Nariman Miraalami, Abbas Ebrahimi, zohreh pourhossein, Ali Ahmad Alow Qotbi.
The Effectof Different LevelsofCitrus Sinensis Peel Extract on Immune System and Blood Parametersof Broilers
Abstract—Theexperiment was conductedto evaluatethe effects ofdifferent levels ofCitrus SinensisPeel Extract (CSPE) on the blood parameters of broilers.Four hundredRoss 308strain day old broiler in acompletelyrandomized design withfivetreatments(four replicatespertreatmentandeach replicatehad20chicks) werecategorized. Eachtreatment used either regulatory diet including1000 ppm and 1250 ppm CSPEin the drinking water and intwoperiods of1stto 21st day and 1stto 42nd dayandbase dietwithout anyadditive for six weeks. Dataanalysis was performed usingSAS softwareandmean comparison was conducted byDuncan method. The mean titer of Newcastle Disease (ND) in days7 and14was not significantly different(p>0.05).While the mean of ND titer on days28, 35 and42was significantly different(p<0.05).The mean of Avian Influenza (AI) titer on days4, 14, 28 and42was significantly different(p<0.05).Theresultsdeterminedthat using different levels of CSPE has significant effects on blood parameters (P<0.05) but thetriglycerideconcentrationintheexperimentaltreatmentwas not significantly different(P>0.05).Theresultsdeterminedthatcholesterol, Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL), Low Density Lipoprotein (VLDL) and LDL/HDL at the rearing periodwassignificantlyinfluencedby experimental treatments (P<0.05).
Keywords—Blood parameters, Broilers, Citrus Sinensis Peel Extract, Immune System.
I.INTRODUCTION
T
HEsuccessful poultry production is based on feeding, breeding, marketing, management and the well developed immune status of the birds. Immunity means the power of resistance, against the pathogenic micro-organisms. Such power of defense has two main sources, natural and induced. Schat and Myers stated that the natural immunity in the avian is concentrated in egg yolk, which discharges its antibodies directly into the intestinal lumen and assures protection of the young bird against infections[1].
Beneficial effects of bioactive plant substances in animal nutrition may include the stimulation of appetite and feed intake, the improvement of endogenous digestive enzyme secretion, activation of immune responses and antibacterial, antiviral andantioxidant actions[2].
consumer demand for natural preservatives has increased, whereas the safety aspect of chemical additives has been questioned. Essential oils and extracts obtained from many plants have recently gained a great popularity and scientific interest[3]-[4]-[5].Essential oils from many medicinal plants were also exhibited antimicrobial activity against many pathogenic microbes [6]-[7]-[8].Phenolic compounds present in essential oils have been recognized as the bioactive components for the antimicrobial activity. Most plant phenolic compounds are classified as Generally Recognized as Safe (GRAS) substances, therefore they could be used to prevent growth of many food-born and food spoilage microorganisms in foods. Citrus fruits belong to six genera (Fortunella, Eremocitrus, Clymendia, Poncirus, Microcitrus and Citrus). The genus Citrus includes several important fruits such as citrus sinensis, mandarins, lime, lemons and grape fruits. Citrus essential oils are present in fruit peel in great quantities. This layer consists of the epidermis covering the excerpt consisting of irregular parenchymatous cells, which arecompletely enclosing numerous glands or oil sacs. Citrusessential oils are a mixture of volatile compounds and mainly consisted of monoterpene hydrocarbons [9].
citrus sinensisconstitutes about 60% of the total citrus world production. A large portion of this production is addressed to the industrial extraction of citrus juice which leads to huge amounts of residues, including peel and segment membranes.Peels represent between 50 to 65%of total weight of the fruits and remain as the primary byproduct. If not processed further, it becomes west produce odor, soil pollution, harborage for insects and can give rise toserious environmental pollution [6]-[10].
