molecules

Article
Phytochemical and Nutraceutical Screening of Ethanol and
Ethyl Acetate Phases of Romanian Galium verum
Herba (Rubiaceae)
Alexandra-Denisa Semenescu 1,2 , Elena-Alina Moacă 1,2 , Andrada Iftode 1,2, *, Cristina-Adriana Dehelean 1,2 ,
Diana-Simona Tchiakpe-Antal 3 , Laurian Vlase 4 , Ana-Maria Vlase 5 , Delia Muntean 6,7 and Raul Chioibaş 8,9

1 Department of Toxicology, Drug Industry, Management and Legislation, Faculty of Pharmacy, “Victor Babes”
University of Medicine and Pharmacy Timisoara, 2nd Eftimie Murgu Square, 300041 Timisoara, Romania;
alexandra.scurtu@umft.ro (A.-D.S.); alina.moaca@umft.ro (E.-A.M.); cadehelean@umft.ro (C.-A.D.)
2 Research Centre for Pharmaco-Toxicological Evaluation, “Victor Babes” University of Medicine and
Pharmacy, 2nd Eftimie Murgu Square, 300041 Timisoara, Romania
3 Department of Pharmaceutical Botany, Faculty of Pharmacy, “Victor Babes” University of Medicine and
Pharmacy Timisoara, 2nd Eftimie Murgu Square, 300041 Timisoara, Romania; diana.antal@umft.ro
4 Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, “Iuliu Hatieganu”
University of Medicine and Pharmacy, 8th Victor Babes Street, 400347 Cluj-Napoca, Romania;
laurian.vlase@umfcluj.ro
5 Department of Pharmaceutical Botany, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and
Pharmacy, 8th Victor Babes Street, 400347 Cluj-Napoca, Romania; gheldiu.ana@umfcluj.ro
6 Department of Microbiology, Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy
Timisoara, 2nd Eftimie Murgu Square, 300041 Timisoara, Romania; muntean.delia@umft.ro
7 Multidisciplinary Research Center on Antimicrobial Resistance, “Victor Babes” University of Medicine and
Pharmacy, 2nd Eftimie Murgu Square, 300041 Timisoara, Romania
8 Department of Surgery I, Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy,
2nd Eftimie Murgu Square, 300041 Timis, oara, Romania; office@medcom.ro
9 CBS Medcom Hospital, 12th Popa Sapca Street, 300047 Timisoara, Romania
* Correspondence: andradaiftode@umft.ro; Tel.: +40-742-426421
Citation: Semenescu, A.-D.; Moacă,
E.-A.; Iftode, A.; Dehelean, C.-A.;
Abstract: Galium species are used worldwide for their antioxidant, antibacterial, antifungal, and
Tchiakpe-Antal, D.-S.; Vlase, L.; Vlase,
antiparasitic properties. Although this plant has demonstrated its antitumor properties on various
A.-M.; Muntean, D.; Chioibaş, R.
Phytochemical and Nutraceutical
types of cancer, its biological activity on cutaneous melanoma has not been established so far.
Screening of Ethanol and Ethyl Therefore, the present study was designed to investigate the phytochemical profile of two extracts
Acetate Phases of Romanian Galium of G. verum L. herba (ethanolic and ethyl acetate) as well as the biological profile (antioxidant,
verum Herba (Rubiaceae). Molecules antimicrobial, and antitumor effects) on human skin cancer. The extracts showed similar FT-IR
2023, 28, 7804. https://doi.org/ phenolic profiles (high chlorogenic acid, isoquercitrin, quercitrin, and rutin), with high antioxidant
10.3390/molecules28237804 capacity (EC50 of ethyl acetate phase (0.074 ± 0.01 mg/mL) > ethanol phase (0.136 ± 0.03 mg/mL)).
Academic Editor: H. P. Vasantha
Both extracts showed antimicrobial activity, especially against Gram-positive Streptococcus pyogenes
Rupasinghe and Staphylococcus aureus bacilli strains, the ethyl acetate phase being more active. Regarding the
in vitro antitumor test, the results revealed a dose-dependent cytotoxic effect against A375 melanoma
Received: 26 October 2023
cell lines, more pronounced in the case of the ethyl acetate phase. In addition, the ethyl acetate phase
Revised: 17 November 2023
stimulated the proliferation of human keratinocytes (HaCaT), while this effect was not evident in the
Accepted: 24 November 2023
case of the ethanolic phase at 24 h post-stimulation. Consequently, G. verum l. could be considered a
Published: 27 November 2023
promising phytocompound for the antitumor approach of cutaneous melanoma.

Keywords: antioxidants; DPPH free radical; total phenolic content; FT-IR profile; cytotoxicity;
Copyright: © 2023 by the authors. melanoma; HaCaT cells
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons 1. Introduction
Attribution (CC BY) license (https://
Nowadays, more and more emphasis is placed on the production of herbal medicines
creativecommons.org/licenses/by/
for the treatment of human diseases [1–4] due to old ethnopharmacological knowledge and
4.0/).

Molecules 2023, 28, 7804. https://doi.org/10.3390/molecules28237804 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 7804 2 of 29

continuous therapeutic research by scientists. Since ancient times, plants have been used as
support in the biomedical domain due to their never-ending pharmaceutical and medical
properties, useful in both the prevention and treatment of various diseases. Therefore,
plant-based medicinal products continue to attract the attention of researchers around the
world due to their beneficial effects on human health as well as minimum side effects in
human organisms [5]. According to the World Health Organization (WHO), medicinal
plants contribute to obtaining many traditional medicines, which are useful for the primary
healthcare needs of millions of people, especially those from developing countries [6,7].
Throughout time, the number of studies involving medicinal plants has increased,
emphasizing potential beneficial effects in severe pathologies like cardiovascular diseases,
diabetes, pulmonary and brain diseases, and cancer [8–13]. Through their mechanism of
action in the human body, medicinal plants are responsible for the protective effects due
to their capacity to reduce oxidative stress (to reduce intracellular reactive oxygen species
(ROS)) and protect cells against the harmful damage caused by H2 O2 [14,15]. Therefore,
an intact skin barrier leads to a strong defense against various factors that could create
dysfunctionality, thus forming a start for various skin disorders, such as skin cancer [16]. It
is well known that skin cancer, especially cutaneous melanoma, has an increased incidence
rate globally, being one of the most frequent cancers among young people. Besides genetic
factors, it is assumed that environmental ones contribute more to the development of
melanoma, like exposure to ultraviolet (UV) radiation from the sun and from tanning
lamps and beds [17,18]. In this context, there is an enormous need to find promising
alternatives to ensure skin integrity maintenance, including the development of beneficial
products for skin disease therapy. Regarding cutaneous melanoma, it is urgent to develop
novel anticancer compounds, especially based on plants, thus overcoming the side effects
after administration of cytotoxic agents currently used for metastatic melanoma (increased
resistance of melanoma cells) [19]. Plant extracts are the perfect candidates for use as alter-
natives to conventional treatments due to their plethora of natural bioactive compounds
and phytochemicals with favorable bioactivity for many human ailments. Plants represent
a rich, never-ending natural source of antioxidant compounds, able to counteract oxidative
stress and mitigate its effects on individuals’ health. Between antioxidant compounds,
polyphenols are considered of major relevance regarding the antioxidant effect for many
medicinal plants. The higher the number of phenolic compounds present in an extract,
the stronger the supplementary effects of the extract, such as synergistically, additively, or
antagonistic actions [20–22], that influence the total capacity of the extract to neutralize free
radicals [23–25].
Among the medicinal plants that have attracted the attention of scientists in recent
years, with a long history as a traditional healing plant, is the Galium species. Galium sp.
belongs to the Rubiaceae family, representing a safe, accessible, and efficient natural health
remedy, proven by its representative bioactive compounds like iridoid glycosides [26],
terpenes [27], phenolic acids [28], flavonoids [29], monoterpene glycosides [30], phytos-
terols, anthraquinones [31], saponins [32], aldehydes, alcohols, small amounts of tannins,
waxes, pigments, essential oils [33], and vitamin C [34]. Across the world, there are about
667 species of Galium, including in Africa, Asia, North America, and Europe, where over
one-third of the species are distributed [35]. In Romania, the genus Galium is represented
by approximately 38 species, of which 6 have yellow flowers [36–39]. Among the species
from Romania, the best known is G. verum L. (lady’s bedstraw), next to G. mollugo L.
(hedge bedstraw), G. aparine L. (cleavers or stickyweed), and G. odoratum L. (syn. Asperula
odorata, woodruff) [40]. Due to the beneficial effects of Galium spp. reported over time,
nutraceuticals (galenic remedies and supplements) are nowadays available on the market
that are supposed to contribute to improving some health issues involving inflammation,
detoxication, or the immune system. The most well-known pharmacological activities of
Galium species have been reported in a previous study and refer to the biological effect of
Galium verum L. extract internal and external administration [40]. In traditional medicine,
G. verum L. has been used for its depurative, diuretic (for bladder and kidney irritation),
Molecules 2023, 28, 7804 3 of 29

laxative, antirheumatic, and sedative actions [26,38], as well as for healing wounds and
gingival inflammations [41], epilepsy and hysteria [42]. In addition, in Romanian tradi-
tional medicine, nutraceuticals from several Galium spp. have been used for different
cosmetic formulations to prevent the actions of oxidative stress on the skin [40]. Several
scientific studies have reported that the representatives of the Galium genus possess a wide
range of biological properties, including antioxidant, detoxicant, antimicrobial, antifun-
gal, antihaemolytic, cardio- and hepatoprotective, immunomodulatory, and anticancer
properties [43–52]. In addition, other scientific studies reported that Galium verum L. ex-
tracts have been used to treat skin disorders, as exogenous treatment in psoriasis, and as a
treatment in the healing of delayed diseases. Moreover, it was shown that these species
have beneficial effects on cancer ulcers, tongue cancer, and breast cancer. Currently, the
extract of Galium verum L. is recommended for therapy in rheumatic diseases and cysti-
tis [27,44,53]. To the best of our knowledge, no study in the scientific literature reports the
in vitro antitumor effect of Galium verum L. extracts on cutaneous melanoma. Based on the
findings reported previously and considering the lack of information, the present study
aims to report for the first time the preliminary results regarding the in vitro antitumor
effect of G. verum L. extracts on skin disorder, more precisely, the in vitro biological effect
on malignant melanoma.
In this context, the current study aimed to outline the phytochemical profile of two
extracts obtained from the aerial part of Galium verum L. plant material acquired from a
local natural products store, including the individual phenolic compounds content and
the antioxidant screening, together with a preliminary biological evaluation regarding the
antimicrobial activity and the in vitro anticancer effect. The novelty of the study consists in
the fact that, although this plant species has shown great antitumor potential on various
cancer cell lines, its efficacy in skin cancer has not yet been established. Therefore, one of
the objectives of the present study was to assess the anticancer effect of Galium verum L. on
the human skin cancer cell line A375, as well as on the healthy human keratinocyte cell
line (HaCaT).

2. Results
2.1. Phytochemical Analysis
After the concentration of both Galium verum L. extracts, the extraction yield was
calculated, followed by Fourier transform infrared spectroscopy (FT-IR) characterization. In
addition, the phenolic composition and antioxidant potential to establish the phytochemical
profile of the obtained extracts were determined.

2.1.1. The Extraction Yield

The extraction was carried out using a conventional extraction procedure based on
maceration of Galium verum L. plant material for 24 h at room temperature, followed by
sonication (at 25 ◦ C for 30 min), then filtration and concentration of a specific volume of
each extracted sample. The extraction yield calculated from 50 mL of each Galium verum L.
extract was 13.95% for GvEtOH extract and 4.65% for GvEtOAc extract, respectively. By
analyzing the values obtained regarding the two extracts, it is clear that the final extraction
yield depends on the type of extraction solvent, the plant:solvent ratio, and the amount
of vegetal material taken into account. It was chosen to work with dried vegetal material
instead of the fresh one because the dried plant has a lower amount of water as compared
to the fresh one, water which is lost together with volatile compounds in the drying step.

2.1.2. FT-IR Investigations

The molecular fingerprint of the Galium verum L. extracts resulting from the signal
recorded by each molecule or chemical structure at a specific wavenumber is depicted
in Figure 1.
 2.1.2. FT-IR Investigations
The molecular fingerprint of the Galium verum L. extracts resulting from the signal
Molecules 2023, 28, 7804 4 of 29
recorded by each molecule or chemical structure at a specific wavenumber is depicted in
Figure 1.

