Journal of Food Processing & Technology

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1 Journl of Food Processing & Technology ISSN: Journl of Food Processing & Technology Gortzi et l. 04, 5:8 DOI: 0.47/ Reserch Article Open Access Study of Antioxidnt nd Antimicroil Activity of Chios Mstic Gum Frctions (Neutrl, Acidic) Before nd After Encpsultion in Liposomes Olg Gortzi *, Vsilios Athnsidis, Stvros Lls, Ionn Chinou nd John Tsknis Deprtment of Food Technology, Technologicl Eductionl Institute of Thessly, Term N. Temponer str., GR-400, Krdits, Greece Division of Phrmcognosy nd Chemistry of Nturl Products, Deprtment of Phrmcy, University of Athens, Pnepistimiopolis-Zogrfou, GR-577, Athens, Greece Deprtment of Food Technology, Technologicl Eductionl Institute of Athens, Agiou Spyridonos str., GR-0, Egleo, Athens, Greece Astrct Mstic is well-known nturl resin from the trunk nd rnches, of Pistci lentiscus vr. chi (Ancrdicee), which is grown s endemic only in the Greek islnd of Chios. During this work, totl mstic gum extrct ws prepred fter removl of the contined insolule polymer in order to meliorte soluility nd enhnce in vivo ctivity. To overcome the drwcks (i.e soluility, iovilility, etc.) of mstic gum extrcts (cidic nd neutrl frction), the selection of suitle crrier is crucil. Three different methods of preprtion, thin-film evportion, freezing-thwing, nd ethnol injection were used for the preprtion of liposomes consisting of Phosphtidylcholine (PC) nd Cholesterol (CH). The effect of PC: CH molr rtio on the percentge of mstic extrct encpsulted ws investigted. Mstic gum extrcts components-liposomes interction ws studied using Fourier trnsform infrred (FT-IR) spectroscopy. The effects of different preprtion methods on the physicochemicl properties of colloidl systems were evluted y mens of surfce morphology, field emission Scnning Electron Microscopy (SEM), nd size distriution using prticle size nlyzer. For the determintion of the ntioxidnt ctivity two methods were used: I) The Rncimt method where the protection fctor ws determined for ech smple nd compred with known ntioxidnts. II) Differentil Scnning Clorimetry (DSC) where the temperture of oxidtion for ech smple ws determined. Moreover, the crude extrct (EtOAc-MeOH) of mstic, s well s, its cidic nd neutrl frctions ws ssyed ginst pnel of 9 humn nd food pthogenic grm (±) cteri nd fungi. Keywords: Chios mstic gum; Antioxidnt ctivity; Antimicroil ctivity; Encpsultion; Liposomes Introduction Chios mstic gum, the resin otined s n exudte from the trunk nd rnches of Pistci lentiscus vr. chi, hs found extensive use in phrmceuticl products nd s nutritionl supplement. The orl sorption of crude resin (contining high percentge of n insolule nd sticky polymer of poly-β-myrcene) is poor due to its low wter-soluility nd reduces the iovilility of the contined ctive compounds []. It hs een used in trditionl Greek medicine for vrious gstrointestinl disorders while the plnt hs een mentioned y fmous ncient Greek physicins (Dioscorides, Theophrstos, etc.) recommending its heling properties. Therefore, the ojective of the present study ws to determine the possiility to encpsulte Chios mstic gum in liposomes. The methods of liposome preprtion nd chrcteriztion were lso determined. Finlly, the ntioxidnt stility nd ntimicroil ctivity of the liposomes preprtions were evluted. Three different methods of preprtion, thin-film evportion, freezing-thwing, nd ethnol injection were used for the preprtion of liposomes consisting of Phosphtidylcholine (PC) nd Cholesterol (CH). The effect of molr rtio on the percentge of mstic extrct encpsulted ws investigted. Mstic gum extrcts componentsliposomes interction ws studied using Fourier Trnsform Infrred (FT-IR) spectroscopy. The effects of different preprtion methods on the physicochemicl properties of colloidl systems were evluted y mens of surfce morphology, field emission Scnning Electron Microscopy (SEM), nd size distriution using prticle size nlyzer. For the determintion of the ntioxidnt ctivity