Secondary Metabolite Profile, Antioxidant Capacity, and Mosquito Repellent Activity of Bixa orellana...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 409826 10 pageshttpdxdoiorg1011552013409826

Research ArticleSecondary Metabolite Profile Antioxidant Capacity andMosquito Repellent Activity of Bixa orellana from BrazilianAmazon Region

Annamaria Giorgi12 Pietro De Marinis1 Giuseppe Granelli1

Luca Maria Chiesa3 and Sara Panseri3

1 Department of Agricultural and Environmental Sciences-Production Landscape Agroenergy Via Celoria 2 20133 Milan Italy2 Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment GeSDiMontUniversita degli Studi di Milano Via Morino 8 Edolo 25048 Brescia Italy

3 Department of Veterinary Science and Public Health Universita degli Studi di Milano Via Celoria 2 20133 Milan Italy

Correspondence should be addressed to Sara Panseri sarapanseriunimiit

Received 12 November 2012 Revised 12 February 2013 Accepted 20 March 2013

Academic Editor A Hamid A Hadi

Copyright copy 2013 Annamaria Giorgi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The Brazilian flora was widely used as source of food and natural remedies to treat various diseases Bixa orellana L (Bixaceae) alsoknown as annatto urucu or achiote is a symbol for theAmazonian tribes that traditionally use its seeds as coloured ink to paint theirbodies for religious ceremoniesThe aimof this studywas to investigate the volatile organic compounds (VOCs) profile ofB orellanafresh fruits (in vivo sampled) dried seeds wood bark and leaves analyzed with Headspace solid-phase microextraction coupledwith gas chromatography and mass spectrometry A screening on phenolic content (the Folin-Ciocalteu assay) and antiradicalactivity (DPPH assay) of seeds was also conducted In addition the repellent properties of seed extracts againstAedes aegypti L wereinvestigated Volatile compounds detected in B orellana samples consisted mainly of sesquiterpenes monoterpenes and arenes120572-humulene is the major volatile compound present in seed extracts followed by D-germacrene 120574-elemene and caryophylleneB orellana proved to be a good source of antioxidants Preliminary data on repellency against A aegypti of three different driedseed extracts (hexane ethanol and ethanolwater) indicated a significant skin protection activity A protection of 90 and 73 forhexane and ethanolwater extracts was recorded

1 Introduction

The Brazilian Amazon regions represent one of the worldrsquosrichest pockets of biodiversity Rich in endemic plantstropical forests are known to host thousands of ediblemedicinal and aromatic plant species [1] These regions arealso populated by numerous Indio tribes renowned for theirtraditional knowledge in using herbs as a source of food andnatural remedies for different illnesses [2] In 2006 BrazilrsquosMinistry of Health established a policy regulating the useof medicinal plants and phytotherapeutic resources (PolıticaNacional de Plantas Medicinais e Fitoterapicos) aimed atimproving healthcare and sustainable uses of biodiversity[3]

B orellana L (Bixaceae) a shrub native to Central andSouth America also known as annatto urucu or achioteis a symbol for the Amazonian tribes that traditionally useits seeds as coloured ink to paint their bodies for religiousceremonies [2]Moreover it is believed that the originalAztecchocolate beverage contained annatto seeds in addition tococoa [4] The term ldquoannattordquo in industrialized countries iscommonly referred to B orellana seed extract containingcarotenoid-type pigments widely used to dye an assortmentof foods textiles and body care products [5] Recently Borellana extracts have also been shown to be a viable optionfor commercial exploitation as a replacement for syntheticdyes and pigments in dyeing and finishing of leather [6]Two carotenoids give annatto its unique red colour bixin

2 Journal of Chemistry

and norbixin Bixin (methyl (9-cis)-hydrogen-66-diapo-WW-carotenedioate) is the main responsible for the orange-red colour of the seeds and extracts of annatto representapproximately 80 of its total carotenoids [5 7] ThereforeB orellana is a well-known commercial crop The main com-mercial producers are tropical countries including JamaicaMexico and the Philippines with Peru Brazil and Kenyaas the main sources of supply [8] About 80 of the worldproduction which ranges between 10000 and 11000 tonsof seeds per year is utilized by the USA and WesternEurope where it is further processed and used to impart thecharacteristic orange-yellow colour to butter margarine andcheese [9]

It is amatter of fact that almost all the organs ofB orellanaare used in traditional medicine to obtain remedies to treatvarious diseases [10] In Brazil B orellana leaves roots andseed extracts are popular as aphrodisiac medicines as wellas a remedy to treat fevers inflammatory conditions andparasitic diseases The entire plant is used against dysenteryExtracts of leaves bark and roots are reported to be antidotesfor poisoning from Manihot esculenta (Crantz) Jatrophacurcas L andHura crepitans L [11] A decoction of the leavesis used to stop vomiting and nausea as a mild diuretic totreat heartburn prostate urinary difficulties and stomachproblems [10]

Scientific evidences of B orellana bioactivity are nowa-days numerous particularly regarding seed and leaf extractsAnticonvulsant antidiabetic cardioprotective and antimi-crobial activities of different B orellana extracts have beenproved [12ndash14] Fleischer et al demonstrated the antimi-crobial activity of B orellana leaf and seed extracts againsta range of Gram-positive and Gram-negative bacteria andfungi [15] Moreover some pharmacological studies haverevealed that B orellana extracts possess an antiprotozoaland anthelmintic effect [16] Due to their broad antioxidantand antibacterial activities polar extracts from B orellanaleaves and seeds have been recently proposed as alternativenatural preservatives in food matrices [17] Phytochem-ical investigations have revealed the presence of almosttwenty-five types of chemical compounds from B orellanadifferent extracts with the following major componentscarbohydrates carotenoids steroids proteins flavonoidsterpenoids phenolics tannins glycosides alkaloids detectedonly in the leaf and anthroquinones only in the seeds[15 18]

Despite being widely used and studied little is knownabout B orellana volatile organic compounds (VOCs) com-position especially regarding the terpene profile fromvariousplant organs Moreover even if B orellana seed extractsclaim to repel insects little data for a clear validation isavailable Therefore the present research provides the firstinvestigation onB orellana chemical composition of differentplant organs (fruits leaves bark wood and seeds) and thevolatile profile sampled from living plants growing in anAmazonian Reserve (in vivo sampling) during an Italian-Brazilian cooperation project In addition a preliminaryinvestigation on the repellent properties of seed extractsagainst A aegypti L was conducted

2 Materials and Methods

21 Plant Samples B orellana plants growing in proximity ofthe Upper Rio Guama Reserve located in the municipality ofCapitao Poco Para State Brazil (46∘591015840548210158401015840 O 1∘52101584040810158401015840S 152m asl) were identified and the volatile compoundsemissions of fruits were in vivo sampled in February 2011during a mission of an Italian-Brazilian cooperation projectDry seeds roots leaves bark and wood were bought atBelem at the Ver-o-Peso commercial district from themost frequented herbalist shop (1∘27101584088410158401015840S 48∘30101584089910158401015840O)Identification of the in vivo sampled plants and purchasedmaterials was participatory and based on traditional knowl-edge of Indios Tembe a tribe living in Itaputyr a village in theupper Rio Guama reserve [19 20]

