P ocesses 2025, 13, 374 h ps://doi.o g/10.3390/p 13020374
A icle
NADES-in-Oil Emulsions En iched wi h Essen ial Oils o
Cosme ic Applica ion
Da id S. F ei as
1
, Diana Rocha
1
, Joana San os
1
, Jenni e No o
2
, Tânia D. Ta a es
3
, Ma a O. Teixei a
3
,
Daniela A aújo
4
, Joana Cas o
4
, Ca ina Almeida
4
, Sónia Sil a
1,4,5
, Helena P. Felguei as
3
, A u Ribei o
1,5
,
A mando Venâncio
1,5
, A u Ca aco-Paulo
1,5
and Ca la Sil a
1,5,
*
1
Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal;
da ids ei [email protected] (D.S.F.); [email protected] (D.R.); joanasan os587@ho mail.com (J.S.);
[email p o ec ed] (S.S.); a u ibei [email protected] (A.R.); a [email protected] (A.V.);
a u @deb.uminho.p (A.C.-P.)
2
I3Bs—Bioma e ials, Biodeg adables and Biomime ics Resea ch G oup, A epa k—Pa que de Ciência e
Tecnologia, Zona Indus ial da Gand a, Ba co, 4805-017 Guima ães, Po ugal; jenn[email p o ec ed]minho.p
3
Cen e o Tex ile Science and Technology (2C2T), Uni e si y o Minho, Campus de Azu ém,
4800-058 Guima ães, Po ugal; [email p o ec ed].p (T.D.T.);
ma aoli ei a eixei [email p o ec ed].p (M.O.T.); helena. elguei as@de .uminho.p (H.P.F.)
4
I.P—Na ional Ins i u e o Ag a ian and Ve e ina y Resea ch (INIAV), Rua dos Lagidos,
4485-655 Vila do Conde, Po ugal; daniela.a aujo@inia .p (D.A.); joana.cas o@inia .p (J.C.);
ca ina.almeida@inia .p (C.A.)
5
LABBELS—Associa e Labo a o y, 4710-057 B aga, Po ugal
* Co espondence: [email p o ec ed]o.p
Abs ac : This esea ch aims o explo e he po en ial bene i s o na u al deep eu ec ic
sol en s (NADES) in o mula ing anslucen NADES-in-oil emulsions (TEs) con aining
essen ial oils (EOs) o cosme ic applica ions. The TEs in es iga ed in his s udy a e based
on p e ious o mula ions, consis ing o 50 w .% egg phospha idylcholine (EPC), 20 w .%
e hanol, 20 w .% oli e oil (OlO), hyme oil (TO), o o egano oil (O O), and 10 w .% NADES
made om a 1:1 o 1:4 a io o lac ic acid and glyce ol (LA). These emulsions exhibi high
an ioxidan ac i i y, a ibu ed o he e penes p esen in he essen ial oils, such as hymol
in TO and ca ac ol in O O. The TEs con aining TO and O O demons a ed a mo e luid
consis ency, along wi h a mo e appealing ex u e and ag ance compa ed o he OlO
con ol. Addi ionally, hese emulsions exhibi ed he abili y o pe mea e pig skin, as well
as signi ican an i ungal and an ibac e ial ac i i y, and low oxici y in he Galle ia
mellonella la al model. O e all, he indings expand he po en ial applica ions o NADES,
pa icula ly in he de elopmen o anslucen emulsions wi h EO o ea ing mic obial
skin and nail in ec ions.
Keywo ds: na u al deep eu ec ic sol en s (NADES); anslucen NADES-in-oil emulsions;
essen ial oils; cosme ics
1. In oduc ion
Deep eu ec ic sol en s (DES) a e a ype o sol en s o med by he combina ion o wo
o mo e componen s ha became liquid a oom empe a u e. These sol en s ha e unique
p ope ies such as low apo p essu e, high conduc i i y, and unable iscosi y. DES ha e
gained a en ion in a ious indus ies due o hei eco- iendly na u e and po en ial
applica ions [1–7]. The special p ope ies o DES ha e made hem inc easingly popula in
he cosme ics indus y o e he yea s. They a e a choice o sus ainable cosme ic p oduc s
Academic Edi o : Ib ahim Abu-
Reidah
Recei ed: 13 Decembe 2024
Re ised: 20 Janua y 2025
Accep ed: 28 Janua y 2025
Published: 29 Janua y 2025
Ci a ion: F ei as, D.S.; Rocha, D.;
San os, J.; No o, J.; Ta a es, T.D.;
Teixei a, M.O.; A aújo, D.; Cas o, J.;
Almeida, C.; Sil a, S.; e al.
NADES-in-Oil Emulsions En iched
wi h Essen ial Oils o Cosme ic
Applica ion. P ocesses 2025, 13, 374.
h ps://doi.o g/10.3390/p 13020374
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/license
s/by/4.0/).
P ocesses 2025, 13, 374 2 o 21
because hey a e biodeg adable and en i onmen ally iendly [8]. Howe e , when
selec ing DES o he de elopmen o a cosme ic p oduc , ce ain ac o s mus be
conside ed. Despi e hei p omising p ope ies, he use o NADES in cosme ics p esen s
ce ain challenges, including hei ela i ely low s abili y in some o mula ions, po en ial
incompa ibili y wi h o he cosme ic ing edien s, and limi ed egula o y amewo ks ha
go e n hei use [8–10]. O e coming hese ba ie s equi es u he esea ch in o he long-
e m efficacy and sa e y o NADES-based p oduc s. The e o e, i is necessa y o examine
he long- e m efficacy and eliabili y o DES in speci ic applica ions, as well as i s po en ial
incompa ibili y wi h ce ain cosme ic ing edien s [8,11].
