Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.971796
SHORT COMMUNICATION Effect of Carum copticum essential oil on growth and aflatoxin formation by 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 (Received 16 September 2014; final version received 21 September 2014) The objectives of this study were to determine the antiaflatoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The C. copticum EO exhibited noticeable inhibition on dry mycelium and synthesis of aflatoxin B1 (AFB1) by Aspergillus flavus, completely inhibiting AFB1 production at 4 mL/mL. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 mL/mL with values of 5.01 ^ 67% for A. flavus and 5.98 ^ 54% for Aspergillus niger. The results indicated that the percentage of infected fruits is significantly ( p , 0.01) reduced by the EO at 168C for 30 days. In this case, the oil at 100 mL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. Thus, the EO of dill could be used to control food spoilage and as a potential source of food preservative. Keywords: Carum copticum; essential oil; antiaflatoxin B1; food preservative
1. Introduction Fungal contamination of food products is a chronic problem in developing countries which results in a decline in quality and quantity. The food and agriculture organisation has estimated that 25% of the world’s food crops are affected by mycotoxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins (Prakash et al. 2012). Among these, aflatoxins, produced by the Aspergillus flavus, are the most prominent mycotoxins occurring in culinary products. A. flavus is a major storage fungi found regularly in important cereal grains cultivated and stored throughout the world (Frisvad 1995), which produce aflatoxins B1, B2, G1 and G2. Ingestion of food contaminated with aflatoxins may cause chronic and acute toxicity including hepatotoxicity, teratogenicity, immunotoxicity and even death (Peraica et al. 1999). Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth (Sengul et al. 2009). Indeed, natural crude extracts and biologically active compounds from plant species used in traditional medicine may represent valuable sources for such new preservatives (Al-Fatimi et al. 2007). Carum copticum is an aromatic, grassy, annual plant grown in Iran. The seeds of C. copticum have several therapeutic effects, including diuretic, anti-vomiting, analgesic, antiasthma and antidyspnoea effects. In Persian folk medicine, the fruits of C. copticum were used as a diuretic, anti-vomiting, carminative and antihelmentic agent (Zargari 1988). Mohagheghzadeh et al. (2007) showed that C. copticum has two chemotypes, thymol and carvacrol. In this study, the effect of C. copticum essential oil (EO) on growth on aflatoxin formation by five fungal strains
*Email: [email protected] q 2014 Taylor & Francis
2
M. Kazemi
(A. flavus, Aspergillus candidus, Aspergillus oryze, Aspergillus terreus and Aspergillus niger) was evaluated 2. Results and discussion 2.1. Extraction yield, total phenolic contents and total flavonoid contents As shown in Table S1, the extraction yield of C. copticum ranged from lowest 98.44 ^ 04 mg g21 (control) to highest 125.81 ^ 83 mg g21 (nano-iron chelate (200 mg L21)). Among the three C. copticum extracts, C. copticum treated with nano-iron chelate at 200 mg L21 showed the highest total phenolic content (318.46 ^ 63 mg g21) and showed the highest total flavonoid content (156.34 ^ 05 mg g21). Furthermore, the total phenolic and total flavonoid contents exhibited the descending order: C. copticum extract (treated with nano-iron chelate 200 mg L21) . C. copticum extract (treated with nano-iron chelate 100 mg L21) . C. copticum extract (treated with control). These results showed that the total phenolic and total flavonoid contents have an obvious variation in various concentrations. 2.2. Chemical composition of C. copticum EO Hydrodistillation showed that C. copticum seeds contained 1.2% (v/w) EO. Results of GC/MS analysis of the EO (Table S1) indicate that g-terpinene was the main monoterpene hydrocarbon, with a content of 33.12%. The GC/MS analysis of C. copticum oil revealed 14 compounds representing 84.79% of the total oil; g-terpinene was the main constituent (33.12%), followed by p-cymene (21.00%), thymol (15.87%), a-pinene (5.90%), b-phellendrene (3.54%), b-myrcene (0.90%), b-fenchyl alcohol (0.76%), b-pinene (0.70%), 4-terpineol (0.63%), a-thujene (0.60%), ethylene methacrylate (0.54%), limonene (0.51), hexadecane (0.41%) and nonadecane (0.31%). The constituents of the obtained EOs of C. copticum treated with nano-iron chelate are presented in Table S2. Fourteen components were identified in untreated plants and 11 components in nano-iron chelate-treated plants (Table S2). The yield of the C. copticum EO was 1.20% in control, 1.84% (100 mg L21) and 2.61% (200 mg L21). Nano-iron chelate significantly increased the yield of EO (Table S2). Srivastava et al. (1999) detected 11 compounds in the C. copticum, with carvacrol (45.2%) and q-cymene (42.0%) being the major constituents. Kobraee et al. (2011) reported that nano-iron foliar application enhanced soybean yield by influencing the number of seeds per plant and seed weight. Therefore, iron deficiency in soils could be a restricting factor of yield and extremely decreases crop yield quality. Lu et al. (2002) have shown that application of nano-fertilizers could increase the nitrate reductase enzyme in soybean (Glycine max L.), increase its abilities of absorbing and utilising water and fertilizer, promote its antioxidant system, and, in fact, accelerate its germination and growth. 