research article antioxidant activity and volatile and...

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 536490 6 pageshttpdxdoiorg1011552013536490

Research ArticleAntioxidant Activity and Volatile and PhenolicProfiles of Essential Oil and Different Extracts of Wild Mint(Mentha longifolia) from the Pakistani Flora

Tahseen Iqbal Abdullah Ijaz Hussain Shahzad Ali Shahid ChathaSyed Ali Raza Naqvi and Tanveer Hussain Bokhari

Institute of Chemistry Government College University Faisalabad Faisalabad 38000 Pakistan

Correspondence should be addressed to Abdullah Ijaz Hussain abdullahijazgcufedupk

Received 29 May 2013 Revised 13 August 2013 Accepted 28 August 2013

Academic Editor Shao-Nong Chen

Copyright copy 2013 Tahseen Iqbal et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The antioxidant activity and free radical scavenging capacity of the essential oil and three different extracts of wildly grownMenthalongifolia (M longifolia) were studied The essential oil from M longifolia aerial parts was isolated by hydrodistillation techniqueusing Clevenger-type apparatus The extracts were prepared with three solvents of different polarity (n-hexane dichloromethaneandmethanol) using Soxhlet extractor Maximum extract yield was obtained with methanol (126 g100 g) while the minimumwithdichloromethane (350 g100 g)The essential oil content was found to be 107 g100 g A total of 19 constituents were identified in theM longifolia oil using GCMSThemain components detected were piperitenone oxide piperitenone germacrene D borneol and120573-caryophylleneThe total phenolics (TP) and total flavonoids (TF) contents of the methanol extract ofM longifolia were found tobe significantly higher than dichloromethane and hexane extractsThe dichloromethane and methanol extracts exhibited excellentantioxidant activity as assessed by 221015840-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability bleaching 120573-carotene andinhibition of linoleic acid peroxidation assays The essential oil and hexane extract showed comparatively weaker antioxidant andfree radical scavenging activities The results of the study have validated the medicinal and antioxidant potential of M longifoliaessential oil and extracts

1 Introduction

Free radicals are considered to initiate oxidation that leads toaging and causes diseases in human beings [1 2] Moreoveractivated oxygen incorporates reactive oxygen species (ROS)which consists of free radicals (1O

2 O2

∙minus ∙OH ONOOminus)and nonfree radicals (H

2O2 NO and RndashOOH) [3] ROS

are liberated by virtue of stress and thus an imbalance isdeveloped in the body that damages cells in it and causeshealth problems [2 4] Moreover oxidation in processedfoods enriched with fats and oils during storage leads tospoilage and quality deterioration [5]

The use of synthetic antioxidants such as butylated hydro-xyanisole (BHA) and butylated hydroxytoluene (BHT) andtertiary butylhydroquinone (TBHQ) have been restrictedbecause of their carcinogenicity and other toxic proper-ties [3 6] Thus the interest in natural antioxidants has

increased considerably Natural antioxidants can be phenoliccompounds (tocopherols flavonoids and phenolic acids)and carotenoids (lutein lycopene and carotene) Growingevidence has shown an inverse correlation between the intakeof dietary antioxidants and the risk of chronic diseases suchas coronary heart disease cancer and several other aging-related health concerns [1 7]

Natural antioxidant compounds exhibit their antioxidantactivity by various mechanisms including chain breakingby donation of hydrogen atoms or electrons that convertfree radicals into more stable species and decomposing lipidperoxides into stable final products [1] Different in vitroassays simply provide an idea of the protective efficacy ofthe test model Thus it is necessary to use at least twomethods depending on the expected antioxidant potentialandor on the origin of the substance Most commonly usedmethods for the determination of antioxidant activity of plant

2 Journal of Analytical Methods in Chemistry

essential oils and extracts are 22-di(4-tert-octaphenyl)-1-picrylhydrazyl (DPPH∙) radical scavenging assay inhibitionof linoleic acid peroxidation and bleaching of 120573-carotene inlinoleic acid system assays DPPH radical scavenging assay isthe most popular and frequently used for the determinationof antioxidant activity of essential oils and plant extracts[1 7 8] Bleachability of 120573-carotene in linoleic acid systemis another simple reproducible and time efficient methodfor rapid evaluation of antioxidant properties [1 7 8]Measurement of inhibition of linoleic acid peroxidation isalso an effective method for the assessment of antioxidantactivity of the plant samples

