SUPPLEMENTARY MATERIAL
Effect of Carum copticum essential oil on growth and aflatoxin formationby Aspergillus strains
M.Kazemi
Department of Horticultural Science, College of Agricultural Science and Natural Resources, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran
Corresponding author: E-mail:
Abstract
The objectives of present study were to determine the antiaflatoxin B1activity in vitro of the essential oilextracted from the seeds of Carum copticumand to evaluate its antifungal activity in vivo asa potential food preservative.The Ca.copticumEO exhibited noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B1 (AFB1) by A. flavus, completely inhibiting AFB1 production at 4 μL/mL. Ca.copticumEos showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 μL/mL with values of 5.01±67 % for A. flavusand 5.98±54 % for A.niger. The results indicated the percentage ofinfected fruits is significantly (p0.01) reduced by essential oil at 16 °C for 30days. In this case, the oil at 100 μL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control.Thus, theessential oil of dill could be used to control food spoilage as a potential source of food preservative.
Keywords: Carum copticum; essential oil; antiaflatoxin B1; food preservative.
1. Experimental
Two separate sets of experiments were conducted in a completely randomized design. In the first set, the effect of exogenous application of nano iron chelate (100and200 mgL-1 ) in early flowering stage on components of Eos of Ca. copticumwas evaluated.The second set included investigating the effect ofplant Eos treated with nano iron chelat at 200 mgL-1onantiaflatoxin B1activity.
1.1Plant materials and nano iron chelate treatments
Seeds of Ca. copticumwere sown in Jefe pot in experimental greenhouse of Ilam, Iran. Plants at flowering stage (2013-2014) were sprayed with distilled water as a control, and nano iron chelate at 100and200 mgL-1. All sprays solution were sprayed to the point of run off. The experiment was arranged in completely randomized block design with three replications for each treatment. At seed stage of Ca. copticumwere harvested and air dried at ambient temperature in the shade.
1.20il isolation and identification of the oil components
The plant was identified by Mr. Esmaili, and the voucher specimen was deposited at private herbarium of Dr F. Esmaili (voucher no. 121). The Ca. copticumseeds were ground and the resulting powder was subjected to hydrodistillation for 3 hours in an all glass Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia (European Pharmacopoeia, 1975). The obtained essential oils were dried over anhydrous sodium sulphate and after filtration, stored at +4 ºC until tested and analysed.The GC/MS analyses were executed on a Hewlett–Packard 5973N gas chromatograph equipped with a column HP-5MS (30 m length × 0.25 mm i.d., film thickness 0.25 lm) coupled with a Hewlett–Packard 5973N mass spectrometer. The column temperature was programmed at 50 ºC as an initial temperature, holding for 6 min, with 3 ºC increases per minute to the temperature of 240 ºC, followed by a temperature enhancement of 15 ºC per minute up to 300 ºC, holding at the mentioned temperature for 3 min. Injector port temperature was 290 ºC and helium used as carrier gas at a flow rate 1.5 ml/min. Ionization voltage of mass spectrometer in the EI-mode was equal to 70 eV and ionization source temperature was 250 ºC. Linear retention indices for all components were determined by coinjection of the samples with a solution containing homologous series of C8-C22 n-alkanes and comparing them and their mass spectra with those of authentic samples or with available library data of the GC/MS system (WILEY 2001 data software) and Adams libraries spectra (2001).
1.3 Total phenolic determination
Total phenolic contents in seeds Ca. copticum were determined by Folin–Ciocalteu method (Jimoh, Sofidiya, & Afolayan, 2007). The total phenolic content was expressed as gallic acid equivalents (GAE) (mg g_1).
1.4Total flavonoid determination
Total flavonoid contents in seeds Ca. copticum were measured as described previously (Piccolella et al., 2008). The total flavonoid content was calculated as rutin equivalents (mg g-1).
1.5 A. flavus growth and analysis of aflatoxin B1
The anti-aflatoxigenic efficacy of Ca.copticumon A. flavus was studiedfollowing Reddy et al.(1970); Bankole et al. (2005) and Kumar et al. (2007).The amount of AFB1 present in the sample was calculated according tothe formula by Sinha et al. (1993): AFB1 content (µg/ml) = D× M/ E × l × 1000. where D is the absorbance, M is the molecular weight of aflatoxin(312), E is the molar extinction coefficient (21, 800), and l is the pathlength (1 cm cell was used).
