Journal of Ethnopharmacology 301 (2023) 115779

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Towards a modern approach to traditional use of Helichrysum italicum in

dermatological conditions: In vivo testing supercritical extract on artificially
irritated skin
Svetolik Maksimovic a, *, Milica Stankovic b, Sonja Roganovic b, Ivana Nesic b,
Jelena Zvezdanovic c, Vanja Tadic d, Irena Zizovic e
a
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11120, Belgrade, Serbia
b
University of Nis, Faculty of Medicine, Dr. Zorana Djindjica 81, 18000, Nis, Serbia
c
University of Nis, Faculty of Technology, Bulevar Oslobodjenja 124, 16000, Leskovac, Serbia
d
Institute for Medical Plant Research “Dr. Josif Pancic “, Tadeusa Koscuska 1, 11000, Belgrade, Serbia
e
Wroclaw University of Science and Technology, Faculty of Chemistry, Wybrzeze Wyspianskiego 27, 50-370, Wroclaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Helichrysum italicum has been widely used in traditional medicine to treat al­
Helichrysum italicum lergies, colds, cough, skin, liver and gallbladder disorders, inflammation, infections, and sleeplessness.
Supercritical extract Furthermore, it possesses considerable wound healing and skin protective properties, documented by several in
Cotton gauze
vivo studies performed on animals. However, there is a lack of experimental evidence supporting its potential as a
Polypropylene
Skinprotective activity
topical agent tested by human clinical trials.
In vivo human studies Aim of the study: The study aimed to investigate the skin protective activity of cotton gauze and polypropylene
non-woven fabric, impregnated with H. italicum extract by the integrated supercritical CO2 extraction-
supercritical solvent impregnation process.
Materials and methods: The integrated process of supercritical CO2 extraction of H. italicum and the impregnation
of cotton gauze and polypropylene non-woven fabric was performed under 350 bar and 40 ◦ C with and without
the addition of ethanol as a cosolvent. Impregnated textile materials were tested in vivo for their bioactivity on
irritated human skin. Randomized in vivo studies performed involved assays of both safety and efficacy of the
impregnated textiles. The effects were evaluated using the in vivo non-invasive biophysical measurements of the
following skin parameters: electrical capacitance, transepidermal water loss, melanin index, erythema index, and
skin pH.
Results: Both cotton gauze and polypropylene non-woven fabric were impregnated with H. italicum extracts under
supercritical conditions with considerable values of the impregnation yield (1.97%–4.25%). The addition of
ethanol as a cosolvent during the process caused significant changes in the incorporated extracts’ impregnation
yield and chemical profile. Both impregnated textile materials were safe, evaluated by their testing on the human
skin with no cause of any irritation and redness. However, efficacy studies revealed that polypropylene non-
woven fabric impregnated with H. italicum extract with ethanol as a cosolvent, possessed significantly greater
potential for skin protection than the other investigated samples.
Conclusions: The present study demonstrated the feasibility of the combined supercritical extraction and
impregnation process in developing materials for topical application based on H. italicum extract. The results of in
vivo studies performed on human volunteers confirmed the suitability of H. italicum active components to be a
part of human skin protective preparations because of their ability to maintain the skin unimpaired. Traditionally
claimed applications as a medicinal plant capable of regenerating skin have been scientifically proven, in
addition to employing green technology in obtaining the impregnated materials with a broad spectrum of
utilization.

* Corresponding author.
E-mail address: smaksimovic@tmf.bg.ac.rs (S. Maksimovic).

