F O L I A V E T E R I N A R I A5, 2 , 3 - 4 : 1 3 5 - 1 3 92, 0 0 8
www.uvm.sk
BIOLOGICALEFFECTSOF
ROSEMARY(rRosmarinas
officinalis1,./ BSSENTIALOIL
(A Review)
Faixov6,Z.r, Faix, S.2
rUniversity of Veterinary
Medicine, Komensk6ho73, 041 g1 Ko5ice
2lnstitute of Animal Physiology
Slovak Academyof Sciences,Solt6sovej4, 040 0l KoSice
The Slovak Republic
[email protected]
ABSTRACT
Many herbs and plant extractsare addedto the diet not
only for their aromaticpropertiesbut they havebeenidentified
as a sourceof variousphytochemicals,
manyof which possess
an importantbiologicalactivity.Resultsof many experiments
showed
that rosemary
essential
oil hadantimicrobial,
antioxidant,
anti-carcinogenic,
cognition-improving
and certainglucoselevel
loweringpropertieswhich makesit usefulas a naturalanimal
feedadditive.This reviewdescribes
themostimportantbiological
activitiesof rosemary(Rosmarinus
olficinatisL.) essentialoil
in animalsand humans.In vitro and,in vivoeffectsof rosemarv
essentialoil are discussed.
Key words:essentialoil; metabolism;rosemary
INTRODUCTION
The useof antibioticsas growthpromotersin animalfeed
is facingreducedsocial acceptance
due to the appearance
of residuesand resistantstrainsof bacteria;antibioticuse
hasbeenbannedin the EuropeanUnion sinceJanuary2006
(Regulationl83l /2003/EC).
Many herbsand plant extractshaveantimicrobialactivities
and antioxidantpropertieswhich makethem usefulas natural
animalfeedadditives.Si et al. (28) reportedthan cinnamon
has antimicrobialactivity againstSalmonellaserotypeTyphymuriumDT 104of swinein vitro study.Thesefindingsare in
agreemew
n ti t h t h e r e p o r t so f P r a b u s e e n i v a sa n e t a l .
(25). They reportedthat cinnamonclove,geranium,lemon,
orangeand rosemaryoils exhibitedsignificantantibacterial
propertiesagainstboth gram-negative
and gram-positive
bacteria in vitro.
Antioxidanteffectof aromaticplantsis dueto the presence
of hydroxylgroupsin their phenoliccompounds
(30).
Most essential
oils are classified
as GenerallyRecognized
as Safe(GRAS)and havebeenapprovedfor food and beverage
consumption
by US Food and Drug administration
(31).
Plantextracts(aniseoil, cinnamonoil, andgarlicoil) may
allowone to manipulatewith rumenmicrobialfermentationby
decreasing
total volatilefatty acid concentrationand reducing
ammoniaN concentration
(6).
Essentialoils derivedfrom sage,rosemary,
thymeand other
herbswere shownto inhibit osteoclastactivity and increase
bone densityin vitro (26).
Recentlyit was reportedthat some essentialoils, such
as cinnamon,havethe potentialto influencefavourablythe
insulinsystemand aflfectbeneficially
bloodglucoseand lipid
metabolism
in peoplewith type 2 diabetes(17).
The greatestattention from among all herbs and spices
actingas biologically
activecompounds
has beenfocusedon
rosemary.
The objectivesof the current review were, therefore.to
describebiologicalactivitiesof rosemary(Rosmarinus
officinalis
l.) essential
oil in animalsand humans.
Essentialoil composition
The essential
oil of rosemarycontainsseveralcompounds
at a rather differentconcentrations.
It is characterized
by
two or three major componentsat fairly high concentrations
(20 to 70%) compared
presentin traces.
to othercompounds
135
Generally,these major componentsdetermine the biological
propertiesof the essentialoil and can act in synergicmanner
oils might be more effective against food-borne pathogens%nd
spoilagebacteriawhen applied to readyto use food containing
a high protein level at acidic pH, as well as lower levels of
or regulate one another.
D e b e r s a c e t a l . ( 8 ) r e p o r t e dt h a t t h e m a j o rc o m p o n e n ot f
fats or carbohydrates.
dried leavesof Rosmarinusofficinalis (L.), supplied by Robertet
( G r a s s eF
, rance)w
, a sm o n o t e r p e n e
o x i d e 1 , 8c i n e o l e( 3 6 . 1 % ) .
