Essential oil of Xylopia aethiopica from Cameroon: Chemical composition, antiradical and in vitro antifungal activity against some mycotoxigenic fungi

2.1 Plant material

Dried fruits of X. aethiopica were collected in Bafoussam (West Cameroon) in July 2015. They were identified by Mr Nikio Kaji, a botanist of the National Herbarium of Yaoundé, Cameroon (located in West Africa between latitude 6°00′N and longitude 12°00′E with the maximum altitude of 4095 m above sea level). The voucher specimen of this plant were deposited (53821/HNC).

The samples were dried at 40 °C for 48 h in a ventilated dryer and then crushed using a mixer to have small particle size used for extraction.

2.2 Fungal strains

The fungal strains used include Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus versicolor and Fusarium oxysporium. They were chosen because of their high frequency of contamination of food products and their involvement in human and animal pathology. All strains were provided by the Microbiology Laboratory of the National High School of Agro-Industrial Sciences of the University of Ngaoundéré, Cameroon.

Throughout the study, the fungal strains were maintained at 4 °C in tubes containing inclined Sabouraud Dextrose Agar medium (SDA) supplemented with chloramphenicol. For their reactivation, the fungal strains were transferred to the surface of a fresh SDA medium, contained in Petri dishes. The Petri dishes were incubated for 7 days at 25 ± 2 °C. After incubation, fungal spores were scraped from the surface of each dish with a sterile material, and introduced into a sterile tween solution (1%), further adjusted to give a final concentration of 10⁶ spores/ml before being seeded uniformly to the surface of SDA medium. The Petri dishes were incubated for an additional 48 h at 25 ± 2 °C, and a mycelial disk was removed for the antifungal test (Yaouba et al., 2011).

2.3 Extraction of essential oil

100 g of dried and crushed plant material was introduced into a flask and 500 ml of distilled water was added. The Clevenger was adapted to the upper part of the flask and the mixture was boiled at 95 °C for 4 h (Oliveira et al., 2017). After condensation of the heterogeneous vapours of water, the essential oil harvested was subsequently dried with an anhydrous sodium sulfate column and stored at 4 °C in a hermetically sealed brown glass vial until used. The extraction yield was calculated and estimated as a percentage (v/w) based on the weight of the dried fruit used for extraction.

2.4 Chemical analysis of essential oil

Analysis of essential oil was performed by gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC/MS).

2.5 Antifungal activity

The antifungal assays were carried out using the incorporation method as described by De Billerbeck et al. (2001). Due to the non-miscibility of the essential oil with water and thus with the culture medium, an emulsification of the essential oil was previously carried out using an aqueous solution of dimethylsulphoxide (DMSO). The essential oil is then incorporated at a different volume into the Sabouraud Dextrose Agar (SDA) medium to have desired concentrations (1000 ppm, 2000 ppm, 3000 ppm, 4000 ppm, 5000 ppm, 6000 ppm and 7000 ppm). The medium thus supplemented is poured into Petri dishes of 90 mm in a proportion of 20 ml and left for about 15–30 min for solidification. For each fungal strain, a mycelial disc of 6 mm in diameter is taken from a preculture of 48 h and seeded directly in the center of each dish. The positive control of fungal growth is carried out according to the same procedure for dishes containing the culture medium not supplemented with essential oil. The dishes are incubated in an inverted position at 25 ± 2 °C and the diameter is measured along two perpendicular lines passing through the center of the plate, at regular time intervals (48 h) until the stop of control radial growth. The minimum inhibitory concentration (MIC) was determined and is equivalent to the lowest concentration of essential oil in which no growth was observed. The following formula was used to calculate the antifungal index (AI): $$\mathit{AI}(\%)=\left(1-\frac{\mathit{Dt}}{\mathit{Dc}}\right)\times 100$$

Dt = diameter of growth zone in the test plate

Dc = diameter of growth zone in the control plate.

The discs exhibiting no fungal growth were seeded again in Petri dishes containing the SDA medium not supplemented with essential oil. Seven days after incubation at 25 ± 2 °C, the Petri dishes in which there was no resumption of growth are noted, which makes it possible to determine the minimum fungicidal concentration (MFC).

2.6 Evaluation of antiradical activity

2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay was performed to assess the antiradical activity of essential oil (Brand-Williams et al., 1995). Different volumes of essential oil were introduced into tubes, and the final volume was made up to 2 ml by addition of the methanolic solution of 100 μM DPPH so as to have essential oil concentrations range from 9.77 to 80,000 µg/mL. The BHT used as a reference antioxidant was evaluated according to the same procedures. The reaction mixture is subsequently incubated in the dark and at room temperature for 60 min. Absorbance is measured at 517 nm using a spectrophotometer (RAYLEIGH VIS-723N). The percentage of free radical trapping (AA) is calculated relative to the control containing only 100 μM DPPH solution and methanol, using the following formula: $$\mathit{AA}(\%)=\left(1-\frac{\mathit{ODt}}{\mathit{ODc}}\right)\times 100$$

ODc = Absorbance of control

ODt = Absorbance of test

AA (%) = Antioxidant Activity

The SC₅₀ representing the concentration of antiradical substance allowing for trapping 50% of the free radical and obtained graphically from the plot of the trapping curves of the free DPPH radical was used as an element of comparison of the antiradical activity.