Mentha longifolia (wild mint) belongs to genus Mentha(family Lamiaceae) and grows widely throughout the tem-perate regions of the world [8] The different herbal and foodproducts fromMentha species have been in use since ancienttimes for the treatment of heart burns indigestion colicflatulence coughs and flu nausea irritable bowel syndromegall-bladder and bile ducts herpes and certain skin infec-tions including acne and pigmentation [7ndash9] Research workon plants from different regions resulted in the innovationof biologically active substances [8 10] Therefore the studywas conducted to investigate the chemical composition andantioxidant and antimicrobial activities of essential oil andthree different extracts fromM longifolia native to dry regionof Pakistan

2 Materials and Methods

21 Collection and Pretreatment of Plant Material Aerialparts of wild mint (M longifolia L) were collected duringMay-June from South Punjab Pakistan The specimens werefurther identified and authenticated by a taxonomist DrQasimAli (Assistant Professor) Department of Botany Gov-ernment College University Faisalabad Collected specimenswere dried at 35∘C in a hot air oven (IM-30 Irmec Germany)and grinded to 80 mesh and stored in polyethylene bags atminus4∘C

22 Chemicals and Reagents Linoleic acid 221015840-diphenyl-1-picrylhydrazyl gallic acid Folin-Ciocalteu reagent ascorbicacid trichloroacetic acid sodium nitrite aluminum chlorideammonium thiocyanate ferrous chloride ferric chloridepotassium ferricyanide butylated hydroxytoluene (990)and homologous series of C

9ndashC24

n-alkanes and variousreference chemicals used to identify the constituents wereobtained from Sigma Chemical Co (St Louis MO USA) Allother chemicals (analytical grade) that is anhydrous sodiumcarbonate ferrous chloride ammonium thiocyanate chloro-form and methanol used in this study were purchased fromMerck (Darmstadt Germany) unless stated otherwise Allculture media and standard antibiotic discs were purchasedfrom Oxoid Ltd (Hampshire UK)

23 Isolation of Essential Oil The oven-dried and groundfennel seeds (80mesh) were subjected to hydrodistillation for4 h using a Clevenger-type apparatus The obtained essentialoil was dried over anhydrous sodium sulfate filtered andstored at minus4∘C until analyzed

24 Preparation of Extracts Ground (80 mesh) M longi-folia sample (100 g) was subjected to extraction for 4 husing Soxhlet unit The plant materials were extracted insequence with three solvents of different polarity that isn-hexane dichloromethane and methanol The extracts wereconcentrated under vacuum at 45∘C using a vacuum rotaryevaporator (NndashN Series Eyela Rikakikai Co Ltd TokyoJapan) and stored at minus4∘C until used for further analyses

25 Analysis of the Essential Oil

251 Gas ChromatographyMass Spectrometry Analysis TheM longifolia essential oil composition was determined onAgilent-Technologies (Little Falls CA USA) 6890NNetworkgas chromatographic (GC) system equippedwith anAgilent-Technologies 5975 inert XL Mass selective detector andAgilent-Technologies 7683B series autoinjector Compoundswere separated on HP-5 MS capillary column (30m times025mm film thickness 025120583m Little Falls CA USA)A sample of 10 120583L was injected in the split mode withsplit ratio 1 100 Helium was used as a carrier gas at aflow rate of 15mLmin For GCMS detection an electronionization system with ionization energy of 70 eV was usedThe column oven temperature was programmed from 80∘Cto 220∘C at the rate of 4∘Cmin initial and final temperatureswere held for 3 and 10min respectively Mass scanningrange was 50ndash550mz while the injector and MS transferline temperatures were set at 220 and 290∘C respectivelyAll quantifications were done by a built-in data-handlingprogram of the equipment used (Perkin-Elmer Norwalk CTUSA) The composition was reported as a relative percentageof the total peak area

252 Compounds Identification The components of the Mlongifolia essential oil were identified by comparison of theirretention indices relative to (C