https://doi.org/10.1016/j.jep.2022.115779
Received 11 July 2022; Received in revised form 27 September 2022; Accepted 28 September 2022
Available online 3 October 2022
0378-8741/© 2022 Elsevier B.V. All rights reserved.
S. Maksimovic et al.
1. Introduction
The genus Helichrysum consists of an estimated 600 species
belonging to the sunflower family (Asteraceae). Helichrysum italicum
(Roth) G. Don is commonly known as curry plant, everlasting or
immortelle. It is an aromatic shrub 30–70 cm high. It has yellow flowers
and blossoms between May and June. It grows in dry, rocky, or sandy
areas of the Mediterranean regions (Viegas et al., 2014). Due to the
variety of secondary metabolites present in the isolates, among which
the most common are terpenes and phenolic compounds (flavonoids,
acetophenones, phloroglucinols, coumarins and coumarates, phenolic
acids, and esters), H. italicum has been reported to possess a wide range
of pharmacological activities (Viegas et al., 2014; Lourens et al., 2008;
Maksimovic et al., 2017).
The most frequently reported traditional uses of H. italicum are
related to respiratory, digestive, and skin inflammatory conditions.
Moreover, skin protective properties seem to be the best documented
therapeutic effects of H. italicum, which was confirmed by several in vivo
studies performed with topical application of H. italicum extracts (Viegas
et al., 2014). Examples of ethnopharmacological use of H. italicum for
the treatment of skin disorders found in the literature include the use of
H. italicum flowers in the form of decoction against alopecia (Ballero
et al., 2001) and against skin inflammation in the form of an infusion
and use of flowers, leaves, and steams as a part of wound healing powder
(Viegas et al., 2014). There are also some data related to the skin pro­
tective effects of H. italicum isolates. However, this statement is based
mainly on data about the traditional use of H. italicum extracts and in
vitro and in vivo animal studies. At the same time, there is a lack of
human clinical assays of the mentioned effects. Experiments carried out
in guinea pigs with the flavonoid fraction isolated from the crude
H. italicum ethanolic extract indicated that the photoprotective and ac­
tivity against UVB-induced erythema were largely due to the action of
this flavonoid complex (Facino et al., 1988). Three new acetophenone
glucosides, isolated after extraction of H. italicum aerial parts by meth­
anol, showed anti-inflammatory activity in a 12-O-tetradecanoylphorbol
13-acetate (TPA)-induced mouse ear edema test (Sala et al., 2001).
Furthermore, the results obtained on the acute edemas induced by TPA
and ethyl phenylpropionate in the mouse ear, by serotonin and phos­
pholipase A2 (PLA2) in the mouse paw, on chronic inflammation induced
by repeated application of TPA in the mouse ear and on the delayed-type
hypersensitivity induced by sheep red blood cells suggest that the
anti-inflammatory activity of H. italicum methanolic extracts can be
explained by multiple effects, including inflammatory enzyme inhibi­
tion, free-radical scavenging activity, and corticoid-like effects (Sala
et al., 2002). In addition, seven previously isolated compounds from
H. italicum methanolic extract (Sala et al., 2001) were studied in
different in vivo experimental models of chronic inflammation. Only
4-hydroxy-3-(3-methyl-2-butenyl)acetophenone showed significant
improvement in chronic inflammation (Sala et al., 2003a). Finally, three
flavonoids, gnaphaliin, pinocembrin, and tiliroside, isolated from
H. italicum methanolic extract, were studied in vivo in different models of
inflammation. The most active compound was tiliroside, while pino­
cembrin was the only flavonoid that exhibited anti-inflammatory ac­
tivity in the sheep red blood cell-induced delayed-type hypersensitivity
reaction. Anti-inflammatory activity of the flavonoids was connected to
their antioxidant properties (Sala et al., 2003b).
Supercritical fluids have gained scientific attention in the last several
decades due to their unique properties, such as high density and diffu­
sion coefficients, low viscosity, and the absence of surface tension. The
most used fluid in a supercritical state is carbon dioxide because of its
favorable critical parameters (31 ◦ C, 7.38 MPa), non-toxicity, avail­
ability, and low price. Supercritical carbon dioxide (scCO2) is used on
the industrial scale in extraction and impregnation, offering numerous
advantages over conventional processes, such as complete waste
effluent elimination, avoidance of organic solvents usage, and high en­
ergy efficiency, production of solvent-free products under mild
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Journal of Ethnopharmacology 301 (2023) 115779
temperature conditions, etc. Supercritical fluid extraction (SFE) allows
for producing high-quality extracts whose composition can be tailored
by appropriate temperature and pressure conditions. Supercritical sol­
vent impregnation (SSI), on the other hand, provides the possibility of
solid impregnation with active compounds soluble in scCO2. Unlike
liquids, supercritical fluids have no surface tension and quickly pene­
trate the solid matrix, allowing for the active component deposition
throughout its volume.
The pharmacological activity of scCO2 extracts of H. italicum was
reported only in a few articles and refers mainly to the antioxidant ac­
tivity investigated by different in vitro methods (Poli et al., 2003; Costa
et al., 2015; Molnar et al., 2017). Mentioned findings on considerable
antioxidant activity of supercritical extracts are important related to
their application in the treatment of skin disorders since there is a
connection between the antioxidant and anti-inflammatory activity of
secondary plant metabolites (Tadic et al., 2008; Ravipat et al., 2012;
Maher et al., 2015; Sala et al., 2002, 2003b).
The application of cotton gauze and polypropylene (PP) non-woven
fibers as medicinal textiles is well-known. Recently, SSI was demon­
strated to be a suitable technique for the impregnation of these materials
with thymol as an antibacterial agent (Milovanovic et al., 2013; Mar­
kovic et al., 2015). The impregnated gauze (Milovanovic et al., 2013)
provided strong antimicrobial activity against tested strains of Escher­
ichia coli, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis,
and Candida albicans, while impregnated PP fibers (Markovic et al.,
2015) achieved 99.9% reduction of S. aureus, E. coli, and C. albicans.
Furthermore, Chen et al. (2017) used PP fibers for SSI with polysiloxane
containing quaternized N-halamine moieties as an efficient antibacterial
coating against S. aureus and E. coli.
The integration of supercritical fluid extraction (SFE) and SSI pro­
cesses proved convenient when the active substance incorporated in the
solid carrier is a supercritical extract (Zizovic, 2017). This concept has
been recently proposed by Fanovich et al. (2013), and it was successfully
applied to the impregnation of cotton gauze and PP fibers with thyme
extract (Ivanovic et al., 2014) and PP fibers with hop extract (Zizovic
et al., 2014). Furthermore, in our previous studies, H. italicum extract
was used as an active substance for the impregnation of corn starch
xerogels by the integrated SFE-SSI process to obtain the formulations for
possible oral application (Maksimovic et al., 2018), as well as for the
impregnation of cotton gauze and PP fabric by the same process (Mak­
simovic et al., 2021).
This study presents a continuation of our recently published article
