BIOLOGICAL ACTIVITY OF HYPERICUM PERFORATUM L. (HYPERICACEAE): A REVIEW

Hypericum perforatum L. (St. John’s wort) is a medicinal plant that has been intensively studied by clinicians, pharmacolo-gists, and chemists. It has resulted in the publication of both original articles and a number of reviews devoted to the general spectrum of the biological activity of its extracts and the separate chemical components of this species. Unlike many other known medicinal plants, the pharmacological study of which is accompanied by the establishment of new (or rediscovered) structures of chemical compounds, the dynamics of the present study of H. perforatum is mostly associated with a detailed study of the mechanisms of its therapeutic effect and less with the search for new components. The aim of this work is to review and analyze the data on the biological activity of extracts and individual compounds of Hypericum perforatum L. (Hypericaceae), or St. John’s wort, published in the scientific literature over the past 10 years. Materials and methods. To collect and analyze the information, such electronic databases as PubMed, Scopus, Web of Sci -ence, Google Scholar, and other available resources have been used. The following keywords and word combinations were used for search in the databases for 2010–2020: “ Hypericum perforatum ”, “St. John’s wort”, “the biological activity of St. John’s wort”, “hypericin”, “hyperforin”. Results. The review provides information on antidepressant, neuroprotective, nootropic, anxiolytic activity, antibacterial, cytotoxic, anti-inflammatory properties, analgesic, hypoglycaemic effects, and other types of activity of H. perforatum ex -tracts, as well as individual compounds (hypericin, hyperforin, amentoflavone, and others) isolated from this species. It is well known that the secondary metabolites of St. John’s wort are naphthodianthrons, flavonoids and other phenolic compounds, several classes of lipophilic substances including phloroglucinol derivatives and terpenoids. Apart from extracts and their fractions, the biological activity of photoreactive naphthodianthrone hypericin and hyperforin (a phloroglucinol derivative) has been studied in detail. This review provides an analysis of published data from 2010 to 2020 on the biological activity of St. John’s wort. At the present time H. perforatum is primarily well-known for its antidepressant-like properties, which are confirmed by numerous pharmacological studies and clinical trials. Still there is no consensus on the effective treatment of severe or even moder ate depression with St. John’s wort. This review also provides information on the neuroprotective, nootropic, antiepilep tic, anxiolytic, antimicrobial, antiviral, antiprotozoal, antitumor, cytotoxic, analgesic, anti-inflammatory and other effects of H. perforatum extracts, as well as its individual compounds. Conclusion. Despite the popularity of H. perforatum as a plant with an antidepressant-like activity, intensive research work continues to be carried out to elucidate the molecular mechanisms of the actions of extracts and individual compounds in disorders of the nervous system. Studying its antibacterial, antiviral, and cytotoxic activity may also open up some great prospects, along with determining the possibility of using St. John’s wort in metabolic disorders, genitourinary disorders, and other fields of medicine. monophosphate; – human – unpigmented melanoma – a disintegrin-like and metal loprotease with thrombospondin type 1 motif 8, 9; BDNF – brain-derived neurotrophic factor; CaMK-IV – calcium/calmod -ulin-dependent protein kinase; cAMP – cyclic adenosine monophosphate; – lymphocytic leukemia line; COX – cyclooxygenases; – cAMP element-binding –


