Step by step removal of hyperforin and hypericin: activity profile of different Hypericum preparations in behavioral models

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 and constituents of Hypericum perforatum inhibit the binding of various ligands to recombinant receptors expressed with the Semliki Forest virus system

Extracts, fractions and constituents of Hypericum perforatum were studied for in vitro receptor binding with various ligands to recombinant CNS receptors expressed with the Semliki Forest virus expression system. For this purpose we have prepared membranes of CHO cells with high density of several opioid, serotonin, estrogen, histamine, GABAA, neurokinin and metabotropic glutamate receptors, respectively. A lipophilic Hypericum fraction revealed relatively potent inhibition to the binding of the mu-, delta- and kappa-opioid and the 5-HT6 and 5-HT7 receptors. Moreover, Hypericum constituents such as the naphthodianthrones, hypericin and pseudohypericin, and the phloroglucinole hyperforin inhibited both binding to the opioid and serotonin receptors in the lower micromolar range. Estrogen binding was 50% inhibited by the biflavonoid I3,II8-biapigenin at micromolar concentration. The lipophilic Hypericum fraction provided a less potent inhibition of the neurokinin-1 receptor binding compared to the opioid and serotonin receptors. A total ethanolic Hypericum extract potently inhibited GABAA binding at approximately 3 micrograms/ml. This inhibition is however not specific to Hypericum, since extracts of plants like Valeriana officinalis and Passiflora incarnata showed similar inhibitions. Binding to neither histamine nor metabotropic glutamate receptors was affected by Hypericum extracts. These results support the hypothesis that several active constituents of Hypericum might in a synergistic way contribute to its antidepressant effect in the central nervous system.     

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.     

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.     

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.     

Fast high-performance liquid chromatographic analysis of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.     

Oral bioavailability of active principles from herbal products in humans. A study on Hypericum perforatum extracts using the soft gelatin capsule technology.

The oral absorption of two known active principles of Hypericum perforatum, namely hyperforin and hypericin, was studied in an open, single dose, two-way, randomized, cross-over study involving 12 healthy subjects (six males and six females). Alcoholic Hypericum extract (300 mg, containing 5% hyperforin and 0.3 % hypericin) was administered in the morning after 12 hours fasting. The formulation was administered as softgel capsules containing, inter alia, soya oil together with the herbal extract. A second standard formulation in two piece hard gelatin capsules was also used for comparison purposes. Blood was sampled from the subjects at different times after drug administration and the plasma was analysed according to published analytical methods for the determination of hyperforin and hypericin. Peaks of plasma concentration, Cmax of hyperforin were 168.35 ng/ml +/- 57.79 for the soft gelatin formulation (CV=34.32, n=12) and 84.25 ng/ml +/- 33.51 for the hard gelatin capsule (CV=39.77, n=12). The Tmax values for hyperforin were 2.50 h +/- 0.83 for the soft gelatin formulation compared to 3.08 h +/- 0.79 for the reference formulation, whereas the total AUC were respectively 1482.7 h x ng/ml +/- 897.13 and 583.65 h x ng/ml +/- 240.29. As for hypericin, plasma levels were detectable in approximately half of the subjects treated. However also in this case the soft gelatin capsules exhibited a higher individual absorption when compared with the corresponding data for the hard gelatin capsules.     

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection

An analytical procedure was developed for the simultaneous determination of total hypericin (protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.     

Treatment with Hypericum perforatum L. does not trigger decreased resistance in Staphylococcus aureus against antibiotics and hyperforin.

Most scientific investigations of Hypericum perforatum L. (Saint John's wort) concentrated on its antidepressant activity. Only recently, its antibacterial activity against multiresistant Staphylococcus aureus led to speculations regarding the use of hyperforin, an antibacterial principle of hypericum, as an antibiotic. In the present investigation, we show that Staphylococcus aureus is able to acquire a resistance against hyperforin which did not lead to a cross resistance against clinically used antibiotics. Resistance development does not take place, however, at concentrations as low as they are found in human blood plasma during antidepressant treatment with 900 mg Hypericum extract/day.     

