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Terpenoids in plants are mainly synthesized by terpene synthases (TPSs), which play an important role in plant-environment interactions. Gossypium arboreum is one of the important cotton cultivars with excellent pest resistance, however, the biosynthesis of most terpenoids in this plant remains unknown. In this study, we performed a comparative transcriptome analysis of leaves from intact and Helicoverpa armigera-infested cotton plants. The results showed that the H. armigera infestation mainly induced the JA signaling pathway, ten TPS genes were differentially expressed in G. arboreum leaves. Among them, a novel terpene synthase, GaTPS1, was heterologously expressed and functionally characterized in vitro. The enzymatic reaction indicated that recombinant GaTPS1 was primarily responsible for the production of (E)-α-bergamotene. Moreover, molecular docking and site-directed mutagenesis analysis demonstrated that two amino acid residues, A412L and Y535F, distinctly influenced the catalytic activities and product specificity of GaTPS1. The mutants GaTPS1-A412L and GaTPS1-Y535F resulted in a decrease in the proportion of products (E)-α-bergamotene and D-limonene, while an increase in the proportion of products (E)-β-farnesene, α-pinene and β-myrcene. Our findings provide valuable insights into understanding the molecular basis of terpenoid diversity in G. arboreum, with potential applications in plant metabolism regulation and the improvement of resistant cotton cultivars., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

mGlu2/3 Receptors (LY354740) in Anxiety mGlu2/3 receptors when activated decrease glutamate excitation on limbic synapses involved in anxiety. The orally active agonist compound LY354740 (or prodrug LY544344) was active in animal and human models of stress/anxiety. Later clinical studies showed efficacy in generalized anxiety in patients, validating this mechanism clinically. However, the compound was terminated due to rodent seizures in long-term toxicology studies., (Copyright © 2024. Published by Elsevier Inc.)

The present study aimed to examine the effect of Calotropis procera (Aiton) and its major cardenolides; calotropin and gomphogenin on ovariectomy-induced osteoporosis in rats. Osteoporotic rats were orally treated with C. procera alcoholic extract (100 mg/kg), calotropin (CLT; 100 μg/kg) and gomphogenin (GPG; 100 μg/kg) for 14 consecutive days. Bone resorption/formation biomarkers; bone specific alkaline phosphatase (BALP), osteoprotegerin (OPG) and nuclear factor-κβ ligand (RANKL) as well as serum calcium and phosphorus were assessed 24 h after last doses of treatments. Serum levels of estradiol (E2) and catalase were also measured. Oral treatment with C. procera extract, CLT and GPG caused E2 restoration to normal level with a marked regulation in the RANKL/OPG axis. Serum phosphorus and calcium were up-leveled whereas BALP was downregulated. Histopathological examination, bone histomorphometric analysis and immunohistochemical staining for osteopontin (OPN) inspection further emphasized the aforementioned outcomes. The results revealed the superiority of CLT and to a lesser extent GPG osteoporotic effect over C. procera extract. Molecular docking of the two compounds on ER-α and RANKL/OPG complex showed noteworthy binding affinities which also confirmed the supremacy of CLT due to the additional hydrogen bonding of the hydroxyl groups of the sugar moiety with RANKL/OPG complex. Finally, it is concluded that CLT and GPG from C. procera hinder bone turnover by decreasing osteoclastic bone cells activity and increasing calcium mineralization thus suppressing bone remodeling and preventing bone infirmity in OVX osteoporotic rats directly via binding to RANKL/OPG complex and ER-α and indirectly through elevating level of E2., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

