Article

Preliminary Analysis of Bilberry NaDES Extracts as Versatile

Active Ingredients of Natural Dermocosmetic Products: In Vitro
Evaluation of Anti-Tyrosinase, Anti-Hyaluronidase,
Anti-Collagenase, and UV Protective Properties
Milica Martinović 1 , Ivana Nešić 1 , Ana Žugić 2 and Vanja M. Tadić 2, *

1 Department of Pharmacy, Faculty of Medicine, University of Niš, Boulevard Dr. Zorana Djindjića 81,
18108 Nis, Serbia; milica.martinovic@medfak.ni.ac.rs (M.M.); ivana.nesic@medfak.ni.ac.rs (I.N.)
2 Department for Pharmaceutical Research and Development, Institute for Medicinal Plant Research “Dr. Josif
Pančić”, Tadeuša Koscuška 1, 11000 Belgrade, Serbia; azugic@mocbilja.rs
* Correspondence: vtadic@mocbilja.rs

Abstract
Bilberry (Vaccinium myrtillus L.) fruits represent the recognized wellspring of bioactive
compounds with various documented bioactivities. Although bilberry leaves are often
treated as industrial by-products, they also represent a valuable source of phytochemicals
with potential dermocosmetic applications. In this study, extracts of bilberry fruits and
leaves were prepared using both conventional solvents (water and 50% ethanol) and natu-
ral deep eutectic solvents (NaDES) as green, biodegradable alternatives. The aim of this
study was to examine the UV protective activity and inhibitory potential of those extracts
against some enzymes (tyrosinase, hyaluronidase, collagenase) that are important in terms
of skin conditioning and skin aging. The results of in vitro tests have shown the superiority
of NaDES extracts compared to conventional extracts regarding all tested bioactivities.
In addition, bilberry leaves extracts were more potent compared to fruit extracts in all
cases. The most potent extract was bilberry leaf extract made with malic acid–glycerol,
Academic Editor: Maria Iorizzi
which exhibited strong anti-tyrosinase (IC50 = 3.52 ± 0.26 mg/mL), anti-hyaluronidase
Received: 27 June 2025
(IC50 = 3.23 ± 0.30 mg/mL), and anti-collagenase (IC50 = 1.84 ± 0.50 mg/mL) activities.
Revised: 21 July 2025
The correlation analysis revealed correlation between UV protective and anti-tyrosinase, UV
Accepted: 24 July 2025
Published: 1 August 2025 protective and anti-collagenase as well as between anti-hyaluronidase and anti-collagenase
activity. UV protection and anti-tyrosinase activity correlated significantly with chloro-
Citation: Martinović, M.; Nešić, I.;
Žugić, A.; Tadić, V.M. Preliminary genic acid and hyperoside contents in extracts. The extracts with the best activities also
Analysis of Bilberry NaDES Extracts as demonstrated a good safety profile in a 24 h in vivo study on human volunteers.
Versatile Active Ingredients of Natural
Dermocosmetic Products: In Vitro Keywords: sun protection; anti-tyrosinase; anti-hyaluronidase; anti-collagenase; NaDES;
Evaluation of Anti-Tyrosinase, Anti- anti-age; enzyme; inhibition; bilberry
Hyaluronidase, Anti-Collagenase, and
UV Protective Properties. Plants 2025,
14, 2374. https://doi.org/10.3390/
plants14152374
1. Introduction
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
Healthy skin is elastic and radiant, and has youthful and fresh appearance, while
This article is an open access article damaged skin looks pale, tired, and dehydrated, which is especially evident in the thinnest
distributed under the terms and areas, such as areas around the eyes. Over recent decades, the cosmetic industry has
conditions of the Creative Commons undergone significant changes, driven by an improved understanding of healthy skin
Attribution (CC BY) license physiology and advancements in research methodologies. As a result, there has been
(https://creativecommons.org/
progress in the discovery of new active ingredients and carriers, based on well-studied
licenses/by/4.0/).

Plants 2025, 14, 2374 https://doi.org/10.3390/plants14152374

Plants 2025, 14, 2374 2 of 21

mechanisms of action, leading to noticeable advancements in the development of modern

cosmetic and dermocosmetic products. In addition, there is a growing emphasis on the
sustainability, biodegradability, and environmental compatibility of cosmetic products as
a new trend in the industry, aligning with rising consumer awareness about health and
ecological impact. The increasing demand for “green” and “natural” cosmetics stems not
only from environmental concerns but also from safety issues, as conventional cosmetic
ingredients such as UV filters, fragrances, and preservatives are associated with adverse
effects, including allergic contact dermatitis [1].
As a rich source of bioactive substances, such as flavonoids, tannins, anthocyanins,
carotenoids, and vitamins, plant extracts exhibit significant antioxidant properties. Further-
more, they serve as functional ingredients that can have additional dermocosmetic effects,
including anti-inflammatory, antibacterial, UV protective, hypopigmenting, emollient, and
moisturizing effects. Their multifunctionality makes them valuable ingredients in formula-
tions designed to protect the skin from environmental stressors, combat aging, hydrate dry
skin, treat acne, and improve overall skin condition [2].
Bilberry (Vaccinium myrtillus L., Ericaceae) is a low-growing shrub abundant in pheno-
lic compounds with known anti-inflammatory, antioxidant, and metabolic health effects.
While bilberry fruits (BFs) are commonly used in traditional medicine, the leaves (BLs)
and seeds are typically discarded as industrial by-products. However, recent studies have
revealed that BLs contain high level of bioactive phytochemicals and exhibit significant
antioxidant activity. Moreover, they show promising skin-rejuvenating activity by en-
hancing the expression of genes in fibroblasts and stimulating synthesis of hyaluronic
acid and gluthatione [3]. As for dermocosmetic purposes, the use of BF is traditionally
associated with antiseptic, adstrigent, and tonic activities [4]. The studies have confirmed
skin-lightening, anti-inflammatory, anti-aging, antioxidant, UV protective, antimicrobial
and chemo-preventive effects on BF [5]. In addition, both Vaccinium Myrtillus Fruit
Extract and Vaccinium Myrtillus Leaf Extract are listed in the Cosing Database as skin-
conditioning agents [6].
In our previous research, we prepared extracts of BF and BL using natural deep
eutectic solvents (NaDES), green solvents formed by combination of sugars, amino acids,
and other plant metabolites, resulting in a mixture with a melting point lower than each of
their individual components. Such extracts are environmentally friendly, biodegradable,
safer, richer and more effective compared to extracts obtained using conventional solvents.
Previous reports have shown that NaDES can enhance solubility, stability, bioavailability,
and even dermal delivery of active compounds from the extracts [7]. In addition, NaDES
have been used in topical formulations, like gels, creams of emulgels, and improved drug
loading and skin penetration [8].
In this study, NaDES composed of alpha-hydroxy acids (AHAs) and polyols were
used: malic acid + glycerol (MG), tartaric acid + sorbitol (TS), and citric acid + sorbitol
(CS). NaDES not only facilitate the extraction of active compounds but also contribute
beneficially to the biological activity of the final product—unlike organic solvents, which
are toxic, or ethanol, which can irritate the skin, or water, which often requires the addition
of preservatives. Our previous study indicated that NaDES extracts had higher polyphenol
content and exhibited better antioxidant activity compared to water and ethanol extracts [9].
Numerous enzymes such as tyrosinase, hyaluronidase, collagenase, elastase, and oth-
ers are present in the skin, and contribute to its structure and function. When overexpressed,
they can contribute to skin aging and pigmentation disorders. Their activity may increase
naturally over time or be induced by external stress factors such as UV radiation, making
their inhibition a promising anti-aging strategy [10]. In addition, excessive exposure to UV
radiation may lead to accelerated skin aging due to the remodeling of the skin immune
Plants 2025, 14, 2374 3 of 21

