Production of stabilized quercetin aqueous suspensions bysupercritical fluid extraction of emulsions

1 PRODUCTION OF STABILIZED QUERCETIN AQUEOUS SUSPENSIONS BY SUPERCRITICAL FLUID EXTRACTION OF EMULSIONS Gyö gy Lé ai, Ángel Ma ín*, Es he de Paz, So aya Rod íguez-Rojo, Ma ía José Coce o High P essu e P ocesses G oup.- Chemical Enginee ing and En i onmen al Technology– Uni e si y o Valladolid, Doc o Me gelina s/n 47011 Valladolid (Spain) Tel: +34 9831840711, e-mail: [email p o ec ed] (Á. Ma ín) 2 PRODUCTION OF STABILIZED QUERCETIN AQUEOUS SUSPENSIONS BY SUPERCRITICAL FLUID EXTRACTION OF EMULSIONS Gyö gy Lé ai, Ángel Ma ín*, Es he de Paz, So aya Rod íguez-Rojo, Ma ía José Coce o High P essu e P ocesses G oup.- Chemical Enginee ing and En i onmen al Technology– Uni e si y o Valladolid, Doc o Me gelina s/n 47011 Valladolid (Spain) Tel: +34 9831840711, e-mail: [email p o ec ed] (Á. Ma ín) Abs ac Que ce in is a la onoid wi h highly p omising bioac i i y agains a a ie y o diseases, due o i s s ong an ioxidan , an i i al and an ihis aminic e ec , bu hese applica ions a e limi ed by he low solubili y o que ce in in gas oin es inal luids and he co espondingly low bioa ailabili y. The objec i e o his wo k is o p oduce encapsula ed que ce in pa icles in sub-mic ome ic scale, in o de o inc ease hei low bioa ailabili y. These pa icles we e p oduced by ex ac ion o o ganic sol en om oil in wa e emulsions by Supe c i ical Fluid Ex ac ion o Emulsions (SFEE). Due o he apid ex ac ion o o ganic sol en by his me hod, he dispe se o ganic phase becomes apidly supe sa u a ed, causing he p ecipi a ion o que ce in pa icles in sub-mic ome ic scale, encapsula ed by he su ac an ma e ial. Two di e en biopolyme s (Plu onic L64 ® poloxame s and soy bean leci hin) we e used as ca ie s and su ac an ma e ials. In expe imen s wi h Plu onic, needle que ce in pa icles we e ob ained a e SFEE ea men , wi h pa icle sizes a ound 1 µm and poo encapsula ion e iciency. In case o soy leci hin, que ce in-loaded mul i esicula liposomes we e ob ained, wi h a mean pa icle size a ound 100 nm and a ound 70% encapsula ion e iciency o que ce in, wi hou p esence o seg ega ed que ce in c ys als. Keywo ds: supe c i ical ca bon dioxide, encapsula ion; que ce in, micelle, liposome, emulsion, an ioxidan ac i i y 3 1. INTRODUCTION Que ce in (3,3’,4,4’,5,7-pen ahyd oxy la one, chemical s uc u e p esen ed on Figu e 1) is a bio la onoid, a ailable in a ious ui s, ege ables and oils. I can sca enge eac i e oxygen species, and down- egula e lipid pe oxida ion due o i s ion chela ing and i on s abilizing e ec [ 1 ]. Fu he mo e i can p omo e he oxida ion o Fe2+ o Fe3+, which is less e ec i e in gene a ing ee adicals. These e ec s o que ce in may be explained by i s o-diphenol B- ing s uc u e [ 2 ] and he abili y o dona ing π elec ons om he benzene ing, while i is emaining ela i ely s able [ 3 ]. I has also an i-p oli e a i e e ec s in a wide ange o human cance cell lines [ 4 ]. Due o hese p ope ies, que ce in is a highly p omising ac i e compound agains a wide a ie y o diseases. (FIGURE 1) A majo limi a ion o he clinical applica ion o que ce in is i s low bioa ailabili y, ha makes i necessa y o adminis a e high doses (50 mg/kg) [ 5 ]. Due o he low wa e solubili y o que ce in, i has a minimal abso p ion in he gas oin es inal ac , and i s o al bioa ailabili y is lowe han 17% in a s [ 6 ] and lowe han 1% in humans [ 7 ]. Di e en app oaches ha e been p oposed in li e a u e o inc ease he bioa ailabili y o que ce in. Mulholland e al. [ 8 ] syn hesized a wa e -soluble de i a i e o que ce in, bu i s bioa ailabili y was only 20%. Also, o inc ease he bioa ailabili y o his poo ly wa e soluble compound, d ug loaded solid lipid nanopa icles could be a p omising al e na i e, and he complexa ion o que ce in wi h leci hin and cyclodex in in aqueous solu ion has been es ed [ 9 , 10 ]. Li e al. [ 11 ] p oduced leci hin encapsula ed que ce in by emulsi ica ion and low- empe a u e solidi ica ion, wi h o e 90% d ug en apmen e iciency in sphe ical pa icles o an a e age diame e o 155 nm was obse ed. He e ogeneous mo phologies we e ob ained wi h a co-exis ence o addi ional colloidal s uc u es, like micelles, liposomes, 4 supe cooled mel s, d ug nanopa icles, which caused a ce ain sca e in he pa icle size dis ibu ion, wi h pa icle