Figure 1. FT −IRspectra
FT−IR spectraof
ofethanol
ethanol(A)
(A)and
andethyl
ethylacetate
acetate(B) Galiumverum
(B)Galium verum L.
L. extracts.
extracts.

The
The results
resultsof
ofthe
theFT-IR
FT-IRanalysis
analysisforfor
both concentrated
both Galium
concentrated verum
Galium L. ethanol
verum extract
L. ethanol ex-
and
tractethyl acetate
and ethyl phasephase
acetate are provided in Table
are provided 1.
in Table 1.

Table 1.1. Peak

Table Peak values
values and
and functional
functional groups
groups of
of ethanol
ethanol and
and ethyl acetate Galium
ethyl acetate Galium verum
verum L. extracts
extracts
recorded
recorded in in the
the spectrum.
spectrum.

Wavenumber (cm−1 ) WavenumberFunctional

(cm−1) Functional Groups
Groups Bond Bond
GvEtOH GvEtOAc GvEtOHGvEtOH
GvEtOAc GvEtOH
GvEtOAc GvEtOAc GvEtOHGvEtOH GvEtOAc
GvEtOAc
OH stretch
3412.08 -
3412.08 Alcohol - Alcohol- - OH stretch
-
-
(H-bonded)
(H-bonded)
Alkane/ Alkane/ C-H stretching/ C-H stretching/
2926.01 2924.09 2926.01 Alkane/
2924.09 Alkane/ C-H stretching/ C-H stretching/
Alcohol (acid) Alcohol (acid)
Alcohol (acid)Alcohol (acid) OH stretch
OH stretch OHOH stretch
stretch
2852.72 2852.72
2852.72 Alkane
2852.72 Alkane
Alkane
AlkaneC-H stretching
C-H stretching C-H C-H stretching
stretching
1735.93 1734.01 Carbonyl Carbonyl C=O stretch C=O stretch
1735.93 1734.01 Carbonyl Carbonyl C=O stretch C=O stretch
C=O stretch
1654.92 - Amide/Alkene - C=O stretch -
1654.92 - Amide/Alkene - C=C stretching -
C=C stretching
1604.77 1604.77 Cyclic alkene/ Cyclic alkene/ C=C stretching C=C stretching
1604.77 1604.77 Cyclic alkene/ Cyclic alkene/ C=C stretching C=C stretching
Aromatic com- Aromatic com-
1516.05 Aromatic
1516.05 Aromatic C=C stretch C=C stretch
1516.05 1516.05 pounds pounds C=C stretch C=C stretch
compounds compounds
Alkane (meth- Alkane (meth-
Alkane (methylene Alkane (methylene C-H bending/ C-H bend-
C-H bending/C-H bending/C=C
1458.18 1463.97 ylene ylene
1458.18 1463.97
group)/Aromatics group)/Aromatics C=C stretchC=C stretch (in stretching/C=C
(in ring) (in ring)
group)/Aromat- group)/Aromat-
1367.53 1379.10 Alkane Alkane -C-H bendingring) stretch
-C-H (in ring)
bending
ics ics
1259.52 1261.45 1367.53 Acids
1379.10 AlkaneAcids Alkane C-O stretch-C-H bending C-O -C-H stretch
bending
1170.79 1172.72 Alcohol1261.45
1259.52 (tertiary) Alcohol
Acids (tertiary) Acids C-O stretching
C-O stretch C-OC-O stretching
stretch
Alcohol (ter-
Alcohol Alcohol (ter-
1074.35 1091.71 Alcohol1172.72
1170.79 (primary) C-O stretch
C-O stretching C-O C-Ostretch
stretching
(secondary)
tiary) tiary)
1045.42 - Anhydride Alcohol (pri-
- AlcoholCO-O-CO
(sec- stretching -
1074.35 1091.71 C-O stretch C-O stretch
Alkane mary)
Alkane ondary)
C=C bending/ C=C bending/
914.26 981.77 (disubstituted (disubstituted
1045.42 - Anhydride - =C-H CO-O-CO
bending stretch-=C-H bending -
(trans))/Alkenes (trans))/Alkenes ing
856.39 - Alkane - C=C bending -
Molecules 2023, 28, 7804 5 of 29

Table 1. Cont.

Wavenumber (cm−1 ) Functional Groups Bond

GvEtOH GvEtOAc GvEtOH GvEtOAc GvEtOH GvEtOAc
Alkane Alkane
C=C bending/ C=C bending/
810.10 804.32 (trisubstituted)/ (trisubstituted)/
C-Cl stretching C-Cl stretching
Halo compounds Halo compounds
Alkane
C=C bending/
763.81 - (trisubstituted)/ - -
C-Cl stretching
Halo compounds
Alkane Alkane
(disubstituted (disubstituted C=C bending/ C=C bending/
719.45 721.38
(cis))/ (cis))/ C-Cl stretching C-Cl stretching
Halo compounds Halo compounds
630.72 - Halo compounds - C-Cl; C-Br stretching -
599.86 - Halo compounds - C-Cl; C-Br stretching -
551.64 - Halo compounds - C-Cl; C-Br stretching -

The FT-IR analysis recorded strong absorption bands, especially in the case of GvEtOH
extract, at around 3400, 1700, and 1070 cm−1 . The first important, strong, and well-
defined band is located at 3412.08 cm−1 and can be attributed to the O-H stretching
vibration (hydroxyl groups (H-bonded)) present in water or flavones contained in the
ethanolic extract of concentrated Galium verum L. plant material. The bands located around
2920 cm−1 in both extracts can be assigned to the O-H stretching in acid functional groups
or to saturated aliphatic C-H stretching bonds (bands around 2852.72 cm−1 ), suggesting
the occurrence of aromatic ring attachment. The bands around 1730 cm−1 present in both
extracts indicate the presence of carbonyl functional groups, and the band recorded at
1654.92 cm−1 from the GvEtOH extract indicates that the C=C group is present in the
alkenes, but it could also be assigned to the C=O stretching vibration of amide functional
groups. The band recorded at 1604.77 cm−1 in both extracts suggests the presence of
C=C stretching vibration of cyclic alkenes. The medium–weak intensity absorption peaks
recorded between 1300 and 1600 cm−1 revealed the presence of the following functional
groups: C=C stretch (in a ring) and C-H bending from aromatic compounds and alkanes
(methylene group). The bands recorded around 1260 cm−1 are assigned to the stretching
vibration of the C-O functional group, most probably from the aromatic carbonyl acids.
Bands between 1070 and 1170 cm−1 are assigned to primary, secondary, and tertiary
alcohols, and the bands recorded between 800 and 1045 cm−1 are attributed to the C=C
bending, C-H bending, and CO-O-CO stretching vibration present in alkanes (di- and
trisubstituted/alkenes and anhydrides functional groups. The region between 550 and
810 cm−1 is specific for the out-of-plane stretching vibration of halo compounds (C-Cl and
C-Br stretching), as well as to the bending vibration of C=C functional groups present in
the alkane aromatic compounds.

2.1.3. Liquid Chromatography Mass Spectrometry (LC-MS)

Table 2 presents the polyphenolic content of the Galium verum L. extracts (ethanolic
and ethyl acetate phases) obtained by LC-MS analysis.
LC-MS analysis revealed seven phenolic compounds in both extracts of Galium verum L.,
identified as major components in the analyzed extracts. Quercitrin was the only phenolic
compound that was below the limit of quantification in the GvEtOH extract. Obtained
results indicated that isoquercitrin and rutin (two important flavonoids) were the most
abundant quantified compounds in GvEtOH extract compared with chlorogenic acid and
isoquercitrin (a phenolic acid and a flavonoid), which were the most abundant quantified
compounds in GvEtOAc extract. Therefore, the GvEtOAc extract was richer in terms of
 Molecules 2023, 28, 7804 6 of 29

phenolic compounds than GvEtOH, except 4-O caffeoylquinic acid, rutin, quercetol, and
luteolin. In both extracts, one can observe that the amount of flavonoids is higher than that
of phenolic acids, quantified in Table 2.

Table 2. Polyphenolic compounds of both extracts analyzed by LC-MS.

GvEtOH
MS Qualita- Concentration
Compound Name UV Identified
tively Identified (µg/mL)
Chlorogenic acid Yes Yes 8.027
4-O caffeoylquinic acid Yes Yes 0.172
Isoquercitrin Yes Yes 17.765
Rutin Yes Yes 14.811
Quercitrin No Yes -
Quercetol Yes Yes 1.275
Luteolin Yes Yes 0.260
GvEtOAc
Chlorogenic acid Yes Yes 10.216
4-O caffeoylquinic acid Yes Yes 0.096
Isoquercitrin Yes Yes 20.384
Rutin Yes Yes 1.896
Quercitrin Yes Yes 6.722
Quercetol Yes Yes 0.779
Luteolin Yes Yes 0.191

The chemical structures of the polyphenols found in both extracts were designed
Molecules 2023, 28, 7804 with the KingDraw Chemical Structure Editor (http://www.kingdraw.cn/en/ accessed 7 of 31
on 16 November 2023) and are presented in Figure 2. One can also observe the organic
functional groups identified with FT-IR analysis.

Figure
Figure2.2.Chemical
Chemicalstructures
structuresof
of the
the main polyphenolsfound
main polyphenols foundininboth
bothGalium
Galiumverum
verum
L. L. extracts
extracts after
after
LC-MS analysis.
LC-MS analysis.

Table33reveals
Table revealsthe
theresults
results for
for the
the identification
identificationand
andquantifications
quantificationsofof
catechins from
catechins from
both Galium verum L. extracts, analyzed by LC-MS. One can observe that epicatechin
both Galium verum L. extracts, analyzed by LC-MS. One can observe that epicatechin andand
gallic acid were the only polyphenolic compounds detected in Galium verum L. extracts in
low concentrations. The rest of the polyphenolic compounds were below the detection
limit.

Table 3. Catechins content of Galium verum L. extracts by LC-MS.

Molecules 2023, 28, 7804 7 of 29

gallic acid were the only polyphenolic compounds detected in Galium verum L. extracts in
low concentrations. The rest of the polyphenolic compounds were below the detection limit.

Table 3. Catechins content of Galium verum L. extracts by LC-MS.

Extract Concentrations (µg/mL)

Epicatechin Catechin Syringic Acid Gallic Acid Protocatechuic Acid Vanillic Acid
GvEtOH 1.10 ND 1 ND 1 ND 1 ND 1 ND 1
GvEtOAc ND 1 ND 1 ND 1 0.34 ND 1 ND 1
1 ND—not detected

2.1.4. Total Phenolic (TPC) and Flavonoid Contents (TFC)

Depending on the solvent used for the extraction of polyphenolic compounds from
Galium verum L. herba, the content of the total phenols slightly varied. The TPC in GvEtOH
extract was 1.30 mg GAE/g dry extract as compared with the TPC in GvEtOAc extract of
1.39 mg GAE/g dry extract. Regarding the total flavonoid content, the same slight variation
was observed. The TFC in GvEtOH extract was 1.42 mg CE/g dry extract as compared
with TFC in GvEtOAc of 1.37 mg CE/g dry extract.

