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Synergistic action between fractions of essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against Penicillium expansum
Food Control 23 (2012) 377e383
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Synergistic action between fractions of essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against Penicillium expansum J. Nguefacka, *, O. Tamguea, J.B. Lekagne Dongmoa, C.D. Dakolea, V. Lethb, H.F. Vismerc, P.H. Amvam Zolloa, A.E. Nkengfackd a
Department of Biochemistry, Faculty of Science, University of Yaoundé I, Box 812, Yaoundé, Cameroon The Danish Seed Health Centre for Developing Countries Royal Veterinary and Agricultural University, Copenhagen Thorvaldsensvej 57, DK-1871 Frederiksberg C, Denmark Medical Research Council, PROMEC Unit, Tygerberg, Cape Town, South Africa d Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, Box 812, Yaoundé, Cameroon b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2011 Received in revised form 21 July 2011 Accepted 2 August 2011
Penicillium expansum is a mould that causes the rotting of several fruits and vegetables, especially apples onto which it also synthesizes some dangerous mycotoxins. The degree of synergism between fractions of essential from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris was evaluated against two mycotoxin producing strains of P. expansum. The antifungal activity determined by dilution method and expressed as a Number of Decimal Reduction of the colony forming units per ml (NDR cfu) showed that the essential oils extracted from O. gratissimum was significantly (P < 0.05) more active against P. expansum than those extracted from C. citratus and T. vulgaris. Fractions enriched with oxygenated terpenes were significantly (P < 0.05) more active than their respective essential oils, whereas most of the fractions enriched with terpene hydrocarbons, were significantly (P < 0.05) less active. The fungicidal activity of mixtures of fractions from the same essential oils or from two different essential oils showed that there exist synergistic, additive and antagonistic effects between fractions of the three essential oils tested against both fungal strains. The synergistic effects observed could be exploited in order to maximize the antimicrobial activity of essential oils and to minimize the concentrations of essential oil required to produce a given antimicrobial effect without any alteration of the food test. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Antifungal Essential oil fractions Synergism Natural food preservative
1. Introduction Penicillium expansum is a post-harvest fungus responsible for the deterioration of fruits and vegetables in general, and of apples particularly on which it causes so-called “blue rot”. This fungus synthesizes roquefortine C, citrinin, chaetoglobosins and the mycotoxin, patulin, which toxicity to humans and animals has been demonstrated (Frisvad & Thrane, 1995; Rosenberger, 2003). Fusarium, Aspergillus and Penicillium species are mycotoxin producing fungi responsible for about 50% of post-harvest losses in tropical and under-developed countries (Jeffries & Jeger, 1990). About 25% of food commodities are contaminated by mycotoxins and it constitutes a threat for the health and the economy of many countries (FAO, 2003). Restrictions imposed by food industries and regulatory agencies on the use of some synthetic food additives have led to renewed interest in searching for alternatives, as natural
* Corresponding author. Tel.: þ237 99 73 21 73. E-mail address: [email protected] (J. Nguefack). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.08.002
antimicrobial compounds, particularly those from plants origin (Delaquis & Mazza, 1995; Hammer, Carson, & Riley, 1999). Natural substances in general and essential oils with their constituents in particular, have been proven to be active against several agents responsible for the deterioration of stored and conserved food commodities (Hammer et al., 1999; Nguefack, Budde, & Jakobsen, 2004; Nguefack, Leth, Amvam, & Mathur, 2004; Nguefack et al., 2007). Several studies have reported results on their preservative action (Burt, 2004; Nielsen & Rios, 2000; Pauli, 2001). Essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris are known to have several active compounds, including geranial, neral, thymol, terpinen-4-ol, linalool, carvacrol (Amvam et al., 1998; Ndoyé, 2001, 319 pp; Nguefack et al., 2007). Bio guided fractionation of these essential oils led to fractions more active than the original oil (Nguefack et al., 2007). However, little is known about the synergistic actions between some of these identified active fractions. This study was initiated to determine the level of synergism or antagonism between some active bio guided fractions of essential oils obtained from Cymbopogon citratus, Ocimum gratissimum and Thymus
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vulgaris against two strains of Penicillium expansum as well as correlation between the chemical composition of fractions and their antimycotic indexes. 2. Materials and methods 2.1. Plant material and oil isolation The essential oils tested were isolated by the hydrodistillation method using a Clevenger-type apparatus (Lamaty, Menut, Bessiere, Zollo, & Fekam, 1987). They included dry leaves of Cymbopogon citratus (DC) Stapf, Ocimum gratissimum L. and Thymus vulgaris L., collected in Yaoundé for C. citratus and O. gratissimum, in Bafoussam for T. vulgaris, in November 2009 and specimens confirmed at the Cameroon National Herbarium in Yaoundé according to the deposited Voucher specimens (No Dang 18628/ SRF/Cam.-1968, Letouzey 5817/SRF/Cam.-1966 and Westphal 42851/HNC-1978, respectively). The recovered oils were dried over anhydrous sodium sulphate and stored in darkness between 4 and 6 C. The yields of the oils as percent of plant material weight were as follows: 0.8%, 0.7% and 0.6% for the essential oil from C. citratus, O. gratissimum and T. vulgaris, respectively. These plant species were selected on the basis of previous knowledge on their antifungal and antibacterial activities (Nguefack, Leth et al., 2004; Nguefack, Budde et al., 2004; Nguefack, Somda, Mortensen, & Amvam Zollo, 2005; Nguefack et al., 2007). 2.2. Fungal strains and cultures The cultures used in this study were obtained from the Medical Research Council, PROMEC Unit, Cape Town, South Africa, i.e. two strains of Penicillium expansum, MRC 6935 and MRC 6939. The cultures were grown on Potato Dextrose Agar (PDA) at 25 C until good growth was obtained.
a Bürker-Türk counting chamber (Heamocytometer). The mixture in eppendorf tubes were incubated for 3 h with vortexing every 1 h. Upon incubation, three ten-fold serial dilution was made and 50 ml aliquots from each dilution were plated on PDA in Petri dishes of 90 mm using a bent glass rod. Six replications of 3 repetitions each were made. After 72 h of incubation under a diurnal cycle of 12 h light and 12 h darkness at 24e25 C, the colony counts from each plate was expressed as Number of Colony Forming Units per ml (N), calculated as the formula: N ¼ Nc 20 dilution factor, where Nc, represent the N CFU recorded from the culture plate. The fungicidal activity was determined and expressed as Number of Decimal Reduction of colony forming units per ml (NDR cfu) and calculated using the following formula:
NDR CFU ¼ log
" # Nþ N0
Nþ: Number of Colony Forming Units per ml from supplemented media with oils, No: Number of Colony Forming Units per ml from control with non-supplemented media. An active fraction was the fraction, which activity was superior or equal to that of the correspondent complete oil. Fractions to be used for the assessment of the synergy were chosen on the basis of their high activities and high proportions. The degree of synergy between essential oil fractions was assessed by comparing the NDRcfu obtained with mixed fractions to those obtained with each individual fraction. Mixture effects were qualified as synergistic, antagonistic and additive when the NDRcfu obtained with the mixed fraction were respectively superior, inferior and equal to the arithmetic sum of NDR cfu obtained with each individual fraction at the same concentration (1000 ppm) (Davidson & Parish, 1989; Pandey, Chandra, Tripathi, & Dixit, 1983). 2.5. Analysis of essential oils and fraction components