The use o he na u al e sion o DES (NADES), in which all cons i uen s a e o
na u al o igin, could make he use o hese sol en s in he cosme ic indus y e en mo e
appealing, pa icula ly in p oduc s bea ing he sus ainable seal. Cu en ly, he e a e
epo ed s udies ha add ess he use o NADES in lo ions, c eams, se ums, emulsions,
shampoos, among o he s [8,11]. Vasylie e al. [12] p oposed NADES composed o choline
chlo ide: 1,2-p opanediol o lac ic acid as p omising sol en s o ex ac ing an ioxidan
bioac i e compounds om oma o pomace. This con i med hei applica ion in cosme ic
o mula ions. This s udy led o he de elopmen o a na u al oil-in-wa e emulsion
o mula ion ha includes he di ec applica ion o NADES con aining he na u al ex ac
[12]. Ano he s udy p oposed he emo al o co k ex ac i es wi h NADES and he di ec
applica ion o he ex ac s (con aining NADES) in cosme ic o mula ions. The
o mula ions p oduced showed high an ioxidan ac i i y and did no demons a e oxic
effec s on ke a inocy e cells [13].
Recen ly, we designed a s udy add essing he use o NADES (wi h o wi hou
ex ac ) o c ea e he i s anslucen NADES-in-oil emulsions (TEs) p oposed o
cosme ic pu poses [14]. The NADES used in his s udy we e composed o lac ic acid and
glyce ol (LA:GLY) in a ios 1:1 and 1:4. TEs we e p epa ed by simply mixing 50 w .% Egg-
phospha idylcholine (EPC), 20 w .% e hanol, 20 w .% oli e oil, and 10 w .% LA:GLY (wi h
o wi hou ex ac ). These TEs demons a ed ema kable an imic obial capaci y, which,
coupled wi h hei low oxici y, enhances hei po en ial o pha maceu ical and cosme ic
applica ions. The de elopmen o new TEs offe s dis inc ad an ages o e adi ional
emulsions in cosme ic applica ions. These emulsions p o ide imp o ed s abili y,
enhanced inco po a ion o bioac i e compounds, and supe io an imic obial and
an ioxidan p ope ies, making hem a p omising candida e o eco- iendly cosme ic
o mula ions [14]. Despi e hei po en ial, challenges emain in he long- e m s abili y o
NADES-based o mula ions, hei compa ibili y wi h o he ing edien s, and he need o
u he egula o y app o als in cosme ic applica ions. Thus, unde s anding and
o e coming hese ba ie s is essen ial o hei widesp ead adop ion [8,10].
In he cu en s udy, we elied on p e iously published TEs composi ion as a s a ing
poin and eplaced oli e oil wi h essen ial oils om Thymus ulga is and O iganum ulga e
aiming o mainly inc emen hei an imic obial effec . The essen ial oils we e selec ed due
o hei po en an imic obial and an ioxidan p ope ies, as well as hei high con en o
phenolic compounds, such as hymol and ca ac ol, which ha e been shown o effec i ely
comba skin- ela ed pa hogens [15]. The cha ac e iza ion o essen ial oils was ca ied ou
using LC-MS (liquid ch oma og aphy–mass spec ome y) and GC-MS (gas
ch oma og aphy–mass spec ome y) echniques, alongside he assessmen o phenolic
and la onoid con en , chela ing powe , and an ioxidan ac i i y. This s udy also
e alua es he iscosi y, ex u e, and o ganolep ic p ope ies o he p oduced TEs.
Addi ionally, he pe mea ion le els in po cine skin, as well as he an i-elas ase and an i-
y osinase ac i i ies o he TEs, we e in es iga ed as po en ial cosme ic and
pha maceu ical candida es. The an imic obial p ope ies o bo h essen ial oils and TEs
we e assessed agains a ange o mic obial s ains commonly associa ed wi h skin and nail
P ocesses 2025, 13, 374 3 o 21
diseases. Finally, he oxici y o TEs was analyzed using an in i o la al model (Galle ia
mellonella). The p ima y objec i e o his s udy was o de elop and cha ac e ize
anslucen NADES-in-oil emulsions (TEs) inco po a ing hyme and o egano essen ial
oils, wi h a ocus on hei an imic obial, an ioxidan , and ansde mal pe mea ion
p ope ies, o assess hei po en ial as sus ainable cosme ic and pha maceu ical
candida es.
2. Ma e ials and Me hods
2.1. Ma e ials
The ollowing chemicals and ma e ials we e used in he s udy: DL-Lac ic acid (80%),
e hanol (99.8%), dime hyl sul oxide (DMSO) (99.9%), glyce ol (99%), sodium dodecyl
sul a e (SDS) (99%), Folin–Ciocal eu eagen , sodium ni a e (98%), sodium ca bona e
(99%), anhyd ous sodium hyd oxide (99%), que ce in (99%), gallic acid (99%), 1,1-
diphenyl-2-pic ylhyd azyl (DPPH) (99%), 2,2′-azino-bis(3-e hylbenzo hiazoline-6-
sul onic acid) diammonium sal (ABTS) (99%), T olox (99%), 2,4,6-T is(2-py idyl)-s-
iazine (TPTZ) (98%), po assium pe sul a e (98%), hyd ochlo ic acid (37%), i on (II)
chlo ide (98%), i on (III) chlo ide (98%), EDTA (98%), Fe ozine (98%), kojic acid (99%),
and L-α-phospha idylcholine egg yolk (EPC) (95%) we e ob ained om Me ck (Ge many)
o TCI Chemicals (Eu ope) and used wi hou u he pu i ica ion. Addi ional ma e ials
included oli e oil Pingo Doce (Pingo Doce – Dis ibuição Alimen a , S.A., Lisboa,
Po ugal), essen ial oils om Thymus ulga is and O iganum ulga e (Can inho das
A omá icas®, Vila No a de Gaia, Po ugal), coconu oil (dōTERRA®, Pleasan G o e, UT,
USA), and a ious mic obiological cul u e media including yp icase soy b o h (TSB),
yp icase soy aga (TSA), nu ien b o h (NB), and nu ien aga (NA) (VWR, Radno , PA,
USA), Müelle –Hin on b o h (MHB) (CondaLab, Spain), and po a o dex ose aga (PDA)
(F ilabo, Maia, Po ugal). Bac e ial s ains used in he s udy, including S aphylococcus
au eus (ATCC 6538), S aphylococcus epide midis (ATCC 35984), Esche ichia coli (ATCC
25922), and Klebsiella pneumoniae (ATCC 4352), we e sou ced om he Ame ican Type
Cul u e Collec ion (ATCC, USA). Fungal s ain T ichophy on men ag ophy es (MUM 0808)
was ob ained om he Mico eca da Uni e sidade do Minho (MUM, B aga, Po ugal).
