Chemical Profile and Antimicrobial Activity of Syagrus oleracea (Mart.) Becc. Oils and Oils Extracted from Speciomerus revoili (Pic.) Larvae

Background: The antimicrobial effect of oils extracted from Syagrus oleracea (Mart.) to prove the action and obtain data for the future development of therapeutic products. Objectives: The present study was to determine the chemical profile and the antimicrobial potential of oils extracted from Syagrus oleracea (Mart.) Becc. almonds and Speciomerus revoilie (Pic.) larvae to obtain scientific information to prove the action and obtain data for the future development of therapeutic products. Materials and Methods: The separation, identification and quantification of fatty acids in oils extracted from the larvae of S. revoili (Coleoptera, Chrysomelidae) and almonds of S. oleracea (Arecaceae) were performed by gas chromatography coupled with mass spectrometry. The determination of antibacterial activity was performed using microdilution in plates. Results: In the oils extracted from S. revoili and S. oleracea , saturated chain fatty acids, the presence of hexanoic acid (only in almonds), and caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and those of unsaturated chain, oleic acid (omega-9) and linoleic acid (omega-6). S. revoili oil showed satisfactory results against Escherichia coli and Staphylococcus aureus .


INTRODUCTION
Syagrus oleracea Mart. Becc., has traditionally been used in the form of root extracts for back pain and flower syrups for bronchitis. [1] In the pulp of the fruits are present phenolic compounds that have antioxidant and antimicrobial activities and in the oils of the almonds, saturated and monounsaturated fatty acids. [2,3] Members of the Bruchida family and mainly attack almonds from plants of the Arecaceae family, such as the species Pachymerus nucelorum and Speciomerus revoili. [4] In Brazil, traditional communities use Pachymerus nucleorum larva oil, popularly known as coró, to treat cracks in the heel, swelling, wounds, seborrheic dermatitis, inflammation, thrombosis, newborn's navel and earache. [5] Thus, the objective of the work was to determine the chemical profile and the antimicrobial potential of oils extracted from almonds of S. oleracea and larvae of S. revoili.

Fruit Processing
The fruits were washed, dried for 24 hr, separated from the almond shells. The obtained larvae were washed with distilled water and stored (-20°C) for later identification and analysis.

Sample preparation: hydrolysis and oil methylation
The analyzes of the three extractions and the hydrolysis and methylation of the oils were analyzed followed by the method described Sande et al., 2018. [6]

Analysis Method: Gas Chromatography coupled to Mass Spectrometer
The analyzes were performed on Gas Chromatographer HP7820A (Agilent) equipped with flame ionization detector. The column used was the Innowax (HP) 15m x 0.25mm x 0.20µ with temperature gradient was used: 70°C (0 min.), 7°C/min. up to 240°C; Injector (Split of 1/30) at 250°C and detector at 260°C. Hydrogen was used as drag gas (3mL/min.). The sample injection volume was 1L. The data acquisition program applied in the analysis was EZChrom Elite Compact (Agilent). The quantitative analysis was done by standardization of area by CG-FID. The identification of peaks was made by comparing retention times with pure patterns of meatilated fatty acids SUPELCO37 and by Gas Chromatography coupled to Mass Spectrometer (CG-MS).

Tested micro-organisms
Reference strains gram negative of E. coli (ATCC 8739) and gram positive of S. aureus (ATCC 25923) were used. Sterility tests were performed for the culture medium and oils.

Standardization of the inoculum
The microdilution plate test [7] started by standardizing the inoculum on a McFarland scale at 0.5 (1.5x10 8 CFU/mL). The inoculum was inserted in Tryptic Soy Brothe nutrient broth and then transferred to Elisa plate wells at a concentration of 5x10 5 CFU/mL.

Standardization of oils
Oils at concentrations of 240μL, 120μL, 60μL/mL and crude oil (without diluition, as used popularly) were used to determine the minimum inhibitory concentration, as positive controls amoxicillin (S. aureus) and tetracycline (E. coli) (16μ/mL, 8μ/ mL and 4μ/mL) and negative control TBS + Tween 80. The standardization of the oils was carried out in TSB nutrient broth together with Tween 80 and the oils [7] (NCCLS, 2003). 20μL were transferred to sterile microplate wells with a final volume of 180μL and incubated for 24 hr. Then it was verified whether there was growth or inhibition of micro-organisms with the addition of 20μL of 1% triphenyl tetrazolium chloride. The assays were performed in three replications.

Statistical analysis
ANOVA and Tukey's test were performed and p<0.05 were considered significant results.

Oils from S. oleracea almonds and S. revoili larvae
The yields found were location 1: 5.3g (45.30%) of larva oil and 20.29g (22.05%) of almond oil; location 2: 0.8g (41.89%) of larva oil and 9.98g (19.70%) of almond oil and location 3: 1.5g (32.47%) of larva oil and 9.63g (16.95%) of almond oil. Two unsaturated fatty acids and the other saturated ones were identified. Variations in fatty acid concentrations were observed and hexanoic acid was found only in almond oils. The main fatty acids observed were saturated chains: hexanoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid and those of the unsaturated chain: oleic acid (omega-9) and linoleic acid (omega-6) ( Table 1).
Higher concentrations of caprylic acids, capric acid and lauric acid were observed in the almond oil of S. oleraceae of myristic, palmitic, oleic and linoleic acids in the oil of S. revoili. Saturated chain fatty acids were found in high concentration in almond oil (86.0%) ( Table 1).

Antimicrobial Activity
In the microdilution plate test, using crude oil of S. revoili, a bacteriostatic effect was observed, since after transfer to plates with culture medium, a small growth of bacteria was observed. In the other dilutions (240μL, 120μL and 60μL) no inhibition was observed. The positive controls (antibiotics) amoxicillin and tetracycline inhibited micro-organisms at a concentration of 4μg/ mL and in the negative control there was bacterial growth.
Medium-chain fatty acids, such as lauric and long-chain oils, are responsible for the inactivation of gram positive and gram negative bacteria. [12] The antimicrobial effects occur due to interferences in the cell wall of micro-organisms and in the mechanisms of bacterial virulence. [13] CONCLUSION Hexanoic, caprylic, capric, lauric, myristic, palmitic, stearic and unsaturated oleic and linoleic fatty acids were identified. Only the crude oil of the larva of the S. revoili beetle showed bacteriostatic activity against gram-negative strains of E. coli and S. aureus. Larva oil as it is popularly used can have an effect because it is used directly in the ear without any dilution, as observed in the experiments, however, further studies must be performed.

ACKNOWLEDGEMENT
To the State University of Montes Claros, to the Postgraduate Program in Biotechnology and CAPES (Higher Education Improvement Coordination).