Posted by sdb on June 27, 2006, at 18:44:55 [reposted on June 27, 2006, at 23:57:50 | original URL]
In reply to Re: hypericum - paroxetine panic maze study (long), posted by sdb on June 27, 2006, at 18:36:55
hpa axis an new approach for crh-1 antagonists?
Flavonoids of St. John’s Wort Reduce HPA Axis Function in the Rat
V. Butterweck1, M. Hegger2, H. Winterhoff2
1 Department of Pharmaceutics, University of Florida, Gainesville, FL, USA
2 Institute of Pharmacology and Toxicology, Münster, Germany
Abstract
Material and Methods
Acknowledgements
References
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AbstractA common biological alteration in patients with major depression is the activation of the hypothalamic-pituitary-adrenal (HPA) axis, manifested as hypersecretion of adrenocorticotropic hormone (ACTH) and cortisol. The hyperactivity of the HPA axis in depressed patients can be corrected during clinically effective therapy with standard antidepressant drugs such as imipramine, indicating that the HPA axis may be an important target for antidepressant action. We previously showed that a methanolic extract of St. John’s wort (SJW) and hypericin, one of its active constituents, both have delayed effects on the expression of genes that are involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis [1], whereas the phloroglucinol derivative hyperforin was inactive in the same model [2]. Since flavonoids of SJW are also discussed as active constituents it was of interest to determine whether these compounds can modulate HPA axis function. Imipramine (15 mg/kg), hypericin (0.2 mg/kg), hyperoside (0.6 mg/kg), isoquercitrin (0.6 mg/kg) and miquelianin (0.6 mg/kg) given daily by gavage for two weeks significantly down-regulated circulating plasma levels of ACTH and corticosterone by 40 - 70 %. However, none of the compounds tested had an effect on plasma ACTH and corticosterone levels after chronic treatment (daily gavage for 8 weeks). Our data suggest that besides hypericin, flavonoids of SJW play an important role in the modulation of HPA axis function. Furthermore, the results support the hypothesis that flavonoids are involved in the antidepressant effects of SJW.
Hypericum perforatum (Clusiaceae) commonly known as St. John’s wort (SJW), is used in many countries for the treatment of mild to moderate forms of depression. Several clinical studies provide evidence that SJW is as effective as conventional synthetic antidepressants [3], [4]. A series of bioactive compounds has been detected in the crude material (for review see [5]). The pharmacological activity of SJW extracts has been reviewed previously [6]. Recent reports have shown that the antidepressant activity of Hypericum extracts can be attributed to the phloroglucinol derivative hyperforin [7], to the naphthodianthrones hypericin and pseudohypericin [8], and to several flavonoids [9], [10]. The role and the mechanisms of these different compounds is still a matter of debate. But, taking these previous findings together, it is likely that several constituents are responsible for the clinically observed antidepressant efficacy of SJW.
In patients with depression, a considerable number of endocrine findings is reported, most prominent are the changes in the hypothalamic-pituitary-adrenal (HPA) axis. Hypersecretion of ACTH and plasma cortisol have been reported in 40 - 50 % of patients suffering from depression [11], [12]. Normalization of the hyperactive HPA system occurs during successful antidepressant pharmacotherapy of depressive illness [13].
Previous work in rats established that long-term treatment with imipramine, fluoxetine, idazoxan, and phenelzine reduces HPA axis activity with delayed onset (after 2 weeks) [14], [15]. Based on the results of Brady et al. [14], [15] we recently studied the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, a methanolic SJW extract, and hypericin on the expression of genes that may be involved in the regulation of the HPA axis [1]. Our data showed that imipramine, SJW extract, and hypericin given daily for 8 weeks but not for 2 weeks significantly decreased levels of CRH mRNA in the PVN. Comparable to imipramine the SJW extract as well as hypericin reduced plasma ACTH and corticosterone levels after 2 weeks of daily treatment, but not after 8 weeks [1].
Because some authors emphasized hyperforin as the major active principle of SJW extract we recently determined whether daily administration of a lipophilic CO2 extract enriched with hyperforin, and a hyperforin derivative (hyperforin trimethoxybenzoate; TMB) had effects on the levels of the above-mentioned mRNAs in a manner similar to the methanolic SJW extract and hypericin in the short-term/long-term administration paradigm [2]. However, a lipophilic CO2 extract as well as hyperforin-TMB failed to affect gene transcription involved in HPA axis control and did not alter plasma levels of ACTH and corticosterone [2].