two methods were used: I) The Rncimt method where the protection fctor ws determined for ech smple nd compred with known ntioxidnts. II) Differentil Scnning Clorimetry (DSC) where the temperture of oxidtion for ech smple ws determined. Moreover, the crude extrct (EtOAc-MeOH) of mstic, s well s, its cidic nd neutrl frctions ws ssyed ginst pnel of 9 humn nd food pthogenic grm (±) cteri nd fungi. Experimentl SPME procedure & GC-MS nlysis The HS-SPME ws performed with Croxen/polydimethylsiloxne coted fier (75μm coting) from Supelco, ttched in mnul SPME fier holder (570-U, Supelco, Germny). For SPME extrction, g of powdered mustic gum in glss vil (5 ml) closed with PTFE coted silicone ruer septum ws used. The GC-MS nlysis ws crried out using Hewlett Pckrd GC-MS system operting on EI mode (ioniztion energy 70 ev) equipped with n HP 5MS 0 m x 0.5 mm x 0.5 μm film thickness cpillry column. In the injector port the temperture ws set t 00ºC nd the SPME fier ws inserted using splitless mode. The fier ws removed fter 0min, in order to completely relese ll sustnces. The crrier gs ws helium (flow rte ml/min) nd the temperture progrm strted t 40 C for min nd grdully incresed to 50 C t rmp of 4 C/min nd then to 50 C t 0 C/min. Identifiction ws succeeded y using spectrl lirry (wiley 75) nd comprison with iliogrphy. *Corresponding uthor: Olg Gortzi, Deprtment of Food Technology, Technologicl Eductionl Institute (T.E.I.) of Thessly, GR-400, Krdits, Greece, Tel: ; Fx: ; E-mil: ogortzi@teilr.gr Received June 5, 04; Accepted Septemer 06, 04; Pulished Septemer 09, 04 Cittion: Gortzi O, Athnsidis V, Lls S, Chinou I, Tsknis J (04) Study of Antioxidnt nd Antimicroil Activity of Chios Mstic Gum Frctions (Neutrl, Acidic) Before nd After Encpsultion in Liposomes. 5: 55. doi:0.47/ Copyright: 04 Gortzi O, et l. This is n open-ccess rticle distriuted under the terms of the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl uthor nd source re credited.

2 Before nd After Encpsultion in Liposomes. 5: 55. doi:0.47/ Pge of 5 Methods of liposome preprtion Thin-Film Evportion (TFE): The method used ws modifiction of tht reported y Du et l. (0). Lipid phse consisting of mixture of PC (0 mg/ml) nd CH ( mg/ml) ws dissolved in chloroform. Then, the solvent ws removed under reduced pressure using rotry evportor t 5 C. Finlly, thin film of dry lipid ws formed on the wll of the flsk. This thin film ws dissolved in n queous phse (HO d.d. + 0.% NCl) t 5 C. To complete the dispersion vortex mixer nd n ultrsonic th were used. Then, the mixture ws heted t 5 C for 45 min. The liposoml system ws centrifuged for 0 min t 5000 rpm nd the superntnt phse ws freeze dried for h. After MLV (Multi Lmellr Vehicles) preprtion the dispersion ws sujected to soniction for 0 min in order to form SLV (Single Lmellr Vehicles) liposomes. Freezing-Thwing (FT): The method used ws modifiction of tht reported y Du et l. (0). After MLV preprtion the dispersion ws sujected to soniction for 0 min in order to form SUV (Smll Unilmellr Vehicles) liposomes. Ethnol Injection (EI): The method used ws modifiction of tht reported y Du et l. []. After MLV preprtion the dispersion ws sujected to soniction for 0 min in order to form SUV liposomes. Methods of chrcteriztion FT-IR: Spectr in the trnsmission mode were crried out t the region of cm-. Potssium romide (KBr) pellets were prepred y gently mixing 6 mg smple powder with 0 mg KBr using n hydrulic press. SEM: Smples were lyophilized, coted with gold nd plldium using vcuum evportor, nd exmined using SEM t 0 kv ccelerting voltge. Size distriution: Liposome prticle size ws nlyzed t 5 C. Methods for determining ntioxidnt ctivity Rncimt: Aout.0 g of smple oil were weighed into the glss vessel nd the pproprite mount for ech smple liposome or known ntioxidnt (BHT or α-tocopherol) ws dded. The conditions were set t 90ºC nd 5 L/h. The protection fctor (PF) ws clculted s PF = (induction time with ntioxidnt)/(induction time without ntioxidnt). A protection fctor greter thn one indictes inhiition of lipid