2.3. Essential oil fractionation Forty (40) g of each oil were chromatographed on silica gel as packing material (40 g, silica gel 60e200 mesh, column size 30 750 mm). A gradient of hexane and ethyl acetate in hexane was used as eluent (each eluent was prepared as 250 mL). The polarity of the solvent was increased by increasing volumes of ethyl acetate from 5% to 100% (v/v) and methanol 100%. The composition of each fraction was examined on silica gel TLC plates with hexane/ ethyl acetate (3:1(v/v)) as the developing solvents. The plates were then placed on UV for the visualization of the components of each fraction and further revealed by iodine vapours. Fractions showing similar patterns were combined. 2.4. Evaluation of the antifungal properties of the essential oils and their fractions The assessment of the fungicidal activity of complete essential oil or their fractions was done following the broth dilution method (Benjilali, Tantaoui-Elaraki, Ayadi, & Ihlal, 1984), coupled to the colony counts. The essential oil or fractions were mixed at different proportions (100, 75/25, 50/50, 25/75%, v/v) to meet 1000 ppm as final concentration. This value of 1000 ppm was recorded from the previous work as the minimum inhibitory concentration of all the three essential oils against six strains, two each of Penicillium expansum, Penicillium verruscosum and Aspergillus ochraceus (Nguefack et al., 2009). To the essential oil or fractions mixed in a PDA broth Difco, were added a conidial suspension of the fungus at a ratio of 9:1 (v/v). The number of conidia in the suspension was adjusted to approximately 107 conidia ml1 for each strain using
The samples were analysed by gas chromatography coupled to mass spectrometry (GC/MS). GC/MS analysis was performed using Hewlett-Packard GC 6890A equipped with a HP-5MS (Cross-linked Methyl Siloxane) fused column (30 m 0.25 mm, film thickness 0.25 mm) and interfaced with a quadrupole detector (Model 5973); temperature programmed at 40 300 C (7 C/min); injector temperature 220 C; temperature of transfer line 280 C, carrier gas Helium at a flow rate of 1 mL/min; injection type split, 1:20 (1 mL of a 1:10 ethyl acetate solution); ionization voltage 70 eV; electron multiplier, 1400 eV; mass range 33e500. The identification of components was assigned on the basis of comparison of their retention time indices and their mass spectra with those given in the literature (Wiley library), or from those of authentic samples. 2.6. Statistical analysis The data were subjected to the analysis of variance (ANOVA) and the separation of means with least significance difference (LSD), using the One Way ANOVA and the parametric T test of StudentNewman-Keuls at 95% level of confidence. 3. Results and discussion 3.1. Efficacy comparisons of essential oils, individual and mixed fractions In general, the fractionation of the three essential oils provided 26 fractions of which 9 were isolated from C. citratus, 9 from O. gratissimum and 8 from T. vulgaris. Characteristics and activities
J. Nguefack et al. / Food Control 23 (2012) 377e383
of these essential oils and their individual fractions are presented in Table 1. The essential oil of O. gratissimum (NDRcfu ¼ 0.47 and 0.80 against the strains MRC 6935 and MRC 6939 respectively) was significantly (P < 0.05) more active than that of C. citratus (NDRcfu ¼ 0.36 and 0.48 against the strains MRC 6935 and MRC 6939 respectively) and T. vulgaris (NDRcfu ¼ 0.16 and 0.18 against the strains MRC 6935 and MRC 6939 respectively) at 1000 ppm. The fractions CC1B, CC1C, CC2, from C. citratus; OG1B, OG1C, OG2 from O. gratissimum and TV1B, TV2 of T. vulgaris were significantly (P < 0.05) more active than their respective original essential oils against both fungus strains. The fractions CC1C (NDRcfu ¼ 3.34 and 5.02 against MRC 6935 and MRC 6939 respectively), OG1C (NDRcfu superior to 6 against both fungus strains) and TV1B (NDRcfu superior to 6 against both fungus strains) were the most active with average activities respectively at least 10, 11 and 36 fold superior to that of their respective original essential oils. The other fractions namely, CC1A,CC3, CC4, CC5, CC6,CC7 OG1A, OG3, OG4, OG5, OG6, OG7, TV1A, TV3 TV4, TV5,TV6 and TV7 were significantly (P < 0.05) less active than their respective original essential oils (Table 1). Among the 7 mixed and tested fractions of C. citratus (Table 2), only the mixture CC1A-CC1B showed a NDRcfu (0.91 for MRC 6935) superior to the arithmetic sum of the NDRcfu (0.5 for MRC 6935) obtained with each of these individual fractions against the two tested strains, thus revealing the existence of a synergistic effect between these two fractions. The mixtures CC1AeCC1C, CC1AeCC2, CC1BeCC1C, CC1BeCC2 and CC1CeCC2 presented an antagonistic effect varying with the proportions of each fraction in the mixture and the strains tested. The mixture of active fractions CC1BeCC2 was significantly (P < 0.05) the least active against both fungus strains
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with NDRcfu of 0.17 < 0.65 for MRC 6935, notably in the proportions 50/50 v/v, meaning an antagonistic effect. The mixture of three active fractions CC1BeCC1CeCC2 presented a NDRcfu (1.56 for MRC 6935) almost equal to the arithmetic sum of the NDRcfu (1.54 for MRC 6935) obtained with each of these fractions, thus revealing the existence of an additive effect. For O. gratissimum’’essential oil, the mixture of fractions OG1AeOG1B showed a NDRcfu (0.77 for MRC 6935) higher than the arithmetic sum of the NDR (0.57) obtained with each of these fractions (Table 3). This means the existence of a synergistic effect between these fractions. An antagonistic effect was observed with the other mixtures of fractions. This antagonistic effect was more pronounced with the mixtures OG1AeOG2 and OG1BeOG2 and varied with the proportions of each fraction in the mixture, and the particular strains of P. expansum. Mixtures TV1AeTV1B, TV1AeTV2 and TV1BeTV2 of T. vulgaris presented NDRcfu lower than the arithmetic sum of those obtained individually with each of the fractions (Table 4). This reveals the existence of an antagonistic effect between the fractions. The mixture of fractions TV1BeTV2 was the least active against the two strains of P. expansum. Mixtures of non-active fractions CC1AeOG1A (Fig. 1A) and CC1AeTV1A (Fig. 1B) presented synergistic effect in contrast to those of high activity such as fractions CC1CeOG1C and CC1CeTV1B that presented antagonistic effect. 3.2. Components of essential oils and active fractions The essential oil from C. citratus was mainly constituted of oxygenated monoterpenes (OMT) (63.9%) among which 58.9% of
Table 1 Characteristics of plant essential oils and their fractions and activity against Penicillium expansum. Solvent system (%)
Colour
Aspect
Proportion (%)
P. expansum strains (NDR cfu per ml)
CC1A CC1B CC1C CC2 CC3 CC4 CC5 CC6 CC7 CC
H (100) H (100) H (100) H/EA (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Yellow Pale yellow Pale yellow Orange yellow Orange yellow Orange Orange Orange Pale yellow
Fluid Fluid Fluid Fluid Fluid Viscous Viscous Viscous Viscous Fluid
17.4 16.5 47.8 10.8 3.8 1.5 1.5 0.5 0.5 100
0.22 0.78 3.34 0.51 0.19 0.12 0.19 0.11 0.11 0.36
0.02b 0.06g 2.31i 0.04e 0.04b 0.05a 0.04b 0.02a 0.03a 0.04c
0.37 0.89 5.02 0.79 0.21 0.15 0.24 0.11 0.12 0.48
0.06cd 0.30f 1.70h 0.16e 0.03b 0.03ab 0.02b 0.03a 0.02a 0.03d
OG1A OG1B OG1C OG2 OG3 OG4 OG5 OG6 OG7 OG
H (100) H (100) H (100) H/EA (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Clear yellow Clear yellow Orange yellow Orange yellow Orange yellow Clear yellow Clear yellow Dark yellow Yellow
Fluid Fluid Fluid Fluid Fluid Fluid Viscous Viscous Viscous Fluid
43.7 8.9 29.4 7.2 4.3 4.9 0.4 0.2 0.8 100
0.22 0.91 >6.0 0.94 0.16 0.14 0.10 0.07 0.06 0.47
0.04b 0.14h 0.00j 0.06h 0.03ab 0.02ab 0.02a 0.02a 0.02a 0.04d
0.33 0.82 >6.0 1.21 0.19 0.16 0.13 0.09 0.09 0.80
0.22c 0.04ef 0.00i 0.14g 0.04b 0.02ab 0.04ab 0.02a 0.04a 0.03e
TV1A TV1B TV2 TV3 TV4 TV5 TV6 TV7 TV
H (100) H (100) H/A (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Yellow Dark orange Dark orange Dark orange Dark orange Orange yellow Dark orange Orange yellow
Fluid Fluid Fluid Fluid Fluid Fluid Viscous Viscous Fluid
54.1 31.4 7.9 1.4 3.6 0.7 0.4 0.4 100
0.13 >6.0 0.62 0.11 0.10 0.09 0.07 0.07 0.16
0.04a 0.00j 0.16f 0.06a 0.04a 0.02 0.03a 0.02a 0.02ab
0.15 >6.0 0.42 0.14 0.12 0.11 0.09 0.08 0.18
0.05ab 0.00i 0.07cd 0.04ab 0.07a 0.04a 0.05a 0.04a 0.03b
MRC 6935
MRC 6939
NDR cfu ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean standard deviation of 3 replications; each replication is the mean of 3 repetitions. Values within the same column followed by different letters are significantly different (P < 0.05); Fractions of Cymbopogon citratus (CC): C1A, CC1B, CC1C, CC2 to CC7; Fractions of Ocimum gratissimum (OG): OG1A, OG1B, OG1C, OG2 to OG7; Fractions of Thymus vulgaris (TV): TV1A, TV1B, TV2 to TV7.
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Table 2 Activities of Cymbopogon citratus essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions (%)
CC CC1A CC1B CC1C CC2
100 100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 1:3e1:3e1:3
P. expansum strains (NDR cfu per ml) MRC 6935
CC1AeCC1B
CC1AeCC1C
CC1AeCC2
CC1BeCC1C
CC1BeCC2
CC1CeCC2 CC1BeCC1CeCC2
0.36 0.22 0.78 3.34 0.51 0.75 0.91 0.88 0.31 0.59 0.81 0.22 0.27 0.35 0.70 1.61 1.62 0.59 0.17 0.21 1.63 0.53 0.53 1.56
0.04b 0.02a 0.06d 2.31fg 0.04c 0.07d 0.03e 0.03e 0.02ab 0.01c 0.07de 0.04a 0.04ab 0.02b 0.08d 0.17f 0.17f 0.10c 0.05a 0.05a 0.19f 0.04c 0.09c 0.25f
MRC 6939 0.48 0.39 0.89 5.02 0.79 0.86 1.05 0.89 0.88 0.93 1.07 0.56 0.63 0.67 1.43 1.43 1.56 0.80 0.40 0.54 0.89 0.26 0.24 1.62
0.03bc 0.06b 0.31d 1.70h 0.16d 0.07d 0.10e 0.03d 0.11d 0.10de 0.15e 0.06c 0.04cd 0.05cd 0.29efg 0.31efg 0.25fg 0.08d 0.03b 0.05c 0.13d 0.01a 0.02a 0.17fg
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different (P < 0.05); Cymbopogon citratus (CC) essential oil fractions: CC1A, CC1B, CC1C, CC2.
