Background
Allium species, especially Allium vegetables are characterized by their high content in thiosulfinates (TNs). The TNs or alkane (ene) thial-S-oxide are formed by the action of the enzyme alliinase (E.C.4.4.1.4) from their respective S-alk (en) yl cysteine sulfoxides. However, depending on the Allium species and the environmental conditions, the TNs formed are degraded to various polysulfides including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, vinyldithiins, (E)- and (Z)-ajoene [1]. The organosulfur compounds in Allium are water-soluble (S-allylcysteine and S-allylmercaptocysteine) and lipid-soluble compounds (alliin, diallyl sulfide, triallyl sulfide, diallyl disulfide, diallyl polysulfides) [2, 3]. TNs exhibit different degrees of antimicrobial activity and are found to be effective antioxidants in terms of scavenging [1].
In damaged plant cells, notably those of Capparidaceae and Euphorbiaceae families, glucosinolates (GLs) are transformed by the endogenous myrosinase (EC 3.2.1.147) to produce a number of compounds depending on the precursor glucosinolate and the environmental conditions [4]. GLs form a variety of hydrolysis products including isothiocyanates, oxazolidine-2-thiones, nitriles, epithionitriles, and thiocyanates [5]. Certain GLs (sulforaphane and other isothiocyanates) are well known for their diverse biological activities ranging from bactericidal, nematocidal, fungicidal, insecticidal, antioxidant, antimutagenic, antiproliferative and allelophatic properties [6, 7].
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Oxidation is a chemical reaction involving the transfer of an electron from electron rich to electron deficient entity. The electron deficient molecule is termed an oxidizing agent [8]. An antioxidant is a substance capable of preventing or slowing the oxidation of other molecules. Substances which protect biomolecules from free radical-mediated damage both in vitro and in vivo fall under this category. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during irradiation by UV light, X and γ rays, products of metal-catalyzed reactions, which are present as pollutants in the atmosphere, produced by neutrophils and macrophages during inflammation process, by-products of mitochondria-catalyzed electron transport reactions and other mechanisms [9]. ROS at higher concentration (termed oxidative stress) are important mediators of cell structure damages including lipids, membranes, proteins and nucleic acids [10]. The harmful effects of ROS are balanced by the antioxidant action of non-enzymatic antioxidants in addition to antioxidant enzymes [11]. Thus, ROS are important mediators that provoke or sustain inflammatory process and consequently, their neutralization by antioxidants and free radical scavengers can attenuate inflammation [12, 13]. Both inflammation and free radical damage are inter-related aspects that influence each other.
Inflammation is a protective and defense mechanism of the body to a physical, chemical or biological agression. It is important when it does not last for long. Its main purpose is to destroy the injurious agent and/or minimize its harmful effect by limiting its propagation [14]. Inflammatory response occurs in two distinct phases: an acute and a chronic. The acute phase is characterized by local vasodilatation, increased capillary permeability and release of inflammatory mediators like histamine, serotonin and prostaglandins. The Chronic phase is characterized by infiltration of leukocytes and phagocytic cells [15]. This results in tissue degeneration and fibrosis. In the market, most of the available conventional drugs to relieve oxidative stress and to treat inflammatory-related diseases are either steroidal or non-steroidal anti-inflammatory chemicals. Though these drugs are potent enough, long-term administration is required for treatment of chronic diseases. Furthermore, these drugs might lead to severe side effects such as gastric intolerance, bone marrow depression, water and salt retention due to prolonged use [16]. It is in this light that natural remedies with very little side effects, proven efficacy and safety are sought for as substitutes for chemical therapies. Therefore, sulfur-containing plants selected for this study were chosen by virtue of their use in traditional medicine in West Cameroon in the treatment of several inflammatory skin disorders and inflammatory-related diseases.