22 Standards and Chemicals 22-diphenyl-1-picrylhydrazyl(DPPH) the Folin-Ciocalteu reagent sodium carbonatemethanol and ethanol were purchased from Sigma-AldrichMilan Italy

Volatile compounds used as references were purchasedfrom Sigma-Aldrich-Fluka from the General and Flavoursand Fragrances catalogue Milan Italy

23 Superfine Grinding (SFG) In order to obtain a represen-tative plant sample a superfine powder was prepared fromB Orellanarsquos different organs (seeds wood bark and leaves)using mechanical grinding activation in an energy intensivevibrational mill Three g of dry plant material samples wereground in a high intensity planetary mill Retsch (modelMM 400 Retsch GmbH Retsch-Allee Haan) as previouslyreported [21] The mill was vibrating at a frequency of 25Hzfor 4min using two 50mL jars with 20mm stainless steelballs Precooling of jars was carried out with liquid nitrogenin order to prevent the temperature from increasing duringthe grinding processThe speed differences between balls andjar resulted in the interaction of frictional and impact forcesreleasing high dynamic energies The interplay of all theseforces resulted in the very effective energy input of planetaryball mills

24 Seed Extracts Preparation Three different B orellanaseed extracts were prepared to evaluate antioxidant capacityand phenolic content

25 Traditional Infusion Aliquots of 1 g from B orellana seedpowder were infused for 15min with 100mL of freshly boileddistilled water and cooled to room temperatureThe infusionwas then filtered usingWhatmanNo 4 filter paper in order toremove plant residues and the aliquots were stored at +4∘Covernight until the analysis

26 Exhaustive Extraction (H2Oand EtOH) Aliquots of 03 g

of B orellana seed powder were subjected respectively tosequential extraction using 8mL of distilled water sonicatedfor 10 minutes macerated for 1 h and then sonicated withan ultrasonic cleaning bath (Branson 2510 20KHz 120W)for a further 30min at room temperature The procedure

Journal of Chemistry 3

was repeated eight times to obtain a final volume of 64mL ofextract This similar procedure was repeated for new samplesusing 8mL of EtOH solution Extracts were then filteredusing Whatman No 4 filter paper and the aliquots werestored at +4∘C until further analysis

27 22-Diphenyl-1-Picrylhydrazyl (DPPH) Assay Antioxi-dant capacity of the extracts was measured by the stableradical DPPH as previously reported [21] Each extractwas diluted with methanol to five different concentrationsbetween 009 and 074mgmLminus1 The DPPH concentrationin methanol was 01 g Lminus1 One mL of extract solution(sample) or methanol (control) was mixed with 1mL ofDPPH and the absorption was recorded after 20min in thedark at 517 nm (UVVIS spectrophotometer model 7800JascoMilan Italy) Antioxidant capacity (AC) was calculatedaccording to the following equation

AC [] = 100 times119860119888ndash119860119904

119860119888

(1)

where 119860119904is the absorbance of the sample and 119860

119888is the

absorbance of the control The concentration of extractscausing a 50 decrease of DPPH solution absorbance (IC

50)

was calculated by linear regression of the concentration-response plots where the abscissa represented the concentra-tion (mgmLminus1) of the extracts and the ordinate of the averagepercentage of AC from three separate determinations Theantioxidant capacity of all B orellana extracts was expressedas 1IC

50(mean value)

28 Determination of Total Phenol Content The amount oftotal phenols in B orellana extracts was determined using theFolin-Ciocalteu reagent according to the modifiedmethod ofSlinkard and Singleton 1977 using Gallic acid as a standardas previously reported [21] Distilled water (1550 120583L) wascombined and vortexed with 50120583L of sample (46mg drywmL) and 100120583L of Folin-Ciocalteursquos reagent Then 300 120583Lof Na

2CO3(20) was added to reach a final concentration

of 005mgmLminus1 the mixture was vortexed The absorbanceof all samples was measured at 765 nm using a UVVISspectrophotometer (model 7800 Jasco Milan Italy) afterincubation at 40∘C for 30min Quantification was doneon the basis of the standard curve of Gallic acid (solutionof Gallic acid 10 EtOH 025ndash10 120583gmL) The calibrationequation for Gallic acid was 119910 = 25509119909 + 00096 1199032 =09986The results were expressed as milligrams of gallic acidequivalents (GAE) per gram of dry weight Measurementswere taken in triplicate and expressed as mean value

29 Seed Extracts for Mosquito Repellency Activity To eval-uate mosquito repellency activity of B orellana differentextracts from powder seeds were prepared using a 5-stepextractiondrying procedure (30mL solvent for each step)using three different solvents ethanol ethanolwater (50 50vv) and hexane as reported in Table 1 Dry residues werestored at 4∘C until resuspension in 5mL of solvent for skinapplication (Table 2)

Table 1 Different extraction conditions of B orellana seed powderfor mosquito repellency evaluation

Extraction solvent Sample weight (g) Dried extract (g)Hexane 2026 0091EtOH 2034 0178EtOHH2O (50 50) 2002 0569

Table 2 B orellana seed extracts for mosquito repellency evalua-tion

Extraction solvent Resuspension solvent(5mL)

Final solution(mgmL)

Hexane Acetone 182EtOH EtOH 356EtOHH2O (50 50 vv) EtOHH2O (50 50 vv) 1138

210 Repellent Activity Test The method of percentage ofprotection in relation to the dose was used [22] Hundredblood-starved Aedes aegypti females (4ndash8 days old) reared at25∘C 80 RH and an LD cycle of 1212 hours were kept ina net cage (45times 30times 45 cm) Before each test the untreatedforearm (control) of some experimenters was exposed tomosquitoes in the test chamber Once the experimentersobserved five mosquito landings on the untreated arm theyremoved their arm from the chamber The arms of the testperson were cleaned with ethanol washed with unscentedneutral soap thoroughly rinsed and allowed to dry 10minbefore extracts application After air drying the arms of thetested person only 25 cm2 ventral side of the skin on eacharm was treated and exposed while the remaining area wascovered by rubber gloves and apposite covering braceletsThe different extracts of B orellana seeds (at three differentconcentrations each) were applied until complete coverturesof the skin in three different trials The control and treatedarms were introduced serially into the cage The number oflandings was counted over 3min at 1100 am at 200 pm andat 600 pm to evaluate differences in mosquito appetite Theexperiment was conducted by 3 human volunteer testers andreplicated three times at each concentration All experimentswere carried out at a temperature of 27 plusmn 1∘C and RH 75 plusmn5 under laboratory conditions No skin irritation from thetested seed extracts was observed

