An Evaluation of the DNA-Protective Effects of Extracts from Menyanthes trifoliata L. Plants Derived from In Vitro Culture Associated with Redox Balance and Other Biological Activities

Menyanthes trifoliata L. is a valuable medical plant found in Europe, North America, and Asia, which grows on peat bogs and swamps. It has long been used in folk medicine as a remedy for various ailments. This is the first report to demonstrate the protective antioxidant and anti-inflammatory properties of aqueous methanolic extracts derived from the aerial parts (MtAPV) and roots (MtRV) of in vitro grown plants on human umbilical vein endothelial cells (HUVECs). It describes the influence of the tested extracts on the expression of antioxidant (HO-1, NQO1, NRF2, kEAP1, and GCLC) and inflammation-related genes (IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ) in cells stimulated with H2O2 or LPS, respectively. In addition, M. trifoliata extracts were found to moderately affect the growth of certain bacterial and fungal pathogens, with the strongest antibacterial effect found against Pseudomonas aeruginosa and Enterococcus faecalis. M. trifoliata extracts demonstrated protective effects against mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage caused by ROS, decreasing the numbers of mtDNA lesions in the ND1 and ND2 genes and nDNA damage in the TP53 and HPRT1 genes and reducing cleavage in PARP1- and γ-H2A.X-positive cells. The root extract of in vitro M. trifoliata (MtRV) appears to have better anti-inflammatory, antioxidant, antimicrobial, and protective properties than the extract from the aerial part (MtAPV). These differences in biological properties may result from the higher content of selected phenolic compounds and betulinic acid in the MtRV than in the MtAPV extract.


Introduction
Health-promoting properties of plants have been used in the prevention and therapy of many human diseases for thou-sands of years. Currently, it is estimated that 300,000 plant species exist worldwide [1]; however, relatively few have confirmed therapeutic or protective properties. Fortunately, modern methods and equipment allow much faster and 2.2. M. trifoliata Aerial Part and Root Extract Preparation. The plant extracts from aerial parts and roots of M. trifoliata plants grown in vitro were prepared according to Sitarek et al. [15]. Briefly, 10 g of dry weight of aerial parts and roots was used for extraction. The plant material was extracted for 15 minutes with 80% (v/v) aqueous methanol (500 mL) at 35°C using an ultrasonic bath and then for 15 minutes with 300 mL of the same solvent. Finally, the extracts were filtered, evaporated under reduced pressure, lyophilized to dryness, and then kept dry in the dark until further use. The yields (w/w) of the extracts were 52.8% and 50.4% of initial plant dry weight for aerial parts (MtAPV) and roots (MtRV) of M. trifoliata plants, respectively.  2.5. Cell Viability. The MTT assay was employed to measure the viability of HUVECs treated with different concentrations of LPS or H 2 O 2 and the MtRV and MtAPV extracts of M. trifoliata plants grown in vitro. Briefly, cells were seeded at 1 × 10 4 cells per well in 96-well culture plates and were left overnight before being treated for attachment. The cells were incubated for 24 hours with MtRV and MtAPV extracts over the concentration ranges 0-5 mg/mL. The inflammatory process was initiated with 1 μg/mL LPS, and separately, the antioxidant response was stimulated with 50 μM H 2 O 2 . In the next experiment, the cells were incubated with 1 mg/mL MtRV and MtAPV extracts or pretreated with LPS or H 2 O 2 for one hour and then treated with plant extracts for 24 hours. After completing the incubation, the cells were washed once and incubated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at 37°C for four hours. The MTT was then carefully removed and DMSO (100 μL) added to each well. The mixture was vortexed at low speed for five minutes to fully dissolve the blue crystals. Absorbance was measured at 570 nm with a reference at 630 nm using a BioTek Synergy HT Microplate Reader (BioTek Instruments, Winooski, VT, USA). Independent experiments were repeated in triplicate. Cell viability was expressed as a percentage relative to the untreated (control) cells, which was defined as 100%.
2.6. Gene Expression. HUVECs were first incubated in the presence of LPS or H 2 O 2 for one hour and then with 1 mg/mL MtRV and MtAPV extracts for 24 hours. Total RNA was then extracted using an ISOLATE II RNA Mini Kit, according to the manufacturer's instructions. cDNA was synthesized from total RNA using a High-Capacity cDNA Reverse Transcription Kit. A sample of 1 ng total RNA was used as a template in a total volume of 10 μL, following the manufacturer's instructions. Next, gene expression was analyzed by TaqMan Probe-Based Real-Time PCR Assay. TaqMan probes (Life Technologies) were used to analyze 10 genes (HO-1, NQO1, kAEP1, GCLC, IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ), and beta actin (ACTB) was included as the reference gene. qRT-PCR was performed using TaqMan® Real-Time PCR Master Mix (Life Technologies) and a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) working on CFX Manager™ Software (version 3.1). The thermal cycling conditions were as follows: 10 minutes of polymerase activation at 95°C, followed by 40 cycles of 30 s denaturation at 95°C and 60 s annealing/extension at 60°C. Each sample was run in triplicate. The basal expression level was calculated using the C t method [16].

