Karyotype Analysis of Hypericum Perforatum L.

A karyotype study was made on Hypericum perforatum using plants differentiated in vitro with different ploidy level. The chromosomes of this species are small, morphologically similar, median and submedian. In the basic chromosome set the most distinguishable is chromosome number 1 which was subjected to detail analysis. It was found that there are two types of this chromosome which contribute differentially in diploid, triploid and tetraploid plants.     

Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John's wort (Hypericum perforatum L. cv. Anthos) plants

Hypericum perforatum cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from ¹⁴C-tryptophan was recovered as ¹⁴C-indoleacetic acid, ¹⁴C-tryptamine, ¹⁴C-5-hydroxytryptophan, ¹⁴C-serotonin and ¹⁴C-melatonin in the treated St. John's wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light. Received: 9 November 1999 / Revision received: 16 December 1999 / Accepted: 21 December 1999     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

Somatic embryogenesis and plant regeneration from fragments of immature inflorescences and coleoptiles of durum wheat

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of serotonin and melatonin in plant morphogenesis: Regulation of auxin-induced root organogenesis in in vitro-cultured explants of st. John's Wort (Hypericum perforatum L.)

Summary St. John's wort (Hypericum perforatum cv. Anthos) is a medicinal plant with historical and anecdotal evidence of efficacy as an anti-depressant. Recent research has demonstrated an active biosynthetic pathway leading to the production of the mammalian neurohormone melatonin in St. John's wort plantlets. The objective of the current study was to assess the physiological role of melatonin and related indoleamines in plant morphogenesis. In the initial experiments, two of the indoleamines; serotonin and melatonin, were supplemented to the culture medium. In subsequent research, six inhibitors of auxin and indoleamine action or transport, 2,3,5-triiodobenzoic acid, p-chlorophenoxyisobutyric acid, p-chlorophenyl-alanine, d-amphetamine, fluoxetine (Prozac^TM), and methylphenidate (Ritalin^TM), were included in a culture medium in the presence or absence of the auxin, indoleacetic acid (IAA). The rate of de novo root and shoot organogenesis and the endogenous concentrations of auxin and indoleamines were determined in cultured explants. Significant reductions in de novo root regeneration were found to correspond with decreases in the pool of both IAA and melatonin. An increase in the endogenous concentration of melatonin was correlated with an increase in de novo root formation, and increased serotonin levels corresponded to increased shoot formation on the explants. Our findings provide the first evidence that a balance of the endogenous concentration of serotonin and melatonin may modulate plant morphogenesis in vitro.     

贯叶连翘的水培及其代谢产物检测

水培可诱导贯叶连翘组培苗生根能力强,根活力也增加;生根苗在1/6MS培养液中培养6周后的金丝桃素(HP)、假金丝桃素(PHP)和贯叶金丝桃素(HF)含量分别比基质[腐质土 蛭石(1:1)]中培养的提高10.13%、16.00%和61.36%。     

Factors affecting growth of cell suspension cultures of hypericum perforatum L. (St. John's wort) and production of hypericin

Summary Use of Hypericum perforatum L., commonly known as St. John's wort, has increased recently due to the pharmaceutical potential of hypericin, found in its leaves. Hypericin has been reported to effect a natural treatment for mild and moderate depression by increasing the concentration of neurotransmitters in the central nervous system. We have developed a novel cell culture system for in vitro growth and production of this species, suggesting a possible technology for large-scale production of hypericin. Leaf explants grown in Murashige and Skoog salts supplemented with 2,4-dichlorophenoxyacetic acid (0.90 μM) and kinetin (0.11 μM) gave maximum percentage callus formation compared to other medium treatments evaluated. Hypericin localization in cell phase and leaves was found to vary, with cell phase accumulating hypericin in special organelles and leaves accumulating it in vacuoles. Light and dark conditions, with cell aggregate size, played important roles in growth and hypericin production in cell suspension cultures.     