The antioxidant properties of plant extracts have been due to their polyphenol contents [11]. So plants containing high level of polyphenol have a great importance as natural antioxidants. The citrus peel and seeds are very rich in phenolic compounds, such as phenolic acids and flavonoids. The peels are richer in flavonoids than seeds [12].Since a citrus fruit is peeled, peel and seeds are not used, it is necessary to estimate these by products as natural antioxidants in foods. Flavonoids of citrus have been show to be powerful antioxidant and free radical scavengers [11]. Synthetic antioxidant is used to suppress the development of rancidity in fat and oil. The synthetic antioxidant are know to have toxic and carcinogenic effects on human health [13].
citrus sinensis peelextract contains significant amount of beta-carotene [14]. Citrus peelconsists of significant antioxidant activity compounds that attributable to minor-occurring flavones [15].Hesperidins, the most important flavanone of citrus sinensis peel, has antioxidant and diuretic effects in rats [16].
Furthermore, its constituents may counteract enzymatic lipid peroxidation processes [17].
Ascorbic acid plays a modulating role during stress in guinea pigs [18]. Vitamin C or polyphenols increased the antioxidant enzymes in red blood cells [19].
II.MATERIAL AND METHODS
A.Materials
1. Animals
Four hundred day-old chicks of Ross308 werepurchasedandtransferredto theexperiment place. The averageweight ofbroilers was 43.5gandbreeders were 38th weeks ofage.
2. Plant material
40g of citrus sinensis peel were mixed in 320ml of 72% ethanol and was put in 50°Cwater bath for 3 hours.The acquired suspension centrifuged in 3000rpm for 10 min.The upper liquid was filtered by wathman No 42 filter paper and concentrate with router evaporator set.The concentrate were dried under the labcabinet in room temperature [20].
3.Diet
The composition ofbasaland its nutrient in the starterandgrowerperiodsare showninTABLESI and II. Basaltablesbased onNRC wereset[21].
B.Methods
1. Treatment
Studied treatments were included:
Treatment 1: Control treatment included standard diet without additive materials.
Treatment 2: Standard diet +1000 ppm citrus sinensis peel extract during 1st- 21st day.
Treatment 3: Standard diet +1000 ppm citrus sinensis peel extract during 1st- 42nd days.
Treatment 4: Standard diet +1250 ppm citrus sinensis peel extract during 1st- 21st day.
Treatment 5: Standard diet +1250 ppm citrus sinensis peel extract during 1st- 42nd days.
2. Data Collection
i. HItest (Haemagglotination Inhibition test)
On days7,14, 28, 35 and 42the blood sample was taken from one bird. HItest was used to determine vaccine titersofNDandAI.
ii. Evaluation blood parameters
To evaluate the effectsofusing citrus sinensispeelextract in the dietonbloodand plasmaparametersinday 42nd, beforeslaughteringthechickensweretaken bloodandthenslaughteringwas done. Blood samples werethenimmediatelytransferredto thelaboratoryandsampleswerecentrifugedat3000rpmfor 20 minandplasmawasseparatedand kept in thetemperature- 20° Cfollowedbydefrostby spectrophotometerweretested.
Table I. Used diets during experimental periods(%)
Grower / Starter / IngredientCorn / 58.69 / 54.32
Soybean meal / 31.87 / 39.43
Oyster shell / 0.79 / 0.90
Corn oil / 5.83 / 2.16
DL-Methionine / 0.22 / 0.20
L-Lysine / 0.05 / 0.07
DCP / 1.68 / 2.05
Salt / 0.37 / 0.37
premix1 / 0.50 / 0.50
Total / 100.00 / 100.00
1Vitamin and mineral premixes supplied per kg diet: retinol 3800 mg,
cholecalciferol 125 mg, _-tocopherol 36mg, menadione 3_0 mg, thiamine
2_5 mg, riboflavin 6_0mg, pyridoxine 2_5 mg, cobalamin 12 mg, folic acid
1_5 mg, nicotinic acid 20 mg, pantothenic acid 15 mg, biotin 100 mg, cobalt
300 mg, copper 16 mg, iron 102 mg, iodine 1_2 mg, manganese 95 mg,
selenium 300 mg and zinc 80 mg.
