Phenolics Profiling by HPLC-DAD-ESI/MSn of the Scientific Unknown Polygonum hydropiperoides Michx. and Its Antioxidant and Anti-Methicillin-Resistant Staphylococcus aureus Activities

Polygonum hydropiperoides Michx. is an Asian native plant species that is also widely distributed in the Americas. Despite its traditional uses, P. hydropiperoides is scarcely scientifically exploited. This study aimed to chemically characterize and investigate the antioxidant and antibacterial activities of hexane (HE-Ph), ethyl acetate (EAE-Ph), and ethanolic (EE-Ph) extracts from aerial parts of P. hydropiperoides. The chemical characterization was performed through HPLC-DAD-ESI/MSn. The antioxidant activity was assessed by the phosphomolybdenum reducing power, nitric oxide inhibition, and the β-carotene bleaching assays. The antibacterial activity was determined by the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration followed by the classification of the antibacterial effect. Chemical characterization revealed the expressive presence of phenolic acids and flavonoids in EAE-Ph. An increased antioxidant capacity was revealed in EAE-Ph. Regarding antibacterial activity, EAE-Ph showed weak to moderate property against 13 strains tested with MIC values ranging from 625 to 5000 µg/mL, with bactericidal or bacteriostatic effects. Glucogallin and gallic acid stand out as the most relevant bioactive compounds. These results suggest that P. hydropiperoides is a natural source of active substances, supporting this species’ traditional use.


Introduction
The World Health Organization (WHO) declared antimicrobial resistance (AMR) as one of the major, serious, and complex public health problems of the 21st century, and gave recommendations for the research, discovery, and development of new antibiotics, mainly for the treatment of multidrug-resistant (MDR) bacteria, specially to the bacterial species that are most commonly isolated from infections around the world and that express resistance genes. Among them, as a high-priority pathogen, methicillin-resistant, vancomycin-intermediate, and vancomycin-resistant Staphylococcus aureus needs to be noteworthy [1,2]. These strains are related to skin and soft tissue infections, particularly the complicated forms of these processes [3].  As displayed in Figures 1 and 2, it was possible to detect 39 peaks in the EAE-Ph and EE-Ph chromatographic profiles. Among them, 32 (82%) could be tentatively identified as shown in Supplementary Materials (Table S1).

Phenolic Acids
Compound 3 was tentatively identified as gallic acid based on its deprotonated molecular ion at m/z 169 and base peak at m/z 125. Compound 2, with more 162 Da, corresponded to galloyl glucose. Compound 29, with 169/125 fragmentation, was tentatively identified as a gallic acid derivative.
Compound 9 displayed the neutral loss of 162 Da to yield dihydroxybenzoic acid at m/z 153, [16] being tentatively identified as a dihydroxybenzoic acid-hexoside.
Compound 8 was characterized as catechin by comparison with an analytical standard. Compound 17, [M − H] − at m/z 441, suffered the neutral loss of 152 Da (galloyl moiety) to yield (epi)catechin at m/z 289, so it was tentatively identified as (epi)catechin-O-gallate.
Compounds 14 and 15 suffered neutral losses of 132 and 146 Da, respectively, which corresponded to pentoside and deoxyhexoside moieties. The aglycone was present at m/z and it did not reveal antibacterial activity. Compounds were numbered according to their order of elution, keeping the same numeration in both extracts.  As displayed in Figures 1 and 2, it was possible to detect 39 peaks in the EAE-Ph and EE-Ph chromatographic profiles. Among them, 32 (82%) could be tentatively identified as shown in Supplementary Materials (Table S1).

Phenolic Acids
Compound 3 was tentatively identified as gallic acid based on its deprotonated molecular ion at m/z 169 and base peak at m/z 125. Compound 2, with more 162 Da, corresponded to galloyl glucose. Compound 29, with 169/125 fragmentation, was tentatively identified as a gallic acid derivative.
Compound 9 displayed the neutral loss of 162 Da to yield dihydroxybenzoic acid at m/z 153, [16] being tentatively identified as a dihydroxybenzoic acid-hexoside. Compound 10 suffered the neutral loss of 162 Da (hexoside) to yield dihydrokaempferol at m/z 287 (main fragment ion at m/z 259) [18].
Compounds 14 and 15 suffered neutral losses of 132 and 146 Da, respectively, which corresponded to pentoside and deoxyhexoside moieties. The aglycone was present at m/z As displayed in Figures 1 and 2, it was possible to detect 39 peaks in the EAE-Ph and EE-Ph chromatographic profiles. Among them, 32 (82%) could be tentatively identified as shown in Supplementary Materials (Table S1).