Evidence-based drug--herbal interactions

Due to the growing use of herbals and other dietary supplements healthcare providers and consumers need to know whether problems might arise from using these preparations in combination with conventional drugs. However, the evidence of interactions between natural products and drugs is based on known or suspected pharmacologic activity, data derived from in vitro or animal studies, or isolated case reports that frequently lack pertinent information. The usefulness of such information is questionable. More recently an increasing number of documented case reports, in vivo studies, and clinical trials have evaluated herbal-drug interactions. Results have sometimes been contradictory and more research is needed. Since there is a lack of rigorous studies that can establish the clinical significance of herb-drug interactions, an evidence-based evaluation of the current literature concerning commonly used herbal-drug interactions, as well as other dietary supplements, was conducted.     

Isolation and activity of the photodynamic pigment hypericin

Abstract. Hypericin, a photodynamic pigment, occurring in members of the Hypericaceae, can induce photosensitivity in grazing animals. The pigment has been isolated from the glandular trichomes located on the calyx of Hypericum hirsulum. Hypericin is shown to be capable of sensitizing the photo-oxidation of methyl linolenate. This activity is reduced in the presence of crocin, a carotenoid. Evidence for the generation of singlet molecular oxygen by hypericin is provided by the monitoring of oxygen consumption during the photosensitized oxidation of imidazole. Rates of oxygen consumption were modified by deuterium oxide and sodium azide. The photodynamic action of hypericin on pea leaf discs results in the promotion of photo-oxidative damage, measured by pigment loss and ethane production. These results are discussed in relation to the possible function of hypericin within the plant and the role of photo-dynamic reactions in nature.     

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos')

 St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N′-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l^–1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders. Received: 19 March 1999 / Revision received: 5 July 1999 · Accepted: 17 August 1999     

A review of clinical and experimental observations about antidepressant actions and side effects produced by Hypericum perforatum extracts

Hypericum perforatum is an herbaceous perennial plant, also known as "St. John's wort", used popularly as a natural antidepressant. Although some clinical and experimental studies suggest it has some properties similar to conventional antidepressants, the proposed mechanism of action seems to be multiple: a non-selective blockade of the reuptake of serotonin, noradrenaline and dopamine; an increase in density of serotonergic and dopaminergic receptors and an increased affinity for GABAergic receptors; moreover, the inhibition of monoaminoxidase enzyme activity has been involved. In any case, the increase of monoamine concentrations in the synaptic cleft resembles several actions exerted by clinically effective antidepressants. In the present article, we review some of the controversial evidence derived from clinical and experimental studies suggesting that H. perforatum exerts antidepressant-like actions, and we also review some of its side effects, such as nausea, rash, fatigue, restlessness, photosensitivity, acute neuropathy, and even episodes of mania and serotonergic syndrome when administered simultaneously with other antidepressant drugs. All of the foregoing suggests that H. perforatum extracts appear to exert potentially significant pharmacological activity involving several neurotransmission systems supposed to be involved in the pathophysiology of depression. However, little information regarding the safety of H. perforatum is available, including potential herb-drug interactions. There is a need for additional research on the pharmacological and biochemical activity of H. perforatum, as well as its side-effects and its several bioactive constituents to further elucidate the mechanisms of antidepressant actions.     

Extracts of St. John's wort and various constituents affect beta-adrenergic binding in rat frontal cortex

The present study was designed to get further insight into the mode of antidepressant action of extracts prepared from St. John's wort (SJW) and relevant active constituents. Down-regulation of central beta-adrenergic receptors (beta-AR's) has been widely considered a common biochemical marker of antidepressant efficacy. Although previous studies have reported a beta-AR down-regulation for SJW extracts, in vivo studies that compare the effects of SJW extracts with those of relevant active constituents on beta-AR density have not been done yet. We used quantitative radioligand receptor-binding-studies to examine in rats the effects of short-term (2 wks) and long-term (8 wks) administration of different SJW extracts and constituents on beta-AR binding in rat frontal cortex. The effects were compared to those of the standard antidepressants imipramine and fluoxetine. [125I]CYP binding to beta-AR was found to be decreased after short as well as after long-term treatment with imipramine (36%, 40%). Short-term treatment with fluoxetine decreased the number of beta-adrenergic receptors (17%) while long-term treatment with fluoxetine elicited an increase (14%) in beta-AR-binding. This effect was comparable to that of the lipophilic CO2 extract which decreased beta-AR-binding (13%) after two weeks and slightly increased the number of beta-AR's after 8 weeks (9%). Short-term treatment with the methanolic SJW extract decreased beta-AR-binding (14%), no effects for this extract were observed after 8 weeks. Treatment with hypericin led to a significant down-regulation (13%) of beta-AR's in the frontal cortex after 8-weeks, but not after 2 weeks, while hyperforin (used as trimethoxybenzoate, TMB), and hyperoside were ineffective in both treatment paradigms. Compared to the SJW extracts and single compounds the effect of imipramine on beta-AR-binding was more pronounced in both treatment paradigms.     