Table II. Nutrients analysis of used diets during experimental periods
Ingredient / Starter / GrowerEnergy (ME) (kcal/kg) / 2900.00 / 3200.00
Cp / 22.16 / 19.20
Lysine (SID)% / 1.15 / 0.96
Methionine (SID)% / 0.50 / 0.48
Met+Cys (SID)% / 0.83 / 0.78
Threonine (SID)% / 0.79 / 0.71
Calcium% / 1.00 / 0.85
Ava.Phosphorus% / 0.50 / 0.42
DCAB mEq/kg / 236.00 / 202.00
C.Statistical Analysis
Data obtained immune system and blood parametersare expressed as mean ± SEM respectively. Differences between the control and thetreatments in these experiments were tested for significanceusing analysis of variance followed by Duncan’s test. A probability of P<0.05 was considered significant.
III. RESULTS
A. ND Titer
Table 3 showed that the mean of NDin days7 and14were not significantly different(P>0.05).While the mean of ND titers on days28, 35 and42were significantly different(P<0.05). In day 35, 28 and 42, the lowest ND titer was related tothecontroltreatmentand its highest titer was concerned to 1250 ppm CSPEtreatment up to the end of rearing period.
B. AI Titer
Table 4 showed the mean of AI titer of treatment in different days. Theresultsdeterminedthatthe mean of AI titers on days7, 14, 28 and42were significantly different(P<0.05). In days 7and 14, the lowest AI titer was related tothecontroltreatmentand its highest titer was concerned to 1250 ppm CSPEtreatment up to the end of rearing period. In days 28, the lowest AI titer was related tothecontroltreatmentand its highest titer was concerned to 1000 ppm CSPEtreatment up to the end of rearing period. In day 42, the lowest AI titer was related tothecontroltreatmentand its highest titer was concerned to 1250 ppm CSPEtreatment up to the end of rearing period. The mean of AI titer on day35was no significantly different(P>0.05).
3. Blood Parameters
Table 5 showed the mean of cholesterol, triglyceride,LDL, HDL, VLDL and LDL/HDL of treatment in different days.
According to the resultsofthisstudy, thecholesterolconcentrationintheexperimentaltreatmentwas significantly different(P<0.05).
Lowestcholesterolwasrelatedto treatment consumedCSPE1000 ppm treatment 1stto 42nd day andthe highestamount of cholesterolwererelated to thecontroltreatment.
According to the resultsofthisstudy, thetriglycerideconcentrationintheexperimentaltreatmentwas not significantly different(P>0.05).
Lowesttriglyceridewasrelatedto consumed1250 ppm treatment 1stto 42nd dayandthe highestamount of triglyceridewererelated to theCSPE1000 ppm treatment 1stto 21st day.According to the resultsofthisstudy, theHDLconcentrationintheexperimentaltreatmentwas significantly different(P<0.05). LowestHDLwasrelatedto control treatment andthe highestamount of HDLwererelated to CSPE1250 ppm treatment 1stto 42nd day.According to the resultsofthisstudy, theLDLconcentrationintheexperimentaltreatmentwas significantly different(P<0.05). LowestLDLwasrelatedto treatment consumedCSPE1250 ppm 1stto 42nd dayandthe highestamount of LDLwererelated to control treatment.
According to the resultsofthisstudy, the VLDLconcentrationintheexperimentaltreatmentwas significantly different(P<0.05). Lowest VLDLwasrelatedto treatment consumedCSPE1250 ppm 1stto 42nd dayandthe highestamount of VLDLwererelated to CSPE1000 ppm 1stto 21st day.
According to the resultsofthisstudy, theLDL/HDLconcentrationintheexperimentaltreatmentwas significantly different(P<0.05). LowestLDL/HDLwasrelatedto treatment consumedCSPE1250 ppm 1stto 42nd dayandthe highestamount of LDL/HDLwererelated to control treatment.