In addition, AFB1 inhibition was calculated as follows:Inhibition (%) =1−X /Y× 100.where X is the mean concentration of AFB1 in the treatment and Y isthe mean concentration of AFB1 in the control.
1.6Fungitoxic spectrum of Ca.copticum EO
The fungitoxic spectrum of Ca.copticumessential oil was evaluatedagainst five fungal strains (Aspergillus flavus, Aspergillus candidus, Aspergillusoryze,Aspergillusterreus, Aspergillus niger) using the poisoned food technique (Prakash et al.,2010).The percent inhibition of fungal
growth was calculated by the following formula:% mycelial inhibation=dc-dt/dc×100
dc:average diameter of fungal colony in control sets.
dt:average diameter of fungal colony in treatment sets
1.7 Antifungal effect of Ca.copticum on wound-inoculated cherry tomatoes and in the conservationof cherry tomatoes
To assess the potency of Ca.copticumoil in controlling fungaldecay of cherry tomatoes caused by food spoilage fungi, antifungal effect of Ca.copticum on wound-inoculatedand healthy cherry tomatoes following previously publishedmethod (Tian et al., 2011).Fourdifferent concentrations (25, 50,75, and 100 μL/mL) of Ca.copticumsolution and sterilized water were prepared as described above. Thepercentage of infected fruits was recorded after 30 days at 16 °C. Alltreatments consisted of 3 replicates with 20 fruits per replicate.
1.8Statistical analysis
The results are presented as mean±S.D and statistically analyzed by oneway analysis of variance (ANOVA) followed by Duncan test.
References
Adams, R.P. (2001). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured, Carol Stream, IL, p. 469.
Bankole, S.A., Joda, A.O., & Ashidi, J.S. (2005). The use of powder and essential oil ofCymbopogon citratus against mould deterioration and aflatoxin contamination of‘egusi’ melon seeds. Journal of Basic Microbiology, 45, 20–30.
European Pharmacopoeia (1975). Vol. 3, Maisonneuve S. A., Sainte–Ruffine.
Jimoh, F.O., Sofidiya, M.O., & Afolayan, A.J. (2007). Antioxidant properties of the methanolextracts from the leaves of Paullinia pinnata. Journal of Medicinal Food, 10, 707–711.
Kumar, R., Dubey, N.K., Tiwari, O.P., Tripathi, Y.B., Sinha, K.K. (2007). Evaluation of som essential oils as botanical fungitoxicants for the protection of stored foodcommodities from fungal infestation. Journal of the Science of Food and Agriculture, 87, 1737–1742.
Piccolella, S., Fiorentino, A., Pacifico, S., D’Abrosca, B., Uzzo, P., & Monaco, P. (2008). Antioxidant properties of sour cherries (Prunus cerasus L.): role of colorless phytochemicals from the methanolic extract of ripe fruits. Journal of Agricultural and Food Chemistry, 56, 1928–1935.
Reddy, T.V., Viswanathan, L., Venkitasubramanian, T.A. (1970). Thin layer chromatography
of aflatoxins. Analytical Biochemistry, 38, 568–571.
Sinha, K.K., Sinha, A.K., & Prasad, G. (1993). The effect of clove and cinnamon oils on growth and aflatoxin production by Aspergillus flavus. Letters in Applied Microbiology, 16,114–117.
Tian, J., Ban, X. Q., Zeng, H., He, J. S., Huang, B., & Wang, Y. W. (2011). Chemicalcomposition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. International Journal of Food Microbiology, 145, 464-470.
Table S1 Effect of nano iron chelate on extraction yields, total phenolic contents and total flavonoid contents of Ca.copticum extracts.Extract / Extraction yielda / Total phenolicb / Total flavonoidc
1 / Control / 98.44±04 / 261.11±59 / 106.45±81
2 / Nano iron chelate (100 mgL-1 ) / 100.31±70 / 298.10±16 / 118.98±43
3 / Nano iron chelate (200 mgL-1 ) / 125.81±83 / 318.46±63 / 156.34±05
The data are expressed as mean±SD. aExpressed as mg of extract per gr dry material. bExpressed as mg of gallic acid per gr dry extract. cExpressed as mg of rutin per gr dry extract.