Antioxidant properties
Many monoterpeneand sesquiterpene
hydrocarbonswere present in largeamounts(32.2%). The authorsidentified c_pinene,
camphene,limonene, myrcene and
B-pinene,B-caryophylene,
p-cymene.They alsodetectedthe monoterpeneketonecamphor
( 1 2 . 8 % ) a n d m o n o t e r p e n ea l c o h o l s( 9 . 6 % ) , s u c h a s b o r n e o l .
Afl these compoundsamounted to 93.4% (wlw) of essential
oil. All remaining substanceswere minor volatile compounds
and six of them were still unidentified.
In the dichlormethane extract (DCME) from rosemary
leavesonly 28.1% of the compoundspresentwere quantified.
The main compoundscharacterizedin DCME were flavonoids
and phenolic diterpenes.Two flavones were identified as cirs!
maritin and genkwantin.Six structuresof phenolic diterpenes
were detectedin that extract, namely carnosic acid (14.j %) and
its methylatedform (7.4%), carnosol(3.8 %), rosmanol(1.4%),
epirosmanol methyl ether (0.8 %) and epirosmanol (traces).
Dichlormethane extracted also other lipophilic chemicals,
probably correspondingto pigments,sterolsand hydroxylated
fatty acids (8).
Water-solubleextract from rosemary leaves was rich in
rosemarinicacid ( 1.3%) and flavonoids(3%). A wide rangeof
oxygenatedmonoterpeneswere identified: oxides,such as l,g cin_
eole, and alcohols,such as borneol and c-terpineol.
Chemical composition of rosemary essentialoil can vary
betweenregionsand it dependsmostly on climate, soil com_
position, plant organ, age and stage of vegetativecycle.
Cytotoxicity of rosemary essential oils
Oxygenatedmonoterpenesof essentialoil seem to exhibit
a variabledegreeofcytotoxicity. As typical lipophilic substances,
they passthrough the cell wall and cytoplasmicmembraneand
disrupt their structure.In bacteria,the membranedamageis
relatedto the loss of ions and reduction in membranepotential,
collapseof the proton pump and ATp depletion. This cytotoxic
property is used to treat individuals affected by some human
or animal pathogensor parasites(20).
B o z i n e t a l . ( 5 ) t e s t e d a n t i m i c r o b i a la n d a n t i o x i d a n t
activities of the essential oils of rosemary (Rosmarinus
ffici_
nalis L.) and sage (Salvia officinatis L.). Their antimicrobial
activity was tested against 13 bacterial strains and 6 fungi,
including Candica albicans and 5 dermatocytes. The highest
antibacterial activity of both essential oils was expressedon
E. coli, Salmonella typhi, S. enreritidis and Shigetta sonei. Essential oil of rosemary exhibited a significant rate of antifungal
activity. Fu etal. (10) reported that essentialoils from clove
and rosemaryalone and in combination exerteda significant
antimicrobial effect against Staphylococcus
epidellnidis,Escherichia coli and Candica albicans.
The cytotoxic effect of rosemary is of great importance
in preservationof agricultural or marine products. The antimicrobial efficacy of plant essentialoils depends on food
c o m p o s i t i o nG
. utie rrez et al. (14) found out that essential
r36
Free radicals/reactiveoxygen speciesare associatedwith
many biologicalphenomena,such as infllammation,aging,and
carcinogenesis.The antioxidant activity of polar extracts of
rosemaryis relatedto the content of phenolic compounds(i. e.
carnosol,carnosicacid). Constituentsin rosemaryhave shown
a varietyof pharmacologicalactivitiesfor cancerchemopreven_
tion and therapy in in vitro and,in vivo models (29).
Cigarettesmoke, containing reactiveoxygen specles,may
activate the second step of benzo(a) pyrene metabolic way
and thus may be partially responsiblefor the formation of
t h e c r i t i c a ll u n g t u m o r i g e n i ca d d u c t .A l e x a n d r o v e t a t . ( l )
reported that cigarettefilter containing rosemary extract caused
higher than 70% decreasein benzo(a) pyrene adducts levels
arising due to cigarettesmoke in MCF cells. This approach
may reduce the lung cancer risk in addicted smokers.
