Toward enhanced hyperforin production in St. John's wort root cultures.
ABSTRACT: During the past decades, several trials targeted a stable, sustainable and economic production of St. John's wort (Hypericum perforatum) extract. The value of this extract stems from its use to treat depression and skin irritation due to its hyperforin content. Previously, hyperforin-forming in vitro root cultures were established. Here, detailed growth and production kinetics have been analyzed over 40 days of cultivation. In the first 10 days, sucrose was completely hydrolyzed to glucose and fructose. The ammonium consumption supported the increase in the biomass and hyperforin production. When sucrose was replaced with glucose/fructose, the linear growth phase started 6 days earlier and resulted in a higher space-time-yield. The maximum hyperforin production was 0.82 mg L-1 day-1, which was 67 % higher than in the sucrose-supplemented standard cultivation. Buffering the sucrose-supplemented medium with phosphate caused a 2.7-fold increase in the product to biomass yield coefficient. However, the combination of monosaccharides and buffering conditions did not cause an appreciable improvements in the production performance of the shake flask approaches. A potential scalability from flask to lab-scale stirred bioreactors has been demonstrated. The results obtained offer a basis for a scalable production of hyperforin and a sustainable source for a tissue culture-based phytomedicine.
Project description:Hyperforin is a pharmacologically active component of the medicinal plant Hypericum perforatum (St. John's wort), recommended as a treatment for a range of ailments including mild to moderate depression. Part of its action has been attributed to TRPC6 channel activation. We found that hyperforin induces TRPC6-independent H(+) currents in HEK-293 cells, cortical microglia, chromaffin cells and lipid bilayers. The latter demonstrates that hyperforin itself acts as a protonophore. The protonophore activity of hyperforin causes cytosolic acidification, which strongly depends on the holding potential, and which fuels the plasma membrane sodium-proton exchanger. Thereby the free intracellular sodium concentration increases and the neurotransmitter uptake by Na(+) cotransport is inhibited. Additionally, hyperforin depletes and reduces loading of large dense core vesicles in chromaffin cells, which requires a pH gradient in order to accumulate monoamines. In summary the pharmacological actions of the "herbal Prozac" hyperforin are essentially determined by its protonophore properties shown here.
Project description:OBJECTIVES:Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). MATERIALS AND METHODS:We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. RESULTS:We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. CONCLUSIONS:Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks.
Project description:N-methyl-D-aspartate receptor (NMDAR) modulators induce rapid and sustained antidepressant like-activity in rodents through a molecular mechanism of action that involves the activation of Ca<sup>2+</sup> dependent signaling pathways. Moreover, ketamine, a global NMDAR antagonist is a potent, novel, and atypical drug that has been successfully used to treat major depressive disorder (MDD). However, because ketamine evokes unwanted side effects, alternative strategies have been developed for the treatment of depression. The objective of the present study was to determine the antidepressant effects of either a single dose of hyperforin or lanicemine vs. their combined effects in mice. Hyperforin modulates intracellular Ca<sup>2+</sup> levels by activating Ca<sup>2+</sup>-conducting non-selective canonical transient receptor potential 6 channel (TRPC6) channels. Lanicemine, on the other hand, blocks NMDARs and regulates Ca<sup>2+</sup> dependent processes. To evaluate the antidepressant-like activity of hyperforin and lanicemine, a set of <i>in vivo</i> (behavioral) and <i>in vitro</i> methods (western blotting, Ca<sup>2+</sup> imaging studies, electrophysiological, and radioligand binding assays) was employed. Combined administration of hyperforin and lanicemine evoked long-lasting antidepressant-like effects in both naïve and chronic corticosterone-treated mice while also enhancing the expression of the synapsin I, GluA1 subunit, and brain derived neurotrophic factor (BDNF) proteins in the frontal cortex. In Ca<sup>2+</sup> imaging studies, lanicemine enhanced Ca<sup>2+</sup> influx induced by hyperforin. Moreover, compound such as MK-2206 (Akt kinase inhibitor) inhibited the antidepressant-like activity of hyperforin in the tail suspension test (TST). Hyperforin reversed disturbances induced by MK-801 in the novel object recognition (NOR) test and had no effects on NMDA currents and binding to NMDAR. Our results suggest that co-administration of hyperforin and lanicemine induces long-lasting antidepressant effects in mice and that both substances may have different molecular targets.
