Evaluation of anticancer and antimicrobial activities of the Polygonum maritimum ethanol extract

Polygonum maritimum is a traditional herbal remedy that produces abundant flavonoid secondary metabolites. The ethanol extract of P. maritimum aerial parts (POM) was chemically characterized and tested for antimicrobial properties and cytotoxicity. Results of LC-MS/MS analysis showed high contents of gallic acid, epigallocatechin gallate and catechin, and significant amounts of quercetin-3-O-galactoside and quercetin-3-O-glucoside. Evaluation of the antifungal properties revealed that POM induced notable growth inhibition of Alternaria alternata (34.3%), Penicillium spp. (30.6%), Fusarium semitectum (20.2%) and Aspergillus spp. (19.6%). Evaluation of cytotoxicity against human hepatoma HepG2 cells included monitoring the effects of both POM alone and its combination with cytostatic doxorubicin (Dox). Cell viability, apoptosis and cell cycle distribution and the expression of antioxidant enzymes (superoxide-dismutases SOD1 and SOD2 and catalase) were determined. A dose-dependent decrease in cell viability was detected, but a remarkably stronger effect was obtained when POM and Dox were applied in combination as compared to individual treatments. IC50 values were determined to be 393 μg/mL (POM) and 2.24 μg/mL (Dox) in combination, but 1153 μg/mL (POM) and 12.56 μg/mL (Dox) in a single treatment. The value of the Loewe index, determined for IC50, was notably lower than 1 (LI=0.51), clearly indicating synergism of POM and Dox. Additionally, POM and POM +Dox induced early/late apoptosis and G2/M cell cycle arrest. Furthermore, POM increased, while Dox decreased the expression levels of SODs and catalase. The obtained results encourage further examination of the potential use of POM in modern phytotherapy.


INTRODUCTION
The genus Polygonum belongs to the family Polygonaceae, one of the dominant families in the flora of most countries, especially in the Northern Hemisphere [1].Being native to Europe, Asia and Africa, Polygonum herbs have been used as traditional medicines in many different cultures for treating dysentery, articular pain, inflammation and diabetes [2][3][4].As reported in recent publications, plants belonging to Polygonum genus are known to produce a large number of biologically important secondary metabolites, such as flavonoids, anthraquinones, stilbenoids, glycolipids and terpenes [1,[5][6][7].These secondary metabolites are largely viewed as potential sources of novel anticancer and antiallergic agents, antiinflammatory drugs, antibiotics, insecticides and herbicides.The pharmacological effects of Polygonum spp.include antioxidant, antibacterial, antifungal, neuroprotective, antidiabetic and antiinflammatory activities [1,[5][6][7][8].Furthermore, the species Polygonum maritimum, commonly known as sea knotgrass, has been described for its antioxidant activities [1].It contains quercetin, quercitrin and catechin, known for their numerous bioactivities [9].
Considering the existence of biodegrading and storage-contaminating fungi in foods, as well as fungal toxicity and potential pathogenicity, there is a constant need for their growth control.Since synthetic substances targeting plant fungal pathogens can produce toxic residues, the preservative activity of herbs and spices has received notable attention [10].In addition, the resistance of fungi to conventional antimycotics further stimulates the search for natural inhibitors [11].
The use of cytostatics in treating different cancers is burdened with numerous problems, including many side effects and the frequent development of drug resistance in tumor cells [12,13].
Taking into account the abovementioned facts, the aims of this study were to examine the cytotoxic and antifungal properties of the ethanolic extract of P. maritimum (POM).Its cytotoxicity was assessed in human hepatocarcinoma HepG2 cells and included an evaluation of its effect on antioxidant enzyme expression and its potential to modulate the cell's response to the common cytostatic doxorubicin (Dox).To the best of our knowledge, none of this has been previously reported.Furthermore, the antifungal effect was examined against Penicillium spp., Alternaria alternata, Aspergillus spp., Fusarium semitectum and Fusarium oxysporum by monitoring growth inhibition and mycelial decolorization.

