Research ArticleSpasmolytic and Antibacterial Activity of Two Citrus sinensisOsbeck Varieties Cultivated in Mexico
Amanda Saacutenchez-Recillas1 Ana Ly Arroyo-Herrera1 Jesuacutes Alfredo Araujo-Leoacuten2
Emanuel Hernaacutendez Nuacutentildeez3 and Rolffy Ortiz Andrade1
1Laboratorio de Farmacologıa Facultad de Quımica Universidad Autonoma de Yucatan Merida YUC Mexico2Laboratorio de Cromatografıa Facultad de Quımica Universidad Autonoma de Yucatan Merida YUC Mexico3CONACYT Departamento de Recursos del Mar CINVESTAV-Unidad Merida Merida YUC Mexico
Correspondence should be addressed to Rolffy Ortiz Andrade rolffycorreouadymx
Received 1 December 2016 Accepted 7 February 2017 Published 5 March 2017
Academic Editor Vincenzo De Feo
Copyright copy 2017 Amanda Sanchez-Recillas et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Antibacterial activity on ATCC strains of Escherichia coli Salmonella enterica Salmonella enteritidis and Salmonella choleraesuisand spasmolytic effect on contraction on rat ileum tripswere determinate Eight organic extracts (hexanic andmethanolic) of albedo(mesocarp) and flavedo (pericarp) of two varieties (Valencian and National) of Citrus sinensis (L) Osbeck of Yucatan Mexico werestudied Additionally chromatographic fingerprints were obtained and correlated with their pharmacological effects MAN MAVand HFN extract caused inhibition against S choleraesuis (MIC 1000120583gmL) and S enteritidis (MIC 1000 120583gmL) Regarding thespasmolytic effect the Valencian extracts variety was more efficient on spontaneous contraction HAV (119864max = 5198 plusmn 198)MAV (119864max = 3598 plusmn 142) HFV (119864max = 6891 plusmn 414) and MFV (119864max = 5128 plusmn 259) versus National variety HAN (119864max= 4380 plusmn 632) MAN (119864max = 1462 plusmn 169) HFN (119864max = 6487 plusmn 304) and MFN (119864max = 3101 plusmn 392) Chromatographicfingerprints of HFV and HFN were found to have some similar signals that belong to monoterpenes whereas for HAN and HAVsimilar signals were found belonging to fatty acids and triterpenoids Methanolic extracts showed signals of (1) furfural (2) furfuralacetone (3) furfuraldehyde and (4) 120573ndashsitosterol compounds Flavedo portion ofC sinensis possessed spasmolytic effect on rat ileumstrips and antibacterial activity against Salmonella strainsThis species is source for obtaining bioactive compoundswith therapeuticpotential in the treatment of infectious diarrhea
1 Introduction
Diarrhea is the passage of three or more loose or liquid stoolsper day or more frequently than normal for the individualIt is usually a symptom of gastrointestinal infection whichcan be caused by a variety of bacterial viral and parasiticorganisms [1] Severe diarrhea leads to fluid loss and maybe life threatening particularly in young children and peoplewho are malnourished or have impaired immunity [1] Thiscondition is the second leading cause of death in childrenevery year Many drugs are used as treatment for diarrheahowever two or more of them are usually required forthe treatment thus their continuous use causes bacterialresistance and the subsequent loss of antibacterial efficacy [23] Natural products are an alternative for obtaining bioactive
compoundswith antidiarrheal activity [4] Since immemorialtimes the entire orange fruit plant including fruits themselvesleaves flowers peels and juice had been used for agriculturepurposes nutrition and traditional medicine Citrus sinensis(L) Osbeck or sweet orange is consumed all over the worldfor being an excellent source of vitaminC and for its powerfulantioxidant properties that build up the immune systemTheorange fruit is composed of three sections an external layer(peel) named flavedo epicarp or exocarp a white portionbelow the exocarp named albedo or mesocarp and theinnermost portion the endocarp that contains vesicles withjuice and seeds [5 6] Orange is a good source of vitaminsminerals and other nutrients Many phytochemicals likelimonoids synephrine hesperidin flavonoid polyphenolspectin and enough folacin calcium potassium thiamine
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 3960837 7 pageshttpsdoiorg10115520173960837
2 Evidence-Based Complementary and Alternative Medicine
niacin and magnesium are also present in it [7] These bio-logically active compounds prevent arteriosclerosis cancerkidney stones and stomach ulcers and cause a reduction incholesterol levels and high blood pressure promoting humanhealth thus it possesses anti-inflammatory antibacteriallarvicidal and antifungal activity [7ndash11] Reports suggest ahigh content of bioactive metabolites in leaves flowers andfruits but few studies describe pharmacological effects ofalbedo and flavedo The present study was undertaken inorder to confirm the possible dual effect spasmolytic andbactericide of albedo and flavedo of two varieties of Csinensis cultivated in Yucatan
2 Materials and Methods
21 Chemicals and Drugs Papaverine HCl dimethyl sulfox-ide (DMSO) and amikacin were purchased from Sigma-Aldrich Co (St Louis MO USA) Ethylic ether n-hexaneand methanol were purchased from High-Purity Co (Mon-terrey NL Mexico) ACS grade Stock solutions of extractswere made using distilled water and freshly prepared thesame day of experimentation
22 Plant Material Fruits of Citrus sinensis (L) OsbeckNational and Valencian varieties were collected in a croplocal in Akil Yucatan Mexico (20∘1491015840N and 98∘2011015840W) inDecember 2009 Plantmaterial was authenticated by SalvadorFlores Guido PhD from the Botany Department of Facul-tad de Veterinaria y Zootecnia of Universidad Autonomade Yucatan (UADY) Voucher herbarium specimens wereobtained and a specimen plant was deposited at UADYrsquosherbarium ldquoAlfredo Barrera Marınrdquo
23 Extraction Firstly mature fruits were washed withdistilled water and then flavedo portion (peel) was sepa-rated mechanically to get the albedo portion (white portionbeneath the peel) The juice was extracted and the vegetalmaterial was dried in a herbal desiccator at 50∘C for threedays to be grounded Dried albedo (210 g) and flavedo (425 g)of Citrus sinensis National and Valencian varieties were suc-cessively extracted in a soxhlet apparatus first with n-hexaneand then with methanol the solutions were concentrated todryness in a rotary evaporator (BUCHI RII Switzerland) at45∘C Extraction yields ( drymass) were for hexanic extractof albedo National variety (HAN 174) methanolic extractof albedo National variety (MAN 405) hexanic extract offlavedo National variety (HFN 61) methanolic extract offlavedo National variety (MFN 250) hexanic extract ofalbedo Valencian variety (HAV 179) methanolic extractof albedo Valencian variety (MAV 389) hexanic extractof flavedo Valencian variety (HFV 30) and methanolicextract of flavedo Valencian variety (MFV 262) To carryout the experiments all extracts were dissolved in a mixtureof water DMSO Final concentration of DMSO inside theorgan chamber never exceeded 01
24 Antimicrobial Activity241 Bacterial Cultures The microorganisms used inthe present investigation included reference strains from
American Type Culture Collection (ATCC) Escherichia coli(ATCC 128) Salmonella enterica (ATCC 14028) Salmonellaenteritidis (ATCC 22177) and Salmonella choleraesuis (ATCC10708) The bacterial stock cultures were incubated for 24 hat 37∘C on nutrient agar Stocks cultures were retained atminus20∘C to use
242 Antibacterial Activity by the Method of MicrodilutionPlateMethod The eight organic extracts (HANMANHFNMFN HAV MAV HFV and MFV) of Citrus sinensis (L)Osbeck National and Valencian varieties were evaluated forantimicrobial activity against Escherichia coli Salmonellaenterica Salmonella enteritidis and Salmonella choleraesuisusing the microdilution minimum inhibitory concentration(MIC) and minimum bacterial concentration (MBC) assays[12 13] TheMBC was defined as the lowest recorded organicextract concentration of the MIC wells in which bacteriafailed to grow All procedures were performed so as to ensuresterility
