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255
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS)
ISSN (Print) 2313-4410, ISSN (Online) 2313-4402
© Global Society of Scientific Research and Researchers
http://asrjetsjournal.org/
Antioxidant and Antifungal Activities of Cocoa Butter
(Theobroma cacao), Essential Oil of Syzygium aromaticum
and a Combination of Both Extracts against Three
Dermatophytes
Patience Mekemzeu Fankema, Sylvie Nguikwie Kwangab, Modeste Lambert
Samezac, François Tchoumbougnangd, Raymond Tchabonge, Leopold
Tatsadjieu Ngounéf, Pierre Michel Jazet Dongmog*
a,b,c,e,gDepartment of Biochemistry, Faculty of science, University of Douala, P.O. Box 24157, Douala,
Cameroon
dFisheries and Halieutic Science Institute, University of Douala, P.O. Box 7236, Douala, Cameroon
fDepartment of Food Engineering and Quality Control, University Institute of Technology, University of
Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon
gEmail: mjazet@yahoo.com
Abstract
To contribute in the research of better drugs against dermatophytosis, we evaluated the antioxidant and
antidermatophytic activities of cocoa butter, cloves essential oil, and a mixture of both extracts. The cocoa butter
was obtained by boiling the cocoa paste. The essential oil extracted by hydrodistillation was chemically
analysed by gas chromatography and gas chromatography coupled with mass spectrometry. The antioxidant
activity was determined using the DPPH scavenging method, and the antidermatophytic activity was evaluated
using the agar dilution method. The essential oil, majoritary constituated by eugenol (87.62%), β-caryophyllene
(5.88%), and β-bisabolene (4.41%), had an antiradical power (4.22 x 10-2) higher than that of BHT (4.00 x 10-3),
like the cocoa butter and essential oil mixture (6.06 x 10-3). The essential oil was more active than the
griseofulvin: it was fungicidal at 400 ppm against Trichophyton rubrum, and at 900 ppm against Microsporum
gypseumand Trichophyton tonsurans.
------------------------------------------------------------------------
* Corresponding author.
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
256
The cocoa butter activity was low, but the mixture with the essential oil had an important activity with inhibitory
percentages of 78.69 %, 88.27 %, 91.20% against T. rubrum (at 400 ppm), T. tonsurans(at 900 ppm)and M.
gypseum (at 900 ppm)respectively. Cloves essential oil and the mixture with cocoa butter can be used to
formulate new drugs against dermatophytes.
Key words: Antioxidant; antidermatophytic; cocoa butter; cloves; essential oil.
1. Introduction
Infectious diseases, especially dermatophytosis, are a major threat to human health [1,2,3]. The incidence and
mortality from these infections are influenced by the characteristics of the population at risk, the availability of
medical care, distribution of species responsible and prevalence of antimicrobial resistance [4,5]. For centuries,
the treatment of diseases was done with different formulations and extracts of plants for their medicinal
properties. Thus, fungal infections have been successfully managed using medicinal plants [6,7,8,9]. Moreover,
infectious diseases are treated by modern medicines such as antibiotics and antifungals. However, these drugs
are not always accessible to poor communities in developing countries [10]. In addition, most of these drugs
have low antimicrobial spectrum, a long duration of treatment, side effects, and their wide spread excessive use
leads to resistance of microorganisms. For example, ketoconazole previously used in the treatment of some
dermatophytes causing ringworm is now rarely used in case of severe systemic fungal infection, because of its
hepatotoxic effects [11]. The upsurge of the resistance of fungal strains is one of the barriers to the successful
treatment of microbial diseases. It increased the universal demand for herbal medicine that is now an integral
part of primary care in most countries [12]. Dermatophytic infections are associated with many oxidative
reactions that may be responsible of the production of free radicals, which contribute to the increase of body
lesions. Meanwhile, synthetic antioxidants usually used have side effects for the organism (the Butylated
hydroxytoluene is a carcinogen molecule). Therefore, it is essential for man to find an alternative to these
treatments. Plants have bioactive metabolites (alkaloids, saponins, flavonoids, tannins and phenolic
compounds), which are responsible for their therapeutic potential [13]. These are a good source of anti-infective
agents, and antioxidants and are a natural reservoir of new biologically active molecules to be discovered.