There are limited studies that report the physiological levels of H 2 S in the eye. The currently available UV/Vis methods lack the required sensitivity and precision. Hence, the purpose of this study was to develop and validate a sensitive and robust pre-column derivatization LC-MS/MS method to measure changes in H 2 S levels in tissues from isolated porcine eyes. H 2 S was derivatized and an LC-MS/MS method was developed to monitor the derivatized product, Sulfide-dibimane (Sdb) using a reverse phase Waters Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm). H 2 S quantification was performed using multiple-ion reaction monitoring (MRM) in positive mode, with the transitions of m/z 415.0 → m/z 223.0 for Sdb and m/z 353.0 → m/z 285.0 for internal standard (griseofulvin). This method provided a suitable way to quantify H 2 S and was then successfully adapted to measure H 2 S levels in isolated porcine iris-ciliary body tissues previously treated in the presence or absence of varying concentrations of lipopolysaccharide (LPS, 5-100 ng/ml), a pro-inflammatory agent. Isolated iris-ciliary bodies (ICB) from porcine eyes were cut into quadrants of approximately 50 mg and homogenized using a 1:3 volume of homogenizing buffer. H 2 S in the supernatant was then derivatized with monobromobimane and quantified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Understanding the interactions between different environmental pollutants is necessary in ecotoxicology since environmental contaminants never appear as single components but rather in combination with other substances. Heavy metals and pesticides are commonly detected in the environment, but the characterization of their mixture toxicity has been inadequately explored. This research aimed to elucidate the mixture impacts of the heavy metal lead (Pb) and the pesticide procymidone (PCM) on the hook snout carp (Opsariichthys bidens) using an array of biomarkers. The data showed that Pb and PCM possessed almost equivalent acute toxicity to the animals, with 4-days LC 50 values of 120.9 and 85.15 mg L -1 , respectively. Combinations of Pb and PCM generated acute synergistic effects on O. bidens. The contents of malondialdehyde (MDA), antioxidative (SOD), apoptotic (caspase-9), and detoxifying enzymes glutathione S-transferase (GST) and cytochrome P450 (CYP450) significantly changed after most of the mixture exposures compared with the baseline level and the corresponding individual exposures. This suggests the induction of oxidative stress, cell damage, and detoxification dysfunction. The expressions of eight genes (mn-sod, cu-sod, p53, cas3, erβ1, esr, ap, and klf2α) associated with oxidative stress, cell apoptosis, immune response, and hormonal functions exhibited pronounced changes when challenged with the mixture compared to the individual treatments. This indicates the occurrence of immune dysregulation and endocrine disorder. These findings provide an overall understanding of fish upon the challenge of sublethal toxicity between Pb and PCM and can be adopted to evaluate the complicated toxic mechanisms in aquatic vertebrates when exposed to heavy metal and pesticide mixtures. Additionally, these results might guide environmental regulation tactics to protect the population of aquatic vertebrates in natural ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hypericum perforatum L. (Clusiaceae), commonly known as St. John's wort, has a rich historical background as one of the oldest and most widely studied herbal medicines. Hyperforin is the main antidepressant active ingredient of St. John's wort. In recent years, hyperforin has attached increasing attention due to its multiple pharmacological activities. In this review, the information on hyperforin was systematically summarized. Hyperforin is considered to be a lead compound with diverse pharmacological activities including anti-depression, anti-tumor, anti-dementia, anti-diabetes and others. It can be obtained by extraction and synthesis. Further pharmacological studies and more precise detection methods will help develop a value for hyperforin. In addition, structural modification and pharmaceutical preparation technology will be beneficial to promoting the research progress of hyperforin based innovative drugs. Although these works are full of known and unknown challenges, researchers are still expected to make hyperforin play a greater value., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Procymidone (PCM) is a fungicide commonly used to prevent and control plant diseases, and it is also an environmental endocrine disruptor that has a typical anti-androgen effect on the function and/or structure of the vertebrate reproductive system. The activation of the unfolded protein response (UPR) will fold the protein correctly to ensure the cell's survival. PCM regulates GRP78 by affecting the level of hormones, and there is a regulatory relationship between the UPR, the circRNAs and the miRNAs. In vivo experiments, PCM (suspended in soybean oil) was orally administered to adolescent female mice for 21 days in 3 different doses of 50 mg kg -1 day -1 (low dose), 100 mg kg -1 day -1 (medium dose) and 200 mg kg -1 day -1 (high dose) to cause ovaries and uteruses damage, and in vitro experiments, various doses of PCM from 0.33 × 10 -5 (low dose) to 1 × 10 -5 (medium dose) then 3 × 10 -5 M (high dose) were used to induce injury on the ovaries and uteri of the mice. We found out that both in vivo and in vitro, PCM caused dose-dependent damages to the ovaries and uteri, increased their circRNA Scar levels and decreased circZc3h4 abundance. Also, all UPR signaling pathways in the low-dose group and some in the middle-dose group were activated. It is speculated that UPR may antagonize the partial ovarian and uterine damage in adolescent mice induced by PCM at doses less than NOAEL via changes in circZc3h4 and circRNA Scar., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To assess the cost-effectiveness of 30 mg of mirogabalin versus no treatment or 300 mg of pregabalin in patients with diabetic peripheral neuropathic pain (DPNP) from a third-party perspective in Taiwan., Methods: A Markov model, developed with cycles of 2-week and a 1-year timeframes, consisted of 3 health states: mild, moderate, and severe pain. Average daily pain score (ADPS) was assessed at the end of each 2-week cycle. All patients entered the model in moderate (4 ≤ ADPS < 7) or severe (7 ≤ ADPS ≤ 10) pain health states. At the end of each cycle, patients remained in their assigned health state or transitioned to a different health state according to their pain score change. Efficacy data were informed by the pivotal phase III clinical trial (J303, NCT02318706) or by a network meta-analysis. Utility values were obtained from published literature, and resource use and cost data from Taiwanese clinical experts and Taiwan National Health Insurance Administration. One-way sensitivity, scenario, and probabilistic analyses were conducted to test the robustness of the results., Results: A head-to-head analysis demonstrated that 30 mg of mirogabalin is a cost-effective treatment option versus placebo for DPNP. The base-case deterministic analysis estimated quality-adjusted life years (QALY) gains of 0.02 at an incremental cost of 9697 New Taiwan dollars (NT$) (equal to $323) versus placebo (incremental cost-effectiveness ratio [ICER]: 489 310 NT$/QALY [$15 860/QALY]). Mirogabalin was also cost-effective compared with 300 mg of pregabalin (ICER: 18 476 NT$/QALY [$600/QALY]). Sensitivity and scenario analyses results confirmed the robustness., Conclusion: Economic analysis suggests that mirogabalin 30 mg, a potent and selective α2δ ligand, is a cost-effective treatment option for DPNP in Taiwan, with an ICER below the willingness-to-pay threshold., (Copyright © 2020. Published by Elsevier Inc.)