(Maksimovic et al., 2021), in which the feasibility of the integrated
SFE-SSI process in impregnating cotton gauze and PP fabric with
H. italicum extracts under 350 bar and 40 ◦ C was proven. The study
included evaluating the influence of adding ethanol as a cosolvent on the
impregnation yield and chemical profile of the extracted and impreg­
nated compounds (Maksimovic et al., 2021). Therefore, this work fo­
cuses on the in vivo investigation of cotton gauze and PP fabric
impregnated with H. italicum extract for their safety and efficacy in
treating artificially irritated human skin. To the best of our knowledge,
for the first time, high pressure processed active principles from
H. italicum were used as an agent with potential positive human skin
protective effect. Besides, selected medicinal textiles (cotton gauze and
PP fabric) were impregnated with the extracts using scCO2. As previ­
ously mentioned, in SSI, impregnation occurs in the whole solid (fiber)
volume due to the absence of surface tension in the supercritical phase
that penetrates the polymer matrix easily. That is an essential advantage
of SSI over conventional methods where impregnation is only superfi­
cial. Therefore, investigated medicinal textile + extract systems provide
prolonged active substances release and are of interest for impaired skin
dressings design.
S. Maksimovic et al.
2. Materials and methods
2.1. Plant material
Helichrysum italicum (Roth) G. Don (the plant name was checked with
http://www.theplantlist.org/on 24/02/2021) flowering aerial parts
were collected from the region of Konavle (the southern part of Croatia,
42.585951◦ N, 18.263743◦ E) in the period 2015–2019, dried in the air,
and kept away from direct sunlight at room temperature. A voucher
specimen was numbered 16,282 and deposited at Herbarium of Institute
of Botany and Botanical Garden Jevremovac, Faculty of Biology, Uni­
versity of Belgrade (Belgrade, Serbia). The average water content of
flowers (8.1 wt%) was determined using Moisture Analyzer, Mettler-
Toledo, HB43–S.
2.2. Chemicals and reagents
Sterile cotton gauze with a weaving density of 17 threads/cm2 was
produced by Niva, Serbia. PP non-woven fabric (40 g/m2) was used for
impregnation with H. italicum extract. Commercial CO2 (purity 99%)
was purchased from Messer-Tehnogas, Serbia. Commercial ethanol (vol.
96%) was purchased from ZORKA Pharma-HEMIJA d.o.o., Sabac,
Serbia. Sodium lauryl sulphate (purity >95%) was purchased from TCI
Chemicals Europe. Standards for HPLC analysis were purchased from
Extrasynthese, Genay Cedex France (arzanol, naringenin, hesperetin,
rutin, hyperoside, isoquercetin, kaempferol-3-O-glucoside, tiliroside,
quercetin, kaempferol, luteolin, chrysin, neochlorogenic acid, chloro­
genic acid, caffeic acid, p-coumaric acid, ferulic acid, cynarine, gallic
acid, protocatechuic acid, homovanillic acid, gentisic acid, pyrogallol,
coumarin).
2.3. The integrated SFE-SSI process
The integrated process of H. italicum extraction and impregnation of
cotton gauze and PP fabric was performed under the pressure and
temperature of 350 bar and 40 ◦ C, respectively, in a High-Pressure
Extraction Adsorption (HPEA) 500 unit (Eurotechnica GmbH) for 5 h.
The plant material to textile mass ratio was approximately 10. At the end
of each experiment, the system was decompressed at a rate of 35 bar/
min. In the experiments with ethanol, a 1:10 ethanol/scCO2 mass ratio
was used. The integrated process and chemical analysis assays were
previously described in detail (Maksimovic et al., 2021).
2.4. In vivo studies
2.4.1. Safety study design
The safety profile was evaluated through a randomized in vivo study
on healthy human volunteers in a short-term 1 h-study under occlusion,
using the measurements of the following parameters: electrical capaci­
tance (EC), transepidermal water loss (TEWL), erythema index (EI) and
skin pH. Parameters were measured before (baseline values) and 30 min
upon cessation of 1 h occlusive treatment. Fourteen healthy female
volunteers (mean age 23.3 ± 2.4 years) were recruited. The flexor side
of their forearms was treated with investigated samples with supercrit­
ical extracts of H. italicum using a precisely delineated and marked
cardboard ruler (with empty spaces in the form of rectangles, 16 cm2
each). Two additional sites were left as a non-treated control under
occlusion and the site for baseline measurements. After sample appli­
cation, the site was immediately covered with Parafilm® and with cot­
ton adhesive Sensifix® tapes. Volunteers were informed of the study and
instructed not to use any skin cleansing or skincare product on the test
sites for the whole week before the study, and during the experiment. All
subjects had healthy skin with no known allergy.
2.4.2. Efficacy study design
The randomized, double-blind study was performed in order to
3
Journal of Ethnopharmacology 301 (2023) 115779
evaluate the efficacy of the investigated samples. The effects were
evaluated using the in vivo non-invasive biophysical measurements of
five skin parameters: electrical capacitance (EC), transepidermal water
loss (TEWL), melanin index (MI), erythema index (EI), and skin pH.
The study was conducted on the 14 healthy volunteers of both sexes
(11 females and 3 males, mean age 24.3 ± 1.2 years), without a past or
present history of skin diseases or known allergy. Participants were
informed not to use any skin products on the tested sites one week before
the study and during the experiment. Previous studies demonstrated the
skin of the volar forearms as the most suitable for non-invasive bio­
physical measurements (Bazin and Fanchon, 2006). The measurements
were performed baseline (prior to the irritation), after the artificially
induced skin irritation, and after treatments with samples, according to
the guidelines from the literature (Berardesca, 1997; Pinagoda et al.,
1990; Parra and Paye, 2003; Rogiers, 2001). For the purpose of inducing
irritation, 60 μL of 12% sodium lauryl sulphate (SLS) water solution was
used (Tupker et al., 1997), on five skin sites under patch occlusion. The
solution was pipetted on the piece of filter paper (4 cm × 4 cm), which
was fixed to the skin with Parafilm® and cotton adhesive Sensifix® tape.
Measurements were taken 24 h after the occlusion, which lasted for 12 h,
was removed. Then, four skin sites in the form of rectangles on the volar
forearms, with an approximate area size of 16 cm2, were chosen for
applying the samples. One site on the left forearm was served as un­
treated but irritated control (UCO), and one site on the right forearm was
untreated control (UC). Samples were attached to the skin with cotton
adhesive Sensifix® tape. Each measurement was done 1 h after
removing the sample from the skin (after 1, 2, and 3 days of the
treatment).
The in vivo studies were approved by the local Ethics Committee of
the Medical faculty (Nis, Serbia), protocol code 12-6316-2/8 from June
16, 2016, in accordance with the Declaration of Helsinki, with a written
informed consent form signed by all volunteers. The original form of the
Ethical approval letter is given in Fig. S1 of the Supplementary material.
Before the measurements, volunteers were acclimatized for at least 30
min under controlled ambient conditions (21 ± 1 ◦ C and 50 ± 5% RH).
Parameter EC was measured using the probe Corneometer® CM 825,
TEWL was measured using Tewameter® TM 300, EI and MI using
Mexameter® MX 18 and pH using Skin-pH-Meter® 905 (all probes are
integral part of the device Multi Probe Adapter MPA® 9, Courage +
Khazaka Electronic GmbH, Germany).