Theobroma cacao known as cocoa, is a plant whose products are widely used: the chocolate for its organoleptic,
nutritional and stimulant qualities and cocoa butter, used in pharmacy and cosmetics primarily for its
moisturizing, nourishing and antiseptic properties on the skin and hair [14]. Besides this plant, Syzygium
aromaticum, commonly called clove, is an aromatic plant used in traditional medicine because of its many
medicinal properties. It has certainly been the subject of several scientific works [15,16,17,18,19], but few of
these works have addressed antidermatophytic properties of the plant essential oil.
2. Material and methods
2.1. Vegetable material
Cocoa capsules and dried cloves buds (Syzygium aromaticum)were collected at Penja, on June and July 2016
respectively.
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
257
These plants were identified at the National Herbarium of Cameroon, in Yaounde, with references number of
cocoa and clove trees being 35970TW/CAM and 2008SKR/CAM.
2.2. Fungal material
Two referenced dermatophytes: Microsporum gypseum(E1420), Trichophyton rubrum (BDO23) and one isolate:
Trichophyton tonsurans were studied. They were obtained from the Laboratory of Microbiology and
Antimicrobial Substances of the University of Dschang.
2.3. Hydrodistillation
The best way to extract clove essential oil was the hydrodistillation [16,20,21].
250g of clove buds were macerated into a liter of water for 8 hours and then introduced in a Clevenger apparatus
[22] for hydrodistillation for 8 hours.
Two phases were obtained: an organic phase, which was the essential oil, and a water-soluble phase, constituted
by water and essential oil. The essential oil was separated from water in the second phase using hexane, by a
liquid-liquid extraction [16]. The hexane was evaporated through a rotary evaporator at 80°C, and the purified
essential oil was added to the previous organic phase. The essential oil obtained was dried using anhydrous
sodium sulphate and kept it in a refrigerator at 4°C. The extraction yield was calculated by the following
formula:
2.4. Extraction of cocoa butter
The cocoa capsules were opened; all the beans were removed and fermented for 5 days, then dried in a sterilizer
at 50°C for 12 hours. The dried beans were roasted, and their husk removed and mashed to obtain a paste. 2500g
of cocoa paste were cooked with 5L of water in order to obtain the cocoa oil as described by [23]. The oil was
filtered, treated three times with distillated water to keep out remains, and completely dehydrated with
anhydrous sodium sulphate. The storage was in a dark and dry place. At room temperature, the oil solidified and
became cocoa butter.
The extraction yield was calculated by the following formula:
2.5. Chemical composition analysis of the essential oil
Yield (%) =
()
()
× 100
Yield (%) =
()
()
× 100
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
258
The essential oil was analyzed by gas chromatography and gas chromatography coupled with mass spectrometry
[24].
2.5.1.Gas chromatography (GC)
The oil was analyzed on a chromatographic Variant type CP 3380, equipped with a flame ionization detector
and a fused silica capillary column (30 m x 0.25 mm) coated with a DB5 film thickness of 0.25μm. The
operating conditions were as follows; the temperature of the injector and detector was programmed at 200℃; the
temperature of the oven from 50℃ to 200℃at 5℃/min; with nitrogen gas vector of 1mL/min. The linear
retention indices of the components were determined relatively by the retention time of a series of n-alkanes and
the percentage compositions were obtained from electronic integration measurements without taking into
account relative response factors.
2.5.2. Gas Chromatography/Mass Spectrometry (GC/SM)
GC/MS analysis was performed using a Hewlett-Packard apparatus equipped with an HP1 fused silica column
(30m x 0.25mm, film thickness 0.25μm) and interfaced with a quadrupole detector (GC- quadrupole MS system,
model 5970). Column temperature was programmed from 70 -200°C at 10°C/min; injector temperature was
200°C. Helium was used as a carrier gas at a flow rate of 0.6 ml/min. The mass spectrometer was operated at
70eV.