sizes spanning he ange om 20 nm o 500 nm. The abso p ion a e o que ce in loaded solid lipid nanopa icles was s udied by in-si u pe usion me hod in a s, ob aining a 6- old ela i e inc ease in bioa ailabili y, compa ed o unp ocessed que ce in. Supe c i ical luids a e ano he p omising al e na i e in he p ocessing o na u al bioac i e compounds, such as que ce in, because hey allow ca ying ou he encapsula ion p ocess a nea ambien empe a u es, and in an ine a mosphe e, hus a oiding he he mal deg ada ion o oxida ion o he p oduc and educing i s con amina ion wi h o ganic sol en s. Se e al au ho s ha e s udied he p ocessing o que ce in by supe c i ical luid echnologies. Due o he low solubili y o que ce in in supe c i ical ca bon dioxide [ 12 ], Supe c i ical An isol en (SAS) expe imen s ha e been pa icula ly success ul. By SAS p ocessing o pu e que ce in, c ys alline pa icles wi h pa icle sizes in he mic ome e ange (1 – 6 m) ha e been ob ained [ 13 , 14 , 15 ]. F aile e al. [ 16 ] p oduced que ce in pa icles encapsula ed wi h Plu onic F127 by SAS echnology. As in p e ious wo ks, SAS-p ocessed pu e que ce in c ys allized as needle like pa icles, meanwhile que ce in co-p ecipi a ed wi h Plu onic had a o ally di e en sphe ical mo phology, indica ing ha Plu onic F127 was able o success ully encapsula e que ce in. Highe pa icle sizes we e ob ained when he que ce in / Plu onic mass a io was inc eased, due o he possible agg ega ion o he polyme shells. Ob ained mo phologies indica e, ha que ce in pa icles ac ed as nuclea ion si es o he o ma ion o a polyme ilm, and his ilm o polyme es ained he g ow h o que ce in pa icles abo e he mass a io o 1/1 = que ce in / Plu onic. Wi h his encapsula ion me hod, he solubili y o que ce in in simula ed in es inal luid was inc eased by a ac o o 8. Supe c i ical Fluid Ex ac ion o Emulsions (SFEE) echnology can be conside ed as an e olu ion o SAS echnology, which is especially sui able o encapsula e poo ly wa e soluble 5 d ugs in an aqueous suspension. The p ocess consis s o o ming an oil-in-wa e emulsion, con aining he wa e -insoluble d ug in he dispe sed o ganic phase. By SFEE, he o ganic sol en is ex ac ed om his emulsion by he supe c i ical sol en , which should ha e high a ini y o he o ganic sol en and a low a ini y o he ac i e compound o in e es . Due o he solubili y di e ences, he supe c i ical sol en quickly ex ac s he o ganic sol en om he emulsion, leading o he apid supe -sa u a ion o ac i e compound, and hence a as p ecipi a ion. Meanwhile in he SAS an isol en p ecipi a ion me hod pa icle nuclea ion and g ow h occu ac oss he whole solu ion olume, in he case o SFEE he o ma ion o pa icles is con ined wi hin he emulsion d ople s. This es ains he size o he pa icles ob ained, ha can be one o de o magni ude smalle han pa icles p oduced by solu ion p ecipi a ion [ 17 ]. F. Ma ea e al. s udied he p ecipi a ion o β-ca o ene by con inuous SFEE in o de o model he p ocess [ 18 ]. In his s udy submic o- and nano-pa icles we e ob ained wi h a esidual o ganic con en as low as 1 ppm. The ob ained pa icle size dis ibu ion was di ec ly ela ed wi h he d ople size dis ibu ion o he ini ial emulsion, while esidual o ganic con en depended on he p ocess pa ame e s, such as he p essu e and he empe a u e. Model esul s showed ha he sa u a ion o o ganic phase d ople s wi h CO2 caused a apid an isol en e ec , which in he con inuous implemen a ion o he p ocess can ake place du ing he d op ly ime, while he elimina ion o he esidual o ganic sol en was much slowe . Based on expe imen al and model esul s, a wo-s ep p ocess s a egy can be p oposed. The i s s ep would in ol e con ac be ween emulsion and CO2, o ensu e he sa u a ion o he dispe se phase, in o de o achie e p ecipi a ion by an isol en e ec . A second s ep would in ol e an ex ended con ac be ween CO2 and emulsion, in o de o elimina e he emaining o ganic sol en . This s ep migh be slowe han he i s , because once he pa icles a e o med, 6 emulsion des abiliza ion is no longe a p oblem. In a subsequen wo k, San os e al. ex ended his app oach o he p ecipi a ion o lycopene [ 19 ]. The aim o his s udy is o apply he Supe c i ical Fluid Ex ac ion o Emulsion p ocess o he encapsula ion o que ce in. Based on he a ailable in o ma ion, wo di e en ca ie ma e ials ha ha e been ound o inc ease he wa e solubili y and he bioa ailabili y o que ce in ha e been es ed: Plu onic block copolyme s, and soybean leci hin. The in luence o he main p ocess pa ame e s has been s udied, including p ope ies o he ini ial emulsion, ex ac ion ime and ex ac ion condi ions. The pe o mance o he p ocess has been e alua ed analysing he encapsula ion e iciency and pa icle size and mo phology o he inal aqueous suspensions. 