Hilke Winterhoff

Long-term effects of St. John's wort and hypericin on monoamine levels in rat hypothalamus and hippocampus.

Hypericum perforatum L. (St. Johns wort) is one of the leading psychotherapeutic phytomedicines and, because of this, great effort has been devoted to clarifying its mechanism of action. Chronic effects of St. Johns wort and hypericin, one of its major active compounds, on regional brain amine metabolism have not been reported yet. We used a high-performance liquid chromatography system to examine the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, Hypericum extract or hypericin on regional levels of serotonin (5-HT), norepinephrine, dopamine and their metabolites in the rat brain. We focused our interest on the hypothalamus and hippocampus, as these brain regions are thought to be involved in antidepressant drug action. Imipramine (15 mg/kg, p.o.), Hypericum extract (500 mg/kg, p.o.), and hypericin (0.2 mg/kg, p.o.) given daily for 8 weeks significantly increased 5-HT levels in the hypothalamus (P<0.05). The 5-HT turnover was significantly lowered in both brain regions after 8 weeks of daily treatment with the Hypericum extract (both P<0.05). Consistent changes in catecholamine levels were only detected in hypothalamic tissues after long-term treatment. Comparable to imipramine, Hypericum extract as well as hypericin significantly decreased 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the hypothalamus (P<0.01). Our data clearly show that long-term, but not short-term administration of St. Johns wort and its active constituent hypericin modify levels of neurotransmitters in brain regions involved in the pathophysiology of depression.

Step by step removal of hyperforin and hypericin: activity profile of different Hypericum preparations in behavioral models

Herbal extracts of Hypericum perforatum L. (St. Johns wort, SJW) are now successfully competing for status as a standard antidepressant therapy. Because of this, great effort has been devoted to identifying the antidepressive active compounds. In the present study we used the following strategy to evaluate the relative pharmacological importance of various extract components: 1. preparation of an hydroalcoholic SJW extract containing both hyperforin (3.2%) and hypericin (0.15%) (extract A); 2. step by step removal of hyperforin and hypericin led to the following extracts: Extract B, devoid of hyperforin but still containing hypericin (0.14%) and Extract C, free of hypericin and hyperforin but enriched in flavonoids ( approximately 12%). We characterized the in vivo activity profile of all three preparations using the tail suspension test (TST) in mice and the forced swimming test (FST) in rats as screening models. We further investigated the activity of pure hyperforin. Extract B and C (500 mg/kg each) as well as pure hyperforin (8 mg/kg) significantly shortened immobility time in the TST after acute pre-treatment whereas extract A was inactive. In the FST all three extracts decreased immobility time in a dosage of 500 mg/kg after acute as well as after repeated treatment. The present results clearly show that an SJW extract free of hyperforin and hypericin exerts antidepressant activity in behavioral models, supporting our working hypothesis that flavonoids are part of the constituents responsible for the therapeutic efficacy of SJW extracts. We also could show that hyperforin contributes to the beneficial properties of SJW extract, confirming the hypothesis that the crude SJW extract contains several constituents with antidepressant activity.

St John's wort, hypericin, and imipramine: a comparative analysis of mRNA levels in brain areas involved in HPA axis control following short-term and long-term administration in normal and stressed rats.

Clinical studies demonstrate that the antidepressant efficacy of St Johns wort (Hypericum) is comparable to that of tricyclic antidepressants such as imipramine. Onset of efficacy of these drugs occurs after several weeks of treatment. Therefore, we used in situhybridization histochemistry to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, Hypericum extract, and hypericin (an active constituent of St Johns wort) on the expression of genes that may be involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Imipramine (15 mg kg−1), Hypericum (500 mg kg−1), and hypericin (0.2 mg kg−1) given daily by gavage for 8 weeks but not for 2 weeks significantly decreased levels of corticotropin-releasing hormone (CRH) mRNA by 16–22% in the hypothalamic paraventricular nucleus (PVN) and serotonin 5-HT1A receptor mRNA by 11–17% in the hippocampus. Only imipramine decreased tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (by 23%), and only at 8 weeks. The similar delayed effects of the three compounds on gene transcription suggests a shared action on the centers that control HPA axis activity. A second study was performed to assess the effects of long-term imipramine and Hypericum administration on stress-induced changes in gene transcription in stress-responsive circuits. Repeated immobilization stress (2 h daily for 7 days) increased mRNA levels of CRH in the PVN, proopiomelanocortin (POMC) in the anterior pituitary, glutamic acid decarboxylase (GAD 65/67) in the bed nucleus of the stria terminalis (BST), cyclic AMP response element binding protein (CREB) in the hippocampus, and TH in the locus coeruleus. It decreased mRNA levels of 5-HT1A and brain-derived neurotrophic factor (BDNF) in the hippocampus. Long-term pre-treatment with either imipramine or Hypericum reduced to control levels the stress-induced increases in gene transcription of GAD in the BST, CREB in the hippocampus, and POMC in the pituitary. The stress-induced increases in mRNA levels of CRH in the PVN and TH in the locus coeruleus were reduced by imipramine but not by Hypericum. The stress-induced decreases in BDNF and 5-HT1AmRNA levels were not prevented by either drug. Taken together, these data show: (1) that Hypericum and hypericin have delayed effects on HPA axis control centers similar to those of imipramine; and (2) that select stress-induced changes in gene transcription in particular brain areas can be prevented by long-term treatment with either the prototypic tricyclic antidepressant imipramine or the herbiceutical St Johns wort. However, imipramine appears to be more effective in blocking stress effects on the HPA axis than the plant extract.