IV.DISCUTION
Livingbodyisconstantlybeingconfrontedwithpathogensandtheimmune systemtask is tofight thesefactors. Strengthen the immune systeminorder toboost itsperformanceto fightinfectious agents, canhelptomakethis systemwork better. A studyshowed that themajority of plants antioxidant capacity may benot just attributedtovitaminsE, Cand β- carotenes, but affiliatedwithothercomponentssuch aspolyphenolwithstrongantioxidant[22]. Herbal extract isasanantioxidant and according tostudies,generallyfruitwithhighantioxidantproperties have more antioxidant. Andshownthat most ofthese antioxidants in nature are phenolic acid, and flavonoeid[23].
Phenoliccompoundsarelargely inplants. Andtheyimportanceinfoodqualityandis a agent for color and tasteinmanyplants[20]. Results fromthis studyshowed that meanofNDwas notsignificantly different at days7and14(P>0.05). While the meantiters ofNDwere significantly different at 28, 35and42 days old (P<0.05).In 28, 35 and 42 days old, the lowesttiter of NDwas related to control treatment and the highest rate was related to 1250 ppm CSPE treatmentuntil the end of rearing period.
AImeantiterson days14 and 28were significantly different(p<0.05). In the days 7, 14, 28 and 42 were significantly different (P<0.05). In days of 7 and 14, the lowesttiter of AIwas related to control treatment and the highest rate was related to 1250 ppm CSPEtreatmentuntil the end of rearing period. In the day 28, the lowesttiter of AIwas related to control treatment and the highest rate was related to 1000 ppm CSPEtreatment up to day 42. In the day 42, the lowesttiter of AIwas related to control treatment and the highest rate was related to 1250 ppm CSPEtreatment up to day 42. The meantiter ofAIwas notsignificantly different at day 35 (P>0.05).
VitaminCfound incitrus sinensisincreaseshumoralresponse and cellularresponse as well as increasedbird'sresistancetoinfectionsfromEscherichia coliandmaycobacterum avium, NDandinfectious bursaldisease andMarekdisease[24]. Inthisstudy,likelydue tovitamin C,citrussinensis peelelevatedantibodytiteragainstNDandAIviruseswhich is due tohigh immunityin poultry. Thisfactorcan beattributedtoincreased activity ofT lymphocyteandB lymphocyte. Vitamin Efound incitrus sinensis, also have significant influenceon theantibodyproduced by theNDandAI [25].
AddingvitaminE feed increase production ofTCR2 +cells,which are generators of lymphocyte T (CD4), andimproved humoral immunesystemresponsethroughmore activeof lymphocyteB. IncreasinglymphocyteT andlymphocyte Bcellsandincreasingin antibodytitle at theage of20 to 40daysbyaddingpropolisinbroilerdietsis probablyrelated toa positive effect of propolison the growth ofthelymphoidorgans,especiallythebursa of fabricius[26].
Giurgenet al.showed that using30g ofpropelsinthe broiler dietfor 21 daysincreasedantibodyproductionin the blood compared withthecontrolgroup [27].
Wojcik et al. studied the effect oflevamisoland isoprenosin on specificimmuneparametersaftervaccination turkeyswithnewcastle Diseasevirus and conclude that LevamisolandIsoprinosinedrug increasedantibodylevels [28]. However, the effectofisoprinosinedrugson theimmunecellularandlevamisoleeffectonhumoralimmunityis more. Lassila et al. examined Levamisoleffectonhumoraland cellular immuneofhealthychickencompared to a thymus-dependent antibodyand a thymusindependentantibody and conclude that Levamisoldrugis capable to increasebothhumoraland cellularimmunityinhealthychickens. This isprobablymediated byactivation oflymphocyte T cells. Thatultimately affected on antibodyresponseagainst thethymus-dependent antibody [29].
Table III. Anti-ND haemagglutination-inhibition(log2) titers of broilers fed with different citrus sinensis peel extract sources
Means with the same letter are not significantly different (P<0.05).