Galle ia mellonella la ae we e aised in da kness on a die o pollen g ains and beeswax,
eaching app oxima ely 250 mg in weigh .
2.2. Me hods
2.2.1. NADES P epa a ion
NADES DL-lac ic acid:glyce ol was p epa ed in mola p opo ions o 1:1 and 1:4
acco ding o he me hod desc ibed by F ei as e al. [14,16]. The NADES we e p epa ed by
igo ously mixing he cons i uen s a empe a u es be ween 25 °C and 100 °C. Wi hin one
hou , a clea solu ion was o med. The eu ec ic mix u e was hen s o ed a oom
empe a u e o u he use.
2.2.2. T anslucen NADES-in-Oil Emulsions (TEs) P epa a ion
TEs we e p epa ed a oom empe a u e (20 °C) by combining 50 w .% o EPC wi h
20 w .% e hanol un il a hick liquid was ob ained (a ound 5 min). Then, 20 w .% o ei he
oli e oil, hyme oil, o o egano oil was inco po a ed and s i ed un il he mix u e achie ed
a uni o m ex u e (a ound 5 min). Las ly, 10 w .% o NADES was added and mixed o 5
min un il he TE was ully o med (Table 1) [14].
P ocesses 2025, 13, 374 4 o 21
Table 1. Essen ial oils and anslucen emulsion (TE) abb e ia ions.
Essen ial Oil Abb e ia ion TE-Oil
Oli e oil OlO TE-OlO
Thyme oil TO TE-TO
O egano oil O O TE-O O
Thyme:o egano oil (50:50
w .%) TO:O O TE-TO:O O
2.2.3. To al Phenolic Con en
The o al phenolic con en in oils and TE was measu ed using he Folin–Ciocal eu
eagen , op imized o a 96-well pla e o ma [13,14,17]. B ie ly, 2 µL o he sample (1
mg/mL in DMSO) was mixed wi h 10 µL o Folin–Ciocal eu eagen , ollowed by 120 µL
o dis illed wa e and agi a ion. A e one minu e, 40 µL o 15% Na2CO3 and 28 µL o wa e
we e added, and abso bance a 750 nm was eco ded wi h a mic opla e eade (BioTek,
SYNERGY H1M2). The o al phenolic con en was calcula ed using a gallic acid calib a ion
cu e (0.001–1 mg/mL) and exp essed as mg GAE pe g am o sample.
2.2.4. To al Fla onoid Con en (TFC)
The TFC o oils and TEs was measu ed using a me hod adap ed o a 96-well pla e,
ollowing Zhishen e al. [14,18]. To each well, 10 µL o sample, 80 µL o wa e , and 3 µL
o 10% NaNO2 we e added and incuba ed o 5 min. Then, 3 µL o 20% AlCl3, 40 µL o 1
M NaOH, and 64 µL o wa e we e added. Abso bance was measu ed a 510 nm using a
BioTek SYNERGY H1M2 mic opla e eade (Agilen , San a Cla a, CA, USA). TFC was
quan i ied using a que ce in s anda d cu e and exp essed as mg QE pe g am o sample.
2.2.5. An ioxidan Ac i i y
DPPH Assay
The ee adical sca enging ac i i y o oils and TEs was measu ed using he DPPH
assay adap ed o a 96-well pla e [13,14,17]. To 10 µL o sample (0.001–1 mg/mL in DMSO),
140 µL o DPPH solu ion (400 µM in e hanol) was added, and abso bance a 515 nm was
moni o ed o 60 min. T olox se ed as a s anda d, and DMSO was used as a blank. The
DPPH educ ion pe cen age (DPPH ) was calcula ed acco ding o Equa ion (1), whe e A
deno es he abso bance eco ded a s eady s a e, and Ai signi ies he ini ial abso bance
measu ed a ime = 0 min.
𝐷𝑃𝑃𝐻𝑟 (%) = Ai − A
Ai × 100. (1)
The EC50 alue was de e mined om he DPPH cu e, he TEC50 deno es he du a ion
equi ed o achie e a s eady-s a e discolo a ion a he EC50 concen a ion, and an i adical
efficiency (AE) was calcula ed as:
𝐴
𝐸= 1
EC50 ×TEC50. (2)
The an ioxidan ac i i y was exp essed as T olox equi alen s (TE) pe g am o
sample:
𝑇𝐸 = EC50 T olox
EC50 Sample. (3)
ABTS Assay
P ocesses 2025, 13, 374 5 o 21
The ABTS•+ clea ance assay was conduc ed as pe Re e al. [19], wi h some
modi ica ions [14]. A solu ion o 7.4 mM ABTS and 2.6 mM po assium pe sul a e was
p epa ed and le o eac in he da k o 12–16 h. The ABTS•+ solu ion was dilu ed o an
abso bance o 0.7 ± 0.1 a 734 nm. Samples (0.01–1.0 mg/mL in DMSO) we e mixed wi h
140 µL o ABTS•+ solu ion and incuba ed o 6 min in he da k. Abso bance was measu ed
a 734 nm. The inhibi ion a e was calcula ed as:
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (%) = A0 − Ai
A0 × 100, (4)
whe e A0 and Ai a e he abso bances o he con ol and sample, espec i ely. T olox was
used as he s anda d.
Fe ic Reducing An ioxidan Powe (FRAP) Assay
The FRAP assay was used o assess he educing powe o oils and TEs [14,17]. The
FRAP eagen was p epa ed by mixing FeCl3, TPTZ, and ace a e buffe in a 10:1:1 a io.