9ndashC24) n-alkanes either with

those of published data or with authentic compounds [1112] Compounds were further identified and authenticatedusing their complete mass fragmentation data compared tothe NIST02L and WILEY7nL mass spectral libraries andpublishedmass spectra andwherever possible by coinjectionwith authentic standards (120572-pinene 120573-pinene limonenecis-120573-ocimene 120575-terpinene 18-cineol linalool borneol120572-terpineol thymol piperitenone piperitenone oxide 120573-caryophyllene germacrene D calamenene cis-jasmone andcaryophyllene oxide) [1 13 14]

26 Antioxidant Activity

261 Determination of Total Phenolics (TP) and Total Flavo-noids (TF) Contents Amounts of total phenolics (TP) andtotal flavonoids (TF) in the M longifolia extracts weredetermined using Folin-Ciocalteu reagent method and alu-minum chloride colorimetric assay respectively as reportedpreviously [15]

262 DPPH Radical Scavenging Assay 221015840-Diphenyl-1-picrylhydrazyl (DPPH) free radical assay was carried outto measure the free radical scavenging activity as reported

Journal of Analytical Methods in Chemistry 3

previously [9] Briefly M longifolia essential oil extractspiperitenone compound and BHT concentrations inmethanol (1ndash100 120583gmL) were mixed with 2mL of 90120583Mmethanol solution of DPPH After 30min incubation periodat room temperature the absorbance was read at 517 nmThescavenging () was calculated by the following formula

Scavenging () = 100 times (119860blank minus 119860 sample)

119860blank (1)

where 119860blank is the absorbance of the DPPH solution and119860 sample is the absorbance of the extract solution Extract con-centration providing 50 scavenging (IC

50) was calculated

from the graph plotted between scavenging percentage andextract concentration

263 Antioxidant Activity Determination in Linoleic AcidSystem The antioxidant activity ofM longifolia essential oiland extracts weas determined in terms of measurement of inhibition of peroxidation in linoleic acid system followingthe reported method with some modification [16] Essentialoil and extracts (5mg) were added to a solution mixtureof linoleic acid (013mL) 998 ethanol (10mL) and 10mLof 02M sodium phosphate buffer (pH 7) Total mixturewas diluted to 25mL with distilled water The solution wasincubated at 40∘C for 175 h The extent of oxidation wasmeasured by peroxide value using the colorimetric methodas reported previously [15]

264 Bleaching of 120573-Carotene in Linoleic Acid SystemAntioxidant activity ofM longifolia essential oil and extractswas also assessed by bleaching of 120573-carotenelinoleic acidemulsion system as reported previously [1] Briefly a stocksolution of 120573-carotene-linoleic acid mixture was preparedby dissolving 01mg 120573-carotene 20mg linoleic acid and100mg Tween 40 in 10mL of chloroform (HPLC grade)Thechloroformwas removed under vacuum in rotary evaporatorat 50∘CThen 50mL of distilled water saturated with oxygen(30min 100mL minminus1) was added with vigorous shaking A50mL of this reaction mixture was dispensed to test tubeswith 200120583L of the essential oil or trans-anethole solutionprepared at 40 g Lminus1 concentrations and the absorbance wasimmediately (119905 = 0) measured at 490 nm against a blankconsisting of an emulsion without 120573-carotene Then emul-sion was incubated for 50 h at room temperature and theabsorbance was recorded at different time intervalsThe sameprocedure was repeated with BHT and blank Antioxidantcapacities of the fennel essential oils were compared withBHT and blank

27 Statistical Analysis All the experiments were conductedin triplicate unless stated otherwise and data are presentedas mean plusmn standard deviation (SD) Statistical analysis of thedata was performed by Analysis of Variance (ANOVA) usingSTATISTICA 55 (Stat Soft Inc Tulsa OK USA) softwareand probability value 119875 le 005 was considered to denote astatistically significant difference

Table 1 Yield of M longifolia essential oil and hexane dichloro-methane and methanol extracts

Samples Yield (g100 g)lowast

Essential oil 107 plusmn 010a

n-Hexane extract 730 plusmn 032c

Dichloromethane extract 350 plusmn 021b

Methanol extract 1260 plusmn 070dlowastValues aremeanplusmn SDof three samples ofM longifolia analyzed individuallyin triplicateDifferent letters in superscript represent significant (119875 lt 005) differencewithin solvents

