ارزیابی خاصیت ضد قارچی عصاره گیاه رزماری و تاثیر آن در بیان ژن AFL1 قارچ آسپرژیلوس فلاووس با استفاده از روش Real-Time PCR
الموضوعات :مجتبی محمدی 1 , سید جمال هاشمی 2 , ساسان رضایی 3 , منصور بیات 4
1 - دانشجوی دکتری، گروه میکروبیولوژی، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران
2 - استاد، گروه قارچ شناسی پزشکی، دانشکده بهداشت، دانشگاه علوم پزشکی تهران، تهران
3 - استاد، گروه قارچ شناسی پزشکی، دانشکده پزشکی، دانشگاه علوم پزشکی تهران، تهران
4 - استاد، گروه میکروبیولوژی، دانشکده دامپزشکی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران
الکلمات المفتاحية: گیاهان دارویی, آسپرژیلوس فلاووس, کاندیدا آلبیکنس, اپیدرموفایتون فلوکوزم, ترایکوفایتون وروکوزوم,
ملخص المقالة :
سابقه و هدف: رزماری گیاه دارویی مهمی است که تاکنون خاصیت ضد میکروبی آن بر روی قارچ های بیماری زا و توکسین زا کمتر مورد توجه قرار گرفته است. دست یابی به گیاهان دارویی، با توجه به محدود بودن داروهای ضد قارچی و مقاومت حاصل از آنها می تواند حائز اهمیت باشد. این مطالعه با هدف ارزیابی خاصیت ضد قارچی عصاره گیاه رزماری بر روی قارچ های آسپرژیلوس فلاووس، کاندیدا آلبیکنس، اپیدرموفایتون فلوکوزوم، ترایکوفایتون وروکوزوم و تاثیر این عصاره در بیان ژن 1 AFL قارچ آسپرژیلوس فلاووس با استفاده از روش Real -Time PCR انجام شد.مواد و روش ها: در ابتدا قارچ های آسپرژیلوس فلاووس و کاندیدا آلبیکنس بر روی محیط سابرودکستروز آگار و درماتوفیت های ترایکوفایتون وروکوزوم و اپیدرموفایتون فلوکوزوم بر روی محیط مایکوسل آگار با تراکم نیم مک فارلند کشت داده شدند. خاصیت ضد قارچی گیاه رزماری با روش انتشار در دیسک ارزیابی گردید. سپس غلظت موثر عصاره رزماری به کمک ده لوله استاندارد و محیط مایع سابرودکستروز براث محاسبه شد. در نهایت تاثیر عصاره بر بیان ژن 1 AFL با روش RT-PCR مورد بررسی قرار گرفت.یافته ها: عصاره رزماری بر روی انواع گروه های قارچی اثر بازدارندگی داشت. به طوری که متوسط قطر هاله عدم رشد حدود 16 تا 18 میلی متر تعیین گردید. MIC موثر برای قارچ کاندیدا آلبیکنس 4 تا 6 میلی گرم بر میلی لیتر، برای قارچ آسپرژیلوس فلاووس 3 تا 5 میلی گرم بر میلی لیتر و برای درماتوفیت های ترایکوفایتون وروکوزوم و اپیدرموفایتون فلوکوزوم نیز 4 تا 6 میلی گرم بر میلی لیتر تعیین شد. همچنین نتایج Real -Time PCR نیز مهار ژن تولید کننده آفلاتوکسین را در سطح مولکولی به خوبی به اثبات رسانید.نتیجه گیری: نتایج نشان داد که عصاره رزماری می تواند رشد قارچ ها را مختل نماید و به طور قابل توجهی تاثیر مهارکنندگی بر بیان ژن 1 AFL و تولید آفلاتوکسین در قارچ آسپرژیلوس فلاووس دارد.
licorice plant extract on aflR gene expression aflatoxin production in Aspergillus paraziticus
via Real-time PCR. Modares J Med Sci. 2012; 15(3): 63-77.
2. Eaton DL, Groopman JD. The toxicology of aflatoxins. Academic Press New York. 1994:
383-426.
3. Nabili M, Moazeni M, Taghizadeh Armaki M, Asgari MR, Nosrati A, Shokohi T. Diagnostic
tools in fungal infections since classical to molecular era. J Mazandaran Univ Med Sci. 2013;
23(104): 109-129.
4. Khan R, Islam B, Akram M, Shakil S, Ahmad AA, Ali SM, Siddiqui M, Khan AU.
Antimicrobial activity of five herbal extracts against multi drug resistant (MRD) strains of
bacteria and fungus of clinical origin. Molecules. 2009; 14: 586-597.