安徽产石蒜属植物三种酶同工酶的分析

采用聚丙烯酰胺凝胶电泳技术分离同工酶,首次分析了安徽产石蒜属６种植物十一个居群的过氧化物酶、酯酶、苹果酸脱氢酶三种酶同工酶,比较了居群间酶谱差异,并用数量分类方法,对酶谱资料进行了聚类分析（ＵＰＧＭＡ法）。结果表明：１、过氧化物酶、酯酶同工酶酶谱均显示出居群间的差异,而苹果酸脱氢酶同工酶酶谱居群间共性很大。２、酶谱特征显示,三种酶系统在居群间或居群间的差异,但种间居群的酶谱差异明显大于种内居群的差     

Liposome-cell interactions: In vitro discrimination of uptake mechanism and in vivo targeting strategies to mononuclear phagocytes

The interactions of liposomes with cells have been extensively studied to determine their potential use as vehicles for the delivery of drugs in vivo. Since intravenously administered liposomes are, for the most part, cleared by cells of the reticuloendothelial system (RES), considerable effort has been made to take advantage of this phenomenon rather than view it as an obstacle. Indeed, cells of the RES, in particular macrophages, have been shown to play a vital role in homeostasis and in host defence mechanisms against infection and neoplasia.

In this article, we present an overview of liposome-cell interactions, with particular emphasis on the techniques used to monitor the interaction of liposomes with macrophages. Specifically, we discuss methodologies which can be used to differentiate between liposome-cell fusion, adsorption and endocytosis in vitro. In addition, we outline the various strategies that have been employed for both actively and passively targeting liposomes to macrophages in vivo. We also review the rationale and various techniques for designing liposomes for enhanced macrophage uptake, which, in certain cases, results in the selective release of liposome-entrapped compounds in situ.     

St. John''s wort and depression: Efficacy, safety and tolerability-an update

St. John's wort (Hypericum perforatum L.) is a medicinal plant traditionally used, both externally and internally, in all Europe for many pathologies. Paracelsus named it “arnica of the nerves” because of its empirical use in nervous diseases. In the last two decades many studies have proved the efficacy of some St. John's wort extracts in mild to moderate depression and it has been successful as an antidepressant both in Europe and the US. Its high efficacy and tolerability is unquestionable and from the clinical studies the activity is comparable to other antidepressants while lacking major side effects, making it a safe antidepressant.

However, recently its potential to induce the metabolism of co-administered medications has been reported because it may potentate certain enzymes of the cytochrome P450 enzyme system. This resulted in a lowering of serum concentration of a number of concomitant drugs, including warfarin, digoxin, theophylline, cyclosporin, and indinavir. Many drugs and also several common foods and drinks can influence this enzyme system. So, even if its safety has been well established, physicians should be aware that St. John's wort administration might significantly affect other prescribed medicines.     

Differential effects of selegiline on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