Biosynthesis of hyperforin and adhyperforin from amino acid precursors in shoot cultures of Hypericum perforatum

Hyperforin and adhyperforin contribute to the antidepressant effects of Hypericum perforatum. The involvement of branched-chain amino acids in the biosynthesis of hyperforin and adhyperforin was demonstrated in H. perforatum shoot cultures. L-[U-(13)C(5)]Valine and L-[U-(13)C(6)]isoleucine, upon administration to the shoot cultures, were incorporated into acyl side chain of hyperforin and adhyperforin, respectively. Feeding the shoot cultures with unlabelled L-isoleucine at a concentration of 2mM induced a 3.7-fold increase in the production of adhyperforin. The addition of 3mM L-threonine, a precursor of isoleucine, stimulated a 2.0-fold increase in the accumulation of adhyperforin. The administration of L-valine at concentrations of 0-5mM had no stimulating effect on the hyperforin production in H. perforatum shoot cultures.     

Protective effect of Hypericum calabricum Sprengel on oxidative damage and its inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages

The present study shows for the first time the phenolic composition and the in vitro properties (antioxidant and inhibition of nitric oxide production) of Hypericum calabricum Sprengel collected in Italy. The content of hypericins (hypericin and pseudohypericin), hyperforin, flavonoids (rutin, hyperoside, isoquercetrin, quercitrin, quercetin and biapigenin) and chlorogenic acid of H. calabricum, have been determined. The ethyl acetate fraction from the aerial parts of H. calabricum exhibited activity against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 value of 1.6 jig/ml. The test for inhibition of nitric oxide (NO) production was performed using the murine monocytic macrophage cell line RAW 264.7. The ethyl acetate fraction had significant activity with an IC50 value of 102 jig/ml and this might indicate that it would have an anti-inflammatory effect in vivo.     

Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the simultaneous determination of hypericin and hyperforin--the two main active ingredients of St. John's Wort (SJW) extract--in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.     

Detection of hypericins in the "red glands" of Hypericum elodes by ESI-MS/MS

The biologically active naphthodianthrones hypericin and pseudohypericin were detected by electrospray ionization mass spectrometry (ESI-MS/MS) in microsamples from the sepals of Hypericum elodes (Hypericaceae) containing the so-called "red glands", i.e. stipitate glands with red-coloured heads. The occurrence of hypericins in the red glands of H. elodes supports the taxonomic position of the section Elodes within the genus Hypericum and provides evidence that the ability of carrying out the biosynthetic pathway leading to the naphthodianthrone compounds, rather than the absolute amounts produced, should be regarded as a chemical marker of the phylogenetically more advanced sections of genus Hypericum. The biologically active phloroglucinol derivatives hyperforin and adhyperforin, so far found only in H. perforatum, were also detected and evidence for their localization in the sepal secretory canals with large lumen, is given.     

Antioxidant properties of some hydroalcoholic plant extracts with antiinflammatory activity

The hydroalcoholic extracts of Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra which exhibited different anti-inflammatory activities were evaluated for the possible mode of action by studying their antioxidant potential. In the present study we investigated if standardized hydroalcoholic extracts of plants such as Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra produced by Hofigal Stock Company could modulate the respiratory burst of human activated neutrophils, as a consequence of their antioxidant capacity. Their antioxidant properties were measured using a colorimetric assay (Total Antioxidant Status kit). We demonstrated that Hypericum perforatum and Calendula officinalis hydroalcoholic extracts possessed a significant antioxidant activity while Plantago lanceolata and Glycyrrhiza glabra hydroalcoholic extracts had a minor antioxidant status. Using reactive oxygen species-generating systems (OZ-activated human PMN neutrophils), Calendula officinalis and Hypericum perforatum extracts showed strong reactive oxygen species scavenging property, Hypericum perforatum extract exhibing the highest scavenging activity. These results confirm the potential of Calendula officinalis and Hypericum perforatum investigated hydroalcoholic extracts as medicinal remedies to be used in different inflammatory/allergic diseases. These extracts could be a useful tool for obtaining new antioxidant/anti-inflammatory agents.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

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.     

