Antioxidants, inhibits the growth of foodborne pathogens and reduces nitric oxide activity in LPS-stimulated RAW 264.7 cells of nipa palm vinegar

Schematic overview of the experimental program

Nipa palm vinegar (NPV) samples were from 3 different plantations based on soil salinity (non-saline, slightly saline and strongly saline) of Khanab Nak, Sub-districts of Pak Phanang, Nakhon Si Thammarat, Thailand. NPV samples were determined physiochemical properties and chemical contents, included total phenolic content (TPC), total flavonoid content (TFC), acetic acid content and pH. In addition, they were assessed for antioxidant antibacterial and anti-inflammatory activities, as shown in schematic overview Fig. 1.

Chemicals and reagents

All chemicals and reagents used were of analytical grade. 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2, 4, 6-tri (2-pyridyl)-l, 3, 5-triazine (TPTZ), Folin–Ciocalteu phenol reagent, sodium carbonate, gallic acid, quercetin, sodium chloride and aluminum chloride were purchased from Sigma-Aldrich (MO, USA), whereas 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from Merck (Darmstadt, Germany).

Determination of soil and water salinity

Khanab Nak, Sub-districts of Pak Phanang, Nakhon Si Thammarat, Thailand, has the potential for Nipa palm plantation (Tripathi & Bencure, 2012). It is divided into 10 villages where are all nipa palm planting areas (Fig. 2). Soil and water samples were collected to study the NPV properties produced from planting areas with different salinities. Soil salinity was analyzed using electrical conductivity (EC) and interpreted according to the laboratory procedures of US Salinity Laboratory Staff (US Salinity Laboratory Staff, 1954). Soil EC was reported in the unit of dS/m. The water samples from three nipa palm planting areas, included non-saline, slightly saline and very strongly saline, were collected and measured. The water salinity was determined by the salinity refractometer (Tran instrument, Petro Centre, Singapore) and was expressed in parts per thousand (ppt) which was interpreted into the degree of salinity.

Nipa palm vinegar samples

The nipa palm sap was fermented to NPV by the traditional local method (Chatatikun & Kwanhian, 2020). The procedure, in brief, collect nipa palm sap from the cut stems and placed it in a terracotta tank at room temperature for 40 days to allow the natural fermentation process. Nipa palm vinegar has an acidity of 4% to 5%. This study was performed on three NPV samples from different plantations, including villages 1, 5 and 6 of Khanab Nak Sub-district, based on the different soil salinity levels (Fig. 1). All parts of the nipa palm were authenticated and a voucher number (Nypa fruticans Wurmb, voucher no. 01518) was deposited at Botanic Garden, Walailak University, Nakhon Si Thammarat, Thailand. Samples were collected from the study areas by cooling transportation. The NPVs were aliquoted and stored in the dark at −20 °C until use.

Determination of acidity

Colorimetric titration (AOAC, 2000) was used to determine the acetic acid of NPV. A sample and phenolphthalein were titrated with sodium hydroxide. By color changed to red, the titrant volume was used to calculate the percentage of acetic acid concentration in the samples. In addition, pH of NPV samples was also determined by a benchtop pH meter (Mettler-Toledo, LLC, Columbus, OH, USA).

Determination of total phenolic contents

According to the procedure described in our previous work, the amount of total phenolic in NPV was determined by Folin-Ciocalteu assay (Chatatikun & Kwanhian, 2020). First, 20 µL of samples were mixed with 100 µL of Folin-Ciocalteu reagent and 80 µL of sodium carbonate solution (75 g/L). After the mixture was vortexed for 10 s, it was left for 30 min at room temperature. Absorbance was measured at 765 nm against a blank. Then, the total phenolic content was determined using a calibration curve of gallic acid (0–500 µg/mL) and the results were expressed as microgram gallic acid equivalent per milliliter (µg GEA/mL).

Determination of total flavonoid contents

The amount of total flavonoid in NPV was investigated using the aluminum chloride colorimetric assay described by Yusoff et al. (2015). First, 50 µL of sample or standard solution was added to 10 µL of 10% aluminum chloride solution, followed by 150 µL of 96% ethanol. Then, 10 µL of 1 M sodium acetate was added to the mixture. All reagents were mixed and incubated for 40 min at room temperature, protected from light. The 96% ethanol was used as a reagent blank. The absorbance was read at 415 nm with a BioTek™ Eon™ microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Quercetin (0–500 µg/mL) was used to make a standard calibration curve. Total flavonoid contents were determined from the standard calibration curve and were expressed as micrograms of quercetin equivalents per milliliter (µg QE/mL).

