International Journal of

Molecular Sciences

Article
Healing of Skin Wounds in Rats Using Creams Based on
Symphytum Officinale Extract
Sorin Marian Mârza 1 , Adela Maria Dăescu 1 , Robert Cristian Purdoiu 1 , Mădălina Dragomir 1 , Mariana Tătaru 1 ,
Iulia Melega 1 , Andras-Laszlo Nagy 1,2 , Adrian Gal 1 , Flaviu Tăbăran 1 , Sidonia Bogdan 1 , Mirela Moldovan 3 ,
Emoke Pall 1 , Camelia Munteanu 4, *, Klara Magyari 5, * and Ionel Papuc 1

1 Faculty of Veterinary Medicine, University of Agricultural Science and Veterinary Medicine,

400372 Cluj-Napoca, Romania; sorinmarza@[Link] (S.M.M.); [Link]@[Link] (A.M.D.);
[Link]@[Link] (R.C.P.); [Link]@[Link] (M.D.);
[Link]@[Link] (M.T.); [Link]@[Link] (I.M.);
nagyandras26@[Link] or anagy@[Link] (A.-L.N.); [Link]@[Link] (A.G.);
[Link]@[Link] (F.T.); sidoniabogdan@[Link] (S.B.); [Link]@[Link] (E.P.);
[Link]@[Link] (I.P.)
2 Department of Biomedical Sciences, Ross University School of Veterinary Medicine,
Basseterre P.O. Box 334, Saint Kitts and Nevis
3 Faculty of Pharmacy, Iuliu Haţieganu University of Medicine and Pharmacy, 400012 Cluj-Napoca, Romania;
mmoldovan@[Link]
4 Department of Plant Culture, Faculty of Agriculture, University of Agricultural Science and Veterinary
Medicine, 400372 Cluj-Napoca, Romania
5 Interdisciplinary Research Institute on Bio-Nano-Sciences, Babes, -Bolyai University,
400271 Cluj-Napoca, Romania
* Correspondence: [Link]@[Link] (C.M.); [Link]@[Link] (K.M.)

Abstract: Rosmarinic acid is a well-known natural antioxidant and anti-inflammatory compound,

and it is one of the polyphenolic compounds found in comfrey plants. Comfrey root also contains
allantoin, which helps with new skin regeneration. This study aimed to investigate the healing
Citation: Mârza, S.M.; Dăescu, A.M.; and skin regeneration process of skin wounds in Wistar rats using creams based on comfrey extract
Purdoiu, R.C.; Dragomir, M.; Tătaru, and to correlate the results with active compounds in the extract. The obtained results showed that
M.; Melega, I.; Nagy, A.-L.; Gal, A.; comfrey root is rich in bioactive compounds, including allantoin, salvianolic acid, and rosmarinic acid,
Tăbăran, F.; Bogdan, S.; et al. Healing which are known for their great free radical scavenging activity, and the high antioxidant activity
of Skin Wounds in Rats Using Creams of the extract may be mainly due to these compounds. The obtained extract has an antimicrobial
Based on Symphytum Officinale effect on Staphylococcus aureus (1530.76/382.69), Escherichia coli (6123.01/6123.01), and Pseudomonas
Extract. Int. J. Mol. Sci. 2024, 25, 3099.
aeruginosa (6123.01/6123.01). The macroscopic evaluation and the histological analysis of the skin
[Link]
defects 14 days after the intervention showed faster healing and complete healing in the skin excisions
ijms25063099
treated with oil-in-water cream with 20% extract of comfrey as the active ingredient.
Academic Editors: Alina Maria
Holban and Carmen Curutiu Keywords: Symphytum officinale; antioxidant activity; cytotoxicity; antimicrobial effects; skin wounds

Received: 30 January 2024

Revised: 1 March 2024
Accepted: 5 March 2024
Published: 7 March 2024
1. Introduction
Symphytum officinale (comfrey) is used in folk medicine, especially in the treatment
of skin wounds and fractures. This plant belongs to the Borangiaceae family. It is a tall,
hairy, grassy plant; the flowers are reddish-purple, sometimes pinkish-white, and are
Copyright: © 2024 by the authors.
arranged in unipolar cymes, and it grows in damp places, by meadows, at the water’s
Licensee MDPI, Basel, Switzerland.
edge [1]. Comfrey has countless uses in pathological conditions such as sprains, bone
This article is an open access article
injuries, rheumatism, liver problems, gastritis, ulcers, gout, skin injuries, hematomas, and
distributed under the terms and
thrombophlebitis. Comfrey tea is recommended for hepatic disturbance. In the sense of
conditions of the Creative Commons
Attribution (CC BY) license (https://
gastritis and ulcers, in Brazil, this tea is widely used. In the USA, root extract is used for skin
[Link]/licenses/by/
problems. In different countries, comfrey is drunk as a tonic beverage [2]. Comfrey leaves
4.0/). and roots are used in concentrations between 5% and 20% in creams, mainly for healing

Int. J. Mol. Sci. 2024, 25, 3099. [Link] [Link]

Int. J. Mol. Sci. 2024, 25, 3099 2 of 22

superficial wounds. Comfrey root contains a wide variety of chemical constituents, such as
carbohydrates, polysaccharides, allantoin, tannins, pyrrolizidine alkaloids, triterpenes, and
phenolic acids (rosmarinic acid, caffeic acid, chlorogenic acid, and p-hydroxybenzoic and
p-coumaric acids), which can affect its application in medicine [1,3].
Phenolic elements originating from herbal plants have garnered a lot of attention
due to their effective anti-inflammatory, antitumor, antioxidant, antiviral, and anticancer
agents [4–8]. The main phenols are rosmarinic acid (RA) and salvianolic acids (SAs),
represented by salvianolic acids I, A, B, and C. Topical or local application of RA to the
skin has demonstrated potential to hasten wound healing and reduce the risk of skin
cancer in murine models [9]. Moreover, RA contains catechol groups that are identical
to dopamine. It has been demonstrated that cream with RA contents will not only have
good tissue adhesion but will also have a variety of activities that promote wound healing
based on the structure and bioactive activities of RA [10]. SAs are found in LPS-stimulated
THP-1-macrophages, and SAs downregulate the protein expression of TLR4, p-p65, and
p-IB and dramatically lower the mRNA expression levels of IL-1, IL-6, and TNF [11].
According to Xiao et al., SAs can reduce and limit the production of ROS by controlling the
Nrf2/Keap1 pathway [12]. In a mouse distal middle cerebral artery occlusion (dMCAO)
model, it was also demonstrated that SA therapy promotes cerebral angiogenesis and
increases microvessel density [13]. Cardiovascular disorders are treated with SA B. In vitro,
it prevents ischemia [14]. SA B also has other pharmacological effects, such as preventing
platelet aggregation and controlling vascular tone [15]. Patients who suffer from systemic
disorders, such as angina pectoris and myocardial infarction, are typically prescribed
Fufang Danshen tablets or Fufang Danshen Dripping Pills, which contain SA B [16]. SA B
has important anti-inflammatory properties and the capacity to block the TGF signaling
pathway [17].
Other phenolic compounds in the root extract of comfrey, such as p-hydroxybenzoic,
caffeic, chlorogenic, and p-coumaric acids, also have beneficial effects on human skin
fibroblasts [18].
Allantoin is another biologically active component of comfrey, which also has antioxi-
dant and antibacterial effects [19,20].
Comfrey polysaccharides are primarily composed of galactose, arabinose, glucose, and
galacturonic acid, indicating that comfrey polysaccharides are non-starch polysaccharides
that may pass to the end of the intestinal tract of monogastric animals and be fermented by
intestinal microflora [21].
Consuming comfrey has been linked to specific instances of hepatotoxic reactions in
people, including liver fibrosis, portal hypertension, and veno-occlusive disorders [22].
On the other hand, when used externally, no negative side effects have been documented.
Pharmacokinetic investigations have revealed low cutaneous absorption [23]. Consequently,
different health organizations do not advise its internal use [24]. However, comfrey is only
sold over the counter in the UK when it is prescribed by medical herbalists. Internal comfrey
use is restricted in the USA, Canada, and other European nations, including Germany,
Denmark, and Austria. Its use should be limited to 4–6 weeks per year, according to the
European Commission [23].
Comfrey has been suggested as an alternative treatment for skin disorders [25], and
comfrey cream has even been proven to be moderately beneficial in treating osteoarthri-
tis [2]. Although the safety evaluations of this plant’s side effects were primarily conducted
in single case reports and based on animal research utilizing high doses, they have not yet
been evaluated in human trials. Therefore, its safety has not been evaluated using human
epidemiological approaches and biochemical markers [26].
Considering the previously described properties, we aimed to obtain a natural-based
cream for wound healing with antioxidant and bactericide properties. The antioxidant
properties are aimed to help control wound oxidative stress and thereby accelerate wound
healing. Bacterial infections are widely known to be extremely detrimental to wound
healing. Superbugs, or germs that are resistant, multiresistant, and panresistant to antimi-
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 22

Int. J. Mol. Sci. 2024, 25, 3099 3 of 22

healing. Bacterial infections are widely known to be extremely detrimental to wound heal-
ing. Superbugs, or germs that are resistant, multiresistant, and panresistant to antimicrobi-
als, are thought to be the biggest issue in recent years. Most of these microorganisms ex-
crobials, are thought to be the biggest issue in recent years. Most of these microorganisms
hibit significant zoonotic potential [27]. Infections with ESKAPE-E, also known as Enter-
exhibit significant zoonotic potential [27]. Infections with ESKAPE-E, also known as En-
obacter
terobacterspp., Staphylococcus
spp., Staphylococcus aureus,
aureus,Klebsiella
Klebsiellapneumoniae,
pneumoniae,Acinetobacter
Acinetobacter baumannii,
baumannii,
Pseudomonas
Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli, are
aeruginosa, Enterobacter spp., and Escherichia coli, are the
the primary
primary causes
causes
of
of these
these pathologies
pathologies [28].
[28]. These
These pathogens
pathogens havehave significant
significant intrinsic
intrinsic resistance
resistance asas well
well asas
the
the potential
potential to
to acquire
acquire resistance.
resistance. The
The ability
ability of
of these
these microorganisms
microorganisms to to bind
bind to
to tissues
tissues
and
and later
later form
form aa biofilm
biofilm makes
makes ESKAPE-E
ESKAPE-E infections
infections challenging
challenging to to avoid
avoid and
and treat
treat [29].
[29].
Three-dimensional
Three-dimensional bacterial communities that develop on both biotic and abiotic surfaces
bacterial communities that develop on both biotic and abiotic surfaces
constitute
constitute bacterial
bacterial biofilm
biofilm [30].
[30]. Thus,
Thus, anan extract
extract from
from the
the roots
roots of
of comfrey
comfrey waswas obtained,
obtained,
from
from which the content of polyphenols and allantoin was evaluated, followed byby
which the content of polyphenols and allantoin was evaluated, followed anan eval-
evalua-
uation of antioxidant and bactericidal properties (Figure 1). The extract was
tion of antioxidant and bactericidal properties (Figure 1). The extract was introduced to an introduced to
an oil-in-water
oil-in-water disperse
disperse system,
system, andand its effect
its effect on wound
on wound healing
healing in Wistar
in Wistar rats evaluated
rats was was eval-
uated
(Figure (Figure 1). Therefore,
1). Therefore, the novelty
the novelty of this
of this studystudy consists
consists in histological
in its its histological evaluation
evaluation of
of two stages of the healing and skin regeneration process, using an oil-in-water
two stages of the healing and skin regeneration process, using an oil-in-water cream based cream
based on comfrey
on comfrey extractextract in different
in different concentrations,
concentrations, an aspect
an aspect that that allows
allows for establishing
for establishing the
the concentration that offers the best
concentration that offers the best results. results.

