Chemical, antimicrobial, and molecular characterization of mortiño (Vaccinium floribundum Kunth) fruits and leaves

Abstract Fruits and leaves of Vaccinium spp. are known for their high content of bioactive compounds, but the chemical and biological characteristics of mortiño (Vaccinium floribundum Kunth) have not been fully described. In this study, the levels of polyphenols, antioxidant capacity, anthocyanins, antimicrobial activity, and genetic variability were determined in mortiño plants. The Folin–Ciocalteu's, ABTS scavenging, pH differential, and well diffusion methods were used to evaluate the levels of polyphenols, antioxidant capacity, anthocyanins, and antimicrobial activity, respectively. The genetic variability was evaluated by sequencing of the matK and rbcl DNA regions. Polyphenol content was up to 229.81 mg gallic acid equivalents/100 g, the average antioxidant capacity was 11.01 mmol Trolox equivalents/100 g, and anthocyanin content was up to 1,095.39 mg/100 g. Mortiño extracts significantly inhibited the growth of Gram‐negative bacteria including Burkholderia gladioli, Burkholderia cepacia, Salmonella Typhimurium, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus, Escherichia coli, and Pseudomona aeruginosa, as well as Gram‐positive bacteria such as Probionibacterium propionicum, Staphylococcus aureus, and Enterococcus faecalis showing greater inhibition halos than those produced by the antibiotic ampicillin. A polymorphic nucleotide was found in position 739 of the matK region. This study shows the potential of mortiño for the food and pharmaceutical industries.


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LLIVISACA et AL. frequently been carried out by sequencing of the matK and rbcL DNA regions (Hilu, 1997). However, identification of mortiño plants has mostly relied on the observation of taxonomic traits without any molecular confirmation (Pérez Flores & Valdivieso Noguera, 2007).
Moreover, no rcbL DNA sequences of this plant species could be found in databases prior to this study.
Recent reports showed that the fruits of mortiño may contain substantial amounts of sugars, antioxidants, vitamins B and C, minerals, and anthocyanins (Lee, 2016). However, studies on polyphenolic compounds, anthocyanins, antioxidant capacity, and antimicrobial activity on leaves of V. floribundum cannot be found in the literature.
The characterization of mortiño leaves has the potential to introduce a novel viable source of pharmaceutical products (Ortiz et al., 2013).
Extracts of Vaccinium spp. have shown significant antimicrobial properties. Extracts of V. corymbosum were able to inhibit the growth of several pathogens including Salmonella spp. (Pervin, Hasnat, & Lim, 2013) and Listeria monocytogenes (Shen et al., 2014). Similarly, Vaccinium macrocarpon extracts caused the inhibition of Bacillus cereus and Micrococcus luteus (Viskelis et al., 2009). However, the antimicrobial properties of mortiño fruits and leaves have not been described.
The objective of this study was to determine the contents of polyphenols, antioxidants, and anthocyanins and assess the antimicrobial potential of mortiño leaves and fruits. Characterization of matK and rbcL DNA regions was also performed.

| Collection and preparation of samples
Mortiño plants of approximately 1.5 m of height were collected from the tundra of the Ecuadorian provinces of Pichincha, Cotopaxi, and Chimborazo. At least 10 plants were collected from each sampled province. Due to difficulties in geographical accessibility, samples from Cotopaxi were only used for molecular analysis. All plants were initially identified by a local guide, and the ID was confirmed by molecular methods as described in Section 2.2. About 50 g of fruit and leaf samples was taken from each plant, but deteriorated fruit samples were discarded. All samples were rinsed with approximately 500 ml of distilled water and lyophilized on a LYOVAC GT2 freezedryer (GEA Group, Düsseldorf, Germany) prior to analysis.

| Genetic characterization
The DNA was extracted from leaf samples exactly as described elsewhere (Azmat et al., 2012), and PCRs were carried out using the primers matK_3f_KIM_f CGTACAGTACTTTTGTGTTTACGAG and matK_1r_ KIM_r ACCCAGTCCATCTGGAAATCTTGGTTC for the matK DNA region, as well as rbcL_F ATGTCACCACAAACAGAGACTAAAGC and rbcL_R GTAAAATCAAGTCCACCRCG for the rbcL DNA region.
All PCRs were run using 1 μl of Go-Taq Green Master Mix (Promega, USA) at 2×, 2 μl of DNA, and 0.5 μl of each forward and reverse primer at 20 ng/μl. Distilled water was added to a final reaction volume of 10 μl. The thermocycler (Eppendorf,nexus GSX1,Spain) program consisted of an initial denaturation at 95°C for 1 min followed by 35 cycles of denaturing at 95°C, annealing at 50°C, and extension at 72°C, followed by a final extension step at 72°C for 1 min (Cobo, Gutiérrez, Torres, & Torres, 2016). The PCR products were then sent for sequencing to a service laboratory, and the resulting sequences were compared with those in the NCBI database using BLAST. A neighbor-joining phylogenetic tree was constructed with 1,000 bootstrap replicates using GENEIOUS (García, Ligarreto, and Adolfo, 2014), and polymorphic nucleotides were estimated by aligning all matK and rbcL DNA consensus sequences using the MUSCLE algorithm in MEGA 7.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013).

