Formulation, characterization and antimicrobial activity of tablets of essential oil prepared by compression of spray-dried powder

Accepted Manuscript Formulation, characterization and antimicrobial activity of tablets of essential oil prepared by compression of spray-dried powder Ioannis Partheniadis, Souzan Vergkizi, Diamanto Lazari, Christos Reppas, Ioannis Nikolakakis PII:

S1773-2247(18)31388-1

DOI:

https://doi.org/10.1016/j.jddst.2019.01.031

Reference:

JDDST 916

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 18 November 2018 Revised Date:

19 January 2019

Accepted Date: 21 January 2019

Please cite this article as: I. Partheniadis, S. Vergkizi, D. Lazari, C. Reppas, I. Nikolakakis, Formulation, characterization and antimicrobial activity of tablets of essential oil prepared by compression of spraydried powder, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/ j.jddst.2019.01.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Spray drying

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EO Emulsion

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EO released (%)

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Antimicrobial activity Disc diffusion zone

ACCEPTED MANUSCRIPT Title: Formulation, characterization and antimicrobial activity of tablets of essential oil prepared by compression of spray-dried powder

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Ioannis Partheniadisa#, Souzan Vergkizib#, Diamanto Lazaric, Christos Reppasd, Ioannis Nikolakakisa*,

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Dept. of Pharmaceutical Technology, cDept. of Pharmacognosy, School of Pharmacy

b

Dept. of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University

of Thessaloniki, 54124, Thessaloniki, Greece

Dept. of Pharmacy, National and Kapodistrian University of Athens, 15772, Athens, Greece

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Authors of equal contribution

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Running Headline: Tablets of spray-dried oregano essential oil

Corresponding author:

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Dr Ioannis Nikolakakis Department of Pharmaceutical Technology School of Pharmacy Faculty of Health Sciences Aristotle University of Thessaloniki Thessaloniki 54124 Greece

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E mail: [email protected] Telephone: +30 2310 997635 +30 6938752485

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Abstract

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Oregano essential oil (EO) was encapsulated by spray drying emulsions stabilised with

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polysaccharides. Both, emulsions and spray-dried powder (SDP) were characterized

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physicochemically and SDP was compressed into tablets with low (10% w/w) and high (20%

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w/w) EO content. Compression behavior, redispersibility and release of EO were studied.

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Antimicrobial activity of reconstituted emulsions was evaluated by disc diffusion and broth

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dilution. SDP showed good EO encapsulation efficiency and retained high carvacrol content.

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SDP with low EO content formed strong tablets, whereas SDP with high EO formed weaker

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tablets but with acceptable mechanical strength and friability. Oil leakage due to compression

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was negligible. Reconstitution of original emulsion was faster from the SDP than the tablets

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and the use of disintegrants did not show improvement. Interestingly, addition of

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croscarmellose sodium (CCS) greatly increased the release rate of EO from tablets despite

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lack of effect on redispersibility, possibly due to fast liquid uptake/swelling by the CCS fibres

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combined with repulsion between negatively charged carboxyl groups on CCS and gum

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Arabic resulting in opening of the microencapsulating wall. Sodium starch glycolate had small

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effect on release. The antimicrobial activity of reconstituted emulsions relative to neomycin

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ranked in the order: E coli
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Key words: oregano essential oil; spray drying; microencapsulation; tablets; in vitro release;

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antimicrobial activity

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1. Introduction Due to the antimicrobial resistance to antibiotics, the interest in natural antimicrobials

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with accepted safety and efficacy has increased (1,2). Oregano essential oil (EO) is active

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against Gram-negative and Gram-positive bacteria (3-6) due to carvacrol in its composition

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and has been found very effective in preventing neonatal diarrhoea syndrome in calves (7). Its

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direct oral delivery is hampered by the irritating taste and for this reason it needs to be diluted

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with water before administration. Oral delivery as a solid tablet dosage form has the

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advantages of easier administration, improved dosing accuracy, better compliance and

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protection against light and humidity (8-11). Furthermore, EO is marketed as diet supplement

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for humans, mainly in the form of soft gelatin capsules, (e.g. Lindens 25 mg, Athina 500 mg)

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which although simple, have disadvantages and limitations such as high manufacturing cost

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due to the low production rate, stability issues due to the presence of plasticizers, increasing

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permeability to moisture and oxygen, and possible interactions with capsule ingredients (12).

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Since EO is sensitive to humidity and light, protection by microencapsulation is desired

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and for this purpose spray drying is a suitable method (8-11, 13). The spray-dried powder

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product (SDP) is generally compressible due to the intraparticle porosity and can be processed

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into a final tablet dosage form with good physicochemical stability, good weight uniformity

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and low production cost. However, the presence of non-compressible oil may adversely affect

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tablet formation due to the adverse effect on compressibility (14).

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Therefore, the aim of this work was to prepare encapsulated EO by spray drying o/w

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emulsions stabilized with polysaccharides that also act as wall materials and tablet binders (8,

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15), followed by compression into tablets. Since the effective EO concentrations vary

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depending on the type and strain of bacteria, severity of infection and age group (16), two SDP

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compositions and corresponding tablets with low (10%) and high (20%) EO were prepared.

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The feed emulsions were characterized for physicochemical properties and stability. The SDPs 3

ACCEPTED MANUSCRIPT were characterized for EO consistency, retention, encapsulation efficiency and physical

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properties (particle size, shape, and morphology). The feasibility of processing SDPs into

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tablets (SDT) was evaluated from compression experiments following FDA guidance for test

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procedures (USP Chapter <1062>) (17). Spray-dried powders and tablets were evaluated also

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for redispersibility and release of EO.

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The main constituent of the EO encapsulating wall material was Arabic gum as it forms

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low viscosity dispersions suitable for spray drying, it is able to stabilize o/w emulsions across

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a wide pH range and also because it makes a good tablet binder (8, 10). Modified starch and

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maltodextrin were used to improve encapsulation, stability and wettability and facilitate spray

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drying (8, 10, 18, 19). Additionally, in order to increase the rate of EO release from the tablets

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two common tablet disintegrants croscarmellose sodium and sodium starch glycolate were

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used (20, 21). Finally, the antimicrobial activity of emulsion reconstituted form tablet was

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tested against Gram-negative and Gram-positive bacteria by disc diffusion (22) and was

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compared with a non-encapsulated EO form and standard neomycin disc. Minimum inhibitory

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concentration (MIC) was further determined using broth dilution and data analysis (23).

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2.1 Materials

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2. Materials and Methods

Oregano essential oil of Greek origin (EO, 85.89±0.06 % w/w carvacrol) from

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Oreganum vulgare (Heracleoticum) was a gift from Ecopharm Hellas, Kilkis, Greece. Arabic

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gum (AG, Spraygum AB) was from Nexira, France. Maltodextrin (MD, Glucidex 21) and

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modified starch (MS, Clearam CH20 20, food grade acetylate di-starch adipate (E1422), were

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from Roquette Italia, offered as gifts from Interallis Chemicals, Greece. Cross-linked

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carboxymethylcellulose sodium (croscarmellose sodium, CCS) with particle size distribution

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parameters D10=11, D50=23, D90=40 µm (determined by optical microscopy) and sodium

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starch glycolate (Primojel, SSG) with D10=13, D50=23, D90=40 µm respectively were gifts

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from AKZO NOBEL (Netherlands) and DFE Pharma (Germany).