Cimicifuga extract BNO 1055: reduction of hot flushes and hints on antidepressant activity

Ethanolic- and isopropanolic-aqueous extracts of Cimicifuga racemosa are used for the treatment of climacteric complaints. As hot flushes and psychic complaints seem to be special targets for Cimicifuga extracts in clinical studies, these parameters were studied in experimental animals. Hot flush equivalents were measured in castrated rats as a quick increase in peripheral temperature with the aid of a transmitter implanted subcutaneously on the ventral side. The hot flush equivalents proved to respond to estrogen and the antidopaminergic drug veralipride but they were also reduced very effectively by Cimicifuga extract BNO 1055 (which is contained in Klimadynon/Menofem). In addition, an ethanolic-aqueous extract of C. racemosa was studied in the tail suspension test (TST), a behavioural test indicative for antidepressant activity. A significant decrease of the period of immobility was observed after treatment with 30 mg/kg body weight (bw) imipramine or with 50 or 100 mg/kg bw Cimicifuga extract. These findings in pharmacological tests-a reduction of the frequency of hot flush equivalents and hints on antidepressant activity of Cimicifuga extracts-are in good agreement with the therapeutical responses in climacteric women.

Compensatory weight gain due to dopaminergic hypofunction: new evidence and own incidental observations

There is increasing evidence for a role of dopamine in the development of obesity. More specifically, dopaminergic hypofunction might lead to (over)compensatory food intake. Overeating and resulting weight gain may be induced by genetic predisposition for lower dopaminergic activity, but might also be a behavioral mechanism of compensating for decreased dopamine signaling after dopaminergic overstimulation, for example after smoking cessation or overconsumption of high palatable food. This hypothesis is in line with our incidental finding of increased weight gain after discontinuation of pharmaceutical dopaminergic overstimulation in rats. These findings support the crucial role of dopaminergic signaling for eating behaviors and offer an explanation for weight-gain after cessation of activities associated with high dopaminergic signaling. They further support the possibility that dopaminergic medication could be used to moderate food intake.

Hyperforin-containing extracts of St John's wort fail to alter gene transcription in brain areas involved in HPA axis control in a long-term treatment regimen in rats.

We previously showed that a methanolic extract of St Johns wort (SJW) (Hypericum) and hypericin, one of its active constituents, both have delayed regulation of genes that are involved in the control of the hypothalamic–pituitary–adrenal (HPA) axis. Hyperforin, another constituent of SJW, is active in vitro and has been proposed to be the active constituent for therapeutic efficacy in depression. We therefore examined if hyperforin has delayed effects on HPA axis control centers similar to those of Hypericum and hypericin. We used in situ hybridization histochemistry to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) oral administration of two hyperforin preparations, fluoxetine (positive control), and haloperidol (negative control) on the expression of genes involved in the regulation of the HPA axis. Fluoxetine (10 mg/kg) given daily for 8 weeks, but not 2 weeks, significantly decreased levels of corticotropin-releasing hormone (CRH) mRNA by 22% in the paraventricular nucleus (PVN) of the hypothalamus and tyrosine hydroxylase (TH) mRNA by 23% in the locus coeruleus. Fluoxetine increased levels of mineralocorticoid (MR) (17%), glucocorticoid (GR) (18%), and 5-HT1A receptor (21%) mRNAs in the hippocampus at 8, but not 2, weeks. Comparable to haloperidol (1 mg/kg), neither the hyperforin-rich CO2 extract (27 mg/kg) nor hyperforin-trimethoxybenzoate (8 mg/kg) altered mRNA levels in brain structures relevant for HPA axis control at either time point. These data suggest that hyperforin and hyperforin derivatives are not involved in the regulation of genes that control HPA axis function.