Allium sativum L. and Allium cepa L. (Liliaceae) commonly known as garlic and onion respectively are bulbous herbs used as food item, spice and medicine in different parts of the world.Allium sativum can also be used in homemade cosmetics. It has been employed for its diverse biological activities including anti-carcinogenic, antiatherosclerotic, antithrombotic, antimicrobial, anti-inflammatory and antioxidant effects [17‐21]. The major beneficial effects of Alliums are ascribed to the high content in organosulfur compounds produced when Allium sativum or Allium cepa tissue is damaged and the odorless precursors are converted by the alliinase enzyme and lachrymatory-factor synthase [22, 23]. The most important sulfur-containing compound in Allium cepa bulbs is the amino acid cysteine and its derivatives. A. cepa also contains high levels of flavonoids mainly quercetin as well as other phenolic compounds [24]. In contrast to Allium cepa, Allium sativum mainly contains allicin derivatives such as ajoene, vinyldithiins, and alkenyl-sulphides such as diallyl disulfide and diallyl trisulfide depending on the type of solvent used for the extraction [22, 25].
Drypetes gossweileri is a dioic plant of the Euphorbiaceae family with an average size of about 30 to 40 m. The color of the stem bark turns from grey-green to yellowish-green and it gives a strong characteristic odor when cut [26]. It has been used in traditional medicine for treating fever, malaria, intestinal worms in Central Africa [27, 28]. Phytochemical studies conducted by Ngouana et al. [29] revealed the presence of metabolites such as alkaloids, phenols, flavonoids, saponines, anthraquinones, tannins, anthocyanins, sterols, lipids and essential oils. Phytochemical analysis of the extracts from previous studies indicated the presence of steroids, triterpenoids, alkaloids, saponins with antimicrobicidal properties [30]. Many research works describe the antibacterial [31, 32], antifungal [29, 33], antioxidant and antiradical [32, 34] effects of D. gossweileri. It is is also used in pest management [35].
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Pentadiplandra brazzeana Baill (Capparidaceae) is a climber found in Western and Central Africa [36], the berries of which are eaten and used as a sweetener of beverages [37]. Root, seed, and leaf extracts of P. brazzeana are known to contain benzyl-, 3-methoxybenzyl-, 4methoxybenzyl-, 3,4-dimethoxybenzyl-, and indole-type glucosinolates [38]; and the essential oil obtained from its roots is mainly constituted of benzylisothiocyanate and benzylcyanide [32, 39‐41]. Its root essential oil (EO) has been proven to possess free-radical scavenging [32, 40, 42], antibacterial [32, 42], antifungal [41, 42] and anti-inflammatory activities [42]. In previous studies, the aqueous extract of P. brazzeana root was shown to possess androgenic activity [43]. Moreover, the crude extract of P. brazzeana root as well as the compounds isolated from it (thiourea and urea) exhibited a moderately strong antiplasmodial activity against two Plasmodium falciparum strains [44].
The antioxidant and anti-inflammatory activities of some Allium species grown in other countries have been analyzed, but no information is available on the in vitro antioxidant activities of red bulb A. cepa and red bulb A. sativum species growing in Cameroon. Antioxidant activities have already been studied using the DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging method and the β-carotene bleaching test [40, 42], but a different method is used here to confirm it. In addition, anti-inflammatory activity has not yet been reported for D. gossweileri stem barks. Moreover, considering that antioxidants and free radical scavengers can also exert an anti-inflammatory effect [13], the essential oil of D. gossweileri stem barks were also assessed for these activities in comparison with that obtained from A. cepa and A. sativum, two plants well-known for their antioxidant and anti-inflammatory activities. Therefore, the purpose of this study was to determine the chemical composition, antioxidant and anti-inflammatory potential of the aforementioned sulfur-containing plants in order to confirm their efficacy in the treatment of inflammatory disorders as claimed in Cameroonian ethnomedicine.
Methods
Chemical reagents and solvents
Folin-Ciocalteu reagent, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and aluminum chloride were purchased from Sigma-Aldrich (Germany). Ascorbic acid and bovine serum albumin were purchased from Sigma-Aldrich (China). All other reagents and solvents were of analytical grade.