Botrytis cinerea is a necrotrophic fungal pathogen that affects a total of 586 genera representing approximately 1400 plant species. This pathogen produces two families of phytotoxins involved in its infection process i.e. botrydial and its relatives, and botcinic and botcineric acids and their relatives, botcinins. The botrydial biosynthetic cluster consists of seven genes, where the gene BcBOT4 encodes a cytochrome P450 monooxygenase that was shown to catalyse regio- and stereospecific hydroxylation at position C-4 of the presilphiperfolan-8-β-ol skeleton. The null mutant bcbot4Δ halted the production of botrydial and its derivatives, and instead accumulated tricyclic presilphiperfolane alcohol and overproduced a significant number of polyketides. A detailed study of the bcbot4Δ mutant led us to the isolation and characterization of five undescribed polyketides, three derived from botcinic and botcineric acids (botcinins H, I, J), one derived from the initial pentaketide (botcinin K), and one cinbotolide derivative (cinbotolide D). Botcinins are tetra-methylated tetraketides biosynthesized by the sequential assembly of a pentaketide (C10) based on an acetate primer unit which is lost through a retro-Claisen type C-C bond cleavage. The structural characterization of botcinin K showed a basic chemical structure corresponding to a botcinin (C14) derivative obtained directly from the original per-methylated pentaketide leading to the biosynthesis of botrylactone and other botcinins, confirming the previously proposed biosynthetic route., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Amomum kravanh is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of A. kravanh fruits was analyzed by gas chromatography-mass spectrometry, and 34 components were identified. 1,8-Cineole (68.42%) was found to be the major component, followed by α-pinene (5.71%), α-terpinene (2.63%), and β-pinene (2.41%). The results of antibacterial tests showed that the sensitivities to the essential oil of different foodborne pathogens tested were different based on the Oxford cup method, MIC, and MBC assays, and the essential oil exhibited the best antibacterial activity against Bacillus subtilis, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. Growth in the presence of Amomum kravanh at the MIC, as measured by monitoring optical density over time, demonstrated that the essential oil was bacteriostatic after 12 h to both B. subtilis and E. coli. Observations of cell membrane permeability, cell constituent release assay, and transmission electron microscopy indicated that this essential oil may disrupt the cell wall and cell membrane permeability, leading to leakage of intracellular constituents in both B. subtilis and E. coli.

