essential oils loaded in nanosystems_a developing strategy for a successful therapeutic approach

7/26/2019 Essential Oils Loaded in Nanosystems_A Developing Strategy for a Successful Therapeutic Approach

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Review ArticleEssential Oils Loaded in Nanosystems: A Developing Strategy fora Successful Therapeutic Approach

Anna Rita Bilia,1 Clizia Guccione,1 Benedetta Isacchi,1 Chiara Righeschi,1

Fabio Firenzuoli,2 and Maria Camilla Bergonzi1

Department of Chemistry, University of Florence, Via U. Schiff , Sesto Fiorentino, Italy Center for Integrative Medicine, Careggi University Hospital, University of Florence, Florence, Italy

Correspondence should be addressed to Anna Rita Bilia; [email protected]

Received January ; Accepted February ; Published May

Academic Editor: Luigi Gori

Copyright Anna Rita Bilia et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Essential oils are complex blends o a variety o volatile molecules such as terpenoids, phenol-derived aromatic components, andaliphatic components having a strong interest in pharmaceutical, sanitary, cosmetic, agricultural, and ood industries. Since themiddle ages, essential oils have been widely used or bactericidal, virucidal, ungicidal, antiparasitical, insecticidal, and othermedicinal properties such as analgesic, sedative, anti-inammatory, spasmolytic, and locally anaesthetic remedies. In this reviewtheir nanoencapsulation in drug delivery systems has been proposed or their capability o decreasing volatility, improving the

stability, water solubility, and efficacy o essential oil-based ormulations, by maintenance o therapeutic efficacy. wo categorieso nanocarriers can be proposed: polymeric nanoparticulate ormulations, extensively studied with signicant improvement o theessential oil antimicrobial activity, and lipid carriers, including liposomes, solid lipid nanoparticles, nanostructured lipid particles,and nano- and microemulsions. Furthermore, molecular complexes such as cyclodextrin inclusion complexes also represent a validstrategy to increase water solubility and stability and bioavailability and decrease volatility o essential oils.

1. Introduction

Spices have been used since antiquity or their perume,medicinal and preservative properties and to impart aromaand avour to ood. Hippocrates, the ather o medicine,prescribed perume umigations and massages with aromaticoils. urpentine was known by the Greeks and Romans or

its properties against lung diseases and biliary lithiasis.Dioscorides saying the best was the white, clear variety. Pliny,Hippocrates, and Galen avoured its properties too. Veniceturpentine was known during the Middle Ages, and the citybecame one o the principal markets or this medicinal drug[]. Te rst distillation o essential oils appeared in the East(India and Persia) [] more than years ago and wasimproved in the th century by the Arabs []. Nevertheless,the rst authentic written account o distillation o essentialoil is ascribed to Villanova (ca. ),a Catalan physician[], and only by the th century, the essential oils (EOs)were being made by pharmacies and their pharmacologicaleffects were described in pharmacopoeias []. By contrast,

their use does not appear to have been widespread in Europeuntil the th century; turpentine, juniper wood, rosemary,spike (lavender), clove, mace, nutmeg, anise, and cinnamonbecame common essential oils. In this century the termessential oil was used or the rst time by Paracelsus vonHohenheim, who named the effective component o a drug,Quinta essential []. By the middle o the th century, the

role o essential oils had been reduced almost entirely to beused in perumes, cosmetics, and ood avourings: rather inpharmaceutical preparations they still represent an importantpart o the traditional medicine and several monographs arereported in the official pharmacopoeias. At present ca. essential oils (EOs) are known, and % o them have com-mercial importance [] or the pharmaceutical, agronomic,ood, sanitary, cosmetic, and perume industries.

2. EOs Chemical Composition

EOs are volatile, limpid, and rarely coloured liquids, lipidsoluble and soluble in organic solvents with a generally lower

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014, Article ID 651593, 14 pageshttp://dx.doi.org/10.1155/2014/651593
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Evidence-Based Complementary and Alternative Medicine

density than that o water. Tey canbe synthesized by all plantorgans, that is,buds,owers, leaves, stems, twigs, seeds, ruits,roots, wood, or bark and are stored in secretory cells, cavities,canals, epidermic cells, or glandular trichomes. Constituentsare lipophilic and highly volatile secondary plant metabolites,reaching a mass below a molecular weight o , that can

be physically separated rom other plant components ormembranous tissue [].Nowadays there are several methods or extracting essen-

tial oils. Tese may include use o liquid carbon dioxideor microwaves, low or high pressure distillation employingboiling water or hot steam. As dened by the InternationalOrganization or Standardization (ISO), the term essentialoil is reserved or a product obtained rom vegetable rawmaterial, either by distillation with water or steam, or romthe epicarp o citrus ruits by a mechanical process, or by drydistillation (ISO , ), that is, by physical means only.Furthermore, essential oils or medical purposes need tocomply with national or international pharmacopoeias.

Te chemical prole o the essential oil products differs

not only in the number and type o molecules but also in theirstereochemical structures, and can be very different accord-ing to the selected method o extraction. Te extractionproduct can uctuate in quality, quantity, and compositionaccording to climate, soil composition, plant organ, age, and

vegetative cycle stage []. Most o the commercialized essen-tial oils are chemotyped by gas chromatography and massspectrometry analysis. Analytical monographs have beenpublished (European Pharmacopoeia, ISO, WHO, Council oEurope) to ensure good quality o essential oils. Te EOs aregenerally complex mixtures o volatile organic compoundsproduced as secondary metabolites in plants; they includehydrocarbons (terpenes and sesquiterpenes) and oxygenated

compounds (alcohols, esters, ethers, aldehydes, ketones, lac-tones, phenols, and phenol ethers) [].

Generally EOs contain about components up tomore than single substances, at quite different concen-trations; two or three are major components at airly highconcentrations (%) compared to others componentspresent in trace amounts. For example, carvacrol (%) andthymol (%) are the major components o the Origanumspecies essential oil.