Treatment / ND7th day / ND14th day / ND28th day / ND35th day / ND42nd dayCONTROL / 5.00a±0.30 / 5.75a±0.46 / 4.75b±0.35 / 5.00b±0.11 / 5.50b±0.38
1CSPE (1000 ppm), 1st- 21st day / 5.50a±0.30 / 6.25a±0.46 / 5.50ab±0.35 / 6.00a±0.11 / 5.75b±0.38
CSPE (1000 ppm), 1st- 42nd day / 5.25a±0.30 / 6.50a±0.46 / 5.75ab±0.35 / 6.00a±0.11 / 6.25ab±0.38
CSPE (1250 ppm), 1st- 21st day / 5.75a±0.30 / 6.50a±0.46 / 5.75ab±0.35 / 6.00a±0.11 / 6.00ab±0.38
CSPE (1250 ppm), 1st- 42nd day / 5.75a±0.30 / 6.50a±0.46 / 6.00a±0.35 / 6.25a±0.11 / 7.00a±0.38
1. CSPE = Citrus Sinensis Peel Extract.
Table IV. Anti-AI haemagglutination-inhibition (log2) titers of broilers fed with different citrus sinensis peel extract sources
Treatment / AI 7thday / AI 14th day / AI 28thday / AI 35th day / AI 42th dayCONTROL / 3.50c±0.27 / 4.25c±0.33 / 3.50b±0.32 / 4.75a±0.31 / 4.50b±0.38
CSPE (1000 ppm), 1st- 21st day / 4.50b±0.27 / 5.00bc±0.33 / 4.75a±0.32 / 4.75a±0.31 / 5.50ab±0.38
CSPE (1000 ppm), 1st- 42nd day / 5.25ab±0.27 / 5.50ab±0.33 / 5.50a±0.32 / 5.50a±0.31 / 5.50ab±0.38
CSPE (1250 ppm), 1st- 21st day / 4.75ab±0.27 / 5.00bc±0.33 / 3.75b±0.32 / 5.25a±0.31 / 5.25b±0.38
CSPE (1250 ppm), 1st- 42nd day / 5.50a±0.27 / 6.50a±0.33 / 5.25a±0.32 / 5.75a±0.31 / 6.50a±0.38
Means with the same letter are not significantly different (P<0.05).
Table V. Plasma citrus sinensis peel extract (mg/dl)
Treatment / Cholesterol / Triglyceride / LDL / HDL / VLDL / LDL /HDLCONTROL / 146.85a±5.92 / 110.20a±8.68 / 75.85a±4.10 / 43.56c±3.87 / 22.04ab±1.85 / 1.75a±0.09
CSPE-1000 ppm (1st- 21st day) / 122.75b±5.92 / 114.00a±8.68 / 35.47b±4.10 / 58.42b±3.87 / 26.22a±1.85 / 0.61b±0.09
CSPE-1000 ppm (1st- 42nd day) / 111.00b±5.92 / 101.50a±8.68 / 38.35b±4.10 / 59.25b±3.87 / 20.30ab±1.85 / 0.66b±0.09
CSPE-1250 ppm (1st- 21st day) / 123.25b±5.92 / 102.18a±8.68 / 29.17b±4.10 / 59.11b±3.87 / 20.43ab±1.85 / 0.51b±0.09
CSPE-1250 ppm (1st- 42nd day) / 117.00b±5.92 / 96.58a±8.68 / 25.73b±4.10 / 72.12a±3.87 / 19.31b±1.85 / 0.36b±0.09
Means with the same letter are not significantly different (P<0.05).