| Total polyphenol content
Total polyphenol content was estimated according to the Folin-Ciocalteu's method (Ortiz et al., 2013). Briefly, 10 ml of 70% acetone was added to 0.2 g of lyophilized sample and liquefied in a blender (Oster Super Deluxe, USA) for 10 min followed by incubation at 24°C for 10 min. The extracts were then filtered in Whatman number 41 papers and stored in amber flasks at 4°C. About 0.5 ml of each extract was diluted with 3.5 ml of distilled water and filtered through an OASIS cartridge (Oasis HLB 6 cc Vac Cartridge; Cod. WAT106202; USA) to remove interfering compounds. About 500 μl of the filtrate was then mixed with 2.5 ml of 2 N Folin-Ciocalteu reagent (Sigma-Aldrich, Germany), homogenized in a vortex apparatus (Boeco brand, model V1 PLUS), and allowed to stand for 2 min at room temperature. The solution was then combined with 2 ml of a 7.5% Na 2 CO 3 solution and incubated in a water bath at 50°C for 15 min. The solution was rapidly cooled in a 4°C ice bath to stop the reaction, and the absorbance was measured at 760 nm in a spectrophotometer (Shimadzu Uv-160 Serial No: 29 D0670; Japan). All samples were prepared and analyzed in triplicated. The results were expressed as gallic acid equivalents (GAEs) using a calibration curve built with the pure standard over the range of 10-100 ppm.

| In vitro antioxidant capacity
The 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid + (ABTS + ) scavenging ability was determined using the method described in a previous report (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006). Briefly, 10 ml of 70% acetone was added to 0.2 g of lyophilized sample and liquefied in a blender (Oster Super Deluxe, USA) for 10 min followed by incubation at 24°C for 10 min. The extracts were then filtered in Whatman number 41 papers and stored in amber flasks at 4°C. Meanwhile, about 96 mg of ABTS (Sigma-Aldrich, USA) was mixed with 16 mg of potassium peroxide disulfate (Sigma-Aldrich, Germany) plus 25 ml of distilled water and allowed to react in darkness at room temperature for 24 hr to form ABTS + . The ABTS + solution was diluted to an absorbance of 0.7 ± 0.02 at 734 nm using ethanol. Extracts (10 μl) were added to 1 ml of ABTS + , and absorbance was measured at 734 nm after 1 and 6 min, respectively. The ABTS reduction was calculated using the equation below, and the results were expressed as mmol Trolox equivalent (TE) per 100 g of sample using a Trolox standard curve built in the range of 12.5%-100%.
where, Cn is the concentration of TEs, I is the percentage of inhibition, df is the dilution factor, V is the volume of the extract, W is the weight of the sample, b is the intercept of the Y axis, and m is the slope.

| Determination of total anthocyanins
The content of anthocyanins was determined by spectrophotometry using the pH differential method exactly as described elsewhere (Sarkis, Tessaro, & Marczak, 2013). For this, two sample suspensions were prepared by combining 1.5 g of lyophilized sample with 500 ml of either 0.2 mol/L hydrochloric acid-potassium chloride buffer (pH 1.0) or 1 mol/L sodium acetate buffer (pH 4.5), respectively. Suspensions were then homogenized and passed through filter paper until a clear solution was obtained. Absorbance was then measured at 520 and 700 nm (Shimadzu UV-160A, Kyoto, Japan) in each solution, and the anthocyanin content was estimated using the following equation (Jiang, Yang, & Shi, 2017): where A = ((Absorbance 520 − Absorbance 700 ) pH 1.0 − (Absorbance 520 − Absorbance 700 ) pH 4.5 ), MW = molecular weight of cyanidin-3glucoside (449.2 g/mol), DF = dilution factor, V = volume of the aliquot, ɛ = molar extinction coefficient of cyanidin-3-glucoside (29,600), l = path length, and M = weight of sample (g). Results were expressed as cyanidin-3-glucoside equivalents. All samples were analyzed in triplicate. and Tryptic Soy Agars (TSAs) and incubated at 37°C. All pathogens were previously identified by sequencing of the 16s rDNA region and by biochemical tests (API 20 NE) and are part of our culture collection of microorganisms. After 24-hr incubation, while in the exponential phase of growth to avoid the selection of atypical variants (Balouiri, Sadiki, & Ibnsouda, 2016), isolates were transferred to a 0.8% saline solution to a final bacterial concentration of 10 5 CFU/ ml or 0.5 turbidity on the McFarland scale measured in a turbidimeter (Biomerieux, Densimat, France) as suggested in previous reports (Shen et al., 2014). Meanwhile, about 500 mg of each lyophilized sample from mortiño fruits or leaves was suspended in 100 ml of 70% ethanol, allowed to soak for 1 hr, and filtered through Whatman 41 paper (Balouiri et al., 2016). Each pathogen suspension was spread-inoculated over the surface of TSA as well as LB and let air dry. Four wells of three millimeter diameter were made in the inoculated media using a sterile puncher, and 50 μl of each sample extract was added into each well. Ampicillin (50 μg/ml) was used as positive control, and 70% ethanol was used as a blank sample (Figure 5a,b).

