* () Rosmarinus officinalis. Aspergillus flavus :&

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1 : 1 (25) (2009)! "# $%& ' * () Rosmarinus officinalis B1 +'&")! ) Aspergillus flavus (1),* -.# 3 Rosmarinus officinalis ) / "# 01 '2 ' "# & B1 +'&")! ) Aspergillus flavus * *$&.( && % ' * *$& + % "# + 8' & B1 +'&")! ) *$& +7 + () 3 9:& ;<= &.( && % ' + % 10 * * %! "# &' Aspergillus flavus * 1 + B1 +'&")! >* ' + &# Aspergillus flavus :&.B1.! "# (1) 121

2 ...Rosmarinus officinalis $% & '( ),* Studying the Effect of Ethanolic Extract of Dried Leaves of Rosemary (Rosmarinus officinalis) on the Growth and Aflatoxin B1 production of Aspergillus flavus M. Abdullatif (1) ABSTRACT The study, investigated the effect of Ethanolic extract of Rosemary (Rosmarinus officinalis) on growth of Aspergillus flavus and aflatoxin B1 production. The inhibitory percentage of growth of A.F. was 11.77, 31.83, 47.78, 61.35, 83.5% at the extract concentrations 2, 4, 6, 8, 10 respectively. Since, the inhibitory percentage of aflatoxin B1 production was: 17.66, 31.53, 51.94, 73.92, respectively. The results suggest that Ethanolic extract of Rosemary may successfully replace chemical fungicides and provide an alternative method to protect the agricultural commodities from aflatoxin B1 production by Aspergillus flavus Key words: Rosemary, Ethanolic extract, Aspergillus flavus, Aflatoxin B1. (1) Department of Horticulture, Faculty of Agronomy, Tishreen University, Syria. 122

3 : 1 (25) (2009) 8 / 0-1!% +', -. # +, # $% *)" B2 B1 /) #, 3%, ", 1 4" %2 B &, ).% & "! 56 $ 78( 1" 9: G2 G1.(Merck Index, 2000) TLC 8 ;#. / (G 6, Carcinogen %-# "- 0)# 7% B1 0- <( )# # ))" < B1!%.(Eaton & Gallagher, 1994) (... 0: <%). < &.) : =:>. 8 < 1, 1% %2 5:; # 1#7# 15! < Aspergillus flavus -. # <=! B1. =# > 0 # 0)# B1 (Abarca et al., 1988) : ;. > C 17 H 12 O 6 O O O O O OCH 3 Aflatoxin B 1 Merck Index, ) 4/(/@/@# LD50.% 8 #,.!. B C: Rosmarinus officinalis $% (2000 &% < #" D &=) &% < = $% # *)" Labiatae.(Brickell, 1990)! E8 F 8 9: G $% C# H)% :'# "=! 5# # ))" F < 06 =) C! $% + ( ) 7# J1989 I- /.-#)! 7! 7% / % 7! ) :%# + 1# 1.5 / F. L 8-%# < 1 #% (Brickell, 1990) K &, $: /'% +.(Polunin, & Huxley, 1987) -# L $%. %. )# <%; $% M =# % # #, L# : (Bown, 1995) #N8"# O7 0)6# )# E F-" < 123

4 ...Rosmarinus officinalis $% & '( ),* 0)Q 0)6# Rosmarol 0)# # $P < ) Rosmarinic )- R D $! )- $% (Duke & Ayensu, 1985). (Holtom & Hylton, 1979) $% 0# $% # $! (Tsai, P.J., Tsai, )6# 0), )6# =# *)" % $# / 7=P S:+ " )" T.H., Ho, S.C., 2006) Carnosic acid, Carnosol, Rosmanol, Rosmariquinone, : '#. # (Aruoma et al., 1992; Basaga, Tekkaya & Citel, 1997) Rosmaridiphenol Penicilium sp., Alternaria -. #% < 1'(. # $ $7R, # $% '( ), ) P < (Alonso, 2004) sp..b1 Aspergillus flavus -. #% <?,= Rosmarinus $% & ) %# ) -. #% -' < = %T # ; 0)# <+ officinalis. B1! B%# ' # Aspergillus () #&' 0-! :& 0-7 4! ) # Sykam 3% HPLC 5) = ;# 7 U Licrospher 100 RP18 (4 4 mm, 5 mm) 3% )# UV/VIS TLC Densitometer Scanner 8 ;# K=. K# 7 U. Camag 3% :A#7 0-7 Buchi 3% <%) #.1 Binder 3% 4..2 <8, +.3 I# 3% =7 07 %-#.4 F256 3% Thin layer chromatography 8 ;# K=..5 Merck! # 0.# TLC 8 ;# K=. 0.6 Saritorius 3% 1#; )) C# #.7 124