The percentage of protection was calculated by using theformula

protection

=

119873∘of landings control ndash119873∘of landings treated

119873∘of landings control

lowast 100

(2)

211 Headspace Solid-Phase Microextraction (HS-SPME)Volatile Compounds Sampling from B orellana Living Plants(In Vivo) B orellana opened fruits were sampled in vivoin order to evaluate the VOCs fingerprint emitted by livingplants Each fruit was enclosed in a customised Teflon cage


(manufactured by SNK Inc Fullerton CA 11times 21 cm) intowhich a manual SPME holder was inserted to extract theheadspace VOCs were collected using 5030120583mDivinylben-zeneCarboxenpolydimethylsiloxane (CARPDMSDVB)StableFlex fibre (Supelco Bellefonte PA) The fibre wasexposed to the plant headspace for 8 h Long sampling timewere selected in order to obtain a comprehensive VOCsprofile emitted by plants Peaks originating from the cage(Teflon material) were also assessed by extracting a blanksample

212 HS-SPME Volatile Compounds Sampling of DifferentDried B orellana Organs (Seeds Wood Bark and Leaves)The HS-SPME extraction conditions were optimised in ourprevious study on the characterisation of Achillea collinaVOCs (selection of SPME fibre sample amount and extrac-tion time) [23] Briefly all samples were prepared byweighingexactly 100 g of B orellana powdered samples of seeds woodbark and leaves respectively in a 20mL glass vial fitted withcap equipped with siliconPTFE septa (Supelco BellefontePA USA) and by adding 1mL of the internal standardsolution (IS) in water (14-cineol 1120583gmL CAS 470-67-7) tocheck the quality of the fibres

At the end of the sample equilibration period (1 h)a conditioned (15 h at 280∘C) 5030 120583m DivinylbenzeneCarboxenpolydimethylsiloxane(CARPDMSDVB) Stable-Flex fibre (Supelco Bellefonte PA) was exposed to theheadspace of the sample for extraction (3 h) by CombiPALsystem injector auto-sampler (CTC analytics Switzerland)Temperature of 30∘C was selected as extraction temperaturein order to prevent possible matrix alterations (oxidation ofsome compounds particularly aldehydes) To keep a constanttemperature during the analysis the vials weremaintained ona heater plate (CTC Analytics Zwingen Switzerland)

213 Gas Chromatography-Mass Spectrometry Analysis ofVOCs HS-SPME analysis was performed using a Trace GCUltra (Thermo-Fisher Scientific Waltham MA USA) GasChromatograph coupled to a quadruple Mass SpectrometerTrace DSQ (Thermo-Fisher Scientific Waltham MA USA)and equipped with an Rtx-Wax column (30m 025mm id025 120583m film thickness Restek USA) The oven temperatureprogramme was from 35∘C hold 8min to 60∘C at 4∘Cminthen from 60∘C to 160∘C at 6∘Cmin and finally from 160∘Cto 200∘C at 20∘Cmin Carry-over and peaks originatingfrom the fibre were regularly assessed by running blank sam-ples After each analysis fibres were immediately thermallydesorbed in the GC injector for 5min at 250∘C to preventcontamination The injections were performed in splitlessmode (5min) The carrier gas helium at a constant flowof 1mLmin An n-Alkanes mixture (C

8ndashC22 Sigma R 8769

Saint Louis MO USA) was run under the same chromato-graphic conditions as the samples to calculate the Kovatsretention indices (KI) of the detected compounds [24] Thetransfer line to the mass spectrometer was maintained at230∘C and the ion source temperature was set at 250∘C Themass spectra were obtained by using amass selective detectorwith the electronic impact at 70 eV a multiplier voltage of

0

1

2

3

4

5

6

Aqueous extract Ethanolic extract Traditional infusion

lowast

lowastlowast

Phenols (mg GAEg dry weight)1IC50

(mLmg)

Figure 1 Antioxidant capacity (DPPH scavenging activity)a andtotal phenol content of different extracts of B orellana seed samples(DW)b Data expressed as mean plusmn standard deviation (119899 = 3) a 11-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity expressed asinhibitory concentration (1IC

50

) lowastlowast significant 119875 lt 005 b Phenolcontent expressed as mg GAEg dry weight lowast significant 119875 lt 005

1456V and by collecting the data at the rate of 1 scansover the mz range of 30ndash350 Compounds were identifiedby comparing the retention times of the chromatographicpeaks with those of authentic compounds analyzed under thesame conditions when available or by comparing the Kovatsretention indices with the literature data The identificationof MS fragmentation patterns was performed either by thecomparison with those of pure compounds or using theNational Institute of Standards and Technology (NIST) MSspectral database Volatile compounds measurements fromeach headspace of the plant extracts were carried out by peakarea normalization (expressed in percentages) All analyseswere done in triplicate

Data is expressed as mean value and standard deviation

3 Results and Discussion

31 Antioxidant Capacity and Total Phenol Content AnalysisResults of the antioxidant capacity and total phenol content(TPC) of all B orellana seed extracts (traditional infusionH2O and H

2OETOH) are presented in Figure 1 The free

radical scavenging activity was evaluated by the 22-diphenyl-1-picrylhydrazyl (DPPH) method which is regarded as aneasy reliable sensitive and rapid method A review byMoonand Shibamoto also reported that almost 90 of antioxidantstudies are conducted with this method [25] We noticedthat the DPPH scavenging activity was significantly greaterfor the seeds extracted with water than for extracts obtainedwith infusion and ethanol It is well known that antioxidantsreact with DPPH free radical and convert it to the stableform Therefore B orellana seeds extracted with H

2O seem


to contain a great amount of antioxidant compounds whichmay act as primary antioxidants able to react with freeradicals as efficient hydrogen donors A broad antioxidantactivity of leaf and seed polar extracts has been recentlyproved using five different test systems by Viuda-Martos etal [17] Cardarelli et al reported that the highest free radicalscavenging capacity of annatto extracts was observed in theextract obtained with the most polar solvents which alsoshowed the highest phenol content [26]

In our conditions the total phenol content resultedsignificantly higher for seed extracts obtained with H

2O than

for infusion or H2OETOH extraction

This result clearly indicates that most of the phenolspresent in the plant are in a polar form The TPC of Borellana seed extracts expressed as Gallic acid equivalentranged from 282mgg to 467mgg Cardarelli et al reporteda lower TPC content that ranged between 030 and 184mg ofGallic acid equivalent per gram of dry seeds (depending onthe solvent used for extraction) while Chiste et al recordedTPC values of 17mg of Gallic acid equivalent per gram ofwet seeds [26 27] The concentration and type of phenolsubstances present in the extracts are influenced by seasonaland environmental factors for example climate and soiltype genetic factors and processing methods such as type ofsolvent used It is noteworthy that thewater extractable phenolcompounds present in B orellana dry seeds are higher thanthat of green beans tomatoes green cabbage green collardand some green leafy vegetables Bioactive phenols are veryinteresting as antioxidants because of their natural origin andtheir ability to act as efficient free radical scavengers able toprotect the cells against the oxidative damage caused by freeradicals [28]