Plant materials and extraction procedure
A. sativum red bulbs, A. cepa red bulbs, D. gossweileri stem barks and P. brazzeana roots were purchased in Yaounde (Mokolo and Mfoundi markets) in August 2013. A. sativum red bulbs and A. cepa red bulbs were harvested at Garoua (North Region of Cameroon). D. gossweileri stem barks and P. brazzeana roots were harvested at Hawae and Ngomedzap (Center Region of Cameroon) respectively by the vendors. Identification was done at the National Herbarium of Cameroon by comparison with voucher specimens 25742/SRF/Cam, 44810/HNC, 25749/SRF/Cam and 42918/SRF/Cam for A. cepa, A. sativum, D. gossweileri and P. brazzeana respectively.
EOs were extracted by hydrodistillation using a Clevenger-type apparatus for 5 h, dried over anhydrous sodium sulfate and then stored at 4 °C until bioassay. The extraction yields were calculated as the ratio of the mass of EO to the mass of the starting plant material and expressed as a percentage (w/w).
Chemical analysis of the essential oils
The EOs were analyzed by gas chromatography and gas chromatography coupled to mass spectrometry as described by Agnaniet et al. [45].
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 radical scavenging and antioxidant activity
In order to determine the radical scavenging and antioxidant activities of the EOs, the following assays were applied: total phenolic content determination, FRAP assay and DPPH assay.
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:
$$ \mathrm{Absorbance} = 0.02\ \mathrm{x}\ \mathrm{ascorbic}\ \mathrm{acid}\ \left(\mu \mathrm{g}\right) + 0.04;\ {\mathrm{R}}^2 = 0.99 $$
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:
$$ \mathrm{Absorbance} = 0.29\mathrm{x}\ \mathrm{ascorbic}\ \mathrm{acid}\ \left(\mu \mathrm{g}\right) + 0.77;\ {\mathrm{R}}^2 = 0.98 $$
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:Where Ablank is the absorbance of the control (containing all reagents except the test sample) and Asample is the absorbance of tested EO solution.
$$ \%\ \mathrm{R}\mathrm{S}\mathrm{A} = \left[{\mathrm{Absorbance}}_{\mathrm{blank}}\hbox{--} {\mathrm{Absorbance}}_{\mathrm{sample}}\Big)/{\mathrm{Absorbance}}_{\mathrm{blank}}\right]\ \mathrm{x}100. $$
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 the anti-inflammatory activity: inhibition of albumin denaturation
Anti-denaturation assay was conducted as described by Biswakanth et al. [51] with slight modifications. The reaction mixture consisted of EO samples at different concentrations and 1 % bovine serum albumin (BSA) fraction prepared in saline phosphate buffer (pH = 7.4). The pH of the reaction mixture was adjusted to 6.8 using small amounts of glacial acetic acid. The test tubes were incubated at 72 ° C for 5 min and then cooled for 10 min. The absorbance of these solutions was determined using a spectrophotometer (Mapada V-1100) at a wavelength of 660 nm. The experiment was performed in triplicate. The percentage inhibition of precipitation (denaturation of the protein) was determined on a percentage basis relative to the control using the formula:
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{inhibition}\ \mathrm{denaturation} = 100\ \hbox{--}\ \left[\left({\mathrm{Absorbance}}_{\mathrm{sample}}/{\mathrm{Absorbance}}_{\mathrm{control}}\right)\ \mathrm{x}\ 100\right] $$
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Statistical analysis
All the grouped data were statistically evaluated with GraphPad InStat software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. P values less than 0.05 were considered to be statistically significant. All the results were expressed as mean ± s.d. The regression equations and correlation coefficients were obtained by the least-square method.
Results and discussion
Extraction yields of essential oils
The extraction yields of the EOs are shown in Table 1. The yield ranged from 0.007 % for A. cepa to 0.2 % for A. sativum bulbs. The extraction yield of red bulb A. cepa was similar to that previously found by Mnayer et al. [52] (0.006 to 0.013 %) while the yield of A. sativum bulbs was similar to that obtained by the same author but, higher (0.09 %) than that observed in a previous study by Khadri et al. [53]. However, the yield obtained in this study represents only half of the yield obtained by Lawrence and Lawrence (0.4 %) [54].