5. Moghtader M, Salari H, Farahmand A. Evaluation of the antifungal effects of the rosemary oil
and comparison whit synthetic borneol and funjicide on the growth of Aspergillus flavus. J Ecol
Natural Environ. 2011; 3(6): 210-214.
6. Rota C, Carraminana JJ, Burillo J, Herrera A. In vitro antimicrobial activity of essential oils
from aromatic plans against selected foodborne pathogens. J Food Prot. 2004; 67: 1252-1256.
7. Flamini G, Cioni PL, Morelli I, Macchia M, Ceccarini, L. Main agronimic-productive
characteristics of two ecotypes of Romarinus officinalis L. and chemical composition of their
essential oils. J Agr Food Chem. 2002; 50: 3512-3517.
8. Couladis M, Tzakou O, Verykokidou E. Screening of some Greek aromatic plants for
antioxidant activity. J Phytother Res. 2003; 17: 194-196.
9. Huang MT, Wang ZY, Ferraro T, Lou YR, Stauber K, Georgiadis C, Laskin JD, Conney A.
Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid.
Cancer Res. 1994; 54: 701-708.
10. Yu J, Chang PK, Ehrlish KC, Cary JW, Bhatnagar D, Cleveland TE, Payne GA, Linz JE,
Woloshok CP, Bennett JW. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ
Microbial. 2004; 70(3): 1253-1262.
11. Jamali A. Expression of aflatoxin genes aflO (omtB) and aflQ (ordA) differentiates levels of
aflatoxin production by Aspergillus flavus strains from soils of pistachio orchards. Res
Microbiol. 2013; 64: 293-299.
12. Yu J, Mohawed SM, Bhatnagar D, Cleveland TE. Substrate-induced lipase gene expression and
aflatoxin production in Aspergillus parasiticus and Aspergillus flavus. J Appl Microbiol. 2003;
95(6): 1334-13420.
13. El-Nagerabi S, Al-Bahry SN, Elshafie A, Al Hilali S. Effect of Hibiscus sabdariffa extract and
Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and
Aspergillus rasiticus strains. Food Cont. 2012; 25(1): 59-63.
14. Sienkiewicz M, Pastuszka M. The potential of use uasil and Rosemary essential oils as effective
antibacterial agent. Molecules. 2013; 18: 9334-9351.
15. Moreno S, Vojnov A. Antioxidant and antimicrobial activities of rozemary extracts linked to
their polyphenol composition. 2006; 40(2): 223-231.
16. Petersen M, Simmonds MSJ. Rosmarinic acid. Phytochem. 2003; 62: 121-125.
17. Lopez-Reyes JG, Spadaro D, Garibaldi A, Gullino ML. Efficacy of plant essential oils on
postharvest control of rots caused by fungi on different stone fruits in vivo. J Food Prot. 2013;
76(4): 631-639.
18. Chifiriuc C, Gumezescu V, saviue C, Lazar V, Andronescu E. Hybrid magnetite nanoparticles/
Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity. Nanoscale Res Lett.
2012; 7: 209.
19. Crawford JM, Kumar V. Liver and biliary tract. Pathologic basis of disease ed. Elsevier
Saundres. Philadelphia. 2005; p: 924.
20. Espinel-Ingroff AV, Pfaller MA. Susceptibility test methods; yeasts and filamentous fungi. In
Manual of clinical microbiology. 9th ed, Murray. ASM Press. Washington. 2007; p: 1972.
21. Masuda T, Inaba Y, Maekawa T, Takeda Y, Tamura H, Yamaguchi H. Recovery mechanism of
the antioxidant activity from carnosic acid quinine, an oxidized sage and rosemary antioxidant.
J Agric Food Chem. 2002; 50: 5863-5869.
22. Farzaneh M, Ahmadzadeh M, Hadian J, Tehrani AS. Chemical composition and antifugal
activity of the essential oils of three species of Artemisia on some soil-borne phytopathogens–
commun Agric. Apple Biol Sci. 2006; 71(3): 1327-1333.
23. Lopez-Malo A, Barreto-Valdivieso J, Palou E, Martin FS. Aspergillus flavus growth response
to cinnamon extract and sodium benzoate mixtures. Food Control. 2007; 18(11): 1358-1362.