The eight organic extracts were diluted to a concentrationof 1000 120583gmL with DMSO as diluent The microtiter plateswere prepared adding 100 120583L of sterile nutrient broth intoeach well Thereafter the organic extracts and the positivecontrol (Amikacin 125 120583gmL) were added at a volume of100 120583L The organic extracts were serially diluted to reachconcentrations of 1000 500 250 125 625 312 156 78 3919 097 and 048 120583gmL Negative control was also included(nutrient broth with DMSO)
Finally 100120583L of inoculum (at 05McFarland) was addedto each well and the plates were incubated for 24 h at 37 plusmn1∘C After incubation 100 120583L of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride) at 002 wasadded to the reaction mixture and then it was incubated at37 plusmn 1∘C in orbital shaking for 30 minutes The interactionof the microorganisms (when viable) with INT gives rise to acolor change from colorless to a reddish-pink colorThe wellswith the lowest dilution changes observed were considered asthe MIC for these tested samples
25 Ex Vivo Pharmacological Assay
251 Animals HealthymaleWistar rats were used andmain-tained under standard laboratory conditions with free accessto food and water All animal procedures were conducted inaccordance with our Federal Regulations for Animal Exper-imentation and Care [14] and approved by the InstitutionalAnimal Care and Use Committee All experiments werecarried out using six animals per group All animals of thestudy were sacrificed by cervical dislocation
252 General Procedures Rats (250ndash300 g bodyweight)werekilled and abdominal dissection was carried out to extractthe ileum It was cleaned from excrement and adjacent andconnective tissue and then cut into strips 2 cm long Thenthe tissue sections were assembled using stainless steel hooksunder an optimal tension in chambers at 37∘C containingKrebs-Henseleit solution (KHS composition mMNaCl 119KCl 46 KH2PO4 12 MgSO4 12 CaCl2 15 NaHCO3 20and glucose 114 pH 74) constantly bubbledwith anO2 CO2
Evidence-Based Complementary and Alternative Medicine 3
(95 5) mixture Changes in tension were recorded by forcetransducers Grass-FT03 (AstromedWestWarwick RI USA)connected to analyzer MP150 (BIOPAC 41 InstrumentsSanta Barbara CA USA) as described previously by Estrada-Soto et al 2010 [15]
253 Rat Ileum Assay Tissue segments (asymp2 cm) were placedin organ baths containing 14mL of KHS All tissues wereassembledwith stainless steel hooks under an optimal tensionof 1 g in organ baths with KH solution After equilibration(15min) a 10min control period was recorded The eightorganic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) positive control (Papaverine phosphodi-esterase inhibitor) and vehicle (DMSO 1) were added tothe bath in a volume of 100 120583L Subsequently cumulativeconcentration-response curves were obtained for each tissuesegment with half-logarithm unit increments (Papaverine097rarr100 120583gmL and extracts 97rarr1000 120583gmL) The effectof organic extracts and positive control on spontaneouscontraction of ileum rings was determined by comparingthe mean of the muscular tone and frequency inscribedby tissue contractions before and after addition of the testmaterials Muscular tone was calculated from the tracingsusing Acknowledge Software (BIOPAC 41)
26 Data Analysis The experimental results are expressed asmean of five experiments plusmn standard error of mean (SEM)Concentration responses curves (CRC) were plotted andexperimental data in the CRC were adjusted using the fit-sigmoidal (Hill equation) in the program Microcal Origin86 (Microcal Software Inc USA) Statistical analysis wasconducted using one-way ANOVA followed by Tukeyrsquos posthoc test 119901 lt 005 was considered to imply significance of thepharmacological effects in the experiments
27 Chromatographic Fingerprint Analysis All the extractswere derivatized with boron trifluoride-methanol (BTFMeOH) for gas chromatography analyses 5mg of eachextract was added to 10mL of BTFMeOH and the mixturewas heated under reflux for 20min and then the solutionswere partitioned with 5mL of n-hexane 1120583L of the hexanesolution (5mgmL) containing the extract was injected insplit mode (50 1 ratio by 1min) in a gas chromatograph(Agilent Technologies 6890N USA) equipped with a massselective detector (5973Network Agilent Technologies USA)and a fused silica capillary column (JampWGC columns USA)of 30m times 025mm times 025 120583m coated with cross-linked 5phenyl-95 methyl polysiloxane High purity (gt99999)helium was used as carrier gas at 08mLmin with constantpressure The oven temperature was programmed 45∘C for3min and then increased 4∘Cmin to 250∘C and stood by for5min then a last increase of 20∘Cmin to 325∘C took placewith a total time of 80min for the analysis of each extract
3 Results
Citrus sinensis (L) O commonly known as ldquosweet orangerdquohas been used for hundreds of years for its medicinal andnutritional properties [7] C sinensis fruits are a main source
of important phytochemical nutrients and for a long timethey have been valued for their wholesome nutritious andantioxidant properties Bactericide activity of flavedo portionhas been investigated previously but albedo (mesocarp) is lessinvestigated
In Yucatan Mexico two varieties of C sinensis L(Osbeck) are knownTheir albedo and flavedowere subject toextraction process exhaustively and subsequently were eval-uated for bactericidal activity and ex vivo spasmolytic effectAdditionally chromatographic fingerprints of each extractwere obtained and helped to correlate the pharmacologicalactivity with the presence of secondary metabolites
31 Antibacterial Activities The antibacterial activities of theeight organic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) of Citrus sinensis (L Osbeck) National andValencian varieties were studied in different concentrations(1000 500 250 125 625 312 156 78 39 19 097 and048 120583gmL) against four bacterial strains (E coli S entericaS enteritidis and S choleraesuis) The results of the antibac-terial activities are presented in Table 1The extracts obtainedfrom Citrus sinensis (L Osbeck) National variety presentedsignificant activity The HFN had antimicrobial activityagainst S enteritidis presenting MIC values of 1000 120583gmLandMAN had activity against S choleraesuis also with aMICof 1000120583gmL Regarding the extracts of Citrus sinensis (LOsbeck) Valencian variety only MAV had activity againstS enteritidis with a MIC of 1000 120583gmL The MBC was notdetermined for any of the strains tested as the requiredconcentrations of the extracts were above the concentrationsexamined
32 Spasmolytic Activity Figure 1(a) shows concentration-response curves (CRC) of spasmolytic effect ofCitrus sinensisNational variety extracts (HAN MAN HFN and MFN)The CRC of hexanic extract of flavedo (HFN) is significantlyshifted to the left when compared to HAN MAN and MFNHFN was more efficient (119864max = 6487 plusmn 304) and potent(EC50 = 300 120583gmL) than HAN (119864max = 4380 plusmn 632 CE50= ND) MAN (119864max = 1462 plusmn 169 CE50 = ND) and MFN(119864max = 3101 plusmn 392 CE50 = ND) On the other handFigure 1(b) shows CRC of spasmolytic effect of C sinensisValencian variety extracts (HAV MAV HFV MFV) Againthe hexanic extract of flavedo (HFV) was more efficient(119864max = 6891 plusmn 414) and potent (EC50 = 28746 120583gmL)compared with HAV (119864max = 5198 plusmn 198 CE50 = ND)MAV (119864max = 3598 plusmn 142 CE50 = ND) and MFV (119864max= 5128 plusmn 259 CE50 = ND) and it was also more powerfulthanHFN (National variety) All extracts of Valencian varietywere more efficient that National variety as shown in Table 2The evaluated extracts were neither more powerful nor moreeffective than Papaverine (phosphodiesterase inhibitor) usedas positive control (119864max = 95)