The relative merits of polyethyleneglycol as a separating agent in the radioimmunoassay of thyroid hormones

Polyethyleneglycol (PEG) has been recommended as a separating agent in the assay of some peptide hormones (Desbuquois, B. and Aurbach, G.D. (1971) J. Clin. Endocrinol. 33, 732) and several substances of low molecular weight (Ratcliffe, J.G. (1974) Br. Med. Bull. 30, 32). In the present study the PEG-separation technique has been modified and adapted for the assay of thyroid hormones. Separation with PEG has the advantage of being cheap, rapid and relatively non-susceptible to disturbances as compared with the charcoal and double-antibody-solid phase techniques. The influence of different buffer systems, varying pH and ionic strength, on the precipitation process with PEG also has been investigated. Of the different systems tested barbital buffer containing 0.1% human serum albumin proved to be the best, preferably in the presence of bovine gamma-globulin. In the radioimmunoassay of T3 variations in pH and ionic strength are of minor importance whereas in the radioimmunoassay of T4 the adherence to a certain pH is recommended. Barbital buffer containing 0.1% bovine serum albumin was inadequate in the T3 radioimmunoassay, while Tris and phosphate buffers did not give satisfying results for either radioimmunoassay.

Tylosin inhibits the steroidogenesis in mouse Leydig cells “in vitro”

Former investigations in rats revealed effects of tylosin on the pituitary gonadal axis. After 15 days treatment the animals showed reduced weights of the seminal vesicles, increased pituitary weights, diminished LH/FSH stores in the pituitary and reduced peripheral levels of LH. To investigate if, in addition, the antibiotic exerts a direct effect on the steroidogenesis, the reactivity of Leydig cells was determined: a) after 8 days pretreatment of the donor mice in vivo; b) after addition of the antibiotic in vitro; c) after addition of the antibiotic in vitro in presence of dibutyryl cyclic AMP or hydrocholesterol. Tylosin caused an inhibition of basal and stimulated testosterone production, all the same if it was applied in vivo or in vitro. Moreover the increase in testosterone production caused by dibutyryl cyclic AMP was inhibited as well as that caused by addition of hydrocholesterol. These data give rise to the suspicion that tylosin inhibits directly intracellular steps of testosterone biosynthesis of Leydig cells.

Effects of Lithospermum officinale and Related Plants on Hypophyseal and Thyroid Hormones in the Rat

AbstractThe antihormonal activity of Lithospermum officinale (Boraginaceae) and Lycopus virginicus and other Lamiaceae has been investigated in the rat. L. officinale cold water freeze dried extracts (FDE) significantly lowered thyroid hormone content in the serum whereas an inactivated extract exhibited a considerable loss of biological activity. The efficacy of different plant extracts greatly depended on the extraction procedure: extraction of powdered leaves with boiling water or ethanol yielded FDEs without thyroid hormone-lowering capacity. The chemical oxidation of a hot-water (100°C) extract by KMnO4 served to reintroduce the antihormonal effectiveness. In a goiter suppression test, the chronic administration of L. officinale FDE greatly suppressed TSH-levels and consequently goiter weight. The antithyrotropic and antithyroidal activity of a variety of plant extracts was accompanied by an additional prolactin diminution. Lithospermum officinale exhibited a strong antigonadotropic effectiveness and...

Embryotoxicity study of propenoic acid, 3-(4-methoxyphenyl)-3-methylbutylester in the wistar rat

3-(4-methoxyphenyl)-3-methylbutylester, propenoic acid (MPMBE; CAS no. 71617-10-2), a UVB-light filter used for sun protection of the skin, was administered once daily by intragastric gavage to pregnant Wistar rats on days 6-15 of gestation. Doses of 0.25 ml/kg/day (study group: D0.25), 0.75 ml/kg/day (D0.75) and 2.25 ml MPMBE/kg/day (D2.25) were applied. A positive control group (Pos) received 15 mg (all trans) retinoic acid/kg/day. MPMBE revealed some toxic effects in the dams of the group receiving the highest dose (D2.25): marked loss of body weight, polydipsia, reduced food consumption and intensified loss and thinning of hair. Only thinning or partial loss of hair without any other harmful effects was seen in the dams of group D0.75. An increase in embryonic deaths was striking in group D2.25; the living foetuses revealed signs of retarded development, but no major external or internal anomalies as signs of teratogenicity. This study was not able to present any teratogenic effects induced by MPMBE in the offspring, even at very high oral doses (> 2 g/kg/day) that caused substantial toxicity in the dams. In conclusion, concerning embryotoxicity an oral dose of 250 mg/kg/day MPMBE is regarded as a safe no-observed-effect level, whereas even 750 mg/kg/day MPMBE--following acknowledged rules--can be judged as a borderline no-observed-adverse-effect level.