3 Results and Discussion

31 Percentage Yield of Essential Oil and Different ExtractsYield (g100 g of dry plant material) of Mentha longifoliaessential oil and n-hexane dichloromethane and methanolextracts is given in Table 1Maximumyield was obtainedwithmethanol (1260 g100 g) The minimum yield was obtainedwith dichloromethane (350 g100 g) The essential oil yieldfrom the aerial parts of M longifolia was found to be107 g100 g Nonpolar extract yield (n-hexane) was foundto be 730 g100 g Tukeyrsquos range test revealed the significant(119875 lt 005) difference among the extract yield with solventsof different polarities Differences in yield of extracts fromdifferent solvents might be attributed to the availability ofextractable component of different polarities

32 Essential Oil Composition The retention indices per-centage composition and identification methods for theessential oil of M longifolia are given in Table 2 Nine-teen compounds 9679 of the total oil were identifiedfrom the oil (Figure 1) The most abundant constituents(gt5) in the essential oil of M longifolia were found tobe piperitenone oxide (283) piperitenone (249) ger-macrene D (816) borneol (596) and 120573-caryophyllene(594) Analyzed essential oil mainly consisted of oxy-genated monoterpenes (6724) followed by sesquiterpenehydrocarbons (1719) monoterpene hydrocarbons (731)and oxygenated sesquiterpenes (505)

The variation in the essential oil composition of Mlongifolia is reported in the literature from different part ofthe world [8 17 18] Our results reported the essential oilcomposition ofM longifolia essential oil from South PunjabPakistan where the weather conditions are very hot and dryVariations in the chemical compositions of essential oil acrosscountries might be attributed to the varied agroclimatic(climatical seasonal and geographical) conditions of theregions isolation regimes and adaptivemetabolism of plants


331 Total Phenolics (TP) and Total Flavonoids (TF) ContentsAmount of total phenolics and total flavonoids is given inFigure 2 The highest TP was found in methanol extract(7143mgg acid of dry plant material measured as gallicequivalent) and the lowest in hexane extract (17mgg acid ofdry plant material measured as gallic equivalent) Similarly


(a) (b) (c) (d) (e)

O

(f)

HO

(g)

OH

(h)

HO

(i)

OH

(j)

O

(k)

O O

(l)

O

O

(m) (n) (o)

(p) (q)

O

(r)

HOO

(s)

Figure 1 Structure ofmajor compounds detected fromM longifolia essential oil (a) 120572-Pinene (b)120573-pinene (c) limonene (d) cis-120573-ocimene(e)120575-terpinene (f) 18-cineole (g) linalool (h) borneol (i)120572-terpineol (j) thymol (k) piperitenone (l) thymol acetate (m) piperitenone oxide(n) 120572-gurjunene (o) 120573-caryophyllene (p) germacrene D (q) calamenene (r) cis-jasmone (s) caryophyllene oxide

01020304050607080

Methanol extract Hexane extract

Dry

pla

nt m

ater

ial (

mg

g)

TPC (measured as gallic acid equivalent)TFC (measured as catechin equivalent)

Dichloromethaneextract

Figure 2 Total phenolics (TP) and total flavonoids (TF) contents ofn-hexane dichloromethane and methanol extracts ofM longifolia

the amount of TF in methanol dichloromethane and hexaneextracts was found to be 1235 47 and 11mgg acid of dryplant material measured as catechin equivalent The effect ofdifferent solvent systems on the amount of TP and TF wassignificant (119875 lt 005) Methanol has been proven as effectivesolvent to extract phenolic compounds [6]

332 DPPH Radical Scavenging Assay The ability of Mlongifolia essential oil anddifferent extract to donate proton toDPPH free radical and change its color fromviolet to yellow isaccessed in this assay Concentration of extracts and essentialoil scavenging 50 of DPPH radical is shown in Table 3IC50

value ranged from 670 to 333 120583gmL Greater IC50

value (maximum radical scavenging activity) was observedwith methanol extract of M longifolia and lesser IC

50value

was recorded with n-hexane extract IC50

values of Mlongifolia essential oil and dichloromethane extracts werefound to be comparable with IC