INTRODUCTION
Despite the fact that Hypericum perforatum L. (St John's wort) has been known for its medicinal properties for more than 2000 years, it has not yet lost its popularity and continues to be studied intensively by clinicians, pharmacologists, and chemists. The indicator of the active interest in H. perforatum is the number of reviews published over the last decade. They are devoted to both the general biological activity profile of its extracts and individual chemical components, as well as to specific types of activity, which are considered in the corresponding sections of this article.
Unlike many other medicinal plants, pharmacological studies of which are accompanied by the determination of the structures of certain new (or rediscovered) chemical compounds, modern research of H. perforatum is more focused on the mechanisms of its therapeutic action, and less on the identification of new components.
It is well known that among the secondary metabolites of St. John's wort, there are naphthodianthrons, flavonoids and other phenolic compounds, several classes of lipophilic substances including phloroglucinol derivatives and terpenoids. Apart from extracts and their fractions, the biological activity of hypericin, a photoreactive naphthodianthrone, and hyperforin -a phloroglucinol derivative, have been studied in most detail [1][2][3][4][5].
Today, H. perforatum is known primarily for its antidepressant-like properties, which have been confirmed by numerous preclinical studies and clinical trials. Still, there is no consensus on the effectiveness of St. John's wort for severe or at least moderate depression (e.g. [2]).
And yet, the aforementioned beneficial properties of H. perforatum, as well as those yet unknown, continue to be studied with unceasing regularity, in different models and within different approaches. A brief (but by no means exhaustive) summary of such studies conducted over the past decade, is presented in this review.
THE AIM of this work is to review and analyze the data on the biological activity of extracts and individual components of Hypericum perforatum L. (Hypericaceae), or St. John's wort, published in the scientific literature over the past 10 years.

MATERIALS AND METHODS
To collect and analyze the information, electronic databases PubMed, Scopus, Web of Science, Google Scholar, and other available resources have been used. The following keywords and word combinations were used for search in the databases for 2010-2020: "Hypericum perforatum", "St. John's wort", "the biological activity of St. John's wort", "hypericin", "hyperforin".
According to a systematic review and a meta-analysis, which included 27 clinical trials, St. John's wort is as effective in the patients with mild-to-moderate depression as are some of the most common synthetic antidepressants, at the same time being better tolerated [14][15][16]. H. perforatum is most effective in patients with mild and moderate depression [15][16][17][18], and in those experiencing pronounced somatization and gastrointestinal symptoms [19]. DOI: 10.19163/2307-9266-2021-9-1-17-31 Volume IX, Issue 1, 2021 Standardized H. perforatum extracts WS 5572, LI 160, WS 5570, ZE 117 were found to be as effective for mild depression as sertraline and imipramine [20,21]. In moderate depression, H. perforatum did not differ in effectiveness from citalopram [22] while surpassing paroxetine [23]. According to a retrospective observational study, IperiPlex ® , a multi-fractionated H. perforatum extract, was significantly more effective in the patients with moderate depression than Nervaxon ® , a mono-fractionated extract [24]. However, H. perforatum effectiveness in patients with moderate and severe depression remains somewhat unclear [25,26].
A multicenter observational study showed the drugs Helarium ® and Helarium-425 ® , both containing extracts of H. perforatum, to be well-tolerated by patients with mild to moderate depression [27]. However, H. perforatum treatment was associated with a greater frequency of some specific adverse events, including damage to the nervous system, reproductive organs, eyes, ears, liver, and kidneys [28]. A case of psychosis has been reported in a patient who self-administered H. perforatum as a herbal infusion [29].
H. perforatum extract potentiated yohimbine toxicity, and exhibited maximal antidepressive activity at 90 mg/kg [14]. Administered over time, both H. perforatum ethanolic extract and fluoxetine were found to affect hippocampal and hypothalamic gene expression in chronically stressed rats. Among those affected were genes coding for a number of biomolecules involved in neuroinflammatory and oxidative stress pathways, and some of those associated with Alzheimer's disease [30].
In 2018, T. Herraiz et al. showed that in various dosage forms, H. perforatum inhibited monoamine oxidase А (МАО-А). Of all the identified plant constituents, quercetin (IC 50 = 3.4 μg/ml) and its glycosides were found to be the most active; hypericin (IC 50 = 17.9 μg/ml) did not contribute significantly to the overall effect of the preparations, and hyperforin failed to show any activity throughout the studied concentration range. According to the authors, taking into account the average content of active ingredients in total preparations with H. perforatum, the observed MAO-A inhibition was most likely an additive effect [31].
When compared to venlafaxine, a serotonin and norepinephrine reuptake inhibitor, hypericin decreased blood corticosterone levels, prevented weight loss and anorexia, reduced anhedonia, and stimulated exploratory behaviour in rats with a chronic unpredictable mild stress (CUMS). Moreover, those effects had a shorter onset in the hypericin-treated group than in the venlafaxine-treated one. At the same time, hypericin affected the metabolism of norepinephrine, serotonin, and excitatory amino acids (glutamate and glutamine) [12]. Hypericin also inhibited calcium ion influx into hippocampal neurons, thus increasing an action potential duration, which could possibly play a role in enhancing a synaptic efficiency [32].

