Chemical composition, in vitro antioxidant and anti-inflammatory properties of essential oils of four dietary and medicinal plants from Cameroon

Gas chromatography

GC analysis was performed on a Varian gas chromatograph, model CP-3380, with flame ionization detector containing two silica capillary columns: HP5 J&W Agilent (5 %-Phenyl-methylpolysiloxane) capillary column (30 m × 0.25 mm i.d. × 0.25 μm film) and Supelcowax 10 (polyethylene glycol) fused capillary column (30 m × 0.25 mm i.d. × 0.25 μm film); N₂ was the carrier gas at 0.8 mL/min; injection type 0.1 μL of pure sample, split ratio 1:100; injector temperature 220 °C, detector temperature 250 °C; temperature program 50-200 °C at 5 °C/min, then kept at 200 °C for 10 min. The linear retention indices of the components were determined relative to the retention times of a series of n-alkanes. The entire set up was coordinated by COMPASS software system that ensured its functioning and follow-up of the chromatographic analysis.

Gas chromatography-mass spectrometry

GC-MS analyses were performed using a Hewlett-Packard GC 5890 series II equipped with a HP5 (5 %-Phenylmethylpolysiloxane) fused silica column (30 m × 0.25 mm; film thickness 0.25 μm) and a DB-Wax fused silica column (30 m × 0.25 mm; film thickness 0.25 μm) interfaced with a quadrupole detector (Model 5972); temperature program (50–200 °C at 5 °C/min); injector temperature, 220 °C; MS transfer line temperature, 180 °C; carrier gas, helium at a flow rate of 0.6 mL/min; injection type, split, 1:10 (1 μL 10:100 CH₂Cl₂ solution); ionization voltage, 70 eV; electron multiplier 1460 eV; scan range, 35–300 amu; scan rate, 2.96 scan/s.

Qualitative analysis

The identification of the constituents was based on comparison of their relative retention times with either those of authentic samples or with published data in the literature [46] and matching their mass spectra with those obtained from authentic samples and/or the NBS75K and Wiley 7th NIST 98 EPA/NIH libraries spectra and literature data [46].

Quantitative analysis

The percentage composition of the essential oils was computed by the normalization method from the GC-FID peak areas, assuming an identical mass response factor for all compounds.

Determination of total phenolic content (TPC)

The phenolic content in EOs was determined according to the method described by Singleton et al. [47] with slightly modifications. In effect, 10 μg/mL of EOs were used in the analysis. The reaction mixtures were prepared by mixing 60 μL of EO, 2 mL of 10 % Folin-Ciocalteu dissolved in water. For the blank, 60 μL of methanol, 2 mL of 10 % Folin-Ciocalteu reagent were mixed in water. After 30 min of incubation at room temperature, the absorbance was measured at 750 nm using a V-1100 spectrophotometer. Ascorbic acid was used as standard. Experiments were carried out in triplicate. The total phenolic content was expressed in μg ascorbic acid equivalent per mg of EO (μg AAE/mg) using the following equation based on the calibration curve:\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \mathrm{Absorbance} = 0.02\ \mathrm{x}\ \mathrm{ascorbic}\ \mathrm{acid}\ \left(\mu \mathrm{g}\right) + 0.04;\ {\mathrm{R}}^2 = 0.99 $$\end{document}Absorbance=0.02xascorbicacidμg+0.04;R2=0.99

Ferric reducing antioxidant power (FRAP) assay

The reducing power of the EOs was determined in accordance with the method reported by Zhao et al. [48] with slight modifications. 1 mL of each EO was mixed with 2.5 mL of 0.2 M phosphate buffer (pH = 6.6) and 2.5 mL of potassium ferricyanide [K₃Fe(CN)₆] (1 %). The mixture was incubated at 50 °C for 20 min. 2.5 mL of 10 % trichloroacetic acid was added and the tubes were centrifuged for 10 min at 3000 rpm. The supernatant (1.25 mL) was mixed with 1.25 mL of distilled water and 0.25 mL of 0.1 % ferric chloride solution. The absorbance was measured at 700 nm in a V-1100 spectrophotometer. Higher absorbance of the reaction mixture indicated greater reducing power. Experiments were carried out in triplicate. The results were expressed in μg ascorbic acid equivalent per mg of EO (μg AAE/mg) using the equation below based on the calibration curve:\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \mathrm{Absorbance} = 0.29\mathrm{x}\ \mathrm{ascorbic}\ \mathrm{acid}\ \left(\mu \mathrm{g}\right) + 0.77;\ {\mathrm{R}}^2 = 0.98 $$\end{document}Absorbance=0.29xascorbicacidμg+0.77;R2=0.98