system and damaging of DNA and skin protein structures. UV rays may also cause local in-
flammation and activation of pro-inflammatory cytokines [11]. Therefore, photoprotection
is an important aspect of proper skin care [12].
The aim of this study was to evaluate the in vitro inhibitory effects of NaDES-based
extracts from bilberry leaves and fruits against tyrosinase, hyaluronidase, and collagenase—
key enzymes involved in melanin production and degradation of extracellular matrix
components like hyaluronic acid and collagen. Additionally, the goal of the study was to
examine the UV protective effect of NaDES extracts and to compare all the determined
activities between NaDES extracts and conventional extracts (water and ethanol), as well as
between BFs and BLs, taking into account that the BL is mostly obtained as BF processing
waste. All assessed biological activities were evaluated with respect to chemical compo-
sition of the prepared extracts. Finally, the aim was also to determine whether there is a
correlation between obtained results of the biological activities and chemical composition
of the extracts.

2. Results and Discussion

In the experiments presented in this paper, ten extracts were prepared (Figure 1) using
ultrasound-assisted extraction (UAE) as the contemporary extraction method, which is
commonly employed with NaDES as extraction solvents. This extraction method is based
on high-frequency ultrasound that causes cavitation and enhances cell wall permeability,
thus increasing mass transfer. In this way, less energy is used, the extraction time is
shortened, and carbon emission is lowered. In addition, obtained extracts have a higher
yield of bioactive compounds [13].

Figure 1. The bilberry fruits (BF) and leaves (BL) extracts prepared using NaDES as green extraction
solvents (TS, CS, MG) and water (W) and 50% ethanol (E) as conventional solvents, used in this study.

Two groups of the prepared extracts—BF (bilberry fruit extracts) and BL (bilberry
leaves extracts) were obtained using five extraction solvents: water (W) and 50% ethanol (E)
as conventional solvents and malic acid–glycerol (1:2 mol/mol) (MG), tartaric acid–sorbitol
(1:2 mol/mol) (TS), and citric acid–sorbitol (1:2 mol/mol) (CS) as NaDES.
Even though solvents that were used were transparent liquids (E, W, TS, CS, MG), the
obtained extracts were colored (Figure 1). Unlike NaDES extracts that were transparent,
ethanol and especially water extracts were not.
Plants 2025, 14, 2374 4 of 21

There are several previous reports of NaDES used as extraction solvents for BF extrac-
tion. For instance, ten different choline chloride-based NaDES were prepared, and choline
chloride–sorbitol (1:1 mol/mol) was selected as the best choice for anthocyanin extraction
and obtaining extracts with promising antioxidant and antimicrobial activity [14]. In addi-
tion, other NaDES made of choline chloride–glycerol–citric acid (1:4:1 mol/mol/mol) were
as efficient as conventional organic solvent (methanol–water–formic acid) for anthocyanin
extraction [15]. Choline chloride-based NaDES were also used for the extraction of bilberry
peels [16]. However, choline chloride is restricted for use in cosmetics due to Regulation
(EC) No 1223/2009, so other NaDES should be used for preparing extracts intended for
cosmetic purposes [17].
When it comes to BL, in previous studies, 18 NaDES were prepared and tested for BL
extraction. The best extraction was achieved using following NaDES: lactic acid–sodium
acetate–water (3:1:2), and choline chloride–oxalic acid (1:1) [18].
To the best of our knowledge, so far NaDES extracts of BF and BL were not tested for
UV protective, anti-tyrosinase, anti-hyaluronidase, and anti-collagenase activity.

2.1. HPLC Analysis

The predominant phenolic compounds identified by HPLC analysis in tested extracts
included flavonols (rutin, hyperoside, quercetin-3-O-glucoside), hydroxycinnamic acids
(chlorogenic acid), flavanols (epicatechin, procyanidin B2), p-hydroxybenzoic acids (pro-
tocatechuic acid), and anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside,
cyanidin-3-O-galactoside). The results of the chemical (HPLC) analysis of bilberry fruit and
leaves extracts were presented in Table 1.

Table 1. Phenolic compounds identified in the analyzed extracts using HPLC. (Within each row,
means labeled with different letters (e.g., a, b, c, d, e) differ significantly at p < 0.05. Means that share
the same letter are not significantly different from one another).

Phenolic Compounds (mg/g) TS-BF CS-BF MG-BF E-BF W-BF

a a a b
Chlorogenic acid 1.53 ± 0.11 1.51 ± 0.01 1.47 ± 0.37 0.83 ± 0.03 0.86 ± 0.06 b
Protocatechuic acid 1.48 ± 0.39 ab 1.30 ± 0.28 ab 1.35 ± 0.15 ab 1.03 ± 0.02 ab 0.94 ± 0.04 b
Delphinidin-3-O-glucoside 1.07 ± 0.02 a 0.99 ± 0.05 a 1.01 ± 0.05 a tr tr
Hyperoside 0.49 ± 0.01 a 0.50 ± 0.07 a 0.57 ± 0.06 a 0.53 ± 0.03 a 0.15 ± 0.03 b
Cyanidin-3-O-glucoside 0.19 ± 0.01 a 0.18 ± 0.01 a 0.19 ± 0.02 a tr tr
Cyanidin-3-O-galactoside 0.10 ± 0.04 a 0.11 ± 0.03 a 0.10 ± 0.03 a tr tr
Phenolic Compounds (mg/g) TS-BL CS-BL MG-BL E-BL W-BL
Chlorogenic acid 19.48 ± 0.92 ab 22.48 ± 1.57 ab 21.17 ± 1.75 ab 22.51 ± 0.50 a 8.37 ± 0.06 c
Procyanidin B2 7.57 ± 1.16 a 15.77 ± 0.68 b 18.59 ± 1.23 c 14.41 ± 0.80 b nd
Quercetin-3-O-glucoside 6.76 ± 0.16 a 8.29 ± 0.12 b 8.02 ± 0.08 b 9.58 ± 0.22 c 4.88 ± 0.20 d
Rutin 3.16 ± 0.05 a 3.60 ± 0.26 bc 3.24 ± 0.10 ab 3.79 ± 0.12 c 1.93 ± 0.15
Hyperoside 2.21 ± 0.07 a 2.85 ± 0.09 b 2.57 ± 0.11 c 3.31 ± 0.10 d 1.32 ± 0.11 e
Quercitrin 0.69 ± 0.04 ab 0.90 ± 0.06 cd 0.76 ± 0.05 ad 0.99 ± 0.10 c 0.56 ± 0.07 be
Epicatechin 0.51 ± 0.09 a 1.74 ± 0.08 b 1.61 ± 0.27 b 1.92 ± 0.13 b nd

