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     

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.     

Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort)

Hypericum perforatum L. (St. John's wort) is an herbal remedy widely used in the treatment of mild to moderate depression. Hypericin, a photosensitive napthodianthrone, is believed to be the compound responsible for reversing the depression symptoms. In this study, novel in vitro cell culture systems of H. perforatum were used to monitor the effect of elicitation on cell growth and production of hypericin. A dramatic increase in cell growth and hypericin production was observed after exposure to jasmonic acid (JA). However, other elicitors such as salicylic acid (SA) and fungal cell wall elicitors failed to show any stimulatory effect on either cell growth or hypericin production. Cell cultures treated with JA and incubated in the dark showed increased growth and hypericin production as compared to the cultures grown under light conditions. Jasmonate induction in dark conditions played an important role in growth and hypericin production in cell suspension cultures, to our knowledge an undocumented observation.     

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%。     

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.     

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.     

St. John's wort (Hypericum perforatum) counteracts cytokine-induced tryptophan catabolism in vitro

St. John's wort (Hypericum perforatum) is an ancient folk remedy that has antiviral and antibacterial properties. Anti-inflammatory effects of the plant have been described and the application of H. perforatum extract as an effective antidepressant is well established. In this study we assayed the effect of H. perforatum extract on cytokine-induced tryptophan degradation in human peripheral blood mononuclear cells. Simultaneously, changes in the production of the immune activation marker neopterin were monitored. Both these biochemical pathways are triggered by interferon-gamma. Our results show that extracts of H. perforatum strongly down-regulate mitogen-mediated tryptophan degradation in a dose-dependent manner. This effect seems to be based on a suppressive activity of H. perforatum on activated immunocompetent cells, resulting in a diminished production of interferon-gamma. In line with this finding, neopterin synthesis was strongly down-regulated by the plant extract. Our results suggest that the reduction of tryptophan degradation by H. perforatum might be important in the action of the plant as an antidepressant.     

Production of St. John's wort plants under controlled environment for maximizing biomass and secondary metabolites

Summary St. John's wort (Hypericum perforatum L.) is a medicinal plant used in the treatment of neurological disorders and has been recently shown to have anticancer potential. The principle medicinal components of St. John's wort are hypericin. pseudohypericin, and hyperforin. One of the problems associated with medicinal plant preparations including St. John's wort is the extreme variability in the phytochemical content, mostly due to environmental variability, and biotic and abiotic contamination during cropping. The current study was undertaken to transplant St. John's wort plants from in vitro bioreactors in a closed controlled environment system (CCES) with CO₂ enrichment for the optimized production of biomas and phytochemicals. The growth and levels of hypericin, pseudohypericin, and hyperforin in plants grown in CCES were compared with those of the greenhouse and in vitro-grown plants. The environmental parameters in the greenhouse were found to be variable whereas in the CCES these parameters were controlled. Generally, all the growth parameters and hypericin and psendohypericin levels were significantly higher in the plants grown in the CCES. These results provide the first indication that growing St. John's wort plants, under CO₂ enrichment in a closed environment system can enhance the biomass and medicinal contents. The adaptation of this growing system may be useful for the production of optimized products of St. John's wort and other medicinal species.     

St. John's wort (Hypericum perforatum L.) and kindling epilepsy in rabbit.

Effects of different extracts of Hypericum perforatum L. on the kindling epileptic discharges were analyzed. The experiment was carried out on Chinchilla rabbits with chronically implanted electrodes in cortical structures and hippocampus. In our study we used water, n-butanol and ether fractions (mass concentrations 0.1 g/ml) of crude ethanol extract of Hypericum perforatum. The particular extracts were given intramuscularly in single dose of 1 ml/kg BW. The bioelectric activity was registered before and after applications of each extracts. The obtained results show that the effect depends on the constituents present in particular fractions. The repression of epileptic activity was in correlation with the polarity of plant constituents. Most polar constituents that remained in water fraction exerted highest antiepileptic activity in all (100%) animals tested. Substances present in butanol fraction repressed the epileptic manifestations in 40% of animals with kindling epilepsy, whereas lipid-soluble constituents in ether fraction potentated the epileptic activity.     

Biogeographic variation in genetic variability,apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range

Background and Aims

St. John''s wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges.

Methods

Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93 %), while lower frequencies of hexaploid (6 %), diploid (<1 %) and triploid (<1 %) individuals were also identified.