33 Fingerprint Chromatogram Analysis of Extracts Manycompounds eluted from capillary gas chromatography ineach extract so the chromatograms were divided into threesections The first one includes chemical compounds withthe lowest molecular weights (MW) as monoterpenes and
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Minimum inhibitory concentration (MIC) and bactericidal (MBC) of hexane and methanolic extracts evaluated against strains ofbacteria the values are concentrations (120583gmL)
Microorganism
Extract Escherichia coli Salmonella enterica Salmonella enteritidis Salmonella choleraesuisMIC MBC MIC MBC MIC MBC MIC MBC
HAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 1000 gt1000HFN gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000MFN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000HAV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAV gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000HFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000AK 125 ND 125 ND 125 ND 125 NDHAN hexanic albedo of C sinensis var National MAN methanolic albedo of C sinensis var National HFN hexanic flavedo of C sinensis var National MFNmethanolic flavedo of C sinensis var National HAV hexanic albedo of C sinensis var Valencian MAV methanolic albedo of C sinensis var Valencian HFVhexanic flavedo of C sinensis var Valencian MFV methanolic flavedo of C sinensis var Valencian AK Amikacin
01 1 10 100 1000100
80
60
40
20
0
HANHFNMAN
MFNPapaverine (control)
sp
asm
olyt
ic eff
ect
National variety (휇gmL)
lowast
lowast
lowastlowast
lowastlowast
(a)
01 1 10 100 1000100
80
60
40
20
0
HAVHFVMAV
MFVPapaverine (control)
S
pasm
olyt
ic eff
ect
lowast
lowast
lowast
lowastlowast
Valencian variety (휇gmL)
(b)
Figure 1 Concentration-response cumulative curves for spasmolytic activity of extracts of (a) National variety and (b) Valencian varietyof Citrus sinensis albedo and flavedo on spontaneous contraction of ileum rat strips Values are expressed as the percentage of inhibition ofcontractile responses calculated as the mean plusmn SEM from six animals lowast119901 lt 005
sesquiterpenes the second part consists of compounds withmiddle MW mainly fatty acids between C14 to C18 and thelast section consists of compounds with the highest MWcontaining triterpenoidic molecules According to the spas-molytic effect and antibacterial activity of hexanic flavedoextracts from both varieties were significantly more efficientand potent on spasmolytic effect than other extracts Wecompared fingerprints of hexanic extracts Chromatographicfingerprint analysis of HFV and HFN suggests the presenceof monoterpenes (81 382) fatty acids (867 542)and triterpenic (52 76) compounds respectively Thecomparison of chromatographic fingerprints of Valencian(HFV) and National (HFN) hexanic flavedo extracts showedsimilar signals in the chromatogram (Figure 2(a))
Themonoterpenes (1) 138-p-methatriene (2) D-limone-ne (3) dehydrocymene and (4) thujol were eluted in the firstsection (low MW) around minutes 10 to 25 In the secondsection (middleMW 30 to 45min) fourmain fatty acids werefound (5) palmitic (6) linoleic (7) oleic and (8) stearic Inthe last section (high MW 50 to 80min) four signals wereidentified as triterpenoids (9) 120573-sitosterol acetate (10) 120573-sitosterol (11) stigmastan-3-5-diene and the last one (12) 120573-tocopherol
The fingerprint analysis from hexanic albedo extracts(HAN and HAV) only showed a few signals in the secondsection of the chromatogram (Figure 2(b)) These signalswere identified as the same fatty acids found in flavedoextracts however two more signals were identified in the
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Spasmolytic effect on rat ileum trips of two varieties of Citrus sinensis L Osbeck
Figure 2 Comparative fingerprint chromatograms (a) hexanic flavedo fraction (b) hexanic albedo fraction and (c) methanolic fraction foralbedo and flavedo
last section corresponding to 120573-Sitosterol and Stigmastan-3-5-diene only representing 2 of the total area of the chro-matogramThe albedo extracts fingerprints (HAN andHAV)were significantly different from flavedo extracts especiallyin the first section which corresponded to small moleculeswith monoterpenic structure The fingerprints of HAV andHAN (albedo extracts) that were less efficient on the ex vivoassay revealed monoterpenes (19 22) fatty acids (973965) and triterpenoids (08 13)
On the other hand albedo (MAN andMAV) and flavedo(MFN and MFV) methanolic extracts (Figure 2(c)) showedsimilar signals In these extracts (MAN MAV MFN and
MFV) the main signals were oxidative forms for carbo-hydrates such as (1) furfural (2) furfural acetone and (3)furfuraldehyde and the last signal was identified as (4) 120573-sitosterol
4 Discussion
The methanolic extracts of both varieties showed higheryields in the extraction process compared to hexanic extractsIt has been described that metabolic content is greater inmedicinal plants extracts obtained with solvents of mediumto high polarity such as methanol [16] Waxes and essential
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
2 Evidence-Based Complementary and Alternative Medicine
niacin and magnesium are also present in it [7] These bio-logically active compounds prevent arteriosclerosis cancerkidney stones and stomach ulcers and cause a reduction incholesterol levels and high blood pressure promoting humanhealth thus it possesses anti-inflammatory antibacteriallarvicidal and antifungal activity [7ndash11] Reports suggest ahigh content of bioactive metabolites in leaves flowers andfruits but few studies describe pharmacological effects ofalbedo and flavedo The present study was undertaken inorder to confirm the possible dual effect spasmolytic andbactericide of albedo and flavedo of two varieties of Csinensis cultivated in Yucatan
2 Materials and Methods
21 Chemicals and Drugs Papaverine HCl dimethyl sulfox-ide (DMSO) and amikacin were purchased from Sigma-Aldrich Co (St Louis MO USA) Ethylic ether n-hexaneand methanol were purchased from High-Purity Co (Mon-terrey NL Mexico) ACS grade Stock solutions of extractswere made using distilled water and freshly prepared thesame day of experimentation
22 Plant Material Fruits of Citrus sinensis (L) OsbeckNational and Valencian varieties were collected in a croplocal in Akil Yucatan Mexico (20∘1491015840N and 98∘2011015840W) inDecember 2009 Plantmaterial was authenticated by SalvadorFlores Guido PhD from the Botany Department of Facul-tad de Veterinaria y Zootecnia of Universidad Autonomade Yucatan (UADY) Voucher herbarium specimens wereobtained and a specimen plant was deposited at UADYrsquosherbarium ldquoAlfredo Barrera Marınrdquo
23 Extraction Firstly mature fruits were washed withdistilled water and then flavedo portion (peel) was sepa-rated mechanically to get the albedo portion (white portionbeneath the peel) The juice was extracted and the vegetalmaterial was dried in a herbal desiccator at 50∘C for threedays to be grounded Dried albedo (210 g) and flavedo (425 g)of Citrus sinensis National and Valencian varieties were suc-cessively extracted in a soxhlet apparatus first with n-hexaneand then with methanol the solutions were concentrated todryness in a rotary evaporator (BUCHI RII Switzerland) at45∘C Extraction yields ( drymass) were for hexanic extractof albedo National variety (HAN 174) methanolic extractof albedo National variety (MAN 405) hexanic extract offlavedo National variety (HFN 61) methanolic extract offlavedo National variety (MFN 250) hexanic extract ofalbedo Valencian variety (HAV 179) methanolic extractof albedo Valencian variety (MAV 389) hexanic extractof flavedo Valencian variety (HFV 30) and methanolicextract of flavedo Valencian variety (MFV 262) To carryout the experiments all extracts were dissolved in a mixtureof water DMSO Final concentration of DMSO inside theorgan chamber never exceeded 01
24 Antimicrobial Activity241 Bacterial Cultures The microorganisms used inthe present investigation included reference strains from