2.2. UV Protective Activity

Ultraviolet (UV) radiation is one of the most impactful external factors—collectively
referred to as exposomes—that contribute to skin damage and accelerate the aging process.
Even the term “photoaging” was established to emphasize this relationship. Since pho-
toaging affects both epidermis and dermis, regular use of sunscreens is recommended for
delaying this process [19].
There are various methods for in vitro assessment of UV protective activities of
herbal extracts that are commonly used due to simplicity, cost-effectiveness, as well as
ethic and safety concerns. In this study, we applied a dilution method to measure ab-
Plants 2025, 14, 2374 5 of 21

sorbance of diluted solutions and calculate the Sun Protection Factor (SPF) using Mansur
Equation (1) [20].
The results of our study revealed that NaDES extracts exhibited higher UV protective
potential compared to water and ethanol extracts (p < 0.05) (Figure 2). Sole solvents (W, E,
TS, CS, MG) showed no significant absorption in UV spectrum, so they were not portrayed
in Figure 2.

Figure 2. UV protective activity (SPF) of tested bilberry fruits (BF) and leaves (BL) extracts prepared
using NaDES as extraction solvents (TS, CS, MG) and water (W) and 50% ethanol (E) as conventional
solvents. As control, 8% homosalate (HMS) was used. (Different letters indicate statistically significant
differences (p < 0.05). Any extracts that share the same letter are not significantly different from
each other).

The enhanced efficacy of BF extracts prepared with NaDES can be attributed to the
presence of anthocyanins, which are responsible for UV protective potential of BF [5].
Anthocyanins detected in tested extracts were delphinidin-3-O-glucoside, cyanidin-3-O-
galactoside, and cyanidin-3-O-glucoside (Table 1). Unlike water and ethanol extracts,
where they were present in traces, they were detected in NaDES extracts (MG, TS, and
CS) in notable amounts (Table 1), which might explain why NaDES BF extracts (MG-BF,
TS-BF, and CS-BF) had similar SPFs, higher than the SPF of water and ethanol BF extracts
(p < 0.05).
Previous studies have shown that BF extracts can protect human keratinocytes (HaCaT
cells) from UVA-induced lipid peroxidation and glutathione depletion [21]. Furthermore,
BF reduced reactive oxygen species (ROS) generation and apoptosis induced by UVA rays
as well as UVB rays mediated lipid peroxidation in HaCaT keratinocytes [22,23]. SPF values
for bilberry extracts reported in the literature were different depending on the solvent used.
While lipophilic chloroform extract showed SPF 3.6 [24], which is close to the SPF measured
in our study, some other authors even measured a 10 times higher SPF (34.5) [25]. However,
a high value of SPF like this is unusual for herbal extracts.
Interestingly, the results of our study also indicated that leaves have more potent UV
protection potential compared to fruits, as all BL extracts exhibited higher SPF values and
a higher phenolic content than their corresponding BF extracts (p < 0.05). The highest
measured SPF in the study was detected for MG-BL (8.12 ± 0.42) followed by CS-BL
(7.62 ± 0.20).
To the best of our knowledge, there are no studies where the UV protective activity of
BL was estimated. However, there are studies that indicate the protective role of flavonoids
Plants 2025, 14, 2374 6 of 21

and cinnamic acids against UV radiation in plants [26] which might explain why BL extracts
richer in phenolic compounds (Table 1) have higher SPF values compared to BF.
To sum up, the SPF calculated using the in vitro dilution method and Mansur
Equation (1) showed higher UV protective potential of bilberry leaves compared to bilberry
fruits. In addition, NaDES extracts showed significantly higher SPF values than water
and ethanol extracts (p < 0.05), likely due to a higher content of anthocyanins and other
phenolic compounds. The extract with the highest calculated SPF was MG-BL.

2.3. Anti-Tyrosinase Activity

Tyrosinase is an enzyme that plays important role in melanogenesis, which, to an
excessive extent, leads to abnormal melanin accumulation, and thus the formation of
various types of freckles, age spots, hyperpigmentation, and melasmas. Although melanin
serves as a natural photoprotective pigment and natural skin UV filter, its overproduction
leads to undesirable aesthetic effects [10].
The anti-tyrosinase assay used in the study was based on the formation of char-
acteristically colored dopachrome, resulting from the reaction between L-DOPA and
mushroom tyrosinase [10]. The positive control used in the study was ascorbic acid
(IC50 = 0.34 ± 0.18 mg/mL).
All BL extracts have shown better tyrosinase inhibitory activity compared to corre-
sponding BF extracts (p < 0.05). The reason for this might be higher phenolic content
in BL extracts. For instance, among phenolic compounds identified by HPLC analysis,
chlorogenic acid as tyrosinase inhibitor [27] was identified in an almost 20 times greater
amount in BL extracts than in BF extracts (Table 1). Also, hyperoside, identified as an tyrosi-
nase inhibitor by Li et al. [28], was detected in higher concentrations in BL extracts (from
1.32 ± 0.11 to 3.31 ± 0.10 mg/g) than in BF extracts (from 0.15 ± 0.03 to 0.57 ± 0.06 mg/g)
(Table 1). In addition, other compounds known for tyrosinase inhibitory activity were
identified in significant amounts in BL extracts, like quercetin-3-O-glucoside [29], rutin [30],
and procyanidin B2 [31].
NaDES extracts exhibited superior tyrosinase inhibitory activity compared to con-
ventional extracts, with the exception of TS-BL (Figure 3). Among all the extracts, the
best inhibitory activity was observed for MG-BL whose IC50 was 3.52 ± 0.26 mg/mL.
Within both the BF and BL groups, MG-extracted samples consistently showed the greatest
inhibitory effect (p < 0.05).
While W and E as negative controls did not inhibit tyrosinase, MG, TS, and CS
showed low inhibitory activity with IC50 values higher compared to the IC50 values of
corresponding extracts (Figure 3). So far, previous studies have shown that malic and citric
acid have tyrosinase-inhibitory potential [32,33]. Additionally, glycerol has been shown to
enhance tyrosinase inhibition when used with flavonoid complexes, as demonstrated in
Sophora japonica L. extracts [34].
The hypopigmentation effects of bilberry extracts have been previously reported.
For instance, some commercially manufactured juices from BF have shown comparable
tyrosinase inhibitory potential at the concentration of 1 mg/mL as 0.02 mg/mL kojic
acid [35]. Another study showed that water, methanol, methanol–water (1:1, v/v), and
acetone–water (1:1, v/v) bilberry fruit extracts inhibited less than 50% of tyrosinase activity
at 10 mg/mL [36], suggesting that the NaDES approach used in the present study yielded
more potent inhibitory profiles.
Overall, the tyrosinase inhibitory assay revealed that all BL extracts showed sig-
nificantly stronger tyrosinase inhibition than BF extracts and that the NaDES extracts
(especially MG-BL) exhibited the most potent inhibitory activity.
Plants 2025, 14, 2374 7 of 21