| Antibacterial properties
Plates were then incubated for 24 hr at 37°C, and the antibacterial activity was expressed as the zone of inhibition diameter (ZOI) produced by the extracts against the test bacteria. (Pervin et al., 2013) Five replicates were run for each pathogen.

| Statistical analysis
Data of polyphenolic compounds, antioxidant capacity, anthocyanins, and antimicrobial activity were analyzed using Tukey's tests in Minitab 16 (Zhang, Yue, Jiang, Fan, & Gao, 2016), and significance was reported at p < .05. Correspondence analysis was carried out using InfoStat version 20161 to determine potential correlations between the bacterial Gram staining, potential degree of antibiotic resistance, and inhibition halo. For this, pathogens were first classified according to the antibiotic resistance potential into low, medium, high, or critically resistant to antibiotics following the WHO classification (WHO, 2014). Additionally, bacterial inhibition halos were divided into quartiles to obtain the low, medium-low, medium-high, and high halo size ranges.

| Genetic characterization
A total of 20 representative mortiño samples were sequenced, and four characteristic sequences of matK and one of the rbcL DNA regions were uploaded to the NCBI database (Table 1)

| Chemical characterization
Mortiño samples from Pichincha yielded significantly higher concentrations of polyphenols (146.10 ± 13.44 and 204.01 ± 12.50 mg GAEs/100 g for fruits and leaves, respectively) than the samples from Chimborazo (107.37 ± 6.71 and 144.33 ± 1.85 mg GAEs/100 g for fruits and leaves, respectively) and the polyphenol content in leaves was significantly higher than that in fruits for all samples (Figure 2). Conversely, mortiño fruits from Chimborazo showed significantly higher antioxidant capacity than those from Pichincha (12.84 ± 0.52 and 9.16 ± 0.41 mmol TE/100 g, respectively), but no significant differences were observed when comparing mortiño leaves obtained from both provinces.
Additionally, no significant differences were observed when comparing the antioxidant capacity of fruit and leaf samples (Figure 3).
Fruit samples from Pichincha contained significantly higher average anthocyanins levels (1,095.39 ± 19.18 mg/100 g) than the fruit samples from Chimborazo (89.88 ± 0.65 mg/100 g), and anthocyanins levels in fruit were significantly higher than those in leaves for all samples (Figure 4).

| Antibacterial properties
All fruit extracts showed inhibition halos against the pathogens tested, and no growth inhibition was observed when ethanol alone was used. The highest ZOI observed was 40 mm and resulted from testing mortiño fruit extracts against Vibrio vulnificus in TSA, fol- To perform the correspondence analysis, ZOIs were divided into low, medium-low, medium-high, and high halo sizes as determined by the 1st, 2nd, 3rd, and 4th quartile ranges, respectively. Table 2 shows the ranges obtained for leaf, fruit, and ampicillin inhibition. Correspondence analysis showed that the low and high ZOIs grouped with Gram-positive and Gram-negative bacteria, respectively ( Figure 6). Similarly, low and medium-low ZOIs grouped with the medium, high, and critical potential of antibiotic resistance, whereas high ZOIs grouped with the low potential of antibiotic resistance ( Figure 6b).  (Borsch & Quandt, 2010). Similarly, highly heterogeneous climatic conditions have been responsible for variations in populations of Vaccinium meridionale in Colombia (Ligarreto, Patiño, & Magnitskiy, 2011), and a decrease in Vaccinium spp. leaf size proportional to the increase in geographical altitude has been observed (Cecco et al., 2016).