Citrals (Table 5). It also contained a non negligible proportion of sesquiterpene hydrocarbons (STH) (12.3%), of oxygenated sesquiterpenes (OST) (10.6%) and very low quantity of monoterpene hydrocarbons (MTH) (3.4%). Fractionation generated 9 fractions of
Table 3 Activities of Ocimum gratissimum essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions (%)
OG OG1A OG1B OG1C OG2
100 100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75
P. expansum strains (NDR cfu per ml) MRC 6935
OG1AeOG1B
OG1AeOG1C
OG1AeOG2 OG1BeOG1C
OG1BeOG2
OG1CeOG2
0.47 0.22 0.91 >6.00 0.94 0.51 0.77 0.85 1.03 1.31 0.40 0.29 0.31 0.40 1.67 >6.00 0.59 0.63 0.64 >6.00 1.33 0.94
0.04bc 0.04a 0.14de 0.00i 0.06de 0.07bc 0.10d 0.14de 0.07e 0.08f 0.04b 0.04ab 0.05ab 0.04b 0.19g 0.00i 0.12cd 0.13cd 0.10cd 0.00i 0.35fg 0.08de
which the fraction CC1C (Hex 100%) was the most representative (47.8% of the oil total volume) and was enriched with OMT (94.7%) to the detriment of MTH (0.1%). The other eight fractions represented about 53% of the oil total volume. Fraction CC1A was considerably enriched with STH (74.7%), while fractions CC1B and CC2 were enriched with OMT (67.2% and 74.8%, respectively). The essential oil from O. gratissimum contained nearly as much MTH (41.8%) as OMT (44.6%) with a predominance of Thymol (40.6%) and p-Cymene (23.5%) (Table 6). It also contained a low proportion of STH (8.8%) and traces of aromatic compound (0.8%). The fractionation generated 9 fractions among which fractions OG1A and OG1C were the most representative (43.7% and 29.4% of the oil total volume, respectively). It also led to the enrichment of the fraction OG1A with terpene hydrocarbons (90. 4%), especially pCymene (61.8%) and enrichment of fractions OG1C and OG2 with oxygenated terpenes (96.6% and 78.8%, respectively), especially Thymol (92.0% and 55.1% respectively). The essential oil from T. vulgaris contained nearly as much MTH (43.3%) as OMT (43.0%), with predominance of p-Cymene (30.9%) and of Thymol (28.1%) (Table 6). It also contained a non negligible proportion of STH (7.0%). The fractionation generated 8 fractions of which fractions TV1A and TV1B were the largest (54.1% and 31.4% of the oil total volume respectively). While fraction TV1A was mainly constituted of terpene hydrocarbons (95.6%) especially of p-Cymene (71.1%), Fractions TV1B and TV2 were enriched with OMT (90.6% and 82.4%) especially Thymol (68.2% and 39.0%, respectively). The fungicidal activity against P. expansum of essential oils, and their individual fractions expressed in terms of NDRcfu, varied with the chemical composition and concentration. The fungicidal activity displayed by C. citratus, O. gratissimum and T. vulgaris oils could be assigned to their high content in OMT (63.9%; 44.6%; 43.0%, respectively), as reported by Nguefack et al. (2007). This hypothesis was confirmed by the strong activity displayed by fractions CC1B, CC1C, CC2, OG1B, OG1C, OG2, TV1B and TV2 that have been enriched in OMT (67.2; 94.7; 74.8; 76.1; 96.5; 78.8; 90.6; 82.4%, respectively) to the detriment of terpene hydrocarbons. In addition, fractions CC1A,OG1A and TV1A enriched with terpene hydrocarbons (78.0; 90.4; 95.6%, respectively) to the detriment of oxygenated terpenes, exhibited a significantly (P < 0.05) low activity against the two strains of P. expansum as compared to their respective original essential oils. These
MRC 6939 0.80 0.33 0.82 >6.00 1.21 0.56 0.67 0.75 0.70 1.22 0.33 0.21 0.26 0.33 1.49 >6.00 0.51 0.72 0.80 >6.00 1.48 0.94
0.03de 0.02b 0.04de 0.00h 0.14f 0.05c 0.06cd 0.07d 0.08d 0.19f 0.06b 0.02a 0.03a 0.06b 0.30fg 0.00h 0.06c 0.03d 0.06de 0.00h 0.22fg 0.11ef
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different (P < 0.05); Ocimum gratissimum (OG) essential oil fractions: OG1A, OG1B, OG1C, OG2.
Table 4 Activities of Thymus vulgaris essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions
TV TV1A TV1B TV2
100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75
Strains of P. expansum (NDR cfu per ml) MRC 6935
TV1AeTV1B
TV1AeTV2
TV1BeTV2
0.16 0.13 >6.00 0.62 0.44 0.51 0.60 0.20 0.30 0.38 0.24 0.14 0.13
0.02a 0.04a 0.00d 0.16c 0.18bc 0.04c 0.04c 0.03ab 0.01bc 0.06bc 0.04b 0.04a 0.02a
MRC 6939 0.18 0.15 >6.00 0.42 0.37 0.41 0.74 0.17 0.20 0.30 1.11 0.66 0.13
0.03ab 0.05ab 0.00e 0.07b 0.04b 0.05b 0.06c 0.03ab 0.03ab 0.03b 0.18d 0.05c 0.02a
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different P < 0.05; Fractions of Thymus vulgaris (TV) essential oil: TV1A, TV1B, TV2.
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Fig. 1. Antifungal activity expressed as Number of Decimal Reduction of colony forming units per ml standard deviation of mixtures of essential oils fractions against mycotoxin producing strains of Penicillium expansum: MRC 6935 ( ) and MRC 6939 ( ). A) Synergistic effect between fractions CC1A and OG1A; B) Synergistic effect between fractions CC1A and TV1A; CC1A: Fraction of essential oil from Cymbopogon citratus obtained with 100% Hexane; OG1A : Fraction of essential oil from Ocimum gratissimum obtained with 100% Hexane; TV1A : Fractions of essential oil from Thymus vulgaris obtained with 100% Hexane.
observations confirmed the strongest antimicrobial activity of oxygenated terpenes over terpene hydrocarbons as reported by several authors (Dorman & Deans, 2000; Hammer et al., 1999). The presence of compounds such as Bornyl-acetate and Borneol in fractions CC1B, CC2 OG1B, OG2, TV1B and TV2 may lower their activity, and that could explain the relatively low activities displayed by these fractions when compared to that of fractions CC1C and OG1C that don’t contain these compounds (Chalchat, Garry, Michel, Beside, & Malhuret, 1987).The low activity of the fraction OG1B (49.4% Thymol; 5.1% Carvacrol) as compared to fraction OG1C (92.0% Thymol; 0.9% Carvacrol) could also be explained by the decrease in Thymol content to the benefit of its isomer Carvacrol which antimicrobial activity is less than that of Thymol (Dorman & Deans, 2000). The comparison of the activities of the three essential oils showed that the essential oil of O. gratissimum displayed a significantly (P < 0.05) high NDRcfu as compared to essential oils of C. citratus and T. vulgaris. This could be explained by the high concentration of the most active antimicrobial compound, Thymol in the essential oil of O. gratissimum (40.6%) and of T. vulgaris (28.1%) as compared to the activity of Citral found in C. citratus essential oil (21.2% Neral, 37.7% Geranial). Ultee, Bennik, and Mezelaar (2002) showed that, compounds like Thymol and Carvacrol that possess in their structures a system of delocalized electrons (aromatic ring) and a hydroxyl group display higher antimicrobial activity than other constituents of the oil such as Citral that doesn’t possess some. According to this hypothesis, the activity of the essential oil from T. vulgaris should be higher than that from C. citratus but opposite result was observed. This could be explained by the presence in the essential oil of T. vulgaris of Borneol (2.1%) and Bornyl-acetate (3.5%), two constituents whose presence in an EO reduces its activity (Chalchat et al., 1987). The superiority of C. citratus essential oil could also be related to its high content in active antimicrobial compounds like citral (58.9%), as compared to the Thymol content in T. vulgaris oil (28.1%). Many
Table 5 Chemical composition (%) of the essential oil from Cymbopogon citratus (CC) and some of its fractions (CC1A, CC1B, CC1C, CC2). Compounds
RI
CC
CC1A
CC1B
CC1C
CC2
a-Pinene
934 943 984 1016 1024 1090 1120 1154 1169 1225 1238 1252 1300 1330 1359 1381 1403 1427 1455 1465 1477 1481 1484 1486 1491 1518 1536 1558 1578 1597
0.1 0.1 2.5 0.5 0.2 1.1 0.2 0.2 0.9 21.2 0.5 37.7 0.7 1.0 0.4 1.1 0.7 1.8 1.2 0.7 0.2 0.3 1.2 1.3 0.9 1.0 0.3 1.6 8.9 1.7
0.7 1.9 e 0.7 e e e e e e e e e e e e 3.8 2.9 12.6 4.4 2.1 4.1 6.3 23.0 6.9 4.7 3.9 e e e
e e 2.1 0.7 0.9 e 0.1 e e 33.3 0.5 30.3 0.3 1.8 0.9 2.9 0.6 0.9 0.5 0.9 e 2.9 e 1.1 1.7 0.9 e 2.9 3.8 1.0
0.1 e e e e 1.0 e e 3.1 42.2 0.7 45.2 e 1.0 1.5 e e 0.2 e e e e e e e e e e 0.1 e
0.2 e e e 0.4 0.5 1.5 2.0 3.0 29.9 0.4 28.2 1.4 4.7 3.2 e e 0.6 e e e e e e e e e e 7.2 2.3
90.2 3.4 63.9 12.3 10.6
78.0 3.3 e 74.7 e
91.0 3.7 67.2 15.3 4.8
95.1 0.1 94.7 0.2 0.1
85.5 0.6 74.8 0.6 9.5
Camphene Myrcene p-Cymene Limonene Linalol Camphor Borneol Terpinen-4-ol Z-Citral/Neral Geraniol E-Citral/Geranial Bornyl-acetate Neryl acetate Geranyl acetate a-Copaene g-Selinene b-Caryophyllene a-Humulene (E) a-Bisabolene a-Amorphene s-Cadinene a-Selinene Germacrene D b-Selinene D-Cadinene a-Cadinene a-Caryophyllene Selina-6-en-4-ol Sesquiterpene alcohol Total MTH OMT STH OST
RI ¼ Retention Index; MTH ¼ Monoterpene Hydrocarbons; OMT ¼ Oxygenated Monoterpenes; STH ¼ Sesquiterpene Hydrocarbons; OST ¼ Oxygenated Sesquiterpens.