Figure 1. Schematic representation of comfrey extract’s investigation.

2. Results and Discussion

2. Results and Discussion
2.1. Comfrey Extract Structural Evaluation and In Vitro Assays
2.1. Comfrey Extract Structural Evaluation and In Vitro Assays
2.1.1. Determination of Total Polyphenol and Allantoin Content of Comfrey Extract
[Link]
Determination of Total Polyphenol and Allantoin Content of Comfrey Extract
determination of the total content of polyphenols using the Folin-Ciocalteu spec-
The determination
trophotometric method of the totalacontent
indicated value ofof7100
polyphenols
µg/mL inusing the Folin-Ciocalteu
the extract of SyOf. spec-
trophotometric
The UV-Vismethod and FT-IRindicated
spectraa revealed
value of 7100 μg/mL in
the presence ofthe extract SAs,
allantoin, of SyOf.
and RA in the
SyOfThe UV-Vis
extract and FT-IR
(Figure 2). The spectra
UV-Vis revealed
spectrum the (Figure
presence2A) of allantoin, SAs, andatRA
has two maxima 236 inand
the
SyOf
304 nm,extract
with(Figure 2). TheatUV-Vis
the shoulder 340 nm. spectrum
According (Figure
to the2A) has two[31,32],
literature maxima at 236
these and 304
absorption
nm,
bends with
are the shoulder
specific to [Link]
shoulderto at the
217 literature
nm can be[31,32],
assigned these
to theabsorption
presence
bends are specific
of the allantoin to The
[33]. SAs SyOf
and RA. Thehas
extract shoulder at 217
a typical nmspectrum
FT-IR can be assigned
of organicto the presence
compounds,
of the allantoin
which [33]. The
can be assigned toSyOf extract has
the presence a typical FT-IR
of allantoin, SAs, andspectrum
RA. For of example,
organic compounds,
the band at
which
1635 cm −1 be
can canassigned to thetopresence
be assigned of allantoin,
the stretching SAs,ofand
vibration C=O RA. For example,
groups, the bands thebetween
band at
1200 and 900 cm −1 can be attributed to the stretching vibration of C-C, and the band at
1635 cm can be assigned to the stretching vibration of C=O groups, the bands between
−1

1273 cm −1 to the asymmetric stretching

1200 and 900 cm−1 can be attributed tovibration of C-Ovibration
the stretching from SAsof and [Link]
C-C, Thethe
presence
band atof
allantoin
1273 cm istoproven
−1 by the presence
the asymmetric of thevibration
stretching shoulderof at C-O cm−1SAs
1772from dueandto stretching
RA. The vibration
presence
C=O andistoproven
of allantoin the bands
by the atpresence
1171, 1269, and
of the 597 cm−
shoulder at1 1772
assigned to C-C
cm−1 due stretching,vibra-
to stretching C-N
stretching,
tion of C=Oand andC-N bending
to the bandsvibration,
at 1171, 1269,respectively
and 597 [34].
cm−1 assigned to C-C stretching, C-N
Identification
stretching, and C-Nofbending
polyphenolic compounds
vibration, respectively performed by LC-ESI+ -MS analysis,
was[34].
based on maximum
Identification ofabsorption,
polyphenolic elution order, MS
compounds wassignals
performed(molecular
by LC-ESIpeaks andanalysis,
+-MS charac-
teristic fragments), and existing literature data. Six phenolic compounds
based on maximum absorption, elution order, MS signals (molecular peaks and charac- from the class of
hydroxycinnamic
teristic fragments),acids were identified
and existing literaturein data.
the aqueous extract
Six phenolic of SyOf (Table
compounds from1,theFigure
classS1),
of
of which salvianolic
hydroxycinnamic acidwere
acids I andidentified
salvianolic inacid C were present
the aqueous extractin ofthe
SyOfhighest
(Table concentrations:
1, Figure S1),
of which±salvianolic
2382.23 115.23 µg/mL acid Iand
and1641.29 ± 80.06
salvianolic C were respectively.
acidµg/mL, present in theSignificant amounts
highest concentra-
were also recorded for RA (1055.02 ± 42.20 µg/mL) and SA
tions: 2382.23 ± 115.23 μg/mL and 1641.29 ± 80.06 μg/mL, respectively. Significant B (641.83 ± 51.34 µg/mL).
amounts were also recorded for RA (1055.02 ± 42.20 μg/mL) and SA B (641.83 ±caffeic
The lowest concentrations were represented by SA A (279.50 ± 27.93 µg/mL) and 51.34
acid (123.162
μg/mL). ± 12.40
The lowest µg/mL). The were
concentrations total represented
content of phenolic
by SA Acompounds
(279.50 ± 27.93 in the extract
μg/mL) andis
6123.05 µg/mL.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 22

Int. J. Mol. Sci. 2024, 25, 3099 caffeic acid (123.162 ± 12.40 μg/mL). The total content of phenolic compounds
4 ofin
22 the ex-
tract is 6123.05 μg/mL.

Figure 2. UV−Vis (A) and FT−IR (B) spectra of the SyOf extract.
Figure 2. UV−Vis (A) and FT−IR (B) spectra of the SyOf extract.
Table 1. Identification and concentration of phenolic compounds in the sample of comfrey extract,
expressed in µg/mL caffeic
The allantoin contentacidwas
equivalent.
identified using HPLC analysis, and it obtained a consid-
erable concentration of allantoin (8228.023 ± 41 μg/mL) from the class imidazole (Figure
UV
Peak S2). [M + H]+ µg/mL
Rt (min) λmax Compound Subclass
No. (m/z) (Mean ± SD)
(nm) Thus, the obtained results show an increasing concentration of phenolic compounds,
1 13.30 which
322 means that 181comfrey root isCaffeic
rich in bioactiveHydroxycinnamic
acid compounds. In this regard,
123.162 these results
± 12.40
2 15.26 are
360,strongly
240 correlated
539 with the histological
Salvianolic acid Ichanges induced by the
Hydroxycinnamic comfrey
2382.23 cream, which
± 115.23
will be presented later.
3 16.98 360, 250 719 Salvianolic acid B Hydroxycinnamic 641.83 ± 51.34
4 17.87 330 1. Identification
Table 361and concentration
Rosmarinic acid
of phenolic Hydroxycinnamic ± 42.20
1055.02 of
compounds in the sample comfrey extract,
5 18.65 320, 260 in μg/mL495
expressed Salvianolic acid A
caffeic acid equivalent. Hydroxycinnamic 279.50 ± 27,93
6 19.60 320, 260 493 Salvianolic acid C Hydroxycinnamic 1641.29 ± 80.06
UV
Peak [M + H]+ Total Phenolics μg/mL
6123.051
Rt (min) λmax Compound Subclass
No. (m/z) (Mean ± SD)
(nm)
1 13.30 322 181
The allantoin content Caffeic acid using HPLC
was identified Hydroxycinnamic 123.162
analysis, and it obtained ± 12.40
a consider-
2 15.26 360, 240able concentration
539 of allantoin
Salvianolic acid I± 41 µg/mL)
(8228.023 from the class imidazole
Hydroxycinnamic (Figure
2382.23 S2).
± 115.23
3 16.98 360, 250 Thus,719
the obtainedSalvianolic
results show anBincreasing
acid concentration of phenolic 641.83
Hydroxycinnamic compounds,
± 51.34
4 17.87 330 which means 361 that comfrey root is rich
Rosmarinic in bioactiveHydroxycinnamic
acid compounds. In this regard,1055.02
these results
± 42.20
5 18.65 320, 260are strongly
495correlated with the histological
Salvianolic acid A changes induced by
Hydroxycinnamic the comfrey cream,
279.50 which
± 27,93
will be presented later.
6 19.60 320, 260 493 Salvianolic acid C Hydroxycinnamic 1641.29 ± 80.06
Total Phenolics
2.1.2. Cell Viability of Comfrey Extract 6123.051
HaCaT is a commonly used aneuploid immortal keratinocyte cell line derived from
2.1.2.
adultCell Viability
human of Comfrey
skin that underwentExtract
spontaneous transformation [35,36]. Considering that
HaCaT cell viability
HaCaT is influenced
is a commonly by phenolic
used aneuploidcontent, the concentration
immortal keratinocyte of the
cellSyOf
line was cal- from
derived
culated according to the total phenolic content expressed in µg/mL caffeic
adult human skin that underwent spontaneous transformation [35,36]. Considering thatacid equivalent
(382.69 µg/mL, 765.38 µg/mL, 1530.76 µg/mL, 3061.52 µg/mL, and 6123.05 µg/mL). No
HaCaT cell viability is influenced by phenolic content, the concentration of the SyOf was
significant differences were observed between treated and untreated cells when treating the
calculated according to the total phenolic content expressed in μg/mL caffeic acid equiv-
HaCaT cell line with different concentrations of SyOf ethanol extract (Figure 3). Therefore,
alent
SyOf(382.69
ethanolic μg/mL,
extract 765.38 μg/mL,
is not toxic 1530.76
to these humanμg/mL, 3061.52
epidermal cells.μg/mL, and 6123.05 μg/mL).
No significant differences were observed between treated and untreated cells when treat-
ing the HaCaT cell line with different concentrations of SyOf ethanol extract (Figure 3).
Therefore, SyOf ethanolic extract is not toxic to these human epidermal cells.
 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
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Int. J. Mol. Sci. 2024, x FOR
3099 PEER REVIEW 5 of 22
5 of 22