32 Repellent Activity Blood-feeding insects are of greatmedical and veterinary importance due both to their nui-sance value and as vectors of disease [29] Amongst insectsthat feed on humans mosquitoes are of global importance[30] In Amazonian regions the use of plants as insect repel-lents is a common practice among Indio tribes The majorityof Itaputyr interviewed villagers admitted that they could notafford to use synthetic commercial mosquito repellents orinsecticides because of high cost andor discouraged by poorperformance of some of the commercial products Similarfindings have been reported elsewhere in Africa such asGuinea Bissau and Kenya where the majority of the villagerscould not afford synthetic commercial mosquito insecticidesdue to poverty [31 32]

The results of mosquito protection in relation to appli-cation dose of different B orellana dried seeds extracts areshown in Table 3 Skin repellent test showed the best resultsusing a highly concentrated extract Dried hexane extractresuspended in acetone provided the highest total percentageprotection followed by hydroalcoholic extract with 90 and73 of protection respectively

These results support the presence of mosquito repellentcompounds in B orellana seeds particularly in the nonpolarfraction also suggested by the Indio tradition to use the

Table 3 Percentage of protection againstmosquitoes (A aegypti) inrelation to the dose of the different B orellana seed extracts (DW)

Extraction solvent Concentration(mgmL)

Protection()

SD(plusmn)

Hexane182 75 16356 85 181138 90 18

EtOH182 22 13356 45 141138 60 16

EtOHH2O (50 50 vv)182 23 3356 50 101138 73 13

Data expressed as mean plusmn standard deviation (119899 = 3)

seeds to paint their bodies during open air religious and warceremonies

33 Volatile Organic Compounds (VOCs) Analysis Thevolatile compounds extracted from headspaces of differentB orellana organs are presented in Table 4 At present thisrepresents the first report on the characterization of volatilecompounds from different B orellana organs by using theSPME technique The SPME technique has the advantage ofminimizing the sample handling and consequently decreas-ing the loss of volatile compounds It is a simple and fastmodern tool used to characterize the VOCs fingerprint ofaromatic and medicinal plants and offers a valid alternativeto hydrodistillation [33] Moreover this technique couldrepresent a valid tool to analyze volatile compounds emittedby in vivo plants sampling them in situ in a nondisruptiveway Overall 71 volatile compounds were identified in Borellana samples with different proportions amongst thedifferent plant organs

The majority of volatile compounds present in theheadspace from different B orellana organs were sesquiter-penes monoterpenes hydrocarbons and ketones Simpleterpenes have gained attention as agents of communicationand defense against insects and can act as attractants orrepellants

Monoterpenes and sesquiterpenes are major componentsof many spices fruit and flower essential oils having a greateconomic importance [34] In the present study we found avery scarce presence of monoterpenes especially 120572-pinenethat was absent in the in vivo samples This is in contrastwith Galindo-Cuspinera et al that found 120572-pinene to be amajor component isolated in the oil obtained fromB orellanasamples However the volatile compounds were recoveredusing dynamic headspace-solvent desorption sampling andanalyzed using GC-MS [35]

Sesquiterpenes constituted the major group of volatilecompounds found in all the different B orellana extractsanalyzed The major constituents were D-germacrene 120575-ele-mene 120574-elemene followed by 120573-caryophyllene 120572-cary-ophyllene 120574-muurolene and 120575-cadinene


Table 4 Identification of volatile organic compounds (VOCs) by HS-SME-GCMS from headspace of B orellana plant extracts obtainedfrom different plant parts and from the headspace in living plant fruits (in vivo sampling)

RIa Volatile compounds Seedb Woodb Leafb Barkb Fruitb ldquoIn vivordquoc

119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sdFree fatty acids

1285 Acetic acid 003 002 nd mdash 001 mdash nd mdash nd mdash nd mdash1628 2-ethylhexanoic acid nd mdash nd mdash nd mdash nd mdash nd mdash 012 001

Total 003 mdash mdash 001 mdash mdash mdash mdash 012Alcohols

659 Ethanol 118 003 nd mdash nd mdash 002 106 003 nd mdash848 2-methyl-1-propanol 001 mdash nd mdash nd mdash nd mdash 003 mdash nd mdash919 2-pentanol nd mdash nd mdash nd mdash nd mdash 001 mdash nd mdash976 1-penten-3-olo nd mdash nd mdash nd mdash nd mdash 001 mdash nd mdash1040 2-methyl-1-butanol 013 005 nd mdash nd mdash nd mdash 005 mdash nd mdash1175 3-methyl-2-buten-1-ol 001 nd mdash nd mdash nd mdash nd mdash nd mdash1573 2-methoxyphenol 006 nd mdash nd mdash nd mdash nd mdash nd mdash1650 Phenol 001 nd mdash nd mdash nd mdash nd mdash nd mdash

Total 139 mdash mdash mdash mdash 002 116 mdash mdashAldehydes

645 2-methyl-butanal nd mdash nd mdash 001 nd mdash nd mdash nd mdash648 3-methyl-butanal nd mdash nd mdash 001 nd mdash nd mdash nd mdash687 Pentanal nd mdash nd mdash 003 nd mdash nd mdash nd mdash815 Hexanal nd mdash nd mdash 001 nd mdash nd mdash 012 0011034 2-hexenal nd mdash nd mdash 001 nd mdash nd mdash nd mdash1243 Nonanal nd mdash nd mdash nd mdash nd mdash nd mdash 017 0021332 Decanal nd mdash nd mdash nd mdash nd mdash nd mdash 017 0031341 Benzaldehyde nd mdash 220 004 nd mdash nd mdash nd mdash 010 mdash

Total mdash mdash 220 007 mdash mdash mdash mdash 056Ketones

614 2-propanone nd mdash nd mdash 002 nd mdash nd mdash 009 001654 2-methyl-3-butanone nd mdash 071 001 nd mdash nd mdash nd mdash nd mdash685 3-pentanone nd mdash 250 002 nd mdash nd mdash 007 nd mdash701 2-methyl-3-pentanone nd mdash 409 017 nd mdash nd mdash nd mdash nd mdash712 4-methyl-2-pentanone nd mdash nd mdash nd mdash nd mdash nd mdash 029 003852 12-cycloctandione 001 nd mdash nd mdash nd mdash nd mdash nd mdash984 2-heptanone nd mdash nd mdash nd mdash nd mdash 001 nd mdash1121 3-hydroxy-2-butanone nd mdash 069 003 nd mdash nd mdash nd mdash nd mdash1189 6-methyl-5-hepten-2-one 002 nd mdash 001 nd mdash nd mdash 014 0021422 4-tert-butylcyclohexanone nd mdash nd mdash nd mdash nd mdash nd mdash 050 006