Phenolic Acids
Compound 3 was tentatively identified as gallic acid based on its deprotonated molecular ion at m/z 169 and base peak at m/z 125. Compound 2, with more 162 Da, corresponded to galloyl glucose. Compound 29, with 169/125 fragmentation, was tentatively identified as a gallic acid derivative.
Compound 9 displayed the neutral loss of 162 Da to yield dihydroxybenzoic acid at m/z 153, [16] being tentatively identified as a dihydroxybenzoic acid-hexoside.
Compound 8 was characterized as catechin by comparison with an analytical standard. Compound 17, [M − H] − at m/z 441, suffered the neutral loss of 152 Da (galloyl moiety) to yield (epi)catechin at m/z 289, so it was tentatively identified as (epi)catechin-O-gallate.
Compounds 14 and 15 suffered neutral losses of 132 and 146 Da, respectively, which corresponded to pentoside and deoxyhexoside moieties. The aglycone was present at m/z 317, with fragments at m/z 271 and 179 (myricetin), so they were tentatively identified as myricetin glycosides. Compound 32 presented deprotonated molecular ion at m/z 331 and base peak at m/z 316. This fragmentation had been previously reported for mearnsetin [19]. Compound 39 was tentatively identified as isorhamnetin due to the 315→300 fragmentation, and fragment ions at m/z 271, 179, and 151. Compounds 18, 23, 27, and 31 were isorhamnetin glycosides (identification based on the neutral losses previously discussed). Compound 26 also presented the 315/300 fragmentation. However, the additional fragment ions were not consistent with isorhamnetin, so its complete identification was not performed.