Figure 3. Proliferation kinetics of HaCaT cells following 24 h of treatment with different con
Figure Proliferation
[Link] ofkinetics
tionskinetics
SyOf of of HaCaT
ethanolic
HaCaT cells
extract.
cells following 2424
The average
following hh of
cell
of treatment with
vitality obtained
treatment with different concentra-
after testing
different in triplicate wa
concentra-
tions
tionsofofSyOf
SyOfethanolic
ethanolic extract.
to compare The
the
extract. average
results
The cell
to the
average vitality
control
cell obtained
obtainedafter
(untreated
vitality cell testing
testinginintriplicate
cultures).
after triplicatewas
wasused
used
totocompare
comparethe theresults
resultstotothe
thecontrol
control(untreated
(untreatedcell
cellcultures).
cultures).
2.1.3. Antioxidant Activity of Comfrey Extract
[Link]
2.1.3. AntioxidantActivity
ActivityofofComfrey
ComfreyExtract
Extract
It is known that overproduction of free radicals exists due to endogenous and
ItItisisknown
known thatoverproduction
that
enous overproduction
stress, which leadsofoffree
free radicalsexists
to radicals
various existsdue
diseases. due toevaluate
to
To endogenous
endogenous andexog-
and exoge- activity
the antioxidant
nous stress, which
enous stress, which leads
leads
extract, to various
to various
CUPRAC and diseases.
diseases.
FRAP assays To evaluate
To evaluate
were used the antioxidant
the antioxidant activity
(Figure 4). activity ofofthe
The formation the of the c
extract, CUPRAC
extract, CUPRAC and
and FRAPFRAP assays were used (Figure 4). The formation of the colored
copper chelateassays
complexwere used
in the (Figure 4).
CUPRAC Theproved
assay formation
to beof the colored
dependent on the antio
copperchelate
copper chelatecomplex
complexininthe theCUPRAC
CUPRACassay assayproved
provedtotobebedependent
dependenton onthe
theantioxidant
antioxidant
concentration in the SyOf extract, with a calculated sample EC50 of 269.5 μM Tr
concentrationininthe
concentration theSyOf
SyOfextract,
extract,with
withaacalculated
calculatedsample
sampleEC50 EC50ofof269.5
269.5μM Trolox/g
µMTrolox/g
(Figure 4A). The reduction potential of the ferric complex 2,4,6-tris(2-pyridyl)-1,3,
(Figure4A).
(Figure 4A).The
The reduction
reduction potential
potentialbyof
of the
theferric
ferriccomplex
complex 2,4,6-tris(2-pyridyl)-1,3,5-triazine
2,4,6-tris(2-pyridyl)-1,3,5-tria-
zine [Fe (III)-TPTZ] the antioxidants found in Symphytum officinale L. root w
[Fe (III)-TPTZ]
zine [Fe (III)-TPTZ] by the antioxidants
by the found in Symphytum officinale L. root was dependent
pendent on antioxidants
the dose usedfoundin thein Symphytum
experiment, officinale
with an IC50L. of root
324.1wasμMde- Fe2+/g sampl
on the dose used in the experiment, with an IC50 of 324.1 µM Fe2+ /g sample (Figure 4B). A
pendent on the dose used in the experiment, with an IC50 of 324.1 μM Fe 2+/g sample (Fig-
ure 4B). A FRAP value, in the same range interval, of 0.274 ± 0.003 mM/g was prev
FRAP value, in the same range interval, of 0.274 ± 0.003 mM/g was previously reported
ure 4B). A FRAPreportedvalue, infor theSymphytum
same range officinale
interval, of 0.274
root ± 0.003
extract [37].mM/g was previously
It is possible that the antioxida
for Symphytum officinale root extract [37]. It is possible that the antioxidant activity of the
reported for Symphytum
tivity of the officinale
extract root
cameextract
from the[37].SAs
It isand
possible that the
RA found antioxidant
in SyOf extract,ac-which are k
extract came from the SAs and RA found in SyOf extract, which are known for their great
tivity of the extract camegreat
for their fromfree
the radical
SAs and RA foundactivity
scavenging in SyOf[38–40].
extract, which are known
free radical scavenging activity [38–40].
for their great free radical scavenging activity [38–40].

Figure 4. Antioxidant
Figureactivity of the SyOf
4. Antioxidant extract
activity determined
of the by CURAC
SyOf extract (A) and
determined FRAP (B)
by CURAC assays.
(A) and FRAP (B)
Figure 4. Antioxidant activity of the SyOf extract determined by CURAC (A) and FRAP (B) assays.
2.1.4. Antibacterial Activity of Comfrey Extract
2.1.4. Antibacterial Activity of Comfrey Extract
The antibacterial
2.1.4. Antibacterial activity
Activity extract was evaluated using Staphylococcus aureus,
of SyOf Extract
of Comfrey
The antibacterial activity of SyOf extract was evaluated using Staphylococcus a
Staphylococcus aureus
The antibacterial MRSA, Escherichia
activity of SyOfMRSA, coli, was
extract and Pseudomonas
evaluated aeruginosa.
using Using the
Staphylococcus agar
aureus,
Staphylococcus aureus Escherichia coli, and Pseudomonas aeruginosa. Using th
diffusion test, no
Staphylococcus aureusantimicrobial
MRSA, effect was observed, probably due to the low diffusibility
diffusion test,Escherichia coli, and
no antimicrobial Pseudomonas
effect aeruginosa.
was observed, Using
probably the
due toagar
the low diffus
potential of the extract in MH agar and the color of the extract, respectively. Consequently,
diffusion test, nopotential
antimicrobial effect was
of the extract observed,
in MH probably
agar and the colordue to the
of the low diffusibility
extract, respectively. Consequ
the microdilution method was performed where the results were differentiated according
potential of the extract in MH agar and the color of the extract, respectively. Consequently,
 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6

Int. J. Mol. Sci. 2024, 25, 3099 6 of 22

the microdilution method was performed where the results were differentiated accor
to the concentration of the product (calculated based on total phenols) and accordi
to the concentration of the product
the bacterial strain,(calculated based
respectively. MIConindex
total results
phenols) areand according
shown to the
in Table 2. The obta
bacterial strain,results
respectively. MIC index results are shown in Table 2. The obtained results
demonstrated that the ethanolic extract of SyOf has antibacterial action. There
demonstrated thatoncethe
thisethanolic
cream is extract ofother
used, no SyOfantibiotics
has antibacterial
are used action.
againstTherefore, once
possible infections that
this cream is used, no other antibiotics
appear in such [Link] used against possible infections that may appear
in such situations.
Table 2. Assay for the bactericidal effect of the extract.
Table 2. Assay for the bactericidal effect of the extract.
MIC Index
MIC Index
MBC (μg/mL)/MIC (μg/mL)
MBC (µg/mL)/MIC (µg/mL)
Staphylococcus aureus Staphylococcus aureus MRSA Escherichia coli Pseudomonas aeruginos
Staphylococcus aureus4
SyOf Staphylococcus aureus MRSA
2 Escherichia coli 1 Pseudomonas aeruginosa1
SyOf 4 1530.76/382.69 2 1530.76/765.38 1 6123.01/6123.01 1 6123.01/6123.01
1530.76/382.69 MIC,1530.76/765.38 6123.01/6123.01
minimum inhibitory concentration; 6123.01/6123.01
MBC, minimum bactericidal concentration; an MBC
ratio ofconcentration;
MIC, minimum inhibitory less than 4 wasMBC,thought to bactericidal
minimum be bacteriostatic, and anan
concentration; MBC/MIC
MBC/MICratio
ratioof
of greater
less than
than 4 was thoughtthought
to be bacteriostatic, and an MBC/MIC ratio of greater than 4 was thought to be bactericidal.
to be bactericidal.

2.2. Characterization and In Vivo Evaluation

2.2. Characterization of Oil-in-Water
and In Vivo Cream
Evaluation of with Comfrey
Oil-in-Water CreamExtract
with Comfrey Extract
2.2.1. Creams Characterization
2.2.1. Creams Characterization
Viscosity is an important
Viscosity property for topically
is an important applied
property products, applied
for topically as it influences
products, their
as it influ
stability and their ability to sample from the packaging, to spread on the skin,
their stability and their ability to sample from the packaging, to spread on the skin, to to hold
in place and even their and
in place sensory
evenattributes.
their sensory Asattributes.
can be seen Asfrom
can be Figure 5A, the
seen from cream
Figure 5A,isthe cream
a non-Newtonian fluid, with a thixotropic behavior, as the viscosity decreases
non-Newtonian fluid, with a thixotropic behavior, as the viscosity decreases when the whe
shear rate increases. The extent of the thixotropy can be appreciated from the
shear rate increases. The extent of the thixotropy can be appreciated from the flow cflow curve.
Thus, the downThus,curvethe obtained
down curvewhenobtained
the shearwhenratesthedecrease follows
shear rates the upfollows
decrease curve butthe up curv
not very closely, and a small hysteresis area is formed. This behavior may be
not very closely, and a small hysteresis area is formed. This behavior may be attributattributed
to polyacrylamide—a gel former that
polyacrylamide—a gel is included
former thatinisthe cream in
included formula and ensures
the cream formula easyand ensures
spreading on the skin and a rapid recovery of the initial viscosity after application.
spreading on the skin and a rapid recovery of the initial viscosity after application.
Figure 5B presents a rheogram
Figure 5B presents of athe SyOf cream,
rheogram of thewhere the shear
SyOf cream, stress
where theincreases
shear stress incr
when the shear rate increases. Analysis of the plot according to the Bingham
when the shear rate increases. Analysis of the plot according to the Bingham modmodel allowed
for the calculation
lowedof yield stress,
for the which was
calculation of yield D/cm2which
846.4 stress, . Thus,was a low force
846.4 is needed
D/cm 2. Thus, toa low for
remove the cream from a tube or a jar, which confirms the ease of sampling and
needed to remove the cream from a tube or a jar, which confirms the ease of sampling spreading
of the cream. The cream of
spreading canthehold
cream.a shape, but itcan
The cream is easily
hold aspread
shape, when
but it shear stress
is easily is when
spread
applied, a behavior suitable for application to injured skin.
stress is applied, a behavior suitable for application to injured skin.

Figure
Figure 5. Flow curve (A)[Link]
Flow curve (A)
rheogram and
(B) of rheogram (B) of (mean
the SyOf cream the SyOf cream
values ± (mean
SD). values ± SD).

The presence ofThe

SAspresence
and RA in of the
SAscreams
and RA in the
with creams
SyOf with
extract are SyOf extract
confirmed byare confirmed by
UV-Vis
Vis (Figure
and FT-IR spectra and FT-IR6).spectra (Figureabsorption
The specific 6). The specific
bands absorption
of SAs andbands
RAofare
SAs and RA are v
visible
at 236
at 236 and 304 nm and 304
(Figure 6A).nm (Figure
The FT-IR6A). The of
spectra FT-IR spectra revealed
the creams of the creams revealedof
the presence the presen
macadamia nutmacadamia nut oil and
oil and glycyrrhiza glycyrrhiza
glabra glabra
(Figure 6B). The(Figure 6B). The
absorption bandabsorption
at 1746 cm −1 at 1746
band
can be assignedcan be assigned
to the stretchingtovibration
the stretching
of thevibration
C=O groupof the C=O
from group from
macadamia macadamia
nut oil [41] nut oi
and to the b=vibration of the C=O group from glycyrrhizin, the main active compound
of glycyrrhiza glabra [42]. The band at 1648 cm−1 can be assigned to the vibration of the
 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 22
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 22

Int. J. Mol. Sci. 2024, 25, 3099 and to the b=vibration of the C=O group from glycyrrhizin, the main active compound of
7 of 22
and to the b=vibration
glycyrrhiza glabra [42]. of the
TheC=Oband group from
at 1648 cmglycyrrhizin,
−1 can be assignedthe main
to theactive compound
vibration of
of the C=C
glycyrrhiza
bond fromglabra [42]. The[42].
glycyrrhizin band Theat band
1648 cm −1 can be assigned to the vibration of the C=C
at 1648 cm−1 originated from the stretching vibra-
bond from
tion of C=Oglycyrrhizin
groups [42]. The band
from polyacrylamide at 1648 cm
[43], theoriginated
−1
main from the stretching vibra-
C=C bond from glycyrrhizin [42]. The band at 1648 cm−1component
originated of fromSepigel 305 ®. The
the stretching
tion of C=O
absorption groups from polyacrylamide [43], the main component of Sepigel 305 ®. The
vibration ofband
C=O at 1038 cm
groups from−1 can be assigned to the stretching vibration of the C=O group
polyacrylamide [43], the main component of Sepigel 305® .
absorption band at 1038 cm −1 can be assigned to the stretching vibration of the C=O group
fromabsorption
The comfrey and band the
at C-O cm−1 can be
1038stretching vibration
assigned from glycerin.
to the stretchingThe vibration
intensity of ratio
theof the
C=O
from comfrey
1648 and 1038and the
cm bands
−1 C-O stretching
decreases vibration from glycerin.
with thevibration
addition of SyOf The intensity
in the cream, ratio
fromof1.72the to
group from comfrey and the C-O stretching from glycerin. The intensity ratio
1648
1.62and the
1038 cm−1 bands −decreases
1 bands with thetoaddition ofcase
SyOf ofin the 20%.
cream, from 1.72 toan
of thein1648 case
and of1038SyOf cm10% and from 1.72
decreases 1.1 in
with the
the addition SyOf
of SyOf in This indicates
the cream, from
1.62 in the
increase case of SyOf
in intensity 10%
of the and from
1038 10% 1.72
cm−1 and to 1.1
absorption in the
band, case of SyOf
indicating 20%. This
the presence indicates an
of comfrey
1.72 to 1.62 in the case of SyOf from 1.72 to 1.1 in the case of SyOf 20%. This
increase in intensity of the 1038 cm −1 absorption band, indicating the presence of comfrey
in the prepared
indicates creams.
an increase in intensity of the 1038 cm−1 absorption band, indicating the presence
inof
the prepared creams.
comfrey in the prepared creams.