Figure 3. Anti-tyrosinase activity (IC50 values) of tested bilberry fruits (BF) and leaves (BL) extracts
prepared using NaDES as extraction solvents (TS, CS, MG) and water (W) and 50% ethanol (E) as
conventional solvents. As positive control, ascorbic acid was used, while clean solvents (TS, CS, MG,
W, and E were used as negative control. W and E showed no anti-tyrosinase activity). (Different
letters indicate statistically significant differences (p < 0.05). Extracts that share the same letter are not
significantly different from each other).

2.4. Anti-Hyaluronidase Activity

Hyaluronic acid is the main component of the extracellular matrix and probably the
world’s most famous glycosaminoglycan. Owing to its remarkable hygroscopic nature—it
can retain up to 1000 times its weight in water—hyaluronic acid plays a crucial role in
maintaining skin hydration in both the epidermis and dermis and preserving the youthful
and healthy being of the skin. It consists of D-glucuronic acid and D-N-acetylglucosamine
linked via β-1,3 and β-1,4 bonds. The enzyme hyaluronidase, which is naturally present
in the human body, is responsible for breaking down this macromolecule into monosac-
charides. Excessive hyaluronidase activity is associated with reduced skin moisture, loss
of elasticity, and the formation of wrinkles. Therefore, the inhibition of hyaluronidase
represents significant tool in anti-aging strategies [37,38].
The hyaluronidase inhibition assay is based on a turbidimetric method, which
quantifies the turbidity caused by residual hyaluronic acid that remains undegraded by
hyaluronidase in the presence of an enzyme inhibitor (herbal extract). Therefore, greater
turbidity corresponds to stronger hyaluronidase inhibition [10].
The hyaluronidase inhibitory activity was portrayed in Figure 4. The best hyaluronidase
inhibitory activity was observed for BL-MG (IC50 = 3.23 ± 0.30 mg/mL). Across both BF
and BL groups, all NaDES extracts demonstrated lower IC50 values (p < 0.05) than their
water or ethanol counterparts (p < 0.05), indicating enhanced enzyme inhibition. The
components identified in the extracts (Table 1) that might have contributed to this ac-
tivity, as suggested in previous investigations, were chlorogenic acid [39], rutin [40,41],
quercetin-3-O-glucoside [41], epicatechin [42], and protocatechuic acid [43],
Plants 2025, 14, 2374 8 of 21

Figure 4. Anti-hyaluronidase activity (IC50 values) of tested bilberry fruits (BF) and leaves (BL)
extracts prepared using NaDES as extraction solvents (TS, CS, MG) and water (W) and 50% ethanol
(E) as conventional solvents. As control, tannic acid was used. (Different letters indicate statistically
significant differences (p < 0.05). Extracts that share the same letter are not significantly different from
each other).

Among tested solvents, besides water and 50% ethanol, NaDES exhibited very low
inhibiting activity against hyaluronidase (359.83–439.05 mg/mL) indicating minimal in-
fluence on the overall activity of the extracts. To the best of our knowledge, there are no
available data on the influence of AHAs or glycerol on hyaluronidase. However, some
studies report that sorbitol can stabilize hyaluronic acid and enhance its resistance to
enzymatic degradation [44]
Among all extracts, those prepared with 50% ethanol exhibited the weakest activity,
with IC50 values were significantly higher than those of NaDES extracts. Notably, the BL
ethanol extract showed a nearly eightfold higher IC50 value compared to the MG extract.
Our extracts showed better hyaluronidase inhibitory properties than the BF water–
acetone extract from the literature data, which was able to inhibit 90% of enzyme’s activity
at concentration 50 mg/mL [36]. However, our extracts had higher IC50 values compared
to the positive control, tannic acid (IC50 = 0.026± 0.02 mg/mL).
Generally speaking, the turbidimetric assay revealed hyaluronidase inhibitory activity
of bilberry fruit (BF) and leaf (BL) extracts made with all extraction solvents used in the
study. While ethanol extracts had the weakest activity, NaDES extracts, especially BL-
MG, had lower IC50 values and were more active in terms of hyaluronidase inhibition.
Compared to the literature data, these extracts demonstrated stronger inhibitory activity
than some previously tested BF extracts but were still less effective than the positive control,
tannic acid.

2.5. Anti-Collagenase Activity

The collagenase inhibition assay in this study was based on the spectrophotometric
method developed by Zhang et al. [45], which is simple, reliable, and economic. Unlike
other methods that use radiolabeled or fluorescence-labeled collagen, it does not require
Plants 2025, 14, 2374 9 of 21

specialized instruments or generate radioactive waste. This assay is based on the colori-
metric ninhydrin reaction with free amino acids released after the degradation of collagen,
since the absorbance of obtained “Ruhemann’s purple” at 570 nm is proportional to the
concentration of free amino acids [45].
Collagen, a key protein in the extracellular matrix, makes up nearly 75% of the skin’s
dry weight. As the most abundant skin structural protein, it plays a crucial role in preserv-
ing not only the skin’s structure and function but also its youthful appearance. Collagen
provides strength and stability to skin tissue by enabling collagen fibrils to slide and realign,
allowing the skin to stretch and move without losing its integrity or sustaining damage.
Collagenase is the enzyme present in skin responsible for the degradation of collagen, a
process that naturally occurs as a consequence of aging or is stimulated by the influence
of external factors like UV radiation or smoking. As a result, collagen is being degraded
and the skin has a wrinkled and toneless look [46]. Therefore, screening for new natural
collagenase inhibitors is an important strategy in anti-aging skincare research [47].
The results of the collagenase inhibition assay were presented in Table 2. The extracts
of BL had lower IC50 values than BF extracts, indicating that leaves demonstrated better
collagenase inhibition activity compared to the fruits (p < 0.05). The best anti-collagenase
activity was observed for MG-BL, the extract that also exhibited the best UV protective,
anti-hyaluronidase, and anti-tyrosinase activity.
Among phenolic components identified in BF and BL extracts (Table 1), those with
known collagenase inhibition potential were rutin [40], hyperoside [48], protocatechuic
acid [43], chlorogenic acid [49], and quercetin [50].
Furthermore, NaDES extracts showed better inhibitory potential than water and
ethanol extracts, whose IC50 values were very high, indicating that NaDES components
themselves contribute to collagenase inhibition. Malic acid, tartaric acid, and citric acid are
commonly used AHAs—weak organic acids, with hydroxyl group attached to the carbon
in alpha position [51]. The study on the human skin of Japanese subjects revealed that citric
acid can induce the proliferation of collagen I and procollagen II. The same was observed
for other AHAs—glycolic and lactic acid [52]. Additionally, glycerol as a component of used
NaDES was studied as extraction solvent for obtaining extracts with notable collagenase
inhibitory activity [47]. The presence of glycerol in extracts can prevent the denaturation of
collagen [53].