oxidtion. Higher PF vlue indictes etter the ntioxidnt ctivity. DSC: The therml ehvior ws studied y heting of out 5.0 mg of ech individul smple in covered smple pn under nitrogen gs flow. The investigtions were crried out t temperture rnge of C nd heting rte of 0 C min -. Methods for determining ntimicroil ctivity In vitro nticteril studies of crude extrct (EtOAc-MeOH) of mstic, s well s, its cidic nd neutrl frctions were crried out y the dilution method y mesuring the MIC vlues ginst: Stphylococcus ureus, S. epidermidis, Pseudomons eruginos, Enterocter cloce, Klesiell pneumonie, Escherichi coli, Cndid licns, C. tropiclis nd C. glrt (Tle ). Results nd Discussion HS-SPME nd GC-MS nlysis Through HS-SPME nlysis α-pinene (5.6%), verenone (4.0%), β-cymene nd verenene ppered s the most undnt constituents, representing 58% of the totl, mong the 7 identified voltile components of the mstic (Tle ). Encpsultion Efficiency (EE) Trpping efficiencies for (cidic mstic gum extrct) nd (neutrl mstic gum extrct) in colloidl crriers were determined y GC-MS nd the results re presented elow (Tle ). Prticle size distriution The men prticle size nd polydispersity of liposomes otined with different methods nd with nd re summrized in Tle. The freezing-thwing circles cused n mount of entrpped RT Constituents %.5 utne.8. -methyl--uten--ol methyl--uten--l < methylpent--en-4-one < α-pinene verenene β-pinene methyl-5-hepten--one < methylnisole o-cymene.0.87 p-menth-4(8)-ene cis-linlool oxide < trns--cren--ol < trns-β-ocimene < α-terpinene < (7),5,8-o-menthtriene ethyl-,-dimethyl enzene ,-dimethyl-4-ethyl enzene pinocrvone myrtenl.0.0 verenone β-cymene p-menth-,5,8-triene < trns-α-ocimene.0.77 cmphene < p-cymene < unknown 0.5 Tle : Mstic gum nlysis through HS-SPME nd GC-MS

3 Before nd After Encpsultion in Liposomes. 5: 55. doi:0.47/ Pge of 5 nd to lek from the ilyers, which led to lower encpsultion efficiency for FT liposomes. Therefore, FT-IR, Rncimt nd DSC studies re given only for the two other liposome preprtions. FT-IR The chrcteristic functionl groups with their frequencies were determined (Tle ). The first spect ws detected in the virtion nd locted etween 600 nd 000 cm - (Figure ). This nd is relted to O-H ing virtion nd is much more pronounced in the liposomes formultions EI nd TFE thn in pure nd. This indictes tht the OH groups of nd components creted hydrogen onds with the contining phospholipids. These onds re proly relted to the interction mong the O-H groups from nd components nd the polr groups of the phospholipids. A second importnt chrcteristic ws relted to the peks t 6 cm - nd 70 cm -, (Figure ). The first one is relted to the ing of P=O ond of the polr heds of phospholipids. The sorption nd ttriutes to phosphte O-P-O symmetric ing virtions nd cn lso provide importnt clues out hydrtion nd hydrogen onding interctions t the surfces phospholipid ssemlies nd components. Also, in the cse of the ing of C=O of ester groups, oserved modifictions (decreses) reflect differences in the degrees of hydrtion nd/or hydrogen onding to the ester cronyl groups in the cse of liposomes (Tle 4). SEM The SEM photogrph of optimized formultion reveled tht prticles were roughly sphericl. Additionlly, n uniformity ws oserved. An verge prticle size elow 0 nm could e chieved nd reproduced (Figure ). Rncimt method Bsed on the protection fctor, the results re comprtive study of the ntioxidnt ctivity of the smples, liposomes nd known ntioxidnts (BHT or α-tocopherol). Preprtion Method Frctions EE % Men prticle size (nm) Polydispersity FT. ± ± ± ± ± ± 0.00 TFE.6 ± ±.4 0. ± ± ± ± EI 6.8 ± ± ± ± ± ± 0.00 Tle : Effect of different preprtion methods nd loding cpcity on prticle size distriution of nd liposomes (EE%: percentge of encpsultion efficiency) Functionl Group Nmes Alcohol Ester Phosphte ester Type of Virtion O-H C=O P=O Empty TFE TFE TFE Empty EI EI EI Tle : FT-IR interprettion of TFE nd EI preprtions (empty nd & ) : : 5 4 : : 5 4 ABS ABS cm - cm - Figure : Comprtive FT-IR grphs of TFE () nd EI () preprtions.