Generally, these majorcomponents determine the biolog-ical properties o the essential oils. Te components includedifferent groups o distinct biosynthetical origin. Te maingroup is composed o terpenoids, phenylpropanoids, and

short-chain aliphatic hydrocarbon derivatives, which are allcharacterized by low molecular weight. Representative struc-tures are depicted inFigure .

erpenes are made rom combinations o several -carbon-base (C) units called isoprene and orm structurallyand unctionally different classes. Te biosynthesis o theterpenes consists o synthesis o the isopentenyl diphos-phate (IPP) precursor, repetitive addition o IPPs to ormthe prenyldiphosphate precursor o the various classes oterpenes, modication o the allylic prenyldiphosphate byterpenespecic synthetases to orm the terpene skeleton, and,nally, secondary enzymatic modication (redox reaction) othe skeleton to attribute unctional properties to the different

terpenes. erpenoids derive rom the C-building blocksisopentenyl diphosphate (IPP) and its isomer dimethylal-lyl diphosphate (DMAPP) and are generally representedby monoterpenes (C) and sesquiterpenes (C), whilehemiterpenes (C) are quite rare []. erpenes containingoxygen in the orm o hydroxyl, ether, aldehyde, ketone, or

carboxylic moieties are called terpenoids.Te monoterpenes (Figure ) are ormed rom the cou-pling o two isoprene units (C). Tey are the most rep-resentative molecules constituting % o the essential oilsand allow a great variety o structures. Tey consist oseveral unctions including acyclic hydrocarbons (myrceneand ocimene); monocyclic hydrocarbons (limonene, ter-pinenes, p-cymene, and phellandrenes); bicyclic hydrocar-bons (pinenes, camphene, and sabinene); acyclic alcohols(geraniol, linalool, citronellol, lavandulol, and nerol); mono-cyclic alcohols (menthol, -terpineol, and carveol); bicyclicalcohols (borneol, enchol, chrysanthenol, and thuyan--ol);acyclic aldehydes (geranial, neral, and citronellal); acyclicketone (tegetone), monocyclic ketone (menthones, carvone,pulegone, and piperitone); bicyclic ketone (camphor, en-chone, thuyone, and pinocarvone); acyclic esters (lina-lyl acetate or propionate and citronellyl acetate); mono-cyclic esters (menthyl or -terpinyl acetate); bicyclic esters(isobornyl acetate); ethers (,-cineole and menthouran);peroxides (ascaridole); and phenols (thymol, carvacrol).

Te sesquiterpenes are ormed rom the assembly o threeisoprene units (C). Te extension o the chain increasesthe number o cyclisations which allows a great variety ostructures (Figure ). Also sesquiterpenes include hydrocar-bons (azulene, -bisabolene, cadinenes, -caryophyllene, ar-nesenes, and zingiberene); alcohols (bisabolol, -nerolidol,arnesol,-santalol, and patchoulol); ketones (germacrone,

-vetinone, and turmerones); and epoxide (caryophylleneoxide and humulene epoxides).

Other aromatic molecules are phenylpropanoids ormedvia the shikimic acid pathway leading to phenylalanine []and occurring less requently than the terpenes.

Aromatic compounds originated rom the shikimatepathway (phenylpropanoids, Figure ) comprise aldehydes(cinnamaldehyde); alcohols (cinnamic alcohol); phenols(chavicol and eugenol); methoxy derivatives (anethole,estragole, and methyleugenols); methylenedioxy compounds(apiole, myristicin, and sarole).

Nitrogenous or sulphured components suchas glucosino-lates or isothiocyanate derivatives (garlic and mustard oils)

are also characteristic secondary metabolites o diverse aro-matic plants or o processed, grilled, or roasted products. Inaddition, some essential oils contain photoactive moleculeslike coumarins and urocoumarins (Citrus aurantium ssp.bergamia essential oil contains psoralens) and short-chainaliphatic substances such as -octanone and methyl nonylketone (Figure ).

3. Limits and Challenges for the RationalClinical Use of Essential Oils

Te most recent applications o EOs include being asantioxidants and preservatives in ood [], incorporated


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Short-chained aliphatics

MonoterpenoidsSesquiterpenoids

3-Octanone

Limonene Tymol Carvacrol

Menthol

Camphor

Citral

Azulene

Eugenol

Phenylpropanoids

Myristicin

Other constituents

Allicin

-Bisabolol

O

O

HH

O

O

O

O

O

S

SC

NS+O

O

CH3 CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3H3C

H3C

H3C

H3C

H3C H3C

H2COH

OH OH

OH

HO

OCH3

OCH3

-caryophyllene

cis-andtransanethole

Methyl nonyl ketone

Allyl isothiocinate

F : Representative structures typical o essential oils.

into oodstuff packaging material [], andapplication as plantand crop protectants []. raditionally, essential oils havebeen used or many biological properties including bacteri-cidal, virucidal, ungicidal, antiparasitical, insecticidal, andother medicinal properties such as analgesic, sedative, anti-inammatory, spasmolytic, and locally anesthetic remedies[].

At present, promising approaches have been reportedusing essential oils or components thereo in medicinalproducts or human or veterinary use []. Te most effective

way to use most EOs is by external application, as gargles andmouthwashes or inhalation; rarely they are used orally eveni generally regarded as sae (GRAS) to ingest. In this caseo oral administration they are generally diluted with milk,soy milk, or olive oil. opical application is generally sae; theoil is diluted in a ormulation but sometimes can give skinreactions and in particular some oils (specically citrus oils)are UV sensitive andmay cause irritation or darkeningo skinupon exposure to sunlight up to days afer application.

In case o inhalation when using strong oils, limit timein immediate vicinity o an essential oil diffuser as theconcentrated vapours may cause eye irritation, some o themare not recommended or diffusing or direct inhalation.

Tere is adequate evidence suggesting that althoughessential oils are metabolized quickly, their distributionthroughout the body is considered to be relatively high.

Most essential oil components are metabolized and eithereliminated by the kidneys in the orm o polar compoundsollowing limited phase I enzyme metabolism by conjugationwith glucuronate or sulate or exhaled via the lungs as CO

2.

For example, afer oral administration o ()-menthol, %o the original menthol content was excreted renally asmenthol glucuronide [, ]. Te same happens with thymol,

carvacrol, limonene, and eugenol. Afertheiroral administra-tion, sulphate and glucuronide orms have been detected inurine andin plasma, respectively [, ]. Te ast metabolismand short hal-lie o active compounds have led tothe belie that there is a minimum risk o accumulation inbody tissues [].