2.4.3. Statistics
Data were presented as means ± standard error of the means (SEM).
Parameters were expressed as absolute changes to the baseline, and
obtained values for samples were compared mutually and with both
controls UCO and UC, using One-way ANOVA, followed by Tukey’s test.
The values of measured parameters at distinct time points were
compared to baseline values using paired sample t-test. The differences
were accepted as statistically significant at p < 0.05. All statistical an­
alyses were done using the software SPSS for Windows 25.0.
2.5. Analytical procedure
The chemical analysis required a re-extraction of impregnated sub­
stances from the gauze and PP fabric. Impregnated textiles were rinsed
in a chloroform-methanol (7:3 v/v) mixture and mixed for 10 min to
dissolve the adsorbed components. The obtained re-extracts were
further analyzed.
2.5.1. Gas chromatography (GC-FID)
Gas chromatography analysis of the re-extracts was carried out on an
HP-5890 Series II GC apparatus [Hewlett-Packard, Waldbronn (Ger­
many)], equipped with split-splitless injector and automatic liquid
sampler, attached to HP-5 column (25 m × 0.32 mm, 0.52 μm film
thickness) and fitted to flame ionization detector (FID). Carrier gas flow
rate (H2) was 1 mL/min, split ratio 1:30, injector temperature was
S. Maksimovic et al.
250 ◦ C, detector temperature 300 ◦ C, while column temperature was
linearly programmed from 40 ◦ C to 260 ◦ C (at a rate of 4 C/min), and
◦
then kept isothermally at 260 C for 10 min. Solutions of samples dis­
◦
solved in chloroform were consecutively injected in an amount of 1 μl.
Area percent reports, obtained as a result of standard processing of
chromatograms, were used as the base for the quantification analysis.
2.5.2. Gas chromatography – mass spectrometry (GC-MS)
The same analytical conditions as those mentioned for GC-FID were
employed for GC-MS analysis, along with column HP-5MS (30 m × 0.25
mm, 0.25 μm film thickness), using HP G 1800C Series II GCD system
[Hewlett-Packard, Palo Alto, CA (USA)]. Helium was used as a carrier
gas. The transfer line was heated at 260 ◦ C. Mass spectra were acquired
in EI mode (70 eV); in m/z range 40–450. The amount of 0.2 μL of the
sample solution in chloroform was injected. The components of the re-
extracts were identified by comparison of their spectra to those from
Wiley 275 and NIST/NBS libraries, using different search engines.
Calibration was performed using linear n-paraffins mixture (C6–C40) as a
standard. The experimental values for retention indices were deter­
mined using calibrated Automated Mass Spectral Deconvolution and
Identification System Software (Amdis ver. 2.1), compared to those from
available literature (Adams, 2007) and used as an additional tool to
approve MS findings.
2.5.3. High performance liquid chromatography (HPLC)
“Fingerprinting” of the investigated phenolic compounds was ach­
ieved by an Agilent Technologies 1200 HPLC, equipped with Lichros­
pher 100RP 18e column (150 × 3.2 mm, 5 μm), applying gradient
elutions of two mobile phases, i.e. “A/B" (“A"-0.2 M solution of phos­
phoric acid, and “B"-being a pure acetonitrile) at flow–rates 1 mL/min,
with photodiode-array (PDA) detection (UV at 280 nm), always within
140 min. Wining combinations were 89–75% A (0–35 min); 75-60% A
(35–55 min); 60% A, isokratic for 10 min, 60-50% A (65–70 min), at
50% A isocratic for 5 min, and 50-43% A (75–78 min), at 43% A isocratic
for 5 min, 43-23% A (83–85 min min); at 23% A isocratic for 5 min, 75-
60% A (35–55 min); 23-11% A (90–98 min); at 11% isocratic for 7 min,
11-0% A% (89–110 min); isocratic at 0% A for 20 min; 0–11% A%
(130–140 min). The concentrations of investigated samples were 42.00,
41.00, 49.00 and 42.00 mg/mL for H. italicum re-extracts 1–4, respec­
tively. Prior to injection, samples were filtered through PTFE membrane
filter. For standards used in the investigation, the concentrations were:
0.34 mg/mL for protocatechuic acid and gallic acid, 0.74 mg/mL for p-
coumaric acid, 0.52 mg/mL for caffeic acid, 0.21 mg/mL for chlorogenic
acid, 0.17 mg/mL for luteolin, 0.36 mg/mL for isovitexin, 0.54 mg/mL
for apigenin, 0.46 mg/mL for chrysin, 0.30 mg/mL for kaempferol, 0.28
mg/mL for kaempferol-7-O-glucoside, 0.36 mg/mL for quercetin, 0.42
mg/mL for tiliroside, 0.15 mg/mL for isorhamnetin, 0.18 mg/mL for
rutin, 0.46 mg/mL for pyrogallol, 0.36 mg/mL for cynarin, 0.10 mg/mL
for coumarin, 0.38 mg/mL for pinocembrin, 0.28 mg/mL for galangin,
0.22 mg/mL for isoquercetin, 0.30 mg/mL for neochlorogenic acid,
0.36 mg/mL for ferulic acid, 0.25 mg/mL for hyperoside, 0.56 mg/mL
for naringin, 0.58 mg/mL for t-cinnamic acid, 0.32 mg/mL for nar­
ingenin, 0.46 mg/mL for hesperetin, 0.67 mg/mL for arzanol, 0.30 mg/
mL for homovanillic acid, and 0.46 mg/mL for gentisic acid. The volume
of standard solutions being injected, as well as for the tested sample re-
extracts, was 4 μL.
Identification was based on retention times and overlay curves. Once
spectra matching succeeded, results were confirmed by spiking with
respective standards to achieve a complete identification by means of
the so-called peak purity test. Those peaks not fulfilling these re­
quirements were not quantified. Quantification was performed by
external calibration with standards.
2.5.4. Ultrahigh performance liquid chromatography – mass spectrometry
(UHPLC-MS)
The chromatographic separation was performed on the Hypersil gold
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Journal of Ethnopharmacology 301 (2023) 115779
C18 column (50 × 2.1 mm, 1.9 μm) at 25 ◦ C using a Dionex Ultimate
3000 UHPLC + system equipped with a diode array (DAD) detector and
LCQ Fleet Ion Trap Mass Spectrometer, Thermo Fisher Scientific, USA. A
mobile phase consisted from two solvents, 0.1%, v/v, formic acid in (A)
water and (B) methanol yielding a gradient program at 0,250 mL/min
flow rate: 0–2 min (10–30% B), 2–4 min (30–35%), 4–5 min (35–40%
B), 5–8 min (40–50%), 8–9 min (50–80% B), 9–11 min (80–90% B),
followed by isocratic period 11–12 min (90% B), 12–12.1 min (90-10%
B) and finished from 12.1 to 15 min by 10% B.
Absorption UV–Vis spectra were recorded on DAD-detector with a
total spectral range between 200 and 800 nm. Mass spectrometric
analysis was performed using a 3D-ion trap with electrospray ionization
(ESI) in negative ion mode. The ESI–source parameters were as follows:
source voltage 4.5 kV, capillary voltage − 41 V, tube lens voltage − 95 V,
capillary temperature 350 ◦ C, nitrogen sheath and auxiliary gas flow 32
and 8 arbitrary units. MS-spectra were acquired by full range acquisition
of m/z 130–1000, with a tandem mass spectrometry analysis performed
by a data dependent scan – the collision-induced dissociation (CID) of
detected molecular ions peaks ([M-H]–) with normalized collision en­
ergy set at 30 eV.
Samples of re-extracts were dissolved in methanol (0.15 mg/cm) and
filtered through 0.45-μm filter before analysis. Samples volumes were 8
μL. Xcalibur software (version 2.1) was used for instrument control, data
acquisition, and data analysis. Qualitative analysis of the re-extracts was
achieved by using the absorption UV–Vis spectra of the DAD signals and
the mass spectra with the corresponding molecular ion peaks ([M-H]-)
and also characteristic ion fragmentation within selected peaks (MS/
MS), from the corresponding UHPLC chromatograms.
3. Results and discussion
3.1. Impregnation yield and influence of cosolvent addition
The integrated SFE-SSI process resulted in the following values of the