Total 003 799 003 mdash mdash 008 103Sulphur compounds

604 Dimethyl sulfide 025 002 nd mdash nd mdash 001 mdash 031 002 nd mdashTotal 025 mdash mdash mdash 001 031 mdash mdashFurans

639 2-methylfuran 002 271 015 001 mdash 001 mdash nd mdash nd mdash671 2-ethylfuran nd mdash nd mdash 001 mdash nd mdash nd mdash nd mdash

Total 002 271 002 001 mdash mdash


Table 4 Continued


119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sdEsters

618 Methyl acetate 001 mdash nd mdash nd mdash nd mdash nd mdash nd mdash636 Ethyl acetate 051 012 nd mdash nd mdash nd mdash nd mdash nd mdash895 2-methyl-1-butanol acetate 003 mdash nd mdash nd mdash nd mdash nd mdash nd mdash898 3-methyl-1-butanol ecetate 002 mdash nd mdash nd mdash nd mdash nd mdash nd mdash

Total 057 mdash mdash mdash mdash mdashHydrocarbons

746 Toluene nd mdash nd mdash 001 nd mdash nd mdash nd mdash884 Ethylbenzene nd mdash 224 006 nd mdash nd mdash nd mdash 272 003894 o-xylene nd mdash nd mdash nd mdash nd mdash 001 070 003909 p-xylene 002 233 002 nd mdash 001 nd mdash 151 015979 m-xylene nd mdash 073 009 nd mdash nd mdash 001 050 0051200 Dodecan nd mdash nd mdash nd mdash nd mdash nd mdash 067 0031093 Styrene 001 001 615 012 001 003 nd mdash 035 002

1166 2-ethenyl-11-dimethyl-3-methylenecyclohexane nd mdash nd mdash 052 003 nd mdash nd mdash nd mdash

1270 Hydrocarbon nd mdash nd mdash nd mdash nd mdash nd mdash 111 008

1278 Methyl-(1-methylethenyl)-benzene 001 mdash nd mdash nd mdash nd mdash 012 008 nd mdash

Total 004 1145 054 004 014 756Mono-di-terpenes

709 Triciclene 002 nd mdash nd mdash 003 mdash nd mdash nd mdash726 120572-pinene 024 001 397 009 002 001 038 003 015 002 nd mdash735 120572-thujene nd mdash nd mdash nd mdash 002 mdash 006 mdash nd mdash776 Camphene 023 002 455 012 nd mdash 098 005 010 mdash nd mdash842 120573-pinene 010 mdash nd mdash nd mdash 027 002 005 mdash nd mdash876 Sabinene 002 mdash nd mdash nd mdash 009 001 003 mdash nd mdash931 120572-phellandrene nd mdash nd mdash nd mdash 001 mdash 001 mdash nd mdash971 120573-mircene 003 mdash nd mdash 019 003 013 001 041 002 011 001978 120572-terpinene 005 mdash nd mdash 001 mdash nd mdash 006 021 nd mdash1008 D-limonene 013 360 011 008 001 010 001 061 003 065 0031081 Cis-ocimene 217 004 nd mdash 319 005 330 008 1999 037 338 0081099 Trans-ocimene 005 001 nd mdash 005 001 014 002 069 004 037 0021111 Cymene 014 002 nd mdash 003 001 nd mdash 211 003 077 0051127 120572-terpinolene 005 001 nd mdash 001 mdash 001 mdash 028 001 nd mdash1223 Allo ocimene isomer nd mdash nd mdash 001 mdash 002 mdash 073 003 nd mdash1227 Allo-ocimene 002 nd mdash nd mdash nd mdash 010 001 010 0021494 Naphthalene nd mdash nd mdash nd mdash nd mdash nd mdash 485 009

Total 325 1212 359 548 2539 1023Sesquiterpenes

1301 120572-cubebene 039 013 243 024 191 007 119 006 nd mdash 116 0141316 120575-elemene 2201 009 547 018 480 021 439 003 1803 027 372 0171321 120572-ylangene 015 001 nd mdash 064 002 815 002 057 003 nd mdash1328 120572-copaene 253 007 739 004 1144 015 420 003 325 005 280 0081355 120572-gurjunene 009 001 nd mdash 039 001 099 002 nd mdash nd mdash1363 120573-cubebene 065 005 nd mdash 076 002 037 001 068 003 nd mdash


Table 4 Continued


119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd 119899 = 5 sd1387 Sesquiterpene 005 002 nd mdash 010 002 009 002 nd mdash 308 0081390 Sesquiterpene 158 003 nd mdash 100 007 035 006 nd mdash nd mdash1402 120573-caryophyllene 180 003 1102 054 2239 018 668 014 nd mdash 3732 0191410 Aromadendrene 016 001 291 008 321 003 1160 008 nd mdash 185 0071413 Sesquiterpene 022 001 nd mdash nd mdash nd mdash 773 024 nd mdash1424 Sesquiterpene 041 nd mdash 107 002 140 003 096 nd mdash1435 Sesquiterpene 016 nd mdash nd mdash nd mdash nd mdash 212 0031440 Calarene 115 003 nd mdash 045 001 076 001 219 002 nd mdash1452 120572-caryophyllene nd mdash 870 025 1200 007 712 017 nd mdash 716 0081454 Sesquiterpene 426 002 nd mdash 055 001 nd mdash nd mdash nd mdash1462 Isoledene 180 004 nd mdash nd mdash 043 001 341 019 nd mdash1469 Sesquiterpene nd mdash nd mdash nd mdash nd mdash 388 021 nd mdash1471 120574-muurolene 099 001 295 007 155 002 196 002 274 013 192 0071476 120573-cadinene 180 003 210 008 201 002 180 001 241 008 218 0121486 D-germacrene 2783 017 711 016 1009 009 2113 031 729 015 196 0031487 120573-selinene nd mdash nd mdash 083 002 066 002 nd mdash 124 0031496 120572-selinene nd mdash nd mdash 051 003 053 001 181 008 nd mdash1499 120574-elemene 369 004 576 031 1245 019 1118 012 328 009 113 0071516 Sesquiterpene 115 007 nd mdash nd mdash nd mdash nd mdash nd mdash1519 120575-cadinene 214 007 575 043 470 005 517 011 889 013 578 0151527 Sesquiterpene 1807 016 nd mdash 147 003 273 009 023 001 nd mdash

153112344a7-naphthalenehexahydro-16-dimethyl-4-(1-methylethyl)

010 mdash nd mdash 019 001 024 001 076 002 026 002

1537 120572-muurolene 029 mdash nd mdash 022 001 024 002 154 009 046 0021557 Sesquiterpene 082 002 nd mdash 032 002 025 002 196 004 044 0031563 Calamenene nd mdash nd mdash 005 mdash 007 001 019 001 099 0021612 120572-calacorene nd mdash nd mdash 002 mdash 003 001 008 001 064 0011642 Sesquiterpene nd mdash nd mdash nd mdash 005 001 025 002 208 0071663 Humulene oxide nd mdash nd mdash 005 mdash 003 nd mdash nd mdash1667 Nerolidol nd mdash nd mdash 015 001 nd mdash nd mdash nd mdash1691 Spathulenol nd mdash nd mdash 026 002 016 nd mdash 084 0021728 120572-cadinol 001 mdash nd mdash nd mdash nd mdash 011 002 017 002