The action of selegiline, a selective and irreversible inhibitor of monoamine oxidase B, commonly applied in the therapy of Parkinson's disease, on glucose formation was investigated in isolated rabbit hepatocytes and kidney-cortex tubules, maintaining the whole body glucose homeostasis via gluconeogenic pathway activity. An intensive hepatic metabolism of selegiline resulted in formation of selegiline-N-oxide, desmethylselegiline, methamphetamine and amphetamine, whereas during slow degradation of the drug in freshly isolated renal tubules selegiline-N-oxide was mainly produced. At 100 μM concentration selegiline markedly diminished glucose synthesis in isolated renal tubules incubated with dihydroxyacetone or alanine + glycerol + octanoate (by about 60 and 30%, respectively), while at 5 μM concentration a similar degree of inhibition was achieved in renal tubules grown in primary culture under the same conditions (about 40 and 60%, respectively). Moreover, desmethylselegiline and selegiline-N-oxide considerably diminished glucose production in renal tubules whereas selegiline and its metabolites did not affect gluconeogenesis in hepatocytes. Contrary to control animals, following selegiline administration to alloxan-diabetic rabbits for 8 days (10 mg kg⁻¹ body wt. daily) the blood glucose and serum creatinine levels were significantly diminished, suggesting a decrease in renal gluconeogenesis and improvement of kidney functions.

Since in renal tubules selegiline induced a decline in the intracellular levels of gluconeogenic intermediates and ATP content accompanied by a decrease in oxygen consumption in both kidney-cortex and hepatic mitochondria it seems possible that its inhibitory action on renal gluconeogenesis might result from an impairment of mitochondrial function, while an intensive selegiline metabolism in hepatocytes causes decrease of its concentration and in consequence no inhibition of gluconeogenesis. In view of these observations it is likely that an increased risk of selegiline-induced hypoglycemia might be expected particularly in patients exhibiting an impairment of liver function and following transdermal administration of this drug, i.e. under conditions of increased serum selegiline concentrations.     

Molecular cloning and tissue-specific expression of two cDNAs encoding polyketide synthases from Hypericum perforatum

Two previously uncharacterized cDNAs encoding for polyketide synthases (PKSs), designated as HpPKS1 and HpPKS2, were isolated from Hypericum perforatum. The full-length HpPKS1 was 1573bp containing an open reading frame (ORF) of 1161bp encoding for a 386 amino acid protein. The full-length cDNA of HpPKS2 was 1559bp with an ORF of 1182bp encoding for a 393 amino acid protein. The highly conserved catalytic amino acid residues common to plant-specific PKSs were preserved in both genes. HpPKS1 and HpPKS2 exhibited distinct tissue-specific expression patterns in H. perforatum. The HpPKS1 expression was highest in flower buds and lowest in root tissues. The expression of HpPKS2 was found to be high in flower buds and leaf margins and low in leaf interior parts, stems and roots. The expression of the HpPKS1 was found to correlate with the concentrations of hyperforin and adhyperforin while the expression of HpPKS2 showed correlation with the concentrations of hypericins and pseudohypericins in H. perforatum tissues.     

Gene expression-based in vivo and in vitro prediction of liver toxicity allows compound selection at an early stage of drug development

We have analyzed gene expression and histopathology of rat liver treated with a histamine-3 receptor inverse agonist under development for the treatment of obesity 24 h after a single acute administration. While histopathology did not identify a clear liver toxicity, analysis of gene changes strongly suggested the development of toxicity. This prediction was confirmed in a 2-week repeat-dose rat study where prominent liver pathology occurred, while gene changes that lead to the prediction persisted. A subset of these genes was analyzed in vitro in both rat and human hepatocytes to reveal the potential relevancy of the findings for the situation in humans. This comprehensive analysis of the development compound at the gene expression level allowed interpretation of findings of the follow-up compound in a frontloaded 24-h single-dose acute study that was initiated before regular 2-week repeat-dose studies started. The high similarity of the follow-up compound to the lead compound based on gene expression lead to the immediate termination of the development program for this compound series. Our data demonstrate the value of genomics-based early toxicity prediction in short-term in vivo studies for the characterization of compounds to allow prioritization and selection of suited candidates before compound-, animal-, and cost-intensive longer term studies are undertaken.     

In vitro and in vivo interactions of homocysteine with human plasma transthyretin

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer's disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35S-L-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with beta-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys10-S-S-homocysteine was then identified in vivo in plasma from normal donors, patients with end-stage renal disease, and homocystinurics by immunoprecipitation and high performance liquid chromatography/electrospray mass spectrometry. The ratios of transthyretin-Cys10-S-S-homocysteine and transthyretin-Cys10-S-S-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys10-S-S-cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys10-S-S-homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.