Collection and adequacy

Pineapple (Ananas comosus) crowns were collected from the waste found in the city's central supply center. 3.5 kg of pineapple crown leaves were taken and cleaned using a 0.9% saline solution and then weighed.

Drying and grinding

For the drying process, a tray dryer, model PS-ECE-001/PE and series GEN-0412-237 was used for 72 hours at 55 °C. to reduce as much humidity as possible, and then they were milled to obtain a fine flour, for which a grain mill was used.

Extraction and identification of phenolic compounds

In the process of obtaining the oleaginous extract, a Soxhlet equipment was used in a solid-liquid extraction for seven hours using 96% ethanol as solvent, then a simple distillation was performed for 60 minutes to obtain the purest extract and evaporate the solvent, then the yield obtained from the extract was calculated using equation 1:

(Equation 1)

The identification of phenolic compounds was carried out by ultra-high performance liquid chromatography with Orbitrap mass detector (UHPLC-ESI+-Orbitrap-HRMS), the analyzed samples were dissolved in a mixture of methanol: water (1:1 v/v) with formic acid 0.2 % v/v, vortexed (5 min) and sonicated (5 min) and then injected into the chromatographic equipment. As reference standards were used the xanthines: caffeine (Part N° C8960-250G, Sigma-Aldrich), theobromine (Part N° T4500-25G, Sigma-Aldrich) and theophylline (Part N° T1633-25G, Sigma-Aldrich); the catechins: (±)-catechin (C) (Part N° C1788-500MG, Sigma-Aldrich), (-)-epigallocatechin gallate (EGCG) (Part N° E4143-50MG, Sigma-Aldrich), (-)-epicatechin (EC) (Part N° E1753-1G, Sigma-Aldrich), (-)-epicateqin gallate (ECG) (Part N° E3893-10MG, SigmaAldrich), (-)-epigallocateqin (EGC) (Part N° E3768-5MG, Sigma-Aldrich); flavonoids: caffeic acid (Part N° C0625, Sigma-Aldrich), p-coumaric acid (Part N° C9008, Sigma-Aldrich), rosmarinic acid (Part N° 536954-5G, Sigma-Aldrich), quercetin (Part N° Q4951-10G, Sigma-Aldrich), naringenin (Part N° N5893-1G, Sigma-Aldrich), luteolin (Part N° L9283-10MG, Sigma-Aldrich), kaempferol (Part N° K0133-50MG, Sigma-Aldrich), pinocembrin (Part N° P5239, Sigma-Aldrich), apigenin (Part N° A3145-25MG, Sigma-Aldrich); anthocyanins: cyanidin 3-rutinoside (Part No. G36428, Sigma-Aldrich), pelargonidin 3-glucoside (Part No. 53489, Sigma-Aldrich). Ultra-high efficiency liquid chromatograph (UHPLC), Dionex Ultimate 3000 (Thermo Scientific, Sunnyvale, CA, USA), equipped with a binary gradient pump (HP G3400RS), an automatic sample injector (WPS 300TRS) and a thermostatted unit for the column (TCC 3000). The LC-MS interface was electronebulization (ESI) and the mass spectrometer was high resolution with an Obitrap ion current detection system. Operated in positive mode with a capillary voltage of 3.5 kV. A Hypersil GOLD Aq Column (Thermo Scientific, Sunnyvale, CA, USA; 100 x 2.1 mm, 1.9 μm particle size) was used at 30 °C. The mobile phase was A: a solution of 0.2 % v/v formic acid in water, and B: 0.2 % v/v formic acid in acetonitrile. The initial gradient condition was 100 % A, changing linearly up to 100 % B (8 min); it was maintained for 4 min; the return to initial conditions in 1 min; the total run time was 13 min, with three min for post-run. Compound identification was performed using full scan acquisition mode and extraction of ionic currents (EIC) corresponding to the [M+H]+ of compounds of interest, mass measurement with accuracy and precision of Δppm < 1 and using a standard solution-mix of the compounds (certified reference material), for quantification of the analytes of interest the external standardization method was used.

Microbiological analysis

For the microbiological analysis, the methodology proposed by ¹⁷ was followed, with some modifications, in which antimicrobial susceptibility was evaluated by the standardized method of diffusion with discs (Bauer-Kirby Method), using Mueller-Hinton agar according to the standards established by the Clinical and Laboratory Standards Institute Clinical and Laboratory Standards Institute. CLSI (2017). For this, the Staphylococcus aureus strain (ATCC 25923) was used, using as positive control oxacillin (10 µL), and as negative control (DMSO), in the elaboration of the treatments of Ananas comosus was implemented the methodology used by. ¹⁸ and ¹⁹, where the extract was diluted in dimethyl sulfoxide (DMSO), with three replicates for each treatment applied, with the inoculum applied, the plates were prepared at concentrations of 25, 50, 75 and 100 mg/ml respectively see (Table 1)

Likewise the turbidity of the medium was fixed in 0.5 units (1.5x108 UFC/ml) according to the Mc Farland scale pattern, which correlates with the number of bacteria present ²⁰, likewise its absorbance was verified at a wavelength of 625nm, then we proceeded to the sowing in agar, pouring 25 µL of the extract and the controls in filter paper discs (Whatman No. 42), these were placed in the agar plate with a sterile clamp at 15 mm of the edge of the plate, pressing on the agar for its adherence with three repetitions for each applied treatment. 42), these were placed on the agar plate with sterile forceps 15 mm from the edge of the plate, pressing on the agar for adherence with three replicates for each treatment applied, the plates were incubated at 37 °C for 24 h in an inverted manner, then the inhibition halos were measured including the diameter of the discs.