To 10 µL o sample (0.01–1 mg/mL), 290 µL o he eagen was added. A e 10 min a 37
°C in he da k, he abso bance a 593 nm was measu ed using a BioTek SYNERGY H1M2
mic opla e eade . Resul s we e exp essed as mg Fe2+ pe g am o sample.
2.2.6. Chela ing Powe
The e ous ion chela ion o oils and TEs was measu ed using a modi ied me hod
om Bouna i ou e al. [14,20,21]. Samples (0.1–1 mg/mL) we e mixed wi h 2 mM FeCl2
and 5 mM e ozine, and a e 10 min, abso bance a 562 nm was eco ded using a BioTek
SYNERGY H1M2 mic opla e eade . E hanol was used as he nega i e con ol, and EDTA
as he posi i e con ol. Chela ing capaci y was calcula ed using he ABTS me hod and
exp essed as EDTA equi alen s pe olume o sample, wi h iplica e measu emen s.
2.2.7. Ty osinase Inhibi ion Assay
Ty osinase inhibi ion by oils and TEs was measu ed ollowing a modi ied Di Pe illo
e al. [14,22] me hod. To 30 µL o y osinase (1000 U/mL), 70 µL o sample (5–100 µg/mL
in DMSO) and 110 µL o 2 mM L- y osine we e added and incuba ed o 30 min.
Abso bance a 492 nm was eco ded using a BioTek SYNERGY H1M2 mic opla e eade .
Kojic acid was used as a posi i e con ol. Inhibi ion was calcula ed as:
𝑇𝑦𝑟𝑜𝑠𝑖𝑛𝑎𝑠𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = A−B
A× 100, (5)
whe e A and B ep esen he abso bance wi hou and wi h he sample, espec i ely.
2.2.8. Elas ase Inhibi ion Assay
Elas ase inhibi ion by oils and TEs was assessed ollowing a modi ied me hod om
Ndlo u e al. [14,23]. To 25 µL o elas ase (0.3 U/mL), 50 µL o sample (0.25–250 µg/mL in
100 mM HEPES buffe , pH 7.5, wi h 2.5% DMSO) was added and incuba ed o 20 min.
Then, 50 µL o N-Me hoxysuccinyl-Ala-Ala-P o-Val-p-ni oanilide (1 mM) was added,
and he abso bance a 405 nm was measu ed a e 40 min a 25 °C. Kojic acid was used as
a posi i e con ol. The IC50 alue was calcula ed by plo ing elas ase inhibi ion agains
concen a ion.
2.2.9. Iden i ica ion o Compounds P esen in Essen ial Oils
Gas Ch oma og aphy–Mass Spec ome y (GC-MS)
The essen ial oils we e de i a ized p io o GC-MS analysis using anses e i ica ion
wi h py idine and BSTFA. A 100 µL sample (1 mg/mL in py idine) was combined wi h
P ocesses 2025, 13, 374 6 o 21
100 µL o BSTFA and incuba ed a 60 °C o 15–25 min. The p epa ed sample was hen
injec ed in o he GC-MS sys em. GC-MS analysis was pe o med on a SCION SQ1/436 GC
(B uke , Bille ica, MA, USA) wi h a Rxi-5Sil MS column (Res ek; 30 m, 0.25 mm ID, 0.25
µm ilm hickness) unde he ollowing condi ions: o en empe a u e held a 70 °C o 1
min, hen amped a 5 °C/min o 260 °C, ollowed by an ins an aneous inc ease o 300 °C.
Injec o and de ec o empe a u es we e se a 280 °C and 270 °C, espec i ely. Helium
was used as he ca ie gas a 1.0 mL/min. Da a we e collec ed in elec on impac (EI) mode
a 70 eV o e a mass ange o 35–600 m/z, wi h a scan ime o 250 ms. Compound
iden i ica ion was based on mass spec a om Wiley and NIST/EPA/NIH lib a ies and
published spec a. The ela i e abundance o compounds was de e mined om he o al
ion ch oma og am [16].
Liquid Ch oma og aphy–Mass Spec ome y (LC-MS)
The compounds in he oils we e iden i ied using a The mo Scien i ic™ Vanquish™
Flex UHPLC sys em connec ed o a The mo Scien i ic™ O bi ap Explo is™ 120 high-
esolu ion mass spec ome e (The mo Scien i ic™, Wal ham, MA, USA). Da a analysis
was pe o med wi h Xcalibu 4.5 so wa e. A YMC-T ia C18 UHPLC column (YMC,
Law ence, MA, USA) (150 × 2.1 mm, 3 µm) was used, p o ec ed by a gua d column o he
same ma e ial. The app oach was p e iously documen ed [14,24], albei wi h ce ain
a ia ions. The mobile phases we e wa e wi h 0.1% o mic acid (A) and ace oni ile wi h
0.1% o mic acid (B). The g adien sys em an a a low a e o 0.35 mL/min wi h he
ollowing pa ame e s: 0 min, 95% A; 5.4 min, 85% A; 7.9 min, 80% A; 9 min, 80% A; 12.6
min, 70% A; 16.2 min, 50% A; 18 min, 5% A; 19.8 min, 95% A; 22 min, 95% A. The column
was se a 30 °C, he au osample a 15 °C, and he injec ion olume was 10 µL. A Hea ed
Elec osp ay Ioniza ion (HESI) p obe was used o ioniza ion, wi h he ion sou ce se a
350 °C, capilla y empe a u e a 325 °C, and elec osp ay ol age a 3.5 kV (posi i e mode)
o 2.5 kV (nega i e mode). Shea h and auxilia y gases we e se a 50 and 10, espec i ely.
Da a we e collec ed in Full-scan mode and da a-dependen MS2 (dd-MS2). The Q-
O bi ap was calib a ed weekly o 3 ppm mass accu acy, wi h MS2 spec a gene a ed by
agmen a ion using no malized collision ene gy (NCE) a 30%, 60%, and 90%. The
esolu ion was se o 30,000 FWHM o ull scans and 15,000 o MS2.
2.2.10. Cha ac e iza ion and S abili y o TEs
TEs O ganolep ic P ope ies
The o ganolep ic p ope ies (pH, odo , appea ance, and colo ) o he TE we e
e alua ed a 25 °C o a pe iod o 120 days. Pe iodic isual inspec ions we e conduc ed o
assess hese quali ies.
