Chemical Constituents and Antimicrobial and Antioxidant Activities of Essential Oil from Dried Seeds of Xylopia aethiopica

The study aimed to investigate the chemical composition and antimicrobial and antioxidant activities of the essential oil from dried seeds of Xylopia aethiopica. The essential oil was obtained by hydrodistillation and analyzed by GC/FID and GC/MS. The essential oil yield was 1.35%. Forty-nine compounds were identified in the essential oil with 1,8-cineole (16.3%), β-pinene (14.8%), trans-pinocarveol (9.1%), myrtenol (8.3%), α-pinene (5.9%), and terpinen-4-ol (5.6%) as major components. The antimicrobial activity of this essential oil was studied using disk diffusion and broth microdilution methods on four bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) and one fungus (Candida albicans). The essential oil exhibited excellent activity against S. aureus, E. faecalis, and C. albicans and moderate activity against E. coli. Among all strains tested, C. albicans showed the best sensitivity with a MIC of 50 mg/mL. The antioxidant activity was examined using a DPPH-free radical scavenging assay. The essential oil of X. aethiopica showed low antioxidant activity (IC50 = 784.604 ± 0.320 mg/mL) compared to that of ascorbic acid and the reference compound (IC50 = 0.163 ± 0.003 mg/mL). The results indicate that consumption of X. aethiopica seeds can reduce the virulence of food-borne pathogens and their resistance to antibiotics.


Introduction
Free radicals and other reactive oxygen species, which are highly reactive compounds, are produced naturally in the cell and play a key physiological role.However, these unstable compounds can exert negative efects on the immune system if their production is greater than that of antioxidants, which are able to neutralize or scavenge free radicals.Overproduction of free radicals known as oxidative stress is involved in the development of several diseases such as cancer, cardiovascular disease, and Alzheimer's or Parkinson's diseases or in cell ageing [1,2].On the other hand, the proliferation of resistance of bacteria to the available antibiotics exacerbates the damage caused by infectious diseases [3].In 2011, the WHO (World Health Organization) called for intensifed research into new antibacterial drugs to deal with this scourge, but only a few new molecules are under development [4,5].Consequently, there has been considerable and growing interest in identifying new sources of antioxidant and antimicrobial potential from natural, safe, and inexpensive sources [6].

Materials and Methods
2.1.Plant Material.Seeds of X. aethiopica were purchased from Tilène market in Dakar.Tey were authenticated in the Laboratory of Pharmacognosy, Faculty of Medicine, Pharmacy and Odontology of Cheikh Anta Diop University in Dakar (Senegal).

Essential Oil Extraction.
Te seeds were powdered using a Brabender brand mechanical grinder.To extract the essential oil, the sample was hydrodistilled (4 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia [48].After hydrodistillation, the essential oil collected was stored at 4 °C before analyses.Te extraction yield of essential oil was calculated as a percentage (w/w) based on the weight of the dried seeds.

Physical Characteristics of Essential Oils.
Physical examinations regarding density, refractive index, and optical rotation of essential oils were carried out in this work.

Density of Essential Oil.
Te gravimetric method measured the density of essential oil.Te weight of the empty pycnometer on an analytical balance was denoted (P 0 ).Besides, the flled pycnometer with distilled water was weighted (P 1 ), and that of the pycnometer flled with the oil of X. aethiopica was coded (P 2 ).Te following formula was used to calculate the density of the volatile oils: Samples were also analyzed with a Perkin-Elmer Turbo mass detector (quadrupole) coupled to a Perkin-Elmer Autosystem XL, equipped with Rtx-1 and Rtx-wax fusedsilica capillary columns.Te oven temperature was programmed from 60 to 230 °C at 2 °C/min and then held isothermally at 230 °C (35 min): hydrogen was used as carrier gas (1 mL/min).Te following chromatographic conditions were employed: injection volume, 0.2 μL of pure oil; injector temperature, 280 °C; split, 1 : 80; ion source temperature, 150 °C; ionization energy, 70 eV; MS (EI) acquired over the mass range, 35-350 da; scan rate, 1 s.
Te identifcation of the components was based on (a) the comparison of their GC retention indices (RI) on nonpolar and polar columns, determined from the retention times of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data; (b) computer matching with commercial mass spectral libraries 2 Biochemistry Research International [49][50][51] and comparison of spectra with those of our personal library; and (c) comparison of RI and MS spectral data of authentic compounds or literature data.