4 Before nd After Encpsultion in Liposomes. 5: 55. doi:0.47/ Pge 4 of 5 S. ureus S. epidermidis P. eruginos E. cloce K. pnumionie E. coli C. licns C. tropiclis C. glrt Crude extrct Acidic frction Neutrl frction Amphotericin flucytocine Amoxicillin with Clvulnic cid Tle 4: Antimicroil ctivity MIC (mg/ml) of the mstic gum Figure : Empty EI Figure : EI Protection Fctor Protection Fctor ppm 500 ppm 700 ppm 00 ppm 500 ppm 700 ppm 700 ppm 000 ppm 00 ppm : 700 ppm : 000 ppm : 00 ppm : 700 ppm : 000 ppm : 00 ppm BHT 00 ppm -toc 000 ppm ppm 500 ppm 700 ppm 00 ppm 500 ppm 700 ppm 700 ppm 000 ppm 00 ppm : 700 ppm : 000 ppm : 00 ppm : 700 ppm : 000 ppm : 00 ppm BHT 00 ppm -toc 000 ppm Figure : Protection fctor of TFE () nd EI () preprtions nd known ntioxidnts (BHT nd α-tocopherol) s determined y the Rncimt method. DSC Therml oxidtive decomposition of nd, TFE nd EI preprtions (empty nd or liposomes) ws studied y the DSC method using the onset temperture (To) of curves t the point where the uto-oxidtion process egins. The results were in line with those reported y other reserchers who lso suggested tht proved low oxidtion stility. The oxidtive stility of oth extrcts ws incresed fter encpsultion nd depended on the method of preprtion used (TFE nd EI) (Figure ). Antimicroil ctivity All tested extrcts of mstic, s well s, its cidic nd neutrl frctions showed very interesting ntimicroil profile ginst ll 9 ssyed microorgnisms. Moreover, the crude extrct ppered s the most ctive, where it exhiited the strongest ctivity ginst Grm

5 Before nd After Encpsultion in Liposomes. 5: 55. doi:0.47/ Pge 5 of 5 Het Flow (mw)- Endo down Temperture ( C) : : 400 Het Flow (mw)- Endo down Temperture ( C) : : Figure 4: Therml profile of TFE () nd EI () preprtions s determined y the DSC. positive humn pthogenic cteri (MIC vlues mg/ml) (Figure 4). Conclusion During current study, colloidl systems (liposomes) contining nd were successfully prepred with three different methods (TFE, FT, EI). The comprison of preprtion methods (using their physicochemicl properties) reveled tht lipid sed crriers prepred y the TFE nd EI methods showed etter encpsulting efficiency. From the experimentl results it is concluded tht the method of preprtion hs n impct on the relese rte of constituents (i.e. terpenes, pinenes, etc.). The encpsulted frctions of mstic gum (especilly the cidic one) presented higher ntioxidnt ctivity in comprison to the nonencpsulted ones. Acknowledgements The study hs een co-funded y 75% from E.E. nd 5% from the Greek Government under the frmework of the Eduction nd Initil Voctionl Trining Progrm-Archimedes III. References. Prschos S, Mitkou S, Skltsounis AL (0) Chios gum mstic: A review of its iologicl ctivities. Curr Med Chem 9: Du JS, Rn AC, Bhndri AK (0) Liposome: methods of preprtion nd pplictions. Int J Phrmceut Sci Res : 4-0.