2.6. Identification of the essential oil components
The qualitative analysis was made possible by calculating the Kovats index (KI) of each element, based on their
retention time and the retention time of a set of alkanes used as a standard. Thus, the identification was assigned
by the comparison of the KI with those given by literature, and with the stored laboratory mass spectral library
[24].
2.7. Evaluation of the antiradical activity
The antiradical activity of the cocoa butter, the essential oil and the mixture of both substances were evaluated
by measuring the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging power at 517 nm [25]. Butylated
hydroxytoluene (BHT) was used as the reference antioxidant. 20 mg of DPPH and 50 mg of BHT were
dissolved in methanol, in order to obtain a 40mg/L and a 1g/L solution, respectively. Hexane was use to mix
essential oil and cocoa butter. The negative control solution was represented by the DPPH methanolic solution.
The final volume of solution in all the tubes was 2200 mL (Table 1). The optical densities were read at 517 nm
using a spectrophotometer [26], after 2 hours at room temperature. The following parameters were determined:
- The SC50 (50% Scavenging Concentration) was determined graphically: it is the concentration required for
50% DPPH reduction [27].
- The EC50 (Effective Concentration), in grams of extract per mol of DPPH.
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259
Table 1: Evaluation of the antiradical activity of BHT, cloves essential oil (EO), cocoa butter (CB), and the
mixture (EO + CB)
Tested substances
Essential oil (EO)
Cocoa butter (CB)
Mixture (EO + CB)
Concentration of the
solution in standard tubes
(mg/L)
25, 50, 100, 200, 400
1000, 2000, 3000, 4000, 5000
- EO : 25, 50, 100, 200,
400
- CB : 5000
Volume of tested substance
introduced in standard tubes
(μL)
100
200
- - EO : 100
- CB : 100
Volume of DPPH per tube
(μL)
2000
2000
2000
Volume of BHT in the
reference tube (μL)
100
200
200
Reference tube: tube containing only BHT and DPPH.
- The AP (Antiradical Power): expresses the antioxidant as the highest and most effective.
2.8. Evaluation of the antifungal activity
The antifungal activity was performed using three dermatophytes: Microsporum gypseum, Trichophyton rubrum
and Trichophyton tonsurans, following the agar diffusion method [28].
Samples were prepared with the Sabouraud Dextrose Agar supplemented with chloramphenicol (SDA+
chloramphenicol) medium, the EO, and the cocoa butter, in a scale of several concentrations. The Dimethyl
Sulfoxyde (DMSO) was used as the surface acting in essential oil samples, and the Tween 80 in cocoa butter
samples.
Griseofulvin was used as the reference antifungal, and the negative control was represented by the DMSO in
SDA medium. Each sample was prepared three times.
2.8.1. Antifungal activity of the essential oil of Syzygium aromaticumand cocoa butter
CE
50
(g of extract/ mol of DPPH) = SC
50
/ C
DPPH
,(C
DPPH
: Molar Concentration of DPPH in each
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260
A solution was firstly prepared with the essential oil and the DMSO in 1/9 (v/v) proportion; then, different
volumes of the solution were added in the liquid SDA, in order to obtain different concentrated solutions with a
final volume of 10mL.
The initial concentration scales were: 500, 750, 1000, and 2000 ppm; these concentrations were successively
split to determine the accurate Minimal Inhibitory Concentration (MIC) of the essential oil.
The cocoa butter was treated using the same protocol, but the first solution was prepared with CB and tween 80,
at 2.5% of tween, and the initial concentration scales were: 500, 750, 1000, 2000 and 4000 ppm.
All samples were poured in 90 mm Petri dishes, and a mycelium explant of 2 mm was deposited in the middle
of each dish. The dishes were sealed and incubated at room temperature in their inverted position.
2.8.2. Antifungal activity of the essential oil and cocoa butter formulation
The antidermatophytic activity of the essential oil and cocoa butter formulation was carried out during 18 days
of incubation, at 25°C. The two extracts were dissolved in SDA medium at the same concentration (the chosen
concentration corresponded to the Minimal Fungicidal Concentration of the essential oil depending on the
germ).