impregnation yield: 2.53 ± 0.10% for the impregnation of cotton gauze
(sample 1), 4.25 ± 0.50% for the impregnation of cotton gauze with the
addition of cosolvent (sample 2), 3.56 ± 0.43% for the impregnation of
PP fabric (sample 3) and 1.97 ± 0.29% for the impregnation of PP fabric
with the addition of cosolvent (sample 4).
3.2. Results of the in vivo studies
In vivo studies were performed by investigating four types of samples:
sample 1 – cotton gauze impregnated using pure scCO2; sample 2 –
cotton gauze impregnated with the addition of cosolvent; sample 3 – PP
fabric impregnated using pure scCO2; sample 4 – PP fabric impregnated
with the addition of cosolvent.
3.2.1. Safety study
The influence of different samples 1, 2, 3, and 4 on the biophysical
parameters of the skin (EC, EI, TEWL, and pH) after 1 h of exposure is
given in Fig. 1. The parameters are shown as absolute changes in values
compared to baselines (parameter values measured before the study).
The effects of different samples were compared with each other and in
relation to the site with occlusion (significant differences in values are
being marked with &). Significant differences in parameter values after
1 h compared to baseline values are labeled with *.
Based on the obtained results, a significant increase in skin hydration
(parameter EC) was noticed after the application of sample 1 and sample
4 (Fig. 1). This parameter, i.e., electrical capacitance, is the measure of
the stratum corneum hydration. Sample 4 showed the best moisturizing
properties of the surface layer of the skin. The results of the safety study
assessment showed that sample 1 caused a significant increase in TEWL
compared to the baseline values. Also, there is a considerable difference
between the effects of samples 1 and 3.
In sample 4, PP non-woven fabric was used as a solid carrier for
S. Maksimovic et al.
Fig. 1. The influence of samples 1, 2, 3 and 4 on the biophysical parameters of the skin (EC, EI, TEWL and pH) after 1 h exposure. The effects of different samples
were compared mutually and in relation to the site with occlusion (significant differences are being marked with &). Significant differences in parameter values after
1 h compared to baseline are being marked with *.
active principals and ethanol as a cosolvent during the SFE-SSI process.
Both factors contributed to the positive moisturizing effects on the skin.
The addition of cosolvent increased the extraction yield of some polar
active principles. On the other hand, PP fabric might be a better carrier
for these active components.
The measurement of the water evaporating from the skin surface is
an important parameter for the evaluation of the barrier function of the
skin. It is used in all kinds of product application evaluations. It is an
assessment of the water barrier functional integrity of stratum corneum.
A certain amount of water evaporates from the skin surface as a result of
normal skin metabolism. When the barrier function is damaged, the
water loss increases (even when the damages are invisible to the human
eye). As seen in Fig. 1, there was increase in TEWL prior to baseline
values after sample 4 application. The increase in hydration of stratum
corneum and better moisturizing effects might also occur as a result of an
occlusion caused by investigated samples. However, sample 1 had cot­
ton gauze as a carrier, and the cosolvent was not used. There was also a
significant increase in TEWL after sample 1 application, and this might
be the reason for an increase in stratum corneum hydration level, which
can be explained by the hydrophilic properties of the cotton. Finally, an
increase in the concentration of the extract in the samples resulted in a
decrease in TEWL values.
pH values for healthy skin range from 5.0 to 6.0, and in most path­
ological conditions, the value increases. None of the samples showed
statistically significant change in the values of the erythema index and
pH. It can be concluded that all samples were safe after application with
no cause of any irritation and redness.
3.2.2. Efficacy study
The results of the investigated effects of the samples after application
on the human skin pre-treated with SLS in a non-invasive in vivo model
are presented in Fig. 2. Parameters were presented as absolute changes
to baseline at distinct time points. The effects of different formulations
were compared mutually and to the corresponding controls UCO and UC
(significant differences are being marked with &). Significant differ­
ences of parameter values at distinct time points related to baseline
values are labeled with *.
Occlusion with SLS led to a significant decrease of the parameter EC
compared to baseline values (Fig. 2a), causing skin dryness. After two
days of the treatment with samples, there was an improvement in skin
5
Journal of Ethnopharmacology 301 (2023) 115779
hydration after applying samples 2 and 4 (both obtained by extraction
with polar cosolvent). However, after three days of the treatment,
sample 4 showed the best moisturizing potential.
As can be seen in Fig. 2b, SLS-induced irritation caused impairment
on the skin barrier function, increasing the TEWL statistically significant
compared to baseline values. In accordance with that, the application of
samples gave us information about their potential to repair skin barrier
function. There were significant differences in TEWL values between
samples 1 and 3 after one day of treatment, where sample 3 revealed
better efficacy in repairing skin barrier function (which is in line with
safety study results). Furthermore, sample 4 also showed lower TEWL
values measured three days after application. As PP fabric as a carrier
has hydrophobic properties, it was expected that parameter TEWL had
lower values for samples 3 and 4, especially for the one impregnated
without cosolvent (sample 3).
Parameters MI and EI are associated with possible skin colour
change, the appearance of redness, and irritation of the skin. Fig. 2c
showed a decrease in MI values after the application of samples (after
three days of treatment), which was statistically significant for sample 1.
None of the samples caused any irritation, appearance of redness, or skin
discoloration. Parameter EI was significantly increased after SLS-
induced irritation at sites where samples 2 and 3 were applied
compared to the baseline values (Fig. 2d). Sample 4 revealed the best
potential to decrease the erythema index. After two days of treatment
with this sample, EI values were restored to the baselines and were even
lower than baseline values, although not statistically significant. The
control UCO (pre-treated with SLS but without treatment) showed an
increase in the EI value, suggesting that samples had positive effects on
reducing skin irritation.
Parameter pH was increased after the irritation (Fig. 2e). There were
significant differences in pH values between sample 1 and samples 2, 3,
and 4 (after two days of the treatment). Investigated samples showed an
increase in pH values compared to baseline values after three days of the
treatment, which was not statistically significant, except for sample 1.
Skin pH values were significantly increased after the application of
sample 1. The acidic pH value is important for the normal functioning of
the skin-protective layer. Sample 3 revealed the lowest pH values after
application on the irritated skin.
The results of the present study demonstrated that sample 4 prepared
with H. italicum supercritical extract obtained and incorporated in PP
S. Maksimovic et al. Journal of Ethnopharmacology 301 (2023) 115779