2. EXPERIMENTAL SECTION 2.1 Ma e ials Que ce in Hyd a e (C15H10O7xH2O, 95% pu i y, CAS: 849061-97-8) was ob ained om Ac os O ganics (New Je sey, USA). The su ac an ma e ial poly-(e hylene glycol)- block – poly-(p opylene glycol)- block -poly-(e hylene glycol) (Plu onic L64, CAS: 9003-11-6) was ob ained om Sigma Ald ich (S Louis, USA). Soy leci hin was ob ained om Glama-So (SOTYA, Mad id, Spain). E hyl Ace a e (E Ac, CAS: 141-78-6) and me hanol (MeOH, CAS: 67-56-1), wi h a pu i y o 99% and 99.9 %, espec i ely, we e ob ained om Pan eac Química (Ba celona, Spain). Ace oni ile (CAS: 75-05-8); ace ic acid ( e e ence numbe : 211008.1211) wi h a pu i y o 99.9% and 99.5 %, espec i ely, we e ob ained om Pan eac Química (Ba celona, Spain). Ca bon dioxide was p o ided by Ca bu os Me álicos (Ba celona, Spain). 7 2.2 Emulsion p epa a ion and supe c i ical ex ac ion o he emulsion The ini ial emulsion was p epa ed using an Ul a u ax IKA LABOR-PILOT 2000/4 (IKA- WERKE GMBH&CO.KG) high equency mixing de ice wi h a cooling jacke . The equi ed amoun o que ce in was dissol ed in an o ganic sol en (e hyl ace a e), and a equi ed amoun o su ac an ma e ial (Plu onic L64 o leci hin) was dissol ed in wa e , pu i ied by Millipo e Elix. Then hese wo solu ions we e mixed oge he by a magne ic s i ing o 5 minu es, in o de o ob ain a homogeneous dispe sion. A e wa ds, he dispe sion was mixed by he Ul a u ax emulsi ie a 70 Hz equency o a p ede ined ime. To ex ac he o ganic sol en om he ini ially p epa ed emulsion, a ba ch SFEE equipmen – p esen ed on Figu e 2 – was used. The equipmen consis s o wo essels: an ex ac o essel wi h a olume o 85 mL, and a bu e essel wi h a olume o 100 mL. The essels a e loca ed in a he mos a ed o en, and a e sepa able om each o he by wo al es. Fi s ly, he equipmen was p essu ized wi h scCO2 and he mos a ed ( ypically, a 110 ba and 40ºC). A e wa ds 25 mL o he ini ially p epa ed emulsion was injec ed by an HPLC in o he ex ac ion essel. The emulsion was loaded a e he p essu iza ion o he sys em, as o he wise he dis u bances caused by he addi ion o CO2 can spill he emulsion ou om he ex ac ion essel in o he eci cula ion ci cui , making i di icul o ecollec he ea ed emulsion a e he expe imen . Then he HPLC pump was isola ed om he ci cui by closing he al e in i s impulsion, and he CO2 eci cula ion pump was swi ched on, s a ing he ci cula ion o he scCO2 be ween he CO2 bu e essel and he ex ac ion essel. Du ing his p ocess, scCO2 was bubbled h ough he emulsion, in o de o ex ac he o ganic sol en om i . As he scCO2 g adually became sa u a ed wi h o ganic sol en du ing his ba ch ex ac ion p ocess, i was pa ially enewed se e al imes in each expe imen , in o de o inc ease he e iciency o he ex ac ion. To do so, he ex ac ion essel was isola ed om he eci cula ion ci cui by closing he al es in i s inle and ou le connec ions, in o de o 8 main ain he ex ac ion essel a a cons an p essu e, meanwhile he CO2 was enewed in he es o he ci cui , hus minimizing he dis u bances and losses o emulsion by en apmen in CO2 du ing he epea ed dep essu iza ion p ocesses needed o each CO2 enewal. Conside ing ha he olume o he ex ac ion essel is 85 mL and i con ains 25 mL o liquid emulsion, meanwhile he olume o he bu e essel is 100 mL, i is es ima ed ha app oxima ely 60% o CO2 in he ci cui was enewed wi h his p ocedu e. A e se e al cycles, he comple e sys em was slowly dep essu ized, and he aqueous suspension – go om he emulsion by SFEE ea men – was e ie ed om he ex ac ion essel and s o ed o analysis. (FIGURE 2) 2.3 Analy ical me hods 2.3.1 Ini ial emulsion s abili y measu emen by lase di ac ion A Tu biScan Classic lase sca e ing de ice (Fo mulac ion, F ance) was used in o de o de e mine he a e age d ople size in he ini ial emulsion and o cha ac e ize i s possible des abiliza ion by c eaming p ocesses [ 20 ], due o he lowe iscosi y o o ganic d ople s wi h espec o he con inuous aqueous phase. Fo