American Type Culture Collection (ATCC) Escherichia coli(ATCC 128) Salmonella enterica (ATCC 14028) Salmonellaenteritidis (ATCC 22177) and Salmonella choleraesuis (ATCC10708) The bacterial stock cultures were incubated for 24 hat 37∘C on nutrient agar Stocks cultures were retained atminus20∘C to use
242 Antibacterial Activity by the Method of MicrodilutionPlateMethod The eight organic extracts (HANMANHFNMFN HAV MAV HFV and MFV) of Citrus sinensis (L)Osbeck National and Valencian varieties were evaluated forantimicrobial activity against Escherichia coli Salmonellaenterica Salmonella enteritidis and Salmonella choleraesuisusing the microdilution minimum inhibitory concentration(MIC) and minimum bacterial concentration (MBC) assays[12 13] TheMBC was defined as the lowest recorded organicextract concentration of the MIC wells in which bacteriafailed to grow All procedures were performed so as to ensuresterility
The eight organic extracts were diluted to a concentrationof 1000 120583gmL with DMSO as diluent The microtiter plateswere prepared adding 100 120583L of sterile nutrient broth intoeach well Thereafter the organic extracts and the positivecontrol (Amikacin 125 120583gmL) were added at a volume of100 120583L The organic extracts were serially diluted to reachconcentrations of 1000 500 250 125 625 312 156 78 3919 097 and 048 120583gmL Negative control was also included(nutrient broth with DMSO)
Finally 100120583L of inoculum (at 05McFarland) was addedto each well and the plates were incubated for 24 h at 37 plusmn1∘C After incubation 100 120583L of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride) at 002 wasadded to the reaction mixture and then it was incubated at37 plusmn 1∘C in orbital shaking for 30 minutes The interactionof the microorganisms (when viable) with INT gives rise to acolor change from colorless to a reddish-pink colorThe wellswith the lowest dilution changes observed were considered asthe MIC for these tested samples
25 Ex Vivo Pharmacological Assay
251 Animals HealthymaleWistar rats were used andmain-tained under standard laboratory conditions with free accessto food and water All animal procedures were conducted inaccordance with our Federal Regulations for Animal Exper-imentation and Care [14] and approved by the InstitutionalAnimal Care and Use Committee All experiments werecarried out using six animals per group All animals of thestudy were sacrificed by cervical dislocation
252 General Procedures Rats (250ndash300 g bodyweight)werekilled and abdominal dissection was carried out to extractthe ileum It was cleaned from excrement and adjacent andconnective tissue and then cut into strips 2 cm long Thenthe tissue sections were assembled using stainless steel hooksunder an optimal tension in chambers at 37∘C containingKrebs-Henseleit solution (KHS composition mMNaCl 119KCl 46 KH2PO4 12 MgSO4 12 CaCl2 15 NaHCO3 20and glucose 114 pH 74) constantly bubbledwith anO2 CO2
Evidence-Based Complementary and Alternative Medicine 3
(95 5) mixture Changes in tension were recorded by forcetransducers Grass-FT03 (AstromedWestWarwick RI USA)connected to analyzer MP150 (BIOPAC 41 InstrumentsSanta Barbara CA USA) as described previously by Estrada-Soto et al 2010 [15]
253 Rat Ileum Assay Tissue segments (asymp2 cm) were placedin organ baths containing 14mL of KHS All tissues wereassembledwith stainless steel hooks under an optimal tensionof 1 g in organ baths with KH solution After equilibration(15min) a 10min control period was recorded The eightorganic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) positive control (Papaverine phosphodi-esterase inhibitor) and vehicle (DMSO 1) were added tothe bath in a volume of 100 120583L Subsequently cumulativeconcentration-response curves were obtained for each tissuesegment with half-logarithm unit increments (Papaverine097rarr100 120583gmL and extracts 97rarr1000 120583gmL) The effectof organic extracts and positive control on spontaneouscontraction of ileum rings was determined by comparingthe mean of the muscular tone and frequency inscribedby tissue contractions before and after addition of the testmaterials Muscular tone was calculated from the tracingsusing Acknowledge Software (BIOPAC 41)
26 Data Analysis The experimental results are expressed asmean of five experiments plusmn standard error of mean (SEM)Concentration responses curves (CRC) were plotted andexperimental data in the CRC were adjusted using the fit-sigmoidal (Hill equation) in the program Microcal Origin86 (Microcal Software Inc USA) Statistical analysis wasconducted using one-way ANOVA followed by Tukeyrsquos posthoc test 119901 lt 005 was considered to imply significance of thepharmacological effects in the experiments
27 Chromatographic Fingerprint Analysis All the extractswere derivatized with boron trifluoride-methanol (BTFMeOH) for gas chromatography analyses 5mg of eachextract was added to 10mL of BTFMeOH and the mixturewas heated under reflux for 20min and then the solutionswere partitioned with 5mL of n-hexane 1120583L of the hexanesolution (5mgmL) containing the extract was injected insplit mode (50 1 ratio by 1min) in a gas chromatograph(Agilent Technologies 6890N USA) equipped with a massselective detector (5973Network Agilent Technologies USA)and a fused silica capillary column (JampWGC columns USA)of 30m times 025mm times 025 120583m coated with cross-linked 5phenyl-95 methyl polysiloxane High purity (gt99999)helium was used as carrier gas at 08mLmin with constantpressure The oven temperature was programmed 45∘C for3min and then increased 4∘Cmin to 250∘C and stood by for5min then a last increase of 20∘Cmin to 325∘C took placewith a total time of 80min for the analysis of each extract
3 Results
Citrus sinensis (L) O commonly known as ldquosweet orangerdquohas been used for hundreds of years for its medicinal andnutritional properties [7] C sinensis fruits are a main source
of important phytochemical nutrients and for a long timethey have been valued for their wholesome nutritious andantioxidant properties Bactericide activity of flavedo portionhas been investigated previously but albedo (mesocarp) is lessinvestigated
In Yucatan Mexico two varieties of C sinensis L(Osbeck) are knownTheir albedo and flavedowere subject toextraction process exhaustively and subsequently were eval-uated for bactericidal activity and ex vivo spasmolytic effectAdditionally chromatographic fingerprints of each extractwere obtained and helped to correlate the pharmacologicalactivity with the presence of secondary metabolites
31 Antibacterial Activities The antibacterial activities of theeight organic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) of Citrus sinensis (L Osbeck) National andValencian varieties were studied in different concentrations(1000 500 250 125 625 312 156 78 39 19 097 and048 120583gmL) against four bacterial strains (E coli S entericaS enteritidis and S choleraesuis) The results of the antibac-terial activities are presented in Table 1The extracts obtainedfrom Citrus sinensis (L Osbeck) National variety presentedsignificant activity The HFN had antimicrobial activityagainst S enteritidis presenting MIC values of 1000 120583gmLandMAN had activity against S choleraesuis also with aMICof 1000120583gmL Regarding the extracts of Citrus sinensis (LOsbeck) Valencian variety only MAV had activity againstS enteritidis with a MIC of 1000 120583gmL The MBC was notdetermined for any of the strains tested as the requiredconcentrations of the extracts were above the concentrationsexamined