Project description:Microbial induced calcite precipitation (MICP) based on ureolysis has a high potential for many applications, e.g. restoration of construction materials. The gram-positive bacterium Sporosarcina pasteurii is the most commonly used microorganism for MICP due to its high ureolytic activity. However, Sporosarcina pasteurii is so far cultivated almost exclusively in complex media, which only results in moderate biomass concentrations at the best. Cultivation of Sporosarcina pasteurii must be strongly improved in order to make technological application of MICP economically feasible. The growth of Sporosarcina pasteurii DSM 33 was boosted by detecting auxotrophic deficiencies (L-methionine, L-cysteine, thiamine, nicotinic acid), nutritional requirements (phosphate, trace elements) and useful carbon sources (glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, L-alanine). These were determined by microplate cultivations with online monitoring of biomass in a chemically defined medium and systematically omitting or substituting medium components. Persisting growth limitations were also detected, allowing further improvement of the chemically defined medium by the addition of glutamate group amino acids. Common complex media based on peptone and yeast extract were supplemented based on these findings. Optical density at the end of each cultivation of the improved peptone and yeast extract media roughly increased fivefold respectively. A maximum OD600 of 26.6 ± 0.7 (CDW: 17.1 ± 0.5 g/L) was reached with the improved yeast extract medium. Finally, culture performance and media improvement was analysed by measuring the oxygen transfer rate as well as the backscatter during shake flask cultivation.
Project description:Hyperforin, a lipophilic constituent of medicinal herb St John's wort, has been identified as the main active ingredient of St John's wort extract for antidepressant action by experimental and clinical studies. Hyperforin is currently known to activate transient receptor potential canonical (subtype) 6 (TRPC6) channel, increase the phosphorylated CREB (p-CREB), and has N-methyl-D-aspartate receptor-antagonistic effect that convert potential neuroprotective effects in vitro. However, the protective effects of hyperforin on ischemic stroke in vivo remain controversial and its neuroprotective mechanisms are still unclear. This study was designed to examine the effects of intracerebroventricular (i.c.v.) injection of hyperforin on transient focal cerebral ischemia in rats. Hyperforin, when applied immediately after middle cerebral artery occlusion (MCAO) onset, significantly reduced infarct volumes and apoptotic cells, and also increased neurologic scores at 24 hours after reperfusion accompanied by elevated TRPC6 and p-CREB activity and decreased SBDP145 activity. When MEK or CaMKIV activity was specifically inhibited, the neuroprotective effect of hyperforin was attenuated, and we observed a correlated decrease in CREB activity. In conclusion, our results clearly showed that i.c.v. injection of hyperforin immediately after MCAO onset blocked calpain-mediated TRPC6 channels degradation, and then to stimulate the Ras/MEK/ERK and CaMKIV pathways that converge on CREB activation, contributed to neuroprotection.
Project description:5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l<sup>-1</sup> 5-KF were formed from 150 g l<sup>-1</sup> fructose within 23 h, corresponding to a space-time yield of 5.6 g l<sup>-1</sup> h<sup>-1</sup> . Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.
Project description:Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca<sup>2+</sup> signaling in keratinocytes by hyperforin treatment. However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl-<i>?</i>-cyclodextrin- (HP-<i>?</i>-CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP-<i>?</i>-CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca<sup>2+</sup> increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca<sup>2+</sup> waves via TRPC6. This process might facilitate wound closure, because the Ca<sup>2+</sup> response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca<sup>2+</sup> signaling. We also applied hyperforin/HP-<i>?</i>-CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP-<i>?</i>-CD treatment for 24?h improved the stretch-induced Ca<sup>2+</sup> responses and oscillations which failed in atopic skin.