Plant material and extract preparation
Aerial parts of POM were collected along the Adriatic coast, in Ulcinj, Montenegro.The voucher specimen was deposited at the Herbarium of the Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Serbia (BUNS Herbarium) under the reference number 2-1670.The dried plant material (5-15g) was extracted with 80% ethanol (1g per 10 mL) and filtered.The obtained extract was evaporated to dryness in a vacuum evaporator (<45°C).Raw extract was suspended in water and purified by liquid-liquid extraction with petroleum ether.The petroleum ether layer was washed with methanol and the methanol extract was pooled with the aqueous layer.Subsequently, the extract was evaporated and dissolved in DMSO to a final concentration of 100 mg/mL.

Identification of compounds
The phytochemical profile of P. maritimum was evaluated by measuring the total phenolic and total flavonoid contents [14] and determined by quantitative LC-MS/ MS analysis [15].

In vitro antifungal activity
The antifungal activity of the plant extract was determined by the agar plate method [16].The fungi Penicillium spp., Alternaria alternata, Aspergillus spp., Fusarium semitectum and Fusarium oxysporum were obtained from the Institute of Medicinal Plants Research "Dr.Josif Pančić", Pančevo, Serbia.Micromycetes were maintained on potato dextrose agar (PDA).One hundred µL of the extract with a particular concentration were brushed with a glass sterile stick in the PDA substrate.The POM was tested at a concentration of 1 mg/mL against Penicillium spp., Fusarium oxysporum and Alternaria alternata, and at a concentration of 5 mg/mL against Fusarium semitectum and Aspergillus spp.Seven-day-old agar disks (5 mm in diameter) bearing the desired fungus growth were transferred to the center of Petri plates with PDA.The fungus cultures were incubated at 25±2°C for 7 days.The experiment was set for up to four replicates and repeated three times.The percentage of mycelial growth inhibition was calculated according to the following formula: %I=100×(dc×dt)/dc where I -mycelial growth inhibition in %, dc -mycelial growth in control in mm, dt -mycelial growth with POM in mm.

Cell culture
Human hepatocellular carcinoma HepG2 cells (ATCC HB-8065, USA) were grown in William's medium containing 15% FBS, 1% penicillin/streptomycin and 2 mM L-glutamine.The cells were maintained in an incubator at 37ºC with 5.0% CO 2 in a humidified atmosphere.The cells were subcultured at 90% confluence, twice each week, using 0.1% trypsin.Cell viability was determined by the trypan blue dye exclusion method.Cells in the logarithmic growth phase were used in all experiments.

Cytotoxicity assay
The cytotoxic effects of the plant extract and doxorubicin, both as single compounds and in mixture, were assessed by a modified MTT assay [17].Briefly, HepG2 cells were seeded into 96-well plates at a density 2x10 4 cells/well and incubated overnight at 5% CO 2 , 37°C.The cells were exposed to a series of two-fold dilutions of test substances and their combinations to obtain concentrations ranging from 4-0.125 mg/mL of POM, and from 22.8-0.712µg/mL of Dox.After incubation for 24 h, the medium containing test substances was displaced with MTT (final concentration 0.5 mg/mL) and incubated for additional 3 h.At the end of the incubation with MTT, the medium was removed, and the formazan crystals were dissolved in DMSO.The optical density was measured at 570 nm using a microplate reading spectrophotometer (Multiskan FC, Thermo Scientific, Shanghai, China).Three independent experiments were conducted, and external wells were excluded.

Analysis of apoptosis by flow cytometry
HepG2 cells (1x10 6 cells/plate) were cultured with plant extract, POM (0.5 and 1 mg/mL) with and without Dox (2.8 and 5.7 µg/mL) for 24 h.After the treatment, the cells were harvested, washed with PBS, stained with Annexin V_FITC and 7-AAD, and analyzed by fluorescence activated cell sorting (FACS) (Calibur Becton Dickinson, Heidelberg, Germany) flow cytometer and Cell Quest computer software, according to the manufacturer's protocol.

Cell cycle analysis
HepG2 cells were treated as described above.After the treatment and incubation for 24h, cells were harvested and fixed with ice-cold 70% ethanol at -20°C for 30 min.Subsequently, the cells were resuspended in PBS containing propidium iodide and RNase A and incubated for 30 min at room temperature.Distribution of the cells was measured by FACS analysis.