Table 1
Relative percentages of constituents and extraction yields of EOs from A. sativum, A. cepa bulbs, D. gossweileri stem barks and P. brazzeana roots
Components | RI | Relative percentage (%) | |||
|---|---|---|---|---|---|
A. cepa
|
A. sativum
|
D. gossweileri
|
P. brazzeana
| ||
2,4-dimethylthiophene | 884 | - | 0.63 | 0.03 | - |
Diallyl sulfide | 888 | 1.38 | 7.10 | 0.11 | - |
2,5-dimethyl thiophene | 908 | 0.82 | - | - | |
Allyl methyl disulfide | 918 | - | - | 0.01 | - |
Methyl propyl disulfide | 922 | - | 1.19 | - | - |
N,N’-Dimethyl thiourea | 951 | - | - | 0.01 | - |
Benzaldehyde | 962 | - | - | 0.28 | - |
dimethyl trisulfide | 964 | - | 0.58 | - | - |
2-Phenyl furan | 978 | - | 0.48 | - | - |
Octyl aldehyde | 990 | 2.26 | - | - | - |
phenylacetaldehyde | 1026 | 2.69 | - | 0.06 | - |
Limonene | 1029 | - | 0.62 | - | - |
2-propenyl propyl disulfide | 1032 | 5.15 | 0.20 | 0.02 | - |
2,5-dimethyl-1,3,4-thiadiazole | 1037 | - | 0.74 | - | - |
Terpinolene | 1059 | - | - | 0.02 | - |
Phenylmethanol | 1081 | - | - | 0.01 | - |
Diallyl disulfide | 1088 | - | 19.74 | - | |
Trans-propenyl propyl disulfide | 1091 | 2.86 | - | - | - |
Linalool | 1101 | - | 0.88 | - | - |
Dipropyl disulfide | 1109 | 2.71 | 0.62 | - | - |
1-propenyl propyl disulfide | 1114 | 3.77 | - | - | - |
3,5-dimethyl-1,2,4-trithiolane | 1118 | 1.81 | - | - | - |
Campholenol | 1124 | - | - | 0.03 | 0.31 |
Allyl methyl trisulfide | 1149 | - | 12.95 | - | - |
Methyl propyl trisulfide | 1153 | 8.14 | - | - | - |
Benzylcyanide | 1157 | 2.52 | - | 35.72 | 0.86 |
3,4-Dihydro-3-vinyl-1,2-dithiin | 1165 | - | 1.37 | - | - |
Dimethyl tetrasulfide | 1224 | 1.32 | 1.59 | 0.02 | - |
2,5-Dimethylthiazole | 1265 | 4.62 | - | - | - |
Diallyl trisulfide | 1319 | 22.17 | 41.62 | - | - |
3-Methoxyoctane | 1327 | - | 0.62 | - | - |
Dipropyl trisulfide | 1334 | 11.11 | - | - | - |
1-propenyl propyl sulfide | 1345 | 1.26 | - | - | - |
Allyl propyl sulfide | 1377 | - | 1.30 | - | - |
Di-1-propenyl sulfide | 1382 | 1.71 | 2.08 | - | - |
Benzylisothiocyanate | 1393 | - | - | 63,19 | 97.63 |
p-methoxybenzylcyanide | 1395 | - | 0.85 | - | 1,2 |
Β-caryophyllene | 1398 | - | 0.23 | - | - |
2-methyl-3-isothiozolone | 1435 | 2.23 | - | -- | - |
Germacrene D | 1484 | - | - | 0.02 | - |
γ-cadinene | 1506 | - | 0.39 | - | - |
2-methyl-3,4-dithiaheptane | 1527 | 9.88 | - | - | |
Β-sesquiphellandrene | 1550 | - | - | 0.05 | - |
Diallyl tetrasulfide | 1558 | - | 4.22 | - | - |
p-methoxybenzylisothiocyanate | 1575 | 3.55 | - | - | - |
Dipropyl tetrasulfide | 1649 | 8.07 | - | - | - |
Benzyl sulfide | 1856 | - | - | 0.06 | - |
Methyl linolenate | 2013 | - | - | 0.06 | - |
Total identified (%) | 98.77 | 100 | 99.70 | 100 | |
EOs extraction yields (%) | 0.007 | 0.2 | 0.04 | 0.02 | |
As regards D. gossweileri, its stem bark gave a yield of 0.04 %. This yield was different from those obtained by Eyele et al. (0.2 %) [55]; Agnaniet et al. (0.19 %) [56]; Ngono (0.007 and 0.29 %) and [32] Mbouma (0.023 to 0.088 %) [33].