2.1.5. Antioxidant Activity

Figure 3 depicts the degradation kinetics of DPPH free radicals provided by both
Galium verum L. extracts, evaluated each at six different concentrations, as well as by the
standard used: the ethanolic solution of ascorbic acid (vitamin C). One can observe that
the degraded amount of DPPH free radicals after 20 min of incubation period indicated a
12% degradation when the GvEtOH extract was used at the highest concentration tested
(1 mg/mL), while at the same concentration, the GvEtOAc showed 4% degradation of
DPPH free radicals, a degradation potential almost identical with the one observed when
vitamin C was used (3%). The same degradation potential of DPPH free radicals was
observed when using the GvEtOAc extract at a concentration of 0.8 mg/mL, even at
0.5 mg/mL (7%), which indicates that, by using ethyl acetate as an extraction solvent, more
antioxidant compounds are extracted from the dried plant, even at low concentrations.
Nevertheless, both assays showed a similar trend, with GvEtOAc extract presenting the
highest antioxidant potential. In addition, in the case of both extracts, the DPPH free
radicals are consumed in the first 300 s (for the extracts with high concentration, between
0.5 and 1 mg/mL), and then the kinetics of the reaction reach equilibrium. In the case of
the last three concentrations (0.05–0.3 mg/mL), the DPPH free radicals are consumed faster,
reinforcing the assumption that the less concentrated extracts contain a reduced amount of
antioxidants and phenolic compounds.
The antioxidant potential of both Galium verum L. extracts, compared with the
antioxidant potential of ascorbic acid ethanolic solution (vitamin C), is shown in
Table 4. The antioxidant potential percentage obtained for all the concentrations tested
of Galium verum L. extracts (ethanol and ethyl acetate fraction) represents an average
of three measurements ± standard deviation (SD). Further, by linear regression analysis,
between these values and their concentrations, the EC50 was calculated. The EC50 of
Galium verum L. ethanol extract was 0.136 ± 0.03 mg/mL (R2 = 0.92046), and the EC50 of
ethyl acetate fraction was 0.074 ± 0.01 mg/mL (R2 = 0.94174).
One can observe that both Galium verum L. extracts show antioxidant potential well
above 35% in the case of GvEtOH samples, whose values were lower than in the case of the
samples obtained from the ethyl acetate fraction (GvEtOAc), which starts at above 45% at
the smallest concentration tested (0.05 mg/mL). Moreover, it can be observed that in the
case of GvEtOAc extract, the samples of 1 mg/mL and 0.8 mg/mL revealed values (96%)
almost identical to the standard value of Vit C (97%) at a concentration of 0.4 mg/mL.
Considering the results obtained, it can be noticed that the antioxidant potential of all the
samples tested, obtained from both Galium verum L. extracts, are concentration dependent.
 Nevertheless, both assays showed a similar trend, with GvEtOAc extract presenting the
highest antioxidant potential. In addition, in the case of both extracts, the DPPH free rad-
icals are consumed in the first 300 s (for the extracts with high concentration, between 0.5
and 1 mg/mL), and then the kinetics of the reaction reach equilibrium. In the case of the
last three concentrations (0.05–0.3 mg/mL), the DPPH free radicals are consumed faster,
Molecules 2023, 28, 7804 reinforcing the assumption that the less concentrated extracts contain a reduced amount8 of 29
of antioxidants and phenolic compounds.

Figure 3. The time-dependent degradationkinetics

time-dependent degradation kineticsofofDPPH
DPPHfree
freeradicals
radicalsare
areprovided
provided byby
thethe etha-
ethanol
nol (A) and ethyl acetate (B) Galium verum L. extracts as well as by the ethanolic solution of vitamin
(A) and ethyl acetate (B) Galium verum L. extracts as well as by the ethanolic solution of vitamin C
C (black
(black line).
line).

4. The
TableThe antioxidantpotential
antioxidant potential values
of both(%) of Galium
Galium verum
verum L. extracts
extracts,at compared
six concentrations tested
with the as
anti-
compared with vitamin C (standard) and the corresponding
oxidant potential of ascorbic acid ethanolic solution (vitaminEC 50 values.
C), is shown in Table 4. The
antioxidant potential percentage obtained for all the concentrations tested of Galium verum
Examined Extract L. extracts (ethanol and ethyl acetate fraction) represents an average of(mg/mL)
three measure-
Antioxidant Potential AP (%) EC50
Concentration (mg/mL)
ments ± standard deviation (SD). Further, by linear regression analysis, between these
Vitamin
valuesCand
(Standard) GvEtOH
their concentrations, the EC50 wasGvEtOAc GvEtOH
calculated. The EC GvEtOAc
50 of Galium verum L. eth-
1 anol extract was 0.136 ±88.66
0.03 ±
mg/mL
0.003 (R = 0.92046),
2 and the EC50 of ethyl acetate fraction
96.10 ± 0.04
0.8 (R2 = ±
was 0.074 ± 0.01 mg/mL 85.95 0.06
0.94174). 95.91 ± 0.04
0.5 56.21 ± 0.04 93.24 ± 0.04
97.08 ± 0.04 0.136 ± 0.03 0.074 ± 0.01
0.3 56.05 ±
Table 4. The antioxidant potential values (%) of 74.74
0.04 Galium± verum
0.06 extracts at six concentrations tested
0.1 as compared with vitamin C40.87 ± 0.07 and the corresponding
(standard) 51.74 ± 0.04 EC50 values.
0.05 36.96 ± 0.02 45.41 ± 0.03
Examined Extract Con-
Antioxidant Potential AP (%) EC50 (mg/mL)
centration (mg/mL) 2.2. Bioactivity
Vitamin C (Standard) Analysis
2.2.1. Antimicrobial GvEtOH GvEtOAc GvEtOH GvEtOAc
1 88.66 ± 0.003 96.10 ± 0.04
The obtained results for the antibacterial activity of Galium verum L. plant extracts
0.8 against Gram-positive and 85.95 ± 0.06
Gram-negative 95.91 ± 0.04
bacilli strains are presented in Table 5. The
0.5 antimicrobial effect was 56.21 ± 0.04
measured by 93.24 ± 0.04
micro-dilution assay, and the±MIC
97.08 ± 0.04 0.136 0.03 (mg/mL)
0.074 and MBC
± 0.01
0.3 56.05 ± 0.04 74.74 ± 0.06
(mg/mL) determination were assessed. The MIC values obtained for the Galium verum L.
0.1 ethanol extract ranged from 40.8715± 0.07 51.74 as
to 30 mg/mL, ± 0.04
well as for the Galium verum L. ethyl
0.05 acetate extract. 36.96 ± 0.02 45.41 ± 0.03

5. The
TableOne minimum
can observeinhibitory
that bothconcentration
Galium verum (MIC) and the minimum
L. extracts bactericidal
show antioxidant concentration
potential well
(MBC) values.
above 35% in the case of GvEtOH samples, whose values were lower than in the case of
the samples obtained from the ethyl acetate fraction (GvEtOAc), which starts at above 45%
Test Compounds Microbial Strains MIC (mg/mL) MBC (mg/mL)
at the smallest concentration tested (0.05 mg/mL). Moreover, it can be observed that in the
Streptococcus pyogenes (Gram +) 15 30
Staphylococcus aureus (Gram +) 30 30
GvEtOH
Escherichia coli (Gram −) 30 NA 1
Pseudomonas aeruginosa (Gram −) NA 1 NA 1
Streptococcus pyogenes(Gram +) 15 15
Staphylococcus aureus (Gram +) 15 15
GvEtOAc
Escherichia coli (Gram −) 30 NA 1
Pseudomonas aeruginosa (Gram −) NA 1 NA 1
1 NA—no activity (absent antimicrobial activity)
Molecules 2023, 28, 7804 9 of 29

The results obtained revealed that both extracts have only a bacteriostatic effect on the
Gram-negative Escherichia coli strain and no antimicrobial activity on the Gram-negative
Pseudomonas aeruginosa strain. By comparing the two extracts, it seems that the GvEtOAc
extract showed better antimicrobial activity against the Gram-positive bacilli strains used.

2.2.2. Anticancer Potential

Viability Assay
To analyze the ability of the extracts to inhibit cell proliferation, the MTT assay was
performed. In all cases, the viability percentages varied in a concentration-dependent
manner. The effect of Galium verum L. extracts (five concentrations from the two phases)
ethanol and ethyl acetate, the lowest concentration used was 15 µg/mL, followed by
25 µg/mL, 35 µg/mL, and 55 µg/mL was the highest concentration used) was evaluated
on a healthy cell line (skin immortalized keratinocytes—HaCaT) and the human malignant
melanoma cell line A375 and compared with the control group, untreated cells.
Figure 4 shows the effect of GvEtOH and GvEtOAc extracts on HaCaT cells after a
24 h stimulation period. It can be seen that at the lowest tested concentrations (15 and
25 µg/mL), the ethyl acetate phase from G. verum L. produced a significant increase in
cellular viability compared to the control, more precisely 121%, and 103.9%, respectively,
but concerning the ethanolic extract, no increase was observed but a slight decrease in cell
Molecules 2023, 28, 7804 10 of 31
viability of 97.4% and 92.6%; from these data, it can be stated that GvEtOAc can stimulate
the proliferation of a healthy cell line.

Figure 4. Cell
Cellviability
viabilityeffect ofof
effect GvEtOH
GvEtOH and GvEtOAc
and GvEtOAc extracts (15, (15,
extracts 25, 35,
25,45,
35,and
45,55 μg/mL)
and 55 µg/mL)deter-
mined by the
determined byMTT assay,assay,
the MTT 24 h 24
post-stimulation of HaCaT
h post-stimulation immortalized
of HaCaT immortalizedhuman keratinocytes.
human keratinocytes. The
statistical differences between the control and the treated group were analyzed by applying
The statistical differences between the control and the treated group were analyzed by applying the the one-
way ANOVA
one-way ANOVA analysis followed
analysis followedby by
Dunett’s multiple
Dunett’s multiplecomparisons
comparisonspost-test
post-test(*(*pp<<0.05;
0.05;**
** pp << 0.01;
0.01;
*** p < 0.001; **** p < 0.0001).
*** p < 0.001; **** p < 0.0001).

Further, Figure
Further, Figure 55 illustrates
illustrates the
the effect
effect of
of the
the extracts
extracts onon A375
A375 melanoma
melanoma cancer
cancer cells
cells
after 24 h of stimulation. Treatment with the two extracts caused a dose-dependent
after 24 h of stimulation. Treatment with the two extracts caused a dose-dependent decrease de-
crease in tumor cell viability. At the first concentration, the GvEtOH extract
in tumor cell viability. At the first concentration, the GvEtOH extract shows a minor and shows a minor
and insignificant
insignificant increase
increase in cellinviability
cell viability at 100.8%,
at 100.8%, followed
followed by 95.4%,
by 95.4%, while
while at at thedoses,
the same same
doses,
the theacetate
ethyl ethyl acetate phase gradually
phase gradually decreasesdecreases cell viability
cell viability at 90.3%atand
90.3% and Additionally,
86.2%. 86.2%. Addi-
tionally,
at at theconcentration
the highest highest concentration tested,
tested, tumor celltumor celldecreased
viability viability compared
decreased tocompared to
the control
the60.3%
to control
fortoGvEtOAc,
60.3% forwhile
GvEtOAc, while the
the GvEtOH GvEtOH
extract extract
showed showedin
a decrease a decrease in 77.8%.
viability of viabil-
ity of 77.8%.
 after 24 h of stimulation. Treatment with the two extracts caused a dose-dependent de-
crease in tumor cell viability. At the first concentration, the GvEtOH extract shows a minor
and insignificant increase in cell viability at 100.8%, followed by 95.4%, while at the same
doses, the ethyl acetate phase gradually decreases cell viability at 90.3% and 86.2%. Addi-
tionally, at the highest concentration tested, tumor cell viability decreased compared to
Molecules 2023, 28, 7804 10 of 29
the control to 60.3% for GvEtOAc, while the GvEtOH extract showed a decrease in viabil-
ity of 77.8%.

Molecules 2023, 28, 7804 11 of 31

The results obtained indicate that the GvEtOAc extract affects more the skin tumor
cells than the healthy cell line, while in the case of the ethanolic phase, there were less
obvious differences between the effect observed on tumor and non-tumor lines following
the MTT assay.