50value of the piperitenone

a major compound of M longifolia essential oil IC50

valueof methanol extract is significantly (119875 lt 005) better thanhexane and dichloromethane extracts and essential oil andcomparable with synthetic antioxidant BHT

333 Antioxidant Activity Determination in Terms of Inhibi-tion of Linoleic Acid Peroxidation The antioxidants activityhas also been assessed as ability to prevent the oxidation oflinoleic acid Therefore inhibition of linoleic acid oxidationwas also used to assess the antioxidant activity ofM longifoliaextracts and essential oil All extracts and essential oilexhibited appreciable inhibition of linoleic acid peroxida-tion (Table 3) ranging from 99 to 916 Methanol extractshowed maximum antioxidant activity (916) followed bydichloromethane extract (893) which is comparable with


Table 2 Chemical composition ofM longifolia essential oil

Componentsb RIc Molecular mass Compositiona Mode of identificationd Quality ()e

Monoterpene hydrocarbons (731)120572-Pinene 939 136 076 plusmn 006 RT RI MS 97120573-Pinene 979 136 214 plusmn 019 RT RI MS 96Limonene 1029 136 180 plusmn 019 RT RI MS 94cis-120573-Ocimene 1037 136 198 plusmn 011 RT RI MS 97120575-Terpinene 1089 136 063 plusmn 004 RI MS 96

Oxygenated monoterpenes (6724)18-Cineol 1031 154 200 plusmn 017 RT RI MS 98Linalool 1097 154 098 plusmn 010 RT RI MS 98Borneol 1169 154 596 plusmn 044 RT RI MS 96120572-Terpineol 1189 154 117 plusmn 009 RT RI MS 98Thymol 1290 150 285 plusmn 020 RT RI MS 99Piperitenone 1343 150 249 plusmn 134 RT RI MS 97Thymol acetate 1352 192 108 plusmn 008 RI MS 94Piperitenone oxide 1370 166 283 plusmn 16 RT RI MS 96

Sesquiterpene hydrocarbons (1719)120572-Gurjunene 1410 204 111 plusmn 018 RI MS 96120573-Caryophyllene 1421 204 594 plusmn 032 RT RI MS 99Germacrene D 1485 204 816 plusmn 101 RT RI MS 99Calamenene 1540 202 198 plusmn 017d RT RI MS 98

Oxygenated sesquiterpenes (505)cis-Jasmone 1393 164 113 plusmn 011a RT RI MS 96Caryophyllene oxide 1583 220 392 plusmn 0b RT RI MS 97

Total 9679aValues are mean plusmn standard deviation of three samples ofM longifolia essential oil analyzed individually in triplicatebCompounds are listed in order of elution from a HP-5MS column cretention indices relative to C

9ndashC24

n-alkanes on the HP-5MS column dmode ofidentifications RT identification based on retention time RI identification based on retention index MS identification based on comparison of MS datacompared with those from the NIST02L and WILEY7nL mass spectral libraries ematching percentage with the NIST02L and WILEY7nL mass spectrallibraries

Table 3 Antioxidant activity of M longifolia essential oil and n-hexane dichloromethane and methanol extracts

SamplesAntioxidant activitylowast

DPPH IC50(120583gmLminus1)

Inhibition of linoleicacid peroxidation ()

Essential oil 218 plusmn 12c 373 plusmn 13c

n-Hexane extract 333 plusmn 17d 99 plusmn 07a

DCM extract 212 plusmn 17c 893 plusmn 29d

Methanol extract 670 plusmn 03a 916 plusmn 23d

Piperitenone 227 plusmn 15c 313 plusmn 21b

BHT 990 plusmn 02b 909 plusmn 27dlowastValues are mean plusmn standard deviation of three samples of each Thymusspecies analyzed individually in triplicate Mean followed by differentsuperscript letters in the same column represents significant difference (119875 lt005)NT not tested

the activity of BHT standard (906) M longifolia essentialoil and hexane extract showed weaker antioxidant activityPolar extract exhibited significantly (119875 le 005) higher

antioxidant activity than nonpolar extracts which might bedue to the higher concentration of TP and TF contents[15]