Fig. 2. The influence of the irritation per se and samples 1, 2, 3 and 4 after irritation on parameters: a) EC, b) TEWL, c) MI, d) EI and e) pH. The effects of different
samples were compared mutually and to the corresponding controls UCO and UC (significant differences are being marked with &). Significant differences in
parameter values at distinct time points compared to baseline are being marked with *.

fabric in a process with the addition of ethanol as a cosolvent, possessed
a significantly greater potential in increasing skin hydration, repairing
skin barrier function (decreasing TEWL from stratum corneum) and
decreasing MI and EI values of the irritated skin in comparison to other
investigated samples.
3.3. Chemical composition of the re-extracts
3.3.1. Results of the GC-MS analysis
Contents of different compound classes identified by GC-MS analysis
of the H. italicum re-extracts are given in Table 1, while contents of

6
S. Maksimovic et al. Journal of Ethnopharmacology 301 (2023) 115779

Fig. 2. (continued).

profiles, are given in Table 2. Obtained results indicated the affinity of

Table 1
all textile materials for incorporating fatty acids and esters, hydrocar­
Chemical profile of H. italicum re-extracts isolated from the impregnated textiles
bons including waxes, and coumarin and amorphene derivatives. On the
obtained by GC-MS analysis.
other hand, the overall content of terpenes in all re-extracts was
Classes of Identified components’ area percentage in the chromatogram
significantly low. The highest content of active components was recor­
compounds (%)
ded in the re-extract obtained from sample 1 (14.4% of terpenes and
Re-extract Re-extract 2 Re- Re-extract 4 35.8% of coumarin and amorphene derivatives). The addition of ethanol
1 (cotton (cotton gauze extract 3 (PP with
in both cases caused a significant increase in the content of hydrocar­
gauze) with cosolvent) (PP) cosolvent)
bons at the expense of a decrease of fatty acids and esters and coumarin
Monoterpene 1.0 0.5 1.1 0.8
and amorphene derivatives content. A stronger influence of the addition
hydrocarbons
Oxygenated 2.2 1.0 1.3 1.8 of ethanol during the SFE-SSI experiments was noted when cotton gauze
monoterpenes was used as the carrier since the content of hydrocarbons in re-extract
Sesquiterpene 9.0 2.1 4.6 4.3 obtained from sample 2 was 63.4%. Individual components with the
hydrocarbons highest content found in all types of re-extracts were β-selinene from
Oxygenated 2.2 0.7 2.4 2.6
group of terpenes, caproic, linoleic and oleic acid from fatty acids and
sesquiterpenes
Fatty acids and 20.8 13.4 24.6 21.8 esters, 2-α-acetoxy-amorpha-4,7(11)-diene-8-one, 2α-acetoxy-11-
esters metoxy-amorpha-4,7-diene and 2,2-dimethyl-7,8-dimetoxychromanone
Hydrocarbons 28.0 63.4 36.9 46.8 from amorphene and coumarin derivatives respectively, and heptaco­
Coumarin and 35.8 18.2 28.8 21.1
sane, nonacosane and untriacontane from hydrocarbons.
amorphene
derivatives
3.3.2. Results of the HPLC and UHPLC-MS analysis
Results of the HPLC analysis of the H. italicum re-extracts are given in
Total 99.0 99.3 99.7 99.2
Table 3. Comparative chromatograms of all investigated samples are
given in Fig. 3. Re-extracts obtained from textiles impregnated without
individual compounds, which mostly contributed to the chemical the addition of cosolvent were rich in several compounds, among which

7
S. Maksimovic et al. Journal of Ethnopharmacology 301 (2023) 115779

Table 2
List of compounds that mostly contributed to the chemical profile, identified by GC-MS analysis of H. italicum re-extracts isolated from the impregnated textiles.
Compounds RI Identified components’ area percentage in the chromatogram (%)

Re-extract 1 (cotton Re-extract 2 (cotton gauze with Re-extract 3 Re-extract 4 (PP with
gauze) cosolvent) (PP) cosolvent)

1 caproic acid 950 3.4 1.1 1.5 2.0

2 trans-β-caryophyllene 1417 1.5 0.5 0.5 0.4
3 neryl propanoate 1475 1.8 0.4 0.6 1.3
4 β-selinene 1489 5.8 1.2 3.4 3.0
5 methyl laurate 1524 1.8 0.2 2.3 1.2
6 1-hexadecane 1590 0.7 0.2 1.5 1.7
7 methyl tridecanoate 1622 1.6 1.2 0.3 0.7
8 methyl myristoleate 1683 0.4 1.8 0.3
9 xanthorrhizol 1751 1.0 0.3 1.1 1.2
10 methyl pentadecanoate 1813 0.4 1.2 3.2
11 methyl palmitate 1922 3.7 2.6 4.8 4.7
12 2-α-acetoxy-amorpha-4,7(11)-diene-8-one 1985 14.7 7.5 13.2 10.5
13 3,4-dihydro-4,4,5,7,8-penthamethyl coumarin- 2087 1.5 0.7 1.1 0.8
6-ol
14 methyl linoleate 2095 0.4 1.1 0.1 0.3
15 linoleic acid 2132 3.9 3.2 5.0 5.0
16 oleic acid 2141 3.9 1.2 4.5 2.2
17 2α-acetoxy-11-metoxy-amorpha-4,7-diene 2146 13.5 7.2 10.3 7.2
(derivative)
18 2,2-dimethyl-7,8-dimetoxychromanone 2206 6.1 2.8 4.2 2.6
19 tetracosane 2400 3.8 4.3 2.2 2.0
20 hexadecanoic acid, cyclohexyl ester 2439 1.4 1.5 2.1 1.9
21 pentacosane 2500 0.4 5.0 2.0 1.9
22 hexacosane 2600 1.4 8.1 0.7 1.6
23 eicosyl cyclohexane 2669 0.7 2.0 2.7 1.3
24 heptacosane 2700 1.9 9.9 3.8 4.0
26 nonacosane 2900 8.2 16.4 12.7 14.1
26 untriacontane 3100 10.9 17.5 11.3 20.2