Cell Morphology and Confluence

As a component of the antitumor profile of GvEtOH and GvEtOAc extracts, a micro-
Figure 5. Cell
Cellviability
Figure
scopic examination ofeffect
viability HaCaTofof
effect GvEtOH
GvEtOH
(Figure and GvEtOAc
6)and
and A375extracts
GvEtOAc (15, (15,
extracts
cells (Figure25, 35,
7)25, 45,
was 35,and
45,55
performedandμg/mL) deter-
55 after
µg/mL) 24
mined by the
determined byMTT
the assay,
MTT 24 h 24
assay, post-stimulation
h of A375
post-stimulation of human
A375 melanoma
human melanoma cells.cells.
The The
statistical dif-
statistical
h of treatment.
ferences between
differences between thethecontrol
controland
andthe treated
theintreated group
group were
were analyzed by
by applying
analyzedbetween applying the one-way
the one-way
ANOVA Because no followed
analysis significant by changes
Dunett’s viability
multiple were
comparisonsobserved
post-test (** p < 0.01;the*** middle
p < 0.001;con-
****
ANOVA
centrationsanalysis followed
tested, we by Dunett’s
decided to multiple
further comparisons
evaluate and post-test
highlight the p < 0.01; *** p <aspect
(**morphological 0.001;
p < 0.0001).
**** p < 0.0001).
at three of the most suggestive concentrations (15, 35, and 55 μg/mL). A bright field mi-
croscope analysis of confluence and cell morphology was performed after 24 h of stimu-
The results obtained indicate that the GvEtOAc extract affects more the skin tumor
lation to provide
cells than an overview
the healthy cell line,ofwhile
the effects
in theofcase
the two extracts.
of the On healthy
ethanolic phase, skintherecells,
wereGvE-less
tOH
obviousanddifferences
GvEtOAc betweenproduced noeffect
the significant
observed changes in cell
on tumor andmorphology,
non-tumor lines withfollowing
cells re-
maining
the MTT similar
assay. to untreated control cells, and had no negative impact on cells’ adherence
or confluence. For GvEtOAc, at the lowest dose, an increase in cell confluence was ob-
served, while at concentrations
Cell Morphology and Confluence of 35 and 55 μg/mL, a slight decrease in confluence was
seen.As Referring to the of
a component GvEtOH extract,profile
the antitumor a dose-dependent
of GvEtOH and decrease
GvEtOAc in cellextracts,
confluence was
a micro-
observed, with minor cell damage at the concentration of 55 μg/mL,
scopic examination of HaCaT (Figure 6) and A375 cells (Figure 7) was performed after 24 h data that are con-
sistent with the cell viability results.
of treatment.

Figure 6. Morphology and confluence of HaCaT cells following the 24 h of treatment with GvEtOH
and
and GvEtOAc (15, 35,
GvEtOAc (15, 35, and
and 55
55 µg/mL).
μg/mL). The
The scale
scale bars
bars indicate
indicate 200
200 μm.
µm.

Instead, in the case of tumor cells, changes in shape and confluence were visible, de-
pending on the extract and the tested concentration. For the GvEtOH extract, the highest
concentration decreased cell confluence with changes in shape. While cells treated with
35 μg/mL and more with 55 μg/mL of the GvEtOAc extract visibly lost their shape and
became round, several signs of cell death were observed, with the cells detaching from
the plate and decreasing the confluence.
Molecules 2023,
Molecules 2023,28,
28,7804
7804 12
11 of 31
of 29

Figure 7.
Figure 7. Morphology
Morphology andand confluence
confluence of
of A375
A375 cells
cells following
following the
the 24
24 hh of
of treatment
treatment with
with GvEtOH
GvEtOH
and GvEtOAc
and GvEtOAc (15,
(15, 35,
35, and
and 55
55 µg/mL).
μg/mL). The
The scale
scale bars
bars indicate 200 µm.
indicate 200 μm.

Therefore,
Because no differences were observed
significant changes between
in viability were treated
observedand untreated
between the cancer
middlecells de-
concen-
pendingtested,
trations on thewetested dose;to
decided the concentration
further evaluateof and55 highlight
μg/mL had thea morphological
direct impact on the cell
aspect at
three of the most
morphology and suggestive
the numberconcentrations
of cells. (15, 35, and 55 µg/mL). A bright field micro-
scope analysis of confluence and cell morphology was performed after 24 h of stimulation
Nuclear
to provide Morphology
an overviewEvaluation
of the effects of the two extracts. On healthy skin cells, GvEtOH and
GvEtOAc produced
The last step innooursignificant
study was changes in cellthe
to evaluate morphology, with cells remaining
cell death mechanisms similar
underlying the
to untreated control cells, and had no negative impact on cells’ adherence
cytotoxic effect of the tested extracts by examining the appearance of the nuclei of HaCaT or confluence.
For
andGvEtOAc,
A375 cells at the Hoechst
using lowest dose,
33342andye.
increase in cell confluence was observed, while at
concentrations of 35 and 55 µg/mL,
Starting from the fact that the highest a slight decrease in confluence
concentration tested causedwas seen. Referring
a reduction in cell
to the GvEtOH extract, a dose-dependent decrease in cell confluence
viability, we continued to evaluate the Galium verum extracts to identify whether was observed, with
cell death
minor
occurred by apoptosis or necrosis, comparing the effect produced by the first and the cell
cell damage at the concentration of 55 µg/mL, data that are consistent with the last
viability results.
dose of the two phases. This analysis was carried out to outline in more depth the activity
Instead, in the case of tumor cells, changes in shape and confluence were visible,
of the GvEtOH and GvEtOAc extracts on HaCaT and A375.
depending on the extract and the tested concentration. For the GvEtOH extract, the highest
Neither extract visibly affected the healthy skin line at the level of the nuclei. No
concentration decreased cell confluence with changes in shape. While cells treated with
apoptotic bodies, cell shrinkage, or nuclear fragmentation were evident, even at the dose
35 µg/mL and more with 55 µg/mL of the GvEtOAc extract visibly lost their shape and
of 55 μg/mL (Figure 8).
became round, several signs of cell death were observed, with the cells detaching from the
plate and decreasing the confluence.
Therefore, differences were observed between treated and untreated cancer cells
depending on the tested dose; the concentration of 55 µg/mL had a direct impact on the
cell morphology and the number of cells.

Nuclear Morphology Evaluation

The last step in our study was to evaluate the cell death mechanisms underlying the
cytotoxic effect of the tested extracts by examining the appearance of the nuclei of HaCaT
and A375 cells using Hoechst 33342 dye.
Starting from the fact that the highest concentration tested caused a reduction in cell
viability, we continued to evaluate the Galium verum extracts to identify whether cell death
occurred by apoptosis or necrosis, comparing the effect produced by the first and the last
dose of the two phases. This analysis was carried out to outline in more depth the activity
of the GvEtOH and GvEtOAc extracts on HaCaT and A375.
Neither extract visibly affected the healthy skin line at the level of the nuclei. No
apoptotic bodies, cell shrinkage, or nuclear fragmentation were evident, even at the dose of
55 µg/mL (Figure 8).
Molecules 2023, 28, 7804
Molecules 2023, 28, 7804 12 of 13
29 of 31
Molecules 2023, 28, 7804 13 of 31

Figure 8. HaCaT nuclei

Figure stained
8. HaCaT withstained
nuclei Hoechst 33342
with dye33342
Hoechst after dye
24 hafter
of treatment with GvEtOH
24 h of treatment and and
with GvEtOH
Figure (15
GvEtOAc GvEtOAc
8. HaCaT
and (15
55nuclei andThe
stained
µg/mL). 55with
μg/mL). Therepresent
Hoechst
scale bars scale bars
33342 represent
dye 100 24 h100
afterµm. μm.
of treatment with GvEtOH and
GvEtOAc (15 and 55 μg/mL). The scale bars represent 100 μm.
At the tumorAtcell the level,
tumor thecell level, the showed
extracts extracts showed
damage damage
to thetonuclei
the nuclei at the
at the dose
dose ofof 55
At the tumor
μg/mL. cell
In level,
the case the
of extracts
the control showed
cells, damage
the nuclei to the
have
55 µg/mL. In the case of the control cells, the nuclei have a regular shape, uniformly anuclei
regular at the
shape,dose of 55
uniformly colored,
μg/mL. In the case of
without the of
signs control cells, the nuclei
fragmentation, have being
this aspect a regular shape,
exposed uniformly
even colored,
at the lowest
colored, without signs of fragmentation, this aspect being exposed even at theconcentration
lowest
without signs of fragmentation,
of the extracts. For the this aspect being
GvEtOAc phase,exposed
it was even at thethat
observed lowest
at theconcentration
highest concentration,
concentration of the extracts. For the GvEtOAc phase, it was observed that at the highest
of the extracts.
signsForof the
cellGvEtOAc
apoptosisphase,
were itpresent,
was observed that at condensation
with nuclear the highest concentration,
and the appearance of
concentration,
signs of cell signs ofbodies.
apoptosis
apoptotic
cell
wereapoptosis
present,
In contrast,
were
with present,
the nuclear
with nuclear
GvEtOHcondensation
extract induced and condensation
the
less appearance
visible
and
of the and
dysmorphology
appearance of apoptotic
apoptotic bodies.
did notIncause bodies.
contrast, In
the GvEtOH
as significant contrast,
ofextract
changes the GvEtOH
induced
in the less extract
visible
nuclei. induced
dysmorphology
Chromatin less
and
condensationvisible
was rec-
dysmorphology
did not causeorded and did
as significantnot cause
of changes
at the concentration as significant
of in
55 the nuclei.
μg/mL of changes
Chromatin
(Figure in the nuclei.
9), and condensation
changes were was Chromatin
rec- after per-
observed
condensation
orded at the was recorded
concentration
forming at
of the
the Hoechst concentration
55 test.
μg/mL (Figure 9), ofand
55 µg/mL
changes(Figure 9), andafter
were observed changes
per- were
observed
formingafter performing
the Hoechst test. the Hoechst test.

Figure 9. (A) A375 nuclei stained with Hoechst 33342 dye after 24 h of treatment with GvEtOH and
GvEtOAc (15stained
and 55 with
μg/mL) and (B) calculated apoptotic
Figure 9. (A)
Figure 9. (A) A375 A375 nuclei
nuclei stained with Hoechst
Hoechst 33342
33342 dyedyeafter
after24 h ofindex
24ofhtreatment (AI) percentages
with
treatment GvEtOH
with for the highest
and
GvEtOH and
GvEtOAc (15 concentration
and 55 μg/mL) tested
and(75(B)
μg/mL). The apoptotic
calculated yellow arrows indexindicate signs of apoptosis.
(AI) percentages The scale bars rep-
for the highest
GvEtOAc (15
concentration
and
resent55
tested
µg/mL)
100
(75μm.
and
Data The
μg/mL).
(B) calculated
are presented apoptotic
as anindicate
yellow arrows apoptotic index
index
signs
(AI) percentages
(%) normalized
of apoptosis.
for the
to control
The scale
highest
and expressed
bars rep-
concentration
resent 100 μm.tested
Data(75
as mean values ± SD ofThe
areµg/mL).
presented anyellow
three
as arrows
independent
apoptotic indicate
experiments.
index signs
(%) normalized of
to apoptosis.
The statistical The between
differences
control scale bars
and expressed the con-
represent
as mean100 trol ±Data
values
µm. and
SD the
ofaretreated group
presented
three independentaswere analyzed index
anexperiments.
apoptotic by applying
The thedifferences
one-way
(%) normalized
statistical toANOVA
control
betweenand analysis followed by
expressed
the con-
trol and
as mean the Dunett’s
values treated
± SDgroupmultiple
of were
three comparisons
analyzed by
independent post-test
applying(***the
experiments. p <one-way
0.001;
The **** p < 0.0001).
ANOVA
statistical analysis followed
differences by the
between
Dunett’s multiple comparisons post-test (*** p < 0.001; **** p < 0.0001).
control and the treated group were analyzed by applying the one-way ANOVA analysis followed by
Dunett’s multiple comparisons post-test (*** p < 0.001; **** p < 0.0001).

Yellow arrows indicate the results regarding apoptotic characters. The results of
nuclear morphology assessment are also expressed as apoptotic index (AI). A concentration
Molecules 2023, 28, 7804 13 of 29

of 55 µg/mL induced an increase in the percentage of apoptotic index in the A375 cell line
compared to the control, where no signs of apoptosis were detected. For the GvEtOH and
GvEtOAc extracts, the percentages recorded by AI were 14.17% and 29.67%, respectively.