EO compounds are small, at soluble molecules, able topermeate the membranesincluding the skin beore being cap-tured by the microcirculation and drained into the systemiccirculation, which reaches all targets organs [, ]. In general,the respiratory tract offers the most rapid way o entryollowed by the dermal pathway []. opically, aromatherapyEOs can sometimes cause irritation o the skin, especially i


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the oils are not diluted. Some oils, such as bergamot oil, canalso cause photosensitization and induce malignant change.Applying excessive amounts o highly concentrated oils to alarge surace o the skin or on broken skin can result in signi-icant systemic absorption and increase the chance o seriousside effects, such as convulsions because EOs are permeation

enhancers.Besides the high volatility, EOs can easily decompose,owing to direct exposure to heat, humidity, light, or oxygen. Arecent manuscript has reviewed the actors inuencingessen-tial oil stability; specic knowledge on the chemical compo-sition and properties o essential oil is undamental or anadequate use [].

Degradation o EOs constituents is due to oxidation,isomerization, cyclization, or dehydrogenation reactions,triggered either enzymatically or chemically [], stronglyinuenced by the conditions during processing and storageo the plant material, upon distillation, and in the courseo subsequent handling o the oil itsel []. Furthermore,besides organoleptic alterations and viscosity changes, someaged essential oils as well as oxidized terpenoidshave revealedskin-sensitizing capacities [] leading to a hypersensitivityreaction synonymous to allergic contact dermatitis [].

4. Nanoencapsulation Technology

Encapsulation o bioactive compounds represents a easibleand efficient approach to modulate drug release, increase thephysical stability o the active substances, protect them romthe interactions with the environment, decrease their volatil-ity, enhance their bioactivity, reduce toxicity, and improvepatient compliance and convenience [].

A signicantly large part o current literature on theencapsulation o EOs deals with micrometric size capsules,which are used or the protection o the active compoundsagainst environmental actors (e.g., oxygen, light, moisture,andpH), to decrease oil volatility andto transorm the oil intoa powder. Encapsulation in nanometric particles is an alter-native or overcoming these problems but additionally, dueto the subcellular size, may increase the cellular absorptionmechanisms and increasing bioefficacy.

Nanosystemsapplied to the skin are used to acilitate localtherapies even i it is still under discussion o the mechanismso penetration trough skin. It is accepted that topical drugdelivery with nanoparticles targets the nanoparticles into the

deeper layers o skin and generally they do not reach theviable epidermis. Only where the cheratine barrier is compro-mised,however, such as in aged or diseased skin, an enhancedparticle penetration occurs. Te use o nanoparticles providesa sustained and slow release o the active constituents;nanoparticles represent a reservoir. In addition, nanoparti-cles can interact with skin at a cellular level as adjuvantsto enhance immune reactivity or topical vaccine applica-tions.

Hair ollicles and urrows were regarded as insignicantas potential routes or drug delivery, covering less than % othe human skin surace area, but their complex vascularisa-tion and deep invagination with a thinning stratum corneum

Stratum corneum

Viable epidermis

Sebaceus gland

Dermal papilla

Sweat duct

Sweat gland

Sweat pore

Subepidermalcapillary

1 2 3 4

Potential routes of penetration of nanoparticles

F : Skin nanoparticle drug delivery takes place in threemajor sites: stratum corneum surace through intracellular () andintercellular () penetration, urrows (), and openings o hairollicles (). Te nanoparticles are shown in violet.

have led to a reappraisal o this view. Ithas beendemonstratedthat in particular hair ollicles are an efficient reservoir ornanoparticle-based drug delivery and nanoparticles pene-tration can be increased with massage [, ]. Figure shows that the potential sites or skin targeting nanopar-ticles include the surace o the skin, urrows, and hairollicles.

Te alternative routes o administration o EOs arerepresented by oral intake and inhalation.

Within these routes the nanodelivery systems encounterthe mucosal lining o the nasal, lung, oral (sublingual andbuccal) cavity, stomach, and gut. Nanocarriers can improvethe stability o EOs against enzymatic degradation, achievedesired therapeutic levels in target tissues or the requiredduration with a lower number o doses, and might ensurean optimal pharmacokinetic prole to meet specic needs.However, the viscous, elastic, andsticky mucus layer that linesall mucosa tissues (even i with different characteristics) hasevolved to protect the body by rapidly trapping and removingoreign particles and hydrophobic molecules. As a conse-quence, mucoadhesion dened as the ability o nanoparticleto adhere to the mucus enhancing drug absorption canrepresent a valid strategy to enhance the residence time o thenanosystem and enhance absorption and bioavailability othe activeconstituent because it can acilitate transport acrosstheepithelium. Te interaction is generally achieved with nat-

ural or synthetic polymers which canorm hydrogenbondingand hydrophobic or electrostatic interactions with mucin.Te electrostatic interaction is the most effective and it can beachieved using positively charged polymers such as chitosan,being mucin negatively charged [].

Particle size, shape, and surace properties o thenanoparticles play a crucial role in the uptake o nanosizeddelivery systems across the mucosal membrane. Te nanocar-riers with particle size o nm, positive zeta potential,and hydrophobic surace were ound to have preerentialuptake as compared to their counterparts [].

Diverse absorption mechanisms have been establishedandtwo have beenpredominantly used: theparacellular route


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Absorption viachylomicrons

Uptake via MALDendritic cells

Enterocytes

Lymphatic absorption

System circulation

MacrophagesChylomicrons

Mucus

ightjunction

Receptor-mediatedendocytosis and

transcytosismodulationight junction

F : Mechanisms o nanocarriers (violet) enhanced absorption by enteric mucosa.

that is slow and passive and the transport through a lipoidalroute and it is also known as the transcellular process whichis responsible or the transport o lipophilic drugs that show arate dependency on their lipophilicity. Drug also crosses cellmembranes by an active transport route via carrier-mediatedmeans or transports through the opening o tight junctionsinteracting with the tight junction proteins [].