Project description:Resveratrol is a plant-derived aromatic compound with a wide range of beneficial properties including antioxidant and anti-aging effects. The resveratrol currently available on the market is predominantly extracted from certain plants such as grape and the Japanese knotweed <i>Polygonum cuspidatum</i>. Due to the unstable harvest of these plants and the low resveratrol purity obtained, it is necessary to develop a stable production process of high-purity resveratrol from inexpensive feedstocks. Here, we attempted to produce resveratrol from a wide range of sugars as carbon sources by a using the genetically-engineered yeast <i>Scheffersomyces stipitis</i> (formerly known as <i>Pichia stipitis</i>), which possesses a broad sugar utilization capacity. First, we constructed the resveratrol producing strain by introducing genes coding the essential enzymes for resveratrol biosynthesis [tyrosine ammonia-lyase 1 derived from <i>Herpetosiphon aurantiacus</i> (<i>HaTAL1</i>), 4-coumarate: CoA ligase 2 derived from <i>Arabidopsis thaliana</i> (<i>At4CL2</i>), and stilbene synthase 1 derived from <i>Vitis vinifera</i> (<i>VvVST1</i>)]. Subsequently, a feedback-insensitive allele of chorismate mutase was overexpressed in the constructed strain to improve resveratrol production. The constructed strain successfully produced resveratrol from a broad range of biomass-derived sugars [glucose, fructose, xylose, <i>N</i>-acetyl glucosamine (GlcNAc), galactose, cellobiose, maltose, and sucrose] in shake flask cultivation. Significant resveratrol titers were detected in cellobiose and sucrose fermentation (529.8 and 668.6 mg/L after 120 h fermentation, respectively), twice above the amount obtained with glucose (237.6 mg/L). Metabolomic analysis revealed an altered profile of the metabolites involved in the glycolysis and shikimate pathways, and also of cofactors and metabolites of energy metabolisms, depending on the substrate used. The levels of resveratrol precursors such as L-tyrosine increased in cellobiose and sucrose-grown cells. The results indicate that <i>S. stipitis</i> is an attractive microbial platform for resveratrol production from broad types of biomass-derived sugars and the selection of suitable substrates is crucial for improving resveratrol productivity of this yeast.
Project description:Dental caries is an important global health concern and Streptococcus mutans has been established as a major cariogenic bacterial species. Reports indicate that a rare sugar, D?tagatose, is not easily catabolized by pathogenic bacteria. In the present study, the inhibitory effects of D?tagatose on the growth and biofilm formation of S. mutans GS?5 were examined. Monitoring S. mutans growth over a 24 h period revealed that D?tagatose prolonged the lag phase without interfering with the final cell yield. This growth retardation was also observed in the presence of 1% sucrose, although it was abolished by the addition of D?fructose. S. mutans biofilm formation was significantly inhibited by growth in sucrose media supplemented with 1 and 4% D?tagatose compared with that in a culture containing sucrose alone, while S. mutans formed granular biofilms in the presence of this rare sugar. The inhibitory effect of D?tagatose on S. mutans biofilm formation was significantly more evident than that of xylitol. Growth in sucrose media supplemented with D?tagatose significantly decreased the expression of glucosyltransferase, exo???fructosidase and D?fructose?specific phosphotransferase genes but not the expression of fructosyltransferase compared with the culture containing sucrose only. The activity of cell?associated glucosyltransferase in S. mutans was inhibited by 4% D?tagatose. These results indicate that D?tagatose reduces water?insoluble glucan production from sucrose by inhibiting glucosyltransferase activities, which limits access to the free D?fructose released during this process and retards the growth of S. mutans. Therefore, foods and oral care products containing D?tagatose are anticipated to reduce the risk of caries by inhibiting S. mutans biofilm formation.
Project description:<h4>Background</h4>Levan is a well-known homopolymer of fructose composed predominantly of ?-(2, 6) fructofuranosyl linkages in the backbone with occasional ?-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated.<h4>Methods</h4>The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).<h4>Results</h4>The levan yield of BD1707 reached 34.86?g/L with a corresponding productivity of 7.47?g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26?°C and 200?rpm for 112?h in tomato juice supplemented with 172?g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320?×?10<sup>7</sup>?Da at 6?h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809?×?10<sup>6</sup>?Da after 120?h of cultivation.<h4>Conclusion</h4>The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.