An extract of the solid cultures of Sporormiella minimoides (Sporormiaceae) isolated as an endophytic fungus from Hintonia latiflora (Rubiaceae), yielded three polyketides, 3,6-dimethoxy-8-methyl-1H,6H-benzo[de]isochromene-1,9-dione, 3-hydroxy-1,6,10-trimethoxy-8-methyl-1H,3H-benzo[de]isochromen-9-one, and 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone, along with three known compounds, corymbiferone, ziganein, and brocaenol B. Their structures were characterized by spectrometric and spectroscopic methods. So as to be consistent the literature reports, 3,6-dimethoxy-8-methyl-1H,6H-benzo[de]isochromene-1,9-dione and 3-hydroxy-1,6,10-trimethoxy-8-methyl-1H,3H-benzo[de]isochromen-9-one were given the trivial names of corymbiferone C and corymbiferan lactone E, respectively. All isolates were tested as potential human calmodulin (hCaM) inhibitors using the fluorescent biosensor hCaM V91C-mBBr, but only 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone quenched significantly the extrinsic fluorescence of this biosensor, with a dissociation constant (Kd) value of 1.55 μM. Refined docking analysis predicted that 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone could also be bound to hCaM at site I displaying hydrophobic interactions with Phe19 and 68, Met51, 71, and 72, and Ile52 and 63 residues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Allergic contact dermatitis (ACD) is the most prevalent form of human immunotoxicity. It is caused by skin exposure to haptens, i.e., protein-reactive, low-molecular-weight chemical compounds, which form hapten-protein complexes (HPCs) in the skin, triggering the immune system. These immunogenic HPCs are elusive. In this study a series of thiol-reactive caged fluorescent haptens, i.e., bromobimanes, were deployed in combination with two-photon fluorescence microscopy, immunohistochemistry, and proteomics to identify possible hapten targets in proteins in human skin. Key targets found were the basal keratinocytes and the keratins K5 and K14. Particularly, cysteine 54 of K5 was found to be haptenated by the bromobimanes. In addition, elevated levels of anti-keratin antibodies were found in the sera of mice exposed to bromobimanes in vivo. The results indicate a general mechanism in which thiol-reactive haptens generate cryptic epitopes normally concealed from the immune system. In addition, keratinocytes and keratin seem to have an important role in the mechanism behind ACD, which is a subject for further investigations.

This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ( approximately 80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC(50) values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.

Clinical data indicate that hydroalcoholic extracts of Hypericum perforatum might be as valuable as conventional antidepressants in mild-to-moderate depression, with fewer side effects. One clinical trial using two extracts with different hyperforin contents indicated it as the main active principle responsible for the antidepressant activity. Behavioural models in rodents confirm the antidepressant-like effect of Hypericum extracts and also of pure hyperforin and hypericin. A hydroalcoholic extract lacking hyperforin also lacks the antidepressant-like effect. According to pharmacokinetic data and binding studies, it appears that the antidepressant effect of Hypericum extract is unlikely be due to an interaction of hypericin with central neurotransmitter receptors. The main in vitro effects of hyperforin (at concentrations of 0.1-1 microM) are non-specific presynaptic effects, resulting in the non-selective inhibition of the uptake of many neurotransmitters, and the interaction with dopamine D1 and opioid receptors. However, it is still not clear whether these mechanisms can be activated in vivo, since after administration of Hypericum extract brain concentrations of hyperforin are well below those active in vitro. In the rat, Hypericum extract might indirectly activate sigma receptors in vivo (through the formation of an unknown metabolite or production of an endogenous ligand), suggesting a new target for its antidepressant effects., (Copyright 2004 Elsevier Inc.)

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.