Antioxidant property

ABTS radical scavenging activity

ABTS assay was performed as described by Re et al. (1999). Briefly, to obtain ABTS radical cation (ABTS⁺) solution, 7 mM ABTS and 1 mM potassium persulfate solution were mixed and stored in the dark at room temperature for at least 12 h. Then one mL of ABTS+ solution was diluted with approximately 88 mL of ethanol to obtain an absorbance value of 0.70 ± 0.02 at 734 nm. Afterward, 200 µL of diluted ABTS⁺ solution and 50 µL of each NPV sample at the different concentrations (25, 12.5 6.25, 3.125 and 1.56 µL/mL) or Trolox standard solution were mixed and the mixture was kept in the dark at room temperature for 3 min. Then, the mixture absorbance was measured at 734 nm using the spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

DPPH radical scavenging activity

The radical scavenging activity of the NPV was measured in DPPH assay according to the method described earlier (Prasad et al., 2011). Briefly, an aliquot of 50 µL of NPV sample (25, 12.5 6.25, 3.125 and 1.56 µL/mL) was mixed with 50 µL of 0.1 mM DPPH radical solution (prepared in ethanol) in a 96-well plate. The reaction mixture was incubated in the dark at room temperature for 30 min. Afterward, the mixture absorbance was read at 515 nm by a microplate reader against a blank (95% ethanol). Vitamin E (0.001-10 mg/mL) was used as positive controls. The percent of radical scavenged was calculated by employing the following equation:

Radical scavenging activity (%) = $\left(\frac{\mathrm{A blank}-\mathrm{A sample}}{\mathrm{A blank}}\right)\times 100$.

Ferric reducing antioxidant power (FRAP) assay

The antioxidant capacity of the NPV was estimated according to the previously reported method (Re et al., 1999). Briefly, working FRAP was prepared by mixing 300 mM acetate buffer (pH 3.6): 10 mM TPTZ solution in 40 mM HCl: 20 mM ferric chloride solution, in proportion of 10:1:1 (v/v/v). An aliquot 20 µL of appropriately diluted NPV was mixed with 200 µL of freshly prepared FRAP reagent and mix thoroughly. After 30 min incubation at 37 °C, an intense blue color complex was formed. The absorbance of the reaction mixture was recorded at 593 nm against a blank using a spectrophotometer. The calibration curve was prepared by plotting the absorbance at 593 nm versus different concentrations of the standard antioxidant Trolox.

Determination of antimicrobial activity

Antibacterial activity of the NPV was studied using the broth microdilution method by detecting minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) according to the guidelines of Clinical and Laboratory Standard Institute (CLSI, 2019). In this, we mainly focused on the pathogenic enterobacteria associated with food-borne diseases in humans. Vibrio cholera [ATCC39541], Vibrio parahemolyticus [ATCC17802], Salmonella enterica serovar Typhimurium [ATCC14028], S. paratyphi A [ATCC9150], Shigella sonnei [ATCC25931], Staphylococcus aureus [ATCC25923] and Escherichia coli [ATCC25922] were used for examining the antibacterial activity of the NPV. Each microorganism was incubated separately with the same volume of three selected NPVs at different concentrations. The final concentrations of the NPVs were serial diluted among 500 to 7.8 µL/mL. The MIC value of the NPV was determined as the lowest concentration that completely inhibited bacterial growth after 48 h of incubation at 37 °C. For the MBC determination, a portion of liquid (5 µL) from each plate well that exhibited no growth was sub-cultured and then, incubated at 37 °C for 24 h. The lowest concentration that revealed no visible bacterial growth after sub-culturing was taken as MBC.