Fahim et al. (9) found that rosemary ethanolic extract
(0.15g/100g b.w.) given to rats for 3 weeks showed hepatoprotective and antimutagenic effects attributed to the presence
of a relatively high percentageof phenolic compounds with
high antioxidantactivity.
C h e u n g e , T a i ( 1 5 ) s t u d i e da n t i - p r o l i f e r a t i vper o p e r t i e s
of crude extracts of rosemary (Rosmarinus fficinatis L.) in
several human cancer cell lines and their anti-oxidative properties lr? vitro in a mouse RAW 264.7 macrophage/monocyte
cell line. The study showedthat the crude ethanolic rosemary
extract had anti-proliferative effect on human leukemia and
breast carcinomacells.
The body possessesvarious antioxidative systems (free
radical scavengingactivity, FRSA) that prevent oxidative stress,
for example saliva exhibits such an activity.
A t s u m i e t T o n o s a k i ( 2 ) m e a s u r e dt h e t o t a l s a l i v a r y
FRSA induced after sniffing lavender and rosemary essential
oils which are widely used in aromatherapy. Salivary FRSA
valueswere increasedby stimulation with low concentrations
(l 000 times dilution) of lavender or by high concentrations
( l0 times dilution) of rosemary.In contrast,both lavenderand
rosemary stimulationsdecreasedcortisol levels.A significant
inverse correlation was observed between the FRSA value and
the cortisol levelswith each concentration of rosemary stimulation. Thesefindings show that Iavenderand rosemaryenhance
FRSA and decreasethe stress hormone, cortisol, protecting
body from oxidative stress.
Plant extractsrich in polyphenols (pERp) could provide
an important alternative antioxidant but little is known about
their use in ruminants since the antioxidantcapacityof pERp
could be altered by digestiveprocesses.
G l a d i n e e t a l . ( 1 2 ) r e p o r t e dt h a t a s i n g l e a c u t e d o s e
of PERP, given directly into the rumen highly susceptibleto
lipoperoxidation,enhancedthe plasmatotal antioxidantstatus.
The digestiveprocessesof PERP in vivo arc beneficial by improving the biological effect of polymeric proanthocyanidins.
It was found that addition of rosemaryextract dnlayedthe
oxidation of lipid fraction of minced meat balls during storage
in the freezer. The antioxidative effect was related to the concentrationofthe ethanol rosemaryextractin the product (16).
S i m i l a r r e s u l t sw e r e r e p o r t e db y L o p e z - B o t e e t a t . ( 1 9 ) .
In contrast to the above, other experiments have reported
There is a lack of data regarding the effects of dietary
rosemaryextract on immunity.
The results of B ab u er al. (3) indicate that rosemary
dietary extract might not result in general enhancing of the
immune system in young rats and will probably be effective
little or no antioxidant activity of rosemary.
G a l o b a r t e t a l . ( 1 1 ) f o u n d t h a t d i e t a r ys u p p l e m e n t a t i o n
with 500 or 1 000 mg/kg of a commercial rosemary extracr
under some stress condition, such as protein or antioxidant
deficiency.
had no effect on the lipid oxidative stability of eggs enriched
with omega-3fatty acids.
Cognition-enhancingproperties
Effect on rumen microbial fermentation
In dairy cattle, the use of antibiotics as feed additives,
such as ionophore antibiotics,has proved to be a useful tool
to reduce energy (in the form of methane) and nitrogen (in
the form ol ammonia) lossesfrom the diet. However,the use
of antibiotics as feed additives in dairy cows is banned in the
EuropeanUnion. The use of plant extractsappearsas one of
the most natural alternativesto the antibiotic use in animal
nutrition.
C a s t i l l e j o s e l a / . ( 7 ) s t u d i e dt h e e f f e c t so f 1 0 e s s e n t i a l
oils (rosemary,hyssop, sage, tea tree, clove leave. lavandin.
lavender, thyme, oregano and savor), administered in vitro at
three doses (5, 50 and 500mg/l), on rumen fermentationof
high concentrate,feedlot-typediet. The majority ofthe essential
oils modified rumen microbial fermentation and may allowed
one to manipulate with rumen fermentation to improve animal
performance.
Effect on bone
Miihlb auer et al. (22) tested the effects of some common herbs (rosemary,thyme and sage) and their constituent
essentialoils and monoterpeneson bone resorptionin ovariectomized rats. Bone resorption was inhibited by the addition
of I g of powdered leaves of each herb, and the essential oils
extractedfrom sageand rosemary had similar inhibitory effects.