TEs Accele a ed S abili y
The accele a ed s abili y o he TE was assessed using a cen i uge es o 5 g o
ma e ial a 3000 pm o e 5 days, du ing which he empe a u e luc ua ed be ween −20
°C and 25 °C, changing e e y 24 h, commencing a −20 °C. Upon comple ion o
cen i uga ion, he TE we e analyzed o phase sepa a ion.
TEs Size, Dispe si y, and Cha ge
The size and su ace cha ge, exp essed as ze a po en ial, along wi h he dispe si y o
he TE, we e assessed using dynamic ligh sca e ing (DLS) equipmen NANO ZS Mal e n
Ze asize (Mal e n Panaly ical L d., Mal e n, UK) a 25 °C. Fo size measu emen , PDI
and ze a po en ial 5 w .% TE we e dispe sed in e hanol. Six independen measu emen s
P ocesses 2025, 13, 374 7 o 21
we e pe o med o each sample. Mal e n dispe sion echnology so wa e (DTS) was used
wi h mul iple na ow modes (high esolu ion) da a p ocessing.
TEs Tex u e E alua ion
The mechanical consis ency o he TE was measu ed using back ex usion wi h a
ex u e analyze (STABLE MICRO SYSTEMS, Model: TA-HDPlus, Godalming, UK) [14].
A 3 mL sample was placed in he p obe (A/BE Back Ex usion Ring (90° cone)), and
i mness and sp eadabili y we e assessed a 10 mm/s speed, 60 mm dep h, and 10 g con ac
o ce. The p obe e u ned o i s o iginal posi ion a e each measu emen .
TEs Viscoelas ic P ope ies
The iscoelas ic p ope ies o he TEs we e e alua ed using a Disco e y HR1
heome e (TA Ins umen s, New Cas le, DE, USA) wi h a 40 mm pa allel pla e [14]. Shea
iscosi y was measu ed a empe a u es om 20 o 60 °C, using h ee low amps: 0.1 o
300 1/s, held a 300 1/s, and 300 o 0.1 1/s.
The mog a ime ic Analysis (TGA)
TGA was conduc ed on TEs (8–10 mg) using a TGA 4000 (Pe kin Elme , Wal ham,
MA, USA) wi h 13.0 Py is so wa e. Samples we e hea ed om 25 o 600 °C a 10 °C/min
in a ni ogen a mosphe e (20 mL/min). Weigh loss and i s de i a i e we e plo ed agains
empe a u e, wi h calib a ion using Cu ie empe a u es o e e ence ma e ials [14,25].
2.2.11. Skin Pe mea ion S udies
An ex i o pe mea ion s udy was conduc ed h ough ull- hickness pig skin using a
F anz diffusion cell (9 mm o i ice diame e , V-Se ies S i e s o F anz Cells, Pe meGea ,
Helle own, PA, USA) [13,14]. The ecep o compa men con ained 5 mL o 16 mM SDS
solu ion, agi a ed wi h a magne ic s i e a 37 ± 1 °C. App oxima ely 0.5 mL o TE was
applied o he skin. Samples om he ecep o solu ion we e collec ed a 1 o 8 h in e als
and eplaced wi h esh SDS solu ion. The samples we e analyzed spec ally be ween 230
and 750 nm, wi h maximum abso p ion a 270 nm. The concen a ion in he ecep o
compa men was de e mined om he abso bance a λmax. Pe mea ion pe cen age was
calcula ed using:
𝑃𝑒𝑟𝑚𝑒𝑎𝑡𝑖𝑜𝑛 (%) = C e
Ce × 100, (6)
whe e C e is he concen a ion in he ecep o and Ce is he ini ial concen a ion in he
dono (0.1 g/g). Pe mea ion s udies o TEs in pig skin we e pe o med in six independen
expe imen s o ensu e s a is ical obus ness and eliabili y o he esul s.
2.2.12. An imic obial Ac i i y
The an ibac e ial ac i i y o he oils and TEs was e alua ed agains G am-posi i e
bac e ia S. au eus and S. epide midis, as well as G am-nega i e bac e ia E. coli and K.
pneumoniae. Zone o inhibi ion (ZoI) assays, based on he Ki by–Baue me hod [26], we e
pe o med o measu e he an ibio ic effec i eness o each sample. The bac e ial
suspension was p epa ed o a concen a ion o 1 × 107 CFU/mL in TSB (S. au eus, S.
epide midis, and E. coli) o NB (K. pneumoniae), and pla ed on TSA (S. au eus, S. epide midis,
and E. coli) and NA (K. pneumoniae) pla es. 50 µL o each sample we e placed in a 6 mm
ca i y a he cen e o he aga pla es, which we e incuba ed a 37 °C o 24 h. A e
incuba ion, he esul ing ZoI, indica ed by clea a eas o aga whe e bac e ia g ow h was
inhibi ed, was pho og aphed and measu ed. The expe imen was pe o med in iplica e
and epea ed a leas h ee imes o each sample.
P ocesses 2025, 13, 374 8 o 21
The quan i a i e an ibac e ial ac i i y was e alua ed using he mic odilu ion
me hod, adap ed om CLSI and EUCAST s anda ds [27]. Bac e ial inocula we e p epa ed
in TSB (S. au eus, S. epide midis, and E. coli) and NB (K. pneumoniae), espec i ely, and
incuba ed o e nigh a 37 °C wi h shaking a 120 pm. In a 96-well pla e, 50 µL o each
sample was mixed wi h 50 µL o bac e ial suspensions (1 × 106 CFU/mL) in MHB. TEs
we e used a 100 w .%, while oils we e applied a a 20 w .% solu ion a pH 5, consis en
wi h he inal o mula ion concen a ion. Con ols included bac e ial suspensions wi hou
he agen and cul u e medium. Abso bance a 600 nm was measu ed be o e (0 h) and a e
24 h o incuba ion a 37 °C. Se ial dilu ions o he bac e ial suspensions we e pla ed on
TSA o NA and incuba ed o e nigh a 37 °C. Viable bac e ial colonies we e coun ed, and
he esul s we e exp essed as a pe cen age compa ed o he con ol suspensions.