Microbial Strains.
Antibacterial activity of the essential oils of X. aethiopica was carried out by using fve pathogenic strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC27853, and Candida albicans ATCC 24433.All of the strains were grown on Mueller-Hinton agar for the bacteria and Sabouraud dextrose agar with chloramphenicol for yeast.
2.6.Determination of Antibacterial Activity.Te sensibility of the fve pathogenic strains to the essential oils was assayed using the agar disc difusion method [52].Inocula were prepared by diluting overnight cultures in Mueller-Hinton broth (MHB; Oxoid) medium to approximately 10 8 CFU/ mL.Filter paper discs (Whatman disc, 6 mm diameter) were impregnated with 25 μL of the essential oil and placed onto the inoculated Petri dishes containing Mueller-Hinton 2 agar.After incubation at 37 ± 1 °C for 24 h for bacteria, the diameters of inhibition zones were measured (mm) and recorded as the mean ± standard deviation.Each test was performed in triplicate separate.According to the width of the inhibition zone diameter expressed in mm, results were appreciated as follows: not sensitive (−) for diameter equal to or below 8.0 mm, moderately sensitive (+) for diameter between 8.0 and 14.0 mm, sensitive (++) for diameter between 14.0 and 20.0 mm, and extremely sensitive (+++) for diameter equal to or longer than 20.0 mm.For the determination of the minimum inhibitory concentration (MIC), which represents the concentration that completely inhibits the growth of microorganisms, a microdilution broth susceptibility assay was used, as recommended by the National Committee for Clinical Laboratory Standards [53].All tests were performed in MHB supplemented with Tween 80 detergent to a fnal concentration of 0.5% (v/v).Dilutions series were prepared from 3.125 to 100.0 mg/mL of the oil in a 96-well microtiter plate.160 μL of MHB was added onto microplates and 20 μL of tested solution.Ten, 20 μL of 1 × 10 8 CFU/mL of standard microorganism suspension was inoculated onto microplates.Plates were incubated at 37 °C for 24 h.Te same test was performed simultaneously for the growth control (MHB + Tween 80) and sterility control (MHB + Tween 80 + test oil).Te MIC is defned as the lowest concentration of the samples at which the bacterium does not demonstrate visible growth.Te bacterium growth was indicated by turbidity.