3. Discussion
Worldwide, most people emphasize the use of herbs for the treatment of any health
condition, according to the World Health Organization [54]. Therefore, the investigation
of the pharmacological effects of vegetal material represents a continuous concern for
many researchers. Vegetal materials are sources full of natural phytocompounds with
a plethora of beneficial activities for the living organism, such as antimicrobial, anti-
inflammatory, antioxidant, and/or anticancer properties, bioactive substances that could
contribute to the manufacturing of efficient and safe therapeutic drugs [55]. Polyphenols
are the most important class of natural bioactive phytocompound and are considered
the secondary metabolites that are either normally synthesized by plants during their
development or are by-products that occur as a response to the ecological stress factors to
which the plant is subjected (like pollution) [56]. Besides polyphenols, flavonoids, phenolic
acids, anthocyanins, tannins, terpenes, saponins, vitamins, or essential oils are natural
antioxidants with an important role in protecting biological systems against the harmful
consequences of oxidative stress. The plant species from the Rubiaceae family, genus
Galium, represent a valuable source of polyphenolic compounds [38]. In this context, the
present study was conducted to evaluate the phytochemical and biological profile of two
phases of Galium verum L. plant material acquired from a local store by assessing their
phenolic content and their antioxidant potential correlated with their possible relevant
bioactivities, like antimicrobial and in vitro anticancer effects.
It is well known that extraction techniques represent a key factor in collecting as many
polyphenols as possible from a vegetal material. Therefore, the extraction technique is
mainly dependent on the quality of the vegetal material, the solvent used in the process, the
extraction procedures chosen, and the equipment used. These critical parameters will define
the quality of the extract and the extraction yield. To achieve a high extraction yield, the
type of solvent, the plant particle size, the temperature, and the duration of the extraction
process are factors that can be modulated. Regarding the best extraction technique used for
collecting a high amount of polyphenols from plant material, the conventional methods
based on solid–liquid extraction with different solvents are still the most desired and used
techniques by researchers due to their easy-to-use, efficient, and wide-ranging applicability
for any vegetal material [57]. Therefore, the present study employs the classical solid–liquid
extraction method, based on maceration, sonication, filtration, and concentration of the
final product, using both the ethanol 95% and concentrated ethyl acetate as solvents. To
obtain a qualitative extract without traces of heavy metals or toxic compounds due to the
soil components or soil contaminants, the plant material was acquired from a local store.
Based on the results obtained after the extraction yield for both phases was calculated
(13.95% for GvEtOH extract vs. 4.65% for GvEtOAc extract values obtained from 50 mL
extract), one can say that the extraction yield depends mainly on the type of solvent used.
Besides solvent type, in our case, even the amount of the plant material (25 g for GvEtOH
vs. 10 g for GvEtOAc), as well as the plant:solvent ratio, were defining factors for the yield
and the rate of polyphenols obtained [58–60]. Even if it is known that methanol is more
efficient for low-molecular-weight polyphenols extraction and acetone for high-molecular-
weight polyphenols extraction, other solvents like ethanol, ethanol–water, ethyl acetate,
etc., can be considered [57,61] because they are volatile solvents, safe and friendly with
the environment, and often used in pharmaceutical applications. Our results regarding
the extraction yields are higher than the results reported in the literature, most probably
due to the quality of the plant material [34]. The group conducted by Lakić found that the
methanolic extract of Galium verum L. wild growing in two different cities of Serbia had
extraction yield ranging from 5.44 to 7.21% (g of dried extract/100 g sample), depending
on the duration of extraction [34]. A result similar but slightly higher compared with
Molecules 2023, 28, 7804 14 of 29

ours (13.95% in the case of GvEtOH extract) was obtained by Friščić and co-workers [62].
The differences come from the type of solvent used; the authors used 80% methanol
and obtained an extraction yield of 18% by employing the ultrasound-assisted extraction
technique after a long period of plant material maceration.
The investigation methods play an important role in the establishment of a basic,
detailed profile regarding the beneficial therapeutic activity of a plant extract and explain
to what extent one or more compounds contribute to the appearance of the biological
effect. Generally, the biological activity of a plant extract is correlated to the concentration
of phytocompounds. The phytochemical analyses contribute to the detection of plant
compounds, thus preventing their use and improper manipulation from the point of
view of the biological effect. Therefore, in the present study, we performed the analytical
technique of liquid chromatography coupled with mass spectrometry (LC-MS), which
separates, identifies, and quantifies the polyphenols (phenolic acids and flavonoids) from
Galium verum extracts. In addition, to identify the unknown organic, polymeric, and, in
some cases, inorganic compounds or additives and contaminants, we employed another
analytical technique: Fourier transform infrared spectroscopy.
The polyphenolic profile of the two phases was analyzed using liquid chromatography
coupled with mass spectrometry detection (LC-MS). The LC-MS is an analytical method
used for the identification of different classes of polyphenolic compounds by a single pass
of the plant extract on a reverse-phase analytical column in a short time [63]. The outcomes
obtained for both phases of Galium verum L. plant material highlighted the identification
of seven polyphenolic compounds in both Galium verum L. extracts, followed by their
quantification based on their peak area and the calibration curve of their corresponding
standards. Both extracts have shown identical qualitative phenolic profiles regardless
of the type of solvent used for extraction, the only differences being quantitative. Rutin
was found in higher quantity in GvEtOH extract (14.811 µg/mL) compared to GvEtOAc
extract (1.896 µg/mL). Chlorogenic acid and isoquercitrin were more abundant in GvEtOAc
extract (10.216 µg/mL and 20.384 µg/mL) as compared with GvEtOH extract (8.027 µg/mL
and 17.765 µg/mL). The rest of the polyphenols and flavonoid compounds were found
in low concentrations or below the limit of detection (e.g., quercitrin in GvEtOH extract
as compared with 6.722 µg/mL quercitrin found in GvEtOAc extract). It was stated that
rutin, an important flavonoid glycoside, has effectiveness in various diseases such as
inflammatory bowel disease, Alzheimer’s disease, and cancer [64–66], while isoquercitrin
(a flavonoid glycoside) possesses suitable well-known anti-inflammatory effects [67]. Our
findings are in agreement with those reported in previous studies [38,68,69].
As a completion of the polyphenolic profile, the functional group’s fingerprints were
investigated by FT-IR. This analysis can be seen as a plus because the chemical structure of
the phenolic compounds is related to the antioxidant capacity of the extracts, especially
the number of available hydroxyl groups as well as the extract concentration. Although
both phenolic acids and flavonoids are known strong antioxidant compounds, their ability
depends on the chemical structure more than the amount in which they are present in a sam-
ple [70]. Therefore, the most important functional groups were recorded at 3412.08 cm−1 ,
which are assigned to the hydroxyl groups from alcohols, phenols, and carboxylic acids [71].
This band usually signals the presence of flavones, such as rutin. The double band recorded
at 2926.01/2852.72 cm−1 indicates the occurrence of the aromatic ring and alkyl group
attachment to the C-H stretching functional group or indicates the O-H functional groups
from phenolic acids, such as chlorogenic and/or 4-O caffeoylquinic acids. However, at
the same time, the band situated at 2926.01 cm−1 may correspond to the CH3 vibrations,
which exist in the functional groups of chlorophyll present in the Galium verum L. plant
material [72]. The peaks recorded at 1516.05 cm−1 confirm the presence of the aromatic
ring in both extracts [73]. The bands situated at 1259.52 cm−1 and 1261.45 cm−1 correspond
to the aromatic acid ester C-O stretching vibration [74]. These bands are attributed to
flavones like rutin or even to chlorogenic acid since it is the ester of caffeic acid because
flavonoids/flavones contain ester-like functional groups in their structure. The primary,
Molecules 2023, 28, 7804 15 of 29

secondary, and tertiary alcohol functional groups from both extracts are highlighted by
the peaks recorded between 1070 and 1170 cm−1 [74]. The bands recorded between 550
and 810 cm−1 could be the out-of-plane bending vibration from aromatics compounds [75],
which in this case could be specific for aromatic bicyclic monoterpenes or of halo com-
pounds (C-Cl and C-Br stretching). The presence of halo compounds may be due to the
presence of some soil minerals that have been absorbed by the plant or even to some
impurities from the glassware used during extraction, respectively, during characterization.
The total content of phenols and flavonoids was also investigated. The total phenolic
content method is based on the electron transfer reactions between the Folin–Ciocalteu
reagent and phenolic compounds present in both Galium verum L. extracts when a blue-
colored complex is formed and can be quantified spectrophotometrically [76,77]. Our
results regarding the total phenolic and flavonoid contents showed a small content of
polyphenolic compounds but agreed with the results obtained from LC-MS analysis. We
have obtained a TPC of 1.30 mg GAE/g dry extract for the GvEtOH extract as compared
to 1.39 mg GAE/g dry extract for the GvEtOAc extract. In the case of TFC quantification,
1.42 mg CE/g dry extract was obtained for the GvEtOH extract, compared to 1.37 mg
CE/g dry extract for the GvEtOAc extract. It seems that our results are similar to the
results obtained by Danila and co-workers [78] but much lower than the results of other
researchers reported in the scientific literature [38,62,68,79,80]. Similar results but slightly
higher regarding the TPC were reported by Mocan and co-workers [38], although the
authors found an increased amount of chlorogenic acid as well as rutin through LC-MS
analysis. Also, similar but higher results regarding the TPC were reported by Lakic and
co-workers [34] and Vlase and co-workers [81]. Regarding the TFC, Laanet and co-workers
obtained 2.6 mg QE/g extract for an extract of 80% ethanol of Galium verum L. herb. These
differences may be due to the extraction method applied (duration, temperature), the
concentration and type of solvent used, the geographical area and soil content where
the plant has grown (quality of plant material), the amount of the plant material used in
the extraction process, and many other factors that can influence the total phenolic and
flavonoid contents.
It is known that plants are the most abundant and cheapest source of food and medici-
nal cures for humans. For medicinal purposes, the plants are used as a source of antioxidant
compounds, like polyphenols, phenolic acids, and/or flavonoids, capable of scavenging
the free radicals by inhibiting the oxidative stress that leads to a variety of human diseases
(asthma, diabetes, Alzheimer’s and Parkinson’s diseases, atherosclerosis, inflammatory
arthropathies, and cancers). According to Armatu et al. [82], a plant material that has a low
content of antioxidant compounds proves the fact that the plant has a weak polyphenols
content and vice versa. Therefore, a strong antioxidant capacity correlated with a high
content of polyphenols leads to a high potential of plant material to prevent oxidative stress
and to confer excellent anti-inflammatory, antibacterial, and/or antitumor properties. There
are various methods to assess the antioxidant potential of Galium verum L., reported either
as IC50 , original absorbance or % loss, or Trolox/ascorbic acid equivalent [38,40,68,80]. Re-
gardless of the comparison method (direct—through a concentration-dependent expression
or indirect—through comparison with a standard), through the antioxidant compounds
assessment, one can determine the health benefits of a plant material as well as the promis-
ing future properties for the curing disease, regarding the people who consume it. For a
simple and fast estimation of the antioxidant potential, it was employed the DPPH free
radical-scavenging assay (2,2-diphenyl-1-picrylhydrazyl), an in vitro non-cellular assay,
due to its wide use, as well as stability, reliability, and the simplicity of the assay [83]. It
has been showed that the assessed extracts obtained from Galium verum L. herba, acquired
from a local natural products store, were able to reduce the purple stable radical DPPH· to
the yellow colored DPPH-H, reaching 50% reduction with an EC50 of 0.136 mg/mL in the
case of GvEtOH and 0.074 mg/mL in the case of GvEtOAc, respectively. Previous studies
of the antioxidant potential of Galium verum L. extracts showed a similar trend, with lower
IC50 values established in the DPPH test. For instance, Lakić and co-workers found that
Molecules 2023, 28, 7804 16 of 29