Total 943 6159 9558 9395 7224 793aThe Kovats retention index calculated for Rtx-Wax capillary column (25m times 025120583m times 025mm id)bNormalized amount of volatile compounds (percentage) (peak of volatile compoundtotal peak area of all volatile compounds) of B orellana (119899 = 3) obtainedfrom different partscNormalized amount of volatile compounds (percentage) (peak of volatile compoundtotal peak area of all volatile compounds) of B orellana living plantsusing in vivo sampling (119899 = 3)sd plusmn standard deviationnd not detected

Sesquiterpenes were significantly higher than mono-di-terpenes in all plant organs analyzed Leaves bark and seedsshowed higher sesquiterpene concentration than wood andfruits dried or in vivo sampledThe total percentage of VOCsidentified in the wood was rather low only 6159 ascompared to the other plant samples Also Zollo et al foundlow levels of volatile compounds extracted from leaves andwood of B orellanaThe authors identified a great percentage

of ishwarane a sesquiterpene hydrocarbon not found in thepresent research [36]

Elemene and 120575-cadinene have been found in all sam-ples at different percentages Minor sesquiterpenes foundin B orellana extracts that have distinctive aromas includecubebene 120573- and 120575-cadinene Cubebene has been describedas having a fruity sweet citrus-like smell 120573- and 120575-cadin-ene are compounds occasionally used as fixatives in candy


flavours and have a dry-woody slightly medicinal-tarryodour with some similarity to spices in the cumin-thymefamily [37] Some of the monoterpenes and sesquiter-penes found in our extracts have been previously describedas having antimicrobial properties or as being associatedwith pharmacological properties [38] D-germacrene mainlyfound in dried seeds and leaves samples (2783 and 2113resp) showed antimicrobial activity [39]120573-caryophyllene mainly found in in vivo samples

(3732) has been proved to have an anti-inflammatory effectby selectively binding cannabinoid receptors type 2 (CB

2)

[37]Cis-ocimene mainly found in dried fruits samples

(1999) showed antibacterial activity against Gram+ andGramminus (Staphylococcus aureus Escherichia coli Pseudo-monas aeruginosa and Klebsiella pneumoniae) [40]

4 Conclusions

This study has renewed the state of knowledge about Borellana bringing together the existing literature informationand novelties such as VOCs profiling and indications aboutmosquito repellent capacity of seed extracts At presentthis research represents the first investigation on volatilecompounds conducted on different B orellana organs bymeans of a fast sample preparation method In additionSPME was confirmed to be a good technique to analyzevolatile compounds emitted by in vivo plants sampled insitu in a nondisruptive way with consequent potential bigadvantages for phytochemical and ecophysiological studiesparticularly regarding rare andor protected plants Generalscreening confirmed the literature data about phenol com-pounds content and radical scavenging capacity of B orellanaseed extracts

The preliminary results on mosquito repellent activityconfirmed the potential of the plant to protect against Aaegypti representing another theme of future investigationfundamental in linking traditional value of a well-knownplant with scientifically obtained proof of efficacy

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors are grateful to the Itapury Cooperation Projectfor supporting the mission costs

References

[1] C Antweiler ldquoLocal knowledge and local knowing an anthro-pological analysis of contested ldquocultural productsrdquo in the contextof developmentrdquoAnthropos vol 93 no 4ndash6 pp 469ndash494 1998

[2] M Coelho-Ferreira ldquoMedicinal knowledge and plant utiliza-tion in an Amazonian coastal community ofMaruda Para State(Brazil)rdquo Journal of Ethnopharmacology vol 126 no 1 pp 159ndash175 2009

[3] Brasil Polıtica nacional de plantas medicinais e fitoterapicosMinisterio da Saude Secretaria de Ciencia Tecnologia e InsumosEstrategicos Departamento de Assistencia FarmaceuticaBrasılia Brazil 2007

[4] R Rajendran Achiote Bixa Orellana L A Natural Food Colourand Dye AES College of Agricultural Science MangilaoGuam 1st edition 1990

[5] P R N Carvalho andM Hein ldquoUrucum uma fonte de corantenaturalrdquo Coletanea do ITAL vol 19 pp 25ndash33 1989

[6] A T Selvi R Aravindhan B Madhan et al ldquoStudies on theapplication of natural dye extract from Bixa orellana seeds fordyeing and finishing of leatherrdquo Industrial Crops and Productsvol 43 pp 84ndash86 2013

[7] M G Parra and L M Fidalgo ldquoBixa orellana properties andapplicationsrdquo inDrug Plants IV V K Singh and JNGovil Edspp 25ndash35 2010

[8] M P Correa Dicionario das Plantas Uteis Do Brasil e dasExoticas Cultivadas vol 4 Ministerio da AgriculturaIBDF Riode Janeiro Brazil 1978

[9] R A Voeks and A Leony ldquoForgetting the forest assessingmedicinal plant erosion in Eastern Brasilrdquo Economic Botanyvol 58 supplement 1 pp S294ndashS306 2004

[10] M T L A CamargoMedicina Popular Aspectos MetodologicosPara Pesquisa Garrafada Objeto de Pesquisa ComponentesMedicinais de Origem Vegetal Animal e Mineral ALMEDEditora e Livraria Ltda Sao Paulo Brasil 1985

[11] L C di Stasi E M G Santos C M Dos-Santos and C AHiruma Plantas Medicinas da Amazonia UNESP Editora SaoPaolo Brasil 1989

[12] S Patnaik S R Mishra G B Choudhury et al ldquoPhytochem-ical investigation and simultaneously study on anticonvulsantantidiabetic activity of different leafy extracts of Bixa orellanaLinnrdquo International Journal of Pharmaceutical and BiologicalArchives vol 2 no 5 pp 1497ndash1501 2011

[13] K Asokkumar P Jagannath M Umamaheswari et al ldquoCardio-protective activity of Bixa orellana L on isoproterenol inducedmyocardial necrosis in ratsrdquo Journal of Pharmacy Research vol5 no 4 pp 1930ndash1934 2012

[14] A T Selvi M G Dinesh R S Satyanet et al ldquoLeaf and seedextracts of Bixa orellana L exert anti-microbial activity againstbacterial pathogensrdquo Journal of Applied Pharmaceutical Sciencevol 1 no 9 pp 116ndash120 2011

[15] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003

[16] A G Barrio M M M Grueiro D Montero et al ldquoIn vitroantiparasitic activity of plant extracts from panamardquo Pharma-ceutical Biology vol 42 no 4-5 pp 332ndash337 2004

