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Leonurus cardiaca L. as a Source of Bioactive Compounds: An Update of the European Medicines Agency Assessment Report (2010)

Leonurus cardiaca L. (motherwort) is a perennial herb, native to Asia and southeastern Europe, with widespread global occurrence in present days. The plant was historically used as cardiotonic and for treating gynaecological afflictions (such as amenorrhea, dysmenorrhea, menopausal anxiety, or postpartum depression). Although its use in oriental and occidental medicine is relatively well documented, the recent progress registered raises the need for an update of the Medicines Agency assessment report on Leonurus cardiaca L., herba (2010). The current study presents the progress made within the 2010-2018 timeframe regarding the potential applications and scientific evidences supporting the traditional use of motherwort, in the same time suggesting future research opportunities.


Introduction
Leonurus cardiaca L. (common namesmotherwort in English, Echte Herzgespann -Deutsch, agripaume -French, etc) represents a perennial herb belonging to the Lamiaceae family. The plants grow up to 1 m, with the hollow aerial stalks growing from the rhizomes. The leaves are palmately lobed, being covered with stiff hairs. Flowers, grouped in 10-20 clusters in the leaf 's axils of the last 10-15 knots, are pink and about 1 cm long [1]. The plant, original to Asia and southeastern Europe is now world-spread, due to its medicinal use [2][3][4]. The potential application in treating several cardiac disorders, as well as female-specific afflictions, made L. cardiaca a very good candidate for development of alternative treatments, in both traditional eastern and modern medicine [2,5]. Besides the traditional medical use, motherwort is used in some cuisines as condiment in various vegetable soup recipes, particularly the lentil or split peas ones, or for flavoring of beer and tea [6], thus increasing the potential intake of the medicinal plant by the general public.
The current review intends to present the main findings regarding the composition and main biological activities of L. cardiaca, as emerging from the scientific studies published within the 2010-2018 timeframe. The time period was selected in order to complete the very comprehensive "Assessment report on Leonurus cardiaca L., herba" published by the European Medicine Agency [5] with the latest findings. The search methodology involved accessing and evaluating the papers found in the PubMed, ScienceDirect, Wiley Online Library, ACS Publications, and SpringerLink databases (search term "Leonurus cardiaca", 27.12.2018). After the removal of duplicate entries, 283 studies were taken into consideration. Figure 1 describes the distribution of the reviewed works by publication year and type of paper. Most of the information presented in the present work was collected from the "Article" type papers (176 works).

Composition of L. cardiaca
The composition of L. cardiaca was previously presented by the EMA report [5], consisting of furanic diterpenes (labdanes), alkaloids (of special interest being stachydrine), sterols, iridoids, flavonoids, ursolic acid, minerals, and others. Figure 2 presents the constituents of L. cardiaca and their potential biomedical application, as presented by the pre-2010 literature sources.
The data briefly presented in Figure 2 can be completed with the findings from the time period covered by the present review. Rusch et al. [7] identified in the L. cardiaca extract the presence of a chlorinated major iridoid glucoside (7-chloro-6-

Biological Activities of L. cardiaca
The main biological activities of L. cardiaca can be divided in several main categories.

Cardiovascular Action.
The application of L. cardiaca in cardiovascular disorders represents one of the main applications of motherwort products [2]. Ritter et al. [16] evaluated the cardiac and electrophysiological effects of several types of L. cardiaca extracts by epicardial potential mapping and by evaluation of the effect on the cardiac ion currents using different types of cell models. The obtained results suggested that L. cardiaca extract acts as a mixed I Ca.L-(L-type calcium current), I Kr-(rapid delayed rectifier current) antagonist, and I f (funny current, recorded in sinoatrial node cells from guinea pigs) modulator, supporting its application as an antianginal and antiarrhythmic agent.
The cardioprotective potential of ursolic acid (the natural pentacyclic triterpenoid carboxylic acid commonly found in different L. cardiaca formulations) was demonstrated by Liobikas et al. [17]. Their study revealed that the ursolic acid induced uncoupling of oxidative phosphorylation in the heart mitochondria without affecting State 3 respiration rate, in the same time suppressing the H 2 O 2 production in isolated mitochondria, in a dose-dependent manner. The uncoupling of mitochondrial oxidation from phosphorylation, partial inhibition of the mitochondrial respiratory chain, and a reduction in the generation of free radicals in mitochondria were also observed by Bernatoniene et al. [18] in rat heart mitochondria using L. cardiaca ethanol extracts.  The clinical trial conducted by Shikov et al. [19] on fifty patients treated with 1200 mg L. cardiaca oil extract per day for 28 days revealed significant changes in systolic blood pressure, diastolic blood pressure, heart rate, and ECG for patients with stages 1 and 2 arterial hypertensions, accompanied by an improvement of psychoemotional status (anxiety, emotional liability, headache, and sleep disorders), especially visible for stage 1 patients.