the methanolic extracts obtained at different hours of extraction showed an IC50 ranging
from 3.10 µg/mL to 8.04 µg/mL, depending on the geographical area from where the
lady’s bedstraw was collected [34]. In addition, Friščić and co-workers reported an IC50 of
30.72 µg/mL for Galium verum L. extract, 80% in methanol [62]. A similar trend (with lower
IC50 values) has also been reported in previous studies regarding the antioxidant activities
of the extracts obtained from Galium species established by the DPPH test [84,85]. These
differences observed between the values regarding the DPPH free radical test could be the
result of either the concentration of DPPH used, its greater sensitivity to reaction conditions,
the geographical area, the age of the plant, the date of collection, the soil where the plant
was grown, the type and volume of solvent used for compounds extraction/isolation, and
the type and parameters of extraction method employed or the plant material:solvent ratio
used. All these factors may contribute to the obtaining of a high inhibitory concentration
value of the plant’s antioxidants, which are needed to scavenge DPPH free radicals. More-
over, the antiradical capacity of Galium species is highly dependent on their flavonoid
levels. Rutin (quercetin 3-O-rutinoside), which turned out to be the second predominant
flavonoid from Galium verum L. ethanolic extract (GvEtOH), might have contributed to
the observed antioxidant activity (Figure 3A, Table 4), comparable with the antioxidant
activity of GvEtOAc extract [81,86]. Almost identical results with ours (0.136 mg/mL) are
reported by Vlase and co-workers for the DPPH free radical-scavenging activity, recorded
for the 70% (v/v) ethanolic extract of G. verum L. (105.43 µg/mL) [81]. These results may
be the consequence of isoquercitrin (quercetin 3-O glucoside) content instead of quercitrin
(quercetin-3-O-rhamnoside), which was recorded below the detection limit by LC-MS
analysis, in the case of ethanolic extract. This affirmation contradicts the statements made
by Li and co-workers [87], which affirmed that quercitrin exhibited greater activity than
isoquercitrin in an H-donating-based 1,1-diphenyl-2-picrylhydrazyl radical-scavenging
assay. In addition, the results obtained in the case of the ethyl acetate phase support and
strengthen the statement made because, in the case of GvEtOAc extract, the content of
isoquercitrin (20.384 µg/mL) was much higher than the content of quercitrin (6.722 µg/mL).
A possible explanation could be the difference between the two forms of DPPH (DPPH-I
(m.p. 106 ◦ C) is orthorhombic as compared with the DPPH-II (m.p. 137 ◦ C), which is
amorphous)). Based on the results obtained, one may say that the phenolic compounds
contributed significantly to the antioxidant capacity of the Galium verum L. medicinal herbs
due to their high potential to neutralize free radicals. This statement is also confirmed by
Lakić and co-workers [34], who have investigated the amount of total chlorophylls (a + b)
from G. verum L. 80% methanol extract, finding the higher amount of total chlorophylls,
which partially explains the stronger effects of the plant material on the neutralization
of DPPH.
The antimicrobial investigation of the two extracts was performed because, among the
phytochemicals of plant material, polyphenols are the most potent antimicrobial compounds,
especially phenolic acids and flavonoids. Because both extracts of Galium verum L. presented
considerable amounts of polyphenols, the antimicrobial activity was performed through the
disc-diffusion assay. The disc-diffusion assay was carried out against a panel of microorganisms,
including two Gram-positive bacteria, Staphylococcus aureus and Streptococcus pyogenes, and two
Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. In this regard, we investi-
gated the antimicrobial activity against the aforementioned strains involved in skin pathol-
ogy. Our findings showed that both Galium verum L. extracts were effective against both the
Gram-positive and Gram-negative bacilli strains used, especially on Streptococcus pyogenes
(Gram+). On Escherichia coli and Pseudomonas aeruginosa strains, both extracts have no
antimicrobial activity or only bacteriostatic activity, probably because the Gram-negative
strains are more resistant due to the more complex structure of the bacterial wall. The
Pseudomonas aeruginosa strain, against which it did not obtain an antibacterial effect, is a
natural strain resistant to numerous antibiotics, such as narrow-spectrum cephalosporins,
ceftriaxone/cefotaxime, tetracyclines, and trimethoprim-sulfamethoxazole. Our findings
are contradictory to the data described in the literature. For instance, Shynkovenko and
Molecules 2023, 28, 7804 17 of 29

co-workers [32] stated that the Galium verum L. 96% ethanol exhibited high antimicrobial
activity in the Pseudomonas aeruginosa Gram-negative strain as compared with our results,
where it was showed that the 95% ethanolic extract of Galium verum L. has no antimicrobial
activity on the as mentioned bacilli strain. The contradictory results are probably due to
other factors, which might play an essential role in the extraction process. However, Vlase
and co-workers [81] have reported that the nature of the solvent might play an important
role in the antimicrobial activity of ethanolic extract (70%) of Galiumn verum L. In addition,
Ilyina and co-workers [86] complete Vlase’s affirmation by reporting a significant level of
antimicrobial properties of chloroform extract of G. verum L. However, in any case, one can-
not say with certainty that a G. verum L. extract is more or less efficient in Gram-positive or
Gram-negative bacilli strains. However, previous studies reported that the Gram-positive
bacteria were more sensitive in contact with plant extract, a fact also observed in the present
study, probably due to the single layer of the cell walls of Gram-positive bacteria [88,89].
Cultured cell lines represent important biological models for the primary in vitro
screening of nutraceuticals that are supposed to be used as possible anticancer candidates.
The cultured cell lines can be seen as platforms used in preclinical studies to provide a
similar situation suitable for clinical studies. The in vitro methods allow us to investigate
the biological potency of the tested phytocompounds from plant material, as well as their
mechanism of action within the human organism. Phenolic compounds have an important
role in the protection mechanism against pathogens and ultraviolet radiation. Human skin
is exposed to ultraviolet radiation, thus increasing ROS (reactive oxygen species) generation,
excessive release, and inflammation in cells, which are involved in the pathogenesis of
multiple skin disorders like skin cancer. Therefore, many research studies are focused on the
investigation of antioxidant compounds that are known to attenuate the damaging effects
of ROS. There is a wide variety of in vitro models used as screening studies and mechanistic
investigations, but the in vitro assays with human malignant melanoma cells employed
in the present study were chosen to offer preliminary valuable information regarding
the cytotoxicity of the two extracts of Galium verum L. because this plant has not been
investigated so far on skin cancer. In addition, another in vitro assay applied in the current
study referred to programmed cell death, namely apoptosis, with nuclear fragmentation
and formation of apoptotic bodies. These in vitro techniques can only estimate the safety
and anticancer activity of the tested extracts based on Galium verum L. plant material.
According to the scientific literature, it can be observed that the extracts obtained from
Galium verum L. have been studied for their antitumor effect on several cancer cell lines, but
the effectiveness of the plant in skin cancer is not being scored. Melanoma is a malignant
tumor of the skin and has a poor prognosis, mainly due to its high resistance to therapeutic
agents. For this reason, finding new antitumor compounds is necessary to improve the
evolution of the pathology, which represents an international health problem [90].
The in vitro tests performed in the present study demonstrate that Galium verum L.
extracts suggest a potential dose-dependent cytotoxic effect against A375 cells, a more
pronounced activity being recorded for the GvEtOAc phase, while the GvEtOH extract
decreased viability to a lesser extent but considerable. Moreover, the ethyl acetate phase
stimulated the proliferation of human keratinocytes, while this effect was not evident
in the case of the ethanolic phase at 24 h post-stimulation. An important aspect is the
data obtained for non-tumor cells, where the extracts had no significant cytotoxic effect
24 h post-stimulation. Furthermore, knowing that the modification and alteration of cell
morphology are signs that attest to cytotoxicity, in the present study, we exposed this type
of changes due to G. verum L. extracts, especially at the concentration of 55 µg/mL. In
addition, we revealed specific characteristics of apoptosis with the help of the Hoechst 33342
method, through which nuclear changes from condensation, cleavage, and the formation
of apoptotic bodies can be highlighted [91].
Our results regarding the bioactivity of the plant G. verum L. on cancer cells are
consistent with the data found in the literature. For instance, Schmidt et al. [53] studied
the in vitro effect of G. verum L. aqueous extract on chemosensitive laryngeal carcinoma
Molecules 2023, 28, 7804 18 of 29

cell lines (Hep-2 and HLaC79) and on chemoresistant laryngeal carcinoma cell lines with
P-glycoprotein overexpression (Hep2-Tax, HLaC79-Tax), where they highlighted an effect
of inhibiting the growth of both types of cell lines. The extract significantly inhibited the
invasion of Hep-2 and Hep2-Tax cells in the collagen gels and extracellular matrix substrates
tested, the strongest effect being in the Hep2-Tax cell line. On the other hand, the extract did
not affect endothelial tube formation. Furthermore, in this study, gene expression profiling
did not reveal unique patterns of gene stimulation or suppression in HLaC79 and Hep2 cell
lines [53]. Another study reported by Pashapour and his team highlighted the antitumor
potential of Galium verum L. extracts on colon (HT29) and liver (HepG2) cancer cell lines.
More precisely, it was shown that the fractionated extract with chloroform showed cytotoxic
effects on HT29 at the highest concentrations (50 and 100 µg/mL) after a 72 h stimulation;
instead, it increased the viability of HepG2 cancer cells. Regarding the petroleum ether
fraction, it was observed that on the colon cancer line, it possessed cytotoxic action at all
tested doses and on liver tumor cells at the lowest concentration of 3.125 µg/mL. According
to the data presented by the researchers, the fractionated extracts exert cytotoxic action
against the HT29 and HepG2 cell lines [92]. The group led by Shinkovenko emphasized
the immunomodulatory activity of three ethanolic extracts (ethanol 20%, 60%, and 96%)
of lady’s bedstraw. All extracts had a stimulating action on the transformation activity of
immunocompetent blood cells. The strongest immunomodulatory effect was established for
the concentrated extract (96%). Under the influence of this extract, the percentage of blast
transformation of lymphocytes increased 6.77–8.04 times compared to their spontaneous
transformation and 1.14–1.36 times compared to phytohemagglutinin [47]. Moreover, the
same types of ethanolic extracts but from the species G. aparine L. were studied in terms
of immunomodulatory activity. All showed the ability to stimulate the transformation of
lymphocytes, and this time, the 96% ethanolic extract is considered the most active [48].
Furthermore, according to Shi and co-workers, the petroleum ether fraction of the product
obtained from the 60% ethanol extraction of Galium aparine L. can exert anti-proliferative
action in vitro on the leukemia cell line K562. This activity is due to the content rich in
the three identified compounds: β-sitosterol, daucosterol, and dibutyl phthalate. The
research team showed that the three compounds can inhibit the proliferation of the K562
leukemic cells in a concentration- and time-dependent manner. Butyl phthalate showed
the strongest action, followed by β-sitosterol, these being considered the main contributors
to the biological action of G. aparine L. extract [93].
The outcomes reported in the present study do not offer sufficient information regard-
ing the effect of Galium verum L. extracts (ethanolic and ethyl acetate phases) on melanoma
cells; therefore, future studies are needed regarding the antitumor potential of this plant
material on human skin cancer, especially experiments that describe the molecular mech-
anism of the phenolic compounds, thus explaining the antitumor efficiency of ethanolic
and ethyl acetate extracts of Galium verum L. In the current study, we have considered each
Galium verum L. extract as an active ingredient because, in applied ethnomedicine, most of
the biologically active compounds, present in a small amount, remain undetectable [94].
Both Galium verum L. extracts are a mixture of bioactive nutraceuticals, able to mediate
therapeutic activity, but, with all these, their isolation should be performed to better explain
the phytochemical basis of biological effects observed in the current study.
Nevertheless, summarizing the literature data and the results obtained in this study,
Galium verum L. could be considered a promising nutraceutical for the antitumor approach
to skin cancer.