334 Antioxidant Activity Determination in Terms of Bleach-ing of 120573-Carotene in Linoleic Acid System Bleaching 120573-carotene with linoleic acid system as antioxidant activity ofthe M longifolia essential oil and extracts is presented inFigure 3 The greater is the effectiveness of an antioxidantthe slower will be the colour depletion In Figure 3 smallerdecline in absorbance of 120573-carotene indicates a lower rate ofoxidation of linoleic acid and higher antioxidant activity inthe presence ofM longifoliamethanol and dichloromethaneextracts and BHT Hexane extract and essential oil showedpoor antioxidant activity

4 Conclusion

Methanol extracts of Mentha longifolia exhibited excellentantioxidant activity and free radical scavenging capacity fol-lowed by dichloromethane extract essential oil and hexane


0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)

Control BHTEssential oil n-Hexane extractDCM extract Methanol extract

Abso

rban

ce 4

90 nm

Figure 3 Antioxidant activity of M longifolia essential oil andn-hexane dichloromethane and methanol extracts in terms ofbleaching of 120573-carotene-linoleic acid emulsion

extract High TP and TF contents and antioxidant potentialof M longifolia extracts lead to its possible use as a foodpreservative Moreover they may be used in pharmaceuticaland natural therapies for treatment of oxidative stress

Acknowledgment

Grants supports by the Higher Education Commission(HEC) Islamabad Pakistan under the National ResearchProgram for University (NRPU) scheme are highly acknowl-edged

References

[1] A I Hussain F Anwar S T H Sherazi and R PrzybylskildquoChemical composition antioxidant and antimicrobial activ-ities of basil (Ocimum basilicum) essential oils depends onseasonal variationsrdquo Food Chemistry vol 108 no 3 pp 986ndash995 2008

[2] F Shahidi and P K Wanasundara ldquoPhenolic antioxidantsrdquoCritical Reviews in Food Science abd Nutrition vol 32 no 1 pp67ndash103 1992

[3] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjuna Acacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[4] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[5] S A S Chatha A I Hussain J Bajwa and M SagirldquoAntioxidant activity of different solvent extracts of rice bran ataccelerated storage of sunflower oilrdquo Journal of Food Lipids vol13 no 4 pp 424ndash433 2006

[6] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[7] A I Hussain F Anwar S A S Chatha A Jabbar S Mahbooband P S Nigam ldquoRosmarinus officinalis essential oil antipro-liferative antioxidant and antibacterial activitiesrdquo BrazilianJournal of Microbiology vol 41 no 4 pp 1070ndash1078 2010

[8] A I Hussain F Anwar P S Nigam M Ashraf and A HGilani ldquoSeasonal variation in content chemical compositionand antimicrobial and cytotoxic activities of essential oilsfrom four Mentha speciesrdquo Journal of the Science of Food andAgriculture vol 90 no 11 pp 1827ndash1836 2010

[9] A I Hussain F Anwar S Rasheed P S Nigam O Janneh andS D Sarker ldquoComposition antioxidant and chemotherapeuticproperties of the essential oils from two Origanum speciesgrowing in Pakistanrdquo Revista Brasileira de Farmacognosia vol21 no 6 pp 943ndash952 2011

[10] F Anwar A I Hussain S T H Sherazi and M I BhangerldquoChanges in composition and antioxidant and antimicrobialactivities of essential oil of fennel (Foeniculum vulgare Mill)fruit at different stages of maturityrdquo Journal of Herbs Spices andMedicinal Plants vol 15 no 2 pp 187ndash202 2009

[11] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[12] R P Adam Identification of Essential Oils Components byGas ChromatographyQuadrupole Mass Spectroscopy AlluredPublishing Carol Stream Ill USA 2001

[13] F Anwar M Ali A I Hussain and M Shahid ldquoAntioxidantand antimicrobial activities of essential oil and extracts of fennel(Foeniculum vulgare Mill) seeds from Pakistanrdquo Flavour andFragrance Journal vol 24 no 4 pp 170ndash176 2009

[14] K Vagionas K Graikou O Ngassapa D Runyoro and IChinou ldquoComposition and antimicrobial activity of the essen-tial oils of three Satureja species growing in Tanzaniardquo FoodChemistry vol 103 no 2 pp 319ndash324 2007

