Antimicrobial activity of Mahonia aquifolium and two of its alkaloids against oral bacteria.

Extracts or alkaloids isolated from Mahonia aquifolium exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and protozoa. In this study the bacteriostatic and bacteriocidal activities of a M. aquifolium extract and two of its major alkaloids, berberine chloride and oxyacanthine sulphate, were tested in vitro against nine different oral bacteria. Minimum inhibitory concentrations were in the range from < or = 0.0031% to 0.1993% for the M. aquifolium extract, from 0.002% to > 0.125% for berberine chloride, and from 0.0156% to > 0.0625% for oxyacanthine sulphate. The values for the minimum bactericidal concentrations were in the same range, indicating that the test substances most probably acted in a bactericidal manner. The most susceptible bacterium against all three test substances was Porphyromonas gingivalis.

Hypericum perforatum: which constituents may induce intestinal MDR1 and CYP3A4 mRNA expression?

In vitro and in vivo studies suggest that extracts of St John's wort (Hypericum perforatum, L .; SJWE) interact with various drugs, by enhancing their elimination, due to induction of intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp), the gene product of multidrug resistance gene 1 (MDR1/ABCB1). The aim of our study was to identify the major constituents responsible for this induction and their relative importance. Therefore, plant extracts were investigated that vary in these constituents with respect to their effect on mRNA expression of MDR1/CYP3A4. First, different pure constituents of Hypericum perforatum L . were investigated. Secondly, diverse SJWE with different concentrations of hyperforin, quercitrin and hypericin were investigated. The concentrations of hyperforin, hypericin, and quercitrin in the plant extracts were determined by HPLC, and an "artificial extract" containing the same mixture of these constituents was investigated. Different plant extracts, pure constituents or "artificial extracts" were applied to the human colon carcinoma-derived cell line (LS180) and the induction of MDR1 and CYP3A4 expression was analyzed by quantitative RT-PCR. MDR1 and CYP3A4 mRNA expression were both induced by single constituents of SJW such as hypericin and hyperforin in a concentration of 10 microM. Additionally, CYP3A4 mRNA expression was induced by quercitrin. SJW extracts containing hyperforin induced significantly MDR1 mRNA expression, whereas no CYP3A4 induction was observed after treatment with any of the investigated SJWE. These effects could be mimicked by "artificial extracts" containing the same compositions of hyperforin, hypericin and quercitrin as the plant extracts.

Preserved pharmacological activity of hepatocytes-treated extracts of valerian and St. John's wort.

The two herbal extracts valerian (Valeriana officinalis L.) and St. John's wort (Hypericum perforatum L.) were studied for their metabolic changes upon incubation with freshly prepared rat hepatocytes and subsequently analysed phytochemically as well as pharmacologically in vitro. Quantitative HPLC analysis of valerian extracts revealed considerable metabolic activities with regard to sesquiterpenes and iridoids. The amount of acetoxyvalerenic acid decreased 9-fold, while that of hydroxyvalerenic acid correspondingly increased 9-fold due to O-deacetylation. The valepotriates didrovaltrate, isovaltrate and valtrate decreased 2-, 18- and 16-fold, respectively. However, the binding affinities of the incubated extracts to the benzodiazepine and picrotoxin binding site of the GABA (A) receptor were quite similar to those of the non-incubated extracts. Neither valerenic acids nor valepotriates exhibited any significant effect on the two binding sites when tested as single compounds. Therefore, either other constituents represent the active ones or multiple compounds are necessary for the observed inhibitory and allosteric effects at the GABA (A) receptor. Extracts of St. John's wort were less potently metabolised than valerian. The amount of pseudohypericin and the main flavonoids (hyperoside, rutin, isoquercitrin, quercitrin, quercetin and I3,II8-biapigenin) slightly decreased during the 4-h incubation period. Both the antagonist effect at the corticotropin-releasing factor (CRF) type 1 receptor and the binding inhibition at the 5-HT transporter were attenuated during the metabolic treatment. The reduced antagonist effect correlates with the decreasing amount of pseudohypericin known to be a CRF (1) receptor antagonist. In conclusion, the incubation of plant extracts with freshly prepared rat hepatocytes represents a useful approach to study the pharmacological action of metabolised plant extracts. The consistent pharmacological activity of both valerian and St. John's wort is concordant with the known clinical efficacy of pharmacological activities.

Antagonist effect of pseudohypericin at CRF1 receptors.

St. John's wort (Hypericum perforatum L.) is widely used for the treatment of mild to moderately severe depression. However, the nature of its active principles and the exact mode of antidepressant action are still unknown. It has been suggested repeatedly in preclinical and clinical studies that the content of the acylphloroglucinol hyperforin decisively contributes to the antidepressant efficacy of St. John's wort extracts. Experimental studies in vivo also indicate that the naphthodianthrone hypericin may reduce the activity of the hypothalamic-pituitary-adrenal axis. Exacerbated hypothalamic-pituitary-adrenal activity has often been associated with depressive states in patients. Corticotropin-releasing factor (CRF) seems to be a major determinant in the regulation of the hypothalamic-pituitary-adrenal activity via activation of CRF(1) receptors. In the present study, we investigated the CRF(1) receptor antagonist activity of three main constituents of St. John's wort (hypericin, pseudohypericin and hyperforin) by measuring their effect on CRF-stimulated cAMP formation in recombinant Chinese hamster ovary (CHO) cells. As a selectivity test, the compounds were also tested against calcitonin in the same cells. Of the three compounds tested, only pseudohypericin selectively antagonised CRF (K(B) 0.76 microM). Hypericin and hyperforin affected both CRF and calcitonin with similar potencies and the same type of behaviour (competitive antagonism for hypericin, noncompetitive for hyperforin). It is concluded that pseudohypericin is the only real CRF(1) receptor antagonist of the three constituents tested. In addition, evidence is provided that beside hyperforin, both pseudohypericin and hypericin are implicated in the antidepressant efficacy of St. John's wort.

Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely used screening method to measure cell viability and proliferation. When testing the effects of kaempferol on breast cancer cell number (crystal violet staining) and viability (MTT tetrazolium assay) conflicting results were obtained. Cell number decreased but MTT formazan formation increased, suggesting a direct interaction of kaempferol with the MTT tetrazolium reduction. Direct reductive potential was observed in a cell-free system for the presumptive phytoestrogens kaempferol and resveratrol, and extracts of Hypericum perforatum L. and Cimicifuga racemosa L. All agents led to instantaneous dark blue formazan formation in the absence of cells. Additionally, antioxidants such as ascorbic acid, vitamin E and N-acetylcysteine interfered with the MTT tetrazolium assay. When MCF7 and HS578 cells treated with kaempferol were washed before addition of MTT tetrazolium, the direct reduction of dye was reduced significantly. These results indicate that the MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential.