Abstract: Background and Objective: Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. The aim of this investigation was to evaluate the inhibition of growth Aspergillus flavus E73 (A. flavus E73) and AFB1 production by Cuminum cyminum L. (C. cyminum L.) and Coriandrum sativum L. (C. sativum L.) essential oils (EOs) as well their antioxidant and phytotoxicity activities. Methodology: The C. cyminum L. and C. sativum L. EOs were extracted by hydrodistillation. The chemical profile of EOs was identified by GC-MS, antifungal activity was assessed by poisoned food technique and in term Minimal Inhibitory Concentration (MIC) and minimal fungicidal concentration (MFC) and antiaflatoxin effect by broth medium. The antioxidant activity of EOs was determined by DPPH free radical scavenging assay, β-carotene bleaching test and total phenolic content by Folin-Ciocalteu. Phytotoxicity of C. cyminum L. and C. sativum L. EOs were determined for varieties of wheat. The results were analyzed by analysis of variance (one way ANOVA). Results: The GS/MS analysis showed that the major components of C. cyminum L. EO were cuminaldehyde (65.98%), o-cymene (18.40%) and C. sativum L. EO was mainly consisted of linalool (78.86%). The results showed that both the EOs could inhibit the growth of A. flavus E73 in the range of 24.27-84.90% for C. cyminum and 15.09-65.00% for C. sativum. During antiaflatoxin investigation, the oils exhibited noticeable inhibition on dry mycelium weight and synthesis of AFB1 by A. flavus E73. EOs of C. cyminum L. and C. sativum L. revealed complete inhibition of AFB1 at 1.25 and 1.5 mg mL–1, respectively. EOs exhibited inhibitory influence against some fungi. The IC50 values of C. cyminum L. and C. sativum L. EOs were 494.93 and 756.43 μg mL–1, respectively, while, β-carotene/linoleic acid bleaching was 47.68 and 29.29% , respectively. Total phenolic content of C. cyminum L. and C. sativum L. were 10.66 and 6.2 μg mg–1. Additionally, the EOs were non-phytotoxic on the two verities of wheat seeds. Conclusion: The C. cyminum L. and C. sativum L EOs could be good alternative to protect foods.
INTRODUCTION
The plants are a promising alternative because plants produce a variety of components. Many of plants are generally recognized as safe (GRAS) by the Unites States Food and Drug Administration (FDA). Essential oils (EOs) are natural, volatile, complex plant compounds1 which can be obtained from different parts of plant such as flowers, seeds, leaves, bark, herbs, fruits and roots by expression, enfleurage, extraction and method of steam distillation. Some EOs appear as a promising approach for inhibition of aflatoxin production which are synthesized by A. flavus group species. The most important fungi capable to produce the aflatoxins are A. flavus, A. parasiticus 2. Aflatoxins affect cereals, oil seeds, nuts, dry fruits, spices, legumes, fruits, milk and milk derivates3-4. It has been reported that consuming food contaminsated with aflatoxins especially AFB1 can cause hepatic carcinoma and other serious diseases vis teratogenicity, immunosuppression and mutagenicity for human beings and other livestock5. Another attributes have been revealed by EOs in their antioxidant activity as well as non phytotoxic.
Cumin (Cuminum cyminum L.) is herbaceous plant from the Apiaceae family, cultivated basically in Saudi Arabia, India and China6. Cumin considered as the second spice after black pepper7. Cumin seeds are used in cuisines of many countries such as India, Pakistan, North Africa, Srilanka, Cuba and Mexico8. Cumin seeds are used in traditional medicine to treat diseases as toothache, dyspepsia, diarrhea, epilepsy and jaundice9. Cumin seeds are also reported to have antioxidant and antimicrobial activity.
Coriander (Coriandrum sativum L.) is a plant belonging to the Umbelliferae family. It has various uses, in flavouring, perfumes and cosmetic products. In traditional medicine, C. sativum L. have been recommended for dyspepsia, loss of appetite, convulsion and insomnia10. It has been proved that C. sativum L. possesses antimicrobial and antioxidant activities.
The EOs of C. cyminum L. and C. sativum L. are consisted of different amounts and volatile components. Chemotypes have been reported for both plants which can be affected by various parameters such as region, environmental conditions, age of plant, the season and the method of extraction.
The study was undertaken to investigate the chemical composition of the EOs from C. cyminum L. and C. sativum L. and to evaluate their antifungal, antiaflatoxin, antioxidant activity and phytotoxicity.
MATERIALS AND METHODS
Plant material: Essential oils (EOs) were isolated from seeds of C. cyminum and C. sativum L. collected from the Garden of Reghaia, Algiers, Algeria in 2015. The identification of the two species was firstly given based on their morphological appearances and then confirmed by Doctor Mahdid Mouhamed of Laboratory of Vegetal Ecophysiology of Biology, Department in Normal High School, Kouba, Algiers, Algeria.
Extraction of essential oils: Two hundred grams of dried seeds was subjected to hydrodistillation in Clevenger’s apparatus for 3 h. The water traces in the EOs eliminated with anhydrous sodium sulphate (Na2SO4). EOs were weighted and stored at 4°C in for further using.
Essential oil analysis
Gas Chromatography-Mass Spectrometry (GC-MS) analysis: The chemical composition of the EO was analyzed using GC-MS. The EO (10 μL) was dissolved in hexane (100 μL) and 2 μL of the solution was injected into a GC-MS (AGILENT, model 6850 and 7890). The capillary column was DB-5 (length = 30 m×0.25 mm i.d., film thickness = 0.25 μm). Helium was used as the carrier gas at a flow rate of 1.0 mL min–1. The column inlet pressure was 8.07 psi. The GC column oven temperature was increased from 60-245°C at 3°C min–1, with a final hold time of 4 min. The EI-MS operating parameters were as follows: Electron energy, 70 eV; automatic scanning of the mass range 50-550 amu; ion source temperature, 230°C and quadrupole, 150°C.
Identification of the volatile compounds: The identification of the volatile compounds was done by comparing the mass spectra (MS) obtained with the NIST electronic databases as well as with the bibliography of Adam11 in parallel with the use of retention indices (RI) based on series of n-alkane indices (C8-C27) on the capillary column.
Fungal material and preparation of spore inoculum: The aflatoxigenic strain A. flavus E73 utilized in this study was obtained from Laboratoire de Biologie des Systèmes Microbiens (LBSM, Kouba, Algeria). Spore inoculum was prepared from the culture of A. flavus E73 on Petri dish containing Potato dextrose agar (PDA) for 7 days at 28±2°C and spores were obtained by washing petri dish with 20 mL o f 0.1% Tween 80 solution. The number of spores (1×106 spores mL–1) was determined using a hemocytometer slide (depth 0.2 mm, 1/400 mm2) under a light microscope (Motic: BA210, China). The number of spores of 1×106 mL–1 was fixed throughout this study.
Antifungal assay: Antifungal activity of C. cyminum L. and C. sativum L. EOs was tested against the A. flavus E73 following the poisoned food technique12. Different concentrations of EOs were added to 10 mL PDA at 45-50°C to obtain final concentrations (0.25-2 mg mL–1) and poured into petri dishes. Thereafter, 10 μL of spore suspension was spotted in the centre of each Petri dish and were incubated at 28±2°C for 7 days. The controls were prepared in parallel without EO. Measurements were made daily by taking the average of two perpendicular diameters of each colony. The comparison of the dimensions obtained with those of the controls made it possible to calculate the percentage inhibition (% I) at day 7, according to the following formula:
| Da | = | Average diameter of A. flavus E73 growth in the treatment |
| Db | = | Average diameter of A. flavus E73 growth in the control |
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC): The MIC and MFC for A. flavus E73 were assessed by broth method of Shukla et al.13. Different concentrations (0.25-2 mg mL–1) of C. cyminum L. and C. sativum L. EOs were added to 10 mL SMKY broth medium in test tubes. Tubes with only SMKY (sucrose: 200 g, MgSO4.7H2O: 0.5 g, KNO3: 0.3 g, yeast extract: 7 g 1000 mL distilled water) medium (10 mL) used as control. The tubes were inoculated with 10 μL of spore suspension and incubated at 28±2°C for 7 days. The lowest concentration of EOs that did not show any growth of A. flavus E73 during 7 days was considered as the MIC. After 7 days, 100 μL from the tubes, where there was no growth, were subcultured on fresh PDA. The lowest concentration of EOs, where no growth reversal carried out during 7 days of incubation was considered as the MFC.
Evaluation of essential oils as aflatoxin B1 suppressor: According to Mishra et al.14, suspensions (50 μL) of A. flavus E73 were inoculated in 25 mL of SMKY medium supplemented with different concentrations of each EO. Cultures were incubated at 28±2°C. SMKY broth containing only 50 μL of spore suspension as a control. Three repetition of each treatment were occurred. For the extraction of AFB1. The content was filtered (Whatman No. 1) and extracted with 20 mL chloroform (Sigma Aldrich, France). After stirring and then decanting, the chloroform phase was recovered, evaporated and redissolved in 1 mL chloroform. A volume of 50 μL of sample was spotted on a Thin Layer Chromatography (TLC) (Silica gel, Fluka, Germany). The development of the chromatograms was carried out in a standard tank (20×20 cm) previously saturated with the solvent system: Toluene: Iso-amyl alcohol: Methanol (90: 32: 2, v/v/v) (Sigma Aldrich, France). After migration, the plates was removed and dried at 60°C for 24 h. AFB1 were detected by placing the plate in UV transilluminator (360 nm) (CN-6, VILBER LOURMAY, France). The AFB1 appeared as a blue spot.
The mycelia produced in the liquid cultures were removed and washed on Whatman No. 1. The weight of the mycelium was determined after desiccation at 80°C for 12 h. For the quantification of AFB1, the blue spots on TLC plates were scraped out, dissolved in 5 mL cold methanol and centrifuged at 2000 rpm (Jouan E76) for 5 min. The absorbance of the supernatant was made using a UV-Visible spectrophotometer (6705 UV/Vis, JENWAY) at 360 nm. The quantity of AFB1 was calculated according to the formula by Tian et al.15:
| D | = | Absorbance |
| M | = | Molecular weight of aflatoxin (312 g mol–1) |
| E | = | Molar extinction coefficient (21, 800 L mol–1 cm) |
| l | = | Path length (1 cm cell was used) |
Spectrum of fungitoxicity: The A. carbonarius, A. fumigatus, A. niger, A. ochraceus, A. tamari, A. terreus, Fusarium sp., Penicillium sp. and Rhizopus sp., were used in this investigation to study the antifungal activity of C. cyminum L. and C. sativum L. EOs in terms of The MIC and MFC.
Evaluation of antioxidant activity| Ablank | = | Absorbance of the control |
| Asample | = | Absorbance of the sample |
IC50 was calculated from the graph plotting between percentage inhibition and concentration. The IC50 value was defined as the quantity of antioxidant necessary to inhibit DPPH radical formation by 50%. The results were expressed as the mean values ±SD.
β-carotene/linoleic acid bleaching method: A solution consisted of 0.5 mg of β-carotene in 1 mL of chloroform, 25 μL of linoleic acid and 200 mg Tween 40 (Sigma Aldrich, France) was prepared. After elimination of chloroform by rotary evaporator at 40°C, 100 mL of distilled water was added and the mixture was agitated. EOs (2 g L–1) were dissolved in Dimethyl Sulfoxide (DMSO) (Sigma Aldrich, France) and then 350 μL were added to 2.5 mL of the above mixture and incubated in water bath at 50°C, for 2 h with blanks17. BHT was used as a positive control and DMSO as a negative control. The absorbance was estimated spectrophotometrically at 470 nm and the antioxidant activity (% I) was calculated using the formula:
| Aβ-carotene after 2 h | = | Absorbance of β-carotene after 2 h of the experiments |
| Ainitial β-carotene | = | Absorbance of β-carotene at the beginning of the experiments. The results were expressed as the mean values ±SD |
Determination of total phenolic content of EOs: Aliquots of 125 μL of EOs in DMSO were dissolved in 500μL of distilled water and 125 μL of Folin-Ciocalteu reagent 10 times diluted (Sigma Aldrich, France). The mixture was agitated and incubated for 3 min and then 1.25 mL of 7% Na2CO3 was added, adjusting with distilled water to 3 mL. After incubation for 2 h at 25±2°C, the absorbance at 760 nm was measured18. The same procedure was also applied to the standard solutions of gallic acid (25-200 μg mL–1).
Total phenolic contents concentration of the EOs was calculated from the regression equation of the curve established with the standard gallic acid and expressed in micrograms of equivalents, gallic acid per milligram of EO. The results were expressed as the mean values ±SD.
Phytotoxicity assay: The phytotoxicity of EOs was determined for varieties of Triticum aestivum (wheat) viz. AS 81189 A (Ain Abid) and HD 1220 (Hiddab) (Dar El Beida, Algiers, Algeria)19. After surface-sterilizing of wheat seeds with sodium hypochlorite (1%) for 20 min, rinsing and removing empty and undeveloped seeds. Two layers of filter paper were placed on each petri plate and then 10 mL of distilled water were poured. Afterwards, 50 wheat seeds were deposited on the filter paper. Ten microliters of each EO was dropped on Whatman No.1 and placed on the lid. Controls were also prepared but no EO was added. Petri plates were closed with parafilm and incubated at 23±2°C. After 8 days of incubation, the experiment was stopped and the percentage of germination of each variety was determined. The germination rate corresponds to the maximum percentage of germinated seeds in relation to the total seed. After determining the number of seeds that germinated, the lengths of the radicle and the plumule were measured. The results were reported as the mean values ±SD.
Statistical analysis: All experiments were repeated three times and the results were analyzed by analysis of variance (one way ANOVA) and the mean values (±SD) were considered significantly different when p<0.05 using STATISTICA version 6.
RESULTS AND DISCUSSION
Yield and chemical composition of EOs: The yield of C. cyminum and C. sativum EOs was 1.08±0.15 and 0.70±0.19%, respectively. The results of present study were in accordance with these of Kedia et al.20, who observed that the yield of C. cyminum EO was between 0.9 and 1.2%. Ravi et al.21 reported that C. sativum EO yield was 0.82%.
The chemical composition of C. cyminum and C. sativum EO are presented in Table 1. A total of 16 constituents of C. cyminum oil, representing 99.98% of the EO. The major components of EO were cuminaldehyde (65.98%), o-cymene (18.39%), α-methyl-benzene methanol (4.51%), β-pinene (4.38%) and 2-thiophene aldehyde (1.89%). The results of present study differed from previous studies where Algerian C. cyminum EO was mainly consisted of cuminaldehyde and the 1-phenyl-1,2-ethanediol22. Romeilah et al.23 reported that caryophyllene oxide (6.12%), β-pinene (4.89%), geranyl acetate (4.11%) and β-caryophyllene (3.44%) were the most abundant components in C. cyminum EO. Other abundant components in C. cyminum were α-pinene (29.1%), 1, 8-cineole (17.9%) and linalool (10.4%)24.
| Table 1: | Chemical composition of C. cyminum and C. sativum EOs |
RI: Retention indices |
|
In another study of Naeini et al.25, α-pinene (30%), limonene (21%) and 1,8-cineole (18.5%) were the main constituents of C. cyminum EO. On the other hand, chemical composition of present study C. sativum EO constituted of 19 compounds, representing 99.98% of the EO, linalool (78.86%), geranyl acetate (4.54%), γ-terpinene (3.33%), α-pinene (2.70%), p-cymene (2.65%), camphor (2.28%), geraniol (1.12%) and limonene (1.06%). Zoubiri and Baaliouamer26 revealed that linalool (73.11%), p-mentha-1,4-dien-7-ol (6.51%), α-pinene (3.41%) and neryl acetate (3.22%) were the main constituents in Algerian C. sativum EO. Samojlik et al.27 exhibited 14 chemical constituents in C. sativum and its major components were linalool (74.6%), camphor (5.9%), geranyl acetate (4.6%) and p-cymene (4.0%).
Antifungal activity assay: The antifungal activity of C. cyminum and C. sativum EOs is represented in Table 2. A significant activity (p<0.05) has been remarked with increasing the concentration of C. cyminum and C. sativum EOs. As shown, the growth of A. flavus E73 was delayed by 4 days at 1.0 mg mL–1 for C. cyminum and by 1 day at 1.25 mg mL–1 for C. sativum. The percentage inhibition of the growth of A. flavus E73 was reported in the range of 24.27-84.90% for C. cyminum and 15.09-65.00% for C. sativum.
The antifungal mechanism of EO components is not completely clear yet. However, their low-molecular weight and highly lipophilicity make them pass easily through membranes and disrupt cell organization of the fungus28.
C. cyminum EO exhibited good antifungal activity which might be attributed to the dominance of o-cymene and cuminaldehyde. These two volatile compounds have been shown to have strong antifungal activity29-31. The monoterpene hydrocarbons, β-pinene might be also involved in the higher antifungal activity of C. cyminum EO.
| Table 2: | Antifungal activity of C. cyminum and C. sativum EOs on A. flavus E73 |
Values are Mean±SD (n = 3) |
|
| Table 3: | Effect of C. cyminum and C. sativum EOs on dry weight of mycelium and AFB1 production |
Values are Mean±SD (n = 3) |
|
According to De Souza et al.32, pinenes showed antibacterial and antifungal activity. Despite that linalool was found as major constituent, C. sativum EO showed moderate antifungal activity. Stevic et al.33 reported that linalool was dominant in coriander with moderate to good antifungal activity. Mishra et al.14 indicated that linalool was moderately antifungal against A. flavus.
Generally speaking, there is evidence that minor and major components interact in synergistic and antagonistic manner.
The MICs and MFCs of C. cyminum and C. sativum EOs were evaluated by broth dilution. This method allows to EOs to contact closely with fungal spores during the distribution in the medium34. Their study is important to determine the minimum concentration to inhibit fungal growth. The MIC of C. cyminum EO against A. flavus E73 was found at 1.25 mg mL–1 while MIC of C. sativum EO was observed at 1.5 mg mL–1. Results obtained from the MIC test were confirmed with MFC tests, where inhibitory activity of C. cyminum occurred at a concentration of 1.5 and 2.0 mg mL–1 for C. sativum.
It has been shown that the MIC of C. cyminum and C. sativum EOs were higher than that of Cicuta virosa30, Ocimum sanctum35 and C. cyminum L.20. However, it was found to be lower than C. sinensis var. Valencia36, C. sativum33 and some prevalent organic preservatives such as salicylic acid, BHA, BHT, ascorbic acid and gallic acid37.
Efficacy of the EOs on dry mycelium weight and aflatoxin B1 content: AFB1 is the most toxic compound. As a consequence, an attempt was made to assess the effect of C. cyminum and C. sativum EOs to inhibit AFB1 production. Results showed that C. cyminum and C. sativum EOs can inhibit weight of mycelium and the AFB1 synthesis (Table 3). As shown, results revealed that the dry weight of mycelium under the influence of C. cyminum was between 616.33 and 120 mg at concentrations ranged between 0.25 and 1 mg mL–1 compared to the control (905.33 mg) (p<0.05) and it was proportional to concentrations of the EO supplemented in the SMKY medium.
| Table 4: | Antifungal activity of C. cyminum and C. sativum EOs against some fungi |
It is clear that C. cyminum EO showed inhibition of dry weight of mycelium at all concentrations tested where complete inhibition was occurred at 1.25 mg mL–1. The same results were obtained for C. sativum EO, dry weight of mycelium diminished proportionally in concentrations ranging from 0.25-1.25 mg mL–1 (716-265 mg) when compared to the control (905.33 mg) (p<0.05) and the total inhibition was carried out at 1.5 mg mL–1.
AFB1 reduction from SMKY broth medium was dependent on the EO concentration. The treatment of A. flavus with different concentrations of C. cyminum EO caused varying degrees of AFB1 inhibition. It is apparently that C. cyminum EO at concentration of 0.75 mg mL–1 and 1 mg mL–1 decreased significantly (p< 0.05) the quantities of AFB1 to 195.59 μg mL–1 and 57.24 μg mL–1, respectively. The C. cyminum EO exhibited complete inhibition of AFB1 at 1.25 mg mL–1. On the hand, AFB1 inhibition increased with increasing of C. sativum EO concentrations. The EO generated significant inhibition of 381.65, 372.10 and 205.06 μg mL–1 at 0.75, 1 and 1.25 mg mL–1 in comparison with the control (868.25 μg mL–1) (p< 0.05). It should be noted that AFB1 was inhibited completely at 1.5 mg mL–1.
According to the above results, C. cyminum and C. sativum EOs inhibited A. flavus E73 growth and AFB1 production at the same concentrations. Present study findings were similar to the ones of Reddy et al.38, who found that Syzygium aromaticum inhibited A. flavus growth and AFB1 production at 5 g kg–1. Mishra et al.39 indicated that Jamrosa EO showed both antifungal activity and inhibition of AFB1 production at 0.4 μL mL–1, but the inhibition of AFB1 production cannot be completely attributed to reduced fungal growth. There were many studies confirmed this suggestion. Kumar et al.40 reported that Lantana indica EO completely inhibited A. flavus growth and AFB1 production at 1.5 and 0.75 μg mL–1, respectively. Similar types of results were obtained by Srivastava et al.41 where Cinnamomum camphora (C. camphora) and Alpinia galanga (A. galanga) inhibited A. flavus growth at 1000 ppm and the AFB1 production at 500 ppm for A. galanga and 750 ppm for C. camphora. In another study performed by Vilela et al.42, the inhibition of AFB1 required a concentration of Eucalyptus globulus EO greater than was for inhibition of A. flavus and A. parasiticus.
Because of the extramitochondrially biosynthesis of aflatoxins from acetylcoenzyme A during the glucose utilization. Thus, the inhibition of AFB1 production can be attributed to the inhibition of carbohydrate catabolism in A. flavus by acting on some enzymes in order to diminish its capacity of AFB1 production15. Generally, the inhibition mechanism of AFB1 production is not very clear as has been reported by those authors. So, C. cyminum and C. sativum EOs may interfere with some steps in the metabolic pathways of the A. flavus, which controls AFB1 biosynthesis.
Spectrum of fungitoxicity: The fungitoxicity of C. cyminum and C. sativum EOs at concentrations between 0.25 and 2 mg mL–1 was tested. Results of antifungal activity of the EOs are shown in Table 4. The C. cyminum EO inhibited the growth of most fungi at concentration between 0.5 and 1.75 mg mL–1. The highest concentration of this EO was that for A. carbonarus (1.75 mg mL–1) and the lowest was that for A. niger (0.5 mg mL–1). It can be clearly seen that C. cyminum EO showed slightly lower inhibition compared to C. sativum EO which was between 0.5 and 2 mg mL–1, except A. niger, A. fumigates and A. tamari, the inhibition occurred somehow at the same concentration (0.5, 1.0 and 1.5 mg mL–1, respectively). C. sativum EO exhibited antifungal activity against A. carbonarius and Penicillium sp. higher than C. cyminum EO with MIC 1.58 and 1.0 mg mL–1, respectively. Additionally, MFC was determined for C. cyminum and C. sativum EOs. Aligiannis et al.43 demonstrated that antimicrobial activity considered strong when MIC to 0.50 mg mL–1, moderate MIC between 0.6 and 1.5 mg mL–1, weak MIC over 1.5 mg mL–1.
| Table 5: | Antioxidant activity and total phenolic of C. cyminum and C. sativum EOs |
nd: Not determined. Values are Mean±SD (n = 3) |
|
From the results presented herein, C. cyminum and C. sativum EOs exhibited strong, moderate and weak activity. It should be noted that MFC values were greater than MIC values where they were between 1.25 and >2 mg mL–1 for C. cyminum EO, 1.58 and >2 mg mL–1 for C. sativum EO.
Many research works has studied the antifungal activity of EOs. Kedia et al.20 found that C. cyminum EO was active against fungi such as Alternaria alternata, A. niger, A. terreus, Mucor sp., Rhizopus stolonifer and Penicillium species. Prakash et al.37 reported that C. sativum EO exhibited inhibitory effect against A. niger, A. candidus, A. terreus, A. fumigatus, Alternaria alternata, Cladosporium cladosporioides, Fusarium nivale, Penicillium italicum at concentration ranging between 2 and 3 μg mL–1. Stevic et al.33 tested the antifungal activity of C. sativum against some fungi viz., A. flavus, A. niger, Alternaria alternata, 8 species of Fusarium, Penicillium sp., Chaetomium sp., Gliocladium roseum, Curvularia lunata, Verticillium dahliae, Trichoderma viride, Trichothecium roseum, Phomopsis sp., Phoma sp. and Myrothecium verrucaria. The authors found that the EO could inhibit these fungi at concentration between 0.97 and 5.10 mg mL–1. Other EOs have been also tested for their antifungal activity like lemon, orange, mandarin and grapefruit peels30, Bidens pilosa44 and cinnamon leaf45. Overall, based on the efficient antifungal activity of C. cyminum and C. sativum EOs, they would use for inhibition of fungal contamination of food and as consequence, used as plant antimicrobial.
Antioxidant activity: During this investigation, two different methods have been used to evaluate the antioxidant activity of C. cyminum and C. sativum EOs: The DPPH radical scavenging activity and β-carotene/linoleic acid bleaching.
Free radical-scavenging ability of C. cyminum and C. sativum EOs were measured by the DPPH and the obtained results were compared with the standard BHT, which is an efficient synthetic antioxidant agent in food. The DPPH scavenging activity was presented by IC50 value ( Table 5). The IC50 concentration and the antioxidant capacity have inversely proportional values and C. sativum (756.43±12.63 μg mL–1) was established to have the lowest antioxidant capacity while C. cyminum (494.93±8.82 μg mL–1) was found to be the richest. However, C. sativum and C. cyminum EOs exhibited lower antioxidant efficacy than BHT (306.15 ±4.49 μg mL–1) (p<0.05). These results were different of the ones of Romeilah et al.23, which reported that the radical scavenging activity of C. cyminum and C. sativum EOs were 83.59 and 74.72%, respectively at 200 μg mL–1. The IC50 (72.3 μg mL–1) of C. cyminum was lower than IC50 (74.05 μg mL–1) of C. sativum EO. Kedia et al.20 reported that C. cyminum EO showed strong free radical scavenging activity where its IC50 was found to be 0.092 μL mL–1. As well, Prakash et al.37 evaluated the antioxidant activity of C. sativum, showing that the IC50 value of the EO was 2.90 μL mL–1. This difference in DPPH radical scavenging activity could be explained by difference in the chemical composition of C. cyminum and C. sativum EOs.
The capacity of C. cyminum and C. sativum EOs to inhibit lipid peroxidation was tested by the β-carotene/linoleic acid bleaching test. The bleaching mechanism of β-carotene is the result of the formation of hydroperoxides from linoleic acid46. Chew et al.47 reported that the antioxidants in the different natural samples can limit β-carotene bleaching.
The oxidation of β-carotene was stopped by EOs of C. cyminum and C. sativum where values were about of 47.68±0.68 and 29.29±1.19%, respectively, comparable to BHT (94.77±1.61%) (p<0.05) (Table 5). Generally, results of β-carotene bleaching were less than those provided by the radical-scavenging activity.
Total phenolic content: Total phenolics were calculated as μg gallic acid equivalent mg–1 of EO. C. cyminum EO had higher total phenolic content (10.66±0.90 μg mg–1) than C. sativum EO (6.2±0.91 μg mg–1) (Table 5). Rebey et al.48 reported that phenolic content of C. cyminum EO was 18.32 mg g–1. On the other hand, Prakash et al.37 found that phenolic content of C. sativum EO was 4.15μg mg–1.
In comparison with present study results, previous studies showed a significant correlation between the antioxidant activity and total phenolic contents in C. sativum 49, herbs, vegetables and fruits50-51. However, the antioxidant activity of EOs cannot be related just to phenolics but to other compounds such as monoterpene alcohols, ketones, aldehydes and hydrocarbons.
It has been reported that the oxidative stress and peroxidation cause AFB1 production by Aspergillus spp.
| Table 6: | Phytotoxic influence of C. cyminum and C. sativum EOs on seed germination and seedling growth of two varieties of wheat |
Values are means (n = 3) ±SD |
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according to Jayashree and Subramanyam52, Narasaiah et al.53, Zjalic et al.54 and Kim et al.55. EOs have been shown to have antioxidant activity in this investigation. Hence, the inhibition of AFB1 by C. cyminum and C. sativum EOs may be related to their antioxidant nature.
Phytotoxicity assay: Phytotoxicity of the C. cyminum and C. sativum EOs were evaluated for assessing their effect on the germination and seedling growth of AS 81189 A (Ain Abid) and HD 1220 (Hiddab). As given in Table 6, C. cyminum and C. sativum EOs showed no significant effect on the germination of HD1220 (Hiddab) and AS 81189 A (Ain Abid) seeds (p>0.05). The length of radicles and plumules in the seeds tested with C. cyminum and C. sativum EOs was also diminished but the effect of C. sativum EO on the length of radicles and plumules of the seeds of AS 81189 A (Ain Abid) tested with C. sativum EO was greater than the others. However, EOs did not reveal somehow significant phytotoxicity against the seeds. Hence, C. cyminum and C. sativum EOs can be recommended for storing food items or sowing purpose.
CONCLUSION
Although C. cyminum and C. sativum EOs have resulted to possess variety of compounds, antifungal, antiaflatoxin, antioxidant activity, to inhibit fungi growth and show their non phytotoxicity, they require deep evaluation to transform them to more effective and safer preservatives and fungicides in order to decrease using chemical products because nowadays consumer is looking for foods that show more fresh-like and natural characteristics.
SIGNIFICANCE STATEMENT
This study discovers the possible effect of C. cyminum and C. sativum EOs that can be beneficial to control A. flavus, aflatoxin B1 production and fungal spoilage to assess antioxidant activity and using them as food preservative for enhancement of shelf life of stored food commodities. This study will help the researchers to uncover the critical area of natural alternative to apply in food that many researchers were not able to explore. Thus, a new theory on the relation between antioxidant activity of C. cyminum and C. sativum EOs and AFB1 production and possibly other activities, may be arrived at because this point is not completely understood.
ACKNOWLEDGMENTS
This study was financially supported by MESRS of Algeria (Program CNEPRU grant F00520120063). We are thankful to Mr. Tarek Benabdelkhader and Laboratoire de Biotechnologies Végétales appliquées aux plantes aromatiques et médicinales (BVpam) de la Faculté des Sciences et Techniques de Saint Etienne, France for Gas chromatography-mass spectrometry (GC-MS) analysis.