2.3. Efficacy of the EO on dry mycelium weight and aflatoxin B1 content The EO inhibited on A. flavus aflatoxin B1 (AFB1) production as well as mycelial dry weight in a dose-dependent manner and at 0.5 mL/mL, it completely inhibited the production of AFB1 (Table S3). The five different concentrations of EO caused different degrees of inhibition in terms of dry mycelium weight and AFB1 synthesis ( p , 0.01). The EO completely inhibited mycelial production at 4 mL/mL. A. flavus can produce AFB1, so we examined the effect of C. copticum EO on AFB1 production. Our results showed that C. copticum EO can effectively reduce dry mycelium weight and inhibit the synthesis of AFB1 in A. flavus. The antifungal mechanism of EO involves membrane disruption by their lipophilic compounds. The low molecular weight and highly lipophilic components of EO pass easily through cell membranes and cause disruption to the fungal cell organisation (Chao et al. 2005). Likewise, AFB1
Natural Product Research
3
production inhibition efficacy of EO may be because of inhibition of carbohydrate catabolism in A. flavus by acting on some key enzymes, reducing its ability to produce aflatoxins (Tian et al. 2011). 2.4. Fungitoxic spectrum of C. copticum EO In the antifungal assay, the EO of C. copticum exhibited a broad range of antifungal activities inhibiting all the moulds tested at 1. 5 mL/mL, except A. flavus and A. niger. However, at 2 mL/ mL it completely inhibited growth of all tested moulds (Table S4). The finding is in accordance with earlier report (Prakash et al. 2012), suggesting that some fungal species or strains may be reacting differently to specific fungicides/preservatives leading to stimulation of mycotoxin synthesis at certain higher concentrations. The Z C. copticum EO also showed a broad fungitoxic spectrum. The findings thus strengthen the possibility of its economic application at a low concentration for protection of food commodities from spoilage by various fungals. 2.5. Effect of C. copticum on the conservation and healthy cherry tomatoes The effects of C. copticum EOs on fungal infection development in wound-inoculated cherry tomatoes are shown in Table S5. The percentages of decayed cherry tomatoes were significantly ( p , 0.01) reduced in all four treatment groups compared with those of the control groups, and percentages also significantly ( p , 0.01) reduced with increasing concentration of C. copticum. Almost all the cherry tomatoes were spoiled in the absence of EO. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 mL/mL, with values of 15.23 ^ 28% for A. candidus, 6.05 ^ 33% for A. oryze, 5.01 ^ 67% for A. flavus, 16.00 ^ 32% for A. terreus and 5.98 ^ 54% for A. niger. The results obtained using EO in unwounded cherry tomatoes are shown in Table S6. The results indicated that the percentage of infected fruits is significantly ( p , 0.01) reduced by the EO at 168C for 30 days. In this case, the EO at 100 mL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. 3. Conclusion Our data indicate that the EO extracted from C. copticum exhibits potent biological activities, which support their use in traditional medicine. Moreover, results regarding the bioactivities of the main volatile components suggest that the observed activities of the EO are connected to its chemical composition, where g-terpinene, thymol and carvacrol have been found to be the most active compounds. There was a good correlation between total phenol content and antifungal, anti-AFB1 capacity of the extracts. In conclusion, C. copticum extracts appear to contain compounds with anti-AFB1 and antifungal activities. Supplementary material Experimental details relating to this article are available online, alongside Tables S1 –S6. References Al-Fatimi M, Wurster M, Schroder G, Lindequist U. 2007. Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen. J Ethnopharmacol. 111:657–666. Chao LK, Hua KF, Hsu HY, Cheng SS, Lin JY, Chang ST. 2005. Study on the antiinflammatory activity of essential oil from leaves of Cinnamomum osmophloeum. J Agric Food Chem. 53:7274– 7278. Frisvad JC. 1995. Mycotoxins and mycotoxigenic fungi. In: Jayas DS, White NDG, Muir WE, editors. Storage grain ecosystems. New York: Marcel Dekker; p. 251 –258.