Since hyperoside, isoquercitrin and miquelianin have been shown to exhibit antidepressant activity in animal models of depression [9], it was of interest in the present study to examine whether these compounds might also act on the HPA axis after short- and long-term treatment.
The major novel finding of the present study is that the flavonoids hyperoside (quercetin 3-O-galactoside), isoquercitrin (quercetin 3-O-glucoside) as well miquelianin (quercetin 3-O-glucuronide) significantly down-regulated plasma ACTH and corticosterone levels in the short-term treatment paradigm similar to the changes elicited by the prototypic synthetic antidepressant imipramine and the naphthodianthrone hypericin (∼40 % and ∼60 %, respectively) - a finding which has not been reported yet for this class of compounds.
Daily treatment over 2 weeks with the tricyclic antidepressant imipramine caused a significant decrease (p < 0.05) in both ACTH (40 % reduction from baseline) and corticosterone (46 % reduction from baseline) compared to vehicle-treated animals (Fig. [1] a, b). A similar decrease in ACTH and corticosterone levels after two weeks of daily treatment was observed for the naphthodianthrone hypericin (74 % and 43 % reduction from baseline, respectively, p < 0.01) and the flavonoids hyperoside, isoquecitrin and miquelianin (∼40 to ∼60 % reduction from baseline, respectively). The replication of the imipramine and hypericin effect [1] and the addition of significant data based on administration of flavonoids further validate the short-term/long-term treatment paradigm for the assessment of efficacy of candidate antidepressant drugs.
None of the substances caused any significant alteration in plasma ACTH and corticosterone after 8 weeks of daily treatment (Fig. [2] a,b). The functional consequences of these findings are not clear. Probably the relatively high baseline levels of ACTH and corticosterone might be a reason for these dissociative effects after two and eight weeks. It can be speculated that different adrenal responsiveness after both time points might play an important role in mediating the ACTH effects, but this needs to be explored by further experimentation. The fact, that we did not observe significant alterations in plasma ACTH and corticosterone levels after the 8 weeks treatment period with any of the substances confirms our previous findings [1]. However, the imipramine-mediated effects on ACTH and corticosterone after 8 weeks are ambivalent, since some studies have reported decreased levels of ACTH and corticosterone plasma levels after long-term imipramine treatment of rats [14], [16]. One reason for these discrepancies might be the large variability in daily imipramine doses used in the different studies. Some studies using imipramine in rats have used doses of 10 - 30 mg/kg/day [17], [18], others have used lower doses of 5 mg/kg/day [14], [16]. In these studies the substance was given interaperitoneally, whereas in the present study 15 mg/kg of imipramine were given orally by gavage. Interestingly, it has been shown in pharmacokinetic studies that the route of administration has an influence on imipramine levels [19]. During intramuscular or intraperitoneal administration, the parent drug impramine predominated in the plasma, and, conversely, the demethylated metabolite desipramine predominated during oral administration [19]. Thus, it can be speculated that the effects observed in the present study are due to desipramine. However, the imipramine dosage used in the present study was selected from the doses shown in animal models to correlate with antidepressant activity [1].
The potency of the Hypericum extract on HPA axis effects [1] could also be demonstrated with pure hypericin, hyperoside, isoquercitrin and miquelianin. It appears, therefore, that besides hypericin the flavonoids represent a possible major active principle which may contribute to the beneficial effect of Hypericum extract after oral dosing. In a recent in vitro study it could be shown that miquelianin - besides crossing walls from the small intestine - was able to cross the blood-brain barrier as well as the blood-cerebrospinal fluid barrier [20]. This finding gives further evidence for our assumption that flavonoids are able to reach the CNS after oral administration. Since only low doses of flavonoids were necessary to reduce plasma ACTH and corticosterone levels (0.6 mg/kg compared to 20 mg/kg of imipramine) these compounds might be used as a template for the development of new antidepressants in the future. In conclusion, in how far there might be synergistic or additive effects of single flavonoids needs to be established in future investigations.