The roots of P. brazzeana gave a yield of 0.02 % which was similar to those found in previous studies by Koudou et al. [39], Ndoye [40], Ngono [32] and Tchinang et al. [41]. However, this yield is lower than those found by Koudou et al. [39] and Nyegue et al. [42], respectively 0.35, 0.2 and 0.13 %.
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Chemical composition
The results of the chemical analysis are presented in Table 1.
GC-MS analysis of Allium sativum EO revealed 22 components representing 100 % of the total EO. The major components were diallyl trisulfide (41.62 %), diallyl disulfide (19.74 %), allyl methyl trisulfide (12.95 %), diallyl sulfide (7.1 %) and diallyl tetrasulfide (4.22 %). This chemical profile was closed to that obtained by Mnayer et al. [52], but with slight difference in that diallyl disulfide (37.90 %) was predominant compared to diallyl trisulfide (28.06 %), while the allyl methyl trisulfide (7.26 %) represented almost half of its content in the previous study [52]. Furthermore, studies conducted by Khadri et al. [53] showed that allyl methyl trisulfide (34.61 %) and diallyl disulfide (31.65 %) were rather the major constituents of the EO of Allium sativum. However, it is worth noting that, in the two previous studies [52, 53], allyl methyl disulfide was present in the chemical composition of the Allium sativum essential oil, contrarily to this study where it is pratically absent. Nonetheless, different studies on the chemical composition of A. sativum essential oil showed that diallyl disulfide and diallyl trisulfide are the two major compounds of the EO of A. sativum [52, 57, 58].
Twenty one constituents representing 98.77 % of the total EO were identified in the Allium cepa EO. The main components were diallyl trisulfide (22.17 %), dipropyl trisulfide (11.11 %), 2-methyl-3, 4-dithiaheptane (9.88 %), methyl propyl trisulfide (8.14 %), dipropyl tetrasulfide (8.07 %) and 2-propenyl propyl disulfide (5.15 %). The chemical composition of A. cepa used in this study differed greatly from previous reports in which dipropyl disulfide was reported to be the major compound present [52, 57].
Analysis of the chemical compositions of the EO of D. gossweileri stem barks revealed 17 compounds representing 99.70 % of the total EO. Benzylisothiocyanate was the main component, representing 63.19 % followed by benzylcyanide (35.72 %). These results are in agreement with those of Eyele et al. [55], Ngono [32] and Mbouma [33] who documented that benzylisothiocyanate (56.5 to 95.6 %) and benzylcyanide (4.3 to 32.3 %) were also the major compounds of D. gossweileri EO. However, Agnaniet et al., [56], reported that benzylcyanide (73.7 %) was the main compound in D. gossweileri stem bark EO from Gabon, instead of benzylisothiocyanate (5.2 %).
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As for the EO of P. brazzeana roots, four components were identified, representing 100 % of the total oil. The main compound found was benzylisothiocyanate (97.63 %). Benzylisothiocyanate has been reported by Ndoye [40], Nyegue et al. [41], Ngono [32] and Tchinang et al. [41] to be the major compound present in P. brazzeana EO.
It was observed that the chemical composition of the aforementioned essential oils differs greatly among genus, and also among the same species. This is possibly due to analytical techniques, chemotypes, and differences in cultivars of the plants, culture climate and other growth conditions which might affect the chemical composition [38, 59].