5 : 1 (25) (2009) ; )) C# #.8 Watman, no.1 K! &.9 :: 56 $% &,.1 Merck! # B1 Q 0)#.2 Sigma! # Ergosterol 0)#.3 Merck! # < #.4 Merck! # <.5 Merck! #.6-8# 5#.7 : 8* Rosmarinus! "# B& 1 :officinalis Rosmarinus officinalis $% & $6 56 & 4. 9: 2006! "# # < 24 $%" $% 4. - =# ) 40 0 ) < $% & # ; # / 0! 7% $' / # , ' &#! $, / =7 %-# - 7%-.(1-8# Y5# # P # 70) < %70 EP = # & - $! 24 0)# <8 7 / $ ' 60 0 ) / ) # - < P ' 1 K! $ B6 <=# <8# :, # $ =# ) $"#.#! < P 8 # E 0)# 57.)P / =# ) 4 0 ) < $"6 1P %70) EP = # # $% )+! #, $"# ' 7.% 8 8- < P (1.)P / =# ) 4 0 ) $R. : 3 Aspergillus flavus * * C B& 2 / 7)8 Z[8 0# Aspergillus Flavus -. $ Potato Dextrose Agar O -- ;\ ) - / B1 125

6 ...Rosmarinus officinalis $% & '( ),* & "# 6 =# ) 27 0 ) )% 7%6 #, 10 )" (PDA) :<( # 18# 06# 3 # $8 ' 10 6 <; 46, 1 <-# &) < $"6 # ; ) )% autoclave )# ) < $#8 ' 1-8# Y5# # C ->6 =# ) 100 &) / (# ) $.6, 8" )" -8# 5# 46, '.(% ) % C, 8"# / 1-8# Y5# # 20 C) - # &) B# 7"# K % 8"# )% $%6 ' <; & "# # # 2 &) $8 '.(< E # $- &) # $%" +)" $:, 1# 15 0)# =# ) 27 0 ) I, < $"6 1#; 50 7%# % % / $# ' 7%# 4)7 1) $# '' B.7 / =# ) 20U 0 ) $R. %. #"# ' 1-8# Y5# # ,, ; )+! #, # # , ' 7.% 8 8- )# ) $#8.)+! :* C () Ergosterol 0 & D1 3 Aspergillus flavus -. #% / 1!]# Ergosterol 0)# <-" L.% # ', -. #% 0)# S:+ ) # : -. 3 # < De Castrol, Bragagnolo, de Toledo ) -. #% 5- / 9:!, +.(Valentini1, 2002 :."#&'E 1977 F=# Seitz 8- O -. 3 # # $%" $ 2 0)# $- ' %# # # , %" # 1#; 50 :, D 0)# # 1#; 20 46,.Watman 41 K! & / $! ' - 8) 0 ) <=# # / $%6 '! / %2 # # 50 KOH 100 F 46, # 200 B# / ^ # 8%.8) 30 0)# =# ) $:, 8- / G ' 0)# # # %" ), 4. # / ) # /. " ;# 7 / $%8 D (% : %) # 2 HPLC 5) = 126

7 : 1 (25) (2009) 0)# 100 6M 9: $# '' EP $) ' 1) $- (@ 1) C, # ; 100 / Ergosterol.'' $# <- O.8 8- O $ :HPLC F! :' )G& - *' & D1 (# ) Ergostyerol " 0)# # 4 $%8 4( Mobile phase % - :_ -! & C" /%%# 5!%2 9: / # 1 &) )"# ( / % 30 : 70) % % : %, # 3.2 /%%# 7R # # 10 %8 1#%% 282 # - 8) #8 # P) Ergosterol 0)# C" /%%# `!%a, 8) Peak area : * C () B1+'&")! 0 & D1 :."#&'E 1#; 50 D IUPAC-AOAC, O $%" $ Y5# # , # 500 " <-# &) < $"6 %" # ' 8) 30 0)# <8 7 - $%" $b # # # / ) # /! $.1 K! & - $! I. # 1! $ cy ' 4. TLC 8 8- ;# - :TLC 81 8* )G& *' & D1 ;# K=. O! 8- ) $%" < ) # # 1 &- D Thin Layer Chromatography (TLC) 8 8- # # '## &- #.8 8- ;# K=. / %", # 9: (# ) B1 Q C" # #.C" /%%# 5!% / C - 0 < 7" ;# K=. $- I.(1.5:12:88) _ O% 5#:: # 4]# # 8- ;# K=. K# 7 ) $%" < P 254 # - )% " # < <=6 E#P I D 8 # # S:+ # 0"# /%%# 5!% 1#%% /%%# # $%" < O.7%# <=6 E#P.0"# 127