his, a glass ial was illed a 5.5 cm heigh wi h he sample, and inse ed in o he de ice, and he del a back sca e ing (ΔBS%) was eco ded e e y 10 minu es o 8 hou s. Obse a ions o c eaming we e made, and he da a we e compu ed using Mig a ion So wa e Ve sion 1.3, equipped in he Tu biScan [ 21 ]. Typically he slope o he c eam peak hickness kine ics was i s iden i ied. The linea po ion o his slope was zoomed and copied in o he Mig a ion So wa e, om whe e he mig a ion a e (equi alen o he c eaming index), which cha ac e izes he b eakage eloci y and he e o e he ins abili y o he emulsion, is compu ed. The so wa e calcula es he hyd odynamic mean pa icle diame e (equi alen diame e ) om pa icle mig a ion eloci y 9 V [m/s], con inuous phase iscosi y and densi y, dispe sed phase densi y and olume ac ion, using he Gene al Se ling Law [ 22 ]. 2.3.2 Pa icle size dis ibu ion measu emen and mo phological cha ac e iza ion A Mal e n Mas e size 2000 Ligh Sca e ing de ice om Mal e n Ins umen s was used in o de o de ine he pa icle size dis ibu ion in he inal aqueous suspension a e SFEE ea men . This equipmen is able o measu e pa icle size be ween 0.02 – 2000 µm, using a diode lase 4 mW wi h a dual – wa eleng h de ec ion sys em ( ed ligh 633 nm, blue ligh 436 nm). The sample was dilu ed by deionized wa e in he dispe sion uni (Hyd o SM) o ob ain an adequa e le el o lase obscu a ion and p e en mul iple sca e ing e ec s. The e ac i e index o he dispe sed phase (wa e ) is 1.331, meanwhile o que ce in pa icles is 1.823. Each measu emen was pe o med iplica e. Addi ionally, isual obse a ions o he ini ial emulsions and SFEE- ea ed aqueous suspensions we e done by mic oscopy in o de o ob ain in o ma ion abou he mo phology o he ini ial emulsion and he inal suspension, and o con i m he size measu emen s ob ained by lase di ac ion. Two di e en echniques we e used depending on he pa icle size o he analysed samples: op ical mic oscopy, using a Leica mic oscope o ini ial emulsion, and C yo - TEM echnology using a GATAN PB3 C yoplunge equipmen o eeze and ca bon coa he sample. The ca bon coa ed samples we e analysed by a JEOL JEM- FS2200 HRP 200 kV TEM equipmen wi h elec on il e ing, o ob ain mic og aphs o he ozen samples o he inal suspensions a e he SFEE ea men , when leci hin was used as su ac an ma e ial, due o he oo low pa icle size o samples o op ical mic oscopy. Fou ie T ans o m In a ed Spec oscopy (FTIR) measu emen s we e done by ALPHA PLATINUM – ATR de ice equipped wi h a high h oughpu ZnSe ATR c ys al, p oduced by BRUKER. Be o e FTIR measu emen , samples we e d ied in a he mos a ed o en unde 0,1 16 size dis ibu ions. P obably due o he needle mo phology o c ys als ob ained in expe imen s, in e e y cases, he pa icle size dis ibu ions we e mul imodal. Howe e , i mus be no ed, ha due o he needle-like mo phology, he accu acy o pa icle size measu emen s p obably is no high, because he measu ed dimensions o he c ys als depend on he spa ial o ien a ion o c ys als in he measu emen cells du ing measu emen . As epo ed in Table 3, in he expe imen s wi h he bes esul s acco ding o he mean pa icle size ma ked bold, he esidual E Ac con en was unde 300 ppm and he p opo ion o pa icles wi h a size below 10 µm was highe han 30% (V/V%). Acco ding o UV-VIS measu emen s, in e e y case a ound 70% o he ini ially added que ce in was eco e ed in he SFEE ea ed aqueous suspension, co esponding o que ce in concen a ions in he ange o 0.160 – 0.245 g/L, and in e e y cases he que ce in con en was s able up o wo mon hs, wi h samples s o ed in glass ials in idge a 4°C. (TABLE 3) (FIGURE 5) Acco ding o he s a is ical analysis o esul s, p esen ed in he Pa e o cha shown in Figu e 6, inal pa icle size dis ibu ion we e in luenced by he concen a ion o que ce in, acco ding o linea model, and concen a ion o E Ac, acco ding o a quad a ic model. In o de o dec ease he pa icle size, i is necessa y o inc ease he concen a ion o que ce in, and o choose an op imal concen a ion o E Ac. I he ini ial concen a ion o E Ac is high, high esidual o ganic sol en concen a ions a e obse ed in he inal p oduc , which can also in luence he o ma ion o pa icles, meanwhile i he concen a ion o E Ac is oo low, i is possible ha sol en is emo ed and pa icles o med al eady in he i s cycles o he ex ac ion, and pa icles s a o agg ega e o deg ade along