[17] M Viuda-Martos G L Ciro-Gomez Y Ruiz-Navajas et al ldquoInvitro antioxidant and antibacterial activities of extracts fromannatto (Bixa orellana L) leaves and seedsrdquo Journal of FoodSafety vol 32 no 4 pp 399ndash406 2012

[18] B Radhika N Begum and K Srisailam ldquoPharmacognosticand preliminary phytochemical evaluation of the leaves of Bixaorellanardquo Pharmacognosy Journal vol 2 no 7 pp 132ndash136 2010

[19] M L T Nguyen ldquoCultivated plant collections from marketplacesrdquo Ethnobotany Research amp Applications vol 3 pp 5ndash152005

[20] U P Albuquerque Folhas Sagradas as Plantas Liturgicas eMedicinais nos Cultos Afro-Bresileiros Nupeea Recife Brazil2nd edition 2006


[21] A Giorgi S Panseri M S Mattara C Andreis and L MChiesa ldquoSecondary metabolites and antioxidant capacities ofWaldheimia glabra (Decne) Regel from Nepalrdquo Journal of theScience of Food and Agriculture vol 93 no 5 pp 1026ndash10342013

[22] WHO ldquoReport of the WHO informal consultation on theevaluation and testing insecticidesrdquo Tech Rep CTDWHOPESIC961 Control of Tropical Diseases Division WorldHealth Organization Geneva Switzerland 1996

[23] A Giorgi M Madeo G Speranza and M Cocucci ldquoInfluenceof environmental factors on composition of phenolic antiox-idants of Achillea collina Becker ex Rchbrdquo Natural ProductResearch vol 24 no 16 pp 1546ndash1559 2010

[24] L S Anker P C Jurs and P A Edwards ldquoQuantitativestructure-retention relationship studies of odor-active aliphaticcompounds with oxygen-containing functional groupsrdquo Ana-lytical Chemistry vol 62 no 24 pp 2676ndash2684 1990

[25] J K Moon and T Shibamoto ldquoAntioxidant assays for plant andfood componentsrdquo Journal of Agricultural and Food Chemistryvol 57 no 5 pp 1655ndash1666 2009

[26] C R Cardarelli M D T Benassi and A Z Mercadante ldquoChar-acterization of different annatto extracts based on antioxidantand colour propertiesrdquo LWTmdashFood Science and Technology vol41 no 9 pp 1689ndash1693 2008

[27] R C Chiste A Z Mercadante A Gomes E Fernandes J LF D C Lima and N Bragagnolo ldquoIn vitro scavenging capacityof annatto seed extracts against reactive oxygen and nitrogenspeciesrdquo Food Chemistry vol 127 no 2 pp 419ndash426 2011

[28] M C Castello A Phatak N Chandra and M SharonldquoAntimicrobial activity of crude extracts from plant parts andcorresponding calli of Bixa orellana Lrdquo Indian Journal ofExperimental Biology vol 40 no 12 pp 1378ndash1381 2002

[29] A Abdul Rahuman ldquoEfficacies of medicinal plant extractsagainst blood-sucking parasitesrdquo in Nature Helps ParasitologyResearch Monographs H Mehlhorn Ed Springer BerlinGermany 2011

[30] J Fang ldquoEcology a world without mosquitoesrdquo Nature vol466 pp 432ndash434 2010

[31] A Seyoum K Palsson S Kungrsquoa et al ldquoTraditional use ofmosquito-repellent plants in western Kenya and their evalua-tion in semi-field experimental huts againstAnopheles gambiaeethnobotanical studies and application by thermal expulsionand direct burningrdquoTransactions of the Royal Society of TropicalMedicine and Hygiene vol 96 no 3 pp 225ndash231 2002

[32] K Karunamoorthi K Ilango and A Endale ldquoEthnobotan-ical survey of knowledge and usage custom of traditionalinsectmosquito repellent plants among the Ethiopian Oromoethnic grouprdquo Journal of Ethnopharmacology vol 125 no 2 pp224ndash229 2009

[33] A Giorgi S Panseri N N M C Nanayakkara and L MChiesa ldquoHS-SPME-GCMS analysis of the volatile compoundsof Achillea collina evaluation of the emissions fingerprintinduced byMyzus persicae infestationrdquo Journal of Plant Biologyvol 55 no 3 pp 251ndash260 2012

[34] S Terashima M Shimizu S Horie and N Morita ldquoStudieson aldose reductase inhibitors from natural products IV Con-stituents and aldose reductase inhibitory effect of Chrysanthe-mummorifoliumBixa orellana and Ipomoea batatasrdquoChemicaland Pharmaceutical Bulletin vol 39 no 12 pp 3346ndash3347 1991

[35] V Galindo-Cuspinera M B Lubran and S A Rankin ldquoCom-parison of volatile compounds in water- and oil-soluble annatto

(Bixa orellana L) extractsrdquo Journal of Agricultural and FoodChemistry vol 50 no 7 pp 2010ndash2015 2002

[36] P H A Zollo L Biyiti F Tchoumbougnang C Menut GLamaty and P Bouchet ldquoAromatic plants of tropical CentralAfrica Part II Chemical composition and antifungal activityof thirteen essential oils from aromatic plants of CameroonrdquoFlavour and Fragrance Journal vol 13 pp 107ndash114 1998

[37] J Gertsch M Leonti S Raduner et al ldquoBeta-caryophyllene isa dietary cannabinoidrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 26 pp9099ndash9104 2008

[38] J A Shilpi M Taufiq-Ur-Rahman S J UddinM S Alam S KSadhu and V Seidel ldquoPreliminary pharmacological screeningof Bixa orellana L leavesrdquo Journal of Ethnopharmacology vol108 no 2 pp 264ndash271 2006

[39] X S Wang W Yang S J Tao et al ldquoThe effect of 120575-elemeneon hela cell lines by apoptosis inductionrdquo Yakugaku Zasshi vol126 no 10 pp 979ndash990 2006

[40] T Jovanovic D Kitic R Palic G Stojanovic and M RisticldquoChemical composition and antimicrobial activity of the essen-tial oil of Acinos arvensis (Lam) Dandy from Serbiardquo Flavourand Fragrance Journal vol 20 no 3 pp 288ndash290 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of



and norbixin Bixin (methyl (9-cis)-hydrogen-66-diapo-WW-carotenedioate) is the main responsible for the orange-red colour of the seeds and extracts of annatto representapproximately 80 of its total carotenoids [5 7] ThereforeB orellana is a well-known commercial crop The main com-mercial producers are tropical countries including JamaicaMexico and the Philippines with Peru Brazil and Kenyaas the main sources of supply [8] About 80 of the worldproduction which ranges between 10000 and 11000 tonsof seeds per year is utilized by the USA and WesternEurope where it is further processed and used to impart thecharacteristic orange-yellow colour to butter margarine andcheese [9]