Table 2. Anti-collagenase activity (IC50 values) of tested bilberry fruits (BF) and leaves (BL) extracts
prepared using NaDES as extraction solvents (TS, CS, MG) and water (W) and 50% ethanol (E) as
conventional solvents. (Different letters indicate statistically significant differences (p < 0.05). Extracts
that share the same letter are not significantly different from each other. n.d. is an abbreviation for
not detected).

Solvents
IC50 (mg/mL) BF BL
(Negative Control)
TS 3.20 ± 0.03 ab 2.05 ± 0.40 d n.d.
CS 3.67 ± 0.25 bc 2.63 ± 0.14 ad n.d.
MG 3.02 ± 0.36 c 1.84 ± 0.50 e n.d.
W >10 f >10 f n.d.
E >10 f >10 f n.d.
Rutin 0.35 ± 0.13 g (positive control)

The results of our study indicated that none of the solvents used in the study expressed
collagenase inhibition activity (Table 2). However, due to the chemical interactions between
NaDES solvents and compounds in extracts, it is possible that they might have contributed
Plants 2025, 14, 2374 10 of 21

to the observed extracts’ activity. Several studies support the role of NaDES in enhancing
collagenase inhibition. For example, choline chloride–glycerol and choline chloride–urea
extracts of green tea showed excellent collagenase inhibition [54]. Proline–glycerol Jasione
montana extract, rich in phenolic compounds, was an even stronger collagenase inhibitor
compared to gallic acid [55]. Grape pomace extract obtained via the betaine–glucose
mixture demonstrated better collagenase inhibition compared to ethanol extract [56].
Altogether, as observed in the other tests, the bilberry leaves were more potent than
the fruits. However, unlike other performed assays, in case of collagenase inhibition,
water and ethanol extracts exhibited low activity. The most potent inhibitor was MG-BL,
which also was marked as the best candidate in terms of UV protection, tyrosinase, and
hyaluronidase inhibition.

2.6. Correlation Study

The correlation analysis was performed in order to explore relationships between
chemical composition (content of chlorogenic acid and hyperoside, as the compounds
detected by HPLC in all the tested extracts) and biological activities investigated in the
paper—UV protective (SPF), anti-tyrosinase (Tyr), anti-hyaluronidase (Hyal), and anti-
collagenase (Col) activities.
The results of correlation analysis were presented using a correlation matrix (Table 3),
where the color gradient ranged from red as a sign of the strongest positive correlation (r = 1)
to white (no correlation, r = 0) and blue as signs of the strongest negative correlation (r = −1).
Negative correlation was important since some data (anti-tyrosinase, anti-hyaluronidase,
anti-collagenase activity) are presented as IC50 values, where a lower value indicates
higher activity.

Table 3. Correlation matrix (** means p < 0.01, * means p < 0.05).

SPF 1.000
Tyr −0.879 ** 1.000
Hyal −0.321 0.322 1.000
Col −0.733 * 0.512 0.817 ** 1.000
Chlorogenic acid 0.894 ** −0.759 * 0.066 −0.201 1.000
Hyperoside 0.935 ** −0.752 * −0.044 −0.289 0.983 ** 1
SPF Tyr Hyal Col Chlorogenic acid Hyperoside
The color gradient reflects the strength and direction of the correlation ranging from red (strong positive correlation,
r = 1) through white (no correlation, r = 0) to blue (strong negative correlation, r = −1).

According to the correlation matrix, there was statistically significant strong corre-
lation between the SPF and chlorogenic acid content, as well as between the SPF and
hyperoside content, indicating that the SPF value might be proportional to the content
of phenolic compounds. Additionally, the SPF was inversely correlated with IC50 values
for anti-tyrosinase and anti-collagenase activities, suggesting that extracts with higher
UV protection potential also possess greater enzyme-inhibitory effects. Anti-tyrosinase
activity also significantly correlated with content of both chlorogenic acid and hyperoside.
Furthermore, correlation between hyaluronidase and collagenase activity was observed.

2.7. In Vivo Safety Assessment

For the purpose of assessing the irritant potential of the extracts, the method of extract
application under occlusion (so-called patch testing) is commonly used, as occlusion is
expected to enhance the effects on the skin. Patches soaked with the extracts are usually
applied for a period of 24 to 48 h, and the measured values of biophysical parameters are
compared with the baseline values [57].
Plants 2025, 14, 2374 11 of 21

The in vivo safety study on human volunteers, which lasted for 24 h, revealed that
the tested extracts (MG-BL and CS-BF) did not cause any adverse skin reactions. As part
of this study, parameters were measured at four sites on the forearms of volunteers, with
one site marked as an non-treated control site (NC) and one as an non-treated control site
under occlusion (NCO), while filter papers soaked with the chosen extracts (MG-BL and
CS-BF) were applied to the remaining two sites, which were then covered with occlusion
that consisted of Parafilm® and Hartmann Omnifix® E elastic adhesive tape.
The results showed statistically significant changes in almost all parameters at the
NCO site, while parameters did not significantly change at NC, indicating that occlusion
caused changes in skin parameters. At NCO, a statistically significant decrease in pH
was observed, as well as an increase in TEWL (transepidermal water loss), EC (electrical
capacitance), and EI (erythema index).
An increase in TEWL at the NCO (Figure 5) may indicate a slight disruption of the skin
barrier or might be a consequence of elevated hydration under occlusion, since occlusion
on the skin surface prevents water evaporation, thereby causing its accumulation in the
intercellular spaces of the stratum corneum and swelling of the corneocytes. Consequently,
an increase in both TEWL and skin hydration often occurs in occluded sites [58]. However,
considering that a statistically significant increase in hydration was observed at all occluded
sites (NCO, MG-BL, and CS-BF) (Figure 6), it cannot be concluded that the increase in
TEWL at the NCO site is solely the result of increased hydration. In addition, NaDES
extracts did not cause elevation of TEWL, indicating that they did not have a negative
influence on the skin barrier.