2.8.3. Evaluation of mycelium growth inhibition
The mycelial growth was followed by measuring every two days, and at the same hour, two perpendicular
diameters on each Petri dish on the explant level.
The comparison between the mycelial growth in dishes containing antifungal substances and the control dishes
helped us to evaluate the radial inhibition of the mycelium, and calculated the inhibition percentage by the
following formula:
- Dc (cm) = mycelium growth diameter in the control,
- De (cm) = mycelium growth diameter in the dish which contained an antifungal.
The Minimal Inhibition Concentration (MIC) was determined.
2.8.4. Nature of inhibition
The fungicidal or fungistatic activity was evaluated by the transfer of explants from the dishes containing the
medium combined with EO at the MIC, into a sterile medium.
=−
× 100
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261
The substance was fungistatic when the dermatophyte had grown in the new medium during the incubation
period, and it was fungicidal when no mycelium growth was observed.
2.9. Statistical data analysis
The numerical data were introduced in EXCEL (Microsoft, 2010). The data was analyzed with the Stat view
software version 5.0 (SAS Institute Inc., USA).
A one-factor Analysis of Variance (ANOVA) and the non-parametric test of Kruskal-Wallis were used, and the
significance level was below the probability of 0.05.
3. Results
3.1. Essential oil and cocoa butter characteristics
The extracted essential oil and cocoa butter showed different characteristics (Table 2).
Table 2: Essential oil and cocoa butter characteristics
Vegetable material(g)
Product (g)
Yield (%)
Color
Essential oil
1000
96.6
9.66
Pale yellow
cocoa butter
2500
400
16
Yellow
3.2. Chemical composition of the essential oil
The essential oil of S. aromaticum of Penja in Cameroon contained 28 constituents (Table 3), mainly
oxygenated monoterpenes (89.06%) and hydrocarbonated sesquiterpenes (10.86%). Three main constituents
were present: eugenol (87.62 %), β-caryophyllene (5.88 %) and β-bisabolene (4.41 %).
3.3. Antiradical Potential of BHT and Syzygium aromaticum essential oil
The absorbance values read on the spectrophotometer at 517 nm were used to calculate the scavenging
percentages of the radical DPPH by the EO, which were graphically used to determine the EO Scavenging
Concentration 50 which was equal to 2.26 x 10-3 g/L (figure 1).
3.4. Antiradical potential of cocoa butter
The maximal scavenging percentage of cocoa butter was lower than 10% (9.57 ± 1.16%) at 0.73 g/L
concentration (Table 4).
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
262
Table 3: Chemical composition of Syzygium aromaticum essential oil.
Index
KI
Components
Quantity (%)
Monoterpenes
89.10
Hydrocarbonatedmonoterpenes
0.04
1
1019
P- cymene
0.01
2
1087
terpinolene
0.03
OxygenatedMonoterpenes
8.06
3
1027
1,8- cineole
0.03
4
1079
fenchone
0.03
5
1104
camphenol
0.03
7
1212
carveol
0.01
8
1237
geraniol
0.35
9
1353
eugenol
87.62
10
1367
dihydro-eugenol
0.21
11
1445
isoeugenol
0.67
12
1521
eugenylacetate
0.05
Sesquiterpenes
10.87
Hydrocarbonatedsesquiterpenes
10.6
13
1338
δ-elemene
0.09
14
1412
β-caryophyllene
5.88
15
1463
epi-( E)- Caryophyllene
0.05
16
1465
γ-gurjunene
0.02
17
1483
germacrene-D
0.02
18
1489
β-selinene
0.08
19
1511
β-bisabolene
4.41
20
1514
∆-cadinene
0.05
Oxygenatedsesquiterpenes
0.27
21
1545
Elemol
0.02
22
1576
spathulenol
0.14
23
1602
Guaiol
0.02
24
1618
cubenol
0.04
25
1627
γ-eudesmol
0.01
26
1674
β-bisabolol
0.04
Linear components
0.03
27
844
3(E)-hexenol
0.01
28
884
4-heptanol
0.02
KI :Kovats Index
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
263
Figure 1: Variation of scavenging percentage in function of the EO concentration
The SC50 of BHT was graphically determined to be 2.27 x 10-2 g/L.