Determination of radical scavenging and antioxidant activity
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.
Table 2
Total phenolic, and ferric reducing power contents of essential oils of A. cepa, A. sativum, D. gossweileiri, P. brazzeana
TPC (μg AAE/mg) | FRAP (μg AAE/mg) | |
|---|---|---|
Essential oil | Essential oil | |
A. cepa
| 429.33 ± 1.31 b | 2.75 ± 0.02 b |
A. sativum
| 463.38 ± 1.24 a | 5.33 ± 0.01 a |
D. gossweileri
| 365.38 ± 0.66 c | 0.76 ± 0.03 c |
P. brazzeana
| 342.20 ± 0.99 d | 0.08 ± 0.03d |
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.
Table 3
DPPH radical scavenging activity and AAI of A. cepa, A. sativum, D. gossweileri, P. brazzeana essential oils and ascorbic acid
Regression curve’s equations | Coefficient of determination : R2 | SC50(μg/ml) | AAI | |
|---|---|---|---|---|
A. cepa
| y = 192.3 × + 11.96 | 0.927 | 0.20 | 12.626 |
A. sativum
| y = 191.6 × + 12.73 | 0.919 | 0.19 | 12.886 |
D. gossweileri
| y = 194.0 × + 11.10 | 0.931 | 0.20 | 12.821 |
P. brazzeana
| y = 190.2 × + 14.15 | 0.904 | 0.19 | 13.298 |
Ascorbic acid | y = 19.12 × + 12.19 | 0.934 | 1.98 | 1.262 |
Table 4
Relationship between TPC and FRAP, TPC and RSA
Relationship between TPC and FRAP, FRAP = f (TPC) | Relationship between TPC and RSA, SC50 = f (1/TPC) | |||
|---|---|---|---|---|
Regression curve Equation | Coefficient of determination : R2 | Regression curve equation | Coefficient of determination : R2 | |
Essential oil | y = 0.016× - 0.434 | 0.22 | y = 0.782 × + 0.189 | 0.21 |
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.
Determination of the anti-inflammatory activity
Anti-denaturation of bovine serum albumin method was used to evaluate the anti inflammatory property of the EOs of A. cepa, A. sativum, D. gossweileiri and P. brazzeana. The results are summarized in Table 5. All the EOs protected the BSA against heat induced denaturation. The percentage of BSA protection against heat increased with increasing concentration. The present findings show a concentration dependent inhibition of protein (albumin) denaturation by EOs from 3.125 to 125 μg/mL. Sodium diclofenac at the same concentration range was used as the reference drug and it also exhibited a concentration dependent inhibition of protein denaturation. Indeed, the effect of sodium diclofenac against heat denaturation of BSA was found to be about 2 folds lower than that of A. cepa and P. brazzeana EO. P. brazzeana EO was found to be 2 folds more active than D. gossweileri EO. The anti-inflammatory activity of P. brazzeana EO was to a lesser extent comparable to that of A. cepa. These observations were confirmed by comparing their IC50 values.