24. Scorzoni L, Benaducci T, Almeida AMF, Silva DHS, Bolzani VS, Mendes-Giannini MJS.
Comparative study of disk diffusion and micro-dilution methods for evaluation of antifungal
activity of natural compounds against medical yeasts Candida spp. and Cryptococcus sp. Rev
Cienc Farm Basica Apl. 2007; 28: 25-34.
25. Santoyo S, Cavero S, Jaime L, Ibanez E, Senorans FJ, Reglero G. Chemical composition and
antimicrobial activity of Rosmarinus officinalis L. essential oil obtained via supercritical fluid
extraction. J Food Prot. 2005; 68(4): 790-795.
26. Watson AJ, Fuller LJ, Jeenes DJ, Archer DB. Homologs of aflatoxin biosynthesis genes and
sequence of aflR in Asperjillus oryzae and Aspergillus sojae. App Environ Microbial. 1999; 65
(1): 307-310.
27. Iu Y, Zu Y, Chen L, Shi X, Wang Z, Sun S, Efferth T. Antimicrobial activity of clove and
Rosemary essential oils alone and in combination. Phytother Res. 2007; 21: 989-994.
28. Pyun MS, Shin S. Antifungal effects of the volatile oils from Allium Plants against
Trichophyton species and synergism of the oils with ketoconazole. Phytomedicine. 2006; 13:
394-400.
29. Yusuke Matsuzaki T, oshiyuki T, Tatsuji N. Antifungal activity of chimotype essential oil
from Rosemary against Candida albicans. Open J Stomatol. 2013; 3: 176-182.
30. Golshani Z, Davoodi V. Invitro study of antimicrobial effects of Rosemarinus officinalis leaf
extract against some pathogens. Arak Med Univ J. 2013; 16(8): 82-89.
31. Inouye S, Uchida K, Nishiyama Y, Hasumi Y, Yamagushi H, Abe S. Combined effect of
heat, essential oils and salt on the functional activity against Trichophyton mentagrophytes in
foot bath. Nippon Ishinkin Gakkai Zasshi. 2007; 48: 27-36.
32. Pauli A. Anticandidal low molecular compounds from higher plants with special reference to
compounds from essential oils. Med Res Rev. 2006; 26: 223-268.
33. Celiktas OY, Hames KEE, Bedir E, Vardar SF, Ozek T, Baser KHC. Antimicrobial activities
of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and
seasonal variations. Food Chem. 2007; 100(2): 553-559.
34. Omidbeygi M, Barzegar H, Hamidi Z, Naghdibadi H. Antifungal activity of thyme, summer
savory and clove essential oils against Aspergillus flavus in liquid medium and tomato paste.
Food Control. 2007; 18: 1518-1523.
35. Nusier M, Batineh H, Daradkah H. Adverse effects of rosemary (Rosmarinus
officinalis L.) on reproductive function in adult male rats. Exp Biol Med (Maywood). 2007;
232: 809-813.
36. Cheung S, Tai J. Anti-proliferative and antioxidant properties of rosemary Rosmarinus
officinalis. Oncol Res. 2007; 17(6): 525-531.
37. Salem M, Zidan Y. Antifungal activities of two essential oils used in the treatment of three
commercial woods deteriorated by five common mold fungi. Int Biodeterior Biodegred. 2015;
106: 96-98.
38. Schmidt-Heydt M, Abdel-Hadi A, Magan N, Geisen R. Complex regulation of the Aflatoxin
biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water
activity and temperature. Int J Food Microbiol. 2009; 135(3): 231-237.
39. Gallo A, Epifani F, Bonsegna S, Pascale M, Santino A, Perrone G. Analysis of genes early
expressed during Aspergillus flavus colonization of hazelnut. Int J Food Microbiol. 2010; 137:
111-115.
40. Scherm B, Palomba M, Serra D, Marcello A, Migheli Q. Detection of transcripts of the
aflatoxin genes aflD, aflO,and aflP by reverse transcription–polymerase chain reaction allows
differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and
Aspergillus parasiticus. Int J Food Microbiol. 2005; 98(2): 201-210.
41. Gallo A, Solfrizzo M, Epifani F, Panzarini G, Perrone G. Effect of temperature and water
activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium.
Int J Food Microbiol. 2016; 217: 162-169
_||_
licorice plant extract on aflR gene expression aflatoxin production in Aspergillus paraziticus
via Real-time PCR. Modares J Med Sci. 2012; 15(3): 63-77.
2. Eaton DL, Groopman JD. The toxicology of aflatoxins. Academic Press New York. 1994:
383-426.