Pretreatment of glioblastoma cells with hyperforin
and hyperoside hindered lateral mobility of β 1 -adrenergic receptors (β 1 -АR) and caused their internalization. The two compounds reduced cyclic adenosine monophosphate (cAMP) formation by 10% and 15%, respectively, and by 23% and 15% under subsequent cell stimulation with 5 μM dobutamine. Similar effects were observed when cells were pretreated with desipramine, a tricyclic antidepressant [7]. H. perforatum extract and hyperforin were found to increase presynaptic calcium concentrations and thus stimulate the release of the excitatory neurotransmitter glutamate [33].
B. Pochwat et al. found that hyperforin potentiated antidepressant-like activity of lanicemine, a N-methyl-D-aspartate receptor (NMDAR) antagonist, in chronic corticosterone-treated mice as well as in healthy controls. A combination of lanicemine and hyperforin increased the expression of synapsin I, a subunit of glutamate receptor A 1 , and neurotrophin BDNF (brain-derived neurotrophic factor) in frontal cortical neurons. Hyperforin also attenuated cognitive dysfunction caused by dizocilpine, a NMDAR antagonist having marked dissociative and psychedelic properties. Nonetheless, 0.3-10 μm hyperforin did not affect NMDAR electrical activity in vitro [13].
Much less is known about adhyperforin than about its parent compound, hyperforin. However, adhyperforin has also been shown to exert an antidepressant-like activity, stimulate exploratory behaviour, and reduce anhedonia and hypodynamia in rodents. It suppressed norepinephrine, serotonin and dopamine reuptake in vitro, and, just like hyperforin, antagonized reserpine-induced effects in vivo [8,34].