DPPH Radical scavenging assay (RSA)

The free radical scavenging activity of the EOs was evaluated as described by Sanchez et al. [49] with slight modifications, based on the ability of test compounds to neutralize DPPH radical. The conversion of the stable violet DPPH radical into a yellow reduced form (DPPH/H⁺) is observed simultaneously. The test samples were prepared in methanol and 100 μL of each sample (1.25 to 10 μg/mL for essential oils) was added to 1900 μL of freshly prepared 2, 2-diphenyl-1-picrylhydrazyl (DPPH) solution (50 mg/L) in pure methanol. Ascorbic acid was used as a positive control and 2 mL of 50 mg/L DPPH/methanol solution was used as negative control. The content of each preparation was mixed and incubated at room temperature in a dark cupboard for 10 min. The absorbance was measured at 517 nm in a V-1100 spectrophotometer. All tests were carried out in triplicate. The radical scavenging activity (RSA) was calculated as a percentage of DPPH radical scavenging, using the equation here below:\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \%\ \mathrm{R}\mathrm{S}\mathrm{A} = \left[{\mathrm{Absorbance}}_{\mathrm{blank}}\hbox{--} {\mathrm{Absorbance}}_{\mathrm{sample}}\Big)/{\mathrm{Absorbance}}_{\mathrm{blank}}\right]\ \mathrm{x}100. $$\end{document}%RSA=Absorbanceblank–Absorbancesample)/Absorbanceblankx100.Where A_blank is the absorbance of the control (containing all reagents except the test sample) and A_sample is the absorbance of tested EO solution.

The SC₅₀ values (concentration of sample required to scavenge 50 % of free radicals) were calculated from the regression equations derived by the least-square method and prepared from the different concentrations of both ethanol extracts and essential oils. The higher the SC₅₀ the lower the antioxidant activity of the assayed sample. Ascorbic acid was used as reference.

The antioxidant activity was then calculated and expressed as the antioxidant activity index (AAI): AAI = final concentration of DPPH in the control sample (μg.ml⁻¹)/SC₅₀ (μg.ml⁻¹). Scherer and Godoy’s criteria were considered [50] depending on whether the EOs showed weak antioxidant activity (AAI < 0.5), moderate antioxidant activity (AAI, between 0.5 and 1.0), strong antioxidant activity (AAI, between 1.0 and 2.0) and very strong antioxidant activity when AAI > 2.0.

Determination of total phenolic content (TPC)

The Total Phenolic Contents (TPC) of Allium sativum, Allium cepa, D. gossweileiri and P. brazzeana essential oils (EOs) are presented in Table 2.

From our literature review, there is no scientific investigation on the determination of the TPC of the EOs of D. gossweileri stem barks and P. brazzeana roots. The results indicate that the TPC of the EOs ranged from 342.20 ± 0.99 to 429.33 ± 1.31 μg AAE/mg.

The highest level of phenolics were found in both A. sativum and A. cepa EOs (463.38 ± 1.24 and 429.33 ± 1.31 μg AAE/mg), while the lowest contents were recorded in the EOs of D. gossweileri stem barks and P. brazzeana roots (365.38 ± 0.66 μg AAE/mg and 342.20 ± 0.99 μg AAE/mg). These results do not match with those of Abdel-Salam et al. [60] who showed that TPC of red A. sativum are lower than those of red A. cepa.

Generally, EOs possess significant secondary metabolites from plants, particularly the active lipophilic compounds. This might be due to the fact that phenolic compounds are often extracted in higher amounts by using polar solvents such as water [61]. Phenolic antioxidants are products of secondary metabolism in plants and their antioxidant activity is mainly due to their redox properties and chemical structure, which might play an important role in chelating transition metals and scavenging free radicals [62]. Consequently, the antioxidant activities of plant extracts are often explained by their total phenolic and flavonoid contents. Also, the sterical structures of antioxidants or free radicals are known to play a more important role in their abilities to scavenge different types of free radicals [63].

Ferric reducing antioxidant power (FRAP)

The ferric reducing power in the EOs of A. cepa, A. sativum, D. gossweileiri, P. brazzeana is presented in Table 2. Form our literature review; there is no scientific report on the determination of the ferric reducing power of D. gossweileri and P. brazzeana EOs.