Statistical analysis

A 1-factor Anova statistical analysis was performed on the data obtained in each treatment, determining the significant differences between them, for which the inhibition halos present in each treatment were used as a parameter, with a confidence base of 95 % and an error percentage of 5 %, showing the treatment with the best results in the process.

Extraction and identification of phenolic compounds

According to ^22,23 solvent extraction is the most common method for the extraction of phenolic compounds, for which ethanol was used as solvent, using the Soxhlet extraction method, which, according to ²⁴, can be attributed several factors that influence the extraction yield, Among these are the extraction temperature, extraction time and the type of solvent used, in this process it was possible to obtain a volume of 63 ml of extract of dried leaves of pineapple crown, in this case 96% ethanol was used as solvent. An HPLC/DAD type chromatographic analysis was performed using a 25 ml sample of the extract obtained, where a qualitative analysis was performed to identify the presence of phenolic compounds present in the extract, which are reported below (Table 2).

Twenty-seven phenolic compounds were identified, among them caffeine, vanillic acid and p-coumaric acid, these being the ones with the highest concentration. In addition, other structures related to polyphenols, such as gallic acid and quercetin, were detected, These compounds have a high potential to be used as antioxidants and antibacterials, as pointed out in previous studies by ²⁵, and when these results were compared with the extraction process carried out by ²⁶ in which a total of 27 polyphenolic compounds were identified, gallic acid, vanillic acid, protocatechuic acid, 2,5-dihydroxybenzoic acid and 4-hydroxybenzoic acid predominated. In particular, rosmarinic acid was reported as the compound with the highest concentration present in the extract, which is a polyphenol known for its antioxidant properties and has been associated with various health benefits, which according to Ramirez et al. ²⁷, the concentration of rosmarinic acid (RA) increases with increasing K, which can have a significant impact on the phenolic composition, contributing to the antioxidant capacity ²⁸, likewise it can be mentioned that one of the possible mechanisms proposed for the antimicrobial effect of phenolic compounds is that they can break the integrity of the membrane and cause loss of cell integrity and eventual cell death ^{29), (30}.

Microbiological analysis

The absorbance of the S. aureus culture was measured, obtaining an average value of 0.142 on the McFarland scale, which is within the range established by ³¹, in his research on the validation of the membrane filtration technique. The treatments applied and the inhibition halos generated by the Ananas comosus extract in its different concentrations and the replicates of each treatment against the S aureus strain, including the positive and negative controls corresponding to each test, were also observed. Figure 1.

Figure 1 Inhibition halos present in each treatment applied per replicate. 

The diameters of each halo generated were also measured and recorded (Table 3).

It should be noted that the evaluation process was only focused on observing the possible inhibition effect, so the dose-response analysis was not performed, then the average of the inhibition halos generated by the pineapple crown extract in S aureus was calculated, obtaining diameters ranging between 4.5 mm and 6, 0 mm, where the analysis of variance showed that with respect to the positive control and the applied treatments determined the difference, taking into account the p-value, which according to ³², this value indicates the probability of obtaining by chance, a difference greater than that observed in the process, this is how the obtained value of p = 5. 80E-13, which was less than 0.05, so there is a significant difference in the analysis of variance of inhibition halos (Table 4).

On the other hand, the microbiological analysis carried out showed a variability in the inhibition halos which is significant because according to ³³ previous results of inhibition halos of 28 and 18 mm have been reported, as reported by ³⁴ and ³⁵, as well as in the research carried out by ³⁶, establishes that bacteria tend to produce resistance mechanisms against antimicrobials, which in the case of Staphylococcus aureus are related to the activation of cell wall synthesis, with the production of proteins, which once these resistance mechanisms are activated, create a greater firmness against the applied agent. It was also observed that treatment three corresponds to the highest concentration of pineapple crown extract, being the most representative in terms of inhibitory capacity, which indicates a gradual inhibition capacity, with a smaller inhibition halo being observed at lower concentrations of the extract. These results are consistent with the findings reported by ³⁷, who indicated that treatments with higher concentrations usually generate larger inhibition halos, demonstrating a direct and effective action in the suppression of pathogenic microorganisms, so in this research it is established that the antimicrobial activity of the extract is proportional to its concentration.