Other Compounds
Compound 1 was tentatively identified as a disaccharide (two hexoses). It suffered the neutral loss of a hexoside moiety (162 Da) and the fragmentation pattern was characteristic of hexoses [20].
Compounds 35 and 38 were tentatively identified as oxo-dihydroxy-octadecenoic and trihydroxy-octadecenoic acids, considering data from the scientific literature.
These results are in agreement with Nikolaeva, Lavrent'eva, and Nikolaeva findings [22]. These authors studied the phenolic composition of the aerial parts of Polygonum amphibium, Polygonum angustifolium, Polygonum aviculare, and Polygonum divaricatum by HPLC, and reported the presence of caffeic, chlorogenic, gallic, p-cumaric, and protocatechuic acids, the glycosylated flavonoids avicularin, kaempferol glycoside, hyperoside, isoquercetin, and rutin and the aglycones quercetin and kaempferol. Furthermore, ferulic acid was detected in P. amphibium and P. angustifolium, luteolin and luteolin-7-glycoside in P. aviculare and P. divaricatum, and cosmosiin and apigenin in P. amphibium, P. aviculare, and P. divaricatum, which demonstrated the diversity of this chemical class of substances in this genus [22].
In addition, Quesada-Romero et al. [23] reported the presence of quercetin, isorhamnetin, and kaempferol and their derivatives in Persicaria maculosa, a plant species from the Polygonaceae family, also corroborating our findings.
Quantification of individual phenolic compounds was performed using UV chromatograms. Only the 18 most abundant ones were quantified, due to the low signal or overlapping observed in some peaks. The results are given in Table 1.  Table 1 shows the expressive presence of phenolic compounds reaching values of 95 and 56 mg/g of the dry extract of EAE-Ph and EE-Ph, respectively. Phenolic acids such as galloyl glucose or β-glucogallin (43 and 48 mg/g of dry extract in EAE-Ph and EE-Ph, in this order) and gallic acid (21 and 4.2 mg/g of dry extract in EAE-Ph and EE-Ph, respectively). Compound 2 (galloyl glucose or β-glucogallin) was responsible for approximately 85% and 45% of the total individual phenolic content (TIPC) present in EE-Ph and EAE-Ph, in this order. Compound 3 (gallic acid) contributed with 22% of the total phenolics in EAE-Ph and 7% in EE-Ph.
Phenolic compounds or polyphenols constitute a broad group of substances originating from the secondary metabolism of plants (it is estimated that more than 8000 structures are known) and can be found in different natural sources such as fruits, vegetables, nuts, seeds, stems, flowers, honey, and beverages such as coffee, tea, and red wine being responsible for pigmentation and astringency [24][25][26]. They also act as protective agents against the harmful action of UV light, in addition to protecting plant species against parasites and insects [27].
These substances can be present as simple molecules (benzoic and cinnamic acids) to highly polymerized compounds (lignin, melanin, and tannin), with flavonoids representing the most common and widely distributed subgroup [25].
As shown in Table 1, phenolic acids and flavonoids were identified in this study, and the concentrations of total phenolic acids (64.26 and 52.20 mg/g of dry extract in EAE-Ph and EE-Ph, respectively) were visibly higher than those of flavonoids (30.96 and 4.22 mg/g of dry extract in EAE-Ph and EE-Ph, in that order). In their study, Quesada-Romero et al., [23] when evaluating the composition of total phenolics and flavonoids in extracts from aerial parts of P. maculosa (Polygonaceae), obtained concentrations of total phenolics ranging from 9.6 to 14.3 (methanolic extracts) and 13.7 to 16.6 (ethanolic extracts) mg of gallic acid equivalents/100 g of extract; and a lower concentration of total flavonoids ranging from 6.6 to 8.4 (ethanolic extracts) and 8.2 to 8.6 (methanolic extracts) mg of catechin equivalents/100 g of extract, thus corroborating our findings.
Phenolic compounds have a broad range of bioactivities, from antioxidant and anticancer properties to the ability against bacterial strains [27,28]. Regarding to pathogenic bacteria, in recent decades, with the advancement of analytical tools, studies could be carried out with commercialized extracts and/or pure or synthetic phenolic compounds. In 2016, Lima et al. [25] evaluated the in vitro antibacterial activity of gallic, caffeic, and pyrogallol acids against clinical strains of Escherichia coli 06 (EC06; isolated from urine), Pseudomonas aeruginosa 15 (PA15; catheter tip), and Staphylococcus aureus 10 (SA10; rectal swab). According to these authors, the three phenolic compounds tested did not show clinically relevant antibacterial activity, with MIC values ≥ 1024 µg/mL.
The main phenolic compound detected in the tested extracts (43 and 48 mg/g in EAE-Ph and EE-Ph dry extract, in that order) was galloyl glucose or β-glucogallin (Table 1). Pawłowska et al. [29] analyzed the antibacterial and anti-inflammatory activities of the aqueous extract from roots of Bistorta officinalis Delalbre [Polygonum bistorta (L.) or Persicaria bistorta (L.) Samp], a perennial plant also belonging to the Polygonaceae family, and its major phytochemicals, derived from galloyl glucose and flavan-3-ols. Galloyl-glucose derivatives and catechin derivatives were effective against S. aureus  [29].
Gallic acid (3,4,5 trihydroxybenzoic acid) is the second most abundant compound in EAE-Ph and EE-Ph extracts (21 and 4.2 mg/g of dry extract in EAE-Ph and EE-Ph, respectively).This bioactive phytochemical is often present in the human diet and is, therefore, considered "safe" and natural in the context of the food production system and used as a preservative in foods and beverages due to its power to scavenge free radicals (antioxidant capacity); in fact, gallic acid is used in the manufacture of gallic acid methyl ester, gallic acid lauryl ester, and propyl gallate, which are widely used antioxidant food additives [30,31]. In addition to its well-established antioxidant capacity, gallic acid has other pharmacological properties like other phenolic acids, such as antimicrobial, anticancer, anti-inflammatory, and antiulcerogenic [25,31]. It is interesting to notice that several flavonoids identified in P. hydropiperoides are esterified with gallate moieties (Table S1). Indeed, this high amount of free gallic acid can be endogenous of the plant or it can be an artifact resulting from the deesterification of galloyl-substituted catechins and other compounds during the extraction process, or both.
Flavonoids were detected at concentrations of 30.96 and 4.22 mg/g of dry extract in EAE-Ph and EE-Ph, in this order (Table 1). Probably, this expressive difference between EAE-Ph and EE-Ph contributed to the antibacterial effectiveness of EAE-Ph in detriment of EE-Ph. Based on the well-known antibacterial activity of this phytochemical class, many modes of action were reported, especially, the action (damage) in the bacterial membrane.
Catechins (present in concentrations of 8.9 mg/g of dry extract in EAE-Ph) were previously described as acting in the bacterial membrane by the generation of ROS, while quercetin (present in concentrations of 15.13 mg/g of dry extract in EAE-Ph) acts decreasing the proton-motile force in S. aureus strains, increasing the membrane permeability. In both cases, the rupture and death of the bacterial cells were promoted [32].