The an i ungal ac i i y o he oils and TEs was es ed agains T. men ag ophy es. The
ungus was ini ially cul u ed on 2 mL o sloping PDA and incuba ed a oom empe a u e
o 7 days. A e adding 3 mL o PBS, he cell densi y was adjus ed o an op ical densi y
(OD) o 1.0 a 620 nm. The p epa ed ungal suspension was hen sp ead on o PDA pla es,
and 50 µL o each sample was placed in a 6 mm well a he cen e o he pla es. The pla es
we e incuba ed a 37 °C o 4 days. Zones o inhibi ion (ZoI) we e measu ed, wi h
anslucen a eas a ound he wells indica ing ungal g ow h inhibi ion. All expe imen s
we e pe o med in iplica e wi h a leas h ee independen ials.
2.2.13. Galle ia mellonella Assays
The heal h and melaniza ion indices o G. mellonella by oils and TEs we e e alua ed
ollowing he me hod p oposed by F ei as e al. [14]. B ie ly, he effec o oils and TEs on
he heal h index o G. mellonella was e alua ed by applying 5 µL o each sample (oils a 20
w .% in coconu oil and TEs a 100 w .%) o he do sal su ace o la ae. A con ol g oup
ecei ed an equi alen olume o wa e . Ten la ae pe condi ion we e used, and he
la ae we e kep in he da k a 37 °C o 24 h, wi h he ea men epea ed daily o 72 h.
The heal h index was based on la al ac i i y, cocoon p oduc ion, melaniza ion, and
su i al a e [28]. The impac o TEs on melaniza ion was assessed by selec ing la ae
wi h high melaniza ion and applying he TE solu ions e e y 24 h o 72 h. The s udy
ollowed e hical guidelines, ocusing on minimizing animal use and ensu ing s a is ically
meaning ul esul s [29–31].
2.2.14. S a is ical Analysis
S a is ical analysis was pe o med o compa e he esul s among he diffe en TEs.
All analy ical me hods used in his s udy we e pe o med a leas in iplica e. Da a we e
exp essed as he mean ± s anda d de ia ion (SD) o each o mula ion g oup. Fo
compa ison o means, one-way analysis o a iance (ANOVA) was used, ollowed by a
pos hoc Tukey’s es o de e mine signi ican diffe ences be ween he o mula ions. A
signi icance le el o p < 0.05, p < 0.01, p < 0.001, o p < 0.0001 was conside ed s a is ically
signi ican . All s a is ical analyses we e pe o med using O igin 2018.
3. Resul s and Discussion
3.1. TE P epa a ion and Chemical Composi ion
In he p esen in es iga ion, anslucen NADES-in-oil emulsions (TEs) con aining
EOs we e de eloped employing NADES composed o lac ic acid and glyce ol (LA:GLY)
in 1:1 and 1:4 a ios, wi h a pH 5. This o mula ion has been p e iously epo ed; howe e ,
in his s udy, oli e oil was eplaced by essen ial oils om Thymus ulga is and O iganum
ulga e. The oli e oil o mula ion was also p epa ed as a con ol. Simila ly o p e ious
P ocesses 2025, 13, 374 9 o 21
esea ch, hese TEs can be p oduced on a la ge scale and in quan i ies ha can mee
indus ial demands [14]. Table 1 p o ides he abb e ia ions employed in his s udy.
The cha ac e iza ion o he essen ial oils and hei espec i e TEs is c ucial, as i helps
iden i y he p ope ies ha make hem sui able o cosme ic applica ions. To achie e his,
measu emen s o phenolic con en , la onoid con en , chela ing powe , and an ioxidan
ac i i y we e ob ained (Table 2).
As expec ed, bo h he oils and hei espec i e TEs exhibi ed ela i ely low le els o
phenolics and la onoids. Howe e , a no able diffe ence was obse ed be ween he oils:
oli e oil (OlO) showed signi ican ly lowe phenolic and la onoid con en s compa ed o
hyme (TO) and o egano (O O) essen ial oils. This end emained consis en h oughou
he TEs, whe e emulsions con aining oli e oil demons a ed lowe phenolic and la onoid
le els han hose con aining hyme o o egano oils.
The chela ing capaci y o he oils, which e lec s hei abili y o bind me al ions ha
can cause damage o he skin and nails, was also e alua ed (Table 2) [13,17,32,33].
Acco ding o p e ious esea ch, NADES wi h a highe lac ic acid concen a ion (1:1 a io)
ha e a g ea e chela ing capaci y because he molecule can o m a biden a e bond,
esul ing in he o ma ion o I on (II) lac a e. The chela ing impac om LA:GLY 1:4 is he
lowes because glyce ol’s abili y o chela e i on is lowe ed. This e idence explains why
TEs wi h a 1:1 NADES a io has highe chela ing powe han hose wi h a 1:4 a io. The
chela ing powe o oli e, hyme, and o egano oils is compa able, wi h alues anging om
25.2 o 28.6 mgEDTA/gex ac .
The an ioxidan p ope ies o oils and essen ial oils (EOs) we e assessed o p edic
hei po en ial use in an i-aging and nail p o ec ion p oduc s. An ioxidan capaci y was
measu ed using h ee me hods: DPPH (1,1-diphenyl-2-pic ylhyd azyl), FRAP (Fe ic
Reducing An ioxidan Powe ), and ABTS (2,2′-Azino-bis-3-e hylbenzo hiazoline-6-
sul onic acid). These assays offe complemen a y insigh s in o he an ioxidan po en ial o
he oils, wi h each me hod a ge ing diffe en mechanisms o an ioxidan ac ion. As seen
in Table 2, hyme and o egano essen ial oils exhibi ed signi ican ly highe an ioxidan
ac i i y, wi h alues up o i e imes g ea e han ha o oli e oil. The an ioxidan ac i i y
o emulsions con aining hyme and o egano oils (TE-TO, TE-O O, and TO:O O) was
no ably enhanced, ollowing a clea end: TE-OlO < TE-TO < TE-TO:O O < TE-O O. This
end demons a es ha emulsions con aining highe concen a ions o hyme and
o egano oils a e mo e effec i e a neu alizing ee adicals and educing oxida i e s ess,
posi ioning hem as p omising candida es o an ioxidan - ich cosme ic o mula ions.