Western blot analysis
After treatment with the test compounds, the cell lysates were prepared in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail solutions.The protein concentration was determined according to the Lowry method [18] at 750 nm, using bovine serum albumin (BSA) as a standard.Protein expression of CuZn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2) and catalase (CAT) were determined by Western blot analysis.Cell lysates (20 μg of samples per lane for SOD1 and SOD2 and 50 μg per lane for CAT) were poured on 12% SDS-polyacrylamide gels and transferred overnight to 0.45 mm polyvinylidene difluoride (PVDF) membranes after electrophoresis.The membranes were blocked in 5% dry non-fat milk in Tris buffered saline containing 0.1% Tween 20 (TBST) for 1.5 h.Anti-SOD1, -SOD2 and -CAT rabbit polyclonal antibodies (1:2000) were used as primary antibodies.Anti-β actin mouse monoclonal antibody (1:1000) was used as a loading control.Horseradish peroxidase-conjugated secondary goat anti-rabbit polyclonal antibodies (for SOD1, SOD2 and CAT, 1:5000) or rabbit anti-mouse polyclonal antibody (for beta-actin, 1:5000) were used as a secondary antibody.The immunoreactive bands were visualized using the enhanced chemiluminescence detection kit (ECLplus, Amersham, Bucks, UK) on a ChemiDoc-It2 detection system (UVP, Jena, Germany).The intensity of signals was evaluated by the ImageJ program.The amount of protein in each lane was normalized to β-actin.

Statistical analysis
To analyze the type of pharmacokinetic interactions of Dox and the extract in combination, the Loewe additivity model (LI) was used and data from the MTT assay were subjected to statistical analysis with the mixLow package in R-studio [19].Data from apoptosis, cell cycle and Western blotting were analyzed by analysis of variance (ANOVA) using GraphPad Prism software.The level of statistical significance was defined as p<0.05.Microsoft Excel software was used to perform the statistical analyses for antifungal experiments.

Identification of compounds
The obtained results showed that the P. maritimum extract of the aerial parts has a high total phenolic content (483 mg eqv.gallic acid/1g dry extract) but a lower total flavonoid content (1.4 mg eqv.quercetin/1g dry extract).The dominant compounds of the POM extract could be presented in following order: gallic acid > epigallocatechin gallate > catechin > quercetin-3-O-galactoside > quercetin-3-O-glucoside (Table 1).

In vitro antimicrobial activity
Antibacterial screening performed by the microdilution assay against a panel of seven pathogenic strains showed no antibacterial activity (in the given range of concentrations; less than 5 mg/mL).Antifungal activity was evaluated by comparing the growth of mycelia in the presence of POM and in the control sample (untreated).Apart from Fusarium oxysporum, POM inhibited the growth of all other tested strains.
The obtained results showed a notable growth inhibitory effect on Alternaria alternata, Penicillium spp., Fusarium semitectum and Aspergillus spp., with inhibitions determined to be 34.3%, 30.6%, 20.24% and 19.6%, respectively (Table 2).Moreover, depigmentation and morphological changes of the Aspergillus spp.mycelium were additional indications of POM toxicity.

Cytotoxicity assay
The cytotoxic effect of the herbal extract, Dox, and their combinations, evaluated on HepG2 cells, is presented in dose-response curves (Fig. 1).The IC 25 , IC 50 and IC 75 values were determined from these curves (Table 3).
In order to quantify the mode of interaction between the tested substances, the Loewe index (LI) was calculated, with LI<1, LI=1, LI>1 indicating synergistic, additive and antagonistic effects, respectively (Fig. 2).Although antagonism was obtained when the binary mixture constituents were applied in low concentrations (for the IC 25 value LI was 1.56), with the increase in their concentrations clear synergism was obtained (for the IC 50 and IC 75 values, LIs were determined to be 0.51 and even 0.35, respectively).For example, 50% lethality was obtained with strikingly lower doses in the cotreatment (393 μg/mL of POM and 2.24 μg/mL of Dox), compared to the treatment with POM (1153 μg/mL) or Dox alone (12.56 μg/mL).