Figure6.
Figure 6. UV
UV−Vis
−Vis (A)
(A) and
and FT−IR
FT−IR(B)
(B)spectra
spectraof
ofthe
theSimple
SimpleC,
C,SyOf
SyOf10%,
10%,and
andSyOf
SyOf20%
20%creams.
creams.
Figure 6. UV−Vis (A) and FT−IR (B) spectra of the Simple C, SyOf 10%, and SyOf 20% creams.
2.2.2.
2.2.2. Epithelium
Epithelium Bacteriological
Bacteriological Examination
Examination
2.2.2. Epithelium Bacteriological Examination
Pathogenic
Pathogenic bacterial
bacterial microflora
microflora can
can influence
influence the
the process
process of
of skin
skin drainage and regen-
Pathogenic
eration.
eration. A bacterial microflora
A bacteriological
bacteriological can influence
examination
examination was the out
was carried
carried process of skin
out before
before skindrainage
skin defects and induced
defects were
were regen-
induced
eration.
to A
identifybacteriological
whether there examination
was a was
bacterial carried
load on out
the before
skin thatskin defects
could were
to identify whether there was a bacterial load on the skin that could influence the healing
influence induced
the healing
toand
identify
and whetherprocess.
regeneration
regeneration there was
process. a bacterial
After
After load macroscopic
incubation,
incubation, on the skin that
macroscopic could influence
examination
examination showed
showed thethat
healing
round,
and regeneration
smooth,
smooth, small- process.
small- to After
to medium-sized, incubation,
medium-sized, cretaceous macroscopic examination
cretaceous white-pigmented, showed
white-pigmented, non-hemolytic that round,
non-hemolytic colonies
colonies
smooth,
developedsmall-
developed on to
on themedium-sized,
the seedingmedium
seeding mediumcretaceous
(Figure white-pigmented,
(Figure7A,B).
7A,B). Smears
Smears were
were non-hemolytic
taken
taken from
from colonies
the the grown
grown col-
developed
colonies
onies and onstained
and the seeding
stained
bybythemedium
the Gram
Gram (Figure
method
method 7A,B).
andand Smears
examined
examined were taken from the
microscopically.
microscopically. grown col-ex-
Microscopic
Microscopic
onies and stained
examination
amination by the
identified
identified aGram
a bacterial method
bacterial germ
germ ofand examined
of the
the genus
genus microscopically.
Staphylococcus
Staphylococcus Microscopic
intermedius,
intermedius, which isex-
which is
con-
amination
considered identified
a a bacterial
saprophytic
sidered a saprophytic skin [Link]
germ. of the genus Staphylococcus intermedius, which is con-
sidered a saprophytic skin germ.

Figure7.
Figure 7. Representative
Representativemacroscopic
macroscopicimages
imagesof
ofbacterial
bacterialcolonies
coloniesgrown
grownon
onblood
bloodagar
agar(A,B).
(A,B).
Figure 7. Representative macroscopic images of bacterial colonies grown on blood agar (A,B).
In conclusion,
In conclusion, we cancan state
statethat
thatnonopathogenic
pathogenicbacterial microflora
bacterial microflorawas identified
was on
identified
on In
thethe conclusion,
skinskin
thatthat we
could
could can state
have
have that no
influenced
influenced pathogenic
the the bacterial
process
process microflora
of vacuuming
of vacuuming and andwas identified
regeneration;
regeneration; on
the
the bac-
the skinflora
bacterial
terial that could
flora have
thatthat
waswas influenced
identified
identified the
was
was process ofwith
commensal,
commensal, vacuuming
with and regeneration;
a protective
a protective barrier
barrier [Link] bac-
role.
terial flora that was identified was commensal, with a protective barrier role.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 22
Int. J. Mol. Sci. 2024, 25, 3099 8 of 22

2.2.3.
[Link]
MacroscopicExamination
ExaminationofofWound WoundHealing
Healing
The distinctive feature of this study
The distinctive feature of this study was the was the decision
decision to to use
useaamacroscopic
macroscopicanalysis
analysisof
of the region of interest (ROI), as opposed to a statistical analysis, since
the region of interest (ROI), as opposed to a statistical analysis, since the latter would the latter wouldbe
be
useless given that the ROI changes over time. The macroscopic investigation of theofskin’s
useless given that the ROI changes over time. The macroscopic investigation the
skin’s
healinghealing
revealsreveals
that thethat
SyOfthe20% SyOf 20% cream-treated
cream-treated skin defects skin defects regenerate
regenerate more quickly more
than
quickly
the SyOf than
10%the SyOf 10% cream-treated
cream-treated skin flaws, which skin take
flaws,12 which
days totake 12 days(Figures
regenerate to regenerate
8 and 9
(Figures
and Table 8 and 9 and
3). The Table 3).
allantoin andTheRA allantoin and RA
from comfrey from
may becomfrey mayfor
responsible bethe
responsible for
faster wound
the fasterwhen
healing wound healing
using SyOfwhen using SyOf
20% cream. It has20%
been cream. It has been
demonstrated demonstrated
that the RA fromthat the
comfrey
RA from complement
inhibits comfrey inhibits complement
activation both inactivation
vivo and in both in [6],
vitro vivobut
and inknown
it is vitro [6], but
that it bio-
the is
known that the biological activity of comfrey extract can be attributed to
logical activity of comfrey extract can be attributed to the interaction of different active the interaction
of different active
compounds in thecompounds
extract [44].in the
At extract
the site[44]. At the site of inflammation
of inflammation where complement where com-
activa-
plement activationRA
tion is occurring, is occurring,
suppresses RA suppresses activation
complement complement by activation
covalentlyby covalently
reacting withre-
the
acting withcomplement
activated the activatedcomponent
complement C3b component C3b without
without having havingside
the negative the effects
negativeof side
other
effects of other
medications likemedications like and
glucocorticoids glucocorticoids and anti-inflammatory
anti-inflammatory drugs. The pro-
drugs. The pro-inflammatory gene
inflammatory
cyclooxygenase gene cyclooxygenase
2 (COX2) 2 (COX2)
may be strongly may be by
inhibited strongly
RA [6].inhibited by RA [6].

Full-thicknessskin
[Link]-thickness
Figure skindefect
defectininrats
ratsand
andthetheevolution
evolutionofofwound
woundhealing
healingtreated
treatedwithwithSimple
Simple
C,
C,SyOf
SyOf10%,
10%,and
andSyOf
SyOf20%
20% cream
cream andandclear wound
clear wound(none) presented
(none) in 3D
presented at 1,at4,1,7,4,12,7,and
in 3D 12, 14
and
post-surgery days.
14 post-surgery Scale
days. bars:bars:
Scale 6 mm.
6 mm.
 Treatment SyOf 10% SyOf 20% Simple C None
Healing of the wound Healing of the wound Healing of the wound starts Healing begins to
starts to be visible on the starts to be visible on the to be visible from the 12th be visible on day 7
12th day and complete 7th day and complete heal- day, and healing is not com- and complete
Int. J. Mol. Sci. 2024, 25, 3099 9 of 22
healing on the 14th day. ing on the 12th day. plete on the 14th day. healing on day 14.

Percentage of
Figure9.9. Percentage
Figure of wound
wound closure
closurefor
forthe
thedefect
defecttreated with
treated Simple
with C, C,
Simple SyOf 10%,
SyOf andand
10%, SyOf 20%
SyOf
cream
20% andand
cream clearclear
wound (no product).
wound p < 0.05.
(no product). p < 0.05.

2.2.4.
TableHistological Analyses
3. Comparative presentation of the macroscopic examination regarding SyOf 10%, SyOf 20%,
Simple C, and clean
Histological wound (None).
sections were carried out after 8 and 14 days in terms of evaluating the
SyOf regeneration efficiency.
Treatment SyOf 10% SyOf 20% Simple C None
In the histological sections from the cream control group treated with Simple C after
Healing of8the wound
days, Healing
the cutaneous of theiswound
defect Healing of thetissue
filled with granulation wound Healing at
that is covered begins to be
its surface
starts to be by
visible on the starts to be visible on the starts to be visible from the
crust and on the marginal zones of the wound by surface epithelium. The superficial visible on day 7 and
12th day and complete 7th day and complete 12th day, and healing is not complete healing on
part of the granulation
healing on the 14th day.
tissue is highly vascularized
healing on the 12th day.
and highly populated with
complete on the 14th day.
fibroblasts
day 14.
and scattered inflammatory cells (neutrophils, macrophages, and multinucleated giant
cells with foamy cytoplasm). A low amount of collagen fibers was detected here. The
2.2.4. Histological
deeper Analyses tissue is less cellularized by fibroblasts and fibrocytes and
part of the granulation
Histological
isolated inflammatorysections
cellswere
can becarried out after
detected, 8 and 14
including days in terms
neutrophils, of evaluating the
macrophages/multi-
SyOf regeneration
nucleated efficiency.
giant cells, and lymphocytes. The fibers, basically collagen, are more prominent
in thisInregion
the histological
as compared sections
to the from the cream
superficial zonecontrol group treated
of the granulation withThe
tissue. Simple C after
superficial
8 days, the cutaneous defect is filled with granulation tissue that is
epidermis is acanthotic in the marginal zones of the granulation tissue and overlaps with covered at its surface
by peripheral
the crust and on the marginal
zones of the scarzones
tissue,ofsuggesting
the woundthe byre-epithelization
surface epithelium. The
of the superficial
skin (Figure
part of the granulation tissue is highly vascularized and highly populated with fibroblasts
10A).
and In
scattered inflammatory
the histological cellsfrom
sections (neutrophils,
the SyOf macrophages,
10% group after and8multinucleated
days, the cutaneous giant cells
de-
with foamy cytoplasm). A low amount of collagen fibers was detected
fect is fully filled with granulation tissue that practically regenerated all dermis and hy- here. The deeper
part of theThe
podermis. granulation
granulation tissue is less
tissue cellularized
is highly by fibroblasts
vascularized and fibrocytes
and intensely and isolated
cellularized by nu-
inflammatory cells can be detected, including neutrophils, macrophages/multinucleated
merous fibroblasts and scattered fibrocytes, whereas the collagen fibers are discrete. The
giant cells, and
inflammatory lymphocytes.
cells The fibers, and
(basically, neutrophils basically collagen, can
macrophages) are more prominent
be detected on the in su-
this
region as
perficial compared
part to the superficial
of the granulation tissue, atzone of the granulation
the junction with the crust,tissue.
whichThe superficial
is prominent
in the central part of the defect. Some hemorrhages along with inflammatory cellsoverlaps
epidermis is acanthotic in the marginal zones of the granulation tissue and can be
with thejust
detected peripheral
below the zones of the
crust. Thescar
skintissue, suggesting is
re-epithelization the re-epithelization
visible on the margins of theof skin
the
(Figure 10A).
In the histological sections from the SyOf 10% group after 8 days, the cutaneous
defect is fully filled with granulation tissue that practically regenerated all dermis and
hypodermis. The granulation tissue is highly vascularized and intensely cellularized by
numerous fibroblasts and scattered fibrocytes, whereas the collagen fibers are discrete.
The inflammatory cells (basically, neutrophils and macrophages) can be detected on the
superficial part of the granulation tissue, at the junction with the crust, which is prominent
in the central part of the defect. Some hemorrhages along with inflammatory cells can be
detected just below the crust. The skin re-epithelization is visible on the margins of the
defect, whereas the central zone of the defect still has the cell debris of the crust (Figure 10B).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 22