32 Spasmolytic Activity Figure 1(a) shows concentration-response curves (CRC) of spasmolytic effect ofCitrus sinensisNational variety extracts (HAN MAN HFN and MFN)The CRC of hexanic extract of flavedo (HFN) is significantlyshifted to the left when compared to HAN MAN and MFNHFN was more efficient (119864max = 6487 plusmn 304) and potent(EC50 = 300 120583gmL) than HAN (119864max = 4380 plusmn 632 CE50= ND) MAN (119864max = 1462 plusmn 169 CE50 = ND) and MFN(119864max = 3101 plusmn 392 CE50 = ND) On the other handFigure 1(b) shows CRC of spasmolytic effect of C sinensisValencian variety extracts (HAV MAV HFV MFV) Againthe hexanic extract of flavedo (HFV) was more efficient(119864max = 6891 plusmn 414) and potent (EC50 = 28746 120583gmL)compared with HAV (119864max = 5198 plusmn 198 CE50 = ND)MAV (119864max = 3598 plusmn 142 CE50 = ND) and MFV (119864max= 5128 plusmn 259 CE50 = ND) and it was also more powerfulthanHFN (National variety) All extracts of Valencian varietywere more efficient that National variety as shown in Table 2The evaluated extracts were neither more powerful nor moreeffective than Papaverine (phosphodiesterase inhibitor) usedas positive control (119864max = 95)
33 Fingerprint Chromatogram Analysis of Extracts Manycompounds eluted from capillary gas chromatography ineach extract so the chromatograms were divided into threesections The first one includes chemical compounds withthe lowest molecular weights (MW) as monoterpenes and
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Minimum inhibitory concentration (MIC) and bactericidal (MBC) of hexane and methanolic extracts evaluated against strains ofbacteria the values are concentrations (120583gmL)
Microorganism
Extract Escherichia coli Salmonella enterica Salmonella enteritidis Salmonella choleraesuisMIC MBC MIC MBC MIC MBC MIC MBC
HAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 1000 gt1000HFN gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000MFN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000HAV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAV gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000HFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000AK 125 ND 125 ND 125 ND 125 NDHAN hexanic albedo of C sinensis var National MAN methanolic albedo of C sinensis var National HFN hexanic flavedo of C sinensis var National MFNmethanolic flavedo of C sinensis var National HAV hexanic albedo of C sinensis var Valencian MAV methanolic albedo of C sinensis var Valencian HFVhexanic flavedo of C sinensis var Valencian MFV methanolic flavedo of C sinensis var Valencian AK Amikacin
01 1 10 100 1000100
80
60
40
20
0
HANHFNMAN
MFNPapaverine (control)
sp
asm
olyt
ic eff
ect
National variety (휇gmL)
lowast
lowast
lowastlowast
lowastlowast
(a)
01 1 10 100 1000100
80
60
40
20
0
HAVHFVMAV
MFVPapaverine (control)
S
pasm
olyt
ic eff
ect
lowast
lowast
lowast
lowastlowast
Valencian variety (휇gmL)
(b)
Figure 1 Concentration-response cumulative curves for spasmolytic activity of extracts of (a) National variety and (b) Valencian varietyof Citrus sinensis albedo and flavedo on spontaneous contraction of ileum rat strips Values are expressed as the percentage of inhibition ofcontractile responses calculated as the mean plusmn SEM from six animals lowast119901 lt 005
sesquiterpenes the second part consists of compounds withmiddle MW mainly fatty acids between C14 to C18 and thelast section consists of compounds with the highest MWcontaining triterpenoidic molecules According to the spas-molytic effect and antibacterial activity of hexanic flavedoextracts from both varieties were significantly more efficientand potent on spasmolytic effect than other extracts Wecompared fingerprints of hexanic extracts Chromatographicfingerprint analysis of HFV and HFN suggests the presenceof monoterpenes (81 382) fatty acids (867 542)and triterpenic (52 76) compounds respectively Thecomparison of chromatographic fingerprints of Valencian(HFV) and National (HFN) hexanic flavedo extracts showedsimilar signals in the chromatogram (Figure 2(a))
Themonoterpenes (1) 138-p-methatriene (2) D-limone-ne (3) dehydrocymene and (4) thujol were eluted in the firstsection (low MW) around minutes 10 to 25 In the secondsection (middleMW 30 to 45min) fourmain fatty acids werefound (5) palmitic (6) linoleic (7) oleic and (8) stearic Inthe last section (high MW 50 to 80min) four signals wereidentified as triterpenoids (9) 120573-sitosterol acetate (10) 120573-sitosterol (11) stigmastan-3-5-diene and the last one (12) 120573-tocopherol
The fingerprint analysis from hexanic albedo extracts(HAN and HAV) only showed a few signals in the secondsection of the chromatogram (Figure 2(b)) These signalswere identified as the same fatty acids found in flavedoextracts however two more signals were identified in the
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Spasmolytic effect on rat ileum trips of two varieties of Citrus sinensis L Osbeck
Figure 2 Comparative fingerprint chromatograms (a) hexanic flavedo fraction (b) hexanic albedo fraction and (c) methanolic fraction foralbedo and flavedo
last section corresponding to 120573-Sitosterol and Stigmastan-3-5-diene only representing 2 of the total area of the chro-matogramThe albedo extracts fingerprints (HAN andHAV)were significantly different from flavedo extracts especiallyin the first section which corresponded to small moleculeswith monoterpenic structure The fingerprints of HAV andHAN (albedo extracts) that were less efficient on the ex vivoassay revealed monoterpenes (19 22) fatty acids (973965) and triterpenoids (08 13)
On the other hand albedo (MAN andMAV) and flavedo(MFN and MFV) methanolic extracts (Figure 2(c)) showedsimilar signals In these extracts (MAN MAV MFN and
MFV) the main signals were oxidative forms for carbo-hydrates such as (1) furfural (2) furfural acetone and (3)furfuraldehyde and the last signal was identified as (4) 120573-sitosterol
4 Discussion
The methanolic extracts of both varieties showed higheryields in the extraction process compared to hexanic extractsIt has been described that metabolic content is greater inmedicinal plants extracts obtained with solvents of mediumto high polarity such as methanol [16] Waxes and essential
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
Evidence-Based Complementary and Alternative Medicine 3
(95 5) mixture Changes in tension were recorded by forcetransducers Grass-FT03 (AstromedWestWarwick RI USA)connected to analyzer MP150 (BIOPAC 41 InstrumentsSanta Barbara CA USA) as described previously by Estrada-Soto et al 2010 [15]
253 Rat Ileum Assay Tissue segments (asymp2 cm) were placedin organ baths containing 14mL of KHS All tissues wereassembledwith stainless steel hooks under an optimal tensionof 1 g in organ baths with KH solution After equilibration(15min) a 10min control period was recorded The eightorganic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) positive control (Papaverine phosphodi-esterase inhibitor) and vehicle (DMSO 1) were added tothe bath in a volume of 100 120583L Subsequently cumulativeconcentration-response curves were obtained for each tissuesegment with half-logarithm unit increments (Papaverine097rarr100 120583gmL and extracts 97rarr1000 120583gmL) The effectof organic extracts and positive control on spontaneouscontraction of ileum rings was determined by comparingthe mean of the muscular tone and frequency inscribedby tissue contractions before and after addition of the testmaterials Muscular tone was calculated from the tracingsusing Acknowledge Software (BIOPAC 41)
26 Data Analysis The experimental results are expressed asmean of five experiments plusmn standard error of mean (SEM)Concentration responses curves (CRC) were plotted andexperimental data in the CRC were adjusted using the fit-sigmoidal (Hill equation) in the program Microcal Origin86 (Microcal Software Inc USA) Statistical analysis wasconducted using one-way ANOVA followed by Tukeyrsquos posthoc test 119901 lt 005 was considered to imply significance of thepharmacological effects in the experiments