In vitro cytotoxic test

A cell viability test was carried out to evaluate the cytotoxic effects of the NPV on RAW 264.7 cells (ATCC CRL-2278) provided by Dr. Jitbanjong Tangpong. First, the viability was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay (Kumar, Nagarajan & Uchil, 2018). Next, the cells were seeded in a 96-well plate at a cell density of 5  ×10⁵ cells/mL with completed Dulbecco’s modified eagle medium (DMEM) and supplemented with 10% fetal bovine serum (FBS) and 2% of 200 × penicillin/streptomycin and 1% of 100 × amphotericin B. Then the plate was incubated at 37 °C, 5% CO₂ for 24 h. After incubation, the cells were treated with different concentrations of NPV (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 µL/mL) or media alone as negative control and incubated 48 h. Then, 20 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at room temperature for 4 h. Next, a volume of 200 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve any formazan crystals formed and the absorbance of each well was determined at 570 nm using the spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the 50% cytotoxicity concentration (CC₅₀) of NPV was calculated according to the NPV concentration that affects 50% RAW 264.7 cell death.

Inhibition of nitric oxide (NO) production

The inhibitory effects of NPV on NO production were evaluated in LPS-activated murine macrophage RAW 264.7 cells, using a method modified from that previously reported (Tham et al., 2010). RAW 264.7 cells were seeded in 96-well culture plates with 5 ×10⁵ cells/mL in DMEM and incubated at 37 °C, 5% CO₂ for 24 h. The cells were induced with 100 µg/mL LPS in the absence (negative control) or presence of various concentrations of NPV (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 µL/mL). The amount of NO in the cultured medium was measured by the Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 2.5% phosphoric acid). The absorbance was measured at 550 nm and a reference wavelength of 650 nm. The amount of NO in the sample was calculated from a NO standard curve.

Statistical analysis

All experiments were performed in triplicates. The data obtained in the study were manifested as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) was used to examine three performances of different NPV types, while Mann - Whitney U test was used for comparing the mean of two NPV types by using IBM^® SPSS^® statistics version 23. The statistically significant difference was considered when p < 0.05.

Soil and water salinity

The lowest EC value was observed in a soil sample in village 1. The EC value in village 1, 2, 3 and 8 was classified as non-saline. The EC value in village 5, 7 and 9 was classified as slightly saline. The soil sample of village 4 and 10 was grouped as moderately saline. The highest EC value was recorded in the soil sample of village 6 (Table 1). Therefore, in this case, three representative varieties of local NPV products were selected from different soil salinity levels, including village1: non-saline, village 5: slightly saline and village 6: strongly saline, as shown in Table 1. The three NPV samples from villages 1, 5 and 6 were defined as fresh water NPV, brackish water NPV and saline water NPV, respectively, according to the salinity level of surface water (Table 2).

Total phenolic, total flavonoid and acetic acid contents

Total phenolic and total flavonoid contents of the NPV samples produced from 3 cultivated areas with different soil and water salinity levels were analyzed. The result showed that the highest total phenolic compound values were found in brackish water NPV (177.16 ± 0.95 µg GAE/mL), followed by fresh water NPV (167.10 ± 10.15 µg GAE/mL) and saline water NPV (80.31 ± 4.00 µg GAE/mL). Meanwhile, the total flavonoid content values ranged from 1.60–2.27 µg QE/mL (Table 3). The NPV with the highest total flavonoid content values were saline water NPV (2.27 ± 0.01 µg QE/mL), followed by fresh water NPV (1.67 ± 0.01 µg QE/mL) and brackish water NPV (1.60 ± 0.01 µg QE/mL).

The acetic acid content plays as the principal quality gauge of vinegar (Sengun, Kilic & Ozturk, 2020). The fresh water NPV exhibited the highest acetic acid content, followed by saline water and brackish water NPV, with the concentration of acetic acid values being 5.28 ± 0.184, 5.26 ± 0.000 and 4.07 ± 0.000%, respectively. While pH values showed a reverse correlation to % acetic acid in order of low to higher pH were fresh water (2.79 ± 0.05), saline water (2.80 ± 0.10) and brackish water NPV (2.87 ± 0.20), respectively (Table 3).

Antioxidant activity of NPV

Results of antioxidant activity of the NPV samples were compared to each other (Fig. 3). Figure 3A illustrates the effect of three different sources of NPV against ABTS radicals. All the tested samples depicted concentration-dependent radical scavenging activities at a concentration range of 1.56–25 µL/mL. Saline water NPV exhibited the highest scavenging activity of 13.97% at a concentration of 25 µL/mL, followed by brackish water and fresh water NPV, with the 50% scavenging activity (SC50) values being 92.17 ± 1.41, 154.74 ± 19.34 and 265.91 ± 22.81 µL/mL, respectively. Lower SC50 values represented greater scavenging capacities and vice versa.