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2.2 Methods 2.2.1. Preparation of spray-dried encapsulated oregano EO Feed emulsions: For the preparation of emulsions, AG and MD were hydrated

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overnight at 4-5 oC. The glass beaker containing the dispersion was immersed in a heated

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water bath and MS was added after about 20 min when the temperature reached 82 oC,

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followed by homogenization (15000 rpm, 20 min), using ULTRA-TURRAX (Model

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TP18/10S5, IKA, Germany) connected to IKA-TRON DZM1 digital counter. EO was added

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after cooling to 5 oC, followed by further 5 min homogenization. Two emulsions in deionised

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water with i) low 3% w/w EO (27% w/w encapsulating material) or ii) high 6% w/w EO (24%

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w/w encapsulating material) were prepared. Both contained 30% w/w dispersed phase (EO

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plus encapsulating material) selected on the basis of stability, acceptable viscosity and

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minimal losses during spray drying. The composition of the encapsulation material in both

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emulsions was fixed: AG 75%, MS 12.5%, MD 12.5%.

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Spray-drying: 100-mL of each emulsion were spray-dried (B-191, Büchi, Switzerland)

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under the following conditions: feed rate 5 mL min-1, inlet air temperature 180 oC, outlet 117

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gave powder product with 10% w/w EO denoted SDP10 and the high 6% w/w EO emulsion

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product with 20% w/w EO denoted SDP20. Additionally, a non-encapsulated EO dispersion in

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aqueous Tween 20 solution (1% w/w) was prepared for comparison of the antimicrobial

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activities and a spray-dried product with all ingredients but EO was prepared as negative

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control.

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2.2.2. Preparation of oregano EO tablets

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C, aspiration 100% and airflow 600 L/h. After spray drying, the low 3% w/w EO emulsion

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ACCEPTED MANUSCRIPT EO tablets (200 mg) were prepared using an instrumented press (∅8.0 mm flat-edged

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punches, Gamlen D-Series Press, Nottingham, UK) in the range 40-100 MPa at 10 mm/min

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compression rate. Force-displacement profiles were recorded during compression and ejection

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stages. From this profile the elastic recovery was estimated as the % increase in tablet

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thickness after removal of compressive force (P=0) compared to thickness at maximum punch

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penetration. Ejectability was estimated as the maximum ejection pressure (=ejection

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force/curved area of tablet). Preliminary experiments showed that although compression of

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SDP easily formed tablets, they needed some time to be dispersed in water. For this reason,

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5% croscarmellose sodium or sodium starch glycolate were added as disintegrants. Tablets

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made with SDP10 (10% EO) without disintegrant were denoted SDT10 and tablets made with

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SDP20 (20% EO) were denoted SDT20 respectively.

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2.2.3. Characterization of feed emulsions

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Viscosity: The viscosity of the feed emulsions was measured with a rotational

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viscometer (HAAKE Viscotester VT24, Thermo Fisher Scientific, Germany) fitted with a cup

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and-bob NV St sensor, rotating from 0.7 to 22.6 rpm corresponding to shear rates 3.8 to 122

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sec-1. Viscosity was calculated from the linear part of shear stress vs shear rate plots.

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Droplet size: Droplet size distribution was analyzed with optical microscopy (Olympus

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BX41 microscope, Japan and video camera Leica DF295, Germany) and image analysis

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software (Leica Microsystems, Switzerland). The microscope was calibrated with 2 mm length

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(0.01 mm interval) micrometer calibration slide (Leitz Wetzlar, Germany). About 200 particles

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were analysed in 4-5 different fields at x40 magnification. Particle size was expressed as

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equivalent circle diameter (diameter of sphere having the same projected area as the measured

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particles) and was calculated as the arithmetic mean.

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Electrical conductivity and zeta potential (ζ): The type of feed spray-dried emulsions was

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examined by electrical conductivity (Mettler Toledo SevenEasy, Switzerland). Droplet surface 6

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charges were estimated from ζ measurements after dilution to 1% w/w with water (Zetasizer,

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ZEN3600, Malvern Instruments Ltd, Worcestershire, UK). Emulsion stability: This was evaluated using an optical analyzer (Turbiscan® MA

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Classic 2000, Formulaction, France) (850 nm light, scattered at 135o) calibrated for T%=100

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with silicon oil and was 96±0.8% for distilled water. The glass test tube was filled to 70 mm

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height and scanned at 40 µm intervals. Back scattering (BS%) - distance profiles were

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recorded every 8 h. Three measurements of the emulsion properties were taken and mean and

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standard deviations calculated.

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2.2.4. Characterization of the spray-dried products (SDP)

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Composition of essential oregano oil: The composition of unprocessed oregano EO

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and after its extraction from the SDP was analyzed by Gas Chromatography–Mass

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Spectroscopy (Shimadzu GC-2010-GCMS-QP2010, 70 eV) (24-26). It was equipped with a

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split/splitless injector (230 oC) and a fused silica HP-5 MS capillary column (30 m length,

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0.25 mm i.d., film thickness 0.25 µm). The heating program was from 50 oC to 290 oC, at a

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rate of 4 oC/min. Helium was used as the carrier gas at a flow rate of 1.0 mL/min and injection

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volume 1 µL. Retention indices for all EO constituents were determined according to van den

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Dool and Kratz (24) using n-alkanes as standards. Identification of the components was based

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on comparison of their mass spectra with those of NIST21 and NIST107 (25), and by

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comparison of their retention indices with literature data (26). Relative concentrations of the

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components were calculated based on GC peak areas of authentic compounds contained in the

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EO (Sigma Aldrich Chemicals).

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Oil retention and encapsulation efficiency: EO in the SDP was estimated using a

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Clevenger apparatus (15). 5-gram was dissolved in 250 mL water, EO was distilled out and

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extracted with ethyl ether. It was passed through filter paper covered with anhydrous sodium

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sulfate and collected in a pre-weighted glass beaker. The filtrate was left for ether evaporation

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and EO was calculated from the weight difference. Oil retention was expressed as

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RT% = 100 * (EO in feed emulsion / EO in spray-dried powder)

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(1)

For the encapsulation efficiency, surface EO was determined by adding 0.5 g SDP to 10

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mL hexane and shaking for 30 sec, followed by centrifugation (1750g, 10 min). The

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supernatant was analyzed for EO by UV spectroscopy (274 nm, UV-1700, Shimadzu, Japan)

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and encapsulation efficiency was expressed as

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EE% = 100 * (total oil – surface oil / total oil)

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Particle size and shape: Particle size was measured using laser light scattering (632.8

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nm, Spray-Tec 3.20, Malvern Instruments, UK) and was expressed as mean volume diameter.