When compa ing hese TEs wi h o he cosme ic solu ions epo ed in he li e a u e, i is
e iden ha in ce ain cases hey ha e compa able o supe io an ioxidan capabili ies
[8,14,34]. The obus an ioxidan capaci y o hese TEs, demons a ed h ough hei abili y
o sca enge ee adicals and educe oxida i e s ess, is pa icula ly ele an o an i-aging
applica ions, as oxida i e damage is a majo con ibu o o he b eakdown o collagen,
elas in, and o he s uc u al componen s o he skin. This capaci y may help p o ec skin
cells om p ema u e aging, educe he appea ance o ine lines and w inkles, and imp o e
o e all skin elas ici y, making hese emulsions aluable candida es o an i-aging
cosme ic o mula ions.
P ocesses 2025, 13, 374 16 o 21
TE
TE-OlO 1:1 6.5 ± 0.4
a
6.0 ± 0.0
a
** 6.0 ± 0.0
a
** 6.0 ± 0.0
a
** 6.0 ± 0.0
a
**
1:4 7.0 ± 0.5
a
6.0 ± 0.0
a
** 6.0 ± 0.0
a
** 6.0 ± 0.0
a
** 6.0 ± 0.0
a
**
TE-TO
1:1 90.0 ± 0.0
c
* 12.3 ± 0.2
b
100 *** 8.3 ± 0.4
a
100 *** 9.3 ± 1.1
a
100 *** 13.0 ± 0.3
b
100 ***
1:4 90.0 ± 0.0
c
* 8.0 ± 0.6
a
100 *** 8.0 ± 0.3
a
100 *** 10.0 ± 0.8
a
100 *** 15.0 ± 0.7
b
100 ***
TE-O O
1:1 90.0 ± 0.0
c
* 15.0 ± 1.7
b
100 *** 9.7 ± 0.9
a
100 *** 12.7 ± 0.5
b
100 *** 19.3 ± 1.0
b
100 ***
1:4 90.0 ± 0.0
c
* 11.0 ± 0.6
a
100 *** 11.0 ± 0.5
a
100 *** 13.0 ± 0.5
b
100 *** 18.7 ± 1.1
b
100 ***
TE-
TO:O O
1:1 90.0 ± 0.0
c
* 12.0 ± 0.3
a
100 *** 9.0 ± 0.7
a
100 *** 12.3 ± 0.8
b
100 *** 19.0 ± 0.8
b
100 ***
1:4 90.0 ± 0.0
c
* 9.0 ± 0.5
a
100 *** 9.0 ± 0.3
a
100 *** 11.3 ± 0.9
a
100 *** 18.3 ± 0.6
b
100 ***
* To al inhibi ion on 90 mm pla e; ** Con ol sample RI es ; *** Inhibi ion in ela ion o con ol (TE-
OlO) RI es .
Table 4. Pho og aphic ep esen a ion o ZoI ob ained a e an imic obial assay wi h TE wi h NADES
1:1 o he ungus T. men ag ophy es and he bac e ia S. au eus, S. epide midis, E. coli, and K.
pneumoniae. The diame e o he holes (Ø = 6 mm) was included.
T. men ag ophy es S. au eus S. epide midis E. coli K. pneumoniae
TE-OlO
6.5 mm
6 mm
6 mm
6 mm
6 mm
TE-TO
To al
12.3 mm
8.3 mm
9.3 mm
13.0 mm
TE-O O
To al
15.0 mm
9.7 mm
12.7 mm
19.3 mm
TE-TO:O O
To al
11.0 mm
9.0 mm
12.3 mm
19.0 mm
P ocesses 2025, 13, 374 17 o 21
3.5. Effec o NADES-Ex ac s and TEs on he Heal h Index o Galle ia mellonella and on he
Melaniza ion Phenomenon
The la al heal h index is an impo an ool o assessing he o e all heal h o G.
mellonella, aking in o accoun ac o s such as la al ac i i y, cocoon o ma ion,
melaniza ion, and su i al [28]. The eac ion o G. mellonella o speci ic cosme ic p oduc s
o compounds p o ides an indica ion o i s oxici y [14,39]. Assessmen o la al heal h is
c i ical o ensu ing he eliabili y and sa e y o p oposed TEs o u u e cosme ic
applica ions. Figu e 5a shows he heal h index o G. mellonella in he p esence o he
diffe en TEs p oduced. The la al heal h index was ound o be lowe in TEs con aining
essen ial oils (TE-TO, TE-O O, and TE-TO:O O), sugges ing a le el o oxici y in compa ed
o he con ol (TE-OlO). Howe e , despi e he educed la al heal h indices o TE-TO,
TE-O O, and TE-TO:O O, he esul s emain accep able, as hey a e s ill abo e 6.
The melaniza ion s a us o G. mellonella is an impo an indica o o hei heal h, as
high melaniza ion indica es comp omised heal h, while educed melaniza ion sugges s a
s eng hened immune sys em and imp o ed o e all heal h. Figu e 5b demons a es ha
he melaniza ion s a e imp o ed o e ime o all TEs. No ably, he TEs con aining
essen ial oils (TO, O O, and TO:O O) showed sligh ly lowe le els o melaniza ion
compa ed o he con ol (OLO), sugges ing ha OLO has a mo e p o ec i e effec agains
melaniza ion in la ae.
Se e al s udies ha e in es iga ed he heal h index o Galle ia mellonella la ae in
esponse o a ious essen ial oils, including hyme and o egano oils [40,41]. All hese
s udies iden i ied he oxici y o hese oils o his la al model, sugges ing he need o a
educed dosage. In his s udy, a 20 w .% concen a ion o oil was selec ed o ensu e
effec i e mic obial inhibi ion while main aining he heal h index wi hin accep able limi s
o he use o hese new cosme ic solu ions.