Int. J. Mol. Sci. 2024, 25, 3099 defect, whereas the central zone of the defect still has the cell debris of the crust (Figure 10 of 22
10B).
In the histological sections from the SyOf 20% group after 8 days, the skin regenera-
tion In the histological
is complete, sections
including thefrom the SyOf
epidermis and20% group after 8 days, The
dermis/hypodermis. the skin regeneration
newly made epi-
isdermis
complete, including
entirely coversthe
theepidermis and dermis/hypodermis.
area of defect and displays acanthosisTheand
newly madespongiosis.
cellular epidermis
entirely covers the
In the dermis, the granulation
area of defect and is
tissue displays acanthosis
intensely andby
cellularized cellular spongiosis.
fibroblasts In the
and a limited
dermis, the granulation tissue is intensely cellularized by fibroblasts and
number of fibrocytes. The inflammatory infiltration is represented by scattered neutro-a limited number
of fibrocytes.
phils The inflammatory
and macrophages infiltration
in the marginal zone,is represented
which by scattered
are detected groups of neutrophils and
multinucleated
macrophages in the marginal zone, which are detected groups of multinucleated
giant cells belonging to the macrophage line. In the mass of the granulation tissue, isolated giant cells
belonging
clefts withtoathe macrophage
translucent line. Incan
material thebemass of the granulation
visualized, bordered tissue, isolated clefts
by macrophages with
(Figure
a10C).
translucent material can be visualized, bordered by macrophages (Figure 10C).

Figure 10.
Figure 10. (A) (group
(group with
with Simple
Simple C):C): the
the presence
presence of of inflammatory
inflammatory cells,
cells, including
including neutrophils,
neutrophils,
macrophages, and
macrophages, and multinucleated
multinucleated giantgiantcells
cells(arrowheads)
(arrowheads)in in thethe
granulation
granulation tissue (arrow);
tissue (B)
(arrow);
(group with SyOf 10%): the re-epithelization of the skin is visible on the margins
(B) (group with SyOf 10%): the re-epithelization of the skin is visible on the margins of the formerly of the formerly
created defect (black arrows), whereas the central zone of the defect still has the crust (arrowhead)
created defect (black arrows), whereas the central zone of the defect still has the crust (arrowhead)
that is covering the granulation tissue (white arrow); (C) (group with SyOf 20%): the newly created
that is covering the granulation tissue (white arrow); (C) (group with SyOf 20%): the newly created
epidermis (arrowhead) is entirely covering the area of the former defect (suggested by black ar-
epidermis (arrowhead)
rows), whereas is entirely
in the dermis the covering
granulationthe tissue
area of(white
the former
arrow)defect (suggested
is intensely by black by
cellularized arrows),
fibro-
whereas in the dermis the granulation tissue (white arrow) is intensely cellularized
blasts, fibrocytes, and a limited number of inflammatory cells; (D) (group with SyOf 20%): acan- by fibroblasts,
fibrocytes,
thosis and and a limited
cellular number
spongiosis of inflammatory
(arrowhead). cells; (A)
Scale bars: (D)50(group with 200
μm, (B,C) SyOfμm,20%):
(D)acanthosis
100 μm. and
cellular spongiosis (arrowhead). Scale bars: (A) 50 µm, (B,C) 200 µm, (D) 100 µm.
Histological examination of the sections from the control group treated with Simple
Histological
C cream, after 14 examination
days, showedofthat
the the
sections from
surgical the control
wound group treated
was completely with
healed, andSim-
the
ple
epidermis was continuous. The superficial dermis and deep dermis contain manyhealed,
C cream, after 14 days, showed that the surgical wound was completely pilose-
and the epidermis
baceous was11A,B,E,F).
units (Figure continuous. The superficial
Occasionally, withindermis and deep
the deeper dermisdermis contain
and hypoder-
many pilosebaceous units (Figure 11A,B,E,F). Occasionally, within the deeper
mis, a few macrophages admixed with rare lymphocytes and plasma cells are present dermis and
hypodermis, a few macrophages admixed with rare lymphocytes and plasma
(Figure 11D,H). The panniculus carnosus is present in these sections (black star, Figurecells are
present
11A,B). (Figure 11D,H). The panniculus carnosus is present in these sections (black star,
Figure 11A,B).
In the histological sections from the SyOf 10% group after 14 days, the surgical wound
was completely epithelialized, and the pilosebaceous units partly regenerated, especially at
the margins of the defect (thin arrow, Figure 12B,C,F,G). The superficial dermis and deep der-
mis were replaced by a large amount of dense fibrous connective tissue (scar tissue), rich in
thick bundles of collagen fibers oriented parallel to the epidermis (black stars, Figure 12C,G).
The deep dermis was infiltrated by a moderate number of lymphocytes, plasma cells, and
macrophages (Figure 12D,H). An amorphous, basophilic foreign material is observed in the
deep dermis (thick arrow, Figure 12D), without a major inflammatory reaction.
 Int. J.J. Mol.
Int. Mol. Sci.
Sci. 2024,
2024, 25,
25, 3099
x FOR PEER REVIEW 11
11 of 22
of 22

Figure 11. Histological images of the cream control group. The skin defect is completely healed,
covered by an intact epidermis and dermis containing many pilosebaceous units. Focally, the fi-
brous reaction, especially within the deep segment, contains poorly demarcated foci consisting of a
few macrophages admixed with rare lymphocytes and plasma cells (image (H), arrow). H&E stain
(images (A–D)) and Masson’s trichrome (images (E–H)). Scale bares: (A,E) 500 μm, (B,F) 200 μm,
(C,G) 100 μm, (D,H) 50 μm.

In the histological sections from the SyOf 10% group after 14 days, the surgical
wound was completely epithelialized, and the pilosebaceous units partly regenerated, es-
pecially at the margins of the defect (thin arrow, Figure 12B,C,F,G). The superficial dermis
and
Figuredeep
Figure 11. dermis
11. wereimages
Histological
Histological replaced
images of by cream
of the
the a largecontrol
cream amount
control of dense
group.
group. fibrous
The skin
The skin connective
defect
defect tissue
is completely
is completely (scar
healed,
healed,
tissue),
covered rich
by in
an thick
intact bundles
epidermis of
and collagen
dermis fibers
containingoriented
many parallel to
pilosebaceous the epidermis
units. Focally,
covered by an intact epidermis and dermis containing many pilosebaceous units. Focally, the fibrous (black
the fi-
brous reaction,
stars, Figure especially
12C,G). within
The deep the deep segment,
dermis was containsby
infiltrated poorly
a demarcated
moderate foci consisting
number of lympho- of a
reaction, especially within the deep segment, contains poorly demarcated foci consisting of a few
few macrophages
cytes, plasmaadmixedadmixed
cells, and with rare lymphocytes
macrophages (Figure and plasma cells (image (H),basophilic
arrow). H&E stain
macrophages with rare lymphocytes and12D,H).
plasma An
cellsamorphous,
(image (H), arrow). H&E foreign
stain
(images (A–D))
material(A–D)) and
is observed Masson’s trichrome (images (E–H)). Scale bares: (A,E) 500 μm,
in the deep dermis (thick arrow, Figure 12D), without a major inflam- (B,F) 200 μm,
(images
(C,G) 100 and Masson’s
μm, (D,H) 50 μm. trichrome (images (E–H)). Scale bares: (A,E) 500 µm, (B,F) 200 µm,
matory
(C,G) 100reaction.
µm, (D,H) 50 µm.
In the histological sections from the SyOf 10% group after 14 days, the surgical
wound was completely epithelialized, and the pilosebaceous units partly regenerated, es-
pecially at the margins of the defect (thin arrow, Figure 12B,C,F,G). The superficial dermis
and deep dermis were replaced by a large amount of dense fibrous connective tissue (scar
tissue), rich in thick bundles of collagen fibers oriented parallel to the epidermis (black
stars, Figure 12C,G). The deep dermis was infiltrated by a moderate number of lympho-
cytes, plasma cells, and macrophages (Figure 12D,H). An amorphous, basophilic foreign
material is observed in the deep dermis (thick arrow, Figure 12D), without a major inflam-
matory reaction.

Figure 12.
Figure [Link]
Histologicalimages
imagesofofthe
the skin
skin and
and subcutaneous
subcutaneous tissue
tissue from
from the animals
the animals ofSyOf
of the the SyOf
10%
10% group. The moderately hyperplastic epidermis is supported by dermal fibrosis (black star)
group. The moderately hyperplastic epidermis is supported by dermal fibrosis (black star) containing con-
taining few partly regenerated pilosebaceous units (thin arrows, B,C,F,G); Focally, intracellular
few partly regenerated pilosebaceous units (thin arrows, (B,C,F,G)); Focally, intracellular amorphous,
amorphous, basophilic test material in the deep dermis (thick arrow, D) is present. H&E stain (im-
basophilic test material in the deep dermis (thick arrow, (D)) is present. H&E stain (images (A–D))
ages A–D) and Masson’s Trichrome (images E–H). Scale bares: (A,E) 500 μm, (B,F) 200 μm, (C,G)
and Masson’s
100 μm, (D,H)Trichrome
50 μm. (images (E–H)). Scale bares: (A,E) 500 µm, (B,F) 200 µm, (C,G) 100 µm,
(D,H) 50 µm.
In the histological sections from the SyOf 20% group after 14 days, the surgical
In the histological sections from the SyOf 20% group after 14 days, the surgical
wound was healed, covered by a regular epidermis. The pilosebaceous units were present,
wound was healed, covered by a regular epidermis. The pilosebaceous units were present,
and the panniculus carnosus was intact (Figure 13A–D). In the deep dermis, numerous
and the panniculus carnosus was intact (Figure 13A–D). In the deep dermis, numerous
macrophages, plasmaimages
Figure 12. Histological cells, and a few
of the skin eosinophils were tissue
and subcutaneous present (black
from the star, Figure
animals the13D).
macrophages, plasma cells, and a few eosinophils were present (black star, Figureof13D). SyOf
An
An amorphous, basophilic foreign material (interpreted
is supportedasbythe test material) was
amorphous, basophilic foreign material (interpreted as the test material) was present in con-
10% group. The moderately hyperplastic epidermis dermal fibrosis (black present
star)
the
taining few partly of
in the cytoplasm regenerated pilosebaceous
a few macrophages units (thin arrows, B,C,F,G); Focally, intracellular
cytoplasm of a few macrophages (Figure(Figure
13D). 13D).
amorphous, basophilic test material in the deep dermis (thick arrow, D) is present. H&E stain (im-
The obtained histological analysis is summarized in Table S1, for comparison purposes.
ages A–D) and Masson’s Trichrome (images E–H). Scale bares: (A,E) 500 μm, (B,F) 200 μm, (C,G)
In
100 μm, (D,H) 50after
conclusion, μm. only 8 days, following the treatment with SyOf 10% cream, all the
dermis and hypodermis were regenerated. The margins of the defect show evidence of skin
re-epithelialization; however,
In the histological the from
sections central area
the of the
SyOf 20%defect
groupstillafter
contains crustthe
14 days, cellsurgical
debris.
Furthermore, the epidermis, dermis, and hypodermis of the skin had fully regenerated
wound was healed, covered by a regular epidermis. The pilosebaceous units were present, after
14 days. The cutaneous defect is filled with granulation tissue in the histological
and the panniculus carnosus was intact (Figure 13A–D). In the deep dermis, numerous sections
from the creamplasma
macrophages, controlcells,
group,
andwhich is crusted atwere
a few eosinophils the surface
present and(blackcovered by surface
star, Figure 13D).
epithelium in the wound’s marginal zones. Neutrophils, macrophages, and
An amorphous, basophilic foreign material (interpreted as the test material) was presentmultinucleated
giant
in thecells with foamy
cytoplasm cytoplasm
of a few are scattered
macrophages across
(Figure 13D).the superficial area of the granulation
tissue, which is also densely populated with fibroblasts. Here, a small number of collagen
fibers were found. These results are in agreement with those obtained by Dähnhardt
Int. J. Mol. Sci. 2024, 25, 3099 12 of 22