the ones with the highest concentration were pyrogallol and flavonoids
naringenin, hesperetin, and chrysin. The addition of ethanol as cosol­
vent caused the appearance of a certain number of phenolic acids,
among which the most significant were chlorogenic, neochlorogenic,
caffeic, gentisic, p-coumaric and ferulic acid, and flavonols rutin, cyn­
arin, tiliroside, quercetin and kaempferol, and flavone luteolin. The
overall concentration of active components in the re-extract obtained
from sample 2 was significantly higher than in the re-extract obtained
from sample 4 (26.19 mg/gextract and 15.61 mg/gextract, respectively),
which is in accordance with obtained impregnation yields.
Common for all types of re-extracts was the detection of arzanol, the
prenylated heterodimeric pholoroglucinyl α-pyrone. The highest con­
centration of arzanol (8.93 mg/gextract) was obtained in the re-extract
obtained from sample 1, while the addition of ethanol caused a signif­
icant decrease in its concentration at the expense of flavonoids con­
centration increase.
Considering the results of the UHPLC-MS analysis, re-extracts ob­
tained from textiles impregnated without the addition of cosolvent were
rich in micropyrone derivatives, whereas the addition of cosolvent
caused the appearance of flavonoids and their derivatives detected by
HPLC. Arzanol was also identified in all samples. It is important to
mention that both of textile carriers showed almost the same affinity for
identified phenolic compounds, according to overall number of identi­
fied phenolic compounds present in the re-extracts. This could be seen
on the appropriate UHPLC-DAD chromatograms given on Fig. 4, where
the components are: 1 - chlorogenic acid (standard), 2 - gentisic acid
(Babota et al., 2018), 3 - caffeic acid derivative, 4 - myricetin-glycoside
(hexahydroxyflavone-glycoside), 5 - caffeic acid (standard), 6 - dihy­
droxycoumarin derivative, 7 - homovanillic acid, 8 – hyperoside (stan­
dard), 9 - cynarin, 10 - isoquercetin, 11 - dicaffeoylquinic acid isomer, 12
- kaempferol-3-O-glycoside (Czinner et al., 2002), 13 - iso­
rhamnetin-3-O-glycoside (Mari et al., 2014), 14–4,5-O-dicaffeoylquinic
acid methyl ester (Mari et al., 2014), 15 - ferullic acid derivative, 16 -
quercetin-hexoside, 17 - ferullic acid derivative, 18 – rutin (standard),
19 – quercetin (standard), 20 - quercetin-O-(p-coumaroyl glycoside)
(Iwashina and Matsumoto, 2013), 21 – luteolin (standard), 22 -
kaempferol-3 (or 7)-O-glycoside-7 (or 3)-rhamnoside, 24 – tiliroside
(Mari et al., 2014), 26 – kaempferol (Babota et al., 2018), 27 – oleoy­
loxylinalol (Appendino et al., 2007), 28 – jaceosidin (Babota et al.,
2018), 32 - oleoyloxylinalol-isomer, 34 – arzanol (standard), 36 - hel­
italone B, 23, 25, 29, 30, 31, 33, 35, 37–39 - micropyrone derivatives
(Appendino et al., 2007; Werner et al., 2019). Furthermore, represen­
tative ESI-MS/MS spectra of several detected compounds are given in
Figs. S2–S8 of the Supplementary material.
4. Discussion
Safety and efficacy study of treatment of the artificially irritated
human skin with textile materials impregnated with H. italicum extract
by innovative green technology, showed certain skin protective effect of
H. italicum supercritical extract. Furthermore, the application of super­
critical plant extracts as part of different skin protective preparations has
not been widely investigated in vivo. There are reports in the literature
that emphasize the high potential for wound healing of supercritical
extracts of juca (Libidia ferrea) (Dias et al., 2013), Thunbergia laurifolia
(Kwansang et al., 2015), mango leaves (Valor et al., 2021) and Sea
buckthorn (Hippophae rhamnoides L.) (Upadhyay et al., 2009). On the
other side, there are few articles that allow the use of supercritical plant
extracts as anti-irritant agents. Supercritical extracts of black currant
seeds, strawberry seeds, mint, and hops were proven as components of
shower gels that possess positive effects by Vogt et al. (2014). Super­
critical extract of strawberry seeds was also proven as a valuable
component of mild cleansing formulations by Sikora et al. (2015), while
black currant seeds supercritical extract showed positive effects as a part
of formulations for dishwashing (Wasilewski et al. (2016)). Finally,
Tadic et al. (2021) showed beneficial effects on the skin of wild bilberry
seeds supercritical extract as a part of oil-in-water cream. Thus, pro­
duction of high pressure processed H. italicum extract for topical appli­
cation could be of great interest for the future investigations of different
skin disorders.

8
S. Maksimovic et al. Journal of Ethnopharmacology 301 (2023) 115779

Table 3
List of identified compounds and classes detected by HPLC in the H. italicum re-extracts and their concentration (mg/gextract).
Compounds Re-extract 1 (cotton gauze) Re-extract 2 (cotton gauze with cosolvent) Re-extract 3 (PP) Re-extract 4 (PP with cosolvent)

1 Phloroglucinol derivatives, α-pyrones

arzanol 8.93 4.26 5.70 2.22
2 Flavonoids
Flavanones
naringenin 1.00 0.41 0.45 0.23
hesperetin 1.56 0.76 1.99 0.33

Flavonols
rutin 1.93 0.94
hyperoside 0.17 0.33
isoquercetin 0.87 0.86
kaempferol-3-O-glucoside 0.44 0.21
tiliroside 1.44 0.37
quercetin 0.53 0.16
kaempferol 0.80 1.47

Flavones
luteolin 0.50 0.31
chrysin 2.83 0.51 2.51 0.56

3 Polyphenolics
Phenolic acids – trans-cynnamic acid derivatives
neochlorogenic acid 0.75 0.83
chlorogenic acid 0.50 0.35
caffeic acid 0.67 0.32
p-coumaric acid 0.12 0.76 0.05 0.23
ferulic acid 2.19 0.73
cynarine 1.10 0.96

Phenolic acids – p-hydroxybenzoic acid derivatives

gallic acid 0.51 0.19 0.06 0.14
protocatechuic acid 0.34 0.37 0.12 0.43
homovanillic acid 0.28 0.05
gentisic acid 0.24 0.83 0.21

Other polyphenolic compounds

pyrogallol 5.62 5.39 2.17 1.55
coumarin 0.97

Total concentration of phloroglucinol 8.93 4.26 5.70 2.22

derivatives, α-pyrones
Total concentration of flavanones 2.56 1.17 2.44 0.56
Total concentration of flavonols 6.18 4.34
Total concentration of flavones 2.83 1.01 2.51 0.87
Total concentration of trans-cynnamic 0.12 5.97 0.05 3.42
acid derivatives
Total concentration of p-hydroxybenzoic 1.37 1.39 0.23 0.78
acid derivatives
Total concentration of other polyphenolic 5.62 5.39 2.17 2.52
compounds

Total 21.43 25.37 13.10 14.71

Based on the results of the in vivo studies, sample 4, PP fabric
impregnated in the SFE-SSI process with the addition of ethanol, was
proven to be the most appropriate material for the application on irri­
tated human skin from both safety and efficacy aspects. Sample 4 pro­
vided the best skin hydration and repairing skin barrier function
potential among the investigated samples. Accordingly, this result will
be commented in light of the obtained impregnation yields and the
chemical analysis results, and hypotheses for future studies will be
derived.
The integrated SFE-SSI process resulted in a lower impregnation
yield when PP fabric was impregnated with the addition of cosolvent
(1.97%) than the cotton gauze (4.25%), emphasizing the hydrophobic­
ity of PP fibers (unlike the cotton, which possesses hydroxyl groups). In
other words, in the experiments with the cosolvent, the more
hydrophilic extract was obtained that was more attracted to hydrophilic
cotton gauze. On the other hand, PP fabric showed a higher affinity for
the impregnation with H. italicum extract than cotton gauze in experi­
ments without adding ethanol, which is in accordance with the hydro­
phobic nature of both PP fibers and the extract isolated with pure scCO2.
In general, there are two impregnation mechanisms with scCO2. The first
is based on the electrostatic interaction between the functional groups of
the substrate and impregnate (e.g., hydrogen bonding, van der Waals
forces). The second is based on the pure physical entrapment of the
impregnated substance in the solid matrix due to the decompression
when its solubility in carbon dioxide rapidly decreases. The results of
GC-MS, HPLC, and UHPLC-DAD analyses indicated similar chemical
profiles of re-extracts from samples 2 (cotton gauze in the experiment
with ethanol) and 4 (PP fabric in the experiment with ethanol). Keeping

9
S. Maksimovic et al. Journal of Ethnopharmacology 301 (2023) 115779

Fig. 3. Comparative HPLC chromatograms of investigated impregnated samples (presented at 280 nm).