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Particle shape was measured with optical microscopy and was expressed as aspect ratio

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(=highest to shortest Ferret diameter) and roundness index [=perimeter2 / (12.56 * mean

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projection area)]. The last index expresses both geometrical asymmetry and surface

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irregularity and has value 1 for sphere, increasing as the shape deviates from spherical.

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(2)

Surface morphology: This was examined using scanning electron microscopy (20 kV, JSM 840A, Jeol, Japan).

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Moisture content: This was determined by heating 1 g at 105 oC (HR73 Mettler

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Toledo, Switzerland), until the weight difference between two successive 30 sec interval

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measurements was <0.01 g and was expressed on dry basis (%).

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2.2.5. Evaluation of tablets (SDT) from compressed spray-dried EO powder

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Oil leakage: The weight of SDP before compression and of the corresponding ejected

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tablet after wiping gently with filter paper were measured with accuracy ±0.01 mg (balance

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Mettler Toledo, New Classic MF, Switzerland). Leakage was expressed as %weight difference

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after subtracting the weight loss of inert tablets prepared from wall materials only.

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Porosity: Weight (Wt) and tablet dimensions were measured with accuracy ±0.001 g

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(balance UW 220 Shimadzu, Japan) and ±0.01 mm (micrometer Moor and Wright, Sheffield,

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UK) respectively. From the tablet volume (Vt), the porosity (ε) was calculated as ε = 1- [ (Wt/Vt) / ps) ]

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Particle density ps (g/cc) was taken as the weighted average of components (for EO 0.95,

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Arabic gum and maltodextrin 1.42 and for starch 1.48 g/cc) and was for SDP10 1.33 g/cc and

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for SDP20 1.38 g/cc.

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Mechanical strength and friability: Mechanical strength was evaluated as tensile

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strength measured by diametrical loading, using the instrument press described previously but

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operated in fracture mode using a 10 kg load cell. Tensile strength (T) was calculated from

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(27)

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T = 2F / πΦh

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(4)

where F is the breaking load, Φ the tablet diameter and h its thickness. Friability: This was determined according to USP Chapter 1216 (28). Tablets

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corresponding to about 6.5 g were accurately weighted, added to the tester drum and rotated

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for 4 min at 25 rpm. Friability was expressed as the %weight difference of tablets before and

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after the test.

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2.2.6. Redispersibility of spray-dried powders (SDP) and tablets (SDT)

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Redispersibility was evaluated using turbidimetry. Samples corresponding to 2.8 mg

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EO that covered the transmittance range of the instrument from the beginning to the of

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reconstitution was placed in 200 mL distilled water in the vessel of USP II apparatus (Pharma

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Test PTW 2, Hainburg, Germany) at 37±0.5 oC and 100 rpm agitation rate. Aliquots (5mL)

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were withdrawn at time intervals of 2, 5, 10, 15, 20, 30, 45, 60, 75 and 90 min, placed in test

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tubes and analyzed for transmittance (T%, 850 nm) using the optical analyzer described above

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for emulsion stability (Turbiscan® MA Classic 2000). After measurement the samples were

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returned to the dissolution vessel.

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2.2.7. In vitro release of EO from spray-dried powder (SDP) and tablets (SDT) Samples of spray-dried powders and tablets corresponding to 20 mg EO were sealed in

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dialysis cellulose membranes (cut-off 12500, Sigma-Aldrich) and immersed in the vessel of

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USP Apparatus II (Pharma Test PTW 2) containing 400 mL deionised water at 37±0.5 oC and

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100 rpm agitation rate. Aliquots were taken at timely intervals and analyzed for EO by UV

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spectroscopy at 273 nm. Three measurements were taken for each redispersibility and in vitro

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release test and mean and standard deviations calculated.

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2.2.8. Antimicrobial activity of reconstituted emulsion

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Disc diffusion: Bacterial strains of E coli, P mirabilis, Klebsiella sp and S aureus were

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used. These were laboratory isolates from the Microbiology Laboratory, School of Medicine,

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Aristotle University of Thessaloniki. Reconstituted emulsions obtained from EO tablets with

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deionised water containing 3.0% w/w EO (or 30% dispersed phase as in the feed emulsion)

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were tested. A standard 30 µg neomycin antibiotic disc (Oxoid, UK) was used as positive

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control and re-emulsified spray-dried wall material (without EO) as the negative control.

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For each strain an inoculum was spread evenly on the entire surface of a Mueller

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Hinton (MH) agar plate for the Gram-negative bacteria and on blood agar for S aureus.

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Whatman filter paper 6 mm discs containing 5 µL emulsion were placed on agar plates,

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incubated at 37 oC for 18-24 h and the diameter of the inhibition zone was measured. Since 5

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µL or 5 mg emulsion (density of emulsion is close to 1) was placed on each test disc, for the

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emulsion with 3% EO w/w the amount of EO per disc was 150 µg. Therefore, the 3% w/w

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reconstituted emulsion was further diluted to give a second with 0.6% w/w EO corresponding

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to 30 µg per disc, and hence the activity of reconstituted 0.6% w/w EO emulsion could be

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compared with that of neomycin control (30 µg per disc). The antimicrobial activity was then

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expressed as diffusion zone diameter relative to neomycin (%). Furthermore, in order to see if

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processing and encapsulating wall affected EO antimicrobial activity, a simple EO emulsion in

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1% w/w Tween 20 (29), was prepared and compared with the reconstituted emulsions. Broth dilution: Density of the bacterial cultures in log phase growth was adjusted to 0.5

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McFarland turbidity in MH broth corresponding to 1-2 x 108 cells mL-1. This was further

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diluted to 1x106 mL-1 and used within 30 min. Serial two-fold dilutions of a 30% w/w

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reconstituted emulsion in sterile water were prepared. Equal amount of bacterial suspension

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was added to each dilution, vortexed and incubated at 37 oC for 18-20 h. Growth of bacterial

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cells was followed from measurements of optical density (OD) (600 nm, UV-VIS 1700,

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Shimadzu, Japan). For the determination of minimum inhibitory concentration (MIC), the OD

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of test bacterial suspensions containing EO emulsion at different dilutions (or different EO

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concentrations) relative to the OD of a control bacterial suspension which did not contain EO

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was plotted against log (EO concentration). The Gompertz Equation (5) was subsequently

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fitted to the data (5, 30) and constants A, C and b were computed by non-linear regression

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(SigmaPlot 11.0, Systat Software).

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M is the log (EO concentration) at the inflexion point. MIC was calculated from Equation (6).