27 Chromatographic Fingerprint Analysis All the extractswere derivatized with boron trifluoride-methanol (BTFMeOH) for gas chromatography analyses 5mg of eachextract was added to 10mL of BTFMeOH and the mixturewas heated under reflux for 20min and then the solutionswere partitioned with 5mL of n-hexane 1120583L of the hexanesolution (5mgmL) containing the extract was injected insplit mode (50 1 ratio by 1min) in a gas chromatograph(Agilent Technologies 6890N USA) equipped with a massselective detector (5973Network Agilent Technologies USA)and a fused silica capillary column (JampWGC columns USA)of 30m times 025mm times 025 120583m coated with cross-linked 5phenyl-95 methyl polysiloxane High purity (gt99999)helium was used as carrier gas at 08mLmin with constantpressure The oven temperature was programmed 45∘C for3min and then increased 4∘Cmin to 250∘C and stood by for5min then a last increase of 20∘Cmin to 325∘C took placewith a total time of 80min for the analysis of each extract
3 Results
Citrus sinensis (L) O commonly known as ldquosweet orangerdquohas been used for hundreds of years for its medicinal andnutritional properties [7] C sinensis fruits are a main source
of important phytochemical nutrients and for a long timethey have been valued for their wholesome nutritious andantioxidant properties Bactericide activity of flavedo portionhas been investigated previously but albedo (mesocarp) is lessinvestigated
In Yucatan Mexico two varieties of C sinensis L(Osbeck) are knownTheir albedo and flavedowere subject toextraction process exhaustively and subsequently were eval-uated for bactericidal activity and ex vivo spasmolytic effectAdditionally chromatographic fingerprints of each extractwere obtained and helped to correlate the pharmacologicalactivity with the presence of secondary metabolites
31 Antibacterial Activities The antibacterial activities of theeight organic extracts (HAN MAN HFN MFN HAV MAVHFV and MFV) of Citrus sinensis (L Osbeck) National andValencian varieties were studied in different concentrations(1000 500 250 125 625 312 156 78 39 19 097 and048 120583gmL) against four bacterial strains (E coli S entericaS enteritidis and S choleraesuis) The results of the antibac-terial activities are presented in Table 1The extracts obtainedfrom Citrus sinensis (L Osbeck) National variety presentedsignificant activity The HFN had antimicrobial activityagainst S enteritidis presenting MIC values of 1000 120583gmLandMAN had activity against S choleraesuis also with aMICof 1000120583gmL Regarding the extracts of Citrus sinensis (LOsbeck) Valencian variety only MAV had activity againstS enteritidis with a MIC of 1000 120583gmL The MBC was notdetermined for any of the strains tested as the requiredconcentrations of the extracts were above the concentrationsexamined
32 Spasmolytic Activity Figure 1(a) shows concentration-response curves (CRC) of spasmolytic effect ofCitrus sinensisNational variety extracts (HAN MAN HFN and MFN)The CRC of hexanic extract of flavedo (HFN) is significantlyshifted to the left when compared to HAN MAN and MFNHFN was more efficient (119864max = 6487 plusmn 304) and potent(EC50 = 300 120583gmL) than HAN (119864max = 4380 plusmn 632 CE50= ND) MAN (119864max = 1462 plusmn 169 CE50 = ND) and MFN(119864max = 3101 plusmn 392 CE50 = ND) On the other handFigure 1(b) shows CRC of spasmolytic effect of C sinensisValencian variety extracts (HAV MAV HFV MFV) Againthe hexanic extract of flavedo (HFV) was more efficient(119864max = 6891 plusmn 414) and potent (EC50 = 28746 120583gmL)compared with HAV (119864max = 5198 plusmn 198 CE50 = ND)MAV (119864max = 3598 plusmn 142 CE50 = ND) and MFV (119864max= 5128 plusmn 259 CE50 = ND) and it was also more powerfulthanHFN (National variety) All extracts of Valencian varietywere more efficient that National variety as shown in Table 2The evaluated extracts were neither more powerful nor moreeffective than Papaverine (phosphodiesterase inhibitor) usedas positive control (119864max = 95)
33 Fingerprint Chromatogram Analysis of Extracts Manycompounds eluted from capillary gas chromatography ineach extract so the chromatograms were divided into threesections The first one includes chemical compounds withthe lowest molecular weights (MW) as monoterpenes and
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Minimum inhibitory concentration (MIC) and bactericidal (MBC) of hexane and methanolic extracts evaluated against strains ofbacteria the values are concentrations (120583gmL)
Microorganism
Extract Escherichia coli Salmonella enterica Salmonella enteritidis Salmonella choleraesuisMIC MBC MIC MBC MIC MBC MIC MBC
HAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 1000 gt1000HFN gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000MFN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000HAV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAV gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000HFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000AK 125 ND 125 ND 125 ND 125 NDHAN hexanic albedo of C sinensis var National MAN methanolic albedo of C sinensis var National HFN hexanic flavedo of C sinensis var National MFNmethanolic flavedo of C sinensis var National HAV hexanic albedo of C sinensis var Valencian MAV methanolic albedo of C sinensis var Valencian HFVhexanic flavedo of C sinensis var Valencian MFV methanolic flavedo of C sinensis var Valencian AK Amikacin
01 1 10 100 1000100
80
60
40
20
0
HANHFNMAN
MFNPapaverine (control)
sp
asm
olyt
ic eff
ect
National variety (휇gmL)
lowast
lowast
lowastlowast
lowastlowast
(a)
01 1 10 100 1000100
80
60
40
20
0
HAVHFVMAV
MFVPapaverine (control)
S
pasm
olyt
ic eff
ect
lowast
lowast
lowast
lowastlowast
Valencian variety (휇gmL)
(b)
Figure 1 Concentration-response cumulative curves for spasmolytic activity of extracts of (a) National variety and (b) Valencian varietyof Citrus sinensis albedo and flavedo on spontaneous contraction of ileum rat strips Values are expressed as the percentage of inhibition ofcontractile responses calculated as the mean plusmn SEM from six animals lowast119901 lt 005
sesquiterpenes the second part consists of compounds withmiddle MW mainly fatty acids between C14 to C18 and thelast section consists of compounds with the highest MWcontaining triterpenoidic molecules According to the spas-molytic effect and antibacterial activity of hexanic flavedoextracts from both varieties were significantly more efficientand potent on spasmolytic effect than other extracts Wecompared fingerprints of hexanic extracts Chromatographicfingerprint analysis of HFV and HFN suggests the presenceof monoterpenes (81 382) fatty acids (867 542)and triterpenic (52 76) compounds respectively Thecomparison of chromatographic fingerprints of Valencian(HFV) and National (HFN) hexanic flavedo extracts showedsimilar signals in the chromatogram (Figure 2(a))
Themonoterpenes (1) 138-p-methatriene (2) D-limone-ne (3) dehydrocymene and (4) thujol were eluted in the firstsection (low MW) around minutes 10 to 25 In the secondsection (middleMW 30 to 45min) fourmain fatty acids werefound (5) palmitic (6) linoleic (7) oleic and (8) stearic Inthe last section (high MW 50 to 80min) four signals wereidentified as triterpenoids (9) 120573-sitosterol acetate (10) 120573-sitosterol (11) stigmastan-3-5-diene and the last one (12) 120573-tocopherol
The fingerprint analysis from hexanic albedo extracts(HAN and HAV) only showed a few signals in the secondsection of the chromatogram (Figure 2(b)) These signalswere identified as the same fatty acids found in flavedoextracts however two more signals were identified in the
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Spasmolytic effect on rat ileum trips of two varieties of Citrus sinensis L Osbeck
Figure 2 Comparative fingerprint chromatograms (a) hexanic flavedo fraction (b) hexanic albedo fraction and (c) methanolic fraction foralbedo and flavedo
last section corresponding to 120573-Sitosterol and Stigmastan-3-5-diene only representing 2 of the total area of the chro-matogramThe albedo extracts fingerprints (HAN andHAV)were significantly different from flavedo extracts especiallyin the first section which corresponded to small moleculeswith monoterpenic structure The fingerprints of HAV andHAN (albedo extracts) that were less efficient on the ex vivoassay revealed monoterpenes (19 22) fatty acids (973965) and triterpenoids (08 13)