Table S2 Effect of nano iron chelate on chemical composition of Ca.copticum essential oil.Components / aCa.copticumEO (%) / bRetention Index / Identification Methods
Control (%) / Nano iron chelate (100 mgL-1 ) (%) / Nano iron chelate (200 mgL-1 ) (%)
1 / α-thujene / 0.60 / _ / _ / 850 / MS, RI
2 / α-pinene / 5.90 / 1.50 / 1.00 / 855 / MS, RI
3 / β-pinene / 0.70 / 0.30 / 0.10 / 190 / MS, RI
4 / β-myrcene / 0.90 / _ / _ / 920 / MS, RI
5 / P-cymene / 21.00 / 15.00 / 10.00 / 950 / MS, RI
6 / β-phellendrene / 3.54 / _ / _ / 954 / MS, RI
7 / limonene / 0.51 / 1.00 / 1.32 / 960 / MS, RI
8 / terpinene / 33.12 / 46.12 / 40.65 / 980 / MS, RI
9 / 4-terpineol / 0.63 / _ / _ / 1.63 / MS, RI
10 / cis limonene oxide / _ / 0.51 / 1.65 / 1085 / MS, RI
11 / dodecane / _ / 1.12 / 1.50 / 1110 / MS, RI
12 / β-fenchyl alcohol / 0.76 / _ / _ / 1126 / MS, RI
13 / thymol / 15.87 / 19.06 / 30.43 / 1208 / MS, RI
14 / Ethylene methacrylate / 0.54 / 0.49 / 0.26 / 1235 / MS, RI
15 / pentadecane / _ / 0.20 / 1.00 / 1264 / MS, RI
16 / hexadecane / 0.41 / _ / _ / 1285 / MS, RI
17 / nonadecane / 0.31 / _ / _ / 1293 / MS, RI
18 / carvacrol / _ / 3.64 / 10.87 / 1306 / MS, RI
Total / 84.79 / 85.30 / 87.91
Yield / 1.20% / 1.84% / 2.61%
a Percentage composition determined on column HP 5.b The retention Kovats indices were determined on HP 5 capillary column in reference to n-alkanes. MS= Mass Spectroscopy, RI= Retention Index.
Table S3 Efficacy of the different concentrations of Ca.copticum EO on dry mycelium weight and aflatoxin B1 synthesis by A. flavus.Oil concentration
(μL/mL) / Dry mycelium weight (mg) / AFB1
(μg/mL) / Inhibition of AFB1 synthesis
(%)
Control / 367.12±3.1 / 321.15±5.4 / -
1 / 291.34±0.3 / 231.67±4.5 / -
2 / 164.54±3.9 / 97.98±0.8 / -
3 / 95.03±4.0 / 34.9±0.0 / -
4 / 0.0±0.0 / 0.0±0.3 / 100
5 / 0.0±0.0 / 0.0±0.0 / 100
AFB1 = Aflatoxin B1 content (μg/mL). Experiments were carried out in triplicate and the results are expressed as mean±S.D.
Table S4 Fungitoxic spectrum of Ca.copticum EO against five fungalFungal / (%) Percent inhibition
1 (μl/mL) / 1.5 (μl/mL) / 2 (μl/mL)
A.flavus / 55.11±09 / 93.23±64 / 100
A.candidus / 89.11±59 / 100 / 100
A.oryze / 88.19±05 / 100 / 100
A.terreus / 76.34±73 / 100 / 100
A.niger / 66.00±45 / 88.12±20 / 100
Experiments were carried out in triplicate and the results are expressed as mean±S.D.
Table S5 Antifungal effect of Ca.copticum on wound-inoculated cherry tomatoesOil concentration / Infected fruits (%)
(μL/mL) / A.flavus / A.candidus / A.oryze / A.terreus / A.niger
Control / 65.05 / 85.23 / 58.95 / 89.32 / 50.67
25 / 54 / 78.34 / 54.87 / 76.98 / 40.76
50 / 46.76 / 69.56 / 48.56 / 70.45 / 31.76
75 / 22.51 / 35.56 / 18.67 / 46.87 / 16.78
100 / 7.01 / 15.23 / 6.05 / 16 / 5.98
Experiments were carried out in triplicate.
Table S6 Efficacy of Ca.copticum on fungal development in unwounded cherry tomatoes.Oil concentration / Infected fruits (%)
(μL/mL)
Control / 85.45
25 / 70.45
50 / 53.78
75 / 33.12
100 / 19.55
Experiments were carried out in triplicate.