Neuroprotective activities
An ethanolic extract of H. perforatum containing 6.0% of hyperforin stimulated neurite outgrowth in HT22 hippocampal cells, increased their resistance to glutamate toxicity, and inhibited the release of tumor necrosis factor α (TNFα) from macrophages [35].
Pretreatment of PC12 pheochromocytoma cells with an H. perforatum extract increased their viability at toxic concentrations of hydrogen peroxide, and prevented DNA fragmentation [36]. Another H. perforatum extract normalized lateral mobility of integral membrane proteins and phospholipids in glioblastoma cells, which made a more efficient transmembrane signal transduction possible [37]. Ethyl acetate, water, and methanolic extracts of H. perforatum inhibited acetyl-and butyrylcholinesterase; ethyl acetate and water extracts also inhibited tyrosinase [38].
An extract of H. perforatum reduced the severity of oxidative stress in leukocytes obtained from patients with multiple sclerosis. Cell apoptosis was largely prevented through the activation of antioxidant systems and normalization of intracellular calcium levels [39]. A similar effect associated with the blockade of calcium channels by H. perforatum was also observed in rat dorsal root ganglion neurons [40]. Later, some of the active constituents of H. perforatum were found to inhibit TRPM2 and TRPV1 channels, which mediate calcium ion influx under oxidative stress conditions [41].
A hyperforin-enriched (6.0%) extract (4 mg/kg/d×45 d) effectively prevented degeneration of nigral neurons induced in rats by a chronic exposure to rotenone. Quite on the contrary, an extract containing only 0.2% hyperforin and pure quercetin both exhibited significantly lower activity when administered by the same route and at the same doses In the study by S. Valvassori et al., an H. perforatum extract (300 mg/kg /d × 28 d) significantly impaired memory acquisition and object recognition, and decreased the levels of the transcription factors BDNF and NGF (nerve growth factor) in rat hippocampi [47].
In an ex vivo experiment in isolated hippocampal neurons, hyperforin (0.3 μM, 24 h) promoted the formation of stubby dendritic spines and, at the same time, decreased the proportion of thin spines [48]. Interestingly, similar alterations in spine morphology have been observed for classical antidepressant agents such as fluoxetine [49], imipramine, and rolipram [50], but in those cases, they had a significantly slower onset, and most probably differed in nature [48].
Hyperforin activated MEK (mitogen-activated protein kinase), MAPK (mitogen-activated protein kinase), phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/Akt), and calcium/calmodulin-dependent protein kinase (CaMK-IV) in PC-12 pheochromocytoma cells and hippocampal neurons [51]. These changes culminated in the phosphorylation and activation of the transcription factor CREB (cAMP response element-binding protein), which is considered to be a promising therapeutic target for the treatment of Alzheimer's disease [52][53][54]. Moreover, the therapeutic effect of H. perforatum in Alzheimer's disease was seemingly independent of hyperforin concentration in the preparation [55]. Hyperforin was confirmed to exert neuroprotective effects against aluminum maltolate-induced toxicity in PC12 and SH-SY5Y cells [56].
Hyperforin stimulated CREB phosphorylation, induced TRPC6 calcium channel and TrkB BDNF receptor expression in embryonic mouse cortical neurons [57]. The extract of H. perforatum reduced a β-amyloid accu-mulation and increased levels of P-glycoprotein in the brain tissue of transgenic mice with Alzheimer's disease. However, another study found an H. perforatum extract, hyperforin, and high concentrations of quercetin to inhibit P-glycoprotein activity in brain capillary endothelial cells [59]. A methanolic extract of H. perforatum inhibited acetylcholinesterase and reduced glutamate levels, at the same time potentiating noradrenergic and dopaminergic neurotransmission in an aluminum chloride-induced rat model of Alzheimer's disease. The extract caused a decrease in β-amyloid deposition rates and ameliorated oxidative stress in treatment groups [60]. A 28 days-long H. perforatum treatment course inhibited neuroinflammation, lipid peroxidation, and lowered blood proinflammatory cytokines levels in rats subjected to mechanic sciatic nerve injury [61].
An intracerebroventricular administration of hyperforin to rats subjected to middle cerebral artery occlusion, significantly reduced infarct volume and post-stroke neurological deficit. Hyperforin inhibited the calpain-mediated TRPC6 channel degradation, thus maintaining normal CREB activity and, ultimately, increasing neuron viability following ischemia [62].
TRPC6 activation is thought to be a non-essential or, at the very least, not the only mechanism of a hyperforin action [9,34]. For instance, a complete absence of TRPC6 had no effect on inward membrane ion currents in microglial cells treated with a hyperforin solution. Its molecule being highly lipophilic, and its properties depending heavily on pH of the medium, it was suggested that hyperforin acted as a protonophore and induced a transmembrane proton transfer in a channel-independent fashion [34]. However, in an in vivo experiment in mice, its neurotropic activity was blocked completely by a prior administration of either larixyl acetate or MK 2206, which inhibited TRPC6 and PKB, respectively [13].
Amentoflavone and hypericin are thought to have an opposite effect on a MAPK pathway activity compared to hyperforin [63][64][65]. Amentoflavone protected HT22 hippocampal neurons against glutamate-induced excitotoxic injury. Besides maintaining the activity of some of the most important antioxidant enzymes and inhibiting reactive oxygen species generation, it inhibited MAPK phosphorylation [66].
Amentoflavone has been shown to exert a direct effect on cholinergic neurotransmission in the central nervous system. It significantly attenuated scopolamine-induced retrograde amnesia through inhibition of acetylcholinesterase and enhancement of antioxidant enzymes activity, thus perpetuating long-term spatial memory [67].