For instance the increase in the absorption o nanocar-

riers by enterocytes is due to tight junction modulation,receptor-mediated endocytosis and transcytosis, phagocyto-sis via specialized microold cells (M cells) o the Peyerspatches, and other mucosa associated lymphoid tissues(MAL) and lymphatic absorption via chylomicron uptakemechanism rom the enterocytes (mediated by lipase or

various lipid-based drug delivery systems) []. Mechanismo carriers penetration through enteric mucosa is reported inFigure .

5. EO-Loaded Nanodelivery Systems

Nanodelivery systems can be engineered to possess a number

o desirable eatures or therapy, including (i) sustained andcontrolledrelease o drugs locally, (ii) deep tissue penetrationdue to the nanometric size, (iii) cellular uptake and subcel-lular trafficking, and (iv) protection o cargo therapeutics atboth extracellular and intracellular levels.

Nanocarriers can be structured by a great variety omaterial and designs. Tis review is ocused on the organicnanocarrier systems, characterised by high biodegradabilityand biocompatibility, and classied in polymer-based nano-particles and lipid-based nanoparticles. In addition molecu-lar complexes such as inclusioncomplexes with cyclodextrinsare reported. A schematic representation o nanosystemplatorms or EOs is reported inFigure .

.. Polymer-Based Nanocarriers. Polymeric nanocarriers areclassied as nanocapsules and nanospheres. Nanocapsuleshave two compartments: a polymeric wall and a core, whichis commonly oily. Nanospheres are matrix systems. Teessential oil may be conjugated with the polymer (matrix orwall) or in the oily core.

Biocompatible polymers o synthetic origin include poly--cyanoacrylate alkyl esters, polyvinyl alcohol, polylactic

acid, polyglycolic acid, and polylactic glycolic acid. Telatter is usually divided into two classes: polysaccharidesand proteins. Polysaccharides include compounds rom plantorigin (e.g., pectin, cellulose and its derivatives, starch andits derivatives, arabic gum, carrageenan, and alginate) andpolysaccharides rom microbial or animal origin (e.g., xan-than gum and chitosan). Proteins are albumin, gelatine, soyproteins, and casein. Nanoparticles made o polysaccharides,due to their unique properties, are promising carriersto deliver and protect the physiological properties ohydrophilic drugs and have been successully applied as drugdelivery systems []. As natural biomaterials, polysaccha-rides are stable, sae, nontoxic, hydrophilic, and biodegrad-

able. In addition, polysaccharides have abundant resources innature and low cost in their processing. Te release o EOsrom carriers occurs through one o the ollowing processes:dissolution, desorption o the surace-bound/adsorbed unc-tional ingredient, diffusion through the matrix; matrix ero-sion including enzyme degradation, and a combination othese processes [].

Eugenol represents the main constituents o diverse EOsbut it is highly volatile, unstable, and sensitive to oxygen,light, and heat during processing, utilization, and storage.Choi et al. [] reported that encapsulation o eugenol intopolycaprolactone nanoparticles could enhance its stabilityagainst light oxidation.


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Strategies foressential oils

nanoencapsulation

Polymer-based nanocarriers

Molecular complexes

Nanocapsules

Solid lipidnanocapsules (SLN)

Nanostructured lipidcapsules (NLC)

Liposomes

Nano- and microemulsions

Inclusioncomplexes withcyclodextrins

Lipid-based nanocarriers

Nanoparticles

0.56nm

0.70nm

0.88nm

F : Schematic illustration o nanosystem platorms or essential oils.

Eugenol has been also encapsulated into chitosannanoparticles with an average size o less than nm.Loading capacity was % and encapsulation efficiency was%. Te particles had positively charged surace, with a zetapotential value ranging rom +. to +. mV. Te eugenol-

loaded chitosan nanoparticles were thermally stable andcould be useul as antioxidants orvarious thermal processingapplications [].

Chitosan nanoparticles have also been developed withoregano essential oil known or its potent antioxidant andantimicrobial activity. Te obtained nanoparticles exhibiteda regular distribution and spherical shape with size rangeo nm and the encapsulation efficiency and loadingcapacity were about % and %, respectively, when theinitial EO content was .. g/g chitosan. In vitro releasestudies showed an initial burst effect and ollowed by a slowdrug release [].

Alginate/cashew gum nanoparticles were prepared via

spray-drying to encapsulateLippia sidoidesessential oil, richin thymol which has ungicide and bactericide activities.Cashew gum is a biopolymer extracted rom the exudate o

Anacardium occidentale, a common tree o Brazils Northeast-ern region. Te gum main chain is composed o galactose(%), with side-chains o arabinose (.%), glucose (%),rhamnose (.%), and uronic acid (.%). Te averaged sizeso the nanoparticles were in the range nm, and zetapotential values ranging rom to mV. Encapsulatedoil levels varied rom . to .% with an encapsulationefficiency o up to %. Te in vitro release prole showedthat between and % o oil was released within h.Te addition o cashew gum to alginate has proven to be

able to maximize the hydrophilic character o the polymermatrices, allowing a quicker release at a satisactory oilloading. Moreover, the oil release prole revealed that the useo alginate in synergy with cashew gum or EO encapsulationpresents itsel as a potential delivery system with tailored

release rate, loading, and encapsulation efficacy [].Using the same EO rom Lippia sidoides nanoparticles

made o chitosan (a deacetylated orm o chitin, chemi-cally D-glucosamine and N-acetyl-D-glucosamine linked bybeta () linkages) and cashew gum aimed to improveessential oil loading and release proles. Samples designedusing relative ratios, matrix: oil, : ; gum : chitosan, : ;and % gum concentration, showed high loading (.%)and encapsulation efficiency (%), with average sizes inthe range nm. In vitro release proles showed thatnanoparticles presented slower and sustained release. Tenanocarriers presented efficacy against St. aegypti larvae,where the mortality rate was related to the loading values and

gum : chitosan ratios. In particular, samples gum : chitosan : and gum : chitosan : showed, respectively, % and% o mortality afer h, reaching over % o mortality at h. Tese results showed that the gum-chitosan nanoparti-cles were designed andpresent sustained release eatures[].

Te ormation o heat-resistant avour nanocapsules ojasmine essential oil was achieved by gelatin and arabic gum.Teir heat-resistance capability against 80C was evaluatedbyboth structural characteristics (size, polydispersity index,andzeta potential) and avour analysis. Te results showed thatthe nanocapsules were stable at80C or h, even i the GC-MS revealed that jasmine essential oil began to destroy above h [].


