins in the rhizomes of three Ruscus species (Ruscus aculeatus, Ruscus hypoglossum and Ruscus colchicus). Saponins were identified by HPLC-ESI-MS. During the study a new major saponin was detected in the rhizomes of R. hypoglossum and R. colchicus. The structure of the new compound was defined as 1-O-[alpha-L-rhamnopyranosyl-(1-->2)-6-O-acetyl-beta-D-galactopyranosyl]-1beta,3beta,22xi,26-tetrahydroxy-furost-5(6)-en-26-O-beta-D-glucopyranoside (8) by spectral analysis.

High-performance liquid chromatography-electrospray ionization mass spectrometry and multiple mass spectrometry studies of hyperforin degradation products.

The alcoholic extract of the aerial parts of Hypericum perforatum L. finds wide application because of its antidepressant activity. The extract contains a number of constituents with documented biological activity including chlorogenic acid, a broad range of flavonoids, naphthodianthrones and phloroglucinols. Hyperforin and adhyperforin are the major phloroglucinol constituents found in the lipophilic fraction of the extracts. Since the stability of hyperforin has been shown to be limited, an investigation of the hyperforin degradation products using HPLC-electrospray ionization mass spectrometry and multiple mass spectrometry was undertaken.

Determination of ginsenosides in Panax ginseng roots by liquid chromatography with evaporative light-scattering detection.

A liquid chromatographic method was developed and validated for the determination of ginsenosides in Panax ginseng roots by using evaporative light-scattering detection. Eighteen ginsenosides were separated on a reversed-phase C18 column with water-ammonium acetate-acetonitrile as the mobile phase. The method is suitable for the routine determination of ginsenosides in P. ginseng roots and extracts. The validation of the method was comprehensive for efficiency and recovery optimization of the P. ginseng roots extraction, specificity by liquid chromatography/mass spectrometry, linearity, stability, reproducibility, repeatability, intermediate precision, and robustness.

Characterization of proanthocyanidins from grape seeds.

Leucoselect (grape seed selected proanthocyanidins) was analyzed. HPLC thermospray mass spectrometry (TSP-MS) allowed the detection of monomeric flavan-3-ols and dimeric proanthocyanidins. Fractionation over Sephadex LH-20 resin and analysis of the isolated fractions by gel permeation chromatography (GPC) and electrospray mass spectrometry (ESI-MS) led to the complete characterization of the proanthocyanidin constituents of Leucoselect. The analysis revealed the presence of approximately 15% of (+)-catechin (1) and (-)-epicatechin (2), 80% of (-)-epicatechin 3-O-gallate (3), dimers, trimers, tetramers and their gallates and 5% of pentamers, hexamers, heptamers and their gallates.

Liquid chromatography-electrospray mass spectrometric identification of ginsenosides in Panax ginseng roots.

A high-performance liquid chromatographic method was developed for electrospray mass spectrometric analysis of ginsenosides in Panax ginseng roots. The analyses were performed on a reversed-phase C18 column using a binary eluent (aqueous 8 mM NH4OAc, buffered to pH 7 with NH4OH-acetonitrile) under gradient conditions. Twenty-five ginsenosides could be separated and detected. The mass spectra obtained provided information on their molecular masses. A MS-MS experiment was undertaken in order to determine the sugar unit sequences and the aglycone moieties.

Paclitaxel analogues from Taxus x media cv. Hicksii.

The roots of T. x media Rehd. cv. Hicksii gave three novel analogues of paclitaxel modified at the N-acyl residue (N-debenzoyl-N-alpha-methylbutyryl paclitaxel and N-debenzoyl-N-cinnamoyl paclitaxel, 1b and 1c, respectively) or at the ester group at C-2 (2-debenzoyl-2-tigloyl paclitaxel, 1d). Compounds 1b and 1d showed reduced cytotoxicity and tubulin binding compared to paclitaxel, while 1c retained substantial activity in these assays.

Acyl secoiridoids and antifungal constituents from Gentiana macrophylla.

LC-UV-mass spectrometry and bioassay co-directed fractionation of an aqueous acetone extract of the roots of Gentiana macrophylla gave three new chromene derivatives and two novel and six known secoiridoids, along with kurarinone, kushenol I, beta-sitosterol, stigmasterol, daucosterol, beta-sitosterol-3-O-gentiobioside, alpha-amyrin, oleanolic acid, isovitexin, gentiobiose and methyl 2-hydroxy-3-(1-beta-D-glucopyranosyl)oxybenzoate. The structures of the new products were established from spectral and chemical evidence as 2-methoxyanofinic acid and macrophyllosides A-D. The six known secoiridoids were gentiopicroside, sweroside, 6'-O-beta-D-glucosylgentiopicroside, 6'-O-beta-D-glucosylsweroside, trifloroside and rindoside. The new acid (2-methoxyanofinic acid), its methyl ester, kurarinone and kushenol I were shown to be active against the plant pathogenic fungus Cladosporium cucumerinum. The methyl ester and kurarinone inhibited also the growth of the human pathogenic yeast Candida albicans. Structure-activity relationships were studied. Thus, addition of a methoxyl group to the benzene nucleus of anofinic acid (2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid) increased the antifungal activity remarkably whereas glycosylation at the carboxylic moiety was found to remove the activity. Esterification of the new acid induced its activity against C. albicans, but decreased its growth inhibition properties against C. cucumerinum. Hydroxylation of kurarinone at the 3 beta-position removed its activity against C. albicans and decreased the inhibition of C. cucumerinum. In addition, the chemotaxonomic significance of the identified constituents is discussed.

Antifungal principles of Baccharis pedunculata.

Four compounds including a flavone, an acetylenic lactone, a prenylated coumarin, and a 3-methyl ether flavone were isolated from the dichloromethane leaf extract of Baccharis pedunculata (Mill.) Cabr. (Asteraceae). The latter three compounds were identified to be responsible for the antifungal activity against some human pathogenic and phytopathogenic fungi. The most active compound, lachnophyllum lactone, an acetylenic lactone, showed a very high toxicity (LD50 2 micrograms/ml) against human keratinocytes.

Polyphenols from Eriosema tuberosum.

A dichloromethane extract of the roots of Eriosema tuberosum exhibited antifungal activity against Cladosporium cucumerinum and Candida albicans using TLC bioautography. Bioassay-directed fractionation led to the isolation of four new compounds, eriosemaones A-D, together with a known compound, flemichin-D, as the active constituents. Three inactive polyphenols were also isolated after methylation, together with one new chromone, eriosematin. Structures were determined by spectroscopic analysis and from chemical evidence.

Xanthone and antifungal constituents from Monnina obtusifolia.

Three biphenyls and four xanthones have been isolated from the aerial parts of Monnina obtusifolia. The structures were established on the basis of their spectral data and that of some derivatives. The biphenyls have been isolated previously from the same genus. 1,3,6-Trihydroxy-2,5-dimethoxyxanthone is a new natural product, whereas the other xanthones have been described in other species. The antifungal activity of the isolated compounds has been determined.