Nootropic activity
A 2016 meta-analysis confirmed H. perforatum to possess a significant nootropic activity independent of its antidepressant-like activity. The authors suggested DOI: 10.19163/2307-9266-2021-9-1-17-31 that the modulation of 5-HT 2 serotonin receptor activity, dopaminergic, glutamatergic, and γ-aminobutyric acid (GABA)-mediated neurotransmission was among the possible mechanisms of H. perforatum nootropic action [68]. Long-term H. perforatum treatment has been shown to inhibit the release of adrenocorticotropin and, as a result, that of the glucocorticoid corticosterone, which is the main hormonal mediator of chronic stress response in rodents [68,69]. An H. perforatum extract (125,250 or 500 mg/kg/d × 30 d) prevented an increase in corticosterone and TNF-α levels in blood and hippocampus in bilateral ovariectomized rats [70].
The nootropic effects of H. perforatum preparations have been confirmed experimentally using acute [71] and chronic restraint stress models [72], and a model of cognitive deficit associated with diabetes mellitus [73].
H. perforatum preparations have been demonstrated to have a beneficial effect on neuronal synaptic plasticity in animal [74,75] and human studies [76]. A single dose of 250 mg of H. perforatum tabletted dry extract (Remotiv ® ) improved short-term verbal and spatial memory in healthy volunteers. Quite surprisingly, no nootropic effect was observed at the dose of 500 mg, although both doses improved the patients' mood and emotional stability. Similarities to and differences from some other neurotropic agents such as citalopram, bromocriptine, and sulpiride, as well as the inverse dose-dependency of H. perforatum effects suggest that its primary mechanism of action could involve the augmentation of dopaminergic transmission [77].
The effectiveness of H. perforatum for the treatment of autism spectrum disorder is limited. St. John's wort modestly improved irritability, stereotypy and abnormal speech patterns, while clinician ratings on several symptom assessment scales remained unchanged [78].

Antiepileptic activity
Amentoflavone exhibited antiepileptic properties in a number of in vitro and in vivo studies. It attenuated oxidative stress, inhibited neuroinflammation, and increased GABA binding affinity to GABA A receptors [79][80][81]. An ether extract of H. perforatum lowered the seizure threshold and increased the after-discharge duration, while n-butanolic and water extracts, on the contrary, inhibited epileptogenesis [82]. A methanolic extract of H. perforatum reduced seizure duration and mortality in a mouse model of picrotoxin-induced epilepsy [83].

Anxiolytic activity
Anxiolytic properties of H. perforatum are related to its nootropic, neuroprotective and antidepressant-like kinds of activity, and are thought to be mediated by its effects on monoaminergic transmission and neuroinflammatory processes [71,74].
The anxiolytic effect of amentoflavone (25 mg/kg), observed in mice following a single administration, was reduced by pretreatment with flumazenil, a benzodiazepine receptor antagonist. This fact suggested that amentoflavone exerted its anxiolytic effect through the interaction with the benzodiazepine-binding site of the GABA A receptor [84]. This mechanism of action was subsequently confirmed by radioligand binding assays [85].
Crupi et al. revealed that three-week treatment with an H. perforatum methanolic extract decreased anxiety in mice with chronic corticosterone-induced stress [74]. Extract-treated (50 or 100 mg/kg/d × 5 d) mice exhibited higher levels of exploratory activity and were less anxious following six hours of acute restraint stress, although those parameters still fell outside normal ranges [71]. An H. perforatum extract (100 or 200 mg/kg/d × 14 d) ameliorated anxiety and depression in streptozotocin-induced type II diabetic rats [86].

Antimicrobial, antiviral and antiprotozoal activity
Antibacterial activity of H. perforatum has been reviewed by Z. Saddiqe et al. in 2010 [87]. The antibacterial activity of individual compounds and extracts of St. John's wort is somewhat unclear. Aerial parts macerated with olive oil showed little overall activity, and only a few samples were active against Trypanosoma brucei rhodesiense and Staphylococcus aureus [88]; hyperforin (but not hypericin) also moderately inhibited Staphylococcus aureus growth [89]. The aqueous fraction of an ethanolic extract of St. John's wort suppressed Streptococcus sobrinus and Lactobacillus plantarum growth [90], and an alcoholic extract and hypericin were active against Lactobacillus acidophilus, allowing to consider them as potential oral disinfectants [91]. Photoactivated hypericin inhibited Candida albicans, C. parapsilosis, C. krusei [92], and Staphylococcus aureus growth, but did not affect that of Escherichia coli [93]. Hyperforin and a methanolic extract of the aerial parts of the plant were active against Mycobacterium JLS, although hypericin and pseudohypericin were not [94]. An ethyl acetate extract of H. perforatum exhibited antiviral activity against infectious bronchitis virus in vitro and in vivo (IBV strain M41) [95], human influenza virus A/PR/8/34 H1N1 [96], influenza A virus [97], and hepatitis B virus [98].