Figure 5. Average basal TEWL (transepidermal water loss) values as well as values measured after
24 h occlusion in an in vivo safety study for determining the irritant potential of selected plant
extracts (MG-BL and CS-BF), non-treated control (NC), and non-treated control under occlusion
(NCO). Values are presented as mean ± standard deviation, while statistically significant differences
are indicated by the symbol * (p < 0.05).

The investigation of EI (Figure 7) pointed out that there was a statistically significant
increase in EI values only at the NCO site, which supported the assumption of a mild
disruption of the skin barrier and mild irritation due to occlusion.
On the other hand, at the sites under occlusion treated with both NaDES extracts
(MG-BL and CS-BF), no increase in TEWL or statistically significant increase in EI was
observed, leading to the conclusion that the tested extracts, in some way, reduced the
irritation observed at the NCO site. Thus, not only did they show no irritant potential
Plants 2025, 14, 2374 12 of 21

after one day of occlusion, but the results of the in vivo study might indicate prospective
anti-irritant potential.

Figure 6. Average basal EC (electrical capacitance) values as well as values measured after 24 h
occlusion in an in vivo safety study for determining the irritant potential of selected plant extracts
(MG-BL and CS-BF), non-treated control (NC), and non-treated control under occlusion (NCO).
Values are presented as mean ± standard deviation, while statistically significant differences are
indicated by the symbol ** (p < 0.01).

Figure 7. Average basal EI (erythema index) values as well as values measured after 24 h occlusion
in an in vivo safety study for determining the irritant potential of selected plant extracts (MG-BL
and CS-BF), non-treated control (NC), and non-treated control under occlusion (NCO). Values are
presented as mean ± standard deviation, while statistically significant differences are indicated by
the symbol * (p < 0.05).

In addition, no statistically significant changes in pH values at the other tested sites

(Figure 8) were observed, which is important since pH may signalize changes in skin
barrier function [59].
Overall, the non-invasive in vivo study on human volunteers highlighted that tested
NaDES extracts (MG-BL and CS-BF) under occlusion did not disrupt basal values of skin
parameters (pH, TEWL, EC, and EI).
Plants 2025, 14, 2374 13 of 21

Figure 8. Average basal pH values as well as values measured after 24 h occlusion in an in vivo
safety study for determining the irritant potential of selected plant extracts (MG-BL and CS-BF),
non-treated control (NC), and non-treated control under occlusion (NCO). Values are presented as
mean ± standard deviation, while statistically significant differences are indicated by the symbol *
(p < 0.05).

3. Materials and Methods

3.1. Materials
Herbal materials used in this study were bilberry fruits and bilberry leaves (Myrtilli
fructus, Myrtilli folium, Vaccinium myrtillus L., Ericaceae), whose identity was confirmed at
Herbarium of Faculty of Pharmacy, University of Belgrade (Belgrade, Serbia) where the
vouchers specimens were deposited (VML_0921).
Purified water, obtained from the Faculty of Medicine (University of Niš, Niš, Serbia)
and 50% ethanol (v/v), prepared from 96% ethanol (Sigma-Aldrich, St. Louis, MO, USA),
were used as conventional extraction solvents. For NaDES preparation tartaric acid and
malic acid purchased from Centrohem (Stara Pazova, Serbia), as well as sorbitol and
glycerol purchased from Comcen (Beograd, Serbia) were used.
Reference HPLC standards, chlorogenic acid, protocatechuic acid, delphinidin-3-O-
glucoside, hyperoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, procyanidin B2,
quercetin-3-O-glucoside, rutin, quercitrin, and epicatechin (purity ≥ 99%) were purchased
from Extrasynthese (Genay, France).
Biological activities of extracts (UV protective, anti-tyrosinase, anti-collagenase, and
anti-hyaluronidase activity) were assessed using following analytical grade reagents
purchased from Sigma-Aldrich: ethanol (70%, v/v), collagenase, gelatin, hyaluronidase,
hyaluronic acid, bovine serum albumin, and L-DOPA mushroom tyrosinase.

3.2. NaDES Extracts Preparation

NaDES tartaric acid–sorbitol (1:2 mol/mol) (TS), citric acid–sorbitol (1:2 mol/mol) (CS),
and malic acid–glycerol (1:2 mol/mol) (MG), were prepared according to the procedure
that involved heating the two individual components to 80 ◦ C while continuously stirring
them using a magnetic stirrer (IKAMAG, IKA, Verke, Staufen, Germany) for 30–60 min.
Upon melting the mixture, a clear liquid is formed to which distilled water was added at
30% (v/v). The extraction conditions were adjusted after series of trial extractions.
Ultrasound-assisted extraction was conducted for 30 min at 50 ◦ C in a sonication water
bath (Gesellschaft fur Labortechnik, Burgwedel, Germany). The dried powdered plant
material and solvents (TS, CS, MG, water or 50% ethanol) were mixed in mass ratio 1:20,
Plants 2025, 14, 2374 14 of 21

commonly used proportion in the preparation of NaDES extracts [60–62]. All extracts were
centrifuged after extraction and the supernatant was used for further work.

3.3. SPF Calculation Using Mansur Equation

The determination of UV absorption capacity was carried out using the dilution
method based on application of Mansur Equation (1) [63].

320
SPF = CF × ∑ EE(λ) × I(λ) × Abs(λ) (1)
290

EE(λ) indicates the erythemal effect of solar radiation at a specific wavelength, I(λ)
refer to the intensity of sunlight at a certain wavelength, while Abs(λ) is the absorbance
measured by the spectrophotometer at a given wavelength. CF represents the correction
factor. The values of EE(λ) × I(λ) are constants and were calculated by Sayre et al. [64].
The procedure involves making series of dilutions of extracts with ethanol until the
final dilution of 0.2 mg/mL is achieved, after which the absorbance was measured at every
5 nm in the range of 290 to 320 nm using spectrophotometer Evolution 60, Thermo-Fisher
Scientific (Waltham, MA, USA).
As positive control, 8% homosalate ethanol solution was used, whose SPF was
3.8 ± 0.18, while pure solvents (W, E, TS, CS, MG), were used as negative controls.

3.4. HPLC Analysis

For the qualitative and quantitative analysis of polyphenols, an Agilent 1200 HPLC
system equipped with a photodiode array (PDA) detector was employed. The chromato-
graphic conditions were set as in Table 4.

Table 4. Chromatographic conditions for HPLC analysis of anthocyanins, flavonoids, and phenolcar-
boxylic acids.