Recently our results showed that diet supplementation with
graded proportions of rosemaryessentialoil (0; 0.1; 0.05 and
0.025%) causeda significantly lower plasma calcium level in
broiler chicksand the effectof rosemaryessentialoil on plasma
calcium levels tended to be dose-dependent(FaixovAetal.
2 0 0 8 , u n p u b l i s h e dd a r a ) .
Antidiabetic properties
Rosemary (Rosmarinus officinatis) is used in traditional
Turkish folk medicine for the treatment of hyperglycaemia.
B a k i r e I et al. (4) investigatedpotential effect of ethanolic
extract of Rosntarinus o.fficinalisleaveson glucose homeostasis
in rabbits. Results of this study showed that ethanolic extracts
of leavesof Rosmarinusofficinalis (L.) reduced blood glucose
level in normoglycaemic and glucose-hyperglycaemicrabbits.
Repeatedadministration ofl rosemary extract to alloxan-diabetic
rabbits led to a decreasein blood glucose level and significant increasein serum insulin level. However,results of our
experimentwith dietary graded levelsof rosemaryessentialoil
in broiler chicks did not confirm the abovementionedfindings
(Faixovri et al., 2008, unpublisheddata).
Effect on immunity
Severalnon-toxic European herbal specieshave pan-cultural
traditions as treatment for cognitive deficit, including that associatedwith ageing.Particularlypromising candidatespecies
include sage,lemon and rosemary.
The essentialaction of rosemaryessentialoil is in stimulation of the nervous system under sympathetic control resUlting
in improved memorizing and concentrating abilities (27).
Moss el al. (21) assessedthe olfactory impact of essential oils of lavender and rosemary on cognitive performance
and mood of healthy volunteers. They reported that rosemary
produced a significant enhancementof performancein terms
of overall quality of memory and secondarymemory factors,
but also impaired the speedcomponent of memory compared
to the control.
Essentialoils of peppermintand rosemaryhave been fiound
to increasethe activity level of mice (18) and diffusion of
rosemary into the environment encouragedthe dogs to spend
more of their time alert (i. e. standing, moving) than any other
olfactory conditions (lavender,chamomile) ( l3).
Rosemaryessentialoil was found to cause moderate inhibition of acetylcholinesterase
(24). The anti-acetylcholinesterasd
activity of rosemary essential oil is explained by synergic
interaction between 1,8 cineole and 2-pinene in rosemary
essentialoil. Inhibition of acetylcholinesterase
is considered
one of the treatment strategies against several neurologic
disorders,including Alzeimer's disease,senile dementia and
myasthenia gravis.
Adverseeffects of rosemary
Nusier et al. (23) studied the effect of dietary rosemary
(250 and 500mg/kg b.w.) on fertility of adult rats over the
period of 63 days. They reported that sperm motility and
density were significantly decreasedin the cauda epididymidis
and in the testes of rosemary-treated male rats in group 2.
In addition, the treatment markedly increasedthe number of
casesof floetalresorption in female rats impregnated by group
2 males, thereby reducing their fertility.
CONCLUSION
Many in vivo and in yitro experiments investigated
effects of rosemary essentialoil in order to show that
its constituents have important biological properties.
Results of the relevant studies showed that there are
biologicallyactive compoundsin rosemary essential
oil exhibiting cytotoxic, antioxidant, anti-carcinogenic
and cognition-enhancingproperties.They also have the
potential to influence glucoselevel in diabetic patients,
t37
rllodify rumen microbial fermentationand enhancebone
resorption. Rosemary essentialoil, however, does not
enhanceimmune responseand there are reports about
its adverse effects on fertility. Essential oils, despite
their wide use and being familiar to us as fragrances,
constituteeffectivealternativesor complementsto synthetic compoundsproducedby chemicalindustrywithout
showingthe same side effects.
ACKNOWLEDGEMENT
Thestudywaspartially supportedby the grants VEGANo I/
0420/08a No 2/ 7046/27.
REFERENCES
1. Alexandrov,
K., Rojas,M., Rolando,
C., 2006:DNA damageby benzo(a)pyrenein humancellsis increasedby cigarette
smokeand decreased
by a filter containingrosemaryextract,
which lowersfreeradicals.CancerRes..66.11938-11945.