Antitumor and cytotoxic properties
An overview summarizing existing knowledge on the anticancer activity of Hypericum species was published in 2017 [99]. It was established that ultraviolet radiation increased the antiproliferative activity of a water/alcohol extract in human melanoma A375 cell line [100]. Investigations are underway to assess the cytotoxic activity of H. perforatum components in photodynamic therapy. For instance, hyperforin and aristofolin (a synthetic derivative of hyperforin) induced apoptosis of HT-29 colon adenocarcinoma cells subjected to hypericin-mediated photodynamic therapy [101], and death of both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells [102] 1 . Photoactivated hypericin decreased the viability of RINm5F insulinoma cells, human squamous carcinoma cells (SCC) [104], D273 medulloblastoma cells [105], and was effective against anaplastic thyroid cancer [106]. A flower extract inhibited growth and induced apoptosis of K562 (acute erythroid leukemia) cells [107], an ethanolic extract blocked proliferation and induced apoptosis of MCF-7 human breast cancer cells [108], and hyperforin induced death of CLL (chronic lymphocytic leukemia) cells ex vivo [109]. Hypericin exerted a cytotoxic effect on MCF-7 cells [110], promoted expression of genes coding for metalloproteinase family enzymes ADAMTS9 and ADAMTS8 having anti-angiogenic and antitumor properties in MCF-7 cells [111]. Hyperforin induced apoptosis of U937 (human acute myeloid leukemia) cells line [112]. An essential oil showed anti-angiogenic properties [113].

Analgesic, anti-inflammatory and wound-healing properties
There has been published a number of reviews focused on the anti-inflammatory and analgesic properties of H. perforatum extracts and their components [114][115][116][117][118]. A dry extract relieved neuropathic pain in an experimental study [119]. Hypericin inhibited the pro-inflammatory enzyme janus kinase 1 (JAK1) in silico, which could explain its anti-inflammatory properties [120].
Hyperforin inhibited the activity of cyclooxygenases (COX) 1 and 2 and microsomal prostaglandin synthase PGE 2 , which play key roles in inflammation and tumorigenesis [121]. Over the past decade, the investigations of anti-inflammatory properties of the four-component fraction of H. perforatum ethanolic extract containing amentoflavone, quercetin, chlorogenic acid and pseudohypericin, have been continued [122,123]. The extract was found to be devoid of anti-inflammatory activity, in contrast with the four compounds [124]. H. perforatum flowering tops extract suppressed the expression of proinflammatory factors and stimulated that of anti-inflammatory ones in cultured adipocytes [125].
An extract facilitated wound healing properties in a clinical trial [126], and was effective for the treatment of psoriasis, lowering TNFα levels in dermis, endothelial, and dendrite cells [127]. An ethanolic extract prevented lipid peroxidation in neutrophils of patients with Behcet's syndrome [128]. An oil extract was effective for the prevention and treatment of pressure sores [129]. Wound-healing properties of H. perforatum extracts were confirmed in different models [130][131][132][133] including diabetic animals [134][135][136]. An oil extract prevented the narrowing of the oesophageal lumen caused by burn injuries [137], and had anti-inflammatory, anti-angiogenic, and anti-fibroblastic effects when applied after corneal alkali burns [138]. Hyperforin reduced the migration of fibroblasts in 2D and 3D models of artificial skin and was proposed for the treatment of hypertrophic scars [139].