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OD = A + Cexp(-exp(b(x-M)))

MIC = 10(M+1/b)

(6)

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3. Results and discussion

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3.1 Characterization of the feed emulsions

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Physicochemical properties: The quality of the spray-dried EO depends on the

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physicochemical properties of the feed emulsions (31). These are presented in Table 1 and

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Figure 1. Electrical conductivity values (3.7 and 3.3 mS cm-1) confirmed that both 3% w/w

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and 6% w/w emulsions were of o/w type. The emulsification ability was primarily due to

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Arabic gum with its protein part resting on the interface and the carbohydrate part extending 11

ACCEPTED MANUSCRIPT into the aqueous phase (32). Zeta potential (ζ) was estimated as an indicator of emulsion

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stability. From Figure 1a it appears that ζ values followed normal distribution with means -

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24.0 mV and -23.3 mV (Table 1), which are within the previously reported range for

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emulsions stabilized with Arabic gum and are attributed to negatively charged carboxyl groups

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of amino acids and glucuronic acid residues on aminogalactane blocks (32, 33).

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From Figure 1b it appears that for both the low 3% w/w EO and the high 6% w/w

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emulsions the increase of shear stress with shear rate is nearly linear except for a small initial

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part below a shear rate of about 20 s-1, indicating predominantly Newtonian behavior. The

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viscosity of the high EO content emulsion was greater than the low content (73.3 cps vs 61.0

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cps, Table 1). Since both the low and high EO emulsions had similar mean droplet diameters

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(13.2 µm and 13.7 µm, Figure 1c-d and Table 1) the greater viscosity of the last is due to the

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greater number of dispersed droplets. Nevertheless, the higher viscosity of the 6% w/w

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emulsion did not impair the feeding of spray dryer.

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Stability: Back scattering (BS%) - distance profiles of the feed emulsions taken every 8

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h up to 72 h are presented in Figure 2. The position at higher BS% of the lines for the high EO

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content emulsion (Fig 2b) can be explained by their smaller mean droplet diameter (Table 1).

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In both graphs the shifting of the curves downwards to lower BS with time % implies increase

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in droplet diameter. Additionally, a minimum in BS% was formed near the bottom of the test

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tube (~7 mm) after 32 h, indicating slow migration of droplets to the top. An estimate of the

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migration rate was obtained from the slope of the linear plot of the BS% minimum with time

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(not shown). Migration rate values were 0.11 h-1 and 0.14 h-1 respectively for the low and the

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high content emulsions (Table 1).

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Overall, since noticeable changes in the BS% - time profiles of the emulsions appeared

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only after a long time of 32 h, the stability of the feed emulsions is considered satisfactory.

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Since the ζ values of the feed emulsions (-24.0 and -23.3 mV, Table 1) were less than -30 mV,

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ACCEPTED MANUSCRIPT accepted as minimum for purely electrostatic stabilization (34), the good stability of the

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emulsions should be also attributed to the steric carbohydrate layer of Arabic gum protruding

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into the aqueous phase (32).

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3.2 Characterization of spray-dried products

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3.2.1. Composition, retention and encapsulation efficiency

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In Table 2 the composition of unprocessed oregano essential oil (EO) and after its

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extraction from the spray-dried products is presented. Carvacrol content was found to be high

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(85.89%) in the unprocessed and even higher in the extracted EO (89.36% and 90.22% for

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SDP10 and SDP20 respectively). This is due to the relative decrease of the minor ingredients

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cymene, terpinene and other terpenes in the extracted EO (33). Since the molecular weights of

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EO ingredients did not vary significantly (from 134.21 to 150.22), the decrease should be

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attributed to the volatility of p-cymene, γ-terpine and other minor ingredients with

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considerably lower boiling points (between 177 °C and 182 °C compared to 238 °C of

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carvacrol, Table 2).

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3.2.2. Physicochemical characteristics

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Particle size distributions are depicted in Figure 3 and derived parameters are given in

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Table 3 as diameters D10, D50 and D90 corresponding to 10%, 50% and 90% of the distribution.

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The D50 values were 9.9 µm and 11.0 µm and span [(D90-D10)/D50] 2.0 and 1.9, for SDP10 and

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SDP20 respectively. These values are within the expected range for spray-dried particles

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prepared using small scale equipment and similar operating conditions (15, 35). The slightly

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smaller diameter of the SDP particles compared to the droplets of the corresponding feed

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emulsions (13.7 µm and 13.2 µm, Table 1) can be attributed to shrinkage during drying and

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removal of the external hydrated layer. The moisture contents were 3.7% and 3.4% for SDP10

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and SDP20 respectively (Table 3) as expected from similar polysaccharide based compositions

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(35). Furthermore, from Table 3 it appears that oil retention in SDP10 was higher (74.9%) than

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in SDP20 (64.3%). This can be explained by the greater wall material/EO ratio in SDP10,

297

resulting in a thicker wall, and thus offering better protection during spray drying (9).

298

Encapsulation efficiencies of both SDP10 and SDP20 were high (98.3% and 97.9% for SDP10

299

and SDP20 respectively). From the SEMs shown in Figure 4 it appears that the SDP particles consisted of single

301

units or agglomerates with a more or less round shape which is confirmed by their low aspect

302

ratios (1.295 and 1.269, Table 3). However, their surfaces were irregular due to the presence of

303

cavities as indicated by high roundness index values (1.944 and 1.695, Table 3). These cavities

304

are characteristic to spray-dried particles of encapsulated oils (35) and are associated with

305

localized pressures of vapour entrapped inside the drying droplets due to the high drying

306

temperature applied (180 oC). When the pressurized vapor escapes through the semipermeable

307

elastic polysaccharide wall, it causes localized collapse and the formation of cavities (9).

308

3.3. Processing of SDP into tablets

309

There is upcoming interest in developing tablet formulations of essential oils for better

310

stability and commercial benefits (36). Due to their porous structure, spray-dried powders are

311

compressible, and therefore suitable for processing into tablets, providing there is no oil

312

leakage during compression. On the other hand, the presence of high oil content is expected to

313

have a negative effect on compressibility, subsequently reducing the ability for tablet

314

formation (14). For the characterization of tablets intended for oral delivery the FDA has

315

provided guidance for the required test procedures and suitable terminology (USP Chapter

316

<1062>) (17). This was followed in the present study and the results of the required tests are

317

depicted in Figure 5 as compressibility, tabletability and ejectability plots of compression

318

pressure vs porosity, tensile strength and ejection pressure respectively for tablets with 10%

319

(SDT10) and 20% EO (SDT20). Plots of weight loss (leakage), elastic recovery and friability

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300

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ACCEPTED MANUSCRIPT 320

with compression pressure are also shown in the same figure, because these are considered

321

important characteristics of tablet quality. It was seen previously in the discussion of encapsulation efficiency that the

323

polysaccharide wall offered high encapsulation efficiency of EO in the SDPs (Table 3).

324

Therefore, it can be expected that the encapsulating wall forms a dense and coherent structure

325

able to resist stresses during compression, thus preventing EO leakage. This is confirmed in

326

Figure 5a where it can be seen that the %weight loss of tablets during compression was less

327

than 1% which practically means no leakage. It is interesting to notice that the %weight loss

328

decreased with increasing compression pressure implying greater impregnation and adhesion

329

of EO to wall materials at higher pressures.