et al. [45]. Within 4–7 days after using comfrey cream, skin cells began to regenerate more
quickly and began to differentiate sooner toward a normal fine structure with discrete
epidermal strata, keratin, and corneocyte production. This study used foreskin samples
taken from 2- to 5-year-old donors’ foreskins compared to our study, which used rats. The
entire extract, rather than just a few isolated ingredients that might be influencing the overall
impact, is defined as the active component, as is typically the case with herbal therapeutic
treatments. For instance, the phytochemical components of comfrey are known to include
allantoin, RA, and SAs, which are recognized for their anti-inflammatory and regenerative
effects. Moreover, in patients with acute coronary syndrome (ACS), the comfrey ointment
sped up the healing of bruises brought on by enoxaparin injections [46]. In addition, the
study by Lin and his coworkers suggests that SAs promote autophagy, which increases the
survival of random-pattern skin flaps, and that this promotes angiogenesis, apoptosis, and
oxidative stress [47]. Microemulsion containing SA B reduced acanthosis, lessened disease
severity, and inhibited interleukin-23/interleukin-17 (IL-23/IL-17) cytokines, epidermal
proliferation, and enhanced skin moisture in mice. Based on this, SAs might represent a
potential new therapeutic medication for the treatment of psoriasis [48]. Other research has
shown similar changes to ours in the treatment of RA. In this sense, the group of rats that
Int. J. Mol. Sci. 2024, 25, x FOR PEER received
REVIEW RA treatment had wounds that had a more advanced re-epithelialization12and of 22
a
better arrangement of the collagen bundle [9].

Figure 13.
Figure [Link]
Histologicalimages
imagesofofthe
the skin
skin and
and subcutaneous
subcutaneous tissue
tissue from
from the animals
the animals ofSyOf
of the the SyOf
20%
20% group. The skin defect is covered by an intact epidermis. Multiple pilosebaceous units
group. The skin defect is covered by an intact epidermis. Multiple pilosebaceous units are presentare pre-
sent within the defect (A,B,E,F); a mild granulomatous reaction in the deep dermis centered on the
within the defect (A,B,E,F); a mild granulomatous reaction in the deep dermis centered on the test
test material (black star, D) was noted. H&E stain (images (A–D)) and Masson’s trichrome (images
material (black star, (D)) was noted. H&E stain (images (A–D)) and Masson’s trichrome (images
(E–H)). Scale bares: (A,E) 500 μm, (B,F) 200 μm, (C,G) 100 μm, (D,H) 50 μm.
(E–H)). Scale bares: (A,E) 500 µm, (B,F) 200 µm, (C,G) 100 µm, (D,H) 50 µm.
The obtained histological analysis is summarized in Table S1, for comparison pur-
This study has some limitations, such as a small number of individuals and the fact that
we did In
poses. notconclusion, after only
examine toxicity in the8 most
days,important
following organs.
the treatment with SyOf
Even though 10% cream,
the organ all
topology
the dermis and hypodermis were regenerated. The margins of the defect
in rats is very similar to that of humans, we do not know the impact of extrapolating the show evidence
of skinto
results re-epithelialization;
humans. however, the central area of the defect still contains crust cell
debris. Furthermore, the epidermis, dermis, and hypodermis of the skin had fully regen-
erated
3. after 14
Materials anddays. The cutaneous defect is filled with granulation tissue in the histolog-
Methods
ical sections from the cream controlofgroup,
3.1. Preparation and Characterization Comfreywhich is crusted at the surface and covered by
Extract
surface epithelium in the wound’s marginal
3.1.1. Preparation of Extract from Comfrey Roots zones. Neutrophils, macrophages, and mul-
tinucleated giant cells with foamy cytoplasm are scattered across the superficial area of
The raw material (Symphytum radix of Symphytum officinale L.) was harvested in
the granulation
February 2022 intissue, which
the Vâlcea is also
area along densely
the Oltpopulated with fibroblasts.
River, Romania. The root wasHere, a small
dried and
number of collagen fibers were found. These results are in agreement with
powdered, and 50 g of it was used to obtain a polyphenolic extract by refluxing three times those obtained
by Dähnhardt
with 65% ethanol et (v/v)
al. [45].
forWithin
30 min 4–7
at 60days afterextract
◦ C. The using was
comfrey cream,toskin
evaporated cells by
dryness began to
using
regenerate more quickly and began to differentiate sooner toward a normal
a rotavapor. The comfrey concentrated extract (SyOf) was finally dissolved in water and fine structure
with stored
then discreteat epidermal
−20 ◦ C until strata, keratin, and corneocyte production. This study used fore-
analysis.
skin samples taken from 2- to 5-year-old donors’ foreskins compared to our study, which
used rats. The entire extract, rather than just a few isolated ingredients that might be in-
fluencing the overall impact, is defined as the active component, as is typically the case
with herbal therapeutic treatments. For instance, the phytochemical components of com-
frey are known to include allantoin, RA, and SAs, which are recognized for their anti-
inflammatory and regenerative effects. Moreover, in patients with acute coronary syn-
drome (ACS), the comfrey ointment sped up the healing of bruises brought on by enoxap-
arin injections [46]. In addition, the study by Lin and his coworkers suggests that SAs
Int. J. Mol. Sci. 2024, 25, 3099 13 of 22

3.1.2. Determination of Total Polyphenols Content of Comfrey Extract

The total phenolic content of SyOf was determined by the Folin–Ciocalteu spectropho-
tometric method with minor adaptations as described by Singleton [49]. Briefly, a 25 µL
sample (extract) was mixed with 1800 µL of distilled water, 120 µL of Folin–Ciocalteu
reagent, and 340 µL of Na2 CO3 (7.5% in water). After 60 min of incubation in the dark at
room temperature, the absorbance of the samples was measured in a Microplate Reader
(BioTek Instruments, Bad Friedrichshall, Germany) at a wavelength of 750 nm. Results
were expressed as mg of caffeic acid equivalents/100 g SyOf [50].
UV-Vis absorption spectra were recorded using a Jasco V-780 UV-Vis spectrometer
(Jasco, Tokyo, Japan) with a 1 nm spectral resolution, using water as solvent. The FT-IR
absorption spectra were recorded with a Jasco 6200 FT-IR spectrometer (Jasco, Tokyo, Japan)
within the range of 400–4000 cm−1 and a spectra resolution of 4 cm−1 by using the KBr
pellet technique.

3.1.3. Determination of Phenolic Compounds of Comfrey Extract

The phenolic compounds of SyOf were determined by LC-ESI+ -MS method. Agilent
1200 HPLC system equipped with a quaternary pump, solvent degasser, autosampler,
UV-Vis photodiode detector (DAD) coupled with Agilent single quadrupole mass detector
(MS) model 6110 (Agilent Technologies, Santa Clara, CA, USA) was used. Compound
separation was performed on a Kinetex XB C18-100 Å column (4.6 × 150 mm, 5 µm particles
(Phenomenex, Torrance, CA, USA) using the following mobile phases: A, water + 0.1%
acetic acid; B, acetonitrile + 0.1% acetic acid. The gradient below was used for 30 min at
25 ◦ C with a flow rate of 0.5 mL/min. Gradient (expressed in % B): 0 min, 5% B; 0–2 min,
5% B; 2–18 min, 5–40% B; 18–20 min, 40–90% B; 20–24 min, 90% B; 24–25 min, 90–5% B;
25–30 min, 5% B. All bend values fell between 200 and 600 nm. Chromatograms were
recorded at wavelengths λ = 280 and 340 nm. For the MS, the ESI-positive ionization mode
was used under the following working conditions: 3000 V of capillary voltage, 3500 ◦ C,
7 L/min of nitrogen flow rate, 120–1200 m/z, full-scan, 35 psi of nebulizer pressure. Agilent
ChemStation software 6110 was used to collect data.

3.1.4. Determination of Allantoin Compounds of Comfrey Extract

Analysis was carried out using Agilent-1200 liquid chromatograph equipped with
a degasser for solvents, quaternary pump, manual injector, and VWD detector (Agilent-
Techonologies, CA, USA). The separation of allantoin was performed on a reverse-phase
column Luna C18 250 × 4.6 mm, 5 µm particle size (Phenomenex, CA, USA). Acetonitrile
and potassium dihydrogen phosphate 50 mM solution in a ratio of 20:80 at pH = 3.5
was used as the mobile phase. The flow rate was maintained at 0.8 mL/min, column
temperature was T = 25 ◦ C, and the detection was performed at 200 nm. Data acquisition
was performed with the OpenLab–ChemStation (Agilent Techologies, CA, USA), Rev
C.01.09 [144] version.
The comfrey extract was diluted with distilled water, filtered through a nylon fil-
ter (Chromafil Xtra PA-45/13 0.45 µm), and 20 µL was injected into the HPLC system.
The allantoin content was determined using a five-point calibration curve of allantoin
(R2 = 0.9983) in the linearity range 100–1000 µg/mL.
Acetonitrile (HPLC-gradient) and potassium dihydrogen phosphate were provided
by Merck (Darmstadt, Germany), and water was purified with a Direct-Q UV system by
Millipore (Bedford, MA, USA). Standard allantoin (purity ≥ 98%) was purchased from
Merck (Germany).