Repeated examination of the aerial parts of Hypericum perforatum yielded a new degradation product of hyperforin (1) namely deoxyfurohyperforin A (2), together with the previously identified furohyperforin (3), furoadhyperforin (4), furohyperforin A (5a and 5b), pyrano[7,28-b]hyperforin (6) and 3-methyl-4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-oxopropyl)-3-(4-methyl-3-pentenyl)-cyclohexanone (7). The antimicrobial activity of the compounds 3, 5a and 5b, 6 and 7 was tested against Staphylococcus aureus, Candida albicans, Bacillus subtilis and Escherichia coli.

Herbal extracts of Hypericum perforatum L. (St. John's 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.

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca(2+) ionophores: (i). ferutinin, an electrogenic naturally occurring ionophore, and (ii). the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca(2+)] ([Ca(2+)](c)), mitochondrial [Ca(2+)] ([Ca(2+)](m)) and mitochondrial membrane potential (deltapsi(m)) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca(2+)] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca(2+)](c) and [Ca(2+)](m) increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca(2+)](c) in response to ferutinin, [Ca(2+)](m) remained elevated for prolonged periods after the recovery of [Ca(2+)](c) levels despite washout of the compound. Both groups of Ca(2+) ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca(2+) and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca(2+)/2Na(+)-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.

Preparations from St. John's wort extracts are used in the treatment of depression in many countries and represent an accepted alternative to synthetic antidepressants or behavioural therapy. St. John's wort extracts are therefore used in a therapeutic area which extends well beyond the traditional field of herbal medicine. The current status of preclinical and clinical research is summarised. St. John's wort extract has a clear inhibitory effect on the neuronal uptake not only of serotonin, noradrenaline, and dopamine but also of gamma-aminobutyric acid (GABA) and L-glutamate. No other antidepressant shows an approximately equally broad inhibitory profile. In good agreement with the effects in various biochemical models of antidepressant action, many effects in a number of behavioural pharmacology models of antidepressant efficacy could also be demonstrated for St. John's wort extract. Similar doses of John's wort also cause changes in the above-mentioned neurotransmitter systems in the brain. Out of all individual substances of St. John's wort only hyperforin and its structural analogue adhyperforin inhibit the re-uptake of the investigated neurotransmitters. However, hyperforin does not act as a competitive inhibitor at the transmitter binding sites of the transporter proteins but it affects the sodium gradient which then leads to an inhibition of uptake. The broad spectrum of action which characterises St. John's wort extracts has only been described for the pure substance hyperforin. Over the past year a number of good clinical studies have been carried out which confirm the efficacy and tolerability of St. John's wort extracts in mild depressive disorders, even if the therapeutic efficacy has recently been questioned by an American study. All studies have confirmed the good tolerability of St. John's wort extract and the very low frequency of adverse events. However, some drug interactions have been found to occur with St. John's wort extract, a number of which are of clinical relevance. In summary, pharmacological activity and therapeutic efficacy of St. John's wort extract as an antidepressant are supported by a large number of scientific publications. Within the wide range of components in St. John's wort extract, hyperforin plays an important, if not an outstanding role.

Hyperforin is one of the most important active components in St. John's wort (Hypericum perforatum), a botanical dietary supplement used as an alternative treatment modality for mild to moderate depression. A solid-phase extraction (SPE) and an isocratic high-performance liquid chromatography (HPLC) analysis with ultraviolet (UV) detection were developed to determine hyperforin in human plasma samples. Benzo[k]fluoranthene was used as an internal standard. The absolute recovery for hyperforin was more than 89% for plasma concentrations ranging from 25 to 500 ng/ml. The linearity of calibration curves, inter-day and intra-day relative standard deviations were investigated. The limit of detection (LOD) of hyperforin was 4 ng/ml in plasma and the limit of quantitation (LOQ) was 10 ng/ml. Hyperforin concentrations in human plasma following St. John's wort administration were analyzed. The result suggests that this method is rapid, sensitive, reproducible and capable of quantitative analysis of hyperforin plasma concentrations.