Hypolipidaemic and hypoglycaemic properties
Hypolipidaemic and hypoglycaemic properties have been discovered for extracts of the aerial parts of H. perforatum [141][142][143]. A hydroalcoholic (50%) extract of the whole plant at the doses of 100 and 200 mg/kg of body weight per day for 15 days exhibited hypocholesterolaemic properties [144], and similar effects were observed for an aqueous extract of the aerial parts at the dose of 300 mg/kg for 60 days [145]. A methanolic extract and hyperforin prevented pancreatic β-cells from damage by the cytokines iNOS, CXCL9, CXCL10, and COX2, which is associated with the development of type I diabetes [146]. It was found that excessive intake of H. perforatum flower extract, hypericin, and hyperforin could aggravate diabetes and obesity by inhibiting the differentiation of preadipocytes and inducing insulin resistance in mature fat cells [147].

Effects of H. perforatum in genitourinary system disorders
A powder of shoots at the dose of 200 mg/kg (8 weeks) in diabetic nephropathy showed a nephroprotective effect [147]. A methanolic extract of the aerial parts of H. perforatum, hypericin, and hyperforin exhibited spasmolytic activity and modulated detrusor contractile activity in isolated urinary bladder; for hypericin, this effect was associated with an increase in plasma membrane depolarization, and for hyperforin, with a stimulatory effect on the cholinergic system [149]. A hydroalcoholic extract of leaves reduced the size and number of ethylene glycol-induced renal calculi [150].
Clinical trials found out that a powder of St. John's wort given at the doses of 270-330 μg for 2 months reduced hot flashes, menopausal symptoms, and depression [151], and the extract was effective for the treatment of premenstrual syndrome [152]. In an in vitro experiment, the extract (25 μg/mL) and hypericin (7.5 and 75 ng/mL) increased calcium concentration in JEG-3 placental cells [153]. The extract intake (100 mg/kg and 300 mg/kg) from mating till delivery prolonged foetal development and damaged foetal liver due to oxidative stress [154]; at the same doses, the extract worsened ovarian function and decreased fertility [155].

Effects of H. perforatum in maxillofacial injuries
An aqueous extract of the aerial parts activated a bone tissue regeneration in the orthopaedically expanded premaxillary suture, which is performed in orthognathic surgery [156]. Another experiment proved a standardized methanolic extract to restore mandible bone tissue in a stress model [157]; an ethanolic extract activated dental pulp regeneration [158]), and an oil extract improved bone healing after xenograft implantation [159]. Other effects Several H. perforatum extracts prevented acetaminophen-induced liver injury [160,161], a petroleum ether leaf extract had protective effects in a hepatic ischaemia-reperfusion model [162], and another extract accelerated hepatic clearance of technetium-99 [163]. Certain fractions of a water/ethanol extract of the aerial parts of H. perforatum had spasmolytic, bronchodilator, vasorelaxant and cardiotropic activities [164]; H. perforatum polysaccharides and a methanolic seed extract showed antioxidant properties [165,166]. It is assumed that the antioxidant properties underly the photoprotective and anti-inflammatory effects of hyperforin on skin tissues [167]. At the doses of 250 and 500 mg/kg, a H. perforatum dry extract reduced binge eating episode frequency [168]. A leaf extract had antimutagenic properties [169]; the effects of hyperforin were described as antigenotoxic [170] and DNA-protective [171] in different in vitro models. An ethanolic extract of H. perforatum stimulated human osteoblast-like MG-63 cell proliferation in osteoporosis induced by ovariectomy [172]. A hydroalcoholic extract (110 mg/kg for 2 weeks) enhanced cellular immunity [173], and a methanolic extract of the aerial parts, dissolved in olive oil, prevented the development of myringosclerosis after myringotomy [174]. The β-diketone 2,6,9-trimethyl-8-decene-3,5-dione, hyperforatins B, D, and F, 15-epi-hyperforatin D, and 32-epi-hyperforatin E inhibited an acetylcholinesterase activity [175,176], and a methanolic extract stimulated hepatic and renal activities of the cytochromes CYP3a and CYP2c [177].

CONCLUSION
Despite the popularity of H. perforatum as a plant with an antidepressant-like activity, intensive research work continues to be carried out to elucidate the molecular mechanisms of the actions of extracts and individual compounds in disorders of the nervous system. Studying its antibacterial, antiviral, and cytotoxic activity may also open up some great prospects, along with determining the possibility of using St. John's wort in metabolic disorders, genitourinary disorders, and other fields of medicine.