382
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authors have reported that the activity of an essential oil can be expected to relate to its chemical composition, the structural configuration of its constituent components and their functional groups and to possible synergistic or antagonistic interactions between components (Burt, 2004; Dorman & Deans, 2000; Delaquis, Stanich, Girar, & Mazza, 2002; Marino, Bersani, & Comi, 2001). The synergy observed with the mixed fraction CC1AeCC1B and OG1AeOG1B could be explained by the combined effect of the different components brought into the mixture by individual fractions. Dorman and Deans (2000) and Ultee, Kets, Alberda, Hoekstra, and Smid (2000 and Ultee et al. 2002) found that oxygenated compounds like Citral (major constituent of fraction CC1B), Thymol and Carvacrol (major constituents of fraction OG1B) are much more active than terpene hydrocarbons such as p-Cymene (major constituent of fraction OG1A), but the latter swells cell membranes to a greater extent than Citrals, Carvacrol and Thymol do. They therefore suggested that by this mechanism, p-Cymene probably enables Carvacrol to be more easily transported into the cell so that a synergistic effect is achieved when the two are used together. This might explain the synergistic effect observed in the present work. This effect varied according to the proportion of each fraction in the mixture and was more important when these fractions were mixed in the proportion 50/50 v/v, suggesting that at this proportion, there would be a sufficient quantity of terpene hydrocarbons (p-Cymene) that could facilitate the transmembrane transportation of the available oxygenated terpenes (Citrals, Thymol or Carvacrol), as compared to other proportions (25/75 and 75/25 %v/v). The synergistic effect observed with the mixtures of non-active fractions CC1AeOG1A and CC1AeTV1A can be explained by the
increase of the concentration of oxygenated terpenes present in traces in each of the individual fractions, and whose transmembrane transportation could be facilitated by terpene hydrocarbons. Also, this beneficial effect could be due to the absence of Citral whose aldehydic function could possibly react with the hydroxyl group of alcohols and phenols. The antagonistic effect observed with mixed fractions CC1BeCC1C, CC1BeCC2, CC1CeCC2, OG1BeOG1C, OG1BeOG2, OG1CeOG2 and TV1BeTV2 could be related to the presence in these mixtures of Borneol or Bornyl-acetate, both components brought into these mixtures by fractions CC1B, CC2, OG1B, OG2, TV1B and TV2. These components would not only lower the activity of their individual fractions, but also the activity of the mixtures in which they are present. This hypothesis was supported by the fact that the reduction of individual fractions in the mixtures led to a decrease of the antagonistic effect. Also, the highest antagonistic effect was obtained with the mixture of these fractions between them. Mixtures of the most active fractions CC1CeOG1C and CC1CeTV1B revealed antagonistic effect between active fractions. These observations might result from the chemical reaction that could occur between the constituents of the fraction CC1C and those of fractions OG1C and TV1B. Fractions OG1C and TV1B were enriched in terpenes with an alcohol function (Terpinene 4-ol, Borneol, Linalol) or a phenol group (Thymol, Carvacrol). The fraction CC1C was enriched in Citral (Geranial and Neral), representing terpenes with an aldehydic function. Thus, in neutral or acidic medium, the hydroxyl group of alcohols and phenols could react with the carbonyl function to form acetals that are compounds with blocked hydroxyl group and carbonyl function. The terpenes whose functional groups are blocked would be less active than those having
Table 6 Chemical composition (%) of Ocimum gratissimum and Thymus vulgaris essential oils and some of their fractions. Compounds
RI
OG
OG1A
OG1B
OG1C
OG2
TV
TV1A
TV1B
TV2
a-Thujene a-Pinene
924 932 943 974 984 1012 1016 1024 1048 1062 1087 1090 1120 1157 1169 1278 1290 1300 1381 1427 1477 1491 1484 1518 1558 1597
3.6 1.2 0.1 0.4 2.5 2.1 23.5 1.5 6.9 0.7 0.8 0.5 e 0.2 2.3 40.6 0.3 e 1.1 3.6 e 1.9 0.7 0.7 0.8 e
5.2 2.0 0.2 0.7 3.5 e 61.8 2.4 1.0 e 1.5 e e e e e e 1.6 0.5 e 4.2 1.1 0.7 5.5 e
0.21 e e e e 0.3 9.9 e 0.2 5.1 e e 3.9 3.9 8.7 49.4 5.1 e e 2.4 e e e e e e
e e e e e e 0.6 0.2 e 0.2 e 0.2 e e 3.2 92.0 0.9 e e e e e e e 0.5 e
0.4 0.3 0.1 0.2 0.9 0.4 2.6 0.7 1.0 2.7 0.7 2.6 2.6 1.8 12.7 55.1 1.3 e 0.5 e e e 0.2 0.3 0.3 1.4
0.9 0.9 0.9 0.7 1.0 1.2 30.9 0.9 5.9 0.7 e 3.2 1.6 2.1 1.2 28.1 2.6 3.5 0.5 4.6 0.3 e 0.2 0.7 0.7 e
2.1 2.3 2.2 0.5 1.7 e 71.1 1.6 4.8 e e e e e e e e e 0.6 1.9 0.5 0.2 e 0.9 5.2 e
e e e 0.3 e e 0.3 0.3 e 0.3 e 5.3 4.1 1.5 3.0 68.2 2.3 5.9 0.5 e e e e e 1.9 e
e e e e e e 0.1 e e e e 8.2 5.0 13.3 4.5 39.0 8.1 4.3 e 0.9 e e e e 0.1 e
96.0 41.8 44.6 8.8 e 0.8
91.9 76.8 e 13.6 e 1.5
89.1 10.6 76.1 2.4 e e
97.8 0.8 96.5 0.5 e e
88.8 6.6 78.8 1.3 1.4 0.7
93.3 43.3 43.0 7.0 e e
95.6 86.3 e 9.3 e e
93.9 0.9 90.6 2.4 e e
83.5 0.1 82.4 1.0 e e
Camphene b-Pinene Myrcene a-Terpinene p-Cymene Limonene g-Terpinene Trans-Sabinene hydrate 1-methyl-4(1-methylethenyl) benzene Linalool Camphor Bornéol Terpinen-4-ol Thymol Carvacrol Bornyl-acetate a- Copaene b-Caryophyllene a-Amorphene b-Selinene a-Selinene D-Cadinene a-Caryophyllene Sesquiterpene alcohol Total MTH OMT STH OST AC
RI ¼ Retention Index; Fractions of Ocimum gratissimum (OG) essential oil: OG1A, OG1B, OG1C, OG2; Fractions of Thymus vulgaris (TV) essential oil: TV1A, TV1B, TV2; MTH ¼ Monoterpene Hydrocarbons; OMT ¼ Oxygenated Monoterpenes; STH ¼ Sesquiterpene Hydrocarbons; OST ¼ Oxygenated Sesquiterpens; AC ¼ Aromatic compound.
J. Nguefack et al. / Food Control 23 (2012) 377e383
their carbonyl function or their hydroxyl group free. Ultee et al. (2002) showed that the antimicrobial activity of Carvacrol is higher than that of Carvacrol methyl ether because of the blockage of the hydroxyl group of the latter by a methyl group. The antagonistic effect observed with the mixtures CC1AeCC1C and OG1AeOG1C could be explained by a reduction of the oxygenated terpenes concentration. Indeed, the mixture of the non-active fractions CC1A and OG1A (enriched with terpene hydrocarbons) with the active fraction CC1C and OG1C (enriched with oxygenated terpenes) respectively, gave a final solution less concentrated in oxygenated terpenes. Consequently, the activity of the mixture of the two fractions will be lower than that of the active fraction CC1C, justifying the antagonistic effect observed. This was supported by the fact that the activity of the mixture increased when the proportion of the active fraction CC1C or OG1C increased within the mixture. This hypothesis might also explain the antagonistic effect observed with the mixtures OG1AeOG2, TV1AeTV1B and TV1AeTV2, but this could also be explained by the presence of compounds such as Borneol and Bornyl -acetate. The additive effect observed with the mixture of fraction CC1BeCC1CeCC2 would result from a balance between the inhibitory action of Borneol and the activator action of terpene hydrocarbons on Citral’s activity. Generally, the degree of sensitivity of the strains MRC 6935 and MRC 6939 to original essential oils, individual fractions and mixed fractions varied and could be explained by the strain effect often observed for the same essential oil against different microorganisms (Nguefack, Budde et al., 2004; Nguefack, Leth et al., 2004). 4. Conclusion The present work aimed at determining the level of synergism and/or antagonism between some fractions of essential oils of C. citratus, O. gratissimum and T. vulgaris against two mycotoxin producing strains of P. expansum. Among the 23 mixtures of essential oil fractions tested, 4 mixtures (CC1AeCC1B OG1AeOG1B, CC1AeOG1A, and CC1AeTV1A) displayed synergistic effect; one mixture (CC1BeCC1CeCC2), additive effect and 18, antagonistic effect. These effects were correlated to the chemical composition of the essential oil fractions and their mixtures. The synergistic effects observed could be exploited in order to maximize the antimicrobial activity of essential oils and to minimize the concentrations of essential oils required to produce a given antimicrobial effect without any alteration of the food taste. The active fraction mixtures that presented antagonistic effects should be avoided in practical applications. These results constitute a basis for the use of essential oils as food preservatives and should be completed by other works such as the assessment of the pH influence on the synergistic, antagonistic and additive effects; the assessment of the degree of synergy of the three essential oils against other toxin producing agents (bacteria and other fungi); the effect of essential oils and their fractions on the fungus or bacteria toxin production; the study of the toxicity of these essential oils to Humans and animals; the formulation of essential oil-based food preservative. Acknowledgements We thank Pr. Gershenzon J. and Dr. Reichelt M. of the MaxPlanck-Institute of Chemical Ecology (Jena, Germany) for their assistance in the analysis of the chemical composition of essential oils.