Determination of Nuclear DNA (nDNA) Damage and
Mitochondrial DNA (mtDNA) Damage. Total genomic DNA (nuclear and mitochondrial) was isolated from cells treated with 1 mg/mL MtRV and MtAPV extracts for 24 h and then treated with 50 μM H 2 O 2 for one hour using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. DNA concentrations were determined by spectrophotometric measure-ment of absorbance at 260 nm, and the purities were calculated by the A260/A280 ratio using a BioTek Synergy HT Microplate Reader (BioTek Instruments, Winooski, VT, USA). The semilong run quantitative RT-PCR (SLR-qRT-PCR) was used to assess mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage, as described previously, with some modifications [17]. All conditions were described previously [18].
2.8. Cytoplasmic ROS and Mitochondrial ROS Detection. The cells were incubated with 1 mg/mL MtRV or MtAPV for 24 hours and then with 50 μM H 2 O 2 for one hour. DCFDA (molecular probe, Life Technologies) staining was used to detect cytoplasmic ROS. DCFDA was diluted to a 5 μmol/L final concentration with Hanks' balanced salt solution (HBSS), which was incubated with the cells at 37°C for 30 minutes. A MitoSOX Red mitochondrial superoxide indicator was used to measure mitochondrial ROS production. The indicator was diluted to a 5 μmol/L final concentration with HBSS, which was applied to incubate cells at 37°C for 30 minutes. The cells were washed three times with HBSS. The fluorescence emission was measured using a BioTek Synergy HT Microplate Reader. Each sample was run in triplicate.   2.11. Data Analysis. The statistical analysis was performed using GraphPad Prism 5. All experimental values presented in this study were expressed as mean ± standard deviation (SD). The Shapiro-Wilk test to analyze the normality of the distribution of results was used. Next, the results were analyzed for equality of variance using Levene's test. Significant differences were identified using the ANOVA test for multiple comparisons, followed by the Dunnet post hoc test.

Effects of MtAPV and MtRV Extracts on Cell Viability.
Our results showed that the MtRV and MtAPV extracts of M. trifoliata plants did not present any cytotoxic effect on the human umbilical vein endothelial cells (HUVECs) in the tested concentrations (0-5 mg/mL) after 24-hour incubation ( Figure 2).
In further experiments, 1 μg/mL LPS or 50 μM H 2 O 2 was used: these values did not exhibit 50% growth inhibition (IC 50 value) of cells and were used for the induction of inflammation or antioxidant effect, respectively (data not shown). In addition, the effect on cell viability was deter-mined for both extracts of M. trifoliata in combination with LPS or H 2 O 2 . It was found that the HUVECs demonstrated no significant difference in cell viability following treatment with MtRV or MtAPV extracts alone, MtRV or MtAPV in combination with H 2 O 2 , and also MtRV or MtAPV in combination with LPS ( Figure 3).

Gene Expression.
Quantitative real-time RT-PCR was used to measure mRNA levels of the antioxidant genes HO-1, NQO1, NRF2, kAEP1, and GCLC and inflammation genes IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ after treatment with 50 μM H 2 O 2 or 1 μg/mL LPS for one hour and after treatment with the MtRV or MtAPV extract of M. trifoliata for 24 hours. Our results demonstrated that treatment with both extracts at a concentration of 1 mg/mL was able to change the level of antioxidant genes in H 2 O 2 -stimulated HUVECs compared to the controls. Similar results were obtained for inflammatory genes. All the tested genes in the LPS-stimulated cells demonstrated changes in expression following treatment with either extract compared to controls, but better anti-inflammatory properties were observed for the MtRV extract. The results are shown in     (Figures 7(a) and 7(b)).