Table 4: Scavenging percentages of the cocoa butter
CB concentration (g/L)
0
0.045
0.09
0.18
0.36
0.73
% de piégeage
± SD
0.00
0.95
±
0.26a
5.92
±
0.29b
6.37
±
0.18b
6.98
±
0.00c
9.57
±
1.16d
SD: Standard Deviation; a, b, c, and d show the significant difference between scavenging percentages at
different concentrations of CB: two concentrations have the same letter if there is no significant difference
between scavenging percentage values.
3.5. Antiradical potential of the essential oil and cocoa butter mixture
Figure 2 shows the variation of the scavenging percentages of DPPH according to the essential oil and cocoa
butter mixture. This plot allowed the determination of the SC50 of the (CB+EO) mixture, whose value was 15 x
103 g/L.
Figure 2: Variation of scavenging percentage of DPPH with respect to EO and CB concentration.
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 5 10 15 20 25
Scavenging percentage
0
10
20
30
40
50
60
0 5 10 15 20
Scavenging percentage
of the DPPH
SC 50 = 15.10-3 g/L
(CB and EO) Concentration (10-3g/L)
EO Concentration (10-3 g/L)
50
SC
50
= 2.26x10-3 g/L
Scavenging
percentage
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
264
The 50% Efficacy Concentration and the Antiradical Power of each studied substance were calculated using the
SC50 corresponding values (Table 5).
Table 5: SC50, EC50 and AP of BHT, EO, CB and (CB+EO) mixture
Tested substance
SC
50
(g/L)
EC
50
(g of EO /mol of DPPH)
AP
S. aromaticumessential oil
2.26 x 10-3
23.70
4.22 x 10-2
Cocoa butter
ID
ID
ID
(CB+EO) mixture
15. 10-3
165
6.06x10-3
BHT
2.27x10-2
249.70
4.10-3
ID= Indeterminate
The comparison of the SC50, CE50 and AC values by the Kruskal-Wallis test made with the Statview software
version 5.0, showed that they each had a significant difference (H= 8.000 ; p= 0.0460).
3.6. Antidermatophytic activity of the esssential oil
M. gypseum and T. Tonsurans growth was completely inhibited from 1000 ppm, while T. rubrum growth was
completely inhibited at all the chosen concentrations (500, 750, 1000 and 2000 ppm). Based on these results,
intermediate concentrations were defined to determine the accurate MIC value against each dermatophyte.
The data statistical analysis revealed that:
The inhibition percentages of the EO at various concentrations were significantly different;
T. rubrum growth was not influenced by the incubation duration;
The incubation duration significantly influenced T. tonsurans and M. gypseum until the 18th and 20th
day of incubation respectively.
Figure 3 illustrates the variation of the mycelium growth inhibition percentage depending on the EO
concentration, after 18 days of incubation.
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
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Figure 3: Variation of the mycelial growth inhibition percentage depending on the EO concentration, after 18
days of incubation
On the 18th incubation day, T. rubrum was totally inhibited (100% of inhibition percentage) at 400 ppm and
beyond, while the case was observed for T. tonsurans and M. gypseum from 825 and 900 ppm respectively.
There was no growth of T. rubrum during the incubation time in Petri dishes which contained essential oil at
400 ppm. Up to the last incubation day, no growth of T. tonsurans was observed in Petri dishes which contained
essential oil at 825 ppm. No grow was observed with M. gypseum in Petri dishes where the essential oil
concentration was equal to 900 ppm.
3.7. MIC and MFC Determination
The MIC values of Syzygium aromaticum essential oil from Cameroon were 400, 825 and 900 ppm, respectively
against T. rubrum, T. tonsurans and M. gypseum.