8 ...Rosmarinus officinalis $% & '( ),* 400 6M 9: $# '' EP $) I $ /@# 8 )" # C, # d 50 / B O - B1 0)# Limit of Detection 4! /%) ) I.8 8- ;# K=. K# 7 1 9:& :Ergosterol 0 & 1 Aspergillus -. 3 # # Ergosterol E $%.%87 / flavus $% )# $%" < Ergosterol 0)# #, $% & 7 < $%" #, )+! % 0)# % Rosmarinus Ergosterol P) Ergosterol 0)# C" /%%# (1) <% HPLC 5) = ;# 7 - (Peak area) #8 # Ergosterol peak $%& y = x R 2 = / (1) ( ' 128

9 : 1 (25) (2009) $% % " (1) ) E : Aspergillus flavus -. # 0.# Ergosterol 0)# ) *$& : ' %Ergosterol (1) Ergosterol 0 & H / H 0.94 ± ± ± ± ± ± "# : ' % $% & K6 '( & ) # R #% < ' # Ergosterol 0)# < Rosmarinus officinalis )% % #% -' % $> D Aspergillus flavus -..)+! %8# $% & %10 $% & =# % " (2) <% : / Aspergillus flavus -. #% Rosmarinus officinalis Ergosterol ' ' '() $'* $'+,- '(.'/ $'0,1 / Ergosterol ( $* $+,-2 $3*4 $5& (2) ( ' 129

10 ...Rosmarinus officinalis $% & '( ),* :B1 0)# U2 K=. K# 7 - B1 0)# 4! /%) ) EP $% < ; ;#.%88 P) B1 0)# C" /%%# (3) <% TLC 8 ;# K=. K# 7 / (Peak area) #8 # Densitometer scanner (CAMAG) (3) ( ' /@# $% )# $%" < B1 C-. #, $% & 7 < $%" #, )+! % % Rosmarinus B1 C-. $% % " (2) ) E : B1 C-. 130

11 : 1 (25) (2009) (2) ) *$& : ' %B1 +'&")! B1 +'&")! & H /H "# : ' % ± ± ± ± ± ± $% & 0) 16, R% & ) # # B1 C-. -' / Rosmarinus officinalis %92.22 B1 -' % $> D Aspergillus flavus -..)+! %8# $% & %10 )% $% & =# % " (4) <% -. B1 Rosmarinus officinalis : / Aspergillus flavus B1.'&*(,89 :'8;) $'* $+,- (./ $0,1 / B1.&*(,89 ( $''* $+,-2 $3*4 $5& (4) ( ' $% &, / 8 ^=%! f % Aspergillus flavus -. #% $-' ) Rosmarinus officinalis f % B1 $"%# # % %

12 ...Rosmarinus officinalis $% & '( ),* D (Hitokoto et al., 1980) 7 < ) B# %) ^=% / -. #% < E ) 0!# &, &# '( ), / ) S:+ ^=% $!, -. # Apsergillus flavus %41 0 f % B1! B%# &, &# '( ) 2000 Nguyen Amiot Campo F C: #" #, ^=% $!, )8 -. 3%, L" < $% & Botrytis -. Penicillium roquefortii -. #% $6. ), /.+#% B%#! cinerea -. $)# '( )) 1993 Hasan +, < ) < Aspergillus flavus -. #% < Vinclozolin, Iprodione, /@# 250 )% A.F. -. #% $-' $)# S:+ M B1.@ /@# )% K6! B1 # $6. '( )) 7 < 2003 F=# Paranagama ), < -. #% < Cymbopogon citrates #!! $% C-" $ -', / 7=% $!, B1 Aspergillus flavus -. d, < # /@# 0.1 )% #! B1.< # /@# )% -' ) # #% Rosmarinus $% &, & ## R% Aspergillus flavus -. #% -' / 0) 7 officinalis 0)) = '\ 7 < =# -. $)# L" 0)8 ',, '## B1 % $%, ## #.= %2 < *)" C: B1 Aspergillus flavus - #% "# 1-1)#!% )" #P < # L" 7%# <%" < #7#!# #.-8 % $% C <" -8 < $% :+ &. < $% < ". $# / ) "# U1.$# S:+ <. # $ #" '( ) U2 06 = ' # =# $)# =) ) /%" < D B! U3.%T %#\ = 8) "- $# %2 ))# D # $% C-" $ # < $) B! U4.". $# 132