Anti-Inflammatory, Antimicrobial Effect, and Application in Female
Disorders. The anti-inflammatory potential of leonurine (considered by some authors a natural component of L. cardiaca [5]) on E. coli-induced mastitis in mice [33]: their results suggested that leonurine alleviates the histopathological changes, downregulates the levels of proinflammatory cytokines (TNF-and IL-6), upregulates the level of anti-inflammatory cytokine IL-10, and inhibits the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The suggested mechanism involves the inhibition of expression of toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-B) activation and mitogen-activated protein kinases (p38), extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK) phosphorylation. The anti-inflammatory potential of leonurine was also demonstrated by Liu et al. [34] in rat animal models of acute gouty arthritis. The obtained results support the use of leonurine as COX-2, mPGES-1 (microsomal prostaglandin E synthase-1), and 5-LOX (5-lipoxygenase) inhibitor, leading to antiarthritis effects. In the same time, amelioration of monosodium urate crystal-induced inflammation was achieved by decreasing interleukin-1 (IL-1 ) and tumor necrosis factor-alpha (TNF-) production.
The immunomodulatory potential of acetone/water extract of L. cardiaca at a 100 g/ml concentration was assessed by Sadowska et al. [35], revealing a significant reduction of the platelet aggregation in the presence of arachidonic acid, an application that could be beneficial in preventing inflammatory lesions. In the same time, the tested extract did not exhibit proapoptotic activity.
The traditional use of L. cardiaca as an anti-inflammatory and antimicrobial agent was also supported by the results presented by Flemmig et al. [36]. They tested several extracts and components of L. cardiaca for their ability to regenerate the pseudo-halogenating activity of lactoperoxidase (LPO). The results supported the use of components with a 3,4dihydroxyphenylic partial structure (such as caffeic acid derivatives or phenylethanoids) as efficient LPO activity regenerators, as well as the use of L. cardiaca ethanol extract for the same application. The same study also presents the isolation of the compound caffeoylmalic acid, which also revealed moderate LPO activity-regenerating effects.
Micota et al. [37] studied the antimicrobial potential of L. cardiaca acetone-water extract and its component ursolic acid by determining the minimal inhibitory concentration, as well as the antiadhesive and antibiofilm properties against Staphylococcus aureus strain (potential etiological agent of infective endocarditis). Their results (MIC = 6 mg/ml for extract and 0.25 mg/ml for ursolic acid) showed weak biostatic activity of L. cardiaca extract in comparison to ursolic acid, but both preparations possessed antiadhesive potential. The S. aureus biofilm formation was slightly inhibited by the extract (5%), but strongly inhibited by ursolic acid (85%) at concentrations of 3/4 MIC.
The study of Samoilova et al. [38] on the effect of subinhibitory doses of plant extract (including L. cardiaca) on Escherichia coli biofilm formation revealed that motherwort extract showed a synergistic effect with sublethal concentration of streptomycin (30 g/ml), inhibiting the specific biofilm formation. As the study was focused on the capacity of low concentration plant extracts and polyphenols to induce adaptive mechanisms in E. coli, it cannot be considered a truly antimicrobial study.
Micota et al. [39] used subinhibitory doses of L. cardiaca extract to establish its effects on the characteristics of Staphylococcus aureus. The beneficial effect of the extract was observed, such as reduction in staphylococcal adherence, aggregates formation, coagulase activity, protein A expression, or alpha-toxin synthesis. However, some of their findings (e.g., enhancement of staphylococcal tolerance to exogenous hydrogen peroxide after preincubation with the extract) led to the conclusion of a possible risk of adverse effects.
Wu et al. [40] presented the application of leonurine for ameliorating the inflammatory responses in endometritis model in mice. The leonurine treatment suppressed the TNF-and IL-1 mRNA levels in uterus tissues, inhibited lipopolysaccharide-induced TLR4 expression, and reduced the phosphorylated p65 and I B proteins.
The clinical trial conducted by Denham et al. [41] documented the herbal prescribing in usual practice, covering a total of 80 herbs on 141 prescriptions (the most encountered being L. cardiaca -77%) for treatment of symptoms associated with the menopause on 35 subjects. L. cardiaca was mainly prescribed to control hot flushes, as a gynecological tonic and as a relaxant.
In a randomized study conducted on 165 women undergoing cesarean section, motherwort, in combination with oxytocin, proved to be efficient for preventing postpartum hemorrhage [42].
Regarding the use of L. cardiaca in female disorders, the plant is listed as a natural remedy for female reproductive system (anxiolytic, antispasmodic, PMS, and menopausal anxiety) [43], as an emmenagogue, nervine, amenorrhea, analgesic, and uterine astringents/vascular decongestants and for treating adolescent dysmenorrhea [44], for treating menopausal anxiety, and as tranquilizer [45]. Lans et al. [46] present motherwort as a natural cure used in North America from colonial times, due to its tonic, emmenagogue, antispasmodic, and nervine properties, citing pre-1900 works.