4
M. Kazemi
Kobraee S, Shamsi K, Rasekhi B. 2011. Effect of micronutrients application on yield and yield components of soybean. Ann Biol Res. 2:476–482. Lu CM, Zhang CYW, Tao MX. 2002. Research of the effect of nanometer on germination and growth enhancement of Glycine max L. and its mechanism. Soybean Sci. 21:168–172. Mohagheghzadeh A, Faridi P, Ghasemi Y. 2007. Carum copticum Benth & Hook essential oil chemotypes. Food Chem. 100:1217–1219. Peraica M, Radic B, Lucic A, Pavlovic M. 1999. Toxic effects of mycotoxins in humans. Bull WHO. 77:754–766. Prakash B, Singh P, Kumar Mishra P, Dubey NK. 2012. Safety assessment of Zanthoxylum alatum Roxb. essential oil, its antifungal, antiaflatoxin, antioxidant activity and efficacy as antimicrobial in preservation of Piper nigrum L. fruits. Int J Food Microbiol. 153:183–191. Sengul M, Yildiz H, Gungor N, Cetin B, Eser Z, Ercili S. 2009. Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants. Pak J Pharm Sci. 22:102–106. Srivastava M, Saxena A, Baby P. 1999. GC–MS investigation and antimicrobial activity of the essential oil of Carum copticum Benth & Hook. Acta Aliment. 28:291–295. Tian J, Ban X, Zeng H, He J, Huang B, Youwei W. 2011. Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. Int J Food Microbiol. 145:464–470. Zargari A. 1988. Medicinal plants. Vol. 2. Tehran: Tehran University Publications.
SHORT COMMUNICATION Effect of Carum copticum essential oil on growth and aflatoxin formation by 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 (Received 16 September 2014; final version received 21 September 2014) The objectives of this study were to determine the antiaflatoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The C. copticum EO exhibited noticeable inhibition on dry mycelium and synthesis of aflatoxin B1 (AFB1) by Aspergillus flavus, completely inhibiting AFB1 production at 4 mL/mL. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 mL/mL with values of 5.01 ^ 67% for A. flavus and 5.98 ^ 54% for Aspergillus niger. The results indicated that the percentage of infected fruits is significantly ( p , 0.01) reduced by the EO at 168C for 30 days. In this case, the oil at 100 mL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. Thus, the EO of dill could be used to control food spoilage and as a potential source of food preservative. Keywords: Carum copticum; essential oil; antiaflatoxin B1; food preservative
1. Introduction Fungal contamination of food products is a chronic problem in developing countries which results in a decline in quality and quantity. The food and agriculture organisation has estimated that 25% of the world’s food crops are affected by mycotoxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone and fumonisins (Prakash et al. 2012). Among these, aflatoxins, produced by the Aspergillus flavus, are the most prominent mycotoxins occurring in culinary products. A. flavus is a major storage fungi found regularly in important cereal grains cultivated and stored throughout the world (Frisvad 1995), which produce aflatoxins B1, B2, G1 and G2. Ingestion of food contaminated with aflatoxins may cause chronic and acute toxicity including hepatotoxicity, teratogenicity, immunotoxicity and even death (Peraica et al. 1999). Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth (Sengul et al. 2009). Indeed, natural crude extracts and biologically active compounds from plant species used in traditional medicine may represent valuable sources for such new preservatives (Al-Fatimi et al. 2007). Carum copticum is an aromatic, grassy, annual plant grown in Iran. The seeds of C. copticum have several therapeutic effects, including diuretic, anti-vomiting, analgesic, antiasthma and antidyspnoea effects. In Persian folk medicine, the fruits of C. copticum were used as a diuretic, anti-vomiting, carminative and antihelmentic agent (Zargari 1988). Mohagheghzadeh et al. (2007) showed that C. copticum has two chemotypes, thymol and carvacrol. In this study, the effect of C. copticum essential oil (EO) on growth on aflatoxin formation by five fungal strains
*Email: [email protected] q 2014 Taylor & Francis
2
M. Kazemi