383
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Davidson, P. M., & Parish, M. E. (1989). Methods for testing the efficacy of food antimicrobials. Food Technology, 43, 148e155. Delaquis, P. J., & Mazza, G. (1995). Antimicrobial properties of isothiocyanates in food preservation. Food Technology, 49, 73e84. Delaquis, J. P., Stanich, K., Girar, B., & Mazza, G. (2002). Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. International Journal of Food Microbiology, 74, 101e109. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308e316. FAO. (2003). Manuel sur l’application du système d’analyse des risques-points critiques pour leur maîtrise (HACCP) pour la prévention et le contrôle des mycotoxines. In. Série étude FAO: Alimentation et Nutrition, 73 (pp. 1e96). Frisvad, J. C., & Thrane, U. (1995). Mycotoxin production by food-borne fungi. In R. A. Samson, E. S. Hoekstra, J. C. Frisvad, & O. Filtenborg (Eds.), Introduction to food- and airborne fungi (4th ed). (pp. 260e351). Baarn: Centraalbureau voor Schimmelcultures. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology, 86, 985e990. Jeffries, P., & Jeger, M. J. (1990). The biological control of post harvest diseases of fruits. Post Harvest News Information, 1, 365e368. Lamaty, G., Menut, C., Bessiere, J. M., Zollo, P. H. A., & Fekam, F. (1987). Aromatic plants of tropical Central Africa. I. Volatile components of two annonaceae from Cameroon: Xylopia aethiopica (Dunal) A. Richard and Monodora myristica (Gaerth.) Dunal. Flavours and Fragrance Journal, 2, 91e94. Marino, M., Bersani, C., & Comi, G. (2001). Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. International Journal of Food Microbiology, 67, 187e195. Ndoyé, F.B.E.F. (2001). frEtude chimique et evaluation des propriétés antiradicalaires et antioxydantes des huiles essentielles d’espèces aromatiques tropicales en provenance de la province Est du Cameroun. Thèse de Doctorat de l’Université Montpellier II, France. Nguefack, J., Leth, V., Amvam, Z. P. H., & Mathur, S. B. (2004). Evaluation of five essential oils from aromatic plants of Cameroon for controlling food spoilage and mycotoxin producing fungi. International Journal of Food Microbiology, 94, 329e334. Nguefack, J., Budde, B. B., & Jakobsen, M. (2004). Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry. Letters in Applied Microbiology, 39, 395e400. Nguefack, J., Somda, I., Mortensen, C. N., & Amvam Zollo, P. H. (2005). Evaluation of five essential oils from aromatic plants of Cameroon for controlling seed-borne bacteria of rice (Oryza sativa L.). Seed Science and Technology, 33, 397e407. Nguefack, J., Nguikwie, S. K., Fotio, D., Dongmo, B., Leth, V., Nkengfack, A. E., et al. (2007). Fungicidal potential of essential oils and fractions from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris to control Alternaria padwickii and Bipolaris oryzae, two seed-borne fungi of rice (Oryza sativa L.). Journal of Essential Oil Research, 19, 581e587. Nguefack, J., Lekagne Dongmo, J. B., Tamgue, O., Dakole, C. D., Guemdjom, E. F. N., Fotio, D., et al. (2009). Food preservative potential of essential oils and fractions from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against six mycotoxigenic fungus strains. Internatioanl Journal of Food Microbiology, 131, 151e156. Nielsen, P. V., & Rios, R. (2000). Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging with special emphasis on mustard essential oil. International Journal of Food Microbiology, 60, 219e229. Pandey, B. K., Chandra, H., Tripathi, N. N., & Dixit, S. N. (1983). Fungitoxicity of some higher plants with special reference to the synergistic activity amongst some volatile fungitoxicants. Phyto Z, 106, 226e232. Pauli, A. (2001). Antimicrobial properties of essential oil constituents. International Journal of Aromatherapy, 11, 126e133. Rosenberger, D. A. (2003). Control of Penicillium expansum during apple harvest and storage. In Patulin technical symposium. Kissimmee Fla (pp. 18e19). National Center for Food Safety and Technology, Summit III. Ultee, A., Kets, E. P. W., Alberda, M., Hoekstra, F. A., & Smid, E. J. (2000). Adaptation of the food-borne pathogen Bacillus cereus to carvacrol. Archives of Microbiology, 174(4), 233e238. Ultee, A., Bennik, M. H. J., & Mezelaar, R. (2002). The phenolic hydroxyl group of Carvacrol is essential for action against the food-borne pathogen Bacillus cereus. 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Synergistic action between fractions of essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against Penicillium expansum J. Nguefacka, *, O. Tamguea, J.B. Lekagne Dongmoa, C.D. Dakolea, V. Lethb, H.F. Vismerc, P.H. Amvam Zolloa, A.E. Nkengfackd a
Department of Biochemistry, Faculty of Science, University of Yaoundé I, Box 812, Yaoundé, Cameroon The Danish Seed Health Centre for Developing Countries Royal Veterinary and Agricultural University, Copenhagen Thorvaldsensvej 57, DK-1871 Frederiksberg C, Denmark Medical Research Council, PROMEC Unit, Tygerberg, Cape Town, South Africa d Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, Box 812, Yaoundé, Cameroon b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2011 Received in revised form 21 July 2011 Accepted 2 August 2011
Penicillium expansum is a mould that causes the rotting of several fruits and vegetables, especially apples onto which it also synthesizes some dangerous mycotoxins. The degree of synergism between fractions of essential from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris was evaluated against two mycotoxin producing strains of P. expansum. The antifungal activity determined by dilution method and expressed as a Number of Decimal Reduction of the colony forming units per ml (NDR cfu) showed that the essential oils extracted from O. gratissimum was significantly (P < 0.05) more active against P. expansum than those extracted from C. citratus and T. vulgaris. Fractions enriched with oxygenated terpenes were significantly (P < 0.05) more active than their respective essential oils, whereas most of the fractions enriched with terpene hydrocarbons, were significantly (P < 0.05) less active. The fungicidal activity of mixtures of fractions from the same essential oils or from two different essential oils showed that there exist synergistic, additive and antagonistic effects between fractions of the three essential oils tested against both fungal strains. The synergistic effects observed could be exploited in order to maximize the antimicrobial activity of essential oils and to minimize the concentrations of essential oil required to produce a given antimicrobial effect without any alteration of the food test. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Antifungal Essential oil fractions Synergism Natural food preservative
1. Introduction Penicillium expansum is a post-harvest fungus responsible for the deterioration of fruits and vegetables in general, and of apples particularly on which it causes so-called “blue rot”. This fungus synthesizes roquefortine C, citrinin, chaetoglobosins and the mycotoxin, patulin, which toxicity to humans and animals has been demonstrated (Frisvad & Thrane, 1995; Rosenberger, 2003). Fusarium, Aspergillus and Penicillium species are mycotoxin producing fungi responsible for about 50% of post-harvest losses in tropical and under-developed countries (Jeffries & Jeger, 1990). About 25% of food commodities are contaminated by mycotoxins and it constitutes a threat for the health and the economy of many countries (FAO, 2003). Restrictions imposed by food industries and regulatory agencies on the use of some synthetic food additives have led to renewed interest in searching for alternatives, as natural
* Corresponding author. Tel.: þ237 99 73 21 73. E-mail address: [email protected] (J. Nguefack). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.08.002
antimicrobial compounds, particularly those from plants origin (Delaquis & Mazza, 1995; Hammer, Carson, & Riley, 1999). Natural substances in general and essential oils with their constituents in particular, have been proven to be active against several agents responsible for the deterioration of stored and conserved food commodities (Hammer et al., 1999; Nguefack, Budde, & Jakobsen, 2004; Nguefack, Leth, Amvam, & Mathur, 2004; Nguefack et al., 2007). Several studies have reported results on their preservative action (Burt, 2004; Nielsen & Rios, 2000; Pauli, 2001). Essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris are known to have several active compounds, including geranial, neral, thymol, terpinen-4-ol, linalool, carvacrol (Amvam et al., 1998; Ndoyé, 2001, 319 pp; Nguefack et al., 2007). Bio guided fractionation of these essential oils led to fractions more active than the original oil (Nguefack et al., 2007). However, little is known about the synergistic actions between some of these identified active fractions. This study was initiated to determine the level of synergism or antagonism between some active bio guided fractions of essential oils obtained from Cymbopogon citratus, Ocimum gratissimum and Thymus
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J. Nguefack et al. / Food Control 23 (2012) 377e383
vulgaris against two strains of Penicillium expansum as well as correlation between the chemical composition of fractions and their antimycotic indexes. 2. Materials and methods 2.1. Plant material and oil isolation The essential oils tested were isolated by the hydrodistillation method using a Clevenger-type apparatus (Lamaty, Menut, Bessiere, Zollo, & Fekam, 1987). They included dry leaves of Cymbopogon citratus (DC) Stapf, Ocimum gratissimum L. and Thymus vulgaris L., collected in Yaoundé for C. citratus and O. gratissimum, in Bafoussam for T. vulgaris, in November 2009 and specimens confirmed at the Cameroon National Herbarium in Yaoundé according to the deposited Voucher specimens (No Dang 18628/ SRF/Cam.-1968, Letouzey 5817/SRF/Cam.-1966 and Westphal 42851/HNC-1978, respectively). The recovered oils were dried over anhydrous sodium sulphate and stored in darkness between 4 and 6 C. The yields of the oils as percent of plant material weight were as follows: 0.8%, 0.7% and 0.6% for the essential oil from C. citratus, O. gratissimum and T. vulgaris, respectively. These plant species were selected on the basis of previous knowledge on their antifungal and antibacterial activities (Nguefack, Leth et al., 2004; Nguefack, Budde et al., 2004; Nguefack, Somda, Mortensen, & Amvam Zollo, 2005; Nguefack et al., 2007). 2.2. Fungal strains and cultures The cultures used in this study were obtained from the Medical Research Council, PROMEC Unit, Cape Town, South Africa, i.e. two strains of Penicillium expansum, MRC 6935 and MRC 6939. The cultures were grown on Potato Dextrose Agar (PDA) at 25 C until good growth was obtained.