3.1.5. Cytotoxicity of the Comfrey Extract

The human keratinocytes (HaCaT) cell line was kindly provided by the Radiother-
apy, Radiobiology, and Tumoral Biology Laboratory of the “Ion Chiricuţă” Institute of
Oncology Cluj-Napoca, Romania. HaCaT cells were maintained in DMEM culture medium
supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic at 37 ◦ C in a
Int. J. Mol. Sci. 2024, 25, 3099 14 of 22

humidified atmosphere with 5% CO2 . To evaluate the cytotoxic potential of SyOf extract
on HaCaT cells, a concentration of 1 × 105 cells was seeded in 96-well tissue culture plates
in a normal propagation medium. After 24 h of incubation, the HaCaT cells were treated
with different concentrations of SyOf and incubated at 37 ◦ C in a humidified atmosphere
supplemented with 5% CO2 [51]. The concentration of the SyOf was calculated according
to the total phenolic content expressed in µg/mL caffeic acid equivalent (382.69 µg/mL,
765.38 µg/mL, 1530.76 µg/mL, 3061.52 µg/mL, and 6123.05 µg/mL).
Untreated cells (cells kept in a typical propagation medium) served as the negative
control. Cell viability was measured using CCK-8 assay following the manufacturer’s
protocol. For this purpose, after 24 CCK-8 solution was added to each well and incubated
for an additional 1.5 h, the optical density was subsequently determined at 450 nm by
using a BioTek Synergy 2 microplate reader (Winooski, VT, USA). The obtained results were
expressed as a viability percentage relative to the negative control (untreated cells) [51].

3.1.6. Antioxidant Activity of Comfrey Extract

The cupric-ion-reducing antioxidant capacity (CUPRAC) of the extract was determined
according to the protocol described previously by R. Apak et al. [52]. The absorbance was
read at 450 nm against the blank with a JASCO V-630 spectrophotometer (International
Co., Ltd., Tokyo, Japan). The antioxidant activity was expressed in µM Trolox equivalents,
based on the calibration curve y = 0.0025x + 0.0358, R2 = 0.9448.
FRAP assay was used to monitor the extract’s ability to reduce ferric 2,4,6-tris(2-
pyridyl)-1,3,5-triazine [Fe (III)-TPTZ] complex to the intensely blue-colored ferrous complex
[Fe2+ -(TPTZ)2 ]2+ , in acidic medium [53]. FRAP reagent is a mixture of 10 mM/L TPTZ with
40 mM/L ferric chloride in acetate buffer (pH 3.6). FRAP reagent (180 µL) was mixed with
extract (20 µL). The absorbance of the colored complex was read at 593 nm after 30 min of
incubation at 37 ◦ C. Results were calculated and expressed as µM Fe2+ /g extract, based on
the calibration curve y = 1.603x − 0.0014, R2 = 0.9999.

3.1.7. Antimicrobial Assay on Comfrey Extract

The in vitro antimicrobial potential of ethanolic extract of SyOf (65% v/v) was tested
by the agar diffusion test (Kirby–Bauer), according to the European Committee on Antimi-
crobial Susceptibility Testing (EUCAST) guidelines [54]. Four reference bacterial strains
(n = 4), methicillin-susceptible Staphylococcus aureus ATCC 25923, methicillin-resistant
Staphylococcus aureus ATCC 700699 (MRSA), Escherichia coli ATCC 25922, and Pseudomonas
aeruginosa ATCC 27853, were used for this purpose. Overnight bacterial suspensions at a
concentration of McFarland 0.5 were used for testing. Mueller–Hinton (MH) agar plates
were inoculated (overnight culture) by flooding. After drying the plate surface, wells
of 6 mm diameter were cut equidistantly, after which tested solutions were added. The
concentration of the SyOf was calculated according to the total phenolic content expressed
in µg/mL caffeic acid equivalent. Gentamicin discs (10 µg) were used for the reference
control. Testing was performed in triplicate. Results were evaluated after 24 h of incubation
at 37 ◦ C by measuring the diameters of the growth inhibition zones.
The minimum inhibitory concentrations (MICs) were assessed using the microdilu-
tion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines
(2018) [55]. The evaluation was performed using the broth microdilution (2-fold dilution)
method on 96-well plates, in triplicate. Briefly, 100 µL of MH broth was added to each
well of the 96-well plates, and SyOf ethanolic extract and 20 µL of bacterial suspension
(1.5 × 106 CFU/mL) were also added in each well. The plates were incubated at 37 ◦ C
for 18 h. For bacterial growth/inhibition, after 18 h of incubation, 20 µL of MTT solution
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 1.25 mg/mL) was added to
each well. Cultures were incubated for 1 h at 37 ◦ C, and bacterial growth was indicated
by the appearance of a chromogenic reaction (purple color) and inhibition of growth by
clear/yellow staining in the wells. Testing was performed in triplicate. The lowest con-
Int. J. Mol. Sci. 2024, 25, 3099 15 of 22

centration that visibly inhibited bacterial growth was defined as MIC. The results were
interpreted in comparison with the untreated control culture (MH broth) [56].
The minimum bactericidal concentration (MBC) value, which represents the lowest
concentration that prevents bacterial growth, was also assessed. To evaluate MBC values,
100 µL of bacterial suspension was collected from the well where no visible bacterial growth
was observed. The suspensions were inoculated on MH agar plates and incubated for
18 h at 37 ◦ C. The MIC index was also calculated, based on the MBC/MIC ratio. Thus,
an MBC/MIC ratio ≤ 4 was considered bacteriostatic, while an MBC/MIC ratio ≥ 4 was
regarded as a bactericide.

3.2. Preparation and Characterization of Oil-in-Water Cream of Comfrey Extract

3.2.1. Preparation of the Oil-in-Water Cream with Comfrey Extract
The cream was prepared as an oil-in-water (O/W) disperse system using the follow-
ing ingredients: caprylic/capric triglycerides (4%, w/w; Croda, Snaith, UK), macadamia
integrifolia nut oil (5%, w/w), Glycyrrhiza glabra extract, glycerin (5%, w/w; Elemental,
Oradea, Romania), paraffin oil (6%, w/w), cetearyl alcohol (6%, w/w; Vitamar, Bucarest,
Romania), Sepigel 305® (3.5%, w/w; polyacrylamide and C13-14 Isoparaffin and laureth-7,
Seppic, Paris, France), Euxyl PE 9010® (0.5%, w/w; phenoxyethanol and ethylhexylglycerin,
Schülke & Mayr, Norderstedt, Germany), and distilled water (to 100%, w/w). SyOf was
selected as the active ingredient of the O/W cream, at concentrations of 10% and 20% (w/w;
SyOf 10% and SyOf 20%), as it controls the inflammatory process and induces collagen
deposition (1). Also, macadamia nut oil acts as an emollient and regenerative through its
content of monounsaturated acids and contains antioxidants that can reduce inflammation
and oxidative stress in the skin (2), while Glycyrrhiza glabra has anti-inflammatory activity
(3) and was added at a concentration of 1%.
First, the aqueous phase was made by mixing distilled water at a controlled temper-
ature (50 ± 2 ◦ C) together with Sepigel 305 ® , glycerol, and the preservative Euxyl PE
9010® . Sepigel 305 creates a gel texture with distilled water, being a multifunctional vehicle
with thickening, stabilizing, texturizing, and tissue-adhering properties (4). Separately, the
paraffin oil was placed in a water bath at 60 ± 2 ◦ C, together with cetearyl alcohol. This
lipophilic melted mixture was added to the water phase at 50 ± 2 ◦ C under continuous
stirring. After removing this mixture from the water bath, when the previous mixture
reached a temperature below 40 ◦ C, macadamia nut oil and caprylic/capric triglycerides
were added under continuous stirring, which was continued until the cream reached room
temperature. Then, the Glycyrrhiza glabra extract and the SyOf were both homogeneously in-
corporated into the cream composition. For the control, the cream without SyOf (Simple C)
was prepared.

3.2.2. Characterization of the of the Oil-in-Water Cream with Comfrey Extract

The cream viscosity was determined using a cone-and-plate viscometer (CAP 2000+,
Brookfield, Phoenix, AZ, USA). The cream was applied to the flat surface of the viscometer,
the disc was sealed, and the results were recorded, using the following viscometer settings:
hold time 10 s, run time 30 s, spindle 08 at rotations speeds varying from 5 to 50 RPM.
Measurements were performed in triplicate, at 23 ± 1 ◦ C, and the mean value ± standard
deviation was reported.
The obtained creams were characterized by UV-Vis and FT-IR spectroscopy using the
equipment described above.

3.3. In Vivo Evaluation of the Oil-in-Water Cream with Comfrey Extract

3.3.1. Animal Care and Use
In this research, the biological material used was represented by 15 rats, family Muridae,
Wistar-Lewis line, female, weighing 150–180 g. Docility to experimental maneuvers, re-
duced susceptibility to bacterial infection, low degree of spontaneous tumor development,
and good adaptability to captive rearing were the arguments underlying the choice of the
Int. J. Mol. Sci. 2024, 25, 3099 16 of 22

animal model [57]. Rats were purchased from the Experimental Medicine Centre of the
Iuliu Hat, ieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, and housed
and maintained at the Unit for Reproduction and Use of Laboratory Animals of the Faculty
of Veterinary Medicine, Cluj-Napoca, where the experiment was conducted. Maintenance
and feeding conditions were standardized for all rats in the study, ensuring a temperature
of 23 ◦ C, humidity cycles of 55%, and light/dark cycles of 12 h, according to [58], and
the feed administered consisted of standard granulated rodent food. The rats’ access to
food and water was ad [Link] batches were formed to experiment on, each batch
having 5 rats. The experiment was approved by the Bioethics Committee of the University
of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, no. 245/05.04/2021 and
authorized by the Sanitary-Veterinary and Food Safety Directorate, Cluj-Napoca, by Project
Authorization no. 256/13 May 2021.
According to the legislation in force, obtaining bioethics approvals and Sanitary-
Veterinary and Food Safety Directorate authorization is a mandatory practice in conducting
in vivo studies on animals. The results indicating good regeneration in the case of the
cream with SyOf justifies running the experiment.