On the other hand albedo (MAN andMAV) and flavedo(MFN and MFV) methanolic extracts (Figure 2(c)) showedsimilar signals In these extracts (MAN MAV MFN and
MFV) the main signals were oxidative forms for carbo-hydrates such as (1) furfural (2) furfural acetone and (3)furfuraldehyde and the last signal was identified as (4) 120573-sitosterol
4 Discussion
The methanolic extracts of both varieties showed higheryields in the extraction process compared to hexanic extractsIt has been described that metabolic content is greater inmedicinal plants extracts obtained with solvents of mediumto high polarity such as methanol [16] Waxes and essential
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Minimum inhibitory concentration (MIC) and bactericidal (MBC) of hexane and methanolic extracts evaluated against strains ofbacteria the values are concentrations (120583gmL)
Microorganism
Extract Escherichia coli Salmonella enterica Salmonella enteritidis Salmonella choleraesuisMIC MBC MIC MBC MIC MBC MIC MBC
HAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 1000 gt1000HFN gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000MFN gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000HAV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MAV gt1000 gt1000 gt1000 gt1000 1000 gt1000 gt1000 gt1000HFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000MFV gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000AK 125 ND 125 ND 125 ND 125 NDHAN hexanic albedo of C sinensis var National MAN methanolic albedo of C sinensis var National HFN hexanic flavedo of C sinensis var National MFNmethanolic flavedo of C sinensis var National HAV hexanic albedo of C sinensis var Valencian MAV methanolic albedo of C sinensis var Valencian HFVhexanic flavedo of C sinensis var Valencian MFV methanolic flavedo of C sinensis var Valencian AK Amikacin
01 1 10 100 1000100
80
60
40
20
0
HANHFNMAN
MFNPapaverine (control)
sp
asm
olyt
ic eff
ect
National variety (휇gmL)
lowast
lowast
lowastlowast
lowastlowast
(a)
01 1 10 100 1000100
80
60
40
20
0
HAVHFVMAV
MFVPapaverine (control)
S
pasm
olyt
ic eff
ect
lowast
lowast
lowast
lowastlowast
Valencian variety (휇gmL)
(b)
Figure 1 Concentration-response cumulative curves for spasmolytic activity of extracts of (a) National variety and (b) Valencian varietyof Citrus sinensis albedo and flavedo on spontaneous contraction of ileum rat strips Values are expressed as the percentage of inhibition ofcontractile responses calculated as the mean plusmn SEM from six animals lowast119901 lt 005
sesquiterpenes the second part consists of compounds withmiddle MW mainly fatty acids between C14 to C18 and thelast section consists of compounds with the highest MWcontaining triterpenoidic molecules According to the spas-molytic effect and antibacterial activity of hexanic flavedoextracts from both varieties were significantly more efficientand potent on spasmolytic effect than other extracts Wecompared fingerprints of hexanic extracts Chromatographicfingerprint analysis of HFV and HFN suggests the presenceof monoterpenes (81 382) fatty acids (867 542)and triterpenic (52 76) compounds respectively Thecomparison of chromatographic fingerprints of Valencian(HFV) and National (HFN) hexanic flavedo extracts showedsimilar signals in the chromatogram (Figure 2(a))
Themonoterpenes (1) 138-p-methatriene (2) D-limone-ne (3) dehydrocymene and (4) thujol were eluted in the firstsection (low MW) around minutes 10 to 25 In the secondsection (middleMW 30 to 45min) fourmain fatty acids werefound (5) palmitic (6) linoleic (7) oleic and (8) stearic Inthe last section (high MW 50 to 80min) four signals wereidentified as triterpenoids (9) 120573-sitosterol acetate (10) 120573-sitosterol (11) stigmastan-3-5-diene and the last one (12) 120573-tocopherol
The fingerprint analysis from hexanic albedo extracts(HAN and HAV) only showed a few signals in the secondsection of the chromatogram (Figure 2(b)) These signalswere identified as the same fatty acids found in flavedoextracts however two more signals were identified in the
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Spasmolytic effect on rat ileum trips of two varieties of Citrus sinensis L Osbeck
Figure 2 Comparative fingerprint chromatograms (a) hexanic flavedo fraction (b) hexanic albedo fraction and (c) methanolic fraction foralbedo and flavedo
last section corresponding to 120573-Sitosterol and Stigmastan-3-5-diene only representing 2 of the total area of the chro-matogramThe albedo extracts fingerprints (HAN andHAV)were significantly different from flavedo extracts especiallyin the first section which corresponded to small moleculeswith monoterpenic structure The fingerprints of HAV andHAN (albedo extracts) that were less efficient on the ex vivoassay revealed monoterpenes (19 22) fatty acids (973965) and triterpenoids (08 13)
On the other hand albedo (MAN andMAV) and flavedo(MFN and MFV) methanolic extracts (Figure 2(c)) showedsimilar signals In these extracts (MAN MAV MFN and
MFV) the main signals were oxidative forms for carbo-hydrates such as (1) furfural (2) furfural acetone and (3)furfuraldehyde and the last signal was identified as (4) 120573-sitosterol
4 Discussion
The methanolic extracts of both varieties showed higheryields in the extraction process compared to hexanic extractsIt has been described that metabolic content is greater inmedicinal plants extracts obtained with solvents of mediumto high polarity such as methanol [16] Waxes and essential
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
Figure 2 Comparative fingerprint chromatograms (a) hexanic flavedo fraction (b) hexanic albedo fraction and (c) methanolic fraction foralbedo and flavedo
last section corresponding to 120573-Sitosterol and Stigmastan-3-5-diene only representing 2 of the total area of the chro-matogramThe albedo extracts fingerprints (HAN andHAV)were significantly different from flavedo extracts especiallyin the first section which corresponded to small moleculeswith monoterpenic structure The fingerprints of HAV andHAN (albedo extracts) that were less efficient on the ex vivoassay revealed monoterpenes (19 22) fatty acids (973965) and triterpenoids (08 13)
On the other hand albedo (MAN andMAV) and flavedo(MFN and MFV) methanolic extracts (Figure 2(c)) showedsimilar signals In these extracts (MAN MAV MFN and
MFV) the main signals were oxidative forms for carbo-hydrates such as (1) furfural (2) furfural acetone and (3)furfuraldehyde and the last signal was identified as (4) 120573-sitosterol
4 Discussion
The methanolic extracts of both varieties showed higheryields in the extraction process compared to hexanic extractsIt has been described that metabolic content is greater inmedicinal plants extracts obtained with solvents of mediumto high polarity such as methanol [16] Waxes and essential
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
6 Evidence-Based Complementary and Alternative Medicine
oils are among the phytochemicals which have been extractedfrom orange peel and they showed antimicrobial activity
Chanthaphon et al 2008 evaluated the antimicrobialactivity of essential oils of kaffir lime lime pomelo acidlessorange Chugun orange and Nech kumquat [17] obtaininga MIC gt225mgmL for Salmonella sp and E coli Similardata is reported by Ou et al 2015 where antibacterialactivities of cold-pressed and distilled essential oils of Cparadise and C grandis (L) Osbeck resulted in a MIC20mgmL for Salmonella sp and E coli [18] Compared withour results we obtained a lower MIC 1000120583gmL for HFNMAN and MAV on S enteritidis and S choleraesuis Onthe other hand hexanic flavedos of both varieties showedsignificant spasmolytic effect (gt60) on ileum strips isolatedfrom rat when compared to hexanic albedos and methanolicextracts