Flavonoids and
Chromatographic
Anthocyanins Analysis Phenolcarboxylic Acids
Conditions
Analysis
Lichrospher 100 RP-18e column
Column
(250 × 4.6 mm, 5.0 µm particle size)
0.1 M phosphoric acid (phase A)
Mobile phase
pure acetonitrile (phase B).
0–11% B (5 min),
11–15% B (25 min),
11–25% B (35 min),
15–18% B (8 min),
25–40% B (20 min),
Gradient program isocratic 18% B (8 min),
40–65% B (5 min),
18–30% B (4 min),
65–100% B (10 min)
30–100% B (3 min),
100% B (7 min)
Total run time 60 min 70 min
Flow rate 0.8 mL/min 1.0 mL/min
Injection volume 4 µL 10 µL
Column temperature 25 ◦ C 25 ◦ C
λ for PDA detector 520 nm 260 nm
Plants 2025, 14, 2374 15 of 21

Sample preparation for chromatographic analysis was based on the dilution of extracts
with deionized water to a concentration of 25 mg/mL and filtration using PTFE membrane
filters prior to injection.
Compound identification was based on overlaying with retention times and UV-VIS
spectra of standards. Upon successful spectral matching, confirmation was achieved
by spiking with the corresponding standards to ensure complete identification through
a peak purity test. Peaks that did not meet these criteria were excluded from quan-
tification. Quantification was carried out using external calibration with the following
standard concentrations:
• Chlorogenic acid: 0.45 mg/mL;
• Protocatechuic acid: 0.52 mg/mL;
• Delphinidin-3-O-glucoside: 0.39 mg/mL;
• Hyperoside: 0.40 mg/mL;
• Cyanidin-3-O-galactoside: 0.42 mg/mL;
• Cyanidin-3-O-glucoside: 0.42 mg/mL;
• Procyanidin B2: 0.36 mg/mL;
• Quercetin-3-O-glucoside: 0.39 mg/mL;
• Rutin: 0.48 mg/mL;
• Quercitrin: 0.52 mg/mL;
• Epicatechin: 0.40 mg/mL.
The final results were expressed as mg/g of dried drug weight.

3.5. Determination of Anti-Tyrosinase Activity

The anti-tyrosinase activity of the extract was assessed following a modified spec-
trophotometric method, which measures the diphenolase activity of mushroom-derived
tyrosinase using L-DOPA as a substrate. In a 96-well microtiter plate, 50 µL of 2 mM
L-DOPA prepared in phosphate buffer (50 mM, pH 6.8), 50 µL of phosphate buffer (50 mM,
pH 6.8), and 50 µL of the extract solution (also in phosphate buffer, 50 mM, pH 6.8, concen-
tration range 0.5–20 mg/mL) were added to each well. After incubation at 30 ◦ C for 5 min,
50 µL of tyrosinase solution (250 U/mL) was added to each well. The reaction mixture
was then incubated for 10 min at 37 ◦ C. Enzymatic activity was determined by measuring
absorbance at 492 nm (AS ). The blank consisted of the same components with the extract
replaced by corresponding solvent and tyrosinase replaced by buffer. The control (AC )
contained buffer in place of the extract only. L-ascorbic acid was used as positive control,
while pure solvents (W, E, TS, CS, MG) were used as negative controls for the corresponding
extract to determine whether the observed bioactivity was due to the solvent itself or the
extract. This was important since NaDES solvents (TS, CS, MG) are composed of substances
known to possess cosmetic activity.
The tyrosinase inhibitory activity was calculated using Equation (2):

Ac − As
% tyrosinase inhibition = × 100 (2)
Ac

The results were expressed as IC50 values (mg/mL), i.e., the concentration of extract
that led to 50% of tyrosinase inhibition.

3.6. Determination of Anti-Hyaluronidase Activity

The anti-hyaluronidase activity of the extract was evaluated using a turbidimetric
45 min method proposed by the Sigma-Aldrich protocol and used by authors [65,66] with
minor modifications. Dilutions of the extracts were prepared in buffer (concentration range
0.5–20 mg/mL), and the pH of all extract solutions was adjusted to 6.4 using NaOH. The
Plants 2025, 14, 2374 16 of 21

reaction mixture consisted of 50 µL of the extract solution and 100 µL of hyaluronidase

enzyme solution (12.44 U/mL, dissolved in 20 mM phosphate buffer, pH 7.0). Following
10 min incubation at 37 ◦ C, 100 µL of hyaluronic acid solution (0.3 mg/mL, dissolved in
300 mM phosphate buffer, pH 5.3) was added to the mixture. The samples were then incu-
bated for an additional 45 min at 37 ◦ C to allow enzymatic degradation of hyaluronic acid.
After the incubation period, 1 mL of acidic albumin solution in acetate buffer (0.1% albumin,
pH 3.75) was added to precipitate the remaining hyaluronic acid. Therefore, the concen-
tration of undegraded hyaluronic acid is proportional to hyaluronidase inhibition. The
absorbance of the samples (AS ) was measured at 600 nm using an ELISA reader. The blank
consisted of the same components with the extract replaced by the corresponding solvent
and enzyme replaced by buffer. As controls (AC ), a reaction mixture containing buffer in
place of both the extract and enzyme were used. Tannic acid served as a positive control,
while pure solvents (W, E, TS, CS, MG) were used as negative controls for the corresponding
extract to determine whether the observed bioactivity was due to the solvent itself or the
extract. The percentage of inhibition was calculated using the following, Equation (3):

As
% hyaluronidase inhibition = × 100 (3)
Ac

The results were expressed as IC50 values (mg/mL), i.e., the concentration of extracts
that led to 50% of hyaluronidase inhibition.

3.7. Determination of Anti-Collagenase Activity

The anti-collagenase activity of the extract was determined according to the method
described by Zhang et al. [45], which is based on the spectrophotometric quantification
of hydrolytic products released from collagenase substrates (collagen or gelatin) using a
ninhydrin reagent. The ninhydrin reagent was freshly prepared prior to use by mixing
equal volumes of tin(II) chloride solution (1.6 mg/mL in 0.2 M citrate buffer, pH 5) and a
ninhydrin solution (50 mg/mL in DMSO). The ninhydrin reagent reacts with free amino
acids released from gelatin hydrolysis, forming a detectable complex whose absorbance
can be measured at 545 nm. Inhibition of collagenase activity reduces the degradation of
gelatin, resulting in a lower release of free amino acids, and therefore a reduced formation
of the ninhydrin–amino acid complex, which is reflected by lower absorbance values.
The procedure for determining the anti-collagenase activity of the extract was based on
mixing 40 µL of extract solution (at various dilutions concentration range 0.5–20 mg/mL)
with 20 µL of collagenase solution (1 mg/mL, dissolved in reaction buffer: 50 mM Tris-HCl,
5 mM CaCl2 , 1 µM ZnCl2 , pH 7.5) and incubating the mixture at room temperature for
5 min. Then, 30 µL of gelatin substrate solution (2 mg/mL in the same reaction buffer) was
added to initiate hydrolysis. Following 10 min incubation at 37 ◦ C, 90 µL of the termination
buffer (containing 120 mg/mL PEG 4000 and 25 mM EDTA) was added. Subsequently,
90 µL of ninhydrin reagent was added, and the reaction mixture was heated at 80 ◦ C in a
water bath for 10 min. After cooling to room temperature, 100 µL of distilled water was
added to the reaction mixture, and absorbance was measured at 545 nm (AS ). The blank
consisted of the same components with the extract replaced by corresponding solvent and
enzyme replaced by buffer. The negative and positive controls (AC ) were prepared in the
same manner, except that distilled water was used instead of the extract. Rutin was used as
a positive control, while pure solvents (W, E, TS, CS, MG) were used as negative controls
for the corresponding extract to determine whether the observed bioactivity was due to the
solvent itself or the extract.
Plants 2025, 14, 2374 17 of 21