a Bürker-Türk counting chamber (Heamocytometer). The mixture in eppendorf tubes were incubated for 3 h with vortexing every 1 h. Upon incubation, three ten-fold serial dilution was made and 50 ml aliquots from each dilution were plated on PDA in Petri dishes of 90 mm using a bent glass rod. Six replications of 3 repetitions each were made. After 72 h of incubation under a diurnal cycle of 12 h light and 12 h darkness at 24e25 C, the colony counts from each plate was expressed as Number of Colony Forming Units per ml (N), calculated as the formula: N ¼ Nc 20 dilution factor, where Nc, represent the N CFU recorded from the culture plate. The fungicidal activity was determined and expressed as Number of Decimal Reduction of colony forming units per ml (NDR cfu) and calculated using the following formula:
NDR CFU ¼ log
" # Nþ N0
Nþ: Number of Colony Forming Units per ml from supplemented media with oils, No: Number of Colony Forming Units per ml from control with non-supplemented media. An active fraction was the fraction, which activity was superior or equal to that of the correspondent complete oil. Fractions to be used for the assessment of the synergy were chosen on the basis of their high activities and high proportions. The degree of synergy between essential oil fractions was assessed by comparing the NDRcfu obtained with mixed fractions to those obtained with each individual fraction. Mixture effects were qualified as synergistic, antagonistic and additive when the NDRcfu obtained with the mixed fraction were respectively superior, inferior and equal to the arithmetic sum of NDR cfu obtained with each individual fraction at the same concentration (1000 ppm) (Davidson & Parish, 1989; Pandey, Chandra, Tripathi, & Dixit, 1983). 2.5. Analysis of essential oils and fraction components
2.3. Essential oil fractionation Forty (40) g of each oil were chromatographed on silica gel as packing material (40 g, silica gel 60e200 mesh, column size 30 750 mm). A gradient of hexane and ethyl acetate in hexane was used as eluent (each eluent was prepared as 250 mL). The polarity of the solvent was increased by increasing volumes of ethyl acetate from 5% to 100% (v/v) and methanol 100%. The composition of each fraction was examined on silica gel TLC plates with hexane/ ethyl acetate (3:1(v/v)) as the developing solvents. The plates were then placed on UV for the visualization of the components of each fraction and further revealed by iodine vapours. Fractions showing similar patterns were combined. 2.4. Evaluation of the antifungal properties of the essential oils and their fractions The assessment of the fungicidal activity of complete essential oil or their fractions was done following the broth dilution method (Benjilali, Tantaoui-Elaraki, Ayadi, & Ihlal, 1984), coupled to the colony counts. The essential oil or fractions were mixed at different proportions (100, 75/25, 50/50, 25/75%, v/v) to meet 1000 ppm as final concentration. This value of 1000 ppm was recorded from the previous work as the minimum inhibitory concentration of all the three essential oils against six strains, two each of Penicillium expansum, Penicillium verruscosum and Aspergillus ochraceus (Nguefack et al., 2009). To the essential oil or fractions mixed in a PDA broth Difco, were added a conidial suspension of the fungus at a ratio of 9:1 (v/v). The number of conidia in the suspension was adjusted to approximately 107 conidia ml1 for each strain using
The samples were analysed by gas chromatography coupled to mass spectrometry (GC/MS). GC/MS analysis was performed using Hewlett-Packard GC 6890A equipped with a HP-5MS (Cross-linked Methyl Siloxane) fused column (30 m 0.25 mm, film thickness 0.25 mm) and interfaced with a quadrupole detector (Model 5973); temperature programmed at 40 300 C (7 C/min); injector temperature 220 C; temperature of transfer line 280 C, carrier gas Helium at a flow rate of 1 mL/min; injection type split, 1:20 (1 mL of a 1:10 ethyl acetate solution); ionization voltage 70 eV; electron multiplier, 1400 eV; mass range 33e500. The identification of components was assigned on the basis of comparison of their retention time indices and their mass spectra with those given in the literature (Wiley library), or from those of authentic samples. 2.6. Statistical analysis The data were subjected to the analysis of variance (ANOVA) and the separation of means with least significance difference (LSD), using the One Way ANOVA and the parametric T test of StudentNewman-Keuls at 95% level of confidence. 3. Results and discussion 3.1. Efficacy comparisons of essential oils, individual and mixed fractions In general, the fractionation of the three essential oils provided 26 fractions of which 9 were isolated from C. citratus, 9 from O. gratissimum and 8 from T. vulgaris. Characteristics and activities
J. Nguefack et al. / Food Control 23 (2012) 377e383
of these essential oils and their individual fractions are presented in Table 1. The essential oil of O. gratissimum (NDRcfu ¼ 0.47 and 0.80 against the strains MRC 6935 and MRC 6939 respectively) was significantly (P < 0.05) more active than that of C. citratus (NDRcfu ¼ 0.36 and 0.48 against the strains MRC 6935 and MRC 6939 respectively) and T. vulgaris (NDRcfu ¼ 0.16 and 0.18 against the strains MRC 6935 and MRC 6939 respectively) at 1000 ppm. The fractions CC1B, CC1C, CC2, from C. citratus; OG1B, OG1C, OG2 from O. gratissimum and TV1B, TV2 of T. vulgaris were significantly (P < 0.05) more active than their respective original essential oils against both fungus strains. The fractions CC1C (NDRcfu ¼ 3.34 and 5.02 against MRC 6935 and MRC 6939 respectively), OG1C (NDRcfu superior to 6 against both fungus strains) and TV1B (NDRcfu superior to 6 against both fungus strains) were the most active with average activities respectively at least 10, 11 and 36 fold superior to that of their respective original essential oils. The other fractions namely, CC1A,CC3, CC4, CC5, CC6,CC7 OG1A, OG3, OG4, OG5, OG6, OG7, TV1A, TV3 TV4, TV5,TV6 and TV7 were significantly (P < 0.05) less active than their respective original essential oils (Table 1). Among the 7 mixed and tested fractions of C. citratus (Table 2), only the mixture CC1A-CC1B showed a NDRcfu (0.91 for MRC 6935) superior to the arithmetic sum of the NDRcfu (0.5 for MRC 6935) obtained with each of these individual fractions against the two tested strains, thus revealing the existence of a synergistic effect between these two fractions. The mixtures CC1AeCC1C, CC1AeCC2, CC1BeCC1C, CC1BeCC2 and CC1CeCC2 presented an antagonistic effect varying with the proportions of each fraction in the mixture and the strains tested. The mixture of active fractions CC1BeCC2 was significantly (P < 0.05) the least active against both fungus strains
379
with NDRcfu of 0.17 < 0.65 for MRC 6935, notably in the proportions 50/50 v/v, meaning an antagonistic effect. The mixture of three active fractions CC1BeCC1CeCC2 presented a NDRcfu (1.56 for MRC 6935) almost equal to the arithmetic sum of the NDRcfu (1.54 for MRC 6935) obtained with each of these fractions, thus revealing the existence of an additive effect. For O. gratissimum’’essential oil, the mixture of fractions OG1AeOG1B showed a NDRcfu (0.77 for MRC 6935) higher than the arithmetic sum of the NDR (0.57) obtained with each of these fractions (Table 3). This means the existence of a synergistic effect between these fractions. An antagonistic effect was observed with the other mixtures of fractions. This antagonistic effect was more pronounced with the mixtures OG1AeOG2 and OG1BeOG2 and varied with the proportions of each fraction in the mixture, and the particular strains of P. expansum. Mixtures TV1AeTV1B, TV1AeTV2 and TV1BeTV2 of T. vulgaris presented NDRcfu lower than the arithmetic sum of those obtained individually with each of the fractions (Table 4). This reveals the existence of an antagonistic effect between the fractions. The mixture of fractions TV1BeTV2 was the least active against the two strains of P. expansum. Mixtures of non-active fractions CC1AeOG1A (Fig. 1A) and CC1AeTV1A (Fig. 1B) presented synergistic effect in contrast to those of high activity such as fractions CC1CeOG1C and CC1CeTV1B that presented antagonistic effect. 3.2. Components of essential oils and active fractions The essential oil from C. citratus was mainly constituted of oxygenated monoterpenes (OMT) (63.9%) among which 58.9% of
Table 1 Characteristics of plant essential oils and their fractions and activity against Penicillium expansum. Solvent system (%)
Colour
Aspect
Proportion (%)
P. expansum strains (NDR cfu per ml)
CC1A CC1B CC1C CC2 CC3 CC4 CC5 CC6 CC7 CC
H (100) H (100) H (100) H/EA (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Yellow Pale yellow Pale yellow Orange yellow Orange yellow Orange Orange Orange Pale yellow
Fluid Fluid Fluid Fluid Fluid Viscous Viscous Viscous Viscous Fluid
17.4 16.5 47.8 10.8 3.8 1.5 1.5 0.5 0.5 100
0.22 0.78 3.34 0.51 0.19 0.12 0.19 0.11 0.11 0.36
0.02b 0.06g 2.31i 0.04e 0.04b 0.05a 0.04b 0.02a 0.03a 0.04c
0.37 0.89 5.02 0.79 0.21 0.15 0.24 0.11 0.12 0.48
0.06cd 0.30f 1.70h 0.16e 0.03b 0.03ab 0.02b 0.03a 0.02a 0.03d
OG1A OG1B OG1C OG2 OG3 OG4 OG5 OG6 OG7 OG
H (100) H (100) H (100) H/EA (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Clear yellow Clear yellow Orange yellow Orange yellow Orange yellow Clear yellow Clear yellow Dark yellow Yellow
Fluid Fluid Fluid Fluid Fluid Fluid Viscous Viscous Viscous Fluid
43.7 8.9 29.4 7.2 4.3 4.9 0.4 0.2 0.8 100
0.22 0.91 >6.0 0.94 0.16 0.14 0.10 0.07 0.06 0.47
0.04b 0.14h 0.00j 0.06h 0.03ab 0.02ab 0.02a 0.02a 0.02a 0.04d
0.33 0.82 >6.0 1.21 0.19 0.16 0.13 0.09 0.09 0.80
0.22c 0.04ef 0.00i 0.14g 0.04b 0.02ab 0.04ab 0.02a 0.04a 0.03e
TV1A TV1B TV2 TV3 TV4 TV5 TV6 TV7 TV
H (100) H (100) H/A (95/5) H/EA (85/15) H/EA (75/25) H/EA (50/50) H/EA (25/75) EA (100) Water
Light yellow Yellow Dark orange Dark orange Dark orange Dark orange Orange yellow Dark orange Orange yellow
Fluid Fluid Fluid Fluid Fluid Fluid Viscous Viscous Fluid
54.1 31.4 7.9 1.4 3.6 0.7 0.4 0.4 100
0.13 >6.0 0.62 0.11 0.10 0.09 0.07 0.07 0.16
0.04a 0.00j 0.16f 0.06a 0.04a 0.02 0.03a 0.02a 0.02ab
0.15 >6.0 0.42 0.14 0.12 0.11 0.09 0.08 0.18
0.05ab 0.00i 0.07cd 0.04ab 0.07a 0.04a 0.05a 0.04a 0.03b
MRC 6935
MRC 6939
NDR cfu ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean standard deviation of 3 replications; each replication is the mean of 3 repetitions. Values within the same column followed by different letters are significantly different (P < 0.05); Fractions of Cymbopogon citratus (CC): C1A, CC1B, CC1C, CC2 to CC7; Fractions of Ocimum gratissimum (OG): OG1A, OG1B, OG1C, OG2 to OG7; Fractions of Thymus vulgaris (TV): TV1A, TV1B, TV2 to TV7.
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Table 2 Activities of Cymbopogon citratus essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions (%)
CC CC1A CC1B CC1C CC2
100 100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 1:3e1:3e1:3
P. expansum strains (NDR cfu per ml) MRC 6935
CC1AeCC1B
CC1AeCC1C
CC1AeCC2
CC1BeCC1C
CC1BeCC2
CC1CeCC2 CC1BeCC1CeCC2
0.36 0.22 0.78 3.34 0.51 0.75 0.91 0.88 0.31 0.59 0.81 0.22 0.27 0.35 0.70 1.61 1.62 0.59 0.17 0.21 1.63 0.53 0.53 1.56
0.04b 0.02a 0.06d 2.31fg 0.04c 0.07d 0.03e 0.03e 0.02ab 0.01c 0.07de 0.04a 0.04ab 0.02b 0.08d 0.17f 0.17f 0.10c 0.05a 0.05a 0.19f 0.04c 0.09c 0.25f
MRC 6939 0.48 0.39 0.89 5.02 0.79 0.86 1.05 0.89 0.88 0.93 1.07 0.56 0.63 0.67 1.43 1.43 1.56 0.80 0.40 0.54 0.89 0.26 0.24 1.62
0.03bc 0.06b 0.31d 1.70h 0.16d 0.07d 0.10e 0.03d 0.11d 0.10de 0.15e 0.06c 0.04cd 0.05cd 0.29efg 0.31efg 0.25fg 0.08d 0.03b 0.05c 0.13d 0.01a 0.02a 0.17fg
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different (P < 0.05); Cymbopogon citratus (CC) essential oil fractions: CC1A, CC1B, CC1C, CC2.