(A. flavus, Aspergillus candidus, Aspergillus oryze, Aspergillus terreus and Aspergillus niger) was evaluated 2. Results and discussion 2.1. Extraction yield, total phenolic contents and total flavonoid contents As shown in Table S1, the extraction yield of C. copticum ranged from lowest 98.44 ^ 04 mg g21 (control) to highest 125.81 ^ 83 mg g21 (nano-iron chelate (200 mg L21)). Among the three C. copticum extracts, C. copticum treated with nano-iron chelate at 200 mg L21 showed the highest total phenolic content (318.46 ^ 63 mg g21) and showed the highest total flavonoid content (156.34 ^ 05 mg g21). Furthermore, the total phenolic and total flavonoid contents exhibited the descending order: C. copticum extract (treated with nano-iron chelate 200 mg L21) . C. copticum extract (treated with nano-iron chelate 100 mg L21) . C. copticum extract (treated with control). These results showed that the total phenolic and total flavonoid contents have an obvious variation in various concentrations. 2.2. Chemical composition of C. copticum EO Hydrodistillation showed that C. copticum seeds contained 1.2% (v/w) EO. Results of GC/MS analysis of the EO (Table S1) indicate that g-terpinene was the main monoterpene hydrocarbon, with a content of 33.12%. The GC/MS analysis of C. copticum oil revealed 14 compounds representing 84.79% of the total oil; g-terpinene was the main constituent (33.12%), followed by p-cymene (21.00%), thymol (15.87%), a-pinene (5.90%), b-phellendrene (3.54%), b-myrcene (0.90%), b-fenchyl alcohol (0.76%), b-pinene (0.70%), 4-terpineol (0.63%), a-thujene (0.60%), ethylene methacrylate (0.54%), limonene (0.51), hexadecane (0.41%) and nonadecane (0.31%). The constituents of the obtained EOs of C. copticum treated with nano-iron chelate are presented in Table S2. Fourteen components were identified in untreated plants and 11 components in nano-iron chelate-treated plants (Table S2). The yield of the C. copticum EO was 1.20% in control, 1.84% (100 mg L21) and 2.61% (200 mg L21). Nano-iron chelate significantly increased the yield of EO (Table S2). Srivastava et al. (1999) detected 11 compounds in the C. copticum, with carvacrol (45.2%) and q-cymene (42.0%) being the major constituents. Kobraee et al. (2011) reported that nano-iron foliar application enhanced soybean yield by influencing the number of seeds per plant and seed weight. Therefore, iron deficiency in soils could be a restricting factor of yield and extremely decreases crop yield quality. Lu et al. (2002) have shown that application of nano-fertilizers could increase the nitrate reductase enzyme in soybean (Glycine max L.), increase its abilities of absorbing and utilising water and fertilizer, promote its antioxidant system, and, in fact, accelerate its germination and growth. 2.3. Efficacy of the EO on dry mycelium weight and aflatoxin B1 content The EO inhibited on A. flavus aflatoxin B1 (AFB1) production as well as mycelial dry weight in a dose-dependent manner and at 0.5 mL/mL, it completely inhibited the production of AFB1 (Table S3). The five different concentrations of EO caused different degrees of inhibition in terms of dry mycelium weight and AFB1 synthesis ( p , 0.01). The EO completely inhibited mycelial production at 4 mL/mL. A. flavus can produce AFB1, so we examined the effect of C. copticum EO on AFB1 production. Our results showed that C. copticum EO can effectively reduce dry mycelium weight and inhibit the synthesis of AFB1 in A. flavus. The antifungal mechanism of EO involves membrane disruption by their lipophilic compounds. The low molecular weight and highly lipophilic components of EO pass easily through cell membranes and cause disruption to the fungal cell organisation (Chao et al. 2005). Likewise, AFB1
Natural Product Research
3