Hyperforin is currently considered to be the major active antidepressant constituent of the medicinal herb St. John's wort (Hypericum perforatum L.). The mechanism of action however, is still largely unknown, although the involvement of sodium and calcium has been recently inferred. In the present study hyperforin (5 microM) significantly potentiated the release of endogenous aspartate and glutamate from mouse cortical slices when stimulated by veratridine or potassium. Hyperforin (5 microM) also stimulated the release of aspartate, glutamate, serine, glycine and GABA when perfused on its own. Perfusion of the sodium channel blocker, tetrodotoxin (TTX) inhibited the effect of hyperforin, whereas removal of extracellular calcium potentiated the effect. Our observations suggests that hyperforin increases the overflow of neurotransmitters from mouse cerebral cortex possibly through facilitating the entry of sodium into the neurone which leads to the release of calcium from intracellular stores.

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.

A method for the simultaneous extraction of hypericin and hyperforin from a St. John's Wort extract, which is used in case of moderate depressions and skin injuries, from human plasma and serum by liquid-liquid extraction (LLE) with n-hexane-ethylacetate (70:30, w/w) was developed. A reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV, fluorescence (FLD) and mass spectrometric (MS) detection using electrospray ionization (ESI) was used to identify and quantify hypericin and hyperforin in the extracts from blood samples. Linearity was obtained in the ranges 8.4-28.7 ng/ml (hypericin) and 21.6-242.6 ng/ml (hyperforin). Recoveries were between 32.2 and 35.6% for hypericin and 100.1 and 89.9% for hyperforin. Intra-day accuracy and precision for this method ranged between 3.2 and 4.3% and 2.6 and 2.8%, respectively. After validation of the LLE, the method was tested on real plasma samples which were obtained by ingestion of St. John's Wort extract capsules. Blood samples were taken 2, 4, and 6 h after ingestion. Finally, this method proved to be highly suitable for clinical and pharmacologically relevant studies.

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.

Hyperforin, hypericin and pseudohypericin are the main ingredients of St. John's wort extract, which is available over the counter for treatment of mild to moderate depression. To facilitate clinical studies we developed two sensitive HPLC methods for determination of hypericin/pseudohypericin and hyperforin, respectively, in human plasma samples. The achieved limits of quantitation of 0.25 ng/ml for hypericin and pseudohypericin and 10 ng/ml for hyperforin were low enough to allow determination of pharmacokinetic parameters of the substances. Following liquid-liquid extraction of human plasma the samples were separated by isocratic reversed-phase HLPC and analyzed using fluorimetric detection for hypericin/pseudohypericin and UV detection for hyperforin.

The alcoholic extract of the aerial parts of Hypericum perforatum L. finds wide application because of its antidepressant activity. The extract contains a number of constituents with documented biological activity including chlorogenic acid, a broad range of flavonoids, naphthodianthrones and phloroglucinols. Hyperforin and adhyperforin are the major phloroglucinol constituents found in the lipophilic fraction of the extracts. Since the stability of hyperforin has been shown to be limited, an investigation of the hyperforin degradation products using HPLC-electrospray ionization mass spectrometry and multiple mass spectrometry was undertaken.

St. John's wort (Hypericum perforatum) is an herbal compound used in the treatment of burns, bruises, swelling, anxiety, and most recently, mild to moderate depression. The present study was designed to evaluate the antioxidant properties of St. John's wort in both cell-free and human vascular tissue. The experiment was performed initially in a cell-free system using Krebs buffer and a combination of xanthine/xanthine oxidase to initiate the production of the superoxide radical. Additionally, human placental vein was incubated in Krebs buffer without xanthine or xanthine oxidase to study the effects of St. John's wort on human tissue in vitro. Commercially available formulations of St. John's wort, standardized to either hypericin or hyperforin, were dissolved in an alkaline solution, and the following dilutions were made: 1:1, 1:2.5, 1:5, 1:7.5, 1:10, and 1:20. Lucigenin chemiluminescence was used to measure free radical production in both systems. A pro-oxidant effect was seen at the highest concentration, 1:1. Lower concentrations revealed antioxidant properties of the compound. All dilutions below 1:1 in both systems showed a dose-related inverse relationship of superoxide inhibition. The largest suppression was seen at the most dilute concentration, 1:20. The addition of 10(-3) M tiron inhibited the chemiluminescence signal, thereby confirming the production of superoxide. The results of this study suggest that St. John's wort inhibits free radical production in both cell-free and human vascular tissue.