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From the compressibility plot in Figure 5b it appears that a wider porosity range was

331

obtained for SDT10 (0.760 to 0.923) indicating high compressibility compared to SDT20

332

(0.633 to 0.824) which is due to the lower content of non-compressible EO, or higher content

333

of compressible wall material compared to SD20. The tabletability plots in Figure 5c show

334

that the tensile strength of tablets increased with compression pressure in the range 40-100

335

MPa. The highly compressible SDP10 powder formed strong tablets (SDT10) reaching tensile

336

strength of ~1.4 MPa (corresponding to 7 kg fracture load) at the highest applied pressure

337

whereas SDP20 formed weaker tablets reaching ~0.15 MPa (or 1 kg fracture load).

338

Nevertheless, examination by eye did not show any defects on the SDT20 tablets, and they

339

were strong enough to withstand shocks during the friability test giving acceptable weight loss

340

of <0.6% (Figure 5d) which is in compliance with the United States Pharmacopoeia (
341

1216>) (28). Furthermore, from Figure 5e it can be seen that the ejection pressure varied from

342

0.44 to 0.80 MPa for SDT10 and from 0.38 to 0.91 MPa for SDT20. The greater ejection

343

pressures of SDT20 at compressions greater than 80 MPa can be explained by their

344

considerably greater elasticity as seen in Figure 5f, resulting in higher wall friction forces.

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Overall, from the above results it can be said that tablets of spray-dried EO powder with

346

good mechanical strength, acceptable friability and practically no leakage can be produced at

347

compressions between 60 and 100 MPa for SDP10 and between 80 and 100 MPa for SDP20.

348

3.4 Redispersibility In Figure 6 transmittance (T%) – time plots of SDP powder and tablets (with or

350

without disintegrant) are presented. A rapid decrease of T% indicating fast emulsion

351

reconstitution is seen for the SDP powders within the first minutes. The final T% of SDP20 is

352

about 15% less than SDP10 which is due to the higher EO content of SDP20, forming greater

353

number of EO droplets. Reconstitution of the emulsions from SDT is seen to take place at

354

considerably slower rate compared to SDP. The T% - time curves of SDT showed an initial

355

linear decrease which corresponded to slow dissolution (macroscopically seen as melting).

356

This is followed by a slower T% decrease up to 90 min after which no further change was

357

noticed. The T% of SDT10 tablets reached the levels of SDP10 after about 60 min whereas the

358

T% of SDT20 approached constant levels slightly above SDP20 after 90 min.

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The faster reconstitution of emulsion from SDP powders is attributed to the large

360

surface area of SDP particles available to the reconstitution fluid immediately upon

361

immersion, allowing instant wetting of the encapsulating wall and emulsion reconstitution.

362

The delay seen in Figure 6 in the reconstitution curve of tablets is because liquid has to

363

penetrate the tablet matrix first before wetting the entire mass. Therefore, to overcome this

364

step disintegrants CCS, SSG were added in the SDTs by mixing with SDP before compression

365

(20, 21). Surprisingly, from Figure 6 it appears that there is no difference in the reconstitution

366

profiles of SDT regardless of the presence of disintegrant, indicating no effect. Similar

367

observations of lack of disintegrating action of CCS and SSG has been previously reported for

368

hydrophilic tablets containing lactose (37) and can be attributed to the softness of the wet

369

tablet matrix, disabling development of internal disruptive stresses.

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ACCEPTED MANUSCRIPT Microscopic analysis of the reconstituted emulsions from SDP10, SDP20, SDT10 and

371

SDT20 gave respectively mean diameters of: 10.2, 10.1, 9.0 and 8.8 µm respectively which

372

are slightly lower than those of the feed emulsions of 13.7 and 13.2 µm (Table 1). The zeta

373

potential values for the respective reconstituted emulsions were: -24.8±0.7, -21.8±0.4, -

374

24.3±0.7 and -21.6±0.3 mV which confirm the similarity of the reconstituted with the primary

375

feed emulsions.

376

3.5. In vitro release

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Release profiles are shown in Figure 7 for SDP10, SDP20 and the corresponding

378

tablets SDT10 and SDT20 with or without disintegrants. In all cases the tested SDP and SDT

379

samples corresponded to the same amount of EO (20 mg). A short lag phase is noticed in the

380

first 10 min for tablets. The %release increases with time and after 90 min reaches a plateau

381

for SDP10, SDP20, SDT10 and SDT20 with CCS, whereas for tablets without disintegrant or

382

with SSG is still increasing. In general, the formulations appear to fall into three groups of: i)

383

instant release SDP10 powder that released 90% EO in less than 30 min, ii) fast release group

384

of SDP20 and SDT tablets with CCS that released 90% in 60 min and iii) slow release group

385

of SDT tablets without disintegrant or with SSG that released 35-45% and 60% EO

386

respectively after 90 min testing time. The faster release of EO from the SDP10 compared to

387

SDP20 is attributed to the greater hydrophilic wall material/EO ratio resulting in faster wetting

388

and release. For tablets with CCS the release of EO was rapid regardless of EO content

389

(Figure 7), whereas for tablets with SSG or no disintegrant the release was slow and EO

390

content had small effect. Supplementary experiments indicated that the release was

391

independent of compression pressure in the range 60-100 MPa (results are not shown).

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377

392

One reason for the improved release rate of tablets with CCS is the fast water uptake of

393

CCS with each microfibril acting as a hydrophilic channel drawing liquid into the tablet by

394

wicking and swelling, thus accelerating wetting of the encapsulating material and re-

17

ACCEPTED MANUSCRIPT emulsification (37). A second reason that can be proposed is the development of repulsive

396

forces between the negatively charged carboxylic groups of the amino acids and glucuronic

397

acid of Arabic gum and the negatively charged carboxylic groups of CCS with known

398

reactivity in neutral pH (38). Addition of SSG gave only a small improvement of release rate

399

compared to CCS, which may be ascribed to the slower uptake of liquid in the tablet matrix

400

(39), and also to the lower density of carboxylic groups per repeating unit compared to CCS

401

(one carbonyl per repeating unit for SSG compared to three for CCS, Figure 9) (40), and less

402

repulsive forces.

403

3.6. Antimicrobial activity

404

3.6.1. Disc diffusion

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Typical disc diffusion zones obtained for the studied bacterial strains E coli, P

406

mirabilis, Klebsiella sp and S aureus using reconstituted emulsions from tablets of spray-dried

407

powder are presented in Figure 8. Reconstituted inert emulsions with only wall material

408

(without EO) applied to E coli was also included for comparison, and demonstrates absence of

409

inhibition zone. Therefore, the zones formed in the presence of EO are due to its antimicrobial

410

activity.