2. Atsumi,T., Tonosaki,
K.,2007:.Smellinglavender
and
rosemary
increases
freeradicalscavenging
activityanddecreases
cortisol levefin saliva.PsychiatryRes,,150,89-96.
3. Babu,U.S., Wiesenfeld,
P.L., Jenkins,M.Y., 1999:
Effectof dietaryrosemaryextracton cell-mediated
immunity
of youngrats.Plant FoodsHum. Nutr., 53, 169-174.
4. Bakirel,T., Bakirel,U., Keles,O.U,, Ulgen, S.G.,
Yardibi,H., 2008: In vitro assessment
of antidiabeticand
antioxidantactivitiesof rosemary(Rosmarinus
officinalis)in
alloxan-diabetic
rabbits.J. Ethnopharmacol.,
116,64-73.
5. Bozin,B., Mimica-Dukic,
N., Samojlik,I., Jovin,8.,2007:
Antimicrobialand antioxidantpropertiesof rosemaryand sage
(RosmarinusofficinalisL. and SalviaofficinalisL., Lamiaceae)
essential
oils.J. Agric.FoodChem.,55,7879-7885.
6. Busquet,M., Calsamiglia,
S., Ferret,A., Kamel,C.,
2006:Plant extractsaffectin vlrrorumen microbialfermentation. ./. Dairy Sci.,89, 76-771.
7. Castillejos,L., Calsamiglia,S., Martin-Tereso,
J., Ter
Wijlen H., 2007:In vitro evaluationof elfectsof ten essential
oils at threedoseson ruminalfermentation
of high concentratefeedlot-type
diet.Anim.FeedSci.Technol.
Doi:10.1016/j.
anifeedsci.2007.05.037.
8, Debersac,P., Heydel,J.M., Amiot, M.J., Goudonnet,H., Artur,Y., Suschetet,
M., Siess,M. H., 2001:Induction
of cytochromeP450 and/or detoxicationenzymesby various
extractsof rosemary:descriptionof speciflicpatterns.Food
Chem.Tbxicol..39. 907-918.
9. Fahim,F,A., Esmat,A. Y., Fadel,H. M,, Hassan,
K. F.,
1999:Allied studieson the effectof Rosmarinus
officinatisL.
on experimentalhepatotoxicityand mutagenesis.
Int. J. Food
Sci.Nutr., 50, 413-427.
1 0 .F u , Y . , Z n , Y , , C h e n ,L . , S h i ,X . , W a n g ,2 , , S u n ,S . ,
Efferth,T.,2007 Antimicrobialactivityof cloveand rosemary
essential
oils aloneand in combination.Phytother.
Res.,21,
989-994.
138
11. Galobart,J., Barroeta,A. C., Baucells,M. D., Codony,
R., Ternes,W,, 2001: Elfect of dietary supplementationwith
rosemaryextract and alpha-tocopherylacetateon lipid peroxidation in eggs enriched with omega 3-fatty acids. Poult. Sci.,
80, 460-467.
1 2 .G l a d i n e , C . , R o c k , 8 . , M o r a n d , C . , B a u c h a r t , D . ,
Durand, D., 2007: Bioavailability and antioxidant capacity
of plant extractsrich in polyphenols,given as a single acute
dose,in sheepmade highly susceptibleto lipoperoxidation.Br.
J . N u t r . .1 8 . 6 9 1 - 7 0 1 .
1 3 . G r a h a m ,L . , W e l l s , D . L . , H e p p e r , P . G . , 2 0 0 5 : T h e
influence of olfactory stimulation on the behaviour of dogs
housed in a rescue shelter.Appl. Anim. Behaviour.Sci.. 91.
t 4 3 - 1 53 .
14. Gutierrez, J., Barry-Ryan,C., Bourke, P,, 2008: Antimicrobial efficacy of plant essentialoil combinations and
interactions with food ingredients. Int. J. Food Microbiol., 124,
91-97.
15. Cheung, S., Tai, J., 2007: Anti-proliferativeand antioxidant properties of rosemary Rosmarinus officinalis. Oncol.
rRes..17. 525-531.
16. Karpinska,M., Borowski,J., Danowska-Oziewicz,
M.,
2000: Antioxidative activity of rosemary extract in lipid fraction
of minced meat balls during storagein a freezer.Nahrung, 44,
38 - 4 1 .