Chromatographic fingerprint analysis of HFV and HFNsuggests the presence of monoterpenes fatty acids andtriterpenic compounds in higher proportion with respect toHAV and HAN (albedo extracts) and it is related to pharma-cological activity Flavedorsquos extract of national and Valencianvarieties showed an efficient spasmolytic effect possiblyrelated to triterpenic or monoterpene compounds since theyare in greater quantity with respect to hexanic extracts ofalbedo (HAV and HAN) The antibacterial activity is onlyrelated to the amount of monoterpenes since the flavedohexanic extract of national variety (382 of monoterpenes)was more active than flavedo hexanic extract of Valencianvariety (81 of monoterpenes) Antimicrobial propertiesanti-inflammatory activity antiulcer activity and antifungalactivity has been reported in triterpenic compounds [19]Also Prissadova et al 2015 report smooth muscle relaxationof ursolic acid due to reducing the Ca2+ influx [20] Monoter-penes such as 138-p-methatriene d-limonene dehydro-cymene and thujol may be responsible for spasmolytic effectChemical medical and pharmacological literature suggeststhat citrus peel essential oils can be successfully used inmanyaspects of health care [21] Several essential oils are reportedto exhibit spasmolytic activity [22ndash25] De Sousa et al 2008report spasmolytic effect of monoterpene (minus)-carvone Fur-thermore (+)-limonene was efficient in contraction inducedby 60mM of KCl solution [26] Besides Santos et al 2011reported vasorelaxant effect of p-cymene [25] Methanolicextract showed antibacterial activity the main constituent inthe MAN andMAVwas furfural which has been reported tohave antimicrobial activity [27]
5 Conclusion
Citrus sinensis (L) Osbeck National variety had significativespasmolytic ex vivo effect and antibacterial activity dueto presence of monoterpenic and triterpenic compoundsin flavedo portion This species represents an importantsource for obtaining bioactive compounds with therapeuticpotential in the treatment of infectious diarrhea
Competing Interests
We declare that we have no conflict of interests
Acknowledgments
This study was financed by a grant from Programa deMejoramiento del Profesorado (PROMEP-SEP) RegistroSISTPROY-UADYFQUI-2011-0003 Special thanks are due toDr GonzaloMena-Rejon for providing facilities to obtain thepreliminary organic extracts
References
[1] World Health Organization (WHO) Report Diarrhoeal Dis-sease httpwwwwhointmediacentrefactsheetsfs330en
[2] R T Cirz J K Chin D R Andes V de Crecy-Lagard WA Craig and F E Romesberg ldquoInhibition of mutation andcombating the evolution of antibiotic resistancerdquo PLoS Biologyvol 3 no 6 article no e176 2005
[3] S K Bouchillon D J Hoban J L Johnson et al ldquoIn vitroactivity of gemifloxacin and contemporary oral antimicrobialagents against 27247Gram-positive andGram-negative aerobicisolates a global surveillance studyrdquo International Journal ofAntimicrobial Agents vol 23 no 2 pp 181ndash196 2004
[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007
[5] D Goudeau S L Uratsu K Inoue et al ldquoTuning the orchestraselective gene regulation and orange fruit qualityrdquo Plant Sciencevol 174 no 3 pp 310ndash320 2008
[6] L Izquierdo and J M Sendra ldquoCitrus fruits compositionand characterizationrdquo in Encyclopedia of Food Sciences andNutrition B Caballero L Trugo and P Finglas Eds vol 2Academic Press Oxford UK 2003
[7] E Etebu and A B Nwauzoma ldquoA review on sweet orange(Citrus sinensis Osbeck) health diseases and managementrdquoAmerican Journal of Research Communication vol 2 no 2 pp33ndash70 2014
[8] P A Roussos ldquoPhytochemicals and antioxidant capacity oforange (Citrus sinensis (l)Osbeck cv Salustiana) juice producedunder organic and integrated farming system in Greecerdquo Scien-tia Horticulturae vol 129 no 2 pp 253ndash258 2011
[9] P A Tarkang G A Agbor T DeutouArmelle T L R YamtheK David and Y S MengueNgadena ldquoAcute and Sub-ChronicToxicity studies of the aqueous and ethanolleaf extracts ofCitrus sinensis(Linnaeus) osbeck (pro sp) in Wistar ratsrdquo DerPharmacia Lettre vol 4 no 5 pp 1619ndash1629 2012
[10] O D Omodamiro and C J Umekwe ldquoEvaluation of anti-inflammatory antibacterial and antioxidant properties ofethanolic extracts of Citrus sinensis peel and leavesrdquo Journal ofChemical and Pharmaceutical Research vol 5 no 5 pp 56ndash662013
[11] S Madhuri U H Ashwini N S Srilakshmi and K T RPrashith ldquoAntimicrobial activity of Citrus sinensis and citrusaurantium peel extractsrdquo Journal of Pharmaceutical and Scien-tific Innovation vol 3 no 4 pp 366ndash368 2014
[12] AMehreenMWaheed I Liaqat andN Arshad ldquoPhytochem-ical antimicrobial and toxicological evaluation of traditionalherbs used to treat sore throatrdquo BioMed Research Internationalvol 2016 Article ID 8503426 9 pages 2016
[13] S De Rapper G Kamatou A Viljoen and S Van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
Evidence-Based Complementary and Alternative Medicine 7
[14] NORMA Oficial Mexicana NOM-062-ZOO-1999 ldquoEspecifi-caciones tecnicas para la produccion cuidado y uso delos animales de laboratoriordquo httpwwwfmvzunammxfmvzprincipalarchivos062ZOOPDF
[15] S Estrada-Soto D Gonzalez-Maldonado P Castillo-Espana FAguirre-Crespo and J C Sanchez-Salgado ldquoSpasmolytic effectofMentha pulegium L involves ionic flux regulation in rat ileumstripsrdquo Journal of SmoothMuscle Research vol 46 no 2 pp 107ndash117 2010
[16] P Tiwari B Kumar M Kaur G Kaur and H Kaur ldquoPhy-tochemical screening and extraction a reviewrdquo InternationalePharmaceutica Sciencia vol 1 no 1 pp 98ndash106 2011
[17] S Chanthaphon S Chanthachum and T HongpattarakereldquoAntimicrobial activities of essential oils and crude extractsfrom tropical Citrus spp Against food-related microorgan-ismsrdquo Songklanakarin Journal of Science and Technology vol 30no 1 pp 125ndash131 2008
[18] M-C Ou Y-H Liu Y-W Sun and C-F Chan ldquoThe compo-sition antioxidant and antibacterial activities of cold-pressedand distilled essential oils of Citrus paradisi and Citrus grandis(L) Osbeckrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 804091 9 pages 2015
[19] L Hernandez-Vazquez J Palazon andANavarro-Ocana ldquoThepentacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health V Rao Ed chapter 23InTech Rijeka Croatia 2012
[20] N Prissadova P Bozov K Marinkov H Badakov and AKristev ldquoEffects of ursolic acid on contractile activity of gastricsmooth musclesrdquo Natural Product Communications vol 10 no4 pp 565ndash566 2015
[21] E Palazzolo V A Laudicina andM A Germana ldquoCurrent andpotential use of citrus essential oilsrdquoCurrentOrganic Chemistryvol 17 no 24 pp 3042ndash3049 2013
[22] O Prakash V K Kasana A K Pant A Zafar S K Hore andC SMathela ldquoPhytochemical composition of essential oil fromseeds of Zingiber Roseum Rosc and its antispasmodic activityin rat duodenumrdquo Journal of Ethnopharmacology vol 106 no3 pp 344ndash347 2006
[23] A Astudillo E Hong R Bye andANavarrete ldquoAntispasmodicactivity of extracts and compounds of Acalypha phleoides CavrdquoPhytotherapy Research vol 18 no 2 pp 102ndash106 2004
[24] M J Gamez J Jimenez C Navarro and A Zarzuelo ldquoStudy ofthe essential oil of Lavandula dentata Lrdquo Pharmazie vol 45 no1 pp 69ndash70 1990
[25] M R V Santos F V Moreira B P Fraga D P de Sousa LR Bonjardim and L J Quintans ldquoCardiovascular effects ofmonoterpenes a reviewrdquo Brazilian Journal of Pharmacognosyvol 21 no 4 pp 764ndash771 2011
[26] D P De Sousa G A S Junior L N Andrade et al ldquoStructureand spasmolytic activity relationships of monoterpene ana-logues found in many aromatic plantsrdquo Section C Journal ofBiosciences vol 63 no 11-12 pp 808ndash812 2008
[27] S Ahmed and N H Othman ldquoReview of the medicinal effectsof tualang honey and a comparison with Manuka honeyrdquoMalaysian Journal of Medical Sciences vol 20 no 3 pp 6ndash132013
