Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages

Hypericum perforatum (St. John’s wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies established synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of a H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC–MS analysis established the 4 compounds were present in the H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract’s inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study established that a different group of major anti-inflammatory constituents were present in H. gentianoides, while showing that the previously identified 4 compound combination was important for H. perforatum’s anti-inflammatory potential.     

Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation

Changes in phenolic metabolism after elicitation with Colletotrichum gloeosporioides (CG) has been studied in Hypericum perforatum L. (HP) cell suspension cultures. Soluble phenolics were analysed by HPLC-DAD and HPLC-DAD-MS/MS. HP cultures elicited with the CG elicitor showed a significant increase in xanthone accumulation. Xanthone accumulation increased twelve fold when the cells were primed with methyl-jasmonate (MeJ) or salicylic acid (SA), before elicitation. HP cultures exposed only to MeJ produced a set of flavonoids, the flavones which represent a substantial part (approx. 40%) of the total flavonoids accumulated in these cells. The possible importance of xanthones as a component of defence mechanism of HP against biotic stress is discussed.     

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.     

Antioxidant properties and protective effects of a standardized extract of Hypericum perforatum on hydrogen peroxide-induced oxidative damage in PC12 cells

Free radical scavenging and antioxidant activities of a standardized extract of Hypericum perforatum (SHP) were examined for inhibition of lipid peroxidation, for hydroxyl radical scavenging activity and interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). Concentrations between 1 and 50 microg/ml of SHP effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe2+/ascorbate or NADPH system. The results showed that SHP scavenged DPPH radical in a dose-dependent manner and also presented inhibitory effects on the activity of xanthine oxidase. In contrast, hydroxyl radical scavenging occurs at high doses. The protective effect of the standardized extract against H2O2-induced oxidative damage on the pheochromocytoma cell line PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays, caspase-3-enzyme activity and accumulation of reactive oxygen species [2',7'-dichlorofluorescin (DCF) assay]. Following 8-h cell exposure to H2O2 (300 microM), a marked reduction in cell survival was observed, which was significantly prevented by SHP (pre-incubated for 24 h) at 1-100 microg/ml. In a separate experiment, different concentrations of the standardized extract (0.1-100 microg/ml) also attenuated the increase in caspase-3 activity and suppressed the H2O2 -induced reactive oxygen species generation. Taken together, these results suggest that SHP shows relevant antioxidant activity both in vitro and in a cell system, by means of inhibiting free radical generation and lipid peroxidation.     

Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow

Background and Aims Hypericum perforatum

(St. John''s wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixis. Here, hypotheses on the evolutionary origin of St. John''s wort are tested and contrasted with the subsequent history of interspecific gene flow.

Methods

Extensive field collections were analysed for quantitative morphological variation, ploidy, chromosome numbers and genetic diversity using nuclear (amplified fragment length polymorphism) and plastid (trnL-trnF) markers. The mode of reproduction was analysed by FCSS (flow cytometric seed screen).

Key Results

It is demonstrated that H. perforatum is not of hybrid origin, and for the first time wild diploid populations are documented. Pseudogamous facultative apomictic reproduction is prevalent in the polyploids, whereas diploids are predominantly sexual, a phenomenon which also characterizes its sister species H. maculatum. Both molecular markers characterize identical major gene pools, distinguishing H. perforatum from H. maculatum and two genetic groups in H. perforatum. All three gene pools are in close geographical contact. Extensive gene flow and hybridization throughout Europe within and between gene pools and species is exemplified by the molecular data and confirmed by morphometric analyses.

Conclusions Hypericum perforatum

is of a single evolutionary origin and later split into two major gene pools. Subsequently, independent and recurrent polyploidization occurred in all lineages and was accompanied by substantial gene flow within and between H. perforatum and H. maculatum. These processes are highly influenced by the reproductive system in both species, with a switch to predominantly apomictic reproduction in polyploids, irrespective of their origin.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE₂ (KH₂-PGE₂) which has a longer half-life in blood. We examined the effects of PGE₂ infusion and alterations in lipolysis , and of clotting, prolonged storage and hemolysis , on KH₂-PGE₂ immunoreactivity in unextracted human plasma and serum samples. Indeed KH₂-PGE₂ levels rose several hundred fold during infusions of PGE₂ at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH₂-PGE₂ levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH₂-PGE₂ levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH₂-PGE₂ levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH₂-PGE₂ measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores or from disrupted membrane phospholipids . Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Controlled trial of induction of labor by vaginal suppositories containing prostaglandin E2

A group of 84 women at 39 – 43 weeks of pregnancy were randomly allocated to a blind trial of induction of labor with vaginal suppositories containing inert material or either 0.2 mg or 0.4 mg of prostaglandin E₂. The suppositories were self-administered every two hours during waking hours on two successive days until labor started or 15 had been used. Side-effects were absent. Labor was established within 48 hr of insertion of the first suppository in 9.3% of control patients, 65.4% of those treated with 0.2 mg PGE₂ and 85.7% of those treated with 0.4 mg PGE₂. The mean Apgar scores in the three groups were the same. The mean total dose of PGE₂ were 2.0 mg (0.2 mg group) and 2.3 mg (0.4 mg group). It is concluded that vaginal PGE₂ is an effective and acceptable method of inducing labor at term.     