Assessment of Antimicrobial Activity of MtAPV and
MtRV Extracts. The antimicrobial activity of MtRV and MtAPV extracts against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Saccharomyces cerevisiae, and Candida albicans was screened using MIC and MBC/MFC methods. Both the tested extracts showed moderate antimicrobial activity ( Table 2) against various strains at a range of MIC values (150-925 μg/mL) and for MBC/MFC (500-2500 μg/mL). The MtRV extract showed better activity than the MtAPV extract against P. aeruginosa and E. faecalis, with a MIC value of 150 μg/mL. The MtRV extract demonstrated also better antifungal activity than the MtAPV extract for C. albicans and S. cerevisiae, with MIC values of 625 μg/mL and 725 μg/mL, respectively, and with MFC values of 625 μg/mL and 1500 μg/mL, respectively.

Discussion
Plants are well-known to have beneficial effects on human health. Their extraordinary wealth of biologically active compounds makes them indispensable for human life and allow the prevention of many civilization diseases. One plant species that contains many valuable biologically active compounds is Menyanthes trifoliata. Extracts and infusions of this plant are used as anti-inflammatory, diuretic, or cleansing agents, becoming an important element of phytomedicine [20].
This work is the first to demonstrate the protective properties of M. trifoliata extracts derived from aerial parts and roots of plants cultivated in vitro. It examines the antiinflammatory and antioxidant properties of M. trifoliata extracts (MtRV and MtAPV extracts) in human umbilical vein endothelial cells (HUVECs) which had previously been treated with LPS or H 2 O 2 . Inflammation is a very complex physiological phenomenon developing in the tissue under the influence of different stimuli and is mediated by a wide range of specific components. It involves interactions between signal molecules produced by leukocytes, macrophages, and mast cells, as well as by the activation of complements [21][22][23]. A range of studies describe the antiinflammatory properties of plant extracts against a wide spectrum of normal and cancer cells [24][25][26].
One of the aims of the study was to investigate the antiinflammatory properties of M. trifoliata extracts on HUVECs following stimulation by lipopolysaccharide (LPS) for 24 hours. LPS is the main component of the outer membrane of Gram-negative bacteria. It activates mononuclear phagocytes and other cell types by toll-like receptor 4 to promote the secretion of inflammatory mediators including TNF-α, IL-6, and IL-1β [27]. It was found that the aerial part (MtAPV) and root (MtRV) extracts of in vitro-derived M. trifoliata exhibit an anti-inflammatory effect by decreasing the expression of selected genes encoding inflammationassociated cytokines (IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ).
Many reports indicate that M. trifoliata is a source of phenolic compounds such as coumarins, flavonols, or iridoids [11]. Our previous study also showed the presence of various phenolic compounds in the extracts of in vitro-and     in vitro cultures. The anti-inflammatory and antioxidant activity of this plant compound is also well documented in literature [28][29][30].
Our present findings indicated that the tested M. trifoliata extracts bestow a DNA-protective effect on the tested cell line. We speculate that betulinic acid and the identified phenolic acids (syringic acid, sinapinic acid, ellagic acid, and chlorogenic acid) may be responsible for this positive effect, especially as betulinic acid and sinapinic acid have been found to suppress inflammatory cytokines such as IL-6 or TNF-α [31,32]. Betulinic acid (lupane-type triterpene) exhibits a protective effect on mice exposed to lethal doses of LPS and causes a reduction of LPS-induced TNF-α production. In addition, betulinic acid is also known as an inhibitor of IFN-γ production [33]. Kim et al. report the compound to be an anti-inflammatory agent acting via inhibition of the nuclear factor-κB (NF-κB) pathway. Similarly, suppression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) was demonstrated in LPS-activated RAW 264.7 macrophages after betulinic acid treatment [34]. Similar properties have also been described for sinapinic acid [35][36][37], ellagic acid [38], or chlorogenic acid [39].
The present study also analyzes the influence of the two M. trifoliata extracts on oxidative stress-related gene expression. Changes in the expression of genes encoding heme oxygenase 1 (HO-1), quinone dehydrogenase 1 (NQO1), nuclear factor (erythroid-derived-2)-like-2 (NRF2), Kelchlike ECH-associated protein 1 (kEAP1), or glutamatecysteine ligase catalytic subunit (GCLC) cast light on the protective role played by the components of the tested extract in H 2 O 2 -stimulated cells. The MtRV and MtAPV extracts, administered at 1 mg/mL, were found to influence the expression of HO-1, NRF2, kEAP1, NQO1, and GCLC genes in H 2 O 2 -stimulated HUVECs, with the MtRV extract demonstrating better protective properties than the MtAPV.