4. Materials and Methods

4.1. Chemicals, Reagents, and Bacterial Strains
Ethanol 95% (v/v) purchased from Girelli alcool SRL (Milano MI, Italy) and ethyl
acetate (≥99.5%), acquired from Sigma Aldrich (Steinheim, Germany) was used to obtain
the extracts from the aerial parts of Galium verum L. plant material. To investigate the
antioxidant potential through the DPPH method, 2,2-diphenyl-1-picrylhydrazyl (DPPH,
Molecules 2023, 28, 7804 19 of 29

purchased from Sigma Aldrich, Steinheim, Germany) was used. To compare the results of
the extracts, ascorbic acid (acquired from Lach-Ner Company (Prague, Czech Republic))
was used as standard. All chemicals used were of high analytical-grade purity.
The standards used for the LC-MS analysis were as follows: chlorogenic acid, 4-O-
caffeoylquinic acid, rutin, quercetin, quercetol, quercitrin, isoquercitrin were purchased
from Sigma-Aldrich (St. Louis, MO, USA), while luteolin and gallic acid were purchased
from Roth (Karlsruhe, Germany). The standards (+)-catechin, (−)-epicatechin, vanillic acid,
syringic acid, and protocatechuic acid (3,4-dihydroxybenzoic acid) were purchased from
Sigma-Aldrich (Steinheim, Germany), Merck (Darmstadt, Germany), and Alfa-Aesar (Karl-
sruhe, Germany). Methanol and acetic acid of HPLC analytical grade were purchased from
Merck (Darmstadt, Germany). Ultrapure deionized water was provided by a MiliQ system
Milli-Q® Integral Water Purification System (Merck Millipore, Darmstadt, Germany).
The total phenolic content determination was performed using gallic acid 98% and
Na2 CO3 99%, which were procured from Roth (Dautphetal, Germany), and Folin–Ciocalteu
reagent, acquired from Merck (Darmstadt, Germany). The total flavonoid content determi-
nation was conducted using NaNO2 acquired from Merck, AlCl3 98% purchased from Roth,
and NaOH pellets procured from ChimReactiv SRL (Bucharest, Romania). The standard
used for the determination of flavonoid content was (+)-Catechin hydrate 98%, acquired
from Sigma-Aldrich.
For the in vitro experiments, the reagents used were culture medium–high glucose
Dulbecco’s Modified Eagle’s Medium (DMEM) and the cell culture supplement fetal bovine
serum (FBS) and trypsin-EDTA solution were purchased from PAN-Biotech GmbH (Aiden-
bach, Germany). Penicillin/streptomycin (Pen/Strep-10,000 IU/mL), phosphate saline
buffer (PBS), dimethyl sulfoxide (DMSO-solvent), and MTT (3-(4,5-dimethylthiazol2-yl)-
2,5-diphenyltetrazolium bromide) viability kit were procured from Sigma-Aldrich, Merck
KgaA (Darmstadt, Germany).
For the antibacterial potential assaying of the Galium verum extracts, all microorgan-
ism strains were acquired from the American Type Culture Collection (ATCC) (Manassas,
VA, USA). The following aerobic bacterial strains, representative of the human pathogenic
bacteria, were used: two Gram-positive Staphylococcus aureus (ATCC 25923) and Strepto-
coccus pyogenes (ATCC 19615) and two Gram-negative Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853). Initially, all tested bacteria were isolated on Columbia
agar with 5% sheep blood (ThermoScientific, Waltham, MA, USA).

4.2. Cell Culture

In vitro experiments were realized on HaCaT-human keratinocytes (CLS, CVCL_0038)
provided by the Cell Lines Service GmbH (Eppelheim, Germany) and on A375-human
melanoma cell line (ATCC® CRL-1619™) purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). HaCaT and A375 cells were cultured and grown
in DMEM supplemented with 10% FBS and 1% antibiotic mixture (Pen/Strep). The analy-
ses were performed under standard conditions: a humidified atmosphere with 5% CO2
and 37 ◦ C. Cells were stimulated with Galium verum extracts at various concentrations
(15–55 µg/mL).

4.3. Plant Material and Extraction Technique

The plant material (dried aerial parts—herba) of the Galium verum L. species was
purchased from the AdNatura store (S.C. ADSERV S.R.L, Timisoara, Romania, batch no.
11/2022) and kept at room temperature (22 ± 2 ◦ C), and then crushed before being subjected
to extraction using the following types of solvents: ethanol 95% and a mixture of distilled
water and ethyl acetate (≥99.5%), according to the procedure described in the literature,
slightly modified [80].
The extraction procedure was carried out as follows: initially, 25 g of dried and ground
plant product was mixed with 150 mL of 95% ethanol and covered with parafilm, and the
whole mixture was left to maceration for 24 h. After 24 h at room temperature (22 ± 2 ◦ C),
 11/2022) and kept at room temperature (22 ± 2 °C), and then crushed before being sub-
jected to extraction using the following types of solvents: ethanol 95% and a mixture of
distilled water and ethyl acetate (≥99.5%), according to the procedure described in the
literature, slightly modified [80].
Molecules 2023, 28, 7804 The extraction procedure was carried out as follows: initially, 25 g of dried 20 ofand
29
ground plant product was mixed with 150 mL of 95% ethanol and covered with parafilm,
and the whole mixture was left to maceration for 24 h. After 24 h at room temperature (22
± 2extract
the °C), thewasextract was subjected
subjected to ultrasoundto ultrasound
for 30 min,for 30 min,
using using
an Elma an Elma
S120 S120 ultrasonic
Elmasonic Elmasonic
ultrasonic water bath, then filtered through a Whatman grade
water bath, then filtered through a Whatman grade 4 filter paper, followed by another 4 filter paper, followed by
another filtration through a 0.45 μm nylon membrane filter
filtration through a 0.45 µm nylon membrane filter (Agilent Technologies, Santa Clara, (Agilent Technologies, Santa
Clara,
CA, CA, to
USA), USA),
ensure to ensure the sterilization
the sterilization of theofextract.
the extract. To remove
To remove the the solvent
solvent (ethanol),
(ethanol), a
a rotary
rotary vacuum
vacuum evaporator
evaporator (HEIDOLPHLaborata
(HEIDOLPH Laborata4000 4000efficient
efficientWBWBeco)eco)was
wasusedusedatataa
temperatureof
temperature of2525◦°C andaapressure
C and pressureof of6060mbar.
mbar.The Theextract
extractobtained
obtained(hereafter
(hereafterreferred
referred
to as GvEtOH) was stored in a refrigerator at 4◦ °C until further
to as GvEtOH) was stored in a refrigerator at 4 C until further evaluation and use [95]. evaluation and use [95].
Furthermore, the 25 g of plant material residue initially used in the
Furthermore, the 25 g of plant material residue initially used in the first phase of extractionfirst phase of extraction
wasweighed
was weighedat at10 10g,g,over
overwhich
whichaamixture
mixtureof of150
150mLmLof ofdistilled
distilledwater
waterand and200200mLmLof of
ethylacetate
ethyl acetatewas
wasadded,
added,and andthetheErlenmeyer
Erlenmeyerflask flaskwas
wassealed
sealedwithwithparafilm.
parafilm.After
Afteranother
another
24hh of
24 of maceration, the the mixture
mixturewas wassonicated
sonicatedfor for3030min,
min, followed
followed bybya separation
a separation of the
of
twotwo
the phases
phases(first, thethe
(first, ethyl acetate
ethyl acetatephase, followed
phase, followed by the aqueous
by the aqueous phase). TheThe
phase). ethyl ace-
ethyl
tate phase
acetate phase waswascollected,
collected, andandthe extract
the extractwas
wasconcentrated
concentratedusing usinga arotary
rotaryevaporator
evaporatoratata
temperature of 25 ◦
°C (to avoid possible degradation
a temperature of 25 C (to avoid possible degradation of vegetal product) andof vegetal product) and a pressure
a pressure of
130
of 130mbar.
mbar.The Theobtained
obtainedextract
extract(GvEtOAc)
(GvEtOAc)was was kept
kept under
under the same conditions
conditions as asthethe
initialtotal
initial totalalcoholic
alcoholicextract
extract(GvEtOH)
(GvEtOH)[96]. [96].
Forbiological
For biologicalexperiments,
experiments,each eachconcentrated
concentratedextractextractwas wasdiluted
dilutedwithwith0.5%
0.5%DMSO
DMSO
to
toyield
yieldaafinal
finalstock
stocksolution
solutionofof1 1mg/mL.
mg/mL.For Forphytochemical
phytochemicalinvestigation,
investigation,the theextracts
extracts
were
werediluted
dilutedinin95% 95%ethanol
ethanoltotoyieldyieldthethesame
samefinal
finalstock
stocksolution
solutionofof1 1mg/mL.
mg/mL.The Thestock
stock
solutions obtained were kept at 4 ◦ C until further evaluation. The schematic protocol is
solutions obtained were kept at 4 °C until further evaluation. The schematic protocol is
depicted
depictedin inFigure
Figure10. 10.

Figure10.
Figure 10.Schematic
Schematicprotocol
protocolof
ofGalium
Galiumverum
verumL.L.extracts
extractspreparation.
preparation.

After processing the dried plant material and using the conventional methods of
extraction (maceration followed by sonication), we can determine the extraction efficiency,
namely the extraction yield, taking into account the parameters that were used in the
working protocol: extraction time, plant/solvent ratio, contact time between plant and
solvent and solvent concentration. For the extraction yield calculation, 50 mL of each
extract obtained (ethanol and ethyl acetate) was subjected to an evaporator at a constant
temperature of up to 25 ◦ C to avoid phytocompounds degradation. The total volume of
each extract obtained after the extraction procedure was 118 mL in the case of GvEtOH
phase and 156 mL for GvEtOAc phase. The extraction yield was calculated using the
following equation:
mresidue · Vextract
η [%] = · 100 (1)
Vtotal · m plant material
Molecules 2023, 28, 7804 21 of 29

where: η—extraction yield (%); mresidue —the mass of the residue obtained after concen-
tration (g); Vextract —the volume of the vegetal extract samples subjected to concentration
step (mL); Vtotal —the total volume of vegetal extract samples obtained after the extrac-
tion process (mL); m plant material —the amount of the plant material used in the extraction
process (g).

4.4. Phytochemical Screening

4.4.1. FT-IR
To identify the presence of chemical molecules in both Galium verum L. extracts,
the Fourier transform infrared spectroscopy (FT-IR) was employed using a Prestige-21
spectrometer (Shimadzu, Duisburg, Germany). The work conditions were as follows: room
temperature (22 ± 2 ◦ C), a spectral region ranging from 4000 to 400 cm−1 using KBr pellets,
and a resolution of 4 cm−1 . The FT-IR investigation is a qualitative method that allows
us to identify the functional organic groups of the main polyphenols present in the dried
Galium verum extracts based on the perfect match between the recorded absorption bands
of the dried extracts at a specific wavenumber and the absorption bands frequencies from
the library [97].

4.4.2. Liquid Chromatography Mass Spectrometry (LC-MS) Analysis

To identify the polyphenols that are present in both extracts, liquid chromatography
coupled with mass spectrometry (LC/MS) was performed using the Agilent Technologies
1100 HPLC Series system (Agilent, Santa Clara, CA, USA), according to a previously
validated and described method [81,98,99]. The system was equipped with a degasser
(G1322A), binary gradient pump (G13311A), column thermostat, auto-sampler (G1313A),
and UV detector (G1316A). In addition, the HPLC system was also coupled with an
Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). A reverse-phase analytical column
(Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 µm particle) was employed for the separation at
a work temperature of 48 ◦ C. The detection of the compounds present in both extracts
was performed on both UV and MS modes. For the analysis, the UV detector was set
at a wavelength of 330 nm for 17 min. (to detect the polyphenolic acids), and then at
370 nm wavelength for 38 min. (to detect flavonoids and their aglycones). By using an
electrospray ion source in negative mode (capillary +3000 V, nebulizer 60 psi (nitrogen),
dry gas nitrogen at 12 L/min. and dry gas temperature 360 ◦ C), the MS system was put
into operation [58]. To carry out the analysis, a mobile phase, which consists of a binary
gradient formed from methanol and acetic acid 0.1% (v/v), was used. The first elution
(5% methanol), which lasted for 35 min with a flow rate of 1 mL·min−1 for 5 µL injection
volume, started with a binary linear gradient and ended at 42% methanol; then, the isocratic
elution started with 42% methanol and lasted 3 min, followed by the rebalancing of the
column with 5% methanol, which lasted for 7 min [100]. To identify the compounds, the
MS spectra obtained from the standard solution of polyphenols were inserted in a mass
spectra library and compared with the MS spectra/traces of each polyphenol found in
the test solutions, provided by an MS signal used only for qualitative analysis. Based on
the standard-compound spectral match, the polyphenols present in both Galium verum L.
extracts were identified. From MS detection, the UV trace of identified compounds was
used for their quantification. In the case of compounds whose MS spectra overlap, they can
be selectively identified based on the differences between their molecular mass and the MS
spectra obtained from qualitative analysis (MS detection). The quantification and detection
limits for each compound were 0.1 µg/mL. The detection limits were calculated as minimal
concentration, producing a reproductive peak with a signal-to-noise ratio greater than three.
By using an external standard method, the quantitative determinations were performed.
By using ChemStation (vA09.03) and Data Analysis (v5.3) software from Agilent (Santa
Clara, CA, USA), all the chromatographic data were processed [100]. The calibration curves
of their corresponding standards for a five-point plot in the range of 0.1–50 µg/mL, with
suitable linearity (R2 = 0.999), were used to determine the concentration of polyphenols
Molecules 2023, 28, 7804 22 of 29

in Galium verum L. extract samples, and the results were expressed as µg of polyphenolic
compound/mL of Galium verum L. extract.
Using the same analytical conditions described above, catechin, epicatechin, gallic acid,
syringic acid, vanillic acid, and protocatechuic acid were investigated. The only applied
difference was the elution, which started with a different binary gradient and compound
detection in MS mode. The binary gradient started with 3% methanol and lasted over
3 min., followed by 8% methanol for 8.5 min., then 20% methanol for 10 min., and finally
3% methanol to rebalance column. The investigated compounds were quantified based on
their peak area and the calibration curve of their corresponding standards, and the results
were expressed as µg of polyphenolic compound/mL of Galium verum L. extract [58].