Assessment of apoptosis and cell cycle
To determine whether the cytotoxicity of POM and its combination with Dox is related to apoptosis induction and if it is mediated by cell cycle arrest, flow cytometry was applied.The POM extract (500 µg/mL), alone and in combination with Dox (2.8 µg/mL), increased the percentage of cells in late apoptosis (89.85% and 87.11%, respectively; Fig. 3A).Nonetheless, the combination of lower concentrations of POM and Dox increased the number of early apoptotic cells.Furthermore, a statistically significant increase in early and late apoptotic cells was observed after the cell treatment with 1000 µg/mL of POM alone (25.51% and 67.5%, respectively), and in combination with Dox (45.88% and 51.6%, respectively).As expected, Dox increased the number of apoptotic cells as follows: when applied in a lower dose (2.8 µg/mL) only cells in early phase (62.15%) were observed, but when applied in a higher dose (5.7 µg/mL), both early and late phases (61.65% and 12.75%, respectively) were observed.The cell cycle phase distribution after the treatment of cells with POM, Dox and their combinations is presented in Fig. 3B.Lower concentrations of the extract, Dox, and their combination induced cell cycle arrest in G2/M phase, causing a significant decrease in cell number in the G0/ G1 phase.Higher concentrations and their combination did not significantly affect the cell cycle.

Western blot analysis
To examine the effect of POM on the antioxidant enzymes SOD1, SOD2 and CAT, we employed Western blotting.HepG2 cells were treated either with the  extract (500 µg/mL) or with Dox (1.4 µg/mL).The obtained results showed that Dox decreased the expression of CAT, SOD1 and SOD2, while POM extract significantly increased CAT and SOD1 expression but had no influence on SOD2 (Fig. 4).

DISCUSSION
Many reports have highlighted the potential benefits of using Polygonum species in phytomedicine, revealing the close correlation between their phenolic composition and the pharmacological effects they exhibit, including antioxidant, anticancer and antimicrobial properties.In the course of the examination of the biological activities of POM, its antimicrobial potential was examined first.While no antibacterial activity was found, a notable antifungal effect against food-contaminating micromycetes was observed.Absence of an antibacterial potential is contrary to the results of El-Haci et al. [1], who determined an antibacterial activity of P. maritimum extract against some human pathogens.This discrepancy could be attributed to the difference in the process of extraction [20].On the other hand, the obtained antifungal effect is consistent with literature data showing that extracts of various Polygonum species, such as P. hydropiper, P. aviculare, P. punctatum and P. acuminatum produced fungicidal effects [5,[21][22][23][24].In addition, the antimicrobial activity of gallic acid and catechin, being dominant in POM, was also shown [25].
The cytotoxicity of POM was monitored in human hepatoma HepG2 cells, which have been widely used as an in vitro model for assessing drug effectiveness and toxicity [26].Since chemosensitizing phytochemicals can be combined with conventional chemotherapeutic agents to improve cancer treatment and clinical outcomes, we also monitored the effect of its combinations with doxorubicin (Dox).The benefits of this approach include a reduction in the doses of applied chemotherapeutic drugs, which is important in view of the severe toxicity of conventional cytostatics.This can decrease the toxicity without compromising treatment efficacy through the induction of more diverse mechanisms of action [27].Dox-induced toxicity is mediated by reactive oxygen species (ROS) production and consequently increases lipid peroxidation [28].Furthermore, the medical application of Dox is limited due to its side effects, mainly hepatotoxicity and cardiotoxicity, but also due to frequent development of drug resistance in tumor cells [29].Taking into account that the active principals of POM, including the most dominant flavan-3-ols (catechin and epigallocatechin gallate), flavon-3-ols (quercetin and myricetin derivatives) and free gallic acid, possessing an antioxidative potential [25,30], POM could protect against toxicity mediated by Dox-induced ROS, and certainly affect the expression of antioxidative enzymes.In line with this are the results indicating that extracts of P. glabrum and P. orientale provided hepatoprotective activity in normal hepatocytes in vivo [31,32].
The low cytotoxic effect of POM is in agreement with literature data showing that plant species from the Polygonaceae family have an antiproliferative effect on hepatocarcinoma cells, including Bel-7402 and Hepa 1-6 [33].Concerning the effect of combinations (POM+Dox), both antagonistic and synergistic interactions were obtained.This could be attributed to the dual effect of antioxidants, which induce a prooxidative effect at higher concentrations [34].In light of this, POM at lower tested concentration induced an antioxidant effect and consequently reduced the ROS-mediated toxicity of Dox.On the other hand, with increasing POM concentration, its effect became prooxidative and enhanced Dox toxicity, inducing a remarkable synergistic effect.
In order to analyze the mode of obtained cytotoxicity, we monitored the effect on apoptosis and cell cycle arrest.Our results indicate that POM alone and combined with Dox significantly induced early and late apoptosis and G2/M cell cycle arrest.This is consistent with recent studies showing that various extracts of Polygonum species and their active compounds also induced a proapoptotic effect and arrested HepG2 cells in the G2/M phase [35,36].In addition, literature data confirmed that the cytotoxic activity of gallic acid and catechin, as dominant constituents of POM, could also be linked with the proapoptotic effect and G2/M cell cycle arrest [3,25,27,30].The authors emphasized their ability to induce cell cycle arrest in malignant cells, which contributes to cancer cell proliferation inhibition while leaving normal cells unscathed.Furthermore, gallic acid-based indanone derivatives have been shown to exhibit substantial anticancer activity against HepG2 cells [25].
Medications based on plants, including Polygonum extracts, and conventional cytostatic drugs can influence the metabolism of toxic substances by modulating both nonspecific and specific detoxifying enzymes and by disturbing the balance between apoptosis, the regulation of cell death and proliferation [37,38].Herein we monitored the impact of POM and Dox on the modulation of nonspecific detoxifying/ antioxidant enzymes such as SOD1, SOD2 and CAT.The results indicated that POM enhanced, whilst Dox suppressed SOD1 and CAT activities in HepG2 cells.Some studies reported higher expression of SOD enzymes in hepatocytes after treatment with P. orientale and P. multiflorum extracts, which is in accordance with our findings [32,39].The main constituents of POM, gallic acid and catechin, exert their ability to elevate the hepatic levels of CAT, and therefore may provide protection against the deleterious effects of lipid peroxides and hydrogen peroxide [40].Moreover, gallic acid and epigallocatechin gallate affect the level of antioxidant enzymes by stimulating Nrf2 protein expression through the Keap1-Nrf2-ARE signaling pathway in HepG2 cells [41].It is well known that activation of the Nrf2-ARE cellular defense system is an important mechanism for protecting cells against oxidative and xenobiotic stresses [26,41,42].
Considering all the obtained results, the POMinduced dual protective and cytotoxic effects depended mainly on the applied dose.A similar observation was described by another research group that showed that plant extracts had a protective effect on hepatocytes in vivo as a result of enhanced antioxidant enzyme activities and the cytotoxic effect obtained through increased apoptosis and alterations of the cell cycle [43].
In conclusion, the Polygonum maritimum extract could be a significant natural source of biologically active compounds that possess antifungal and cytotoxic properties.It is important to emphasize its ability to potentiate the chemotherapeutic activity of the conventional cytostatic doxorubicin.However, further investigation is needed to elucidate the mechanism of action of POM and to examine its application both as a fungicide and adjuvant in chemotherapy.