Hyperforin

Hyperforin is a polyprenylated acylphloroglucinol derivative from Hypericum perforatum (St. John's wort). It exhibits antidepressant activity by a novel mechanism of action, antibiotic activity against gram-positive bacteria, and antitumoral activity in vivo. However, it also produces drug-drug interactions by activation of the pregnan X receptor. No total synthesis has been described. Some natural and semisynthetic analogues are available to study structure-activity relationships. Enzymatically, the skeleton of hyperforin is formed by isobutyrophenone synthase from isobutyryl-CoA and three molecules of malonyl-CoA. The first prenylation step is catalyzed by a soluble and ion-dependent dimethylallyltransferase. Hyperforin mainly accumulates in pistils and fruits where it probably serves as defensive compound.     

Positive correlations between hypericin and putative precursors detected in the quantitative secondary metabolite spectrum of Hypericum

A spectrum of eight pharmacologically important secondary compounds, all putatively belonging to the polyketide pathway (hypericin, pseudohypericin, emodin, hyperforin, hyperoside, rutin, quercetin, and quercitrin) were analyzed in several hypericin-producing species of Hypericum by LC–MS/MS. Different organs such as leaves, stems and roots of wild-grown plants of Hypericum hirsutum L., Hypericum maculatum Crantz s. l., Hypericum montanum L., Hypericum tetrapterum Fr. collected in Slovakia and of Hypericum perforatum L. collected in India were examined individually. Highest contents of hypericin, pseudohypericin, and emodin were found in H. montanum, suggesting that there are alternative species to H. perforatum with high pharmaceutical value. Amounts of hyperforin and quercetin were highest in H. perforatum, whereas highest contents of hyperoside and quercitrin were found in H. maculatum. A significant positive correlation between hypericin and pseudohypericin as well as between hypericin and emodin was observed by Kruskal’s multidimensional scaling (MDS), indicating a parallel enhancement of emodin as a common precursor in the biosynthetic pathways of hypericin and pseudohypericin. Furthermore, MDS combined with principal component analysis (PCA) revealed strong correlations in the occurrence of pseudohypericin and emodin, pseudohypericin and quercitrin, hypericin and quercitrin, emodin and quercitrin, hyperoside and quercitrin, rutin and quercetin, and, hyperforin and quercetin. On the other hand, rutin showed a negative correlation with emodin as well as with quercitrin. Furthermore, hierarchical agglomerative cluster analysis (HACA) clustered hypericin and pseudohypericin, grouping emodin at equal distance from both. Considerable infraspecific variability in secondary compound spectrum and load of different populations of H. maculatum from Slovakia underscores the need for detailed studies of genotypic variation and environmental factors in relation to polyketide biosynthesis and accumulation.     

Hyperforin depletes synaptic vesicles content and induces compartmental redistribution of nerve ending monoamines

Hyperforin, a phloroglucinol derivative found in Hypericum perforatum (St. John's wort) extracts has antidepressant properties in depressed patients. Hyperforin has a unique pharmacological profile and it inhibits uptake of biogenic monoamines as well as amino acid transmitters. We have recently showed that the monoamines uptake inhibition exerted by hyperforin is related to its ability to dissipate the pH gradient across the synaptic vesicle membrane thereby interfering with vesicular monoamines storage. In the present study we demonstrate that hyperforin induces dose-dependent efflux of preloaded [3H]5HT and [3H]DA from rat brain slices. Moreover, we show that hyperforin attenuates depolarization- dependent release of monoamines, while increasing monoamine release by amphetamine or fenfluramine. It is also demonstrated that preincubation of brain slices with reserpine is associated with dose- dependent blunting of efflux due to hyperforin. Our data indicate that hyperforin-induced efflux of [3H]5HT and [3H]DA reflect elevated cytoplasmic concentrations of the two monoamines secondary to the depletion of the synaptic vesicle content and the compartmental redistribution of nerve ending monoamines.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Simultaneous determination of hypericin and hyperforin in human plasma and serum using liquid-liquid extraction,high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry

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.