Flowers and fruits of St. John's wort collected in different Italian regions were evaluated for their naphtodianthrone (hypericin and pseudohypericin), phloroglucinol (hyperforin and adhyperforin) and flavonol (rutin, quercetin, quercitrin, isoquercitrin and hyperoside) content. The quantitative evaluation was performed by HPLC-DAD. The crude drug collected at the fruit ripening period had the highest content in phloroglucinols and the lowest level of both naphtodianthrones and flavonols. Phloroglucinols peaked in the samples collected in Puglia followed by Lombardia and Veneto, while hypericins and flavonols were highest in the samples harvested in Sardegna and Trentino.

The present paper describe investigations which demonstrate that hyperforin is not the only phloroglucinol derivative in extracts of the medicinal plant Hypericum perforatum L., which possess a biological activity. Hyperforin was the major lipophilic constituent in two different extracts, whereas the amount of adhyperforin was approximately 10 times lower. Adhyperforin, like hyperforin, is a potent inhibitor of the uptake of dopamine, serotonin and noradrenaline. Neither hyperforin nor adhyperforin inhibited binding of the cocaine analogue, [3H]WIN 35,428 to the dopamine transporter. However, the known antidepressives imipramine, nomifensine and fluoxetine all inhibited binding of [3H]WIN 35,428, indicating that hyperforin and adhyperforin do not bind to the same site on the dopamine transporter as these compounds. Furthermore, hyperforin and adhyperforin did not prevent dopamine binding, but inhibited dopamine translocation. Our studies further support recent reports suggesting that the effect of hyperforin on uptake of monoamines is probably not caused by a direct effect of hyperforin on known sites on the transporters.

Hyperforin is a constituent of Hypericum perforatum extracts (St. John's wort, H. perforatum), which have antidepressant action. Hyperforin was extracted from plasma utilising a solid-phase extraction procedure. Chromatography was performed by isocratic reversed-phase high-performance liquid chromatography with UV end-point detection. The calibration curve was linear over the range 0.15-3 microg per ml of plasma. The sensitivity for hyperforin was 4.5 ng on-column. Mean inter- and intra-assay relative standard deviations over the range of the standard curve were less than 5%. The absolute recovery for hyperforin averaged 97.8%.

Background: We studied the effect of a single dose of a formulation of a methanolic extract of Hypericum perforatum (HP), also known as St. John's wort, on plasma concentrations of growth hormone (GH), prolactin (PRL), and cortisol (CORT) in 12 healthy male volunteers., Methods: Subjects received 9 tablets of the finished product Jarsin 300 and placebo in a double-blind, balanced-order, cross-over design., Results: Following HP relative to placebo, there was a significant increase in plasma GH and a significant decrease in plasma PRL. Plasma CORT levels were unchanged., Conclusions: Taken together with data from animal experimental studies, the findings suggest that this dose of HP may increase some aspects of brain dopamine function in humans.

Effects of hyperforin, an acylphloroglucinol derivative isolated from antidepressive medicinal herb Hypericum perforatum (St. John's Wort), on voltage- and ligand-gated ionic conductances were investigated. Whole-cell patch clamp and concentration clamp techniques on acutely isolated hippocampal pyramidal neurons and on cerebellar Purkinje neurons of rat were used. At concentrations between 3 to 100 microM hyperforin induced a dose and time dependent inward current which completely stabilized within a few seconds. Although 1 microM hyperforin inhibited virtually all investigated conductances (GABA > or = I(Ca(N)) > I(Na) > I(Ca(P) > or = AMPA > or = I(K(A)) > NMDA > I(K(DR))), its effects on several of them could not be reversed by repeated washings. Dose response studies revealed that although AMPA induced current is inhibited by hyperforin in a competitive manner, these responses are not completely blocked by very high concentration of the agent. On the contrary, however, NMDA receptor-activated ionic conductance could be completely and uncompetitively inhibited by the agent. Taken together these observation not only reconfirm that hyperforin is a major neuroactive component of hypericum extracts but also demonstrate that this structurally unique and naturally abundant molecule is a potent modulation of mechanism involved in the control of neuronal ionic conductances. Various observed effects of hyperforin do not, however, seem to be mediated by one single molecular mechanism of action of the agent.