Previous studies provide a wealth of information describing the cytoprotective effect of plant phenolic compounds on HUVECs [40][41][42]. Some affect the expression of oxidative stress-related genes; for example, those contained in the green tea extract increase the mRNA level of heme oxygenase-1 (HO-1) in human aortic endothelial cells [43], while the polyphenolic antioxidant fraction from Nymphaea nouchali leaves increases HO-1 and Nrf2 level in RAW 264.7 cells [44]. Recently, special attention has been paid to the  close relationship between inflammation and oxidative stress. The appearance of an excessive amount of free radicals reacts with cell membrane fatty acids, proteins, or DNA, leading to mutation and consequently to the development of many diseases. In this context, searching for natural components that protect cells from adverse changes is extremely desirable. The present study also tested cytoplasmic and mitochondrial ROS levels in H 2 O 2 -stimulated HUVECs after treatment with MtRV and MtAPV extracts. Our results, obtained with dichloro-dihydro-fluorescein diacetate and red mitochondrial superoxide indicator (MitoSOX), found the plant extracts to exert a protective effect by decreasing cytoplasmic and mitochondrial ROS levels in cells. The MtRV extract demonstrated a stronger antioxidant effect than the MtAPV extract. It is known that ROS are produced in epithelial cells by many enzymes, including nicotinamide adenine dinucleotide phosphate oxidase (NOX), nitric oxide synthase (NOS), or xanthine oxidase (XOD), as well as the respiratory chain complex [45]. ROS are extremely important elements that play a crucial role in the initiation of inflammatory processes. Elevated levels of ROS produced by polymorph nuclear neutrophils (PMN) at the site of inflammation can lead to epithelial cell dysfunction and consequent tissue damage [46]. A considerable body of evidence indicates that the excessive cellular production of ROS may be closely related to the induction of inflammation and consequently affect the development of diseases such as Alzheimer's, Parkinson's, or cancer [47]. Our findings confirm that M. trifoliata extracts exhibit protective antioxidant properties, probably due to the presence of identified phenolic compounds and pentacyclic triterpenes, such as betulinic acid, which is consistent with literature data [48]. It should also be noted that the MtRV extract appears to offer the strongest cytoprotective effects.
Nuclear and mitochondrial DNA damage was also quantified by SLR-qRT-PCR amplification of DNA from HUVECs exposed to plant extracts followed by 50 μM H 2 O 2 . Both extracts were found to have a protective effect, resulting in a decrease in the lesion rate for all the tested nuclear and mitochondrial DNA regions after incubation with MtRV and MtAPV extracts compared to cells incubated only with 50 μM H 2 O 2 . Although mitochondrial and nuclear DNA damage, and hence the protective effects of the tested extracts, can be detected by various methods such as Southern blot analysis or comet assay, the present study used a semilong run RT-PCR-based assay. Also, the MtRV extract demonstrated a stronger protective potential against ROS-derived DNA damage than the MtAPV extract.
Plant phenolic compounds are known to bestow a protective effect on DNA against various physical or chemical factors [49][50][51]. A similar study investigating the protective effect of Salvia officinalis and Thymus vulgaris extracts on the DNA of HepG2 cells was performed by Kozics et al. [52]. Their results indicate that H 2 O 2 -induced DNA damage was significantly reduced in cells pretreated with both the tested plant extracts, which were also found to contain phenolic compounds and betulinic acid. Another study examined the effect of a purified Eugenia jambolan leaf and fruit extract on HepG2 cells; the extracts were found to be rich sources of many phenolic compounds and betulinic acid and to exhibit antigenotoxic and antimutagenic effects [53].
Other studies have shown that phenolic compounds, including phenolic acids, can reduce the level of oxidative DNA damage in a variety of cell types [54,55]. However, the present study is first to report the ability of the in vitro-derived M. trifoliata plant extract to protect cells against DNA damage. As noted in previous studies, our results suggest that the protective effect demonstrated by the examined extracts is related to its phenolic compound content, including betulinic acid, and the synergy between extract components.
Flow cytometric analysis showed that M. trifoliata extracts had a protective effect on the tested cells. HUVECs treated with H 2 O 2 generated a higher level of γH2A.Xand cleaved PARP1-positive cells than untreated cells. On the other hand, 24-hour incubation with the studied MtRV and MtAPV extracts exhibited a protective effect against pretreatment with H 2 O 2 by reducing the number of γH2A.Xand cleaved PARP1-positive cells. Poly (ADP-ribose) polymerase 1 (PARP1) is responsible for the ADP-ribosylation process (PARylation), which is a common posttranslational modification of DNA damage. This modification regulates many other biological processes, including the reorganization of chromatin or DNA damage response (DDR). In addition, PARP is a substrate for caspases and is cleaved during apoptosis into 86 and 24 kDa fragments [56,57]. For this reason, PARP1 acts as a good DNA damage sensor [58]. Similarly, the H2A.X protein is a key factor in the repair of damaged DNA and can be phosphorylated on the 139th serine residue in the presence of DNA damage [59]. Phosphorylated protein causes a conformational change in the DNA-H2A.X complex, which allows recruitment of proteins needed to repair DNA injury [60].