[15] A I Hussain S A S Chatha S Noor et al ldquoEffect of extractiontechniques and solvent systems for the extraction of antioxidantcomponents from peanut (Arachis hypogaea L) Hullsrdquo FoodAnalytical Methods vol 5 no 4 pp 890ndash896 2012

[16] S Iqbal M I Bhanger and F Anwar ldquoAntioxidant propertiesand components of some commercially available varieties ofrice bran in Pakistanrdquo Food Chemistry vol 93 no 2 pp 265ndash272 2005

[17] A M Viljoen S Petkar S F van Vuuren A C Figueiredo LG Pedro and J G Barroso ldquoThe chemo-geographical variationin essential oil composition and the antimicrobial properties ofldquowild mintrdquomdashMentha longifolia subsp polyadena (Lamiaceae)in Southern Africardquo Journal of Essential Oil Research vol 18 pp60ndash65 2006

[18] M Gulluce F Sahin M Sokmen H Ozer D Daferera andA Sokmen ldquoAntimicrobial and antioxidant properties of theessential oils and methanol extract from Mentha longifolia Lssp longifoliardquo Food Chemistry vol 103 no 4 pp 1449ndash14562007

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essential oils and extracts are 22-di(4-tert-octaphenyl)-1-picrylhydrazyl (DPPH∙) radical scavenging assay inhibitionof linoleic acid peroxidation and bleaching of 120573-carotene inlinoleic acid system assays DPPH radical scavenging assay isthe most popular and frequently used for the determinationof antioxidant activity of essential oils and plant extracts[1 7 8] Bleachability of 120573-carotene in linoleic acid systemis another simple reproducible and time efficient methodfor rapid evaluation of antioxidant properties [1 7 8]Measurement of inhibition of linoleic acid peroxidation isalso an effective method for the assessment of antioxidantactivity of the plant samples

Mentha longifolia (wild mint) belongs to genus Mentha(family Lamiaceae) and grows widely throughout the tem-perate regions of the world [8] The different herbal and foodproducts fromMentha species have been in use since ancienttimes for the treatment of heart burns indigestion colicflatulence coughs and flu nausea irritable bowel syndromegall-bladder and bile ducts herpes and certain skin infec-tions including acne and pigmentation [7ndash9] Research workon plants from different regions resulted in the innovationof biologically active substances [8 10] Therefore the studywas conducted to investigate the chemical composition andantioxidant and antimicrobial activities of essential oil andthree different extracts fromM longifolia native to dry regionof Pakistan

2 Materials and Methods

21 Collection and Pretreatment of Plant Material Aerialparts of wild mint (M longifolia L) were collected duringMay-June from South Punjab Pakistan The specimens werefurther identified and authenticated by a taxonomist DrQasimAli (Assistant Professor) Department of Botany Gov-ernment College University Faisalabad Collected specimenswere dried at 35∘C in a hot air oven (IM-30 Irmec Germany)and grinded to 80 mesh and stored in polyethylene bags atminus4∘C

22 Chemicals and Reagents Linoleic acid 221015840-diphenyl-1-picrylhydrazyl gallic acid Folin-Ciocalteu reagent ascorbicacid trichloroacetic acid sodium nitrite aluminum chlorideammonium thiocyanate ferrous chloride ferric chloridepotassium ferricyanide butylated hydroxytoluene (990)and homologous series of C

9ndashC24

n-alkanes and variousreference chemicals used to identify the constituents wereobtained from Sigma Chemical Co (St Louis MO USA) Allother chemicals (analytical grade) that is anhydrous sodiumcarbonate ferrous chloride ammonium thiocyanate chloro-form and methanol used in this study were purchased fromMerck (Darmstadt Germany) unless stated otherwise Allculture media and standard antibiotic discs were purchasedfrom Oxoid Ltd (Hampshire UK)

23 Isolation of Essential Oil The oven-dried and groundfennel seeds (80mesh) were subjected to hydrodistillation for4 h using a Clevenger-type apparatus The obtained essentialoil was dried over anhydrous sodium sulfate filtered andstored at minus4∘C until analyzed