In Vitro Antioxidant Activity
The in vitro antioxidant capacity of EAE-Ph, EE-Ph and the standards [gallic acid, quercetin, rutin, Trolox, and butylated hydroxytoluene (BHT)] were assessed by the phosphomolybdenum reducing power, nitric oxide (NO) inhibition, and β-carotene bleaching assays, as can be seen in Table 2. The total antioxidant capacity was evaluated by the phosphomolybdenum reducing power, demonstrating that EAE-Ph and EE-Ph were able to reduce Mo (VI) to Mo (V). In comparison, EAE-Ph and EE-Ph (214.57 ± 23.99 and 230.82 ± 4.60 µg/mg of ascorbic acid equivalents (AAE), respectively) were statistically equal and more effective than the gallic acid standard (160.78 ± 17.04 µg/mg AAE). However, these extracts represent about 50% of the quercetin (424.70 ± 10.99 µg/mg AAE) ability to reduce Mo (VI) to Mo (V). Berwal et al. [33], when analyzing the total antioxidant activity of methanolic extracts of Calligonum polygonoides (Polygonaceae) with the phosphomolybdenum method, revealed an antioxidant capacity under this assay, corroborating our findings.
The inhibition of NO formation revealed that EAE-Ph possesses the most relevant activity, being more effective than the standards gallic acid, Trolox (vitamin E analogue) and the EE-Ph extract, which showed statistically equal values. In addition, corroborating our findings, Choi et al. [34] demonstrated a positive inhibition of the NO production by the methanolic extract from aerial parts of Persicaria senticosa (Polygonaceae) with values of IC 50 equal to 71.8 ± 3.7 µg/mL.
Antioxidant activity was also assessed by the β-carotene bleaching assay. It showed that EAE-Ph (74.44 ± 5.17%) was the most effective sample tested, being better than the standards quercetin (43.33 ± 5.47%), rutin (27.78 ± 7.43%), and BHT (61.11 ± 5.33%) in inhibiting the lipid peroxidation. EE-Ph was less effective than EAE-Ph but was statistically equal to the quercetin standard. Similar results were found by Sahreen, Khan, and Khan [35] who demonstrated the capacity of lipid peroxidation inhibition of methanolic extract and fractions from roots of Rumex hastatus (Polygonaceae). These authors showed that some fractions possess activity comparable with the standard catechin.
Together, these results revealed that EAE-Ph and EE-Ph possess an interesting antioxidant capacity being able to act in different mechanisms and inhibit oxidative stress. Probably this activity is related to the expressive presence of phenolic acids and flavonoids (Table 1), especially in EAE-Ph, the most effective one, that exhibits higher concentrations of these metabolites.