Our results clearly show that M. trifoliata extracts from plants derived from in vitro cultures play an important role in DNA protection of the tested cells. This is consistent with other literature data showing that polyphenol-rich natural products also inhibit the increases in cleaved PARP level [61,62]. Similarly, the level of phosphorylated H2A.X, another determinant of DNA damage, has previously been found to be reduced under the influence of the black tea or Camptosorus sibiricus extract [63] in other cells. It is most likely that the phenolic compounds and pentacyclic triterpene present in the MtRV and MtAPV extracts may be responsible for the biological effect observed in the present study.
The study also examines the antibacterial and antifungal properties of M. trifoliata extracts. The tested MtRV and MtAPV extracts demonstrated stronger antibacterial than antifungal action. It was also shown that the MtRV extract possessed a stronger effect against the tested microorganisms than MtAPV. The best bacterial growth inhibitory effects were obtained against Pseudomonas aeruginosa (ATCC 27853) and Enterococcus faecalis (ATCC29212) for the MtRV extract (MIC = 150 μg/mL). Similar effects were demonstrated for the MtAPV extract against all the tested bacteria (MIC = 250 μg/mL). Also, better fungal growth inhibition was exhibited by the MtRV extract than the MtAPV extract against Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 2601), where the MIC for Candida albicans was 625 μg/mL. Many plant species naturally produce compounds with antimicrobial or antifungal properties [64][65][66]. Ivanišová et al. [67] examined the antimicrobial activity of M. trifoliata leaf extracts, rich sources of polyphenols and flavonoids, and extracts from other plants against three Gram-negative bacteria and two Gram-positive species. The M. trifoliata extract demonstrated the strongest antibacterial activity against Escherichia coli, Salmonella enterica subsp. enterica, Bacillus thuringiensis, Staphylococcus aureus subsp. aureus, and Pseudomonas aeruginosa, which corresponds to our results. This study found M. trifoliata to be the richest source of polyphenols, flavonoids, and phenolic acids among all the analyzed wild plants in the study. The antibacterial and antifungal properties of M. trifoliata extracts have also been investigated by Paudel et al. [68] by testing their influence on pathogenic microorganisms such as S. aureus, E. coli, and C. albicans. In this case, the plant extracts were active only against S. aureus. The authors attribute these differences in antimicrobial activity to variations in antimicrobial metabolites among the tested samples.
The search for new antibacterial compounds has recently become an extremely important issue due to emerging resistance of pathogens to traditional medicines; in this sense, plant-derived compounds can play a potentially important role. They often have specific chemical structures that can inhibit bacterial growth through new mechanisms of action [69]; however, this group of secondary metabolites may also be of great importance in the development of new compounds against human pathogens. Based on data obtained in previous studies [70][71][72], it is likely that betulinic acid may be chiefly responsible for the antibacterial and antifungal effects of the tested extracts [70,73]. Additionally, we suspect that phenolic acids may have a similar effect and show synergistic effects with other components, particularly since the MtRV extract showed the best antimicrobial properties. Generally, the antibacterial and antifungal mechanism of action of many phenolic compounds is not yet fully understood [69]. Some studies indicate that they interact with a range of targets such as DNA, DNA gyrase [74], protein kinases [75], and helicase, among others [69]. Attempts should be made to identify new antimicrobial components, especially as increasing numbers of multidrug-resistant strains are appearing in the environment [76].

Conclusion
The results of our study demonstrate for the first time that MtRV and MtAPV extracts from M. trifoliata plants derived from in vitro cultures showed antioxidant and antiinflammatory effects on human umbilical vein endothelial cells (HUVECs) stimulated by H 2 O 2 or LPS, and of the two, the MtRV extract demonstrated the strongest activities. Additionally, the protective effect was observed which was associated with a decrease in DNA damage, reductions in cytoplasmic and mitochondrial ROS levels, and reduced γH2A.Xand cleaved PARP1-positive HUVEC numbers. Furthermore, the tested extracts demonstrated moderate antimicrobial activities against various pathogenic bacterial and fungal strains. Therefore, this plant may play a potential role in the therapy and prevention of many civilization diseases.

Data Availability
All data are available at the request of the reviewer or other interested persons.

Conflicts of Interest
The authors declare no competing interests.