The percentage of collagenase inhibition was calculated using the following Equation (4):

Ac − As
% collagenase inhibition = × 100 (4)
Ac

The results were expressed as IC50 values (mg/mL), i.e., the concentration of extracts
that led to 50% of collagenase inhibition.

3.8. In Vivo Safety Assessment

The extracts identified as those with best characteristics were evaluated on the skin
of healthy volunteers. For the in vivo testing on human subjects, approval was obtained
from the Ethics Committee of the Faculty of Medicine, University of Niš (approval number
12-10650/2-6 dated 3 October 2022). The participants were fully informed about the
course of the study and signed written informed consent. The study was conducted in
accordance with the Declaration of Helsinki. All participants were healthy volunteers with
no prior history of dermatological conditions. They were instructed not to use any other
cosmetic products on the tested skin area for one week prior to and throughout the in vivo
testing period.
The in vivo testing was conducted using the Multi Probe Adapter System MPA®
9, manufactured by Courage + Khazaka electronic GmbH, Cologne, Germany, consist-
ing of the different probes (Corneometer® CM 825, Tewameter® TM 300, Mexameter®
MX 18 and Skin-pH-Meter® PH 905). All probes were calibrated according to the
manufacturer’s instructions.
The safety profile assessment of the selected extracts was conducted in a double-blind
study which involved 20 healthy volunteers of both gender (25.85 ± 4.74 years). After
applying the square filter paper soaked with extracts (whose pH was set using NaOH at
4.5) in the amount of 0.016 g/cm2 to predefined sites on the volar side of the forearm of
each subject, those sites were covered with an occlusive film (Parafilm® , American National
Can. Co., Chicago, IL, USA) and subsequently secured with self-adhesive tape (Omnifix®
E, Hartmann, Heidenheim, Germany). One site was left as a non-treated control without
occlusion (NC), and one site was marked as non-treated control under occlusion (NCO, a
site where no extract was applied, but which was still covered with the occlusive film and
adhesive tape).
The following parameters were measured: EC (stratum corneum hydration), TEWL
(transepidermal water loss), EI (erythema index), and pH, both before the study began
(baseline values) and 60 min after the removal of occlusion, which lasted 24 h. The results
were presented as mean values ± standard deviation.

3.9. Statistical Analysis

The IC50 values were presented as average result of three measurements ± standard
deviation. The statistical analysis was conducted using software SPSS Statistics version
20 (IBM). Homogeneity of variances was checked with Levene’s test. For analyzing the
influence of extraction solvent or the herbal material on the results, the two-way analysis
of variance ANOVA was used with post hoc analysis while Welch–ANOVA was used for
unequal variances. Student t-test was used for comparing measured in vivo parameters
before and after 24 h of occlusion. The statistically significant differences were considered
at p < 0.05. The Pearson and Spearman correlation analysis were used for comparing
correlation between results, depending on whether data meet the assumptions of normality
and linearity. The Shapiro–Wilk test was used for testing normality while linearity was
tested using scatterplots. All the graphs and tables in the paper were constructed using
Microsoft Office Excel 10 software.
Plants 2025, 14, 2374 18 of 21

4. Conclusions
The results of our study demonstrated better inhibitory activity of NaDES ex-
tracts against some important skin enzymes (tyrosinase, hyaluronidase, and collage-
nase) compared to water and ethanol extracts. In addition, these extracts also exhib-
ited higher UV protective activity (higher SPF). The most potent extract was bilberry
leaves extract made with malic acid–glycerol, which exhibited strong anti-tyrosinase
(IC50 = 3.52 ± 0.26 mg/mL), anti-hyaluronidase (IC50 = 3.23 ± 0.30 mg/mL), and anti-
collagenase (IC50 = 1.84 ± 0.50 mg/mL) activities.
In addition, this study highlighted that bilberry leaves are mostly treated as waste
products in bilberry processing, exhibiting significant potential as a valuable source of
active dermocosmetic ingredients, being more active than bilberry fruit extracts.
Furthermore, correlation analysis revealed correlation between UV protective and
anti-tyrosinase activity, UV protective and anti-collagenase activity as well as between
anti-hyaluronidase and anti-collagenase activity. UV protective and anti-tyrosinase activity
also significantly correlated with the content of both chlorogenic acid and hyperoside
in extracts.
The non-invasive in vivo study conducted on human volunteers demonstrated that
the selected NaDES extracts, when applied under occlusion for 24 h, did not alter the basal
values of skin parameters (pH, TEWL, EC, and EI).
Bearing in mind the results of our preliminary in vitro study, bilberry leaves extract
prepared with malic acid–glycerol mixture have potential for use in cosmetic and/or
dermopharmaceutical anti-aging products as a natural and green raw material obtained
from the waste of bilberry processing in the food industry. However, further in vivo studies
need to be conducted with the aim of assessing the effects of selected extracts on the skin.

Author Contributions: Conceptualization, M.M. and I.N.; methodology, M.M., V.M.T. and I.N.; soft-
ware, M.M.; validation, A.Ž. and V.M.T.; formal analysis, M.M. and V.M.T.; investigation, M.M., I.N.,
A.Ž. and V.M.T.; resources, I.N. and V.M.T.; data curation, M.M.; writing—original draft preparation,
M.M. and A.Ž.; writing—review and editing, I.N. and V.M.T.; visualization, M.M.; supervision, I.N.,
A.Ž. and V.M.T.; project administration, I.N. and V.M.T.; funding acquisition, I.N. and V.M.T. All
authors have read and agreed to the published version of the manuscript.

Funding: Ministry of Science, Technological Development, and Innovation of the Republic of Serbia,
grant numbers: 451-03-137/2025-03/200113 and 451-03-136/2025-03/200003.

Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author.

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

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