production inhibition efficacy of EO may be because of inhibition of carbohydrate catabolism in A. flavus by acting on some key enzymes, reducing its ability to produce aflatoxins (Tian et al. 2011). 2.4. Fungitoxic spectrum of C. copticum EO In the antifungal assay, the EO of C. copticum exhibited a broad range of antifungal activities inhibiting all the moulds tested at 1. 5 mL/mL, except A. flavus and A. niger. However, at 2 mL/ mL it completely inhibited growth of all tested moulds (Table S4). The finding is in accordance with earlier report (Prakash et al. 2012), suggesting that some fungal species or strains may be reacting differently to specific fungicides/preservatives leading to stimulation of mycotoxin synthesis at certain higher concentrations. The Z C. copticum EO also showed a broad fungitoxic spectrum. The findings thus strengthen the possibility of its economic application at a low concentration for protection of food commodities from spoilage by various fungals. 2.5. Effect of C. copticum on the conservation and healthy cherry tomatoes The effects of C. copticum EOs on fungal infection development in wound-inoculated cherry tomatoes are shown in Table S5. The percentages of decayed cherry tomatoes were significantly ( p , 0.01) reduced in all four treatment groups compared with those of the control groups, and percentages also significantly ( p , 0.01) reduced with increasing concentration of C. copticum. Almost all the cherry tomatoes were spoiled in the absence of EO. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 mL/mL, with values of 15.23 ^ 28% for A. candidus, 6.05 ^ 33% for A. oryze, 5.01 ^ 67% for A. flavus, 16.00 ^ 32% for A. terreus and 5.98 ^ 54% for A. niger. The results obtained using EO in unwounded cherry tomatoes are shown in Table S6. The results indicated that the percentage of infected fruits is significantly ( p , 0.01) reduced by the EO at 168C for 30 days. In this case, the EO at 100 mL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. 3. Conclusion Our data indicate that the EO extracted from C. copticum exhibits potent biological activities, which support their use in traditional medicine. Moreover, results regarding the bioactivities of the main volatile components suggest that the observed activities of the EO are connected to its chemical composition, where g-terpinene, thymol and carvacrol have been found to be the most active compounds. There was a good correlation between total phenol content and antifungal, anti-AFB1 capacity of the extracts. In conclusion, C. copticum extracts appear to contain compounds with anti-AFB1 and antifungal activities. Supplementary material Experimental details relating to this article are available online, alongside Tables S1 –S6. References Al-Fatimi M, Wurster M, Schroder G, Lindequist U. 2007. Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen. J Ethnopharmacol. 111:657–666. Chao LK, Hua KF, Hsu HY, Cheng SS, Lin JY, Chang ST. 2005. Study on the antiinflammatory activity of essential oil from leaves of Cinnamomum osmophloeum. J Agric Food Chem. 53:7274– 7278. Frisvad JC. 1995. Mycotoxins and mycotoxigenic fungi. In: Jayas DS, White NDG, Muir WE, editors. Storage grain ecosystems. New York: Marcel Dekker; p. 251 –258.
4
M. Kazemi
Kobraee S, Shamsi K, Rasekhi B. 2011. Effect of micronutrients application on yield and yield components of soybean. Ann Biol Res. 2:476–482. Lu CM, Zhang CYW, Tao MX. 2002. Research of the effect of nanometer on germination and growth enhancement of Glycine max L. and its mechanism. Soybean Sci. 21:168–172. Mohagheghzadeh A, Faridi P, Ghasemi Y. 2007. Carum copticum Benth & Hook essential oil chemotypes. Food Chem. 100:1217–1219. Peraica M, Radic B, Lucic A, Pavlovic M. 1999. Toxic effects of mycotoxins in humans. Bull WHO. 77:754–766. Prakash B, Singh P, Kumar Mishra P, Dubey NK. 2012. Safety assessment of Zanthoxylum alatum Roxb. essential oil, its antifungal, antiaflatoxin, antioxidant activity and efficacy as antimicrobial in preservation of Piper nigrum L. fruits. Int J Food Microbiol. 153:183–191. Sengul M, Yildiz H, Gungor N, Cetin B, Eser Z, Ercili S. 2009. Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants. Pak J Pharm Sci. 22:102–106. Srivastava M, Saxena A, Baby P. 1999. GC–MS investigation and antimicrobial activity of the essential oil of Carum copticum Benth & Hook. Acta Aliment. 28:291–295. Tian J, Ban X, Zeng H, He J, Huang B, Youwei W. 2011. Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. Int J Food Microbiol. 145:464–470. Zargari A. 1988. Medicinal plants. Vol. 2. Tehran: Tehran University Publications.