Results fromthis studyshowed that meanconcentration of cholesterol in control treatment was more than the other treatments and in treatments 1250 ppm CSPE and treatments 1000 ppm CSPE was less than the other treatments. The results ofthisstudy was consistent with the findings of Qureshi et al.who showedplasmacholesterolof chicks fedwithdiets containinglimonene(the active ingredientincitrus sinensis peel extract)in25-100ppm for26 daysand also with the findings of Qureshi et al. who showed that using theextractobtained from thegarlic createsa positive correlationbetweenenzyme activitycoA HMG-reducetasewithplasmacholesterol and reduced it [30]-[31]. Also it consistent with the findings of Mohan et al. who investigated probioticeffectsonserumcholesterolof broiler and observed that use ofprobiotic reducedserumcholesterollevelof132.2 mg/dlin the control groupto 93.3 ml/dl in the groupconsumingtheprobiotic[32].The resultsofthisstudywas consistent with the study of Abdolahi who showedthat using bioplus B2 probioticsin the diets of broilers will reduce theserumcholesterollevel [33].Reduction ofplasma cholesterolduetotheconsumption ofprobioticscan beattributedto thestrength oflactobacilliinthebile degradation [34].Plasmacholesterollevelsinbirdsaffected byfactors such asheredity, nutrition, age, sexandenvironmental conditions. Some compoundssuch ascarbohydrates, vitaminCandsomeplantcompoundscanreducecholesterolinbirds.
Gillilandet al. stated that mechanismsofcholesterol reduction are because of cholesteroldigestedand renovation [35].Lactobacilli stop thebindingoftorinand glycine from bile acid, sothat theprimarybile acidthat are containscholic acid(Taurocholic acid andGlycocholic) andketo desoxi cholicacid(Tauroglycoconodesoxi colic),converted to desoxi cholicacidand litocholic acid, respectivelythataresecondarybile acids. Thesecondarybileacidsdue tobinding toothernon-absorbent materialbecomeinsolubleandareexcretedthrough thefeces,so theabsorption ofbileacids is reduced. It is here that thebarrier of 7-α-dehedrocsilation reaction beremoved andconversion of bloodcholesterolintobileacids(which is aprerequisitefor allsteroid compoundsincludingbileacids) increasedandreducedbloodcholesterol[36].
Results fromthis studyshowed that the average of plasmaLDLconcentrationin the control treatmentwas more than the othertreatmentsandin 1250 ppm CSPE treatmentwas lowerthanothertreatments. The results ofthisstudy is consistent with the findings of Qureshiet al. who showed that rationofgarlic extractinbroiler,lowers the LDL [31].Qureshiet al. while investigated themetabolismoffatin the poultryliver,withextractobtained from garlic studiedits effectonserum lipids, showed positive correlationbetweenenzyme activity of HMG-coA reducetase with total cholesteroland LDLinbroilers [31]. Enzyme activity of HMG-coA reducetase decreased the serumcholesterol inchicks fedwithdiets containingLimonene(the active ingredientin theextract ofcitrus)atlevels of25-100ppmfor26 days [30].Iso flavonincitrus sinensis peelreducethe levelof cholesterol byincreasingLDLreceptoractivityandincreasedLDLcatabolismin theliver[37]. Iso flavon in the consumed extracthasbeen providing thepossiblepresence ofsuch a mechanism for influencing thisextract. Citrus sinensis peelextract islikely tolipid peroxidationand increases antioxidantenzymeactivity.
Results fromthis studyshowed that the average of HDLconcentrationin 1250 ppm CSPE treatment was more than the other treatments and in control treatment was lower than the other treatments. The results ofthisstudy is consistent with the findings of Zhong who showed that hypolipidemicandantioxidantproperties ofArtemisiaspeciesreduced totalcholesterol andincreased HDLlevels [38]. Butwith theresults ofLee et al. who wereused200 ppm carvacrolin the dietand Panda et al. who showed that the use of probiolac probioticsin thebroilerdietfor 6 weekshadnosignificant effectonbloodHDL was inconsistent [39]-[40]. Among thelipids, HDL is thegood fat and infact, high HDLisaprotectivefactor. Themainstrategy used to raise HDL is to use low cholesteroland low fat diets[41]. HDL is needed to transportcholesterolfrom peripheral tissuesto the liver and HDLisbeneficialBloodlipid. Sincethis compoundis madein the liver and sinceprobioticsare notdirectlyassociatedwithliver, is possibleonlythrough theeffectonproduction orinhibitionofinducer productionproduced inhibitors in theintestinal mucosacanaffectonliverproduction. For example,compoundsin the diet such asfat, vitamins, Endols, phenolic compoundscancompletelyaltertheintestinalenzymes activity[36]-[42]-[43].