Table 5
Effect of essential oil on BSA denaturation inhibitory activity, percentage inhibition compared to sodium diclofenac
A. cepa
|
A. sativum
|
D. gossweileri
|
P. brazzeana
| Diclofenac sodium | |
|---|---|---|---|---|---|
Concentration (μg/ml) | Inhibition percentage (%) | ||||
3.125 | 7.33 ± 1.15b | 4.00 ± 0.00a | 4.00 ± 0.00a | 7.56 ± 2.00b | 7.33 ± 0.00b |
6.25 | 9.33 ± 1.15a | 6.00 ± 2.00a | 8.67 ± 2.31a | 16.477 ± 1.15b | 9.33 ± 1.15a |
12.5 | 14.00 ± 2.00a | 14.00 ± 2.00a | 13.33 ± 3.05a | 26.94 ± 2.00b | 14.00 ± 0.00a |
25 | 33.33 ± 1.15c | 22.66 ± 2.30b | 19.33 ± 1.15a | 38.95 ± 1.15d | 18.00 ± 0.00b |
50 | 54.67 ± 3.05c | 38.00 ± 2.00b | 28.00 ± 2.00a | 52.52 ± 1.15c | 24.00 ± 0.00a |
75 | 82 ± 1.04e | 57 ± 1.08c | 42 ± 0.11b | 72.87 ± 0.98d | 36 ± 2.00a |
100 | - | 76 ± 0.91c | 56 ± 0.76b | - | 48 ± 1.00a |
125 | - | 95 ± 1.02c | 70 ± 2.14b | - | 60 ± 1.73a |
Regression curve equations | y = 1.070x + 2.579 | y = 0.753x – 3.637 | y = 0.525x + 3.642 | y = 0.907x + 8.587 | y = 0.430x + 5.090 |
R2 = 0.991 | R2 = 0.976 | R2 = 0.991 | R2 = 0.973 | R2 = 0.982 | |
IC50 value (μg/mL) | 44.31 | 61.577 | 88.30 | 45.66 | 104.44 |
To the best of our knowledge, no study on the in vitro anti-inflammatory properties of the EOs of D. gossweileri has been published. However, [42] reported that the anti-inflammatory activity of the EO of P. brazzeana root is about 100 folds less active (35 ± 5 μg/mL) than that of nordihydroguaiaretic acid (0.23 ± 0.02 μg/mL) against the soybean 5-lipoxygenase enzyme. The IC50 of P. brazzeana EO found in this study is slightly higher (45.66 μg/mL) than that reported by Nyegue et al. [42] using a different method. This means that the EO sample used by Nyegue et al. [42] was slightly more active than the present EO sample. This could be explained by the fact that the EO sample used by the aforementioned author contained more compounds (11) than the EO sample used in this investigation, which contained only 4 components (Table 1). Concerning the antiradical, anti-inflammatory activities of the EO of P. brazzeana and D. gossweileri, these activities might be due to the synergetic action of several components or the contribution of trace components. Nyegue et al. [42] reported that the two main components (benzyisothiocyanate and benzylcyanide) tested individually were found to be less active than the whole essential oil.
Conclusion
In order to valorize some organosulfur plants from Cameroon as dietary, cosmetic and pharmaceutical additives, the chemical composition, in vitro antioxidant and anti-inflammatory activities of the essential oils of A. sativum, A. cepa, D. gossweileri and P.brazzeana were investigated in this study. This paper reveals that the essential oils of D. gossweileri stem barks and P. brazzeana roots as well as those of the red bulbs of A.sativum and A. cepa are all effective antioxidant and anti-inflammatory compounds. The findings of this study support the popular use of these plants against inflammatory skin disorders and inflammatory-related diseases in West-Cameroon, though additional studies are necessary to test other enzymatic systems that trigger the development of the inflammatory process and also address their mechanism of action.
Availability of data and materials
The datasets supporting the conclusions of this article are presented in this main paper. Plant materials used in this study have been identified at the Cameroon National Herbarium where voucher specimens are deposited.
Consent for publication
Not applicable in this section.
Ethic approval and consent to participate
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Acknowledgments
We are profoundly grateful to the University of Yaounde I (Cameroon), the Institute of Medical Research and Medicinal Plants Studies (Cameroon), and the Institute of Biomolecules Max Mousseron (France) for their technical support.
Fundings
The authors declare that they have received no funding for the research reported.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NFFMC, TKTF, and EFX designed the study and were involved in the drafting and correction of the manuscript. NFFMC also coordinated the study. TKTF carried out the plant selection and collection, extraction of essential oils, in vitro antioxidant and anti-inflammatory assays, analysis and interpretation of data. NM performed GC, GC/MS analyses of the essential oils and participated in the correction of the manuscript. AJP and YGAJ carried out radical scavenging assays. TTA also participated in correcting the manuscript. All the authors read the manuscript, critically revised it for important intellectual content and approved the final version of the manuscript.