he emaining ea men ime. On he o he hand, he e is no signi ican ac o in luencing he que ce in eco e y a io. (FIGURE 6) 17 The esul s o he s a is ical analysis indica e, ha he ou come o he p ecipi a ion mainly depended on he concen a ion o que ce in in he o iginal solu ion, which is a de e minan ac o o an an isol en p ecipi a ion om a homogeneous solu ion, and i was no in luenced by he pa ame e s ha signi ican ly in luenced he d ople size o he emulsion, such as he concen a ion o su ac an , o he o ganic/wa e a io. These esul s o he s a is ical analysis ag ee wi h he mo phology p esen ed in he mic og aph o Figu e 3, which as p e iously desc ibed showed la ge c ys alline pa icles o que ce in, which appa en ly we e no encapsula ed o dispe sed by he su ac an ma e ial. Indeed, he mo phologies and sizes – ob ained and shown in his Figu e – a e simila o he esul s epo ed by F aile e al. by p ecipi a ion o que ce in by SAS p ocess om que ce in – ace one homogenous solu ions, changing he ini ial concen a ion o que ce in. In he wo k o F aile e al., mo phology and pa icle size depended on he ini ial concen a ion o que ce in: wi h inc easing que ce in concen a ion, pa icle size dec eased. P ecipi a ed que ce in c ys als also showed needle-like mo phology, agglome a ed in locks o abou 1 µm scale [16]. Conside ing hese esul s, i can be concluded ha Plu onic L64 is no a sui able ma e ial o encapsula e que ce in, as he p ecipi a ion occu ed as in a no mal an i-sol en p ocess, and he su ac an did no p o ide any addi ional con ol o e pa icle size h ough he o ma ion o an emulsion empla e. 3.4 P epa a ion o emulsions using leci hin as su ac an In his sec ion, an expe imen al plan was comple ed, using leci hin as su ac an ma e ial, as epo ed in Table 4, analysing he p ocess pa ame e s (sol en /wa e a io, que ce in concen a ion and leci hin concen a ion) wi h a signi ican in luence ega ding he a e age d ople size in ini ial emulsion, as well as inal pa icle size dis ibu ion and que ce in eco e y in SFEE ea ed aqueous suspensions. 18 As p esen ed in Table 5, he d ople size o he ini ial emulsions p epa ed wi h leci hin, showed smalle a ia ions wi h p ocess condi ions han hose obse ed in expe imen s wi h Plu onic L64 and epo ed in Table 2. Acco ding o he s a is ical analysis o he esul s p esen ed in Table 4, ac o s in luencing he a e age d ople size in ini ial emulsion we e he concen a ion o leci hin and concen a ion o E Ac. The concen a ion o leci hin should be inc eased in o de o dec ease he d ople size in ini ial emulsion, while he concen a ion o E Ac should be dec eased. In addi ion o a smalle d ople size and smalle a iabili y o his size (Table 4 and Table 5), he use o leci hin as su ac an p o ided a be e s abili y o he emulsion. This is shown in he esul s p esen ed in Table 5 ha summa ize he measu emen s o emulsion s abili y, pe o med wi h he Tu biscan appa a us. As p esen ed in his able, he mig a ion a e, which is ela ed o he emulsion des abiliza ion by c eaming e ec , was less han hal e in emulsions p epa ed wi h leci hin han in emulsions wi h Plu onic L64. (TABLE 4) (TABLE 5) 3.5 Supe c i ical Fluid Ex ac ion o Emulsions s abilized wi h Leci hin Table 4 epo s he main esul s ob ained a e SFEE ea men o emulsions p epa ed wi h leci hin. As shown in Figu e 7, a e SFEE ea men , mul imodal pa icle size dis ibu ions we e ob ained, wi h a main peak in he sub-mic ome e size ange, and addi ional peaks a la ge sizes, ha p obably co esponded o a small numbe o la ge que ce in c ys als ha we e no encapsula ed in leci hin, in some cases isible o he naked eye. (FIGURE 7) The mo phology o pa icles ob ained a e SFEE ea men can be obse ed in he TEM mic og aphs p esen ed in Figu e 8. The mic og aphs showed esicles o leci hin o med in he aqueous media, wi hou p esence o seg ega ed que ce in c ys als. The size obse ed, 19 co espond well wi h he measu emen s ob ained by lase di ac ion, epo ed in Table 4. In expe imen s pe o med wi h highe leci hin concen a ion, TEM mic og aphs showed bigge pa icles due o he o ma ion o mul i-laye ed esicles (Figu e 8 C). One expe imen al un was done wi hou que ce in (o he se ings a e he same as in he case o cen um poin uns), in o de o can compa e by TEM he s uc u e o he sys em wi h and wi hou que ce in (Figu e 8 D). Mul i esicula sys em is obse ed