Antioxidant Action.
The antioxidant activity of L. cardiaca products was evaluated using several methods. Sadowska et al. [35] evaluated the antioxidant potential of L. cardiaca extract by ABTS • , DPPH • , and ferric reducing antioxidant power assay, obtaining values of the antioxidant capacity in the range 350±20-455±17 M Trolox/g. Ebrahimzadeh et al. [47] evaluated the antioxidant potential of Iranian native L. cardiaca extract obtained from dried aerial parts by percolation using methanol, by comparison with Grammosciadium platycarpum and Onosma demawendicum extracts. The results obtained by DPPH • assay (IC 50 = 144 ± 12.1 mg/ml), iron reducing assay (results superior to vitamin C in the concentration range 25-100 g/ml), nitric oxidescavenging assay (IC 50 = 0.15 ± 0.01 mg/ml), metal chelating assay (IC 50 = 20 ± 1 g/ml), and scavenging of hydrogen peroxide (IC 50 = 438.2 ± 21.8 g/ml) were correlated with the total phenol content (54.3 ± 1.8 mg gallic acid equivalent/g of extract). Ebrahimzadeh et al. [48] evaluated the correlation between the total phenolic compounds and total flavonoids content and the nitric oxide scavenging properties for 26 Iranian medicinal plants. The authors found good correlation between total phenolic content (54.3 ± 2.71 mg gallic acid equivalent/g of extract) for L. cardiaca aerial parts methanol extract, total flavonoids content (35.2 ± 1.76 mg quercetin equivalents/g of extract), and nitric oxide radical scavenging activity (IC 50 = 0.15 ± 0.007 mg/ml).
Armatu et al. [49] evaluated the antioxidant potential of several extracts obtained from Romanian Lamiaceae species (including L. cardiaca methanol extract) using the DPPH • assay, phosphomolybdenum method, and chemiluminescence assay in relationship with HPTLC fingerprints and total phenolic content. The results obtained by the three antioxidant assays at 5 mg/ml concentration (DPPH • -20%, total antioxidant capacity -approx. 40 mg ascorbic acid equivalents/g and 48% antioxidant activity for the chemiluminescence activity) were correlated with the relatively low total phenolics content (2.8 mg gallic acid equivalents/g of extract).
Jafari et al. [50] evaluated the total phenolic content and antioxidant capacity (DPPH • assay) of different fractions of Iranian L. cardiaca extract. The best results were obtained for the 50:50 metanolic-aqueous fraction (total phenolic content 70.79±4.41 gallic acid equivalents/g of fraction and IC 50 = 53.79 g/ml -DPPH • assay).
The influence of drying method was studied by Yi and Wetzstein [51] using three drying methods (greenhouse sun-drying, 40 ∘ C oven-drying, and 70 ∘ C oven-drying) on 80% methanol and 80% ethanol extracts from the leaves of cultivated L. cardiaca plants. The best results were obtained for ethanol extracts of 40 ∘ C oven-dried plants (total polyphenolics -approx. 70 mg/g GAE, Trolox-equivalent antioxidant capacity -approx. 400 mM/g TE).
Polysaccharides from L. cardiaca extract exhibited a very good scavenging activity of hydroxyl radicals (IC 50 = 6.98 ± 0.87 mg/mL), compared with vitamin C (IC 50 = 7.59 ± 0.94 mg/mL) [13]. Wong et al. [52] evaluated the antioxidant potential and phytochemical composition of aqueous extracts obtained from Malaysian plants. The extract showed a relatively good antioxidant potential (>60% DPPH • scavenging activity at 16 mg/ml, >80% NO scavenging activity at 10 mg/ml, and >85% metal chelating activity at 10 mg/ml) for a phytochemical composition of 12.13 ± 0.12 mg GAE/g dry weight (total phenolics), 9.86 ± 0.15 mg QE/g dw (total flavonoids), and 2.01 ± 0.01 mg CAE/g dw (hydroxycinnamic acids). The study of Ji et al. [53] on the antioxidant effect of plants with therapeutic potential on gynecological diseases revealed no significant influence on the lag phase duration of copper-induced low-density lipoprotein cholesterol (LDL-C) oxidation, the authors suggesting as main reason for the lack of antioxidant activity the solvent used for extraction. Ziyatdinova et al. [54] proposed an alternative method for the evaluation of the antioxidant activity (determined as DPPH • inhibition using differential pulse voltammetry) and compared the values obtained for several medicinal plants with those obtained spectrophotometrically. The results showed better antioxidant effect for the L. cardiaca infusion (32±1%, determined by differential pulse voltammetry, respectively, 33±1%, determined by spectrophotometry) compared with the tincture (70% ethanol, 16.6±0.4%, determined by differential pulse voltammetry, respectively, 17±2% determined by spectrophotometry). Ziyatdinova et al. [55] described a chronocoulometric method for the evaluation of the antioxidant potential of 42 commercial-available medicinal plants tinctures (including L. cardiaca), also establishing a correlation between the antioxidant potential and the total phenolics content. The evaluation revealed a relatively low antioxidant capacity for the L. cardiaca tincture (1.7±0.02 mg quercetin/mL).
The antioxidant potential of L. cardiaca extracts was also briefly presented in other works (for example, the works of Sen and Chakraborty [56] and Krishnaiah et al. [57]).