In Vitro Antibacterial Activity
The in vitro antibacterial activity of HE-Ph, EAE-Ph, EE-Ph, ampicillin (AMP), and chloramphenicol (CHL) was established by determining MIC and MBC values and the classification of the antibacterial effect as bactericidal or bacteriostatic, which are expressed in Table 3. The appropriate controls were performed to ensure that solvents used in the extraction and in the preparation of the solutions had no influence in the bacterial growth, demonstrating that at the tested concentrations it was not able to promote the bacterial inhibition. MIC values for HE-Ph, EAE-Ph, and EE-Ph ranged from 625 to 5000 µg/mL. EAE-Ph was the most effective extract, being active against all tested strains, except Pseudomonas aeruginosa (ATCC 27853). EE-Ph was less effective, being not active against three of the 14 strains tested. HE-Ph was not active against all the strains in the concentration gradient tested.
Regarding the antibacterial effect, EAE-Ph MIC value of 5000 µg/mL for P. aeruginosa (ATCC 9027) was bactericidal, while MIC values of EAE-Ph and EE-Ph for all other strains tested were classified as bacteriostatic.
The antibiotics (AMP and CHL) MIC values are in accordance with CLSI document, validating the assay. AMP and CHL were selected in accordance with the CLSI document that describes the antimicrobials that should be reported to the bacterial species tested. Furthermore, AMP was selected to represent a simple penicillin being able to be a marker to resistance mechanisms, such as the production of beta-lactamases. CHL was selected to represent a non-conventional therapeutic option to the treatment of bacterial infections that possess some resistance to other antimicrobials, but still sensible to this one. In addition, the different mechanisms of action of AMP (action in the bacterial cellular membrane) and CHL (action in the protein synthesis) were considered in the selection process.
As previously mentioned, there are very few reports on the antibacterial activity of P. hydropiperoides, but the existing ones corroborate our findings. Bussmann et al. [37] when analyzing ethanolic and aqueous extracts of this plant species against S. aureus (ATCC 25923) by the broth microdilution method, reported activity of the ethanolic extract against this strain, with a MIC value of 1000 µg/mL, considering it a promising extract. Miranda et al. [38] also evaluated the antibacterial activity of the ethanolic and aqueous extracts of P. hydropiperoides against S. aureus (ATCC 33591) by the diffusion method according to Kirby and Bauer and reported that the ethanolic extract was active against this strain in all alcoholic strengths tested, but mainly in 60% and 100%, at 200 and 500 mg/mL. Bouzada et al. [39] reported the activity of the methanol extract of P. hydropiperoides by the broth microdilution method with MIC values equal to 5000 µg/mL (leaves) and 2500 µg/mL (flowers) for S. aureus (ATCC 6538) and 2500 µg/mL (leaves and flowers) for Salmonella Typhimurium (ATCC 13311).
S. aureus is a Gram-positive coccus species related to community and healthcareassociated infections (HAIs), being responsible for skin and soft tissues infections, food poisoning, and the most complicated clinical situations, such as endocarditis, osteomyelitis, pneumonia, bloodstream infections, and septic shock syndrome [40]. The most complex infections caused by S. aureus are related to the resistance genes expression, especially the mecA, that concedes the mutation on the penicillin-binding protein 2a (PBP2a), promoting the lower affinity to β-lactams such as penicillin, amoxicillin, and methicillin [41]. Considering the MRSA strains, Dzoyem et al. [42] described the antibacterial activity of Polygonum limbatum against MRSA (ATCC 33591). Zuo et al. [43] previously reported the anti-MRSA activity of Polygonum multiflorum (MIC ≤ 1.43 mg/mL) against nine clinical MRSA isolates and methicillin-sensitive S. aureus (ATCC 25923) (MSSA), corroborating our findings. In the present study, five MRSA isolated from blood cultures were tested and MIC values ranged from 625 to 1250 µg/mL to EAE-Ph and were equal to 1250 µg/mL to EE-Ph. According to Simões, Bennett, and Rosa [44], phytochemicals are routinely classified as antimicrobials based on susceptibility tests that produce MIC values of 100 to 1000 µg/mL. On the other hand, Kuete's [45] classification to plant extracts antimicrobial activity demonstrates a moderate antibacterial effect (100 < MIC ≤ 625 µg/mL) of EAE-Ph phytochemicals against MRSA 1664534 (MIC equal to 625 µg/mL) and a weak activity (MIC > 625 µg/mL) of EAE-Ph and EE-Ph against the other strains tested. Probably, this activity is related to galloyl-glucose derivatives as previously discussed, based on Pawłowska et al. [29] reports.
In addition, it has been shown that the combination of antibiotics and extracts can be more effective than the isolated substances [40] and the synergism between antibiotics (such as β-lactams and aminoglycosides or fluoroquinolones) has been used in clinical practice [46]. This is an exciting field of research, enabling available antibiotics, no longer effective due to developed bacterial resistance, to be useful again while in combination with phytochemicals, which themselves cannot be used in monotherapy due to their higher MIC (100-5000 µg/mL) [24].

Extraction Procedures
Selected aerial parts (458 g) were dried in an oven, at 40 • C, with forced air until there was a humidity loss of 90 to 95% [48]. After this, the vegetable material was cut with an industrial knife mill, pulverized on tamis (n. 20), and subjected to static maceration extraction at room temperature with solvents of increasing polarity (hexane, ethyl acetate, and ethanol).
Initially, the pulverized plant material was extracted with hexane P.A. (hexane extract, HE-Ph). The resulting solution was filtered on simple filter paper and concentrated on a rotary evaporator (R-215 Büchi Labortechnik AG, Flawil, Switzerland) at 40 • C. The same procedure was performed over the solid residue with ethyl acetate P.A. (ethyl acetate extract, EAE-Ph) and, subsequently, with ethanol P.A. (ethanolic extract, EE-Ph). From 458 g of dried and pulverized plant material subjected to extraction, it is observed that EE-Ph showed the highest yield (13.9%; 63.5 g), followed by EAE-Ph (1.3%; 5.8 g) and HE-Ph (0.6%; 2.8 g). The three extracts were stored under refrigeration, between 2 and 8 • C and protected from light, until the time of use in chemical and biological tests. The hexane extract was only used in this work in the microbiological assays.