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The results of disc diffusion tests are given in Table 4 as zone diameters. At the 3%

412

w/w EO concentration the non-encapsulated formulation showed slightly higher antimicrobial

413

activity (compare rows B vs D): for E coli (diameter 11.50 vs 10.69 mm), for P mirabilis

414

(12.15 vs 10.28 mm), for Klebsiella sp (10.50 vs 9.61 mm) and for S aureus (9.50 vs 9.30

415

mm). Decreasing the EO concentration from 3.0% to 0.6 % w/w decreased the zone diameter

416

and the decrease was greater for the non-encapsulated resulting in overall lower activity

417

compared to the reconstituted encapsulated emulsion (compare rows A vs C): for E coli (7.78

418

vs 6.21 mm), for P mirabilis (6.50 vs 6.25 mm), for Klebsiella sp (7.70 vs 6.25 mm) and for S

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ACCEPTED MANUSCRIPT 419

aureus (6.79 vs 6.20 mm). Overall, from the above results it can be said that processing of EO

420

(spray drying and compression) had small effect on the antimicrobial activity. Antimicrobial activities (%) of the non-encapsulated and reconstituted (encapsulated)

422

EO emulsions relative to the standard neomycin antibiotic are also presented in Table 4. Both

423

emulsions exhibited antimicrobial activity (35.20-61.72% and 28.1-56.36% respectively of

424

neomycin) increasing in the order E coli < P mirabilis < Klebsiella sp < S aureus. The

425

reconstituted emulsion showed higher relative activity in the cases of E coli, Klebsiella sp and

426

S aureus which may be attributed to protection and preservation of the encapsulated EO active

427

ingredients during the disc diffusion test. The low relative activity for E coli compared to other

428

bacteria may be ascribed to the high response shown by the particular E coli strain used in the

429

study to neomycin (zone diameter of 22.1 mm) that could be associated with the fact that the

430

bacterial strains were clinical samples and individual differences in response to EO or

431

neomycin are possible. Overall, the results showed that the antibacterial activity of oregano

432

EO was retained in the tablets which is attributed to the protection provided by the wall

433

materials.

434

3.6.2. Broth dilution and data processing

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Inhibition profiles obtained by addition of re-constituted emulsion to bacterial cultures

436

are presented in Figure 10 as plots of fractional optical density (OD) against log concentration

437

EO. Additionally, the parameters A, C, b, of the Gompertz Equation (5) derived from non-

438

linear regression (Radj2>0.991) together with MIC values are presented in Table 5. Values of A

439

(lower asymptote to X-axis) approached 0.1 instead of zero because at high EO concentrations

440

there was some turbidity (OD) due to the presence of wall material in broth suspension. For

441

the same reason the values of C (OD difference between the upper and lower asymptote)

442

approached 0.9 instead of 1.

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ACCEPTED MANUSCRIPT The parameter b which is related to the slope of the sigmoidal curves (Figure 10) had

444

highest value for E coli (7.178) indicating rapid response and high sensitivity to EO, followed

445

by S aureus (5.149) and Klebsiella sp (4.419), whereas P mirabilis showed low value (2.653)

446

indicating low sensitivity to EO. MIC values (mg L-1) ranked as follows: E coli (119.8) < S

447

aureus (129.0) < P mirabilis (132.2)
448

comparable or lower than those reported for other wall materials and encapsulation methods

449

(41-43).

450

4. Conclusions

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Development of a tablet solid dosage form of encapsulated oregano EO with

452

polysaccharides is a robust 3-step process comprising preparation of stable feed emulsions,

453

spray drying and finally compression of the spray-dried powder into tablets. Tablets with

454

adequate strength could be made in the compression range 60-100 MPa with practically no oil

455

loss, adequate strength and very low friability. The release profile of EO from tablets prepared

456

from SDP with 10% or 20% w/w EO containing 5% w/w croscarmellose sodium was fast and

457

similar to the profile obtained from spray-dried powder with 20% EO content. Reconstituted

458

emulsion from EO tablets showed excellent antimicrobial activity that was comparable with

459

the non-encapsulated EO form. MIC values for Gram-negative and Gram-positive bacteria

460

were also comparable or lower than values reported in the literature, indicating that the

461

selected wall materials and applied processes of spray drying and compression did not alter

462

the antimicrobial activity. Therefore, since oregano oil is irritating to taste, encapsulation by

463

spray drying and processing into tablet formulation offers a promising dosage form for oral

464

delivery providing an accurate dose that can be delivered in a convenient way.

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Conflicts of Interest: The authors declare no conflict of interest.

5. References 20

ACCEPTED MANUSCRIPT 1.

21 CFR 182.20 - ESSENTIAL OILS, OLEORESINS (SOLVENT-FREE), AND

NATURAL EXTRACTIVES (INCLUDING DISTILLATES). 2012. 2. Franklyne, J.S.; Mukherjee, A.; Chandrasekaran, N. Essential oil micro- and nanoemulsions: promising roles in antimicrobial therapy targeting human pathogens. Letters in applied microbiology 2016, 63, 322-334.

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3. Béjaoui, A.; Chaabane, H.; Jemli, M.; Boulila, A.; Boussaid, M. Essential oil composition and antibacterial activity of Origanum vulgare subsp. glandulosum Desf. at different phenological stages. Journal of medicinal food 2013, 16, 1115-1120. 4. Burt, S.A.; Reinders, R.D. Antibacterial activity of selected plant essential oils against

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Escherichia coli O157:H7. Letters in applied microbiology 2003, 36, 162-167. 5. Lambert, R.J.; Skandamis, P.N.; Coote, P.J.; Nychas, G.J. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of applied microbiology 2001, 91, 453-462.

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6. Lopez-Romero, J.C.; Gonzalez-Rios, H; Borges, A.; Simoes, M. Antibacterial Effects and Mode of Action of Selected Essential Oils Components against Escherichia coli and Staphylococcus aureus. Evidence-Based Complementary and Alternative Medicine 2015, 2015, 1-9. 7. Katsoulos, P.; Karatzia, M.; Dovas, C.; Filioussis, G.; Papadopoulos, E.; Kiossis, E.; Arsenopoulos, K.; Papadopoulos, T.; Boscos, C.; Karatzias, H. Evaluation of the in-field efficacy of oregano essential oil administration on the control of neonatal diarrhea syndrome

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in calves. Research in Veterinary Science 2017, 115, 478-483. 8. da Costa, J.M.G.; Borges, S.V.; Hijo, A.A.C.T.; Silva, E.K.; Marques, G.R.; Cirillo, M.Â.; Azevedo, V.M.d. Matrix structure selection in the microparticles of essential oil oregano produced by spray dryer. Journal of Microencapsulation 2013, 30, 717-727.