V. CONCLUSION
Based onthe resultsofthis study, usingcitrus sinensis peel extractincreasedantibody titer ofND, AI. Therefore,thisaction implying increased herdimmunity by consumption of extract. Also Adding citrus sinensis peelextract onplasmabloodparametersoncholesterol, LDL, VLDL, HDL and LDL/HDL wasasignificant difference (P<0.05). Adding 1250 ppm citrus sinensis peel extract in daily drinking waterreducescholesterol, LDL and increased HDLinthe blood ofbroilers, butdid notsignificantlytriglyceridelevels (P>0.05).
ACKNOWLEDGMENT
We are grateful to the Islamic Azad University, Rasht Branch, Rasht, Iran and Dr. Alireza Seidavi for supports.
References
[1]K. A. Schat, T.J.Myres, “ Avian intestinal immunity,”Crit. Rev. Poult. Biol. 3: pp. 19–34. (quoted from the book Biotecnology in Animal Feeds and Animal Feeding: An overview, Chapter 10, pp. 213, 1991.
[2]R .Fuller, “Probiotics in man and animals,” J. Appl. Bacteriol, 66: pp. 365–378, 1989.
[3]R. A.Holley, D.Patel, “Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials,” Food Microbiol, 22: pp. 273–292, 2005.
[4]S.Burt, “Essential oils: their antibacterial properties and potential applications in foods,” a review. Int. J. Food Microbiol,94: pp. 223–253, 2004.
[5]A. O.Gill, P. Delaquis, P. Russo, and R. A.Holley, “Evaluation of antilisterial action of cilantro oil on vacuum packed ham,”Int. J. of Food Microbiol, 73: pp. 83–92, 2002.
[6]G.Mandalari, R. N. Bennett, G.Bisignano, A.Saija, G.Dugo, R. B.Lo Curto, C. B. Faulds, and K. W.Waldron, “Characterization of flavonoids and pectin from bergamot (Citrus bergamia Risso) peel, a major byproduct of essential oil extraction,” J. Agric. and Food Chem, 54: pp. 197–203, 2006.
[7]R. P. Samy, “Antimicrobial activity of some medicinal plants from India. Fitoterapia,”,76: pp. 697–699, 2005.
[8]B. Wannissorna, S. Jarikasemb, T.Siriwangchaib, andS.Thubthimthed, “Antibacterial properties of essential oils from Thai medicinal plants,”76: pp. 233–236, 2005.
[9]M. Sawamura, U. S. Son, H. S. Choi,M. S. L. Kim, N. T. L. Phi,M.Fears, and C.Kumagai, “Compositional changes in commercial lemon essential oil for aromatherapy,”Int. J. Arom,4: pp. 27–33, 2004.
[10]J. A.Manthey, K.Grohmann, “Concentrations of hesperidin and other orange pel flavonoids in citrus processing byproducts,”J. Agric. Food Chem, 44: pp. 811–814, 1996.
[11]O.Benavente-Garicia, J.Castillo, F. R.Marin, A.Ortuno, andJ. A. Del Rio, “Uses and properties of Citrus flavonoids,”J.Agric and Food Chem, 45: pp. 4505–4515, 1997.
[12]S.Yusof, H.Mohd Ghazali, and G.Swee King, “Naringin content in local citrus fruits,”Food Chemistry, 37: pp. 113–121, 1990.
[13]Z. U. Rehman,“Citrus peel extract – A natural source of antioxidant,”Food Chemistry, 99: pp. 450–454, 2006.
[14]A.Ghazi, “Extraction of beta-carotene from orange peels,” Nahrung Aug, 43: pp. 274–281, 1999.
[15]J. A.Manthey, “Fractionation of orange peel phenols in ultrafiltered molasses and mass balance studies of their antioxidant levels,”J. Agric. Food Chem., 15: pp. 7586–7592, 2004.