Chromatographic Analysis
The phytochemical profile was evaluated using high-performance liquid chromatography with a diode array detector and an ion trap mass spectrometer with an electrospray interface (HPLC-DAD-ESI/MS n ). About 5-10 mg of EAE-Ph and EE-Ph were re-dissolved in 1 mL of methanol, filtered through 0.45 µm nylon filters, and 10 µL of sample was injected. The HPLC system was an Agilent Series 1100 with a G1315B diode array detector and an ion trap mass spectrometer (Esquire 6000, Bruker ® Daltonics, Billerica, MA, USA) with an electrospray interface. Compounds were separated with a Phenomenex Gemini  [49]. DAD chromatograms were recorded at wavelengths of 280, 320, and 350 nm. The most abundant compounds were quantified using the following analytical standards: quercetin (350 nm) for flavonoids (except catechin and its derivatives, which were quantified using catechin at 280 nm), and gallic acid for phenolic acids at 320 nm. Calibration graphs were constructed in the 0.5-100 mg/L range. Peak areas were plotted vs analyte concentration. Detection limits (3σ criterion) were 0.1-0.2 mg/L. Repeatability (n = 10) and intermediate precision (n = 9, three consecutive days) were lower than 4 and 8%, respectively. Chromatograms at each wavelength for the corresponding extracts are shown in Supplementary Materials (Figures S1-S6).

Phosphomolybdenum Reducing Power
The total antioxidant capacity of EAE-Ph and EE-Ph was evaluated by phosphomolybdenum reducing power assay according to Prieto et al. [50], using ascorbic acid as the standard. Gallic acid and quercetin were tested as reference antioxidant substances. This spectrophotometric method is based on the reduction in molybdenum (VI) to molybdenum (V) in the presence of certain substances with antioxidant capacity, with formation of phosphate/molybdenum (V) green complex at acid pH [49]. The extract solutions (300 µL, or 97.82 µg/mL) were added to 2000 µL of the phosphomolybdenic complex reagent and kept in a water bath at 95 • C for 90 min. After cooling, 250 µL was transferred into a 96-well microplate, and absorbance was measured in a microplate reader (Thermo Scientific ™ , Waltham, MA, USA) at 695 nm. The experiment was performed in triplicate. The results were expressed as ascorbic acid equivalent (AAE) ± standard deviation (SD).

Nitric Oxide (NO) Inhibition
The antioxidant activity of EAE-Ph and EE-Ph was also evaluated by the indirect production of NO by Griess' method, according to Green et al. [51], with few adjustments. For NO production, sodium nitroprusside (NPS) (10 mM) in a phosphate buffer (PBS) (10 mM, pH 7.4) was used. Samples (EAE-Ph and EE-Ph) and standards (gallic acid and Trolox-vitamin E analogue) solutions in PBS were prepared, with 1% dimethyl sulfoxide (DMSO), at concentrations ranging from 25 to 400 µg/mL. The assay was performed in 96-well microplates that were incubated in the presence of light at room temperature for 60 min, followed by the addition of the Griess reagent (1% sulfanilamide + 0.1% N-(1naphthyl)-ethylenediamine (NED) in 2.5% phosphoric acid). After 10 min, the absorbance was measured at a wavelength of 540 nm in a microplate reader (Thermo Scientific ™ , MA, USA). Appropriate controls were performed. The experiment was carried out in triplicate. Results were expressed in percentage of inhibition of NO production ± standard deviation (SD).