in all cases (expec o he one wi h highe leci hin concen a ion, Figu e 8 C), wi hou any a ia ion in he mo phology due o he p esence o que ce in, which u he indica es ha seg ega ed c ys als o que ce in we e no o med, and he e o e did no al e he mo phology o he esicles. Rega ding he in luence o p ocess pa ame e s, i expe imen al esul s a e analysed conside ing he a e age pa icle size d0.5, inconclusi e esul s wi h weak dependencies in all p ocess pa ame e s a e ob ained, due o he low ep oducibili y o he ail o he pa icle size dis ibu ion ha p obably co esponds o he ac ion o que ce in ha was no encapsula ed inside leci hin (Figu e 7). In con as , analysing he ac ion o pa icles wi h a size below 1 µm, which can be conside ed as a quan i a i e es ima ion o he p opo ion o pa icles ha showed an app op ia e encapsula ion in leci hin, a a less sensi i e dependency on a ied ac o s was obse ed, and only he concen a ion o que ce in and leci hin p o ed o be signi ican . As p esen ed in Figu e 9Figu e , in o de o ge mo e pa icles unde 1 µm acco ding o a linea model, he concen a ion o que ce in should be dec eased, while he concen a ion o leci hin should be inc eased. Fu he mo e, as p esen ed in Table 4, he mode o he pa icle size dis ibu ion, which co esponds o he mean pa icle size o he sub- mic ome ic pa icles (Figu e 7), p ac ically does no show any a ia ions be ween expe imen s, wi h alues in a ange be ween 130 and 190 nm. The e y small co ela ion be ween his size and he ini ial emulsion p ope ies obse ed in his wo k is in con as wi h he co espondence be ween hese wo alues epo ed in p e ious wo ks [19, 11]. This is 20 p obably due o he small a ia ion o he p ope ies o emulsions p epa ed wi h leci hin wi h p ocess condi ions, desc ibed in sec ion 3.4. The a e age o eco e ed que ce in a e SFEE is 65.4 %, co esponding o concen a ions in he ange o 0.16 – 0.2 g/L, depending on he ini ially added que ce in. This esul is app oxima ely 16 – 20 imes highe han he solubili y o que ce in in pu e wa e (0.01 g/l) [ 26 ], indica ing ha i was possible o subs an ially inc ease he amoun o que ce in ha could be s abilized in he aqueous suspension by encapsula ion in leci hin liposomes. No signi ican ac o was ound o in luence he que ce in eco e y. Acco ding o Figu e 10, leci hin encapsula ed que ce in was s able up o 14 days. This means ha he deg ada ion o que ce in, he c ys alliza ion p ocess o que ce in ou o lyposomes, o g owing o al eady exis ing que ce in c ys als a e e y slow. (FIGURE 8) (FIGURE 9) (FIGURE 10) 3.6 S uc u al cha ac e iza ion Se e al SFEE ea ed aqueous suspensions whe e cha ac e ized by FT-IR spec oscopy in o de o de e mine he mechanism o encapsula ion o que ce in by leci hin. As p esen ed in Figu e 11, pu e que ce in show i s cha ac e is ics peaks [ 27 ] in he ange o 1600 – 1100 cm-1, and OH – phenolic bending: 1400 – 1200 cm-1. Leci hin shows cha ac e is ic peaks [ 28 ] in he ange o 1765 – 970 (a 1765-1720 cm-1 co esponding o C=O, 1200-1145 cm-1 co esponding o P=O, 1145-970 cm-1 co esponding o P-O-C, and 1200-970 cm-1 co esponding o P-O-C + PO2). Acco ding o FTIR spec a’s p esen ed on Figu e 11, i can be obse ed ha he cha ac e is ic peaks o que ce in a e no p esen in he spec a o he SFEE ea ed sample. In con as , a que ce in – leci hin physical mix u e’s spec a shows cha ac e is ic peaks o que ce in a ound 21 1625 cm-1 and 1170 cm-1. This esul indica es ha in he SFEE ea ed sample, que ce in is encapsula ed by leci hin and i is no p esen as seg ega ed c ys als. (FIGURE 11) An XRD spec a o physical mix u e o que ce in/leci hin = 1/75 in mass and expe imen al un 1 and 8 we e measu ed also by XRD, in o de o examine he encapsula ion o que ce in by leci hin. In o de o di ec ly analyse he aqueous suspensions wi hou d ying, samples om expe imen al uns we e disposed on he su ace o a silica slide, and hei spec a was co ec ed as well o he spec a o he pu e silica slide. As isible on he spec a p esen ed on Figu e 12, leci hin p esen s a peak a 20°, meanwhile pu e que ce in has high c ys allini y and p esen s i s cha ac e is ics peaks a 2θ alues: 10.78°, 12.46°, 15.88°, and wo mo e p ominen peaks a 25.66°, and 27.4°.