Citrals (Table 5). It also contained a non negligible proportion of sesquiterpene hydrocarbons (STH) (12.3%), of oxygenated sesquiterpenes (OST) (10.6%) and very low quantity of monoterpene hydrocarbons (MTH) (3.4%). Fractionation generated 9 fractions of
Table 3 Activities of Ocimum gratissimum essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions (%)
OG OG1A OG1B OG1C OG2
100 100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75
P. expansum strains (NDR cfu per ml) MRC 6935
OG1AeOG1B
OG1AeOG1C
OG1AeOG2 OG1BeOG1C
OG1BeOG2
OG1CeOG2
0.47 0.22 0.91 >6.00 0.94 0.51 0.77 0.85 1.03 1.31 0.40 0.29 0.31 0.40 1.67 >6.00 0.59 0.63 0.64 >6.00 1.33 0.94
0.04bc 0.04a 0.14de 0.00i 0.06de 0.07bc 0.10d 0.14de 0.07e 0.08f 0.04b 0.04ab 0.05ab 0.04b 0.19g 0.00i 0.12cd 0.13cd 0.10cd 0.00i 0.35fg 0.08de
which the fraction CC1C (Hex 100%) was the most representative (47.8% of the oil total volume) and was enriched with OMT (94.7%) to the detriment of MTH (0.1%). The other eight fractions represented about 53% of the oil total volume. Fraction CC1A was considerably enriched with STH (74.7%), while fractions CC1B and CC2 were enriched with OMT (67.2% and 74.8%, respectively). The essential oil from O. gratissimum contained nearly as much MTH (41.8%) as OMT (44.6%) with a predominance of Thymol (40.6%) and p-Cymene (23.5%) (Table 6). It also contained a low proportion of STH (8.8%) and traces of aromatic compound (0.8%). The fractionation generated 9 fractions among which fractions OG1A and OG1C were the most representative (43.7% and 29.4% of the oil total volume, respectively). It also led to the enrichment of the fraction OG1A with terpene hydrocarbons (90. 4%), especially pCymene (61.8%) and enrichment of fractions OG1C and OG2 with oxygenated terpenes (96.6% and 78.8%, respectively), especially Thymol (92.0% and 55.1% respectively). The essential oil from T. vulgaris contained nearly as much MTH (43.3%) as OMT (43.0%), with predominance of p-Cymene (30.9%) and of Thymol (28.1%) (Table 6). It also contained a non negligible proportion of STH (7.0%). The fractionation generated 8 fractions of which fractions TV1A and TV1B were the largest (54.1% and 31.4% of the oil total volume respectively). While fraction TV1A was mainly constituted of terpene hydrocarbons (95.6%) especially of p-Cymene (71.1%), Fractions TV1B and TV2 were enriched with OMT (90.6% and 82.4%) especially Thymol (68.2% and 39.0%, respectively). The fungicidal activity against P. expansum of essential oils, and their individual fractions expressed in terms of NDRcfu, varied with the chemical composition and concentration. The fungicidal activity displayed by C. citratus, O. gratissimum and T. vulgaris oils could be assigned to their high content in OMT (63.9%; 44.6%; 43.0%, respectively), as reported by Nguefack et al. (2007). This hypothesis was confirmed by the strong activity displayed by fractions CC1B, CC1C, CC2, OG1B, OG1C, OG2, TV1B and TV2 that have been enriched in OMT (67.2; 94.7; 74.8; 76.1; 96.5; 78.8; 90.6; 82.4%, respectively) to the detriment of terpene hydrocarbons. In addition, fractions CC1A,OG1A and TV1A enriched with terpene hydrocarbons (78.0; 90.4; 95.6%, respectively) to the detriment of oxygenated terpenes, exhibited a significantly (P < 0.05) low activity against the two strains of P. expansum as compared to their respective original essential oils. These
MRC 6939 0.80 0.33 0.82 >6.00 1.21 0.56 0.67 0.75 0.70 1.22 0.33 0.21 0.26 0.33 1.49 >6.00 0.51 0.72 0.80 >6.00 1.48 0.94
0.03de 0.02b 0.04de 0.00h 0.14f 0.05c 0.06cd 0.07d 0.08d 0.19f 0.06b 0.02a 0.03a 0.06b 0.30fg 0.00h 0.06c 0.03d 0.06de 0.00h 0.22fg 0.11ef
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different (P < 0.05); Ocimum gratissimum (OG) essential oil fractions: OG1A, OG1B, OG1C, OG2.
Table 4 Activities of Thymus vulgaris essential oil fraction mixtures at 1000 ppm against Penicillium expansum. Mixtures
Proportions
TV TV1A TV1B TV2
100 100 100 100 75/25 50/50 25/75 75/25 50/50 25/75 75/25 50/50 25/75
Strains of P. expansum (NDR cfu per ml) MRC 6935
TV1AeTV1B
TV1AeTV2
TV1BeTV2
0.16 0.13 >6.00 0.62 0.44 0.51 0.60 0.20 0.30 0.38 0.24 0.14 0.13
0.02a 0.04a 0.00d 0.16c 0.18bc 0.04c 0.04c 0.03ab 0.01bc 0.06bc 0.04b 0.04a 0.02a
MRC 6939 0.18 0.15 >6.00 0.42 0.37 0.41 0.74 0.17 0.20 0.30 1.11 0.66 0.13
0.03ab 0.05ab 0.00e 0.07b 0.04b 0.05b 0.06c 0.03ab 0.03ab 0.03b 0.18d 0.05c 0.02a
NDR cfu per ml ¼ Number of Decimal Reduction of colony forming units per ml standard deviation; Data are the mean SD of 3 replications. Each replication is the mean of 3 repetitions; Values within the same column followed by different letters are significantly different P < 0.05; Fractions of Thymus vulgaris (TV) essential oil: TV1A, TV1B, TV2.
J. Nguefack et al. / Food Control 23 (2012) 377e383
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Fig. 1. Antifungal activity expressed as Number of Decimal Reduction of colony forming units per ml standard deviation of mixtures of essential oils fractions against mycotoxin producing strains of Penicillium expansum: MRC 6935 ( ) and MRC 6939 ( ). A) Synergistic effect between fractions CC1A and OG1A; B) Synergistic effect between fractions CC1A and TV1A; CC1A: Fraction of essential oil from Cymbopogon citratus obtained with 100% Hexane; OG1A : Fraction of essential oil from Ocimum gratissimum obtained with 100% Hexane; TV1A : Fractions of essential oil from Thymus vulgaris obtained with 100% Hexane.
observations confirmed the strongest antimicrobial activity of oxygenated terpenes over terpene hydrocarbons as reported by several authors (Dorman & Deans, 2000; Hammer et al., 1999). The presence of compounds such as Bornyl-acetate and Borneol in fractions CC1B, CC2 OG1B, OG2, TV1B and TV2 may lower their activity, and that could explain the relatively low activities displayed by these fractions when compared to that of fractions CC1C and OG1C that don’t contain these compounds (Chalchat, Garry, Michel, Beside, & Malhuret, 1987).The low activity of the fraction OG1B (49.4% Thymol; 5.1% Carvacrol) as compared to fraction OG1C (92.0% Thymol; 0.9% Carvacrol) could also be explained by the decrease in Thymol content to the benefit of its isomer Carvacrol which antimicrobial activity is less than that of Thymol (Dorman & Deans, 2000). The comparison of the activities of the three essential oils showed that the essential oil of O. gratissimum displayed a significantly (P < 0.05) high NDRcfu as compared to essential oils of C. citratus and T. vulgaris. This could be explained by the high concentration of the most active antimicrobial compound, Thymol in the essential oil of O. gratissimum (40.6%) and of T. vulgaris (28.1%) as compared to the activity of Citral found in C. citratus essential oil (21.2% Neral, 37.7% Geranial). Ultee, Bennik, and Mezelaar (2002) showed that, compounds like Thymol and Carvacrol that possess in their structures a system of delocalized electrons (aromatic ring) and a hydroxyl group display higher antimicrobial activity than other constituents of the oil such as Citral that doesn’t possess some. According to this hypothesis, the activity of the essential oil from T. vulgaris should be higher than that from C. citratus but opposite result was observed. This could be explained by the presence in the essential oil of T. vulgaris of Borneol (2.1%) and Bornyl-acetate (3.5%), two constituents whose presence in an EO reduces its activity (Chalchat et al., 1987). The superiority of C. citratus essential oil could also be related to its high content in active antimicrobial compounds like citral (58.9%), as compared to the Thymol content in T. vulgaris oil (28.1%). Many
Table 5 Chemical composition (%) of the essential oil from Cymbopogon citratus (CC) and some of its fractions (CC1A, CC1B, CC1C, CC2). Compounds
RI
CC
CC1A
CC1B
CC1C
CC2
a-Pinene
934 943 984 1016 1024 1090 1120 1154 1169 1225 1238 1252 1300 1330 1359 1381 1403 1427 1455 1465 1477 1481 1484 1486 1491 1518 1536 1558 1578 1597
0.1 0.1 2.5 0.5 0.2 1.1 0.2 0.2 0.9 21.2 0.5 37.7 0.7 1.0 0.4 1.1 0.7 1.8 1.2 0.7 0.2 0.3 1.2 1.3 0.9 1.0 0.3 1.6 8.9 1.7
0.7 1.9 e 0.7 e e e e e e e e e e e e 3.8 2.9 12.6 4.4 2.1 4.1 6.3 23.0 6.9 4.7 3.9 e e e
e e 2.1 0.7 0.9 e 0.1 e e 33.3 0.5 30.3 0.3 1.8 0.9 2.9 0.6 0.9 0.5 0.9 e 2.9 e 1.1 1.7 0.9 e 2.9 3.8 1.0
0.1 e e e e 1.0 e e 3.1 42.2 0.7 45.2 e 1.0 1.5 e e 0.2 e e e e e e e e e e 0.1 e
0.2 e e e 0.4 0.5 1.5 2.0 3.0 29.9 0.4 28.2 1.4 4.7 3.2 e e 0.6 e e e e e e e e e e 7.2 2.3
90.2 3.4 63.9 12.3 10.6
78.0 3.3 e 74.7 e
91.0 3.7 67.2 15.3 4.8
95.1 0.1 94.7 0.2 0.1
85.5 0.6 74.8 0.6 9.5
Camphene Myrcene p-Cymene Limonene Linalol Camphor Borneol Terpinen-4-ol Z-Citral/Neral Geraniol E-Citral/Geranial Bornyl-acetate Neryl acetate Geranyl acetate a-Copaene g-Selinene b-Caryophyllene a-Humulene (E) a-Bisabolene a-Amorphene s-Cadinene a-Selinene Germacrene D b-Selinene D-Cadinene a-Cadinene a-Caryophyllene Selina-6-en-4-ol Sesquiterpene alcohol Total MTH OMT STH OST
RI ¼ Retention Index; MTH ¼ Monoterpene Hydrocarbons; OMT ¼ Oxygenated Monoterpenes; STH ¼ Sesquiterpene Hydrocarbons; OST ¼ Oxygenated Sesquiterpens.