Abbildung in neuem Fenster zeigenFig. 1 A,B: Adrenocorticotropin (ACTH) and corticosterone levels in plasma in the short-term (2 weeks) treatment paradigm; a = control; b = imipramine; c = hypericin; d = hyperoside; e = isoquercitrin; f = miquelianin. * p < 0.05; ** p < 0.01. Bars indicate ± SEM.
Abbildung in neuem Fenster zeigenFig. 2 A,B:Adrenocorticotropin (ACTH) and corticosterone levels in plasma in the long-term (8 weeks) treatment paradigm; a = control; b = imipramine; c = hypericin; d = hyperoside; e = isoquercitrin. Bars indicate ± SEM.
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Material and MethodsAnimals: Male CD rats (150 - 180 g, Charles River WIGA, Sulzfeld, Germany) were single housed in a 12 h light/dark cycle, with lights off at 19.00 h, at a constant temperature of 25 ± 1 °C and free access to food (Altromin 1324, Altromin Lage, Germany) and tap water. Rats were randomly assigned to the various experimental groups (n = 10/group) and weighed daily. The experimental procedures used in this work were officially approved by the Regierungspräsident, Münster (A 92/99). Animals were decapitated between 9.00 and 11.00 h; the last drug administration was the day before between 16.00 and 17.00 h. Trunk blood was collected on ice-chilled EDTA-coated (10 mL) tubes containing 500 KIU aprotinin/mL, centrifuged, and plasma was frozen at -70 °C until use.
Chronic antidepressant treatment: Imipramine-HCl was purchased from Sigma (Deisenhofen, Germany), hypericin, hyperoside and isoquercitrin from Roth (Karlsruhe, Germany; purity > 95 %). Miquelianin was a gift of Prof. Dr. A. Nahrstedt, Institut für Pharmazeutische Biologie und Phytochemie, WWU Münster.
All substances were administered orally using the gavage technique; imipramine (15 mg/kg), hyperoside (0.6 mg/kg), isoquercitrin (0.6 mg/kg) and miquelianin (0.6 mg/kg) were dissolved in deionized water. All substances contained ethanol in a concentration of 160 μL/10 mL. As hypericin is sparely soluble in water, an ethanolic stock solution was prepared: 5 mg hypericin were dissolved in 2.5 mL ethanol (stock solution). 0.5 mL stock solution was diluted with water to a final concentration of 0.1 mg/kg hypericin. Control animals received deionized water with an ethanol content of 160 μL/10 mL. The final application volume of each solution was 10 mL/kg b. w. Treatment was performed between 16.00 and 17.00 h every day. The hypericin dosage of 0.2 mg/kg was chosen corresponding to [8], the hyperoside, isoquercitrin and miquelianin dosage was chosen because of their demonstrated efficacy in the forced swimming test [9]. Because the amount of miquelianin was limited, this compound was only tested in the 2 weeks treatment paradigm.
Measurement of corticosterone and adrenocorticotropic hormone (ACTH): A radioimmunoassay (RIA) of corticosterone was performed using [125I]corticosterone, antiserum, and the standard solution in a kit from ICN Biomedical (Costa Mesa, CA, USA). The assay was adapted to rat serum conditions. Precipitation was done using a second antibody solid phase. ACTH was measured using a DSL kit (Webster, Texas, USA). Both assays were performed according to manufacturer’s instructions. The inter- and intraassay coefficients of variance for ACTH were 10.6 % and 6.9 %, respectively, with a detection limit of 10 pg/mL. For corticosterone, the inter- and intraassay coefficients of variance were 7.2 % and 4.4 %, with a detection limit of 25 ng/mL.
Statistics: Statistical procedures were performed by use of the STATVIEW statistical software package, version 5.0 (SAS®, USA). All data were expressed as the mean ± SEM. Group mean differences were ascertained with analysis of variance (ANOVA). Multiple comparisons among treatment means were checked with the Student-Newman-Keuls post-hoc test. The results were considered significant if the probability of error was < 5 %.
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AcknowledgementsWe thank Prof. Dr. A. Nahrstedt (Institut für Pharmazeutische Biologie und Phytochemie, WWU Muenster, Germany) for the generous supply of miquelianin. We acknowledge the Steigerwald Arzneimittel GmbH (Darmstadt, Germany) for financial support of this study.
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