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Tymol loaded in zein (a corn prolamine protein)nanoparticles stabilized with sodium caseinate and chi-tosan hydrochloride were prepared and characterized. Inthe absence o sodium caseinate, the particle size and zetapotential o zein nanoparticleswere . nm and+. mV,respectively. Te zeta potential o rein nanoparticles afer

coating with sodium caseinate reversed rom positive tonegative (in the range o. to . mV), while sizewas around nm. Due to the presence o sodium caseinate,the stabilized zein nanoparticles showed a shif o isoelectricpoint rom . to . and had a desirable redispersibility inwater at neutral pH afer lyophilization. Encapsulated thymolwas more effective in suppressing Gram-positive bacteriumthan unencapsulated thymol or a longer time period. Zeinnanoparticles presented a two-phase release prole o the EO.Te authors believe that the rapid rst phase represents theportion o thymol that was in the external phase o the lm;the slower second phaserepresents thymol thatwas containedin the zein particles [].

Polylactic glycolic acid nanocapsules containing eugenolor transcinnamaldehyde both presented a two-phase EOrelease. Te rst phase was rapid (under minutes) andapproximately % o the EO load was detected; the secondrelease phase was prolonged and afer hours % oeugenol and % o transcinnamaldehyde were detected.Considering that PLGA hasa lowdegradation rate, thereleasewas governed mostly by diffusion with a possible inuenceo polymer swelling and bulk erosion. Te rst release phasemay be attributed to the molecules that are adsorbed to thepolymeric wall, while the second release phase represents theEO present in the core o the nanocapsules which diffusesthrough the polymeric wall [].

Polylactic glycolic acid nanocapsules containing car-vacrol have also been produced. Size was about . nm,polydispersity was ., zeta potenzial was ., drugloading was %, and encapsulation efficiency was %. Invitro release prole occurred with an initial burst releaseollowed by a slower release due to the concentration gra-dient. Nanoparticles showed a % release afer h andapproach to completeness afer h with approximately %o carvacrol released. Te effect o carvacrol EO antimicrobialactivity was enhanced because the nanoparticles signicantlyaltered rheological characteristic o bacterial biolms poten-tially acilitating the action o carvacrol [].

Methyl cellulose/ethyl cellulose polymeric nanoparticles

containing thymol attaining the relatively high thymol load-ing level o .% (weight o encapsulated thymol to weighto the thymol-loaded spheres) were able to reduce andpreserve levels o E. coli in an oil/water lotion and in ahydrophilic gel, o P. aeruginosa in an oil/water lotion andoS. aureus in an oil/water lotion and in a water/oil cream.Interestingly, ree thymol was also capable o reducing micro-biologic levels in these ormulations, but the preservationperiod was shorter except or the S. aureus in an oil/waterlotion where ree thymol maintained low microbial levelsor the same period as the nanocapsules. Effective bacterialsuppression by encapsulated thymol was also observed whenused in cream and aqueous gel ormulations [].

.. Lipid-Based Nanocarriers. Lipid-based nanocarriersinclude micro- and nanometric-scaled emulsions and lipidnanoparticles, roughly divided in liposomes, micelles,niosomes, solid lipid nanoparticles (SLN), and nano-structured lipid carriers (NLC). Liposomes and niosomesare colloidal association o amphiphilic lipids that organize

themselves spontaneously in bilayer vesicles and that aresuitable with hydrophilic and hydrophobic compounds.SLN and NLC are solid particles at room and human bodytemperatures that present lipid core, which makes thesecarriers a proper medium or entrapment o lipophiliccompounds, as EO.

As these nanoparticles are composed o lipids and/orphospholipids, they have the ability to interact with severalcell types. So, these carriers can been seen as alternatives ortreatment o microbial inections, due to their capacity ointeraction with inected cells. Furthermore, the associationo EO with lipid nanoparticles has different objectives, butthe main aims are the enhancement o the stability and thesolubility in aqueous media o EO, maintenance or evenenhancement o their biological activity, and drug targeting.

... Micro- and Nanoemulsions. Microemulsions can bedened as homogeneous thermodynamically stable trans-parent dispersions o two immiscibleliquids stabilized by aninteracial lm o suractants. Tey have droplet size above nm and require very low energyto ormulate emulsion,since they orm spontaneously when aqueous, oily, andamphiphilic components are brought intocontact, besideshaving a lower production cost compared to nanoemulsions.One major drawback to microemulsions isthat ormationrequires high suractant concentration, which can causetoxicity when used in pharmaceutical applications.

In contrast nanoemulsions can be prepared using lowersuractant concentrations. Nanoemulsions are ne oil-in-water dispersions, nonequilibrium systems with a sponta-neous tendency to separate into the constituent phases. Nev-ertheless,nanoemulsions may possess a relatively high kineticstability even or several years, due to their very small size,essentially the consequence o signicant steric stabilizationbetween droplets. Tey have droplet covering the size rangeo nm and also reerred to as miniemulsions, ultraneemulsions, and submicrometer emulsions.

Antimicrobial properties o micro- and nanoemulsion arebelieved to result rom the small size o oil particles that havea high surace tension which can use with and subsequently

disrupt the membrane o isolated prokaryotic cells, viruses,and eukaryoticcells o ungi but they do not affect eukaryoticcells o higher organisms. A synergistic antimicrobial effectcould be afforded by including some substances which pos-sess strong antimicrobial activity into the ormula, reducingthe amounts o active substances and detergents used orkilling microorganisms by the conventional method and thecost o raw materials. Furthermore, irritation caused romdetergents in the ormula is not likely to happen when theyare used in low concentrations.

Encapsulation in nanoemulsion-based delivery systemso two antimicrobial compounds, a terpenes mixtureextracted romMelaleuca alternifoliaand D-limonene, deals


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with the issues o ormulation and abrication in order toretain and possibly enhance the antimicrobial activity o theencapsulated compounds.