It is amatter of fact that almost all the organs ofB orellanaare used in traditional medicine to obtain remedies to treatvarious diseases [10] In Brazil B orellana leaves roots andseed extracts are popular as aphrodisiac medicines as wellas a remedy to treat fevers inflammatory conditions andparasitic diseases The entire plant is used against dysenteryExtracts of leaves bark and roots are reported to be antidotesfor poisoning from Manihot esculenta (Crantz) Jatrophacurcas L andHura crepitans L [11] A decoction of the leavesis used to stop vomiting and nausea as a mild diuretic totreat heartburn prostate urinary difficulties and stomachproblems [10]

Scientific evidences of B orellana bioactivity are nowa-days numerous particularly regarding seed and leaf extractsAnticonvulsant antidiabetic cardioprotective and antimi-crobial activities of different B orellana extracts have beenproved [12ndash14] Fleischer et al demonstrated the antimi-crobial activity of B orellana leaf and seed extracts againsta range of Gram-positive and Gram-negative bacteria andfungi [15] Moreover some pharmacological studies haverevealed that B orellana extracts possess an antiprotozoaland anthelmintic effect [16] Due to their broad antioxidantand antibacterial activities polar extracts from B orellanaleaves and seeds have been recently proposed as alternativenatural preservatives in food matrices [17] Phytochem-ical investigations have revealed the presence of almosttwenty-five types of chemical compounds from B orellanadifferent extracts with the following major componentscarbohydrates carotenoids steroids proteins flavonoidsterpenoids phenolics tannins glycosides alkaloids detectedonly in the leaf and anthroquinones only in the seeds[15 18]

Despite being widely used and studied little is knownabout B orellana volatile organic compounds (VOCs) com-position especially regarding the terpene profile fromvariousplant organs Moreover even if B orellana seed extractsclaim to repel insects little data for a clear validation isavailable Therefore the present research provides the firstinvestigation onB orellana chemical composition of differentplant organs (fruits leaves bark wood and seeds) and thevolatile profile sampled from living plants growing in anAmazonian Reserve (in vivo sampling) during an Italian-Brazilian cooperation project In addition a preliminaryinvestigation on the repellent properties of seed extractsagainst A aegypti L was conducted

2 Materials and Methods

21 Plant Samples B orellana plants growing in proximity ofthe Upper Rio Guama Reserve located in the municipality ofCapitao Poco Para State Brazil (46∘591015840548210158401015840 O 1∘52101584040810158401015840S 152m asl) were identified and the volatile compoundsemissions of fruits were in vivo sampled in February 2011during a mission of an Italian-Brazilian cooperation projectDry seeds roots leaves bark and wood were bought atBelem at the Ver-o-Peso commercial district from themost frequented herbalist shop (1∘27101584088410158401015840S 48∘30101584089910158401015840O)Identification of the in vivo sampled plants and purchasedmaterials was participatory and based on traditional knowl-edge of Indios Tembe a tribe living in Itaputyr a village in theupper Rio Guama reserve [19 20]

22 Standards and Chemicals 22-diphenyl-1-picrylhydrazyl(DPPH) the Folin-Ciocalteu reagent sodium carbonatemethanol and ethanol were purchased from Sigma-AldrichMilan Italy

Volatile compounds used as references were purchasedfrom Sigma-Aldrich-Fluka from the General and Flavoursand Fragrances catalogue Milan Italy

23 Superfine Grinding (SFG) In order to obtain a represen-tative plant sample a superfine powder was prepared fromB Orellanarsquos different organs (seeds wood bark and leaves)using mechanical grinding activation in an energy intensivevibrational mill Three g of dry plant material samples wereground in a high intensity planetary mill Retsch (modelMM 400 Retsch GmbH Retsch-Allee Haan) as previouslyreported [21] The mill was vibrating at a frequency of 25Hzfor 4min using two 50mL jars with 20mm stainless steelballs Precooling of jars was carried out with liquid nitrogenin order to prevent the temperature from increasing duringthe grinding processThe speed differences between balls andjar resulted in the interaction of frictional and impact forcesreleasing high dynamic energies The interplay of all theseforces resulted in the very effective energy input of planetaryball mills

24 Seed Extracts Preparation Three different B orellanaseed extracts were prepared to evaluate antioxidant capacityand phenolic content

25 Traditional Infusion Aliquots of 1 g from B orellana seedpowder were infused for 15min with 100mL of freshly boileddistilled water and cooled to room temperatureThe infusionwas then filtered usingWhatmanNo 4 filter paper in order toremove plant residues and the aliquots were stored at +4∘Covernight until the analysis

26 Exhaustive Extraction (H2Oand EtOH) Aliquots of 03 g

of B orellana seed powder were subjected respectively tosequential extraction using 8mL of distilled water sonicatedfor 10 minutes macerated for 1 h and then sonicated withan ultrasonic cleaning bath (Branson 2510 20KHz 120W)for a further 30min at room temperature The procedure




AC [] = 100 times119860119888ndash119860119904

119860119888

(1)


119888is the


50)


50(mean value)













protection

=



lowast 100

(2)









0

1

2

3

4

5

6


lowast

lowastlowast


(mLmg)


50








2O seem




2O than








Protection()

SD(plusmn)

Hexane182 75 16356 85 181138 90 18

EtOH182 22 13356 45 141138 60 16

EtOHH2O (50 50 vv)182 23 3356 50 101138 73 13























Table 4 Continued














Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of






AC [] = 100 times119860119888ndash119860119904

119860119888

(1)


119888is the


50)


50(mean value)













protection

=



lowast 100

(2)









0

1

2

3

4

5

6


lowast

lowastlowast


(mLmg)


50








2O seem




2O than








Protection()

SD(plusmn)

Hexane182 75 16356 85 181138 90 18

EtOH182 22 13356 45 141138 60 16

EtOHH2O (50 50 vv)182 23 3356 50 101138 73 13























Table 4 Continued














Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of










0

1

2

3

4

5

6


lowast

lowastlowast


(mLmg)


50








2O seem




2O than








Protection()

SD(plusmn)

Hexane182 75 16356 85 181138 90 18

EtOH182 22 13356 45 141138 60 16

EtOHH2O (50 50 vv)182 23 3356 50 101138 73 13























Table 4 Continued














Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of






2O than








Protection()

SD(plusmn)

Hexane182 75 16356 85 181138 90 18

EtOH182 22 13356 45 141138 60 16

EtOHH2O (50 50 vv)182 23 3356 50 101138 73 13























Table 4 Continued














Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of



















Table 4 Continued














Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of




Table 4 Continued




010 mdash nd mdash 019 001 024 001 076 002 026 002









2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of






2)



4 Conclusions





Acknowledgment


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of


































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of



Secondary Metabolite Profile, Antioxidant Capacity, and Mosquito Repellent Activity of Bixa orellana...

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Transcript of Secondary Metabolite Profile, Antioxidant Capacity, and Mosquito Repellent Activity of Bixa orellana...