17. Khan, A., Safdar,M., Ali Khan, M. M., Khattak, K. N.,
Anderson,R.A., 2003: Cinnamon improvesglucoseand lipids
of people wit type 2 diabetes.Diabetes Care, 26, 3215-3218.
18. Kovar,K. A., Gropper,8., Ammon, H. T. P., 1987:Blood
levelsof 1,8 cineole and locomotor activity of mice after inhalation and oral administration of rosemary oil. Planta Med.,
53,315-319.
1 9 . L o p e z - B o t eC, , J . , G r a y , J , L , G o m a a , E . A . , F l e g a l ,
C.J., 1998: Effect of dietary administration of oil extracts
from rosemary and sage on lipid oxidation in broiler meat.
Br. Poult. Scrl, 39, 235-240.
2 0 . M o o n , T . , W i l k i n s o n ,J . M . , C a v a n a g hH
, .M., 2006:
Antiparasitic activity of two Lavandula essentialoils against
Giardia duodenalis, Trichomonas vaginalis and Hexamita infata.
Parasitol. Res.,99, 722-278.
21. Moss, M., Cook, J., Wesnes,K., Duckett, P., 2003:
Aromas of rosemary and lavender essentialoils differentially
affect cognition and mood in healthy adults. /nl. J. Neurosci.,
I 13, l 5-3 8.
22. Miihlbauer, R. C., Lozano, A., Palacio, S., Reinli, A.,
Felix, R., 2003: Common herbs, essentialoils and monoterpenes potently modulate bone metabolis. Bone, 32, 372-380.
23.Nusier, M. K., Bataineh, H. N., Daradkah, H. M.,
2007: Adverse effects of rosemary (Rosmarinus officinalis L.)
on reproductive function in adult male rats. Exp. Biol. Med.
(Maywood), 232, 809 813.
24, Orhan, L, Aslan, S., Kartal, M., Sener, 8,, Hiisnii,
Can Baser,K., 2008: Inhibitory effect of Turkish Rosmarinum
officinalis L. on acetylcholinesteraseand butyrylcholinesterase
enzymes.Food Chemistry,108, 663-668.
25. Prabuseenivasan,S,, Jayakumar, M,, Ignacimuthu, S.,
2006: In vitro antibacterial activity of some plant essentialoils.
BMC Complement.Altern. Med., 6, 39-45.
2 6 . P u t n a mS
, . E . , S c u t t ,A . M . , B i c k n e l l K
, ., priestley,
C. M., Williamson,
products
E. M., 2006:Natural
asalternative
treatments
for metabolicbonedisordersandfor maintenance
of
bone health(Reviewarticle).Phytotherapy
Res.,21,99-112.
27.Sanders,C., Diego, M,, Fernandez,
M., Field, T.,
Hernandez-Reif,
M., Roca,A.,2002'.EEG asymmetry
responses
to lavenderand rosemaryaromasin adultsand infants.Int. J.
Neurosci.,
112,1305-1320.
28,Si, W., Gong,J., Chanas,C., Cui, S., Yu, H., Caballero,C., Friendship,R.M., 2006: In yirro assessment
of
antimicrobialactivityof carvacrol,thymoland cinnamaldehyde
towardsSalmonellaserotypeTyphimuriuntDT effectsof pig
diets and emulsificationin hydrocolloids.J. Appt.Microbiol.,
t0l.1282-1291.
29.Shabtay,
A., Sharabani,
H., Barvish,2., Kafta, M.,
Amichay,D., Levy,J., Sharoni,Y., Uskokovic,M.R., Studzinski, G. P., Danilenko,M., 2008: Synergistic
antileukemic
activityof carnosicacid-richrosemaryextractand the l9-nor
Gemini vitamin D analoguein a mousemodelof systemic
acutemyeloidleukemia.Oncology,
75, 203-214.
30.Shahidi,F., Wanasundara,
P.D., 1992:Phenolicantioxidants.Crit. Rev.Food Sci.Nutr.,32, 67-103.
31. USFDA,2006. Everythingaddedto food in the United
States:
a foodadditivedatabase
[online].USFDACentrefor Food
Safetyand AppliedNutrition.Availablefrom http:// vm.cfsan.
fda.gov/dmsieatfus.html
30 August20061.
Iaccessed].
Received
June 25. 2008
r39