[ 29 , 30 ]. Acco ding o he XRD spec a, physical mix u e and he expe imen al uns ha e simila spec a han pu e leci hin has, con o ming ha encapsula ion o que ce in is pe o med wi hou o ma ion o seg ega ed c ys als. (FIGURE 12) 3.7 An ioxidan ac i i y The an ioxidan ac i i y o some o he SFEE ea ed samples was measu ed by ORAC as well. In ag eemen wi h F aile e al., he an ioxidan ac i i y o unp ocessed c ys alline que ce in (p epa ed in E OH (abs.) / wa e milliQ = 1 / 1 in olume) de e mined in his wo k is 6300 ± 300 µM T olox equi alen / g o que ce in [16]. Leci hin an ioxidan ac i i y is also p esen ed p esen ed in Figu e 13, epo ed as µM T olox equi alen / g o leci hin. Howe e , he SFEE ea ed leci hin sample (expe imen al un wi hou que ce in, o he se ings a e he same as in he case o cen um poin uns), has a non-measu able and negligible an ioxidan ac i i y compa ing o leci hin dissol ed in wa e milliQ, ( esul s no shown in Figu e 13). This d as ic educ ion o he an ioxidan ac i i y can be due o a pa ial ex ac ion o low 22 molecula weigh an ioxidan compounds o leci hin du ing he supe c i ical CO2 ea men , such as s e ols. Fu he mo e que ce in – leci hin physical mix u es in di e en mass a ios (que ce in / leci hin = 1 / 36; que ce in / leci hin = 1 / 75 same as he cen um poin s anda d uns; que ce in / leci hin = 1 / 160: equal a io as expe imen al un 9 in mass) we e also p epa ed in a solu ion E OH (abs.) / wa e milliQ = 1 / 1 in olume, in o de o compa e he an ioxidan ac i i y o pu e physical mix u es (wi hou SFEE ea men ) wi h an ioxidan ac i i ies o ea ed samples. As displayed in Figu e 13, his que ce in – leci hin physical mix u es ha e an inc eased an ioxidan ac i i y, compa ing o pu e, unp ocessed que ce in, due o an an ioxidan syne gism e ec : he e a e chain s uc u es o ma ion be ween que ce in and phospha idyl-choline, linked by hyd ogen bonds [2]. The an ioxidan ac i i y o physical mix u es ( epo ed as a unc ion o he amoun o que ce in in he sample) is independen on he mass a io o que ce in / leci hin. Que ce in encapsula ed in leci hin by SFEE ea men also shows a highe an ioxidan ac i i y han pu e que ce in. Howe e , as in he case o pu e leci hin, SFEE ea ed que ce in-leci hin samples show lowe an ioxidan ac i i y han physical mix u es o he wo compounds, again p obably due o he loss o low molecula weigh an ioxidan compounds o leci hin by CO2 ex ac ion. Analyzing he esul s o SFEE ea ed samples, in case o expe imen al un 8, he an ioxidan ac i i y is no changing signi ican ly in he ollowing 10 days a e he expe imen . Fu he mo e, an ioxidan ac i i y o expe imen al un 7 (C) also did no change signi ican ly a e 10 days. An inc eased an ioxidan ac i i y is ob ained in expe imen al un 9 (wi h an inc eased leci hin concen a ion, que ce in / leci hin ~ 1 / 160 in mass a io) compa ing wi h he o he SFEE ea ed samples. (FIGURE 13) 23 4. CONCLUSIONS In his wo k encapsula ed que ce in mic opa icles we e p oduced by SFEE om an ini ially p epa ed oil in wa e emulsion. Two ypes o su ac an ma e ial we e ied: Plu onic L64® and soy bean leci hin. Plu onic is no a sui able ma e ial o encapsula e que ce in because mic ome ic c ys alline pa icles o que ce in we e ob ained wi hou encapsula ion, meanwhile wi h soy bean leci hin mul i esicula sys em was ob ained in sub-mic ome ic scale, wi h an encapsula ion e iciency a ound 70%, encapsula ed que ce in s able up o wo weeks and esidual o ganic con en below 300 ppm, and wi hou p esence o c ys alline que ce in pa icles. The an ioxidan ac i i y o que ce in was enhanced by encapsula ion in leci hin, in ag eemen wi h p e ious epo s ha desc ibe a syne gis ic e ec o hese wo compounds, bu he an ioxidan ac i i y dec eased by SFEE ea men , pe haps due o a pa ial ex ac ion o low molecula weigh ac ions o que ce in by supe c i ical CO2. Acknowledgemen s This esea ch has been unded by he Eu opean Ini ial T aining Ne wo k FP7-PEOPLE 2012 ITN 316959, “DoHip”, and by Jun a de Cas illa y León wi h p ojec VA225U14. Á. Ma ín hanks he Spanish Minis y o Economy and Compe i i eness o a Ramón y Cajal esea ch ellowship. S. Rod íguez-Rojo hanks he Spanish Minis y o Economy and Compe i i eness o a Juan de la Cie a esea ch ellowship. 