Other Applications.
Motherwort was historically used for the treatment of several nervous afflictions, such as depression, anxiety, or stress [57]. Romm [58] classified L. cardiaca as a natural remedy for the treatment of several afflictions, including postpartum depression, while the traditional internal use of motherwort for the treatment of epilepsy was documented by Adams et al. [59]. The potential towards the treatment of anxiety and depressive disorders was evaluated by Rauwald et al. [15,60], by studying the effect of L. cardiaca extract and constituents (isoleosibirin, 7R-chloro-6-desoxyharpagide, lavandulifolioside, stachydrine, and leonurine) on the neuronal receptor gamma-aminobutyric acid (GABA). The extract inhibited the concentration-dependent binding of [(3)H]-SR95531 to the GABA site of the GABA type A receptor with a binding affinity (IC 50 ) of 21 g/ml, suggesting a potential neurological mechanism of action of L. cardiaca, based on interaction to the GABA site of the GABA type A receptor. The individual components tested (except leonurine -IC 50 -15 g/ml) did not exhibit significant activity. Commercially available leonurine was demonstrated by Xu et al. [61] to ameliorate LPS-induced acute kidney injury in mice. The nephroprotective effect was expressed, after 14 days of treatment, by the values of reactive oxygen species (ROS), malonyldialdehyde (MDA), and reduced glutathione (GSH) which were reduced to near control levels, while the lipopolysaccharide-induced tubular damage was significantly ameliorated, decreased renal injury biomarker (KMI-1), and inhibited the nuclear transfer of NF-B p65. A similar nephroprotective effect was registered by Cheng et al. [62] in mouse unilateral urethral obstruction, by suppressing ROS-mediated TGF-/Smad3-induced tubulointerstitial fibrosis and inhibiting NF-B-mediated inflammatory response. Leonurine was also found to ameliorate cognitive disfunction in rats' model [63]. At a 100 concentration, it was found to decrease the oxygen-glucose deprivation-(OGD-) induced brain cell death to approx. 130% (fold of control group), an approx. 50% reduction, compared with the OGD group. Also, leonurine was found to alleviate the impaired spatial learning and memory, as demonstrated through Morris water maze test. Leonurine also decreased the concentrations of glutamate and hydrogen peroxide in hippocampus, ameliorated the impaired long-term depression in hippocampus, improved cognitive function by modulating the N-methyl-D-aspartate receptors-associated proteins, and protected rats from bilateral carotid artery occlusion-induced damage by inhibiting autophagy. The results suggested leonurine as a potential drug candidate for chronic cerebral hypoperfusion.
Ethanolic extract obtained from aerial parts of L. cardiaca was evaluated by Rezaee-Asl et al. [64] as a potential analgesic, using formalin, tail flick, and hot plate tests in mice. The results of the study proved that at 500 mg/kg the extract was able to reduce the formalin-induced pain in the early phase, increase the antinociceptive activity, and significantly influence the reaction time of the animals to the hot plates, supporting the analgesic properties of the extract, action mediated through peripheral, and central inhibitory mechanisms.
Onumah [66] presents L. cardiaca as a potential adjuvant in treating overactive thyroid, due to its action against symptoms associated with hyperthyroidism (palpitations and anxiety). Inhibition of the thyroid-stimulating hormone and the reduction of excess production of thyroid hormones were also presented by Shokri et al. [67], the property being assigned to its content in rosmarinic acid.
Unlike many other plant species, the evaluated literature data (even outside the time period covered by the present review) presents no research regarding the phytosynthesis of metallic nanoparticles using L. cardiaca extracts, although the procedure was presented for L. japonicus [68]. Table 1 summarizes the main biological activities presented.