| D ISCUSS I ON
Sequences of the rcbL region were homogeneous across all sampled provinces, and no polymorphism was observed. The rbcl DNA region has shown lower variability than the matK region in various plant species (Funke, 2016). In this study, polymorphism was only found in matK sequences probably due to the potential of this region to evolve more quickly than other DNA regions (Wicke & Quandt, 2009). Prior to this study, no rcbL sequences of mortiño could be found in available databases.
Various methods are available to determine the antioxidant capacity of plant products including ABTS, 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorption capacity, ferric reducing antioxidant power, among others. Applications in Vaccinium spp. (Connor, Luby, & Tong, 2002;Oszmiański, Kolniak-Ostek, Lachowicz, Gorzelany, & Matłok, 2017) and other fruits such as guava (Thaipong et al., 2006) have yielded comparable results across the different antioxidant capacity methods. Slightly higher antioxidant capacity values were observed in Vaccinium spp. when using ABTS in a recent study (Rodrigues et al., 2011). In this study, ABTS was selected as the method for assessing the antioxidant capacity in mortiño.
The total polyphenol content of mortiño leaves was similar to the 255 mg GAEs/100 g reported for blueberry leaves in previous studies (Venskutonis et al., 2016), but the total polyphenol content and antioxidant capacity of mortiño fruits were significantly lower than the values reported for the fruits of other berries. Fruits Mortiño fruits showed significantly higher anthocyanin levels than the 376.2 mg/100 g observed in V. floribundum fruits analyzed in previous studies (Prencipe et al., 2014). Additionally, the levels of anthocyanins in fruits were significantly greater than those in leaves, but the levels of polyphenols were significantly higher in leaves when compared to fruits. Results are in agreement with previous reports where anthocyanins were observed in fruits of Vaccinium spp.
and not in leaves (Feng et al., 2017), but phenolic acids and flavonols were significantly higher in leaves when compared to fruits (Teleszko & Wojdyło, 2015).  TA B L E 2 Range categories of zone of inhibition diameters (ZOIs) obtained by in vitro tests against all the pathogens described in Figure 5 F I G U R E 6 Correspondence analysis between Gram staining (a) or resistance class (b) vs inhibition halo size. Gram staining (•) was positive or negative, and resistance level (•) was low (Low-R), medium (Medium-R), high (High-R), and critical (Critical-R); leaf ( ), fruit ( ), and ampicillin (○) halo sizes were low, medium-low (M-Low), medium-high (M-high), or high as indicated in Table 2 showed a 11 mm ZOI against S. aureus (Biswas, Rogers, McLaughlin, Daniels, & Yadav, 2013). Additionally, neem leaf extracts inhibited the growth of S. aureus and V. parahaemolyticus with ZOIs of 10 mm for both bacteria (Farjana, Zerin, & Kabir, 2014).
The observed antimicrobial activity is probably due to the highpolyphenol levels in the extracts. Phenolic compounds containing hydroxyl groups have been reported to inhibit the enzymatic activity of microorganisms (Samad, Debnath, Ye, Hasnat, & Lim, 2014). Additionally, phenolic acids such as gallic and caffeic acids are potential inhibitors of proline metabolism in pathogens such as L. monocytogenes (Apostolidis, Kwon, & Shetty, 2008), and phenolic compounds can be complexed with proteins in the outer membrane of microorganisms (Yow, Tang, Chu, & Huang, 2012), potentially resulting in the pathogen's inhibition or death.
Results from the correspondence analysis suggest that Gramnegative pathogens are more susceptible to mortiño leaf and fruit extracts than the Gram-positive ones. The finding of effective antimicrobials against Gram-negative bacteria has been challenging due to the low permeability of the outer membrane in this type of microorganisms (Delcour, 2009). Mortiño leaves and fruits have the potential to become the source of new and effective antimicrobials that can be used in the food and pharmaceutical industries.
Correspondence analysis grouped the ZOI sizes with the expected antibiotic resistance as reported by the WHO (WHO, 2014).
Low ZOI sizes grouped with highly resistant strains and vice versa.
Results support WHO report that suggests that the pathogens tested possess innate mechanisms of resistance to broad-spectrum antibiotics, including ampicillin and other natural antimicrobials (WHO, 2014).

| CON CLUS IONS
Mortiño leaves and fruits contain high levels of antioxidants, polyphenols, anthocyanins, and antimicrobial activity. Mortiño has the potential to become the source of effective bioproducts that can be used in the food and pharmaceutical industries to develop novel nutraceuticals and antimicrobials.

ACK N OWLED G M ENTS
This work was supported by the International Foundation for Science (IFS), Grant No. D/5451-1. This work was part of an interuniversity agreement supported by the VLIR Network Ecuador.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to report.

E TH I C A L R E V I E W
This article does not contain any studies with human or animal subjects.