3. Nabili M, Moazeni M, Taghizadeh Armaki M, Asgari MR, Nosrati A, Shokohi T. Diagnostic
tools in fungal infections since classical to molecular era. J Mazandaran Univ Med Sci. 2013;
23(104): 109-129.
4. Khan R, Islam B, Akram M, Shakil S, Ahmad AA, Ali SM, Siddiqui M, Khan AU.
Antimicrobial activity of five herbal extracts against multi drug resistant (MRD) strains of
bacteria and fungus of clinical origin. Molecules. 2009; 14: 586-597.
5. Moghtader M, Salari H, Farahmand A. Evaluation of the antifungal effects of the rosemary oil
and comparison whit synthetic borneol and funjicide on the growth of Aspergillus flavus. J Ecol
Natural Environ. 2011; 3(6): 210-214.
6. Rota C, Carraminana JJ, Burillo J, Herrera A. In vitro antimicrobial activity of essential oils
from aromatic plans against selected foodborne pathogens. J Food Prot. 2004; 67: 1252-1256.
7. Flamini G, Cioni PL, Morelli I, Macchia M, Ceccarini, L. Main agronimic-productive
characteristics of two ecotypes of Romarinus officinalis L. and chemical composition of their
essential oils. J Agr Food Chem. 2002; 50: 3512-3517.
8. Couladis M, Tzakou O, Verykokidou E. Screening of some Greek aromatic plants for
antioxidant activity. J Phytother Res. 2003; 17: 194-196.
9. Huang MT, Wang ZY, Ferraro T, Lou YR, Stauber K, Georgiadis C, Laskin JD, Conney A.
Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid.
Cancer Res. 1994; 54: 701-708.
10. Yu J, Chang PK, Ehrlish KC, Cary JW, Bhatnagar D, Cleveland TE, Payne GA, Linz JE,
Woloshok CP, Bennett JW. Clustered pathway genes in aflatoxin biosynthesis. Appl Environ
Microbial. 2004; 70(3): 1253-1262.
11. Jamali A. Expression of aflatoxin genes aflO (omtB) and aflQ (ordA) differentiates levels of
aflatoxin production by Aspergillus flavus strains from soils of pistachio orchards. Res
Microbiol. 2013; 64: 293-299.
12. Yu J, Mohawed SM, Bhatnagar D, Cleveland TE. Substrate-induced lipase gene expression and
aflatoxin production in Aspergillus parasiticus and Aspergillus flavus. J Appl Microbiol. 2003;
95(6): 1334-13420.
13. El-Nagerabi S, Al-Bahry SN, Elshafie A, Al Hilali S. Effect of Hibiscus sabdariffa extract and
Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and
Aspergillus rasiticus strains. Food Cont. 2012; 25(1): 59-63.
14. Sienkiewicz M, Pastuszka M. The potential of use uasil and Rosemary essential oils as effective
antibacterial agent. Molecules. 2013; 18: 9334-9351.
15. Moreno S, Vojnov A. Antioxidant and antimicrobial activities of rozemary extracts linked to
their polyphenol composition. 2006; 40(2): 223-231.
16. Petersen M, Simmonds MSJ. Rosmarinic acid. Phytochem. 2003; 62: 121-125.
17. Lopez-Reyes JG, Spadaro D, Garibaldi A, Gullino ML. Efficacy of plant essential oils on
postharvest control of rots caused by fungi on different stone fruits in vivo. J Food Prot. 2013;
76(4): 631-639.
18. Chifiriuc C, Gumezescu V, saviue C, Lazar V, Andronescu E. Hybrid magnetite nanoparticles/
Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity. Nanoscale Res Lett.
2012; 7: 209.
19. Crawford JM, Kumar V. Liver and biliary tract. Pathologic basis of disease ed. Elsevier
Saundres. Philadelphia. 2005; p: 924.
20. Espinel-Ingroff AV, Pfaller MA. Susceptibility test methods; yeasts and filamentous fungi. In
Manual of clinical microbiology. 9th ed, Murray. ASM Press. Washington. 2007; p: 1972.
21. Masuda T, Inaba Y, Maekawa T, Takeda Y, Tamura H, Yamaguchi H. Recovery mechanism of
the antioxidant activity from carnosic acid quinine, an oxidized sage and rosemary antioxidant.
J Agric Food Chem. 2002; 50: 5863-5869.
22. Farzaneh M, Ahmadzadeh M, Hadian J, Tehrani AS. Chemical composition and antifugal
activity of the essential oils of three species of Artemisia on some soil-borne phytopathogens–
commun Agric. Apple Biol Sci. 2006; 71(3): 1327-1333.