Te nanoemulsions based on ood-grade ingredientswere investigated by determining the minimum inhibitoryconcentration (MIC) and minimum bactericidal concen-

tration (MBC) or three different classes o microorgan-isms (Lactobacillus delbrueckii,Saccharomyces cerevisiae, andEscherichia coli). Te increase o the antimicrobial activityresulted in dependance on the ormulation andmean diam-eter o the delivery systems as well as on the microorganismsclass. Additionally, GC-MS analysis revealed that high inten-sity processing or nanoemulsion production may affect thechemicalstability o several active compounds.

Te results o the accelerated shel lie studies show thator both ruit juices afer days, the total inactivation o the

initial microbial load o 3 CFU/mL was already reachedor the terpenes concentrations o . g/L and g/L. At aterpenes concentration o . g/L, microorganism growth is

delayed by days in orange juice and days in pear juice incomparison to the control [].Another study reported the preparation o a sel-

nanoemulsiying drug delivery system or the oral delivery ozedoary turmeric oil, an essential oil extracted rom the dryrhizome o Curcuma zedoaria. Te optimized ormulationconsisting o EO, ethyl oleate, ween , ranscutol P(.:.:.:, /), and loaded with % drug was pre-pared. Upon mixing with water, the ormulation was rapidlydispersed into ne droplets with a mean size o68.3 1.6 nmand zeta potential o41.2 1.3 mV. Te active componentsremained stable in the optimized ormulation stored at25Cor at least months. Following oral administration inrats, both AUC and Cmax o germacrone, a representativebioactive marker o zedoary turmeric oil, increased by .-oldand .-old, respectively, compared with the unormulatedzedoary turmeric oil [].

... Liposomes. Liposomes are one o the most studiedcolloidal delivery systems; in act, they were rst developedor drug delivery purposes as early as s [,].

Liposomes consist o vesicular sel-assembled systemcomprising o one or more bilayers, usually ormed using aphospholipid, surrounding an aqueous core. Liposomes cancontain (i) one bilayer orming unilamellar vesicles (ULV),(ii) several concentric bilayers orming multilamellar vesicles,or (iii) nonconcentric bilayers orming multivesicular vesicles

(MVV).Te size o these structures canbe rather small (intherange o nm) or rather large (exceeding m). Owing tothe presence o the hydrophilic compartment and lipophilicpalisade, they can be used as carriers or both lipophilic andhydrophilic molecules [].

Bioactive compounds compartmentalised in liposomescan be protected against degradation and in case o lipophiliccompounds, liposomal encapsulation can also lead toincreased solubilisation [].

Te effect o liposomal inclusion on the stability andin vitro antiherpetic activity oSantolina insularis EO wasinvestigated. Vesicles were obtained rom hydrogenatedsoya phosphatydilcholine and cholesterol. Formulations were

examined or their stability or over one year monitoring thedrug leakage rom vesicles and the average size distribution.Te stability o the incorporated oil was veried by studyingits quali-quantitative composition. Te antiviral activity wasstudied against herpes simplex virus type (HSV-) by plaquereduction and yield reduction assays. Results showed that

Santolina insularisEO can be incorporated in high amountsin the prepared liposomes, which successully preventedits degradation. Moreover, stability studies pointed out that

vesicle dispersions were stable or at least one year and neitheroil leakage nor vesicle size alteration occurred during thisperiod. Antiviral activity assays demonstrated thatSantolinainsularisessential oil is effective in inactivating HSV- andthat the activity is principally due to direct virucidal effects.Free EO proved to be more effective than liposomal oil and adifferent activity was discovered which related to the vesicularstructure. Te ED() values, signicantly lower when cellswere preincubated with the EO beore the virus adsorption,indicate an intracellular mechanism in the antiviral activity oSantolina insularis[].

Te effect o liposomal inclusion on thein vitroantiher-petic activity oArtemisia arborescens L. EO was investigated.In order to study the inuence o vesicle structure and com-position on the antiviral activity o the vesicle-incorporatedoil, multilamellar (MLV) and unilamellar (SUV) positivelycharged liposomes were prepared. Liposomes were obtainedrom hydrogenated (PH) and nonhydrogenated (P) soyphosphatidylcholine. Formulations were examined or theirstability or over one year, monitoring the oil leakage rom

vesicles and the average size distribution. Te antiviral activ-ity was studied against herpes simplex virus type (HSV-)by a quantitative tetrazolium-based colorimetric method.Results showed that Artemisia EO can be incorporated ingood amounts in the prepared vesicular dispersions. Stabilitystudies pointed out that vesicle dispersions were very stableor at least six months and neither oil leakage nor vesiclesize alteration occurred during this period. Afer one yearo storage oil retention was still good, but vesicle usion waspresent. Antiviral assays demonstrated that the liposomalincorporation o A. arborescens EO enhanced its in vitroantiherpetic activity especially when vesicles were made withPH. On the contrary, no signicant difference in antiviralactivity was observed between the ree and SUV-incorporatedoil. PH MLV showeda higher activity than P MLV (EC

values o . and . g/mL or PH MLV and PMLV,resp.), while no signicant differences o the antiviral activity

were observed between the ree essential oil andSUV vesicles.Incorporation oA. arborescensessential oil in multilamellarliposomes greatly improved its activity against intracellularHSV- [].

A modied technique o rapid expansion o supercriticalsolutions (RESS) was applied to incorporate EO extractedrom Atractylodes macrocephala Koidz into liposomes. Teoptimised entrapment efficiency, drug loading, and averageparticle size o liposomes were ound to be .%, .%,and nm, respectively. Te physicochemical propertiesincluding the entrapment efficiency, dissolution rate, andstability met the characteristic or a pharmaceutical use o thedeveloped ormulation [].


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Carvacrol, thymol, p-cymene, and c-terpinene were iden-tied as major constituents and isolated rom the EOs romOriganum dictamnus L. Te above components were suc-cessully encapsulated in phosphatidyl choline-based lipo-somes and the possible improvement o their antioxidantand antimicrobial activities was tested against our Gram-

positive and our Gram-negative bacteria and three humanpathogenic ungi, as well as the ood-borne pathogen, Lis-teria monocytogenes. In order to investigate any possiblesynergistic or antagonistic effect between carvacrol/thymoland carvacrol/c-terpinene, the antimicrobial activities o themixtures werealso determined beore and aferencapsulationin liposomes. All tested compounds presented enhanced anti-microbial activities afer the encapsulation [].