Prostaglandin E2 produced by microsomal prostaglandin E synthase-1 regulates the onset and the maintenance of wakefulness

This study examined the effect of prostaglandin E₂ (PGE₂) produced by microsomal prostaglandin E synthase-1 (mPGES-1) on circadian rhythm. Using wild-type mice (WT) and mPGES-1 knockout mice (mPGES-1^−/−), I recorded and automatically analyzed the natural behavior of mice in home cages for 24 h and measured brain levels of PGE₂. The switch to wakefulness was not smooth, and sleepiness and the total duration of sleep were significantly longer in the mPGES-1^−/− mice. Moreover, the basal concentration of PGE₂ was significantly lower in the mPGES-1^−/− mice. These findings suggest that PGE₂ produced by mPGES-1 regulates the onset of wakefulness and the maintenance of circadian rhythm.     

Effects of prostaglandin E2 on the micronucleus formation in mouse bone marrow cells by various mutagens

The effects of prostaglandin E₂ (PGE)₂, as trigger of erythroid progenitor cells into the cell cycle, were studied on the induction of micronucleu by various mutagens; with mitomicin C (MMC) the optimal protocol was established. dose-response relationship between PGE₂ doses and micronucleus frequency were observed 30 h after injection of MMC to mice administered PGE₂ 24 h previously. Sensitazion by PGE₂ pretreatment was also found for other mutagens, such as vincristine, 5-fluorouracil, benzo[a]pyrene, 1,1-dimethylhydrazine and 2-naphthylamine. These results support the hypothesis that accelerating the erythropoiesis increases the frequency of micronucleic induced by mutagens.     

Expression of prostaglandin E2 receptor subtypes, EP2 and EP4, in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the distribution and time course of expression of two subtypes of prostaglandin E₂ (PGE₂) receptors, EP2 and EP4, in a rat model of cerebral ischemia and ischemic tolerance. Adult male Sprague-Dawley rats were subjected to either lethal global ischemia (10 min) with or without sublethal ischemic preconditioning (3 min), or ischemia only (3 min). A short 3-min cerebral ischemia and a 3-min ischemia followed by a second lethal ischemia enhanced the expression of EP2 and EP4 receptors in CA1 pyramidal neurons of the hippocampus. In tolerance-acquired CA1 neurons, the immunoreactivities of EP2 and EP4 were upregulated after 4 h and 12 h, respectively. The immunoreactivities were most prominent at 3 days and were sustained for at least 14 days, consistent with results of immunoblotting experiments. However, immunoreactivities for these PGE₂ receptors increased in reactive glial cells in the vulnerable CA1 and hilar regions of rats subjected to lethal ischemia without ischemic preconditioning. Most of the EP2 immunoreactivity occurred in microglial cells and some astrocytes, whereas increased immunoreactivity for EP4 was found only in astrocytes. These data suggest that ischemia and the induction of ischemia tolerance have different regulatory effects on the expression of EP2 and EP4 receptors. Moreover, PGE₂ may exert its unique pathophysiological functions in relation to delayed neuronal death and ischemic tolerance induction in the rat hippocampus via specific PGE₂ receptors.This research was supported by a grant (M103KV010019 04K2201 01930) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.     

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts

Interleukin-17 (IL-17) is a cytokine secreted primarily by T_H-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE₂ production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE₂, in osteoblasts.     

Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E2 production by osteoblasts

Several studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria in plaque and that tobacco smoking may be an important risk factor for the development and severity of periodontitis. The present study was undertaken to determine the effect of nicotine and LPS on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in osteoblasts, and the indirect effect of nicotine and LPS on the formation of osteoclast-like cells. Saos-2 cells were cultured with 10(-3) M nicotine, or 1 or 10 microg/ml LPS and 10(-3) M nicotine, for up to 14 days. The gene and protein expression of M-CSF and OPG were determined using real-time PCR and ELISA, respectively. PGE2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from nicotine and LPS-treated Saos-2 cells and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression increased markedly in cells cultured with nicotine and LPS compared with those cultured with nicotine alone. OPG expression increased in the initial stages of culture with nicotine and LPS but decreased in the later stages of culture. The conditioned medium containing M-CSF and PGE2 produced by nicotine and LPS-treated Saos-2 cells with soluble RANKL increased the TRAP staining of osteoclast precursors compared with that produced by nicotine treatment alone. These results suggest that nicotine and LPS stimulate the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production and that the stimulation is greater than with nicotine treatment alone.     

The use of oral prostaglandin E2 in the management of intrauterine fetal death

12 otherwise healthy patients with intrauterine fetal death 1 to 6 weeks earlier were treated with oral prostaglandin E₂. 9 of the 12 patients delivered within 48 hours after treatment began. 2 others delivered within 48 hours after unsuccessful treatment ceased. In a third patient the cervix relaxed after treatment, and the uterine contents were removed by curettage. No serious complications, such as hemorrhage occurred. The uterus seemed surprisingly responsive to oral prostaglandin E₂ in cases of intrauterine fetal death.     

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E₂ 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E₂ titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E₂ or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E₂ raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.     

Inhibition of prostaglandin E2 formation and histamine action in cancer immunotherapy

The activity of cell-mediated defense systems is stimulated by consecutive formation of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon (IFN). The system is inhibited by interleukin-4 (IL-4) and also by prostaglandin E₂ (PGE₂) and histamine, which are released when the immune system is activated. The inhibition is strong in cancer patients, because PGE₂ is formed in many cancer cells and its formation is stimulated by IL-1. The release of histamine is also stimulated by IL-1. Tus PGE₂ and histamine are feedback inhibitors of cell-mediated immunity. This inhibition can be abolished by inhibitors of the cyclo-oxygenase (e. g. indomethacin) and H-2 receptor antagonists (e. g. cimetidine). This may offer a new option to stimulate the immune system to kill cancer cells.