As shown in Fig. 3B, the highest value obtained with DPPH assay was found in saline water NPV (19.34%), higher than brackish water NPV (17.72%) and fresh water NPV (15.62%) at a concentration of 25 µL/mL. On the other hand, the saline water NPV, at a concentration range of 3.1–12.5 µL/mL, had the lower scavenging activity than brackish water and fresh water NPV, according to the result of SC50 values. SC50 value of saline water NPV (72.53 ± 5.52 µL/mL) was more potent than brackish water (85.29 ±13.28 µL/mL) and fresh water NPV (103.00 ± 22.09 µL/mL). No significant differences were observed between tested samples in the DPPH assay.

Another effective method to evaluate the antioxidant activity of plant polyphenol is FRAP. Like other antioxidant assays, saline water NPV showed a high reducing power (40.05%) followed by brackish water (35.20%) and fresh water NPV (27.13%) at 25 µL/mL (Fig. 3C). All the samples displayed a concentration-dependent activity.

Anti-enteropathogenic bacteria activity

The MIC and MBC of the vinegar samples were assessed against seven pathogens. The study results exhibited that three different sources of the NPV have similar MIC but different MBC values (Table 4). In all cases, the similarity of MIC values was ≤ 7.8 µL/mL among the strains of the seven bacterial species, displaying intraspecies homogeneous susceptibility. Moreover, the highest growth-inhibitory and bactericidal effects were observed in brackish water and saline water NPV against V. cholera, V. parahaemolyticus, S. aureus and E. coli with MIC and MBC of ≤ 7.8 µL/mL. In contrast, fresh water NPV showed the highest bactericidal effect against V. cholera, V. parahemolyticus and S. sonnei. These results could suggest that all three vinegar samples from different soil salinity were effective against both gram-positive and gram-negative bacteria, enteropathogenic bacteria.

The cytotoxicity effect of the NPV on RAW 264.7 cells

The cytotoxic effects of the NPV samples on the viability of the RAW 264.7 cells were performed. Values obtained from MTT assays of fresh water, brackish water and saline water NPV were presented as percent cell viability in Fig. 4A. From the result, percent viability values of cells were revealed to diminish with increased concentration (6.25–100 µL/mL). On the other hand, the lower concentration of three NPV samples (1.56 and 3.125 µL/mL) could enhance cell viability. The fresh water NPV had the highest cytotoxicity on RAW 264.7 cells, with a CC50 value of 39.56 ± 2.16 µL/mL, followed by the saline water NPV with a CC50 value of 39.86 ± 5.34 µL/mL, and the brackish water NPV showed the lowest cytotoxicity with a CC50 value of 42.05 ± 3.68 µL/mL (Table 5). The differences were not significant. NPVs at concentrations ranging from 1.56–100 µL/mL did not cause any cytotoxic effect to RAW 264.7 cells, suggesting the effect of NPVs on inhibition of NO production was not due to its cytotoxicity.

Inhibition of nitric oxide production

The ability of the NPV produced from different sources in reducing the inflammation in LPS-activated RAW 264.7 macrophages was investigated through the ability of the samples to decrease the level of NO. As shown in Fig. 4B, fresh water and saline water-treated cells could reduce the NO level significantly at a concentration of 12.5 and 25 µL/mL in a dose-dependent manner (p < 0.05). However, the degree of NO production was remarkably decreased in fresh water NPV-treated cells more than in saline water NPV-treated cells. In contrast, brackish water NPV had a significant inhibitory effect on LPS-induced NO production at 25 µL/mL (p < 0.05), but not at lower concentrations of 1.56 to 12.5 µL/mL. The anti-inflammatory effects of the treatment were also exhibited by IC50 (50% inhibitory concentration) values in which fresh water NPV showed the lowest IC50 values followed by saline water and brackish water NPV, with the IC50 values being 17.59 ± 0.17, 18.12 ± 0.49 and 28.29 ± 2.64 µL/mL, respectively (Table 5). The lower IC50 value, the more influential the anti-NO production potential of NPVs.