A study examined carvacrol (derivatives) and thymolencapsulated in liposomes to increase their bioavailabilityand stability. Similarly, the endurance to humidity and UVlight was enhanced [].

... SLN Solid Lipid Nanoparticles. Solid lipidnanoparticles(SLN) reer to nanoscale size particles prepared using lipids

that remain solid at room temperature (or/and body temper-ature). Te lipid component may comprise o a broad rangeo lipid and lipid-like molecules such as triacylglycerols orwaxes []. Te diameter o such lipid particles can be alsoquite small, that is, in the range between nm and m.Active ingredients can be solubilised homogeneously eitherin the core o the SLNs or in the outside part []. Te advan-tage o SLNs as delivery system or lipophilic active compo-nents is reported to lie in the immobilisation o active ele-ments by the solid particle structure leading to an increasedchemical protection, less leakage, and sustained release[].

Tis physical property allows a better control o boththe physical (against recrystallisation) and chemical (againstdegradation) stability o the delivered constituents.

Te effect o SLN incorporation on transdermal deliveryandin vitroantiherpetic activity oArtemisia arborescensEOhas been investigated. wo different SLN ormulations wereprepared using the hot-pressure homogenization technique,Compritol AO as lipid, and Poloxamer (SLN ) andMiranol Ultra C (SLN ) as suractants.

One day afer production, the SLN had a size o nm(. polydispersion index) while the particle size o SLN prepared using Miranol Ultra C as suractant was nm(. polydispersion index, PI). Te mean particle size o

the ormulations increased only slightly afer two years ostorage, indicating a high physical stability o both SLN andSLN ormulations. In particular, years afer production,SLN and SLN ormulations showed a mean size o nm(. PI) and nm (. PI). Te PI values were alwayssmaller than . indicating a airly narrow size distributiono the particles. Formulations were examined or their sta-bility or two years by monitoring average size distributionand zeta potential values. Te antiviral activity o ree andSLN incorporated EO was tested in vitro against HerpesSimplex Virus- (HSV-), while the effects o essential oilincorporation into SLN on both the permeation through andthe accumulation into the skin strata were investigated by

using in vitro diffusion experiments through newborn pigskin and an almond oil Artemisia essentialoil solution as acontrol. Results showedthat bothSLN ormulationswere ableto entrap the EO in high yields and that the mean particle sizeincreased only slightly afer two years o storage, indicatinga high physical stability.In vitroantiviral assays showed that

SLN incorporation did not affect the EO antiherpetic activity.Tein vitroskin permeation experiments demonstrated thecapability o SLN o greatly improving the oil accumulationinto the skin, while oil permeation occurred only when theoil was delivered rom the control solution [].

Alhaj and coworkers developed a ormulation based onNigella sativaessential oil into solid lipid nanoparticles SLN.SLN ormulations were prepared using hydrogenated palmoil Sofisan andN.sativaessential oil as lipid matrix, sor-bitol, and water. Data showed a high physical stability or or-mulationsat various storage temperatures during months ostorage. In particular, average diameter oN.sativaessentialoil loaded SLN did not vary during storage and increasedslightly afer reeze-drying the SLN dispersions. Tereore,obtained results showed that the studied SLN ormula-tions are suitable carriers in pharmaceutical and cosmeticelds [].

Frankincense and myrrh are gum resins obtained romthe genera Boswellia and Commiphora, respectively. Bothgenera belong to the amily Burseraceae, which is nativeto Northeast Arica and the Middle East. Frankincenseand myrrh have been used or medical purposes in Chinaand India or thousands o years. Modern pharmacologicalresearch has revealed that essential oils are the primaryeffective components in rankincense and myrrh oil (FMO)that exhibit a broad spectrum o biological activities suchas antimicrobial, anti-inammatory, and antitumoractivities.As with other essential oils, the instability and poor watersolubility o FMO result in poor oral bioavailability, whichlimits its clinical application. Te components o FMO aresensitive to light, air, and high temperature, and FMO stim-ulates the gastrointestinal tract, making it unsuitable or oraladministration. A study has reported the preparation o solidlipid nanoparticles or the oral delivery o rankincense andmyrrh essential oils (FMO). Aqueous dispersions o SLNswere successully prepared by a high-pressure homogeniza-tion method using Compritol AO as the solid lipid andsoybean lecithin and ween as the suractants. RoundSLNs were with a mean size o . nm, a zeta potenzial o. mV, and an encapsulation efficiency o .%. SLN

ormulation increased the antitumor efficacy o FMO in H-bearing Kunming mice. Compritol AO showed reason-able FMO solubilization capacity. Te poorly water-solubledrug FMO was efficiently encapsulated into the nanoparti-cles. Particles prepared under proper ormulation conditionswere spherical with diameters o nm []. Solid lipidnanoparticles (SLNs) o essentialoil oZataria multiora havebeen developed. Te results showed that the encapsulationefficiency was .%. Results o particle size determinationshowed a mean size o nm and SLNs were sphericalas shown by EM. Te DSC curve o sodium dodecylsulate, polyethylene glycol, cetyl alcohol, and EO was differ-ent rom EO containing SLNs, which indicated that the EO


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can interact with the matrix o lipid during the preparation othe SLNs. .% othe essential oil was releasedafer h. Teresults o characterization o the SLNs indicated the potentialapplication o essential oil o Z. multiora loaded SLN ascarrier system [].

.. Molecular Complexes. A simple strategy to deliver activeingredients is by physically complexing them with othermolecules in order to have a better solubility prole and/or anincrease in the chemical stability o the complexed system. Inthis context a molecular complex is reerring to the physicalassociation between a host and a guest (active ingredient)molecule and in the case o EOs the complexes are reportedwith cyclodextrins (CDs).