Explants were taken from Petri dishes containing essential oil at the MIC, put in new dishes containing only
sterile medium, and the whole was incubated at most during 18, 20 and 26 days for T. tonsurans, M. gypseum,
and T. rubrum respectively. Following that, conclusions about the antidermatophytic activity of the cloves
essential oil were that:
Syzygium aromaticum essential oil was fungicidal against M. gypseum and T. rubrum respectively at
900 and 400 ppm;
Syzygium aromaticum essential oil was fungistatic against T. tonsurans at 825 ppm (the explant growth
was recovered after two incubation days in the new medium). But the MCF was found at 900 ppm
concentration.
The comparison of those MIC and MFC values by the Kruskal-Wallis test confirmed their significativity at a
level of 0.05%.
3.8. Antidermatophytic activity of cocoa butter
0
20
40
60
80
100
120
200 300 400 500 750 825 900 1000
Inhibition percentage of the
dermatophytes mycelial
growth
EO concentration (ppm)
T. rubrum
T. tonsurans
M. gypseum
a
b
c
d
e
f
g
h
i
j
k
l
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
266
The incubation duration of each germ in the medium containing cocoa butter was the same as in the medium
with the EO, and mycelial growth was as shown in figure 4.
Figure 4: Inhibition percentages of the dermatophytes growth depending on CB concentration, after 18
incubation days.
The maximal inhibition of cocoa butter against T. rubrum was observed at 500 ppm (18.8%), followed by the
dose of 4000 ppm (14.14%). On the other hand, its maximal inhibition against M. gypseumand T. tonsurans,
(7.72 and 9.28% respectively) was reached with the dose of 4000 ppm.
3.9. Antidermatophytic activity of essential oil and cocoa butter formulation
To formulate the mixture, the essential oil and cocoa butter were introduced in the growth medium at the same
concentration, which corresponded to the MFC of the essential oil against the respective dermatophyte. With
respect to T. rubrum, the extracts were introduced in the medium at 400 ppm each. But for T. tonsurans and M.
gypseum, they were mixed at 900 ppm each. Although the essential oil and cocoa butter formulation did not
completely hinder the growth of the three dermatophytes, its inhibitory action was very important. In effect, the
mixture had a depressive action on the dermatophytes, because it hindered their growth at least for 10 days for
M. gypseum, and 12 days for T. tonsurans and T. rubrum (figure 5).
Figure 5: Variation of the inhibition percentage of the dermatophytes growth, depending on the concentration
of (EO+CB) mixture during 18 days of incubation.
0
5
10
15
20
25
500 ppm 750 ppm 1000 ppm 2000 ppm 4000 ppm
Inhibition percentage of
the dermatophytes
mycelial growth
CB concentration
T. rubrum
T. tonsurans
M. gypseum
0
20
40
60
80
100
120
10 12 14 16 18
Inhibition percentage
of the dermatophytes
mycelial growth
Incubation days
T. rubrum
T. tonsurans
M. gypseum
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
267
The inhibition of T. rubrum by the (EO+CB) mixture at 400 ppm is higher than 70% after 18 days of incubation
(78.69 ± 1.64%). Furthermore, at 900 ppm and after 18 days of incubation, the mixture induced inhibition of
91.20 ± 7.34% and 88.27 ± 11.43% against M. gypseum and T. tonsurans respectively.
3.10. Antidermatophytic activity of the griseofulvin
The inhibition percentages of the three dermatophytes mycelial growth by the griseofulvin (antifungal reference)
after 18 days of incubation are presented on figure 6.
Figure 6: Inhibition Percentages of the three dermatophytes mycelial growth depending on the concentration of
the griseofulvin after 18 days of incubation.
The inhibition of each dermatophyte’s mycelial growth by the griseofulvin was not total since 100% inhibition
was not achieved at any dose level tested (500, 1000, 2000, 4000 ppm).
Griseofulvin was more active against T. rubrum, with an inhibition percentage of 47.65 ± 3.89% after 18 days of
incubation. Then comes T. tonsurans with 30.3 ± 0%, and finally, M. gypseum with 30.18 ± 7.22%.Thus, the
griseofulvin MIC against the three germs was greater than 4000 ppm.