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authors have reported that the activity of an essential oil can be expected to relate to its chemical composition, the structural configuration of its constituent components and their functional groups and to possible synergistic or antagonistic interactions between components (Burt, 2004; Dorman & Deans, 2000; Delaquis, Stanich, Girar, & Mazza, 2002; Marino, Bersani, & Comi, 2001). The synergy observed with the mixed fraction CC1AeCC1B and OG1AeOG1B could be explained by the combined effect of the different components brought into the mixture by individual fractions. Dorman and Deans (2000) and Ultee, Kets, Alberda, Hoekstra, and Smid (2000 and Ultee et al. 2002) found that oxygenated compounds like Citral (major constituent of fraction CC1B), Thymol and Carvacrol (major constituents of fraction OG1B) are much more active than terpene hydrocarbons such as p-Cymene (major constituent of fraction OG1A), but the latter swells cell membranes to a greater extent than Citrals, Carvacrol and Thymol do. They therefore suggested that by this mechanism, p-Cymene probably enables Carvacrol to be more easily transported into the cell so that a synergistic effect is achieved when the two are used together. This might explain the synergistic effect observed in the present work. This effect varied according to the proportion of each fraction in the mixture and was more important when these fractions were mixed in the proportion 50/50 v/v, suggesting that at this proportion, there would be a sufficient quantity of terpene hydrocarbons (p-Cymene) that could facilitate the transmembrane transportation of the available oxygenated terpenes (Citrals, Thymol or Carvacrol), as compared to other proportions (25/75 and 75/25 %v/v). The synergistic effect observed with the mixtures of non-active fractions CC1AeOG1A and CC1AeTV1A can be explained by the
increase of the concentration of oxygenated terpenes present in traces in each of the individual fractions, and whose transmembrane transportation could be facilitated by terpene hydrocarbons. Also, this beneficial effect could be due to the absence of Citral whose aldehydic function could possibly react with the hydroxyl group of alcohols and phenols. The antagonistic effect observed with mixed fractions CC1BeCC1C, CC1BeCC2, CC1CeCC2, OG1BeOG1C, OG1BeOG2, OG1CeOG2 and TV1BeTV2 could be related to the presence in these mixtures of Borneol or Bornyl-acetate, both components brought into these mixtures by fractions CC1B, CC2, OG1B, OG2, TV1B and TV2. These components would not only lower the activity of their individual fractions, but also the activity of the mixtures in which they are present. This hypothesis was supported by the fact that the reduction of individual fractions in the mixtures led to a decrease of the antagonistic effect. Also, the highest antagonistic effect was obtained with the mixture of these fractions between them. Mixtures of the most active fractions CC1CeOG1C and CC1CeTV1B revealed antagonistic effect between active fractions. These observations might result from the chemical reaction that could occur between the constituents of the fraction CC1C and those of fractions OG1C and TV1B. Fractions OG1C and TV1B were enriched in terpenes with an alcohol function (Terpinene 4-ol, Borneol, Linalol) or a phenol group (Thymol, Carvacrol). The fraction CC1C was enriched in Citral (Geranial and Neral), representing terpenes with an aldehydic function. Thus, in neutral or acidic medium, the hydroxyl group of alcohols and phenols could react with the carbonyl function to form acetals that are compounds with blocked hydroxyl group and carbonyl function. The terpenes whose functional groups are blocked would be less active than those having
Table 6 Chemical composition (%) of Ocimum gratissimum and Thymus vulgaris essential oils and some of their fractions. Compounds
RI
OG
OG1A
OG1B
OG1C
OG2
TV
TV1A
TV1B
TV2
a-Thujene a-Pinene
924 932 943 974 984 1012 1016 1024 1048 1062 1087 1090 1120 1157 1169 1278 1290 1300 1381 1427 1477 1491 1484 1518 1558 1597
3.6 1.2 0.1 0.4 2.5 2.1 23.5 1.5 6.9 0.7 0.8 0.5 e 0.2 2.3 40.6 0.3 e 1.1 3.6 e 1.9 0.7 0.7 0.8 e
5.2 2.0 0.2 0.7 3.5 e 61.8 2.4 1.0 e 1.5 e e e e e e 1.6 0.5 e 4.2 1.1 0.7 5.5 e
0.21 e e e e 0.3 9.9 e 0.2 5.1 e e 3.9 3.9 8.7 49.4 5.1 e e 2.4 e e e e e e
e e e e e e 0.6 0.2 e 0.2 e 0.2 e e 3.2 92.0 0.9 e e e e e e e 0.5 e
0.4 0.3 0.1 0.2 0.9 0.4 2.6 0.7 1.0 2.7 0.7 2.6 2.6 1.8 12.7 55.1 1.3 e 0.5 e e e 0.2 0.3 0.3 1.4
0.9 0.9 0.9 0.7 1.0 1.2 30.9 0.9 5.9 0.7 e 3.2 1.6 2.1 1.2 28.1 2.6 3.5 0.5 4.6 0.3 e 0.2 0.7 0.7 e
2.1 2.3 2.2 0.5 1.7 e 71.1 1.6 4.8 e e e e e e e e e 0.6 1.9 0.5 0.2 e 0.9 5.2 e
e e e 0.3 e e 0.3 0.3 e 0.3 e 5.3 4.1 1.5 3.0 68.2 2.3 5.9 0.5 e e e e e 1.9 e
e e e e e e 0.1 e e e e 8.2 5.0 13.3 4.5 39.0 8.1 4.3 e 0.9 e e e e 0.1 e
96.0 41.8 44.6 8.8 e 0.8
91.9 76.8 e 13.6 e 1.5
89.1 10.6 76.1 2.4 e e
97.8 0.8 96.5 0.5 e e
88.8 6.6 78.8 1.3 1.4 0.7
93.3 43.3 43.0 7.0 e e
95.6 86.3 e 9.3 e e
93.9 0.9 90.6 2.4 e e
83.5 0.1 82.4 1.0 e e
Camphene b-Pinene Myrcene a-Terpinene p-Cymene Limonene g-Terpinene Trans-Sabinene hydrate 1-methyl-4(1-methylethenyl) benzene Linalool Camphor Bornéol Terpinen-4-ol Thymol Carvacrol Bornyl-acetate a- Copaene b-Caryophyllene a-Amorphene b-Selinene a-Selinene D-Cadinene a-Caryophyllene Sesquiterpene alcohol Total MTH OMT STH OST AC
RI ¼ Retention Index; Fractions of Ocimum gratissimum (OG) essential oil: OG1A, OG1B, OG1C, OG2; Fractions of Thymus vulgaris (TV) essential oil: TV1A, TV1B, TV2; MTH ¼ Monoterpene Hydrocarbons; OMT ¼ Oxygenated Monoterpenes; STH ¼ Sesquiterpene Hydrocarbons; OST ¼ Oxygenated Sesquiterpens; AC ¼ Aromatic compound.
J. Nguefack et al. / Food Control 23 (2012) 377e383
their carbonyl function or their hydroxyl group free. Ultee et al. (2002) showed that the antimicrobial activity of Carvacrol is higher than that of Carvacrol methyl ether because of the blockage of the hydroxyl group of the latter by a methyl group. The antagonistic effect observed with the mixtures CC1AeCC1C and OG1AeOG1C could be explained by a reduction of the oxygenated terpenes concentration. Indeed, the mixture of the non-active fractions CC1A and OG1A (enriched with terpene hydrocarbons) with the active fraction CC1C and OG1C (enriched with oxygenated terpenes) respectively, gave a final solution less concentrated in oxygenated terpenes. Consequently, the activity of the mixture of the two fractions will be lower than that of the active fraction CC1C, justifying the antagonistic effect observed. This was supported by the fact that the activity of the mixture increased when the proportion of the active fraction CC1C or OG1C increased within the mixture. This hypothesis might also explain the antagonistic effect observed with the mixtures OG1AeOG2, TV1AeTV1B and TV1AeTV2, but this could also be explained by the presence of compounds such as Borneol and Bornyl -acetate. The additive effect observed with the mixture of fraction CC1BeCC1CeCC2 would result from a balance between the inhibitory action of Borneol and the activator action of terpene hydrocarbons on Citral’s activity. Generally, the degree of sensitivity of the strains MRC 6935 and MRC 6939 to original essential oils, individual fractions and mixed fractions varied and could be explained by the strain effect often observed for the same essential oil against different microorganisms (Nguefack, Budde et al., 2004; Nguefack, Leth et al., 2004). 4. Conclusion The present work aimed at determining the level of synergism and/or antagonism between some fractions of essential oils of C. citratus, O. gratissimum and T. vulgaris against two mycotoxin producing strains of P. expansum. Among the 23 mixtures of essential oil fractions tested, 4 mixtures (CC1AeCC1B OG1AeOG1B, CC1AeOG1A, and CC1AeTV1A) displayed synergistic effect; one mixture (CC1BeCC1CeCC2), additive effect and 18, antagonistic effect. These effects were correlated to the chemical composition of the essential oil fractions and their mixtures. The synergistic effects observed could be exploited in order to maximize the antimicrobial activity of essential oils and to minimize the concentrations of essential oils required to produce a given antimicrobial effect without any alteration of the food taste. The active fraction mixtures that presented antagonistic effects should be avoided in practical applications. These results constitute a basis for the use of essential oils as food preservatives and should be completed by other works such as the assessment of the pH influence on the synergistic, antagonistic and additive effects; the assessment of the degree of synergy of the three essential oils against other toxin producing agents (bacteria and other fungi); the effect of essential oils and their fractions on the fungus or bacteria toxin production; the study of the toxicity of these essential oils to Humans and animals; the formulation of essential oil-based food preservative. Acknowledgements We thank Pr. Gershenzon J. and Dr. Reichelt M. of the MaxPlanck-Institute of Chemical Ecology (Jena, Germany) for their assistance in the analysis of the chemical composition of essential oils.
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