Cyclodextrins are natural macrocyclic oligosaccharideswell known or having toroid-shaped structures with rigidlipophilic cavities and a hydrophilic outer surace insuringgood dissolution o the complex in an aqueous environment.Tey are able to enclose highly hydrophobic molecules

inside their hydrophobic cavity, constituting a true molec-ular encapsulation []. Te major advantages o the useo CD-complexation in pharmaceutical applications, oods,cosmetics, and toiletries are protection o the active ingre-dients against oxidation, light induced reactions, decom-position and thermal decomposition, loss by evaporationand sublimation, and elimination or reduction o undesiredtastes/odours, to reduce or prevent gastric-intestinal irri-tation (mainly due to anti-inammatory drugs) or oculardisturbances, prevent drug-drug or drug-additive interac-tions, or even to convert oils and liquid drugs into micro-crystalline or amorphous powders and to reduce microbi-ological contamination, bres, or the elimination o other

undesired components and hygroscopicity []. Moreover,ormation o inclusion complex (IC) increases the guests invivo stability against hydrolysis, oxidation, decomposition,and dehydration, consequently increasing bioavailability andbioefficacy. Tere are three main types o CDs: -, -, and-cyclodextrins, corresponding to , , and glucopyranoseunits linked by-(,) bonds, respectively. Te dimensionso the internal cavity are .. nm and are crucial or theencapsulation o guest molecules [].

In the last years, physicochemical properties and, con-sequently, the inclusion capacity o the natives CD havebeen improved by chemical modication o their hydroxylgroups []. Besides natural cyclodextrins, a growing num-

ber o semisynthetic derivatives and copolymers have beenprepared and are already commercially available. Many othem ound use as structural and chiral selectors, with newproperties given by the type and number o substituents.Te semisynthetic derivatives o cyclodextrins show bettersolubility in water, can decrease and modulate the release rateo watersoluble molecules,are able to enhance the dissolutionrate and the inclusion capacity, and also decrease the sideeffects o some molecules.

Te majority o the publications is concerning theencapsulation o essential oils with -CD and its deriva-tives: randomly methylated--cyclodextrin, hydroxypropyl--cyclodextrin, and low methylated--cyclodextrin.

Te IC o thymol and cinnamaldehyde and -CD wasinvestigated [] in order to study the inuence o wateradsorption by CDs and their complexes on the release oencapsulated compounds. Te results showed that -CDencapsulates efficiently both o them, in a : molar ratio. TeICs were obtained upon mixing the components in aqueous

media and subsequent reeze-drying, as conrmed by differ-ential scanning calorimetry. Te samples were stored at con-stant relative humidity, rom % to %, at 25C. Te releaseo encapsulated compounds was determined ollowing themelting enthalpy o each guest. Water sorption isothermsor -CD and the complexes showed constant and lowwater sorption at RH < %; then the uptake o waterincreased abruptly. Te amount o sorbed water at each RHwas smaller or the complexes than or -CD. Te guestmolecules displaced water molecules rom inside the cavityo-CD. No thymol or cinnamaldehyde release was detectedat RH < %, and it increased abruptly rom % RH,coincidentally with the abrupt increase o absorbed water.Water sorption signicantly affects -CD complexes stability,which is thus governed by the shape o the water sorptionisotherm. Te stability studies showed that the inclusioncomplexes thymol--CD and cinnamaldehyde--CD remainstable up to % RH during long storage times. In act, theguests released rom the -CD complexes were detectablein the region o the water adsorption isotherm at which asharp increase o water content occurred (% RH). Teseresults show the relevance o selecting appropriated storageconditions or hydrophobic avours encapsulated in -CD oror predicting the shel lie o unctional products ormulatedwith nanoencapsulated compounds [].

-Caryophyllene (BCP), a natural sesquiterpene exist-ing in the essential oil o many plants, has exhibited awide range o biological activities such as antimicrobial,anticarcinogenic, anti-inammatory, antioxidant, anxiolytic-like, and local anaesthetic effects. However, its volatility andpoor water solubility limit its application in pharmaceuticaleld. Liu and coworkers investigated and compared the oralbioavailability and the pharmacokinetics o ree BCP andBCP/-CD ICafer a singleoraldoseo mg/kg onrats [].BCP was rapidly released rom inclusion complex and theinvivodata showed that BCP/-CD IC displayed earlier max,higher Cmax and the AUC- h showed approximately .times higher increase than those o ree BCP. Te -CD hassignicantly increased the oral bioavailability o the drug in

rats than ree BCP [].Te essential oil oChamomilla recutita (L.) Rauschert,

syn. Matricaria recutita L., contains up to % ()--bisabolol which contributes to the anti-inammatory proper-ties o camomile oil. Bisabolol is a very lipophilic substance,with a tendency to oxidise decreasing anti-inammatoryactivity ca. %. ()--Bisabolol was ound to orm an inclu-sion complex with -CD in solution as well as in the solidstate. o investigate molecular associations o -CD withpure ()--bisabolol or ()--bisabolol as a component ocamomile EO, Waleczek et al. undertook phase solubility

studies []. Te complex constant was M1 or thepure ()--bisabolol and M1 or ()--bisabolol as


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a constituent o the EO. Te intrinsic solubility o pure

()--bisabolol (. 4 M) and ()--bisabolol as acomponent o the EO (. 4 M) differ signicantly.Computer simulation proved an inclusion complex having astoichiometric composition o : (-CD : drug) [].

Tymol is a monoterpene present in Lamiaceae plants,

specially oreganos and thymes. Cinnamaldehyde (-phenyl--propenal) represents % o the cinnamon EO. Tymoland cinnamaldehyde are requently used as avours, butthey are also becoming increasingly important as naturallyoccurring antimicrobial, antioxidant, and antiseptic agents.As natural and articial avours they are very sensitiveto the effects o light, oxygen, humidity, and high tem-peratures. Te study o Hill et al. [] aimed to elucidatethe physicochemical characteristics o essential oils and-Cyclodextrin (EO--CD) inclusion complexes and theirresulting antimicrobial activity. Cinnamon bark extract, tran-scinnamaldehyde, clove bud extract, eugenol, and a : (transcinnamaldehyde : eugenol) mixture were microencap-

sulated by the reeze-drying method. EO--CD complexeswere characterized or particle size, morphology, polydisper-sityindex(PI),entrapment efficiency, and phasesolubility. Allparticles showed a spherical shape, smooth surace, no sig-nicant differences in size distribution, and strong tendencyto agglomerate. Te entrapment efficiencies ranged rom. to .%, where pure compounds were higher (

essential oils loaded in nanosystems_a developing strategy for a successful therapeutic approach

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