Fig. 4. Representative mirrored UHPLC-DAD chromatograms at 280 nm detection wavelength of: a) re-extracts obtained from samples 1 and 3 and b) re-extracts
obtained from samples 2 and 4.

in mind the lower impregnation yield obtained when PP fabric was
impregnated with the addition of cosolvent (sample 4; 1.97%), and PP
hydrophobic nature, it could be assumed that the physical deposition of
the extract’s components during the decompression is more expressive
in sample 4 than sample 2. That would also mean the migration of the
impregnated components from sample 4 would be easier. Indeed, the
best potential for skin hydration and skin repairing of sample 4 could be
the consequence of the unhampered migration of the active components
from the impregnated PP fabric.
Results of the chemical analyses of re-extracts by GC-MS and HPLC
methods revealed that the re-extract of sample 4 contained numerous
active components. The data about their topical activity can be found in
the literature. Han et al. (2017) confirmed an inhibitory activity of the
essential oil of H. italicum obtained by hydrodistillation on human
dermal fibroblast tissue remodeling-related proteins, suggesting a
wound healing property. Among the identified compounds by GC-MS
from H. italicum, the most abundant were terpenes neryl acetate,
γ-curcumene, and α-pinene. The treatment with α-pinene effectively
prevented UVA-induced photoaging in mouse skin in the work of Kar­
thikeyan et al. (2019). Also, sesquiterpenoid xanthorrhizol, which could

10
S. Maksimovic et al.
be found in H. italicum isolates (Kladar et al., 2015; Marongiu et al.,
2003; Ivanovic et al., 2011) showed significant skin anti-aging effect (Oh
et al., 2009) and anti-inflammatory effect against mouse skin carcino­
genesis (Park et al., 2003; Chung et al., 2007). Finally, the topical
application of coumarin had beneficial effects on different phases of
wound healing in the skin of BALB/c mice, as reported in the work of
Afshar et al. (2020). Among the listed flavonoids, several possess re­
ported topical activity. For example, quercetin was proven to reduce
inflammation by decreasing edema, leukocyte formation, and irritation
(Gupta et al., 2016). Kaempferol acts as a novel agent in treating the
UVB-induced tumorigenesis and photo-inflammation (Lee et al., 2010).
Hesperetin possesses strong anti-inflammatory activity, like the ability
to reduce hyperpigmentation caused by UV rays, producing a whitening
effect (Cho, 2006). Finally, luteolin is very effective in treating arthritis
(Aziz et al., 2018). Furthermore, caffeic and dicaffeoylquinic acid,
quercetin, and kaempferol and their derivatives were identified as a part
of H. italicum ethanolic extracts and showed considerable antioxidant
activity in the work of Facino et al. (1990) and Kladar et al. (2015).
Arzanol is well-known for its strong anti-inflammatory (Appendino
et al., 2007; Bauer et al., 2011) and antioxidant activity (Rosa et al.,
2007; 2011; 2017).
Considering chemical profiles of the samples 2 and 4, obtained by
GC-MS and HPLC analyses, it could be noticed that the re-extract of
sample 4 possessed higher content of terpenes and coumarin and
amorphene derivatives than the same of sample 2 (Table 1), but
significantly lower content of phenolic compounds (Table 2). Within the
phenolic compounds, it could also be noticed that the differential con­
tent of both types of phenolic acids and flavones, flavonols and flava­
nones was significantly lower in re-extract 4 than in re-extract 2
(Table 2). This could lead to the hypothesis of the dominant effect of
certain compound groups on human skin. In the case of sample 4, it
could be assumed that mono- and sesquiterpenes, with the lowest mo­
lecular mass among all identified active components, could play the
most significant role in the topical activity of sample 4. This hypothesis
is supported by the fact that re-extract 4 possessed a significantly lower
concentration of phenolic compounds than re-extract 2.
5. Conclusion
In this work, the skin protective effect of the impregnated textile
materials with supercritical extract of H. italicum was investigated. The
integrated process of supercritical CO2 extraction from H. italicum and
supercritical impregnation of cotton gauze and polypropylene non-
woven fabric with the extract was performed under 350 bar and
40 ◦ C, with and without the addition of ethanol as a cosolvent.
As continuation of the previously published study, in which the
feasibility of the impregnation of cotton gauze and PP non-woven fabric
with the H. italicum extract by the integrated SFE-SSI process was
proven, this work was focused on the explanation of different effects of
impregnated textiles on artificially irritated skin by introducing certain
hypotheses. The hypotheses were proposed with aim to connect the ef­
fects of the impregnated textile materials on irritated skin with chemical
profile of incorporated extracts and physical nature of individual textile
material.
Impregnated textile materials were subjected to the in vivo studies
performed on human volunteers. Results of the safety study indicated
that all impregnated samples were safe after application with no cause of
any irritation and redness. The efficacy study showed that PP fabric,
impregnated with H. italicum extract with the addition of ethanol as a
cosolvent, possessed significantly greater potential in increasing skin
hydration, repairing skin barrier function (decreasing TEWL from stra­
tum corneum), and decreasing MI and EI values of the irritated skin in
comparison to other investigated samples. Physical deposition of the
extract and weak van der Waals attraction forces between the extract’s
active compounds and carrier and hydrophobic PP nature contributed to
the unhampered migration of the active principles from the PP fabric.
11
Journal of Ethnopharmacology 301 (2023) 115779
Furthermore, dominant effect on human skin of mono- and sesquiter­
penes of incoroporated extract in PP fabric, over phenolic compounds,
could also influenced the high topical activity of PP fabric.
The obtained results proved the high-pressure processed H. italicum
extract suitable for developing human skin protective preparations and
indicated PP fabric as an appropriate carrier.
Credit author statement
Svetolik Maksimovic: Conceptualization, Software, Formal anal­
ysis, Investigation, Data curation, Writing – original draft preparation,
Writing – review & editing, Visualization, Project administration; Milica
Stankovic: Software, Formal analysis, Data curation, Writing – original
draft preparation; Sonja Roganovic: Software, Formal analysis, Data
curation, Writing – original draft preparation; Ivana Nesic: Conceptu­
alization, Methodology, Validation, Investigation, Resources, Funding
acquisition; Jelena Zvezdanovic: Software, Formal analysis, Data
curation; Vanja Tadic: Conceptualization, Methodology, Software,
Validation, Formal analysis, Investigation, Resources, Writing – review
& editing, Supervision, Funding acquisition; Irena Zizovic: Conceptu­
alization, Methodology, Validation, Investigation, Resources, Writing –
review & editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Contract No. 451-
03-68/2022-14/200135, No. 451-03-68/2022-14/200003 and No. 451-
03-68/2022-14/200133) and by the Project No. E!13632 in Eureka
Programs.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2022.115779.
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