β-Carotene Bleaching Assay
The antioxidant activity of EAE-Ph and EE-Ph was additionally investigated by βcarotene/linoleic acid system method as described by Koleva et al. [52], with little adjustments. EAE-Ph and EE-Ph (250 µg/mL) and quercetin, rutin, and BHT (25 µg/mL) ethanolic solutions were prepared. The β-carotene/linoleic acid emulsion was made in a round bottom flask protected from light with aluminum foil where were added 1000 µL of β-carotene in chloroform solution (0.20 mg/mL), linoleic acid (25 µL), and Tween 40 (200 mg). After mixing, the chloroform was evaporated using a rotary evaporator (R-215 Büchi Labortechnik AG, Flawil, Switzerland) at 40 • C, and distilled water previously saturated with oxygen for 30 min was added (50,000 µL). Then, 30 µL of EAE-Ph, EE-Ph, quercetin, rutin, and BHT solutions were placed into a 96-well microplate. Then, 250 µL of the emulsion were added and the microplate was maintained at 50 • C for 105 min. The reaction was monitored by discoloration of β-carotene by absorbance reduction measurement at 492 nm, with reading at 15 min intervals for a total of 105 min, using a microplate reader (Thermo Scientific ™ , MA, USA). Appropriated controls were performed. The experiment was made in quadruplicate. The results were expressed in percentage of inhibition of lipid peroxidation (%I) ± standard deviation (SD).

In Vitro Antibacterial Activity
The antibacterial activity was determined by the minimal inhibitory concentration (MIC) using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) M07-A10 [53] and M100-S30 [36] documents. The determination of minimal bactericidal concentration (MBC), followed by the classification of the antibacterial effect as bactericidal or bacteriostatic procedures, was carried out according to Andrews [54].

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) Determinations
As previously described, to determine the MIC values the broth microdilution method was applied according to CLSI M07-A10 guideline [53]. HE-Ph, EAE-Ph, and EE-Ph stock solutions were prepared using water and DMSO, respecting the limit of 1% of this organic solvent in the first well of the microtitration plate [36], at the concentration of 10 mg/mL.
To validate the assay, ampicillin (AMP) and chloramphenicol (CHL) were used as control antibiotics. The antibiotic solutions were prepared at a concentration of 1 mg/mL with their respective solvents/diluents, as described in document M100-S30 [36].
In a sterile 96-well microplate, twofold serial dilutions of extracts (quadruplicate) and antibiotics (triplicate) were prepared in MHB at concentrations ranging from 5000 to 40 µg/mL and 500 to 4 µg/mL, respectively. MIC values above 5000 µg/mL were not determined. Subsequently, 10 µL of standardized bacteria suspension according to the 0.5 McFarland scale were added. After incubation at 35 ± 2 • C for 16 to 20 h under aerobic conditions, 20 µL of 1 mg/mL iodonitrotetrazolium chloride (INT) solution (w/v) was used as an indicator of bacterial growth (any color change from purple to pink was recorded as bacterial growth). Then, this system was incubated for a further 30 min at the same conditions, and the MIC was determined. The appropriate controls were performed. After the determination of MIC values, MBC was established according to Andrews' method [54] by the spreading of 10 µL of suspensions from wells showing no visible bacterial growth on MHA Petri dishes. After incubation at 35 ± 2 • C for 16 to 20 h under aerobic conditions, the presence or absence of bacterial growth was analyzed. MBC was determined as the lowest concentration of dilutions that prevented the visible bacterial growth after subculture on MHA Petri dishes. Bacterial growth or no bacterial growth on MHA revealed a bacteriostatic or bactericidal effect, in this order.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism 7.0 software, data were submitted to analysis of variance (ANOVA) followed by Tukey's test to measure the degree of significance for p < 0.05. Results were expressed as mean ± standard deviation (SD).

Conclusions
The results reported in the present study suggest that Polygonum hydropiperoides is a natural source of active substances, such as phenolic acids and flavonoids, with particular relevance for galloyl-containing compounds, especially in the ethyl acetate extract (EAE-Ph). Gallic acid and β-glucogallin can act in synergic mode since the extract shows higher antioxidant capacity than gallic acid alone. These results add scientific support for the popular use of Polygonum hydropiperoides in the treatment of inflammatory processes and wounds.
Author Contributions: Y.F.G.d.C. was responsible for the conceptualization, methodology, validation, formal analysis, investigation, writing-original draft preparation, writing-review and editing, and visualization; E.J.L.-M., L.S.F. and P.H.S.d.F. were responsible for the methodology, validation, investigation, writing-original draft preparation, writing-review and editing, and visualization; O.V.d.S. and E.S. was responsible for the formal analysis, validation, visualization, writing-original draft preparation, and writing-review and editing; P.C.C. was dedicated to methodology, validation, formal analysis, data curation, writing-original draft preparation, writing-review and editing, and visualization; and M.S.A. was responsible for conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing-original draft preparation, writing-review and editing, visualization, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.