Antioxidant Activity.
Te antiradical activity of the essential oil of X. aethiopica was estimated by way of the 2,2′diphenyl-1-picrylhydrazyl (DPPH) test according to the method described by Scherer et al [54].Ten, the DPPH .solution was prepared by dissolving 4 mg of DPPH radical in 100 mL of methanol, and the solution was stirred in the dark for 1 hour.Ascorbic acid was used as standard.About 0.1 mL aliquots of methanolic solution of the sample or standard at diferent concentrations were each added to 3.9 mL of a DPPH .methanolic solution.After homogenization, the mixture was incubated in the dark at room temperature for 30 minutes, and the absorbance was measured at 517 nm.Te blank sample consisted of 0.1 mL of methanol added to 3.9 mL of DPPH . .Analyses were performed in triplicate.Te radical scavenging activity was expressed as PI � [(A 0 − A 1 )/A 0 ] × 100, where PI was the percentage inhibition, A 0 was the absorbance of the blank, and A 1 , the absorbance in the presence of the sample or standard at diferent concentrations.Te results were also indicated as IC 50 (the concentration of sample required to scavenge 50% of DPPH radicals).
Besides, physical parameters, which are useful for the quality of essential oils, were determined.Ten, a density of 0.902 g•cm −3 was found, allowing the oil to be above the water during extraction.It is comparable to that found by Vyry Wouatsa et al. which was 0.9239 [25].Xylopia's essential oils show a refractive index of 1.334 and an optical rotation of +7.69 °.Tese physical properties are also comparable to those of Vyry Wouatsa et al. which established a refractive index of 1.488, an optical rotation of +6.133, and a density of 0.9239 (Table 1) [25].
Te minimum inhibitory concentration (MIC) was measured to determine the minimum concentration of oil that inhibited the growth of the microbes used in this study.Te MIC helps determine the level of resistance of a particular bacterial strain.Several concentrations were prepared and evaluated only on four strains of three bacteria (E.coli, S. aureus, and E. faecalis) and one fungus (C.albicans) because P. aeruginosa was not sensitive to the essential oil.Among all strains tested, C. albicans showed the best sensitivity, and the MIC measured for the essential oil of X. aethiopica was 50 mg/ mL (Table 3).However, E. coli and E. faecalis are less sensitive to X. aethiopica essential oil (MIC > 100 mg/mL).
Te good antimicrobial activity of X. aethiopica fruit essential oil corroborates previously reported data [13,19,[21][22][23][24][25][26][27][28][29][30].In addition, a number of studies have shown that the antimicrobial activity of an essential oil is closely linked to its main constituents and their interactions with certain minor constituents [57,58].Previously, several studies have demonstrated the antimicrobial properties of essential oils rich in 1,8-cineole against a wide range of microorganisms [59,60].1,8-cineole has been shown to change the shape and size of the bacterial cell (for both Gram-negative and Gram-positive bacteria) [61].Tegang et al. attributed the antimicrobial activity of X. aethiopica essential oil to its high content of bioactive compounds such as β-pinene (32.16%) and α-pinene (7.39%) which have been isolated, purifed, and studied extensively for their antimicrobial activity [19].

Antioxidant Activity.
Te DPPH radical scavenging method was adopted to assess the antioxidant efect of X. aethiopica essential oil, and ascorbic acid was used as a positive control.Based on our results, Xylopia aethiopica exhibited antioxidant activity by scavenging DPPH-free radicals.Tus, the IC 50 value of the essential oil of Xylopia aethiopica was 784.604 ± 0.320 mg/mL.However, this activity is very weak compared to that of the reference ascorbic acid (IC 50 � 0.163 ± 0.003 mg/mL).Besides, chromatographic analysis revealed that the main compound of Xylopia essential oil had a maximum of one hydroxyl group.Tus, this low antioxidant activity of volatile compounds could be due to their low capacity to donate an electron or a hydrogen atom to reduce the DPPH radical.Finally, the complex cluster of organic products in the essential oils and the signifcant amount of monoterpene hydrocarbons could have an antagonistic activity and thus reduce the antioxidant activity.Tis same remark was made by Alitonou et al. [18] and Tegang et al. [19] who reported low antioxidant activity of X. aethiopica essential oils mainly dominated by hydrocarbon compounds.Tey concluded that the activity observed could be due to the presence of one (or more) minority constituents in the essential oil.

Conclusion
Tis study reported the chemical composition and the antibacterial and antioxidant activities of the essential oil from dried seeds of Xylopia aethiopica.Tis essential oil mainly consisted of 1,8-cineole, β-pinene, trans-pinocarveol, myrtenol, α-pinene, and terpinen-4-ol showed excellent activity against S. aureus, E. faecalis, and C. albicans and moderate activity against E. coli.However, it had low antioxidant activity.It may have potential applications in food and pharmaceutical products.

Table 1 :
Yield and physical properties of Xylopia's essential oil.

Table 2 :
Chemical composition of essential oil from the dried seeds of X. aethiopica.

Table 3 :
Antimicrobial activity of the essential oil from X. aethiopica.