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9. Fernandes, L.P.; Turatti, I.C.C.; Lopes, N.P.; Ferreira, J.C.; Candido, R.C.; Oliveira, W.P. Volatile Retention and Antifungal Properties of Spray-Dried Microparticles of Lippia sidoides Essential Oil. Drying Technology 2008, 26, 1534-1542. 10. Jafari, S.M.; Assadpoor, E.; He, Y.; Bhandari, B. Encapsulation Efficiency of Food Flavours and Oils during Spray Drying. Drying Technology 2008, 26, 816-835. 11. Reineccius, G.A. The Spray Drying of Food Flavors. Drying Technology 2004, 22, 1289-1324. 12. Cole, E.T.; Cade, D.; Benameur, H. Challenges and opportunities in the encapsulation of liquid and semi-solid formulations into capsules for oral administration. Advanced drug delivery reviews 2008, 60, 747-756. 13. Bakry, A.M.; Abbas, S.; Ali, B.; Majeed, H.; Abouelwafa, M.Y.; Mousa, A.; Liang, L. Microencapsulation of Oils: A Comprehensive Review of Benefits, Techniques, and Applications. Comprehensive Reviews in Food Science and Food Safety 2016, 15, 143-182.

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Hansen, T.; Holm, P.; Schultz, K. Process characteristics and compaction of spray-

dried emulsions containing a drug dissolved in lipid. International journal of pharmaceutics 2004, 287, 55-66. 15. Alvarenga Botrel, D.; Vilela Borges, S.; Victória de Barros Fernandes, R.; Dantas Viana, A.; Maria Gomes da Costa, J.; Reginaldo Marques, G. Evaluation of spray drying

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conditions on properties of microencapsulated oregano essential oil. International Journal of Food Science & Technology 2012, 47, 2289-2296. 16. Burt, S. Essential oils: their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology 2004, 94, 223-253.

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17. New USP Chapter <1062> Tablet Compression Characterization. 18. McNamee, B.F.; White, L.E.; O'Riordan, E.D.; O'Sullivan, M. Effect of partial replacement of gum arabic with carbohydrates on its microencapsulation properties. Journal of agricultural and food chemistry 2001, 49, 3385-3388.

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19. Zhu, F. Encapsulation and delivery of food ingredients using starch based systems. Food chemistry 2017, 229, 542-552. 20. Caramella, C.; Ferrari, F.; Bonferoni, M.C.; Ronchi, M. Disintegrants in Solid Dosage Forms. Drug Development and Industrial Pharmacy 1990, 16(17), 2561-2577. 21. Desai, P.M.; Liew, C.V.; Heng, P.W.S. Review of Disintegrants and the Disintegration Phenomena. 2016, 105, 2545-2555. 22. Matuschek, E.; Brown, D.F.; Kahlmeter, G. Development of the EUCAST disk

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diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 2014, 20, O255-266. 23. Lambert, R.J.; Pearson, J. Susceptibility testing: accurate and reproducible minimum

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inhibitory concentration (MIC) and non-inhibitory concentration (NIC) values. Journal of applied microbiology 2000, 88, 784-790. 24. van Den Dool, H.; Dec. Kratz, P. A generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography. Journal of Chromatography A 1963, 11, 463-471. 25. Masada, Y. Analysis of essential oils by gas chromatography and mass spectrometry; New York (N.Y.) : Wiley, 1976. 26. Adams, R.P.; Adams, R.P. Identification of essential oil components by gas chromatography, quadrupole mass spectroscopy; Carol Stream, 2001. 27. Fell, J.T.; Newton, J.M. Determination of tablet strength by the diametral-compression test. Journal of pharmaceutical sciences 1970, 59, 688-691. 28. USP Chapter <1216> TABLET FRIABILITY.

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Martin, M.J.; Trujillo, L.A.; Garcia, M.C.; Alfaro, M.C.; Muñoz, J. Effect of emulsifier

HLB and stabilizer addition on the physical stability of thyme essential oil emulsions. Journal of Dispersion Science and Technology 2018, 39, 1627-1634. 30. Mann, C.M.; Markham, J.L. A new method for determining the minimum inhibitory concentration of essential oils. Journal of applied microbiology 1998, 84, 538-544.

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31. Vicente, J.; Pinto, j.; Menezes, J.; Gaspar, F. Fundamental analysis of particle formation in spray drying. Powder Technology 2013, 247, 1–7. 32. Jayme, M.L.; Dunstan, D.E.; Gee, M. Zeta potential of gum arabic stabilized oil-inwater emulsions. Food Hydrocolloids 1999, 13, 459–465.

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33. Bouyer, E.; Mekhloufi, G.; Le Potier, I.; de Kerdaniel Tdu, F.; Grossiord, J.L.; Rosilio, V.; Agnely, F. Stabilization mechanism of oil-in-water emulsions by beta-lactoglobulin and gum arabic. Journal of colloid and interface science 2011, 354, 467-477. 34. Mirhosseini, H.; Tan, C.P.; Hamid, N.S.A.; Yusof, S. Effect of Arabic gum, xanthan

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gum and orange oil contents on ζ-potential, conductivity, stability, size index and pH of orange beverage emulsion. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008, 315, 47-56. 35. Toledo Hijo, A.A.C.; Costa, J.M.G.; Silva, E.K.; Azevedo, V.M.; Yoshida, M.I.; Borges, S.V. Physical and Thermal Properties of Oregano (Origanum vulgare L.) Essential Oil Microparticles. Journal of Food Process Engineering 2015, 38, 1-10.

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36. Ying, D.; Ling Ong, Y.; Jiang Cheng, L.; Sanguansri, L.; Shen, Z.; Augustin, M.A. Compressible extruded granules containing microencapsulated oil powders, 2015. 37. Zhao, N.; Augsburger, L. The influence of product brand-to-brand variability on superdisintegrant Performance. A case study with croscarmellose sodium, 2006.

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38. Huang, W.X.; Desai, M.; Tang, Q.; Yang, R.; Vivilecchia, R.V.; Joshi, Y. Elimination of metformin-croscarmellose sodium interaction by competition. International journal of pharmaceutics 2006, 311, 33-39. 39. Ekmekciyan, N.; Tuglu, T.; El-Saleh, F.; Muehlenfeld, C.; Stoyanov, E.; Quodbach, J.

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Competing for water: A new approach to understand disintegrant performance. International journal of pharmaceutics 2018, 548, 491-499. 40. Rowe, R.C.; Sheskey, P.J.; Cook, W.G.; Fenton, M.E. Handbook of pharmaceutical excipients - 7th edition, 2009. 41. Keawchaoon, L.; Yoksan, R. Preparation, characterization and in vitro release study of carvacrol-loaded chitosan nanoparticles. Colloids and surfaces. B, Biointerfaces 2011, 84, 163-171. 42. Moraes-Lovison, M.; F.P. Marostegan, L.; S. Peres, M.; F. Menezes, I.; Ghiraldi, M.; A.F. Rodrigues, R.; Fernandes, A.; Pinho, S. Nanoemulsions encapsulating oregano essential oil: Production, stability, antibacterial activity and incorporation in chicken pâté, 2016.

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Wang, Q.; Gong, J.; Huang, X.; Yu, H.; Xue, F. In vitro evaluation of the activity of

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microencapsulated carvacrol against Escherichia coli with K88 pili. Journal of applied microbiology 2009, 107, 1781-1788.