23. Lopez-Malo A, Barreto-Valdivieso J, Palou E, Martin FS. Aspergillus flavus growth response
to cinnamon extract and sodium benzoate mixtures. Food Control. 2007; 18(11): 1358-1362.
24. Scorzoni L, Benaducci T, Almeida AMF, Silva DHS, Bolzani VS, Mendes-Giannini MJS.
Comparative study of disk diffusion and micro-dilution methods for evaluation of antifungal
activity of natural compounds against medical yeasts Candida spp. and Cryptococcus sp. Rev
Cienc Farm Basica Apl. 2007; 28: 25-34.
25. Santoyo S, Cavero S, Jaime L, Ibanez E, Senorans FJ, Reglero G. Chemical composition and
antimicrobial activity of Rosmarinus officinalis L. essential oil obtained via supercritical fluid
extraction. J Food Prot. 2005; 68(4): 790-795.
26. Watson AJ, Fuller LJ, Jeenes DJ, Archer DB. Homologs of aflatoxin biosynthesis genes and
sequence of aflR in Asperjillus oryzae and Aspergillus sojae. App Environ Microbial. 1999; 65
(1): 307-310.
27. Iu Y, Zu Y, Chen L, Shi X, Wang Z, Sun S, Efferth T. Antimicrobial activity of clove and
Rosemary essential oils alone and in combination. Phytother Res. 2007; 21: 989-994.
28. Pyun MS, Shin S. Antifungal effects of the volatile oils from Allium Plants against
Trichophyton species and synergism of the oils with ketoconazole. Phytomedicine. 2006; 13:
394-400.
29. Yusuke Matsuzaki T, oshiyuki T, Tatsuji N. Antifungal activity of chimotype essential oil
from Rosemary against Candida albicans. Open J Stomatol. 2013; 3: 176-182.
30. Golshani Z, Davoodi V. Invitro study of antimicrobial effects of Rosemarinus officinalis leaf
extract against some pathogens. Arak Med Univ J. 2013; 16(8): 82-89.
31. Inouye S, Uchida K, Nishiyama Y, Hasumi Y, Yamagushi H, Abe S. Combined effect of
heat, essential oils and salt on the functional activity against Trichophyton mentagrophytes in
foot bath. Nippon Ishinkin Gakkai Zasshi. 2007; 48: 27-36.
32. Pauli A. Anticandidal low molecular compounds from higher plants with special reference to
compounds from essential oils. Med Res Rev. 2006; 26: 223-268.
33. Celiktas OY, Hames KEE, Bedir E, Vardar SF, Ozek T, Baser KHC. Antimicrobial activities
of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and
seasonal variations. Food Chem. 2007; 100(2): 553-559.
34. Omidbeygi M, Barzegar H, Hamidi Z, Naghdibadi H. Antifungal activity of thyme, summer
savory and clove essential oils against Aspergillus flavus in liquid medium and tomato paste.
Food Control. 2007; 18: 1518-1523.
35. Nusier M, Batineh H, Daradkah H. Adverse effects of rosemary (Rosmarinus
officinalis L.) on reproductive function in adult male rats. Exp Biol Med (Maywood). 2007;
232: 809-813.
36. Cheung S, Tai J. Anti-proliferative and antioxidant properties of rosemary Rosmarinus
officinalis. Oncol Res. 2007; 17(6): 525-531.
37. Salem M, Zidan Y. Antifungal activities of two essential oils used in the treatment of three
commercial woods deteriorated by five common mold fungi. Int Biodeterior Biodegred. 2015;
106: 96-98.
38. Schmidt-Heydt M, Abdel-Hadi A, Magan N, Geisen R. Complex regulation of the Aflatoxin
biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water
activity and temperature. Int J Food Microbiol. 2009; 135(3): 231-237.
39. Gallo A, Epifani F, Bonsegna S, Pascale M, Santino A, Perrone G. Analysis of genes early
expressed during Aspergillus flavus colonization of hazelnut. Int J Food Microbiol. 2010; 137:
111-115.
40. Scherm B, Palomba M, Serra D, Marcello A, Migheli Q. Detection of transcripts of the
aflatoxin genes aflD, aflO,and aflP by reverse transcription–polymerase chain reaction allows
differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and
Aspergillus parasiticus. Int J Food Microbiol. 2005; 98(2): 201-210.
41. Gallo A, Solfrizzo M, Epifani F, Panzarini G, Perrone G. Effect of temperature and water
activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium.
Int J Food Microbiol. 2016; 217: 162-169