4. Discussion
The essential oil yield of extraction (9.66%) is different from those obtained by other authors [29,30]who also
used hydrodistillation. In effect, it was more than twice greater than the yield of 3.5% obtained by an author [16]
after the distillation of dried clove buds collected at Tizi Ouzou. This yield was also far greater than 0.18%
obtained with cloves from Benin [15]. As a result of this yield, clove buds essential oil from Cameroon can
easily be available.
Many factors can explain these differences: the plant origin, the plant age, the development stage of the plant at
the harvest [31], the harvest period, the plant treatment after harvest (drying for example), or the methods and
conditions of essential oil extraction [7].
The high amount of eugenol in S. aromaticum essential oil was in accordance with the results of several authors
0
10
20
30
40
50
60
500 ppm 1000 ppm 2000 ppm 4000 ppm
Inhibition percentages of
the dermatophytes mycelial
growth
Griseofulvin concentration
M. gypseum
T. tonsurans
T. rubrum
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
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[32, 33,34]. Some scientists showed eugenol as the main component of S. aromaticum essential oil, followed by
β-caryophyllene [32]. Another one [35] obtained beyond eugenol about 80.8% and β-caryophyllene (10.5%),
two other main components: eugenyl acetate (4.4%) and α-humulene (1.26%). Likewise, some authors
compared the chemical composition of three clove essential oil samples coming from Madagascar, Indonesia
and Zanzibar [19]. They found that each pattern withdrew 10 components, mainly eugenol, β-caryophyllene and
eugenyl acetate.
A study of cloves essential oil from Benin [15], showed 21 components for 99.4%, among which oxygenated
monoterpenes and hydrocarbonated sesquiterpenes were the principal components: eugenol (60.4%), trans β-
caryophyllene (24.0%), eugenyl acetate (10.0%), γ-muurolene (1.4%) and β-sesquiphellandrene (1.7%). These
difference can be explained by the lowest extraction yield of the EO of Benin (0.18%), compared to the one of
Cameroon clove buds (9.66%), which was far higher.
These results showed that from one region to another, there are many chemotypes of clove buds [7], though
eugenol and β-caryophyllene were always found as the traces of cloves essential oil. The cloves essential oil had
the strongest antiradical activity (SC50=2.26. 10-3 g/L, EC50= 23.7g of EO/mol of DPPH and AP= 4.22 x 10-2).
This result was better than the one obtained by some authors [36,37]. This high antiradical activity could be the
fact of the high rate of eugenol (87.62%) in the essential oil from Cameroon. In effect, some authors prooved an
important antiradical activity of eugenol [38,39,40,41]. In fact, the EO antiradical capacity was ten times greater
than the BHT sample and seven times greater than the CB and EO mixture sample. Moreover, the ratio of the
mixture antiradical on that of BHT was 1.5, showing a higher antiradical potential of the CB and EO mixture.
From the antidermatophytic activity, the essential oil of Syzygium aromaticum was more potent than
griseofulvin, the reference antifungal, with MICs of 400 p pm against T. rubrum, 825 ppm against T. Tonsurans
and 900 ppm against M. gypseum. That activity confirmed the antifungal property mentionned by an author [39].
On the other hand, cocoa butter was weakly active, compared to griseofulvin. The supplementation of cocoa
butter by Syzygium aromaticum essential oil increased its antidermatophytic activity against the three studied
dermatophytes, and the (EO + CB) mixture was more active than the griseofulvin. Of the three products (EO,
CB and (EO+CB) mixture), we had shown that the essential oil was the most active, and its activity far
exceeded that of griseofulvin, regardless of the considered dermatophyte.
5. Conclusion
The essential oil of Syzygium aromaticumis a good natural source of antioxidants, and provides a compounding
basis for the treatment of dermatophytosis. Otherwise, cocoa butter can be used successfully to formulate
antidermatophytic drugs that include the essential oil of Syzygium aromaticum.
Acknowledgement
Special thanks to the BiochemistryLaboratory of the Microbiology and Antimicrobial Substances (LAMSA) of
the Department of Biochemistry of the Faculty of Science of the University of Dschang; and the laboratory of
Max Mousseron Biomolecules Institute of the University of Montpellier II.
American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2017) Volume 37, No 1, pp 255-272
269
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