24

ACCEPTED MANUSCRIPT Legends to Figures

Figure 1. Physicochemical properties of primary feed emulsions (3% w/w EO in blue circles, 6% w/w EO in red squares): (a) zeta potential distributions; (b) shear stress vs shear rate plots; (c) optical microscopy images ((c1) low and (c2) high content); (d) cumulative oversize

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distributions from optical microscopy.

Figure 2. Back scattering (%) vs distance profiles of feed emulsions with (a) 3% w/w and (b) 6% w/w oregano essential oil taken every 8 h (time increases top to bottom).

Figure 3. Particle size frequency (%) distribution and cumulative undersize (%) distribution of

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the encapsulated oregano EO spray-dried products SPD10 (10% EO) and SPD20 (20% EO).

dried products SDP10 and SDP20.

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Figure 4. Scanning electron microphotographs of particles of encapsulated oregano EO spray-

Figure 5. Plots showing the compression behavior of SDP10 (black) and SDP20 (red) oregano EO powders during tableting.

Figure 6. Transmittance (T%) of reconstitution medium vs time for oregano EO formulations of spray-dried powder (black), tablets without disintegrant (blue), tablets with croscarmellose

20% w/w (squares) EO.

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sodium (red) and tablets with sodium starch glycolate (green) containing 10% w/w (circles) or

Figure 7. In vitro release profiles of oregano EO formulations of spray-dried powder (black),

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tablet without disintegrant (blue), tablets with croscarmellose sodium (red) and with sodium starch glycolate (green) containing 10% w/w (circles) or 20% w/w (squares) EO.

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Figure 8. Chemical structures of disintegrants. Figure 9. Disc diffusion zones obtained from reconstituted emulsions from tablets of spraydried EO for: E coli (a), P mirabilis (b), S aureus (c) and reconstituted emulsions from inert tablets with only wall material for E coli (d) showing absence of diffusion zone. Figure 10. Inhibition profiles presented as plots of fractional optical density (OD) vs logarithm of oregano EO concentration obtained by addition of two-fold serial dilutions of reconstituted emulsions from SDT10 tablets to bacterial cultures.

25

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Table 1. Physicochemical properties of feed emulsions with low (3% w/w) and high (6% w/w) essential oregano (EO) content that were used for the production of spray-dried EO powders. Property

Feed emulsions

.

6% w/w

Electrical conductivity (mS cm-1)

3.69±0.21

3.35±0.11

Viscosity (cps)

61.0±0.3

73.3±0.4

Mean droplet diameter (µm)

13.7

13.2

Zeta potential (mV)

-24.0±0.6

Migration rate (h-1)

0.11±0.022

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3% w/w

-23.3±0.4

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0.14±0.030

26

ACCEPTED MANUSCRIPT Table 2. Composition of unprocessed oregano essential oil (EO) and after extraction from the low (10%) (SDP10) or high (20%) (SDP20) content spray-dried powders together with oil retention and encapsulation efficiency (mean±SD, n=3).

substances

Boiling

Molecular Unprocessed EO

o

point ( C) weight

(%)

Extracted EO (%)

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Identified

SDP10

SDP20

238

150.22

85.89±0.06

89.36±0.06

90.22±0.64

p-Cymene

177

134.21

5.45±0.04

3.72±0.05

3.47±0.27

γ-Terpinene

182

136.23

3.06±0.02

Thymol

233

150.22

1.99±0.04

-

-

3.61±0.07

2.07±0.01

1.87±0.01

2.19±0.06

2.04±0.08

2.67±0.08

2.40±0.09

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Other terpenes

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Carvacrol

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Boiling points and molecular weights were taken from the literature (https://pubchem.ncbi.nlm.nih.gov)

27

ACCEPTED MANUSCRIPT Table 3. Particle size, shape, moisture content, oil retention and encapsulation efficiency of spray-dried EO with low (SDP10) or high (SDP20) oil content. Property

SDP10

SDP20

D10 D50

4.7 11.0

4.4 9.9

D90 Span

26.0 2.0

23.3 1.9

Particle Shape Aspect ratio Roundness index

1.295 1.944

1.269 1.695

SC

3.7±0.2 64.3±1.5 97.9±0.1

M AN U

3.4±0.2 74.9±0.9 98.3±0.1

AC C

EP

TE D

Moisture content (%) Oil retention (%) Encapsulation efficiency (%)

RI PT

Particle size (µm)

28

ACCEPTED MANUSCRIPT Table 4. Disc diffusion zone diameters obtained from reconstituted oregano EO emulsion (encapsulated form) and simple emulsion in Tween 20 (non-encapsulated form) at two EO concentrations 0.6% w/w and 3% w/w (mean ± standard error, n=3). Data from standard neomycin discs are included for comparison. Oregano EO Zone diameter (mm) in test disc (µg) ________________________________________ E coli P mirabilis Klebsiella sp S aureus

RI PT

Oregano EO in emulsion (w/w)

Disc diffusion 7.75 ± 0.21

B. Reconstituted emulsion 3.0%

150

10.75 ± 0.21

C. Tween 20 emulsion 0.6%

30

D. Tween 20 emulsion 3.0%

150

Neomycin disc

30

6.20 ± 0.08

7.40 ± 0.08

6.75 ± 0.21

10.25 ± 0.21

9.50 ± 0.42

9.25 ± 0.21

SC

30

M AN U

A. Reconstituted emulsion 0.6%

6.21 ± 0.01

6.25 ± 0.12

6.25 ± 0.12

6.20 ± 0.08

11.50 ± 1.23

12.15 ± 0.12

10.50 ± 0.42

9.50 ± 0.42

22.10 ± 0.47

11.20 ± 0.18 17.10 ± 0.47 11.00 ± 0.08

TE D

***

Relative antimicrobial activity (%)

P mirabilis

Klebsiella sp

S aureus

E. Reconstituted emulsions 0.6%

35.21

58.03

45.03

61.72

F. Tween 20 emulsion 0.6%

28.10

55.80

36.55

56.36

AC C

EP

E coli

29

ACCEPTED MANUSCRIPT Table 5. Parameters of the Gompertz equation describing inhibition profiles together with MIC values for the four bacteria tested (mean±SD, n=3) obtained from SDT10 reconstituted emulsion. C

b

M

Ra2

MIC (mg L-1)

E coli

0.038

0.870

7.178

2.078

0.998

119.8±3.3

Klebsiella sp

0.137

0.898

4.419

2.285

0.997

P mirabilis

0.131

0.885

2.653

2.120

0.998

S aureus

0.104

0.858

5.149

2.110

0.991

RI PT

A

193.0±7.5

132.2±3.5

129.0±4.7

AC C

EP

TE D

M AN U

SC

Bacteria

30

CC EP TE D

M AN U

SC

RI P

TE

D

M AN U

S

TE

D

M AN U

ED

M AN U

AC C EP TE D

SC

M AN U

RI PT

EP TE D

M AN U

SC

EP TE D

M AN U

SC

TE

D

M AN U

S

EP TE D

M AN U

SC

R

CE ED

PT

M AN U

SC

RI