13 : 1 (25) (2009) REFERENCES J. & ' D"* 4(& (*.(1989).D"* 3*.+& 4 4: ' & &.(2003).,* -. 4 &.(1987).* Alonso, Jorge. (2004). Tratado de Fitofármacos y Nutracéuticos. Barcelona: Corpus, p: Abarca, M. L.; Brgulat, M. R.; Burguera, M. T.; Cabanes, F. J. (1988). Comparison of some screening methods for aflatoxigenic moulds, Mycopathologia, 104, Aruoma, O. I., Halliwell, B., Aeschbach, R., & Lo liger, J. (1992). Antioxidant and pro-oxidant properties of active rosemary constituents:carnosol and carnosic acid. Xenobiotica, 22(2), Basaga, H., Tekkaya, C., & Acitel, F. (1997). Antioxidative and free radical scavenging properties of rosemary extract. Lebensmittel Wissenschaft und Technologie, 30(1), Bown. D. (1995). Encyclopaedia of Herbs and their Uses, Dorling Kindersley, London. Brickell. C. (1990). The RHS Gardener's Encyclopedia of Plants and Flowers, Dorling Kindersley Publishers Campo, J., Amiot, M., Nguyen, C. (2001). The antimicrobial effect of rosemary extracts. Journal of Food Protection; 63: De Castro1, M.F.P.M.; Bragagnolo, N.; de Toledo Valentini1, S.R. (2002). The relationship between fungi growth and aflatoxin production with ergosterol content of corn grains, Brazilian Journal of Microbiology, 33:22-26 Duke. J. A.; yensu. E. S. (1985). Medicinal Plants of China, Reference Publications, Inc. Eaton, D. L.; Gallagher, E.. (1994). Mechanism of aflatoxin carcinogenesis, Annual Review of Pharmacology and Toxicology, 34, Hasan, H. A. (1993). Fungicide inhibition of aflatoxins, diacetoxyscirpenol and zearalenone production, Folia Microbiol, 38(4):295-8 Hitokoto, H.; Morozumi, S.; Wauke, T.; Sakai, S.; Kurata, H. (1977). Mycotoxin production of fungi on commercial foods, Mycotoxins, Pathotox Publishers, Inc., Chicago, USA Holtom. J. and Hylton. W. (1979). Complete Guide to Herbs, Rodale Press. IUPAC AOAC, , (1990) AOAC Official Methods of Analysis. Merck Index. (2000). CD-ROM Windows, Version 12.3 (CD-ROM), Chapman & Hall/CRC. 133

14 ...Rosmarinus officinalis $% & '( ),* Paranagama P.A, Abeysekera K.H, Abeywickrama K, Nugaliyadde L.(2003). Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus Link. isolated from stored rice, Lett Appl Microbiol. 2003;37(1):86-90 Polunin. O.; Huxley. A. (1987). Flowers of the Mediterranean, Hogarth Press Seitz, L.M.; Mohr, H. E.; Burroughs, R.; Sauer, B. (1977). Ergosterol as an indicator of fungal invasion in grains. Cereal Chem., 54: Tsai, P. J. Tsai, T. H., Ho, S. H. (2006) In vitro inhibitory effects of rosemary extracts on growth and glucosyltransferase activity of Streptococcus sobrinus, Food Chemistry Received 2007 /07/ 08? C Accepted for Publ. 2008/02/ 25? 1 134

Nerium oleander Oleandrin

Nerium oleander Oleandrin 66 55 (4 (008 ( Apocynacea 30 00 08 00 0097 ( 55 Study of the Effect of Collecting Time of Nerium Oleander Leaves on theire Concentration in Damascus and Lattakia Regions M Abdullatif ( ABSTRACT from the