24 REFERENCES [ 1 ]S. Das, A. Mandal, A. Ghosh, S. Panda, N. Das, S. Sa ka , Nanopa icula ed que ce in in comba ing age ela ed ce eb al oxida i e inju , Cu Aging Science, 2008, p. 1 [ 2 ]M. F. Ramadan, An ioxidan cha ac e is ics o phenolipids (que ce in-en iched leci hin) in lipid ma ices, Indus ial C ops and P oduc s 36, 2012, p. 363–369 [ 3 ]A. Pa ma a, K. Singhb, A. Bahadu c, G. Ma angonib, P. Bahadu a, In e ac ion and solubiliza ion o some phenolic an ioxidan s in Plu onic® micelles, Colloids and Su aces B: Bioin e aces 86, 2011, p. 319–326 [ 4 ]Y. Gao, Y. Wang, Y. Ma, A. Yu, F. Cai, W. Shao, G. Zhai, Fo mula ion op imiza ion and in si u abso p ion in a in es inal ac o que ce in-loaded mic oemulsion, Colloids and Su aces B, Bioin e aces, Vol 71, 2009, p. 306 [ 5 ]C. 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Res., 53 (11), p 2014, 4318–4327, DOI: 10.1021/ie5001136 32 Table 3 : Expe imen al plan o s udying he e ec o ac o s o Supe c i ical Fluid Ex ac ion o Emulsions p epa ed wi h Plu onic L64 on inal pa icle size, que ce in eco e y and esidual o ganic sol en concen a ion (Bes esul s acco ding o inal pa icle size and esidual o ganic con en ma ked by Bold) Expe i -men Sol en / wa e a io [ml/ml] Que ce in concen - a ion [w/w%] Plu onic concen - a ion [w/w%] Que - ce in eco e y [%] A e age d ople size in ini ial emulsion [μm] D(0.5) [μm] Unde 10 μm [V/V%] Residu -al E Ac [ppm] 1 (C) 0.30 0.02 1.0 64.7 2.3 778.6 10.2 259 2 (C) 0.30 0.02 1.0 70.0 2.4 150.6 15.0 354 3 (C) 0.30 0.02 1.0 72.8 2.9 462.2 5.0 233 4 0.25 0.02 0.8 64.7 3.0 389.1 15.0 134 5 0.25 0.03 0.8 66.6 2.1 13.4 37.5 170 6 0.25 0.03 0.8 65.6 2.1 0.9 79.5 140 7 0.25 0.02 1.2 70.8 2.7 163.9 15.7 107 8 0.25 0.03 1.2 71.1 1.9 108.4 22.2 1684 9 0.35 0.02 0.8 69.1 3.4 126.2 26.1 2317 10 0.35 0.03 0.8 73.1 3.6 93.6 25.6 2361 11 0.35 0.02 1.2 73.0 2.6 87.3 23.2 2608 12 0.35 0.03 1.2 69.7 2.5 7.8 52.6 1436 13 0.35 0.03 1.2 68.0 3.1 3.2 64.6 1623 33 Table 4 Expe imen al plan o s udying he e ec o ac o s on emulsion d ople size, inal pa icle size, que ce in eco e y and esidual o ganic sol en concen a ion (Cen um poin measu emen s ma ked by (C), bes esul acco ding o inal pa icle size dis ibu ion ma ked by Bold) Expe imen Sol en / /wa e a io [ml/ml] Que ce in concen - a ion [w/w%] Leci hin concen - a ion [w/w%] Que ce -in eco e- y [%] D ople size in ini ial emulsion [μm] D (0.5) [μm] Unde 1 μm [V/V%] Mode [μm] Resi- dual E Ac [ppm] 1 0.20 0.020 2.0 74.1 1.3 0.19 77.7 0.127- 0.172 24 2 0.20 0.028 1.6 49.3 1.4 6.384 36.6 0.134- 0.181 18 3 0.30 0.020 1.6 67.3 1.9 0.257 65.4 0.140- 0.191 23 4 0.30 0.028 2.0 66.9 1.6 0.548 52.8 0.137- 0.186 26 5 (C) 0.25 0.024 1.8 57.7 1.9 0.3 60.0 0.130- 0.177 177 6 (C) 0.25 0.024 1.8 74.0 1.9 0.426 55.1 0.130- 0.177 7 7 (C) 0.25 0.024 1.8 68.2 1.9 0.426 55.4 0.138- 0.158 82 8 0.20 0.020 2.0 37.1 1.5 1.229 47.1 0.138- 0.158 10 9 0.20 0.02 3.3 34.8 1.1 0.631 54.8 0.138- 0.158 62 34 Table 5: S abili y o e hyl ace a e-on-wa e emulsions p epa ed wi h Plu onic L64 and leci hin su ac an s Su ac an ma e ial Plu onic L64 Leci hin Range o sol en /wa e a io [mL/mL] 0.25 – 0.35 0.2 – 0.23 Su ac an concen a ion ange [w/w%] 0.8 – 1.2 1.6 – 2.0 Mig a ion a e be ween [mm/min] 3.37·10-3 – 8.60·10-3 1.58·10-3 – 3.63·10-3 S anda d de ia ion o mig a ion a e 1.67·10-3 6.40·10-4 A e age d ople size [µm] 2.6 1.6 D ople size changing be ween [µm] 1.9 – 3.4 1.1 – 1.9 S anda d de iance o d ople size [µm] 0.48 0.27 35 FIGURES Figu e 1: Chemical s uc u e o que ce in [5] 36 Figu e 2: Schema ic diag am o ba ch Supe c i ical Ex ac ion o Emulsions (SFEE) equipmen 37 Figu e 3: Expe imen s wi h Plu onic L64 su ac an : Op ical mic oscopy pic u e o he ini ially p epa ed emulsion (le ) and o aqueous suspension o que ce in pa icles ob ained by SFEE ea men o he emulsion ( igh ). Inse s show sizes measu ed by an image analysis echnique 38 Figu e 4: Su ace plo o in luencing ac o s on a e age d ople size o ini ial emulsion 39 Figu e 5: Example o mul imodal pa icle size dis ibu ion wi h Plu onic L64: expe imen al un 5 indica ed by ed, expe imen al un 5 indica ed by g een. 0 1 2 3 4 5 6 0,01 0,1 1 10 100 1000 10000 Volume pe cen Pa icle size [µm] Expe imen al un 5 Expe imen al un 6 40 Figu e 6: In luencing ac o s o pa icle size dis ibu ion in SFEE ea ed aqueous suspension 41 Figu e 7: Final pa icle size dis ibu ion o SFEE ea ed emulsions p epa ed wi h leci hin, acco ding o olume pe cen and numbe pe cen 0 2 4 6 8 10 12 14 0 1 2 3 4 5 6 0,01 0,1 1 10 100 1000 Numbe pe cen Volume pe cen Pa icle size [µm] log scale 5 ( C ) Volume pe cen 5 ( C ) Numbe pe cen