Fig. 1 .
Fig. 1.Inhibition of HepG2 cell viability after treatment with POM, Dox and their combination.The tested concentrations of POM, Dox and their combination are presented as values of natural logarithms (LN values).POM was tested in a concentration range from 4-0.125 mg/mL (that corresponded to the range from 1.386 to -2.079 presented in LN values).Dox was tested in a concentration range from 0.0228-0.0007125mg/mL (that corresponded to the range from -3.780 to -7.246 presented in LN values).

Fig. 4 .
Fig. 4. The effect of POM and Dox on the induction of the antioxidative defense enzymes.A -Blots were normalized to β-actin as the internal control.B -Histograms of densitometric analyses of SOD1, SOD2 and CAT levels following treatment of HepG2 cells with POM or Dox (POM 0.5 mg/mL, Dox 1.4 µg/mL); C -untreated control * p≤0.05.

Table 1 .
Results of phenols quantification in the P. maritimum herbal extract.
*given in mg per dry extract (DW)±relative standard deviation of repeatability; nd -not detected -peak not observed, concentration is lower than the limit of detection

Table 2 .
Inhibition of mycelium growth after treatment with POM.
POM was tested at concentrations of 1 mg/mL against Penicillium spp., Fusarium oxysporum and Alternaria alternata and 5 mg/mL against Fusarium semitectum and Aspergillus spp.*nd -not detected -inhibition of growth is not detected

Table 3 .
The cytotoxicity of POM and Dox, either alone or in a binary-drug combination on HepG2 cells.
* Values are presented as IC 25 IC 50 and IC 75 , in µg/mL, from three independent experiments by the MTT assay.IC 25 -75 -the concentration of sample required to inhibit cell survival by 25%, 50% and 75%, respectively.