Dosage and Toxicology
As previously has been presented, L. cardiaca preparations are currently used in the treatment of several conditions. Relative to commercial products, the EMA report cited [5] presents them to be safe, suggesting a duration of use limited to four weeks. The report also presents the adverse effects of an intake of 3.0 grams of a powdered extract per day (diarrhea, uterine bleeding, and stomach irritation). L. cardiaca is listed as a "herb to avoid during pregnancy" [69,70], mainly due to its emmenagogue and uterine stimulation properties. Kaye et al. [71] listed L. cardiaca as a "herbal drug associated with bleeding abnormalities", an aspect to be considered by the anesthesia practitioner. Anadón et al. [72] presented the reduction of platelet aggregation and fibrinogen levels upon intravenous administration of motherwort. L. cardiaca also potentiates antithrombotic and antiplatelet effects, increasing the risk of bleeding. When administered concomitant with benzodiazepines, motherwort can also have a synergistic sedative effect resulting in coma [73].
L. cardiaca was also presented to potentialize the effect of warfarin, by inhibiting platelet aggregation [74].
Related to individual compounds, data are scarce and mainly outside the current review time period: Milkowska-Leyck et al. presented moderate toxicity of lavandulifolioside (LD 50 approx. 1000 mg/kg) and n-butanol extract (LD 50 approx. 400 mg/kg) for intravenous administration, while for oral administration, the toxicity was much lower (LD 50 >2000 mg/kg) [75]; Mitchell and Rook [76] present the potential of L. cardiaca leaves to cause photosensitization and dermatitis; herbal intravenous injection has a LD 50 of 30-60 mg/kg (mice), while the intravenous LD 50 of the total alkaloids of the herb was approx. 570 mg/kg, while the minimal lethal dose of leonurine in frogs (subcutaneous administration) was 400-600 mg/kg [77]. Due to the lack in scientific evidences, most of the sources presenting traditional use of motherwort suggest the strict following of relevant directions on products containing L. cardiaca and the requesting supplemental information from pharmacists, physician, or other healthcare professionals before use [78]. Some chemical components from L. cardiaca aerial parts (pyrrolidine alkaloids, such as stachydrine, cyclic peptide, such as cycloleonurinine or labdane diterpenes, such as leosibiricin) are considered "chemicals of concern" for human health when used in food and food supplements [79].

Conclusions
The use of motherwort (Leonurus cardiaca L.) has been documented since ancient times, especially as a cardiotonic and for the treatment of gynecological conditions. The composition (dominated by furanic diterpenes, alkaloids, sterols, and iridoids) was proved to present a complex biological activity, with cardioprotective, antioxidant, antimicrobial, anti-inflammatory, analgesic, nephroprotective, and antiviral properties, among others.
The current study aimed to present the progress made in the study of motherwort from the date of the European Medicine Agency "Assessment report on Leonurus cardiaca L., herba". According to our findings, most of the literature data focuses on cardioprotective and antioxidant potential of L. cardiaca, although the data also suggest the exploration of new applications. This, in turn, would be a promising research area for future studies. Future research should also be focused on a definitive conclusion regarding the composition of motherwort (especially the presence of leonurine), as well as the opening of new research directions, such as the use of L. cardiaca extracts in nanotechnology (for the phytosynthesis of nanoparticles).

Conflicts of Interest
The authors declare no conflicts of interest.