24 Preparation of Extracts Ground (80 mesh) M longi-folia sample (100 g) was subjected to extraction for 4 husing Soxhlet unit The plant materials were extracted insequence with three solvents of different polarity that isn-hexane dichloromethane and methanol The extracts wereconcentrated under vacuum at 45∘C using a vacuum rotaryevaporator (NndashN Series Eyela Rikakikai Co Ltd TokyoJapan) and stored at minus4∘C until used for further analyses

25 Analysis of the Essential Oil

251 Gas ChromatographyMass Spectrometry Analysis TheM longifolia essential oil composition was determined onAgilent-Technologies (Little Falls CA USA) 6890NNetworkgas chromatographic (GC) system equippedwith anAgilent-Technologies 5975 inert XL Mass selective detector andAgilent-Technologies 7683B series autoinjector Compoundswere separated on HP-5 MS capillary column (30m times025mm film thickness 025120583m Little Falls CA USA)A sample of 10 120583L was injected in the split mode withsplit ratio 1 100 Helium was used as a carrier gas at aflow rate of 15mLmin For GCMS detection an electronionization system with ionization energy of 70 eV was usedThe column oven temperature was programmed from 80∘Cto 220∘C at the rate of 4∘Cmin initial and final temperatureswere held for 3 and 10min respectively Mass scanningrange was 50ndash550mz while the injector and MS transferline temperatures were set at 220 and 290∘C respectivelyAll quantifications were done by a built-in data-handlingprogram of the equipment used (Perkin-Elmer Norwalk CTUSA) The composition was reported as a relative percentageof the total peak area

252 Compounds Identification The components of the Mlongifolia essential oil were identified by comparison of theirretention indices relative to (C

9ndashC24) n-alkanes either with

those of published data or with authentic compounds [1112] Compounds were further identified and authenticatedusing their complete mass fragmentation data compared tothe NIST02L and WILEY7nL mass spectral libraries andpublishedmass spectra andwherever possible by coinjectionwith authentic standards (120572-pinene 120573-pinene limonenecis-120573-ocimene 120575-terpinene 18-cineol linalool borneol120572-terpineol thymol piperitenone piperitenone oxide 120573-caryophyllene germacrene D calamenene cis-jasmone andcaryophyllene oxide) [1 13 14]


261 Determination of Total Phenolics (TP) and Total Flavo-noids (TF) Contents Amounts of total phenolics (TP) andtotal flavonoids (TF) in the M longifolia extracts weredetermined using Folin-Ciocalteu reagent method and alu-minum chloride colorimetric assay respectively as reportedpreviously [15]

262 DPPH Radical Scavenging Assay 221015840-Diphenyl-1-picrylhydrazyl (DPPH) free radical assay was carried outto measure the free radical scavenging activity as reported




119860blank (1)


50) was calculated


















(a) (b) (c) (d) (e)

O

(f)

HO

(g)

OH

(h)

HO

(i)

OH

(j)

O

(k)

O O

(l)

O

O

(m) (n) (o)

(p) (q)

O

(r)

HOO

(s)


01020304050607080


Dry

pla

nt m

ater

ial (

mg

g)








50value















9ndashC24















4 Conclusion



0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)


Abso

rban

ce 4

90 nm



Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




119860blank (1)


50) was calculated


















(a) (b) (c) (d) (e)

O

(f)

HO

(g)

OH

(h)

HO

(i)

OH

(j)

O

(k)

O O

(l)

O

O

(m) (n) (o)

(p) (q)

O

(r)

HOO

(s)


01020304050607080


Dry

pla

nt m

ater

ial (

mg

g)








50value















9ndashC24















4 Conclusion



0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)


Abso

rban

ce 4

90 nm



Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b) (c) (d) (e)

O

(f)

HO

(g)

OH

(h)

HO

(i)

OH

(j)

O

(k)

O O

(l)

O

O

(m) (n) (o)

(p) (q)

O

(r)

HOO

(s)


01020304050607080


Dry

pla

nt m

ater

ial (

mg

g)








50value















9ndashC24















4 Conclusion



0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)


Abso

rban

ce 4

90 nm



Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









9ndashC24















4 Conclusion



0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)


Abso

rban

ce 4

90 nm



Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0010203040506070809

1

0 5 10 15 20 25 30 35 40 45 50Time (h)


Abso

rban

ce 4

90 nm



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

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