Key Results

A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73 %) than sexual (24 %) seed, while hexaploids produced more sexual (73 %) than apomictic (23 %) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe.

Conclusions

The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.     

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.     

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.     

Elevated carbon supply altered hypericin and hyperforin contents of St. John's wort (Hypericum perforatum L. cv `New Stem') grown in bioreactors

For the high frequency selection of salt-tolerant doubled haploids (DHs) using rice anther culture, the efficiency of anther culture was investigated with different genotype, media condition and NaCl concentrations. The six F₁ hybrids obtained by backcross or three-way cross between indica and japonica differed in salt tolerance. The efficiencies of callus induction and plant regeneration was decreased by NaCl concentration and salt tolerance of donor variety, and those in japonicas were higher than those in indicas. The percentages of callus induction in Gyehwa 5 (japonica, tolerant) and IR61633-B-2-2-1 (japonica, sensitive) were 21.1 and 13.5% on agar medium containing 0.3% NaCl, respectively. The plant regeneration of callus derived from anther floating culture in liquid media was less than that on solid medium. In four F₁ hybrids, the frequencies of high salt-tolerant DHs were 21.4 and 8.9% in 0.3% NaCl medium and the control, respectively. The high frequency of salt-tolerant DHs could be selected in the callus induction medium (0.3% NaCl) and in the combinations crossed with salt-tolerant japonica as the third parent.     

Effect of eluent pH on the HPLC-UV analysis of hyperforin from St. John's Wort (Hypericum perforatum L.)

The effect of the pH of the mobile phase in HPLC analysis of hyperforin was investigated. Working with an extract of St. John's Wort (Hypericum perforatum L.) that is rich in hyperforin, significant differences were observed in conventional chromatograms depending on whether the mobile phase was acidic or alkaline. Chromatogram changes were paralleled by changes in the UV spectrum of the hyperforin peak. The structural changes in hyperforin occur in the chromatographic column itself, as has been confirmed by UV spectroscopy performed on a sample of purified hyperforin, which showed that the UV spectrum is indeed dependent on the pH of its environment.     

Short term treatment with St. John's wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse

This investigation was designed to determine whether St. John's wort (SJW)(435 mg/kg/d), a readily available antidepressant, or its purported active constituents hypericin (1 mg/kg/d) and hyperforin (10 mg/kg/d) were able to induce various hepatic cytochrome P450 (CYP450) isoforms. SJW, hypericin and hyperforin were administered to male Swiss Webster mice for four consecutive days and hepatic microsomes were prepared on day 5. None of the three treatments resulted in a statistical change in total hepatic CYP450 (SJW treated 0.95 +/- 0.09 nmol/mg vs control 1.09 +/- 0.14 nmol/mg). Furthermore, the catalytic activities of CYP1A2. CYP2E1 and CYP3A were unchanged from control following all three treatments as determined by ethoxyresorufin O-deethylation, p-nitrophenol hydroxylation and erythromycin N-demethylation respectively. Additionally, western immunoblotting demonstrated that there was no significant change in the polypeptide levels of any of the three isoforms. These results indicate that four days of treatment with moderate to high doses of SJW, hyperforin or hypericin fails to induce these CYP450 isoforms in the male Swiss Webster mouse.     

Modes of reproduction in Australian populations of Hypericum perforatum L. (St. John's wort) revealed by DNA fingerprinting and cytological methods.

Hypericum perforatum L. (St. John's wort) is widely used in homeopathic medicine, but has also become a serious weed in Australia and many other countries. Reproduction in H. perforatum was investigated using markers based on restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP). Between two Australian populations, plants displayed 14 polymorphisms from a total of 22 scorable RFLP markers when genomic DNA was probed with M13 bacteriophage, but individuals within each population exhibited identical RFLP fingerprints. Ninety-four percent of the progeny of four crosses made between the two populations exhibited identical fingerprint and ploidy level to the maternal parent, and probably originated apomictically. Seven seedlings with recombinant RFLP or AFLP fingerprints were found from a total of 121 progeny. Both molecular marker techniques detected the same recombinants from a subset of screened progeny. Cytological analysis showed that the seven recombinants comprised three tetraploids (2n = 4x = 32), three hexaploids (2n = 6x = 48), and one aneuploid (2n - 1 = 31), which suggested that the level of normal reduced embryo sacs was only 2.5%. These results are discussed in relation to the management of invasive populations, and the implications for plant breeding and production of St. John's wort for medicinal purposes.     

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.     

In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.