2.7 Statistical analysis

Each assay was done in triplicate to minimize the experimental error, and the average was calculated. Data were analyzed by Statistica .06, Statistical package (Statsoft, 1995). Differences between means were tested using Duncan Multiple Range Test with p ≤ 0.5.

3.1 Chemical analysis

The extraction yield of the essential oil of X. aethiopica was 4.2%. These results are comparable to those previously obtained by Bakarnga-Via et al. (2014) who found extraction yields of 3.57% and 4.68%, for dry fruits of X. aethiopica from Chad and Cameroon, respectively. But, the extraction yields of the essential oil of dry fruits of X. aethiopica obtained from local market in Keffi, Nasarawa State, Nigeria, was too lower; 1.2% (Olonisakin et al., 2007).

Besides, the analysis of the chemical composition of the essential oil of X. aethiopica allowed to identified 70 compounds listed in their order of elution from DB-5 column, the results are presented in Table 1. All compounds identified are divided into four major chemical classes: monoterpenes hydrocarbons (69.41%), oxygenated monoterpenes (8.42%), sesquiterpenes hydrocarbons (17.58%) and oxygenated sesquiterpenes (1.73%). The major compounds identified are β-pinene (32.16%), β-phellandrene (10.71%), α-pinene (7.39%), α-phellandrene (6.8%) for the hydrocarbon monoterpene class, and finally, the Z-γ-bisabolene (10.07%) belonging to the oxygenated monoterpenes. The chemical composition analysis of X. aethiopica essential oil from Cameroon shows that it is represented only by a single chemotype since β-pinene always appears as the major compound. However, in Sudan, two other chemotypes have been postponed. Indeed, EL-Kamali and Adam (2009), Elhassan et al. (2010) have examined the chemical composition of essential oil of the dry fruits of X. aethiopica obtained from Sudan and found that the major component was the 4-isopropylbenzyl alcohol (16.67%) and 4-terpineol (11.30%), respectively.

Several factors can influence the yield of the essential oil and its chemical composition, including plant age, place and time of harvest, and presence of parasites (Zantar et al., 2015).

3.2 Antifungal activities

3.2.1

3.2.1 Effect of X. aethiopica essential oil on all fungal strains

The effects of X. aethiopica essential oil on mycelial growth of Aspergillus niger, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus flavus and Fusarium oxysporium are expressed in antifungal index and are illustrated in Fig. 1. These figures show that for all fungal strain, the antifungal index increases with the increase in the concentration of essential oil, indicating its antifungal activity. Furthermore, throughout the incubation period and for a defined concentration of essential oil of X. aethiopica, significant differences (p > 0.05) was observed in the antifungal index against A. flavus, F. oxysporium, A. versicolor, A. fumigatus and A. niger. This shows the variability of the resistance level exerted by each strain under the effect of the supplementation of the culture medium with the essential oil.

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Fig. 1

Antifungal Index of X. aethiopica essential oil against: A. flavus (a); A. fumigatus (b); A. niger (c); A. versicolor (d) and F. oxysporium (e).

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3.3 Antiradical activities of essential oil

The DPPH assay was used to evaluated the scavenging activity of the essential oil of X. aethiopica and that of BHT and the results are reported in Table 3. It is apparent from this table that the increase in the concentration of essential oil leads to an increase in the percentage of trapping of the free DPPH radical. This shows that it possesses, like the reference antioxidant, an anti-radical activity. To compare their antioxidant potential, the SC₅₀, which refers to the minimum concentration of anti-radical substance capable of trapping 50% of free radicals was used. The lower its value, the more the substance tested has a good antiradical efficacy. The graphical plot was used to determine the SC₅₀ values of the essential oil and the BHT: 594.58 ± 57.37 μg/mL and 65.03 ± 0.99 μg/mL, respectively. This shows that the anti-radical activity of the essential oil is very low compared to that of the reference antioxidant. As has been shown in several studies, the biological activity of essential oils is closely related to their chemical profile. Thus, the low antioxidant potential of the essential oil of X. aethiopica could be due to its low content of γ-terpinene, p-cymene, thymol known for their considerable antioxidant activity and its high content of monoterpenes hydrocarbons (69.41%) and sesquiterpenes hydrocarbons (17.58%). The terpene compounds are endowed with a low antioxidant activity. They are incapable of giving their hydrogen atom necessary for the reduction of the free DPPH radical, and they have a low solubility in alcoholic medium for DPPH test (Mata et al., 2007).

Konan et al. (2009) showed that the antiradical potential of dry fruits of X. aethiopica essential oil (SC₅₀ = 4100 µg/mL) which originated from Langossou, in Ivory Coast was close to that of the reference antioxidant solution which was the α-tocopherol (SC₅₀ = 4300 µg/mL).