Their ferric reducing capacity ranged from 0.08 ± 0.03 to 2.75 ± 0.02 μg AAE/mg. The highest reducing power was found in A. sativum EO (5.33 ± 0.01 μg AAE/mg), followed by the A. cepa EO (2.75 ± 0.02 μg AAE/mg), D. gossweileiri EO (0.76 ± 0.03 μg AAE/mg) and finally, EO (0.08 ± 0.03 μg AAE/mg) of P. brazzeana. These results are comparable to that of Benkeblia et al. [64] who found that A. cepa and A. sativum showed the highest reducing capacity with 107 and 196 %, respectively. It is worth mentioning that the ferric reducing antioxidant power of D. gossweileiri EO was 10 folds greater than that of P. brazzeana EO. This might be ascribed to the presence of more phenolic compounds [65] in D. gossweileiri than in P. brazzeana. The ability to reduce the ferricyanide complex of Fe³⁺ to the ferrous (Fe²⁺) form might be attributed to the hydrogen donating ability of phenolic compounds [66], which is indicative of the presence of a reducing agent [67]. In addition, the number and position of the hydroxyl groups in phenolic compounds also govern their antioxidant activity [68]. However, Amagase et al. [69] argued that this antioxidant activity could be attributed to the organosulfur compounds.

DPPH Radical scavenging assay (RSA)

Tables 3 and 4 show the radical scavenging activity (RSA) of the EOs of A. cepa, A. sativum, D. gossweileiri and P. brazzeana.

The stable free radical DPPH was used to test the ability of the EOs and ascorbic acid to donate hydrogen ion. The scavenging effects of the EOs on DPPH radical increased as the EO concentration increased. All the EOs were able to reduce the stable free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) to the yellow diphenylpicrylhydrazine with varying degrees of scavenging capacities. Great bleaching action (from purple to yellow) reflected a higher antioxidant activity and thus a lower SC₅₀. The SC₅₀ values increased in the following order: P. brazzeana < A. sativum < D. gossweileri < A. cepa < ascorbic acid. These results indicate that EOs exhibited a significant DPPH radical scavenging activity about 10 folds more active than ascorbic acid. Thus, the EOs were found to be better antioxidants. These results are in agreement with the findings of Agnaniet et al. [56], Ngono [32] and Mnayer et al. [52], but in disaccordance with those found by Ndoye [40], Nyegue et al. [42], Lee et al. [24] and Abdel-Salam et al. [60]. Mnayer et al. [52] showed that Allium sativum EO was a more effective DPPH radical scavenger (SC₅₀ = 7.67 mg/mL) than Allium cepa EO (SC₅₀ = 20.19 mg/mL). On the contrary, Lee et al. [24] and Abdel-Salam et al. [60] reported that Allium cepa EO exhibited higher DPPH radical scavenging activity than A. sativum EO. Moreover, Negue et al. [42] found that P. brazzeana EO exhibited a weak DPPH radical scavenging activity (RSA = 14 % for 1 g/L, SC₅₀ = 1.5 mg/mL and SC₅₀ = 3.23 mg/mL) in comparison with the results of the present study. However, Ngono [32] stated that although the RSA of both the EO of P. brazzeana roots and D. gossweileri stem barks were weak, the RSA of P. brazzeana EO was higher than that of D. gossweileri EO. The major components of the EO (benzylisothiocyanate and benzylcyanide) of these two plants could not be responsible for the antiradical activity, but it might be due to the synergetic action between different components or the contribution of some minor components [41]. It has been shown that benzylisothiocyanate and benzylcyanide tested individually were found to be less active than the whole EO.

Using Scherer and Godoy’s criteria [49], EO showed very strong antioxidant activity (AAI > 2), while ascorbic acid showed a strong antioxidant activity index (1 < AAI < 2).

It is interesting to note that there were weak linear correlations between the total phenolic concentration and the FRAP values or DPPH radical scavenging percentage, with coefficients, R² = 0.22 and 0.21 respectively (Table 4). These results show that most antioxidant compounds might be organosulfur compounds. Indeed, the antioxidant activity of Allium species is attributed partly to its sulfur compounds, which represent the main constituents of these EO [57, 70, 71]. Amagase et al. [69] reported that diallyl polysulphides contributed to the antioxidant properties of the EOs. In addition, the antioxidant activity is also related to other minor bioactive compounds: dietary fibers, microelements (especially selenium) and polyphenols [72] which potentialize antioxidant activity. The different antioxidant activities between these EO might be due to the variability of the chemical composition and sulfides concentration. It might also be attributed to the presence and synergitic action of the different trace compounds.