Figu e 5. The G mellonella heal h index assay (a) and melaniza ion index assay (b) o all he
p oduced TE. The esul s o he la al heal h index (a) in he p esence o TE-ex ac s a 72 h e ealed
s a is ically signi ican diffe ences (p ≤ 0.0001) wi h he espec i e TE-con ols. The melaniza ion
index esul s o he la ae (b) in he p esence o TEs om 0 h o 72 h e ealed s a is ically signi ican
diffe ences (p ≤ 0.05) wi hin he same sample g oup. No s a is ically signi ican diffe ences we e
obse ed be ween he TE-ex ac s and hei espec i e TE-con ols a e 72 h o exposu e.
4. Conclusions
This s udy success ully de eloped NADES-in-oil emulsions (TEs) designed o
pha maco-cosme ic applica ions, le e aging he unique p ope ies o na u al deep
eu ec ic sol en s (NADES) and essen ial oils. The no el y o his wo k lies in he
subs i u ion o adi ional oli e oil wi h essen ial oils om Thymus ulga is ( hyme) and
O iganum ulga e (o egano), combined wi h he use o NADES composed o lac ic acid
P ocesses 2025, 13, 374 18 o 21
and glyce ol a pH 5. The esul ing emulsions demons a ed signi ican an ioxidan
ac i i y, d i en by he e pene compounds hymol and ca ac ol, which a e abundan in
hyme and o egano oils. These indings sugges ha essen ial oils no only enhance he
an ioxidan po en ial o he emulsions bu also p o ide addi ional an imic obial bene i s.
The TEs displayed a o able p ope ies o opical applica ions, including mode a e
skin pene a ion and signi ican an i ungal ac i i y agains T ichophy on men ag ophy es
and an ibac e ial efficacy agains S aphylococcus au eus, S aphylococcus epide midis,
Esche ichia coli, and Klebsiella pneumoniae. The use o he Galle ia mellonella la al model
con i med he low oxici y o he emulsions, while also highligh ing hei po en ial o
p omo e skin heal h by imp o ing la al melaniza ion. These esul s unde sco e he
po en ial o TEs as a sa e and effec i e op ion o he ea men and p e en ion o ungal
and bac e ial skin in ec ions.
The implica ions o his wo k ex end beyond he immedia e scope o opical
he apeu ic applica ions, sugges ing ha NADES-based emulsions en iched wi h
essen ial oils could se e as a p omising pla o m o he de elopmen o ad anced
pha maco-cosme ic o mula ions. These emulsions may offe a mo e sus ainable and
po en al e na i e o con en ional skinca e p oduc s, bene i ing om he syne gy be ween
na u al ing edien s and he s abili y p o ided by he NADES ma ix.
Mo ing o wa d, u u e esea ch should ocus on op imizing he o mula ion
pa ame e s o u he enhance he efficacy and s abili y o he TEs. Addi ionally, clinical
s udies a e needed o con i m he sa e y and effec i eness o hese emulsions in human
skin applica ions. In es iga ing he syne gis ic in e ac ions be ween diffe en essen ial oils
and explo ing he use o o he na u al sol en s could p o ide aluable insigh s in o
expanding he ange o he apeu ic applica ions o NADES-based emulsions in
de ma ology and beyond.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a :
h ps://www.mdpi.com/a icle/10.3390/p 13020374/s1, Figu e S1: TGA deg ada ion s eps (a) and
TGA he mog ams (b) and c) o all he p oduced TEs.; Table S1: Compounds iden i ied in oli e oil
by GC-MS.; Table S2: Compounds iden i ied in hyme oil by GC-MS.; Table S3: Compounds
iden i ied in o egano oil by GC-MS.; Table S4: E alua ion o he inhibi ion o y osinase and elas ase
by essen ial oils and espec i e TE.
Au ho Con ibu ions: Concep ualiza ion, D.S.F., A.C.-P. and C.S.; me hodology, D.S.F., D.R., J.S.,
J.N., T.D.T., M.O.T., D.A., J.C., S.S., H.P.F. and A.R.; da a cu a ion, D.S.F. and C.S.; w i ing—o iginal
d a , D.S.F.; w i ing— e iew and edi ing, D.S.F. and C.S.; supe ision, A.C.-P. and C.S.; unding
acquisi ion, D.S.F., T.D.T., M.O.T., J.C., D.A., S.S., C.A., A.R., A.V., A.C.-P. and C.S. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding: This s udy was suppo ed by he Po uguese Founda ion o Science and Technology (FCT)
unde he scope o he s a egic unding o UIDB/04469/2020 uni , and by LABBELS—Associa e
Labo a o y in Bio echnology, Bioenginee ing and Mic oelec omechanical Sys ems, LA/P/0029/2020
D. S. F. also hanks o FCT o unding (SFRH/BD/147190/2019). T. T. and M. T. hanks o FCT o
unding (2020.06046.BD and 2021.06906.BD). J.C. and D.A. also hank FCT o he CEEC Indi idual
(DOI: 10.54499/2022.06886.CEECIND/CP1737/CT0001, and 2023.06040.CEECIND, espec i ely). S. S.
also hanks FCT o he CEEC Ins i u ional (DOI: 10.54499/CEECINST/00018/2021/CP2806/CT0003).
A.R. hanks FCT o i s con ac unde he CEEC-Indi idual-4 h Edi ion wi h he e e ence
2021.02803.CEECIND (DOI: 10.54499/2021.02803.CEECIND/CP1664/CT0018). The au ho s
acknowledge he suppo o he MIRRI-PT (Polo No e) p ojec (PINFRA04/84445/2020) unded by
Eu opean Regional De elopmen Fund unde No e2020—P og ama Ope acional Regional do
No e.
Da a A ailabili y S a emen : Da a a e con ained wi hin he a icle and supplemen a y ma e ials.
P ocesses 2025, 13, 374 19 o 21
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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