4.4.3. Total Phenolic (TPC) and Flavonoid Contents (TFC)

The total phenolic content of both extracts obtained from the Galium verum L. aerial
part was performed using the Folin–Ciocalteu method, with some modifications [77]. The
method is based on mixing 0.5 mL Galium verum L. extract solution (1 mg/mL) with 2.5 mL
Folin–Ciocalteu reagent, which was previously diluted 1:10. Then, 2 mL of 7.5% Na2 CO3
solution was added in each mixture. Both samples were kept in the dark for 90 min, and
then the absorbance was read versus blank at 750 nm wavelength using a UviLine 9400
Spectrophotometer from SI Analytics (Mainz, Germany). For the quantification of the total
phenolic content, an equation obtained from the calibration curve of gallic acid was used
(R2 = 0.997), using gallic acid solutions of different concentrations (0.05–1 mg/mL). The
total phenolic content of both Galium verum L. extracts was expressed as milligrams of
gallic acid equivalents (GAE) per gram of dry extract (mg GAE/g dry extract).
The total flavonoid content of both extracts obtained from Galium verum L. herba was
conducted using the method described by Masaada and co-workers [101]. Briefly, 250 µL
of 1 mg/mL from each extract solution was mixed with 75 µL of 5% NaNO2 solution.
After 6 min, the mixture was added sequentially 150 µL of 10% AlCl3 and 500 µL of 1 M
NaOH. After that, the total volume was adjusted to 2.5 mL by completing with distilled
water. The absorbance was read at 510 nm wavelength versus blank using a UviLine
9400 Spectrophotometer from SI Analytics. The total flavonoid content was calculated
using an equation obtained from the calibration curve of (+)-Catechin hydrate (R2 = 0.999)
in the range of 0.001–0.05 mg/mL. The results were expressed as milligrams of catechin
equivalents per gram of dry extract (mg CE/g dry extract).

4.4.4. Antioxidant Activity

The antioxidant potential (AP) of Galium verum ethanol and ethyl acetate extracts
(at 6 different concentrations for EC50 determination) was established using a DPPH
free radical-scavenging assay, according to a previously reported method, modified and
developed by our research group [102]. The results obtained were expressed as EC50 value,
which represents the half maximal inhibitory concentration of the antioxidants contained
in Galium verum L. total ethanolic extract as well as in the Galium verum L. ethyl acetate
fraction, needed to scavenge 50% of DPPH free radicals present in the test solutions. Briefly,
an ethanol solution of DPPH 0.1 mM was prepared and kept at 4 ◦ C until further use. A
precise volume of each test sample was added into a quartz test cuvette (10 × 10 mm) with
2.7 mL DPPH 0.1 mM ethanol solution. The absorbance values were read continuously for
20 min using a UviLine 9400 Spectrophotometer from SI Analytics (Mainz, Germany). As
etalon for comparison, it was used ascorbic acid (Vit C) 0.4 mg/mL in 95% ethanol. The
absorbances (test samples, etalon, and control) were measured spectrophotometrically at
517 nm wavelength. For the quantification of the DPPH free radical inhibition percentage,
the below equation was used:

A DPPH − Atest sample

AP (%) = ( ) · 100 (2)
A DPPH
Molecules 2023, 28, 7804 23 of 29

in which the Atest sample is the absorbance of each concentration of Galium verum L. test
sample (from total ethanolic extract and ethyl acetate fraction) in the presence of DPPH
free radical and A DPPH is the absorbance of DPPH free radical (control) without the
Galium verum L. test sample.
By linear regression analysis curve plotting between the inhibition percentages of antioxi-
dant potential (AP%) obtained and the concentrations of each test sample of Galium verum L.
(total ethanolic extract and ethyl acetate fraction), the half maximal inhibitory concentration
(EC50 ) was determined using OriginLab 2020b software (Origin Lab—Data Analysis and
Graphing Software, Szeged, Hungary).

4.5. Bioactivity Screening

4.5.1. In Vitro Antimicrobial Effects
Antimicrobial activities of the Galium verum L. extracts were tested by determina-
tion of minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC). The broth dilution assay was performed according to indications from both the
European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clini-
cal Laboratory and Standard Institute (CLSI) and was extensively described in previous
studies [103–107]. The standardized bacterial inoculum of 0.5 McFarland was diluted in
NaCl 0.85% (bioMérieux, Marcy-l’Étoile, France) to obtain approximately 5 × 105 colony-
forming units/mL (CFU). Then, the bacterial suspension and the tested compounds were
added in Mueller Hinton broth (ThermoScientific, Waltham, MA, USA), supplemented
with blood and β-Nicotinamide adenine dinucleotide (β-NAD) for S. pyogenes, obtaining
dilutions with concentrations of 30, 15, 7.5 and 3.75 mg/mL. After 24 h incubation at 35 ◦ C,
the lowest concentration without visible growth was interpreted as the MIC value. The
MBC was established by sub-cultivating on Columbia agar with 5% sheep blood 1 µL of
suspension from the test tube without visible growth. The lowest concentration that killed
99.9% of the bacteria was considered as MBC. Determinations were performed in triplicate
for each tested strain and each Galium verum L. extract.

4.5.2. Anticancer Potential

Cell Viability Assessment
G. verum L. extracts were evaluated for possible anticancer activity in vitro against the
human skin cancer cell line A375, and in addition, their effect on the non-tumor skin cell
line (HaCaT) was evaluated.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric
assay was applied to evaluate the impact of G. verum extracts on cell viability. Briefly,
cancerous and non-cancerous cells were seeded (1 × 104 cells/well) in 96-well culture
plates and allowed to adhere to the bottom of the well. After they attached, the cells were
stimulated with different concentrations of the two extracts dissolved in DMSO (15, 25, 35,
45, and 55 µg/mL) and incubated for 24 h. The control group was represented by untreated
cells. After the incubation period, the culture medium was exchanged with 100 µL/well of
fresh medium, and then the cells were treated with 10 µL/well of MTT reagent 1 solution
(tetrazolium salt) for 3 h. In the end, the blue formazan crystals obtained were dissolved in
100 µL of solution 2 (solubilization buffer) from the MTT kit and left in contact for 30 min,
as presented in one of our previous publications [108]. Absorbance was determined at
570 nm with a Cytation 5 device (BioTek Instruments Inc., Winooski, VT, USA) to calculate
cell viability. All experiments were carried out in triplicate.

Cell Morphology and Confluence Evaluation

To comprehend the cytotoxic potential effect induced in A375 and HaCaT cells, cellular
morphology and confluence were analyzed after 24 h of stimulation with G. verum L.
extracts. Cells were microscopically examined under bright field illumination. The images
were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA) and interpreted
Molecules 2023, 28, 7804 24 of 29

using Gen5 Microplate Data Collection and Analysis Software (BioTek Instruments Inc.,
Winooski, VT, USA).

Nuclear Staining Evaluation

To determine the type of cell death (apoptosis/necrosis) induced by the analyzed
G. verum L. extracts, the Hoechst 33342 test was performed. Briefly, cells were cultured in
12-well plates (1 × 105 cells/well) and then, at an appropriate level of confluence, were
stimulated with increasing concentrations (15 and 55 µg/mL). After 24 h, the cell medium
was removed, and 500 µL/well of staining solution diluted 1:2000 in PBS was added. After
keeping it at room temperature in the dark for 10 min, the solution was removed and
washed three times with PBS. The pictures were captured using Cytation 1 and processed
using Gen5 Microplate Data Collection and Analysis Software. The apoptotic index was
calculated according to the formula reported by Xu and co-workers [109].

4.6. Statistical Analysis

The results are expressed as mean ± standard deviation (SD). GraphPad Prism soft-
ware version 9.4.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.
com) was used to present statistical data obtained in all the in vitro biological studies. A
one-way ANOVA test followed by Dunnett’s multiple post-test comparisons was utilized
to determine statistical differences in samples. Statistically significant differences between
data were tagged with * p < 0.1, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. OriginLab
2020b software (Origin Lab—Data Analysis and Graphing Software, Szeged, Hungary)
was used to process the statistical data obtained at the antioxidant potential and FT-IR
investigations of Galium verum L. extracts.

5. Conclusions
The present study undertakes an evaluation of the phytochemical and biological profile
of two phases of Galium verum L. plant material, especially their efficacy on skin cancer.
In the present study, two extraction phases (ethanol and ethyl acetate) of Galium verum
L. were prepared and assessed, as well as phytocompound, antimicrobial, and antitumor
properties. The antioxidant screening outcomes showed that, following the evaluation
of several concentrations of Galium verum L. ethanolic extract and ethyl acetate fraction
samples, all the samples tested have significant antioxidant potential in a concentration
dose-dependent manner and an EC50 value quite suitable as compared with polyphenols
content determined through LC-MS analysis. GvEtOAc extract showed an EC50 value much
higher than GvEtOH extract, meaning that the ethyl acetate phase contains a higher amount
of antioxidants than the ethanolic one. Rutin, chlorogenic acid, and isoquercitrin were the
phenolic compounds found in high concentrations in both extracts, and we believe that they
are responsible for the investigated activities in this study. The ethyl acetate phase recorded
more concentrated phenolic compounds than ethanolic one, a result also confirmed by
total phenolic content determination (1.39 mg GAE/g dry extract for GvEtOAc vs. 1.30 mg
GAE/g dry extract for GvEtOH). However, the total quantity of flavonoids resulting from
LC-MS analysis was higher for GvEtOH extract than GvEtOAc extract, a fact also confirmed
by the total flavonoid content determination (1.42 mg CE/g dry extract for GvEtOH vs.
1.37 mg CE/g dry extract for GvEtOAc).
The antimicrobial activity outcomes confirm one more evidence of the effectiveness of
the traditional use of Galium verum L. herba against various pathogens, especially against
the Gram-positive Streptococcus pyogenes and Staphylococcus aureus bacilli strains, the ethyl
acetate phase being more active then ethanolic one. One can affirm that the changes
observed in the antimicrobial activity of Galium verum L. extracts corresponded to the type
of solvent used. Regarding the in vitro antitumor tests performed, the outcomes suggested
that the Galium verum L. extracts showed a potential dose-dependent cytotoxic effect against
A375 melanoma cell lines. The more pronounced activity is again revealed by the ethyl
acetate phase (GvEtOAc).
Molecules 2023, 28, 7804 25 of 29

In summary, we can conclude that our results complete the lack of literature data
with new information concerning the bioactivity of Galium verum L. herba natural product,
especially regarding the antitumor potential on malignant melanoma cells.

Author Contributions: Conceptualization, A.-D.S. and C.-A.D.; methodology, A.-D.S., E.-A.M.,

D.-S.T.-A., L.V., A.-M.V. and D.M.; software, A.I. and R.C.; validation, A.I., C.-A.D. and R.C.; formal
analysis, E.-A.M., D.-S.T.-A., L.V., A.-M.V. and D.M.; investigation, A.-D.S., E.-A.M., D.-S.T.-A.,
L.V., A.-M.V. and D.M.; resources, A.I., C.-A.D. and R.C.; data curation, A.I., C.-A.D. and R.C.;
writing—original draft preparation, A.-D.S. and E.-A.M.; writing—review and editing, A.-D.S. and
E.-A.M.; visualization, A.I., C.-A.D. and R.C.; supervision, A.I., C.-A.D., D.-S.T.-A. and R.C.; project
administration, A.-D.S., C.-A.D. and R.C.; funding acquisition, C.-A.D. and R.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by “Victor Babes” University of Medicine and Pharmacy
Timisoara, grant number Doctoral Grant–GD 2019. The APC was funded by “Victor Babes” University
of Medicine and Pharmacy Timisoara, Doctoral School.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Authors can provide raw data upon request.
Acknowledgments: The in vitro experiments were conducted within the Research Center for Physico-
Chemical and Toxicological Analysis of the “Victor Babes”, University of Medicine and Phar-
macy, Timisoara.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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