3.3.2. Surgical Procedure

The actual surgery was performed only after each rat was weighed and anesthetized
with a mixture of Ketamine and Xylazine (Xylazin Bio 2%, Bioveta, Czech Republic,
6 mg/kg and Ketamine Narkamon Bio, Bioveta, Czech Republic 60 mg/kg), injected
intraperitoneally [59]. After anesthesia, the excision site was prepared for surgery by
trimming the hair in the dorsal thoracic area from T1 to L1, and special attention was paid
to the skin from the region of the withers between the two scapular joints to the level
of the pelvis. After trimming, a depilatory cream was applied to ensure complete hair
removal and left to work for 3 min to avoid burning, and then the depilatory cream was
removed with a special plastic scraper. Antisepsis of the area was achieved by cleaning
it with sterile swabs soaked in a Lifo-Scrub solution (Braun Medical), after which sterile
swabs soaked in 70% sanitary alcohol were used to achieve a better antiseptic effect. Four
dermal excisions were performed in the body area prepared for the intervention, following
the following steps: the rat was fixed in a lateral position, and then the skin in the dorsal
region, along the line of the spine, was clamped and pulled with two hemostatic tweezers,
obtaining a skin fold. Dermal excisions were performed using a biopsy punch, fixed at a
distance of approximately 8 mm from the edge of the skin fold. The 4 dermal excisions
were obtained by gently rotating the biopsy punch through both layers of the skinfold, the
distance between the 2 excisions being approximately 16 mm as a result of the skinfold
unraveling [60]. After dermal excisions were performed, they were cleaned with a sterile
swab. To minimize the constricting effect of the panniculus carnosus muscle on each
excision, a silica ring was applied. Superglue was applied to the side of the silica ring that
came into contact with the skin for immediate fixation, which then allowed them to be
sutured with non-absorbable sutures so that each silica ring was fixed to the skin with 4
symmetrical sutures [60]. For the application of the cream with the comfrey extracts, the
rats were restrained in sterno-abdominal decubitus, with the hind limbs oriented to the
right of the researcher, so that the dorsal region with the 4 excisions remained free for the
application of the experimental product. The dermal excision on the lower left was the
control excision for all 3 batches. A simple cream without any extract was applied to it. The
dermal excision on the upper left side was the excision on which the experimental product
was applied, i.e., SyOf 10% cream and SyOf 20% comfrey extract cream were applied to the
dermal excision on the right side. Nothing was applied to the dermal excision on the lower
right side, considered the control excision, and regeneration occurred spontaneously. After
the application of the simple cream (Simple C) and the experimental product, the rats were
dressed in sterile elastic bandages to avoid wound infection. On the 4th and 7th days of the
experiment, a new application of ointment was made, following the same protocol and in
the same concentrations, after which the rats were bandaged, following the same steps. For
Int. J. Mol. Sci. 2024, 25, 3099 17 of 22

15 days, the rats in this study were monitored for health and skin healing and regeneration.
On day 15 of the experiment, after complete wound healing, each rat was euthanized by
anesthetic overdose and cervical dislocation. Samples of skin tissue were taken from the
site of each dermal excision, covering both the area where the scar had formed and normal
surrounding tissue of approximately 1 cm, for histopathological examination.
Materials used in the survey were: biopsy punch with a diameter of 5 mm—by relaxing
the skin, the excision had a diameter of 6 mm; solutions for antisepsis such as Lifo-Scrub
(Braun Medical) and sanitary alcohol 70%; scalpel with fixed blade; surgical tweezers and
hemostatic tweezers; scissors; needles and non-absorbable sutures (Nyllion 4.0); clipper;
depilatory cream (Veet, France); sterile dressing; elastic bandage and superglue.

3.3.3. Epithelium Bacteriological Examination

The method used is a qualitative one. Samples were taken before skin defects from
3 rats. All rats included in this study received the same feeding, watering, and microclimate
conditions. For bacteriological examination, several steps were followed, such as collection
of biological samples, transport, seeding on special culture media, and identification
of germs based on certain characteristics by performing macroscopic and microscopic
examination. The sampling was carried out on the first day of surgery. The samples were
taken from the skin before trimming, shaving, and disinfecting the area to see if there were
pathogenic bacteria on the skin that could then influence the healing of the skin. After
collection, samples were seeded in Petri dishes on blood-enriched agar and incubated
at 37 ◦ C for 24 h. The results indicate the absence of pathological bacteria and only the
presence of normal ones. Also, in the absence of clinical symptoms such as scratching
or hair loss, the animals were considered clinically healthy without pathological dermal
factors influencing the healing of the skin during the experiment.

3.3.4. Macroscopic Examination of Wound Size Reduction

The pictures of the wounds were taken on days 1, 4, 7, 12, and 14 and were used
to quantify the degree of healing. For the evaluation of the images, free, open-source
software was used (ImageJ 1.54g), and each picture was transformed into an 8-bit picture,
grayscale, at dimensions of 336 × 336 pixels, to have homogeneity. Before image capture,
wound dimensions were measured using a standard ruler, establishing a calibration of
27 pixels per millimeter within the software. A free-hand ROI was traced to include the
wound and the changes in the ROI were evaluated. This ROI enabled the calculation of
pixel value alterations throughout the healing timeline. Based on these selections, surface
plots were generated to provide a visual representation of the healing process, comparing
pixel intensity levels at the wound site against those of the surrounding intact skin surface.
Thus, to have a graphical representation of the wound, a histogram was obtained for each
ROI traced. This methodology was consistently applied across all specimens, ensuring a
standardized approach for image capture and analysis. The same process was used for all
products, and pictures were obtained.

3.3.5. Histological Method

In the middle of the healing period (eight days), after fixation in 10% buffered formalin,
the samples underwent dehydration with alcohol in increasing concentrations (70 O, 96 O,
and 100 O), clarified with 1-butanol (three baths, 1 h each) and embedded in paraffin
blocks. Eventually, 5 µm thick tissue sections were prepared using a LEICA RM2125RT
microtome. Finally, tissue sections were stained by Goldner’s trichrome method for general
histological aspects. The achieved histological slides were examined under an Olympus
BX41 microscope, and the microphotographs were obtained by using a built-in Olympus
U-TV0.35XC-2 T8 camera.
At the end of the experimental period, the animals were killed by cervical dislocation,
and skin samples were harvested for histological examination. Skin samples were fixed in
10% buffered neutral formalin and embedded in paraffin, sections were made at 4 µm, and
 microtome. Finally, tissue sections were stained by Goldner’s trichrome method for gen-
eral histological aspects. The achieved histological slides were examined under an Olym-
pus BX41 microscope, and the microphotographs were obtained by using a built-in Olym-
pus U-TV0.35XC-2 T8 camera.
Int. J. Mol. Sci. 2024, 25, 3099 At the end of the experimental period, the animals were killed by cervical dislocation,
18 of 22
and skin samples were harvested for histological examination. Skin samples were fixed in
10% buffered neutral formalin and embedded in paraffin, sections were made at 4 μm,
and the slides were stained by Haematoxiline–Eosine (HE) and Masson’s trichrome meth-
the slides were stained by Haematoxiline–Eosine (HE) and Masson’s trichrome methods.
ods. The histological sections were examined under an Olympus BX 51 microscope, im-
The histological sections were examined under an Olympus BX 51 microscope, images
ages were
were takentaken
usingusing the Olympus
the Olympus UC 30UC 30 digital
digital camera,
camera, and and
theythey
werewere processed
processed using
using an
an image acquisition and processing program: Olympus Stream
image acquisition and processing program: Olympus Stream Basic. Basic.

3.3.6. Pain
3.3.6. Pain Management
Management
Theprocedures
The proceduresinvolving
involvingthethehandling
handlingofof animals
animals were
were ledled following
following thethe guidelines
guidelines of
of Directive 2010/63/EU, Romanian National Low 43/2014, and ISO
Directive 2010/63/EU, Romanian National Low 43/2014, and ISO 10993-2 Animal Welfare 10993-2 Animal Wel-
fare Requirements.
Requirements Ratswere
[58]. Rats wereanesthetized
anesthetizedin inan
anIsoflurane
Isofluranechamber
chamber(Isoflutek
(Isoflutek1000
1000mg/g,
mg/g,
LaboratoriosKarizoo
Laboratorios KarizooS.A.,
S.A.,Voorschoten,
Voorschoten,The The Netherlands,
Netherlands, 3%)
3%) andand were
were maintained
maintained un-
under
der inhalation
inhalation anesthesia
anesthesia via facial
via facial mask mask
for for preparation
preparation before
before andand during
during surgery.
surgery. Pre-
Preop-
operatively,
eratively, thethe following
following were
were administered
administered subcutaneously:5 5mL
subcutaneously: mLofofNaCl
NaCl0.9%
0.9% (NaCl
(NaCl
0.9% B.
0.9% B. Braun,
Braun, Melsungen,
Melsungen, Germany)
Germany) to to prevent
prevent dehydration.
dehydration. Sterile
Sterile acupuncture
acupuncture needles
needles
(0.16 ×
(0.16 × 20
20 mm
mm (Acimut,
(Acimut,Madrid,
Madrid,Spain))
Spain))with
withcopper
copper handles
handles were
wereinserted 5 mm
inserted 5 mmintointo
the
depression
the depressionon the
ondorsal midline
the dorsal between
midline the seventh
between cervicalcervical
the seventh vertebrae and firstand
vertebrae thoracic
first
thoracic dorsal spinous
dorsal spinous processes,
processes, at acupointat acupoint GV-14 (Governing
GV-14 (Governing vessel)
vessel) and and between
between the
the seventh
seventh lumbar vertebrae
lumbar vertebrae and firstand firstvertebrae,
sacral sacral vertebrae, on the midline,
on the dorsal dorsal midline, at acupoint
at acupoint Bai-hui
Bai-hui
(“Hundred (“Hundred
meetings meetings point”,14).
point”, Figure Figure
The 14). The stimulation
stimulation between between the two acu-
the two acupoints de-
points described before was performed with a constant current pulse
scribed before was performed with a constant current pulse for 50 min (15 min for induc- for 50 min (15 min
for induction,
tion, 30 min
30 min during theduring theand
surgery, surgery,
5 minandafter5 the
minsurgery).
after theWesurgery).
used anWe used an elec-
electroacupunc-
troacupuncture
ture machine made machine made
by Dr. by Dr. Huisheng,
Huisheng, a Xie-JM-3A, a Xie-JM-3A,
the stimulithewere
stimuli
set were
at 40 set
Hz,atand
40 Hz,
the
and the intensity
current current intensity was increased
was increased in a stepwise
in a stepwise fashion
fashion until until atwitch
a muscle musclewastwitch was
observed
observed
(~1–1.5 V)(~1–1.5
[60]. V) [60].

Figure 14. Moxibustion and acupuncture encourage the growth of fibroblasts and neoangiogenesis
during the healing of experimental excisional wounds in adult female Wistar rats.

After the surgery, every third day we performed electroacupuncture for pain manage-
ment and also to improve the healing process. Electroacupuncture reduces local inflamma-
tion and provides blood supply for the skin. Rats were observed daily until completion of
the experiment, and the Grimace scale was used based on changes in a number of facial
action units [60].

3.4. Statistical Analysis

All data reported in cell viability assay and wound regeneration are reported as the
mean ± SD. The triplicate (n = 3) values obtained for cell viability were analyzed by
two-way analysis of variance (ANOVA). Statistical significance was at p < 0.05 in all cases.

4. Conclusions
Comfrey root extract showed a rich content of polyphenolic compounds, especially
SAs and RA, as well as a rich content of allantoin. Referring to the compounds identified in
the analyzed extract and the literature, we can conclude that a large part of the positive
effects is due to both polyphenolic and allantoin compounds. Good antioxidant activity
was obtained for the extracts, which are known for their great free radical scavenging
Int. J. Mol. Sci. 2024, 25, 3099 19 of 22

activity. The antimicrobial effect of comfrey extract on epithelial cell lines demonstrated
health effects, having a bactericidal property on four reference bacterial strains: methicillin-
sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli,
and Pseudomonas aeruginosa. Histological analysis of the skin defects 14 days after the
intervention has shown that in the skin excisions treated with SyOf 20% the healing is
complete, and the pilosebaceous units and the panniculus carnosus muscle are completely
formed compared to those treated with SyOf 10% cream concentration where in the dermis
and hypodermis there are areas of inflammation, compared to the control excisions where
in the deep segment there is an inflammatory reaction with macrophages and lymphocytes.
Thus, the obtained results confirm the wound regeneration effect of comfrey extract.
Still, we cannot exclude the possible synergistic effect since macadamia nut oil and gly-
cyrrhiza glabra also have wound regeneration abilities.

Supplementary Materials: The following supporting information can be downloaded at: https:
//[Link]/article/10.3390/ijms25063099/s1.
Author Contributions: Conceptualization, S.M.M., C.M., K.M. and I.P.; methodology, S.M.M., A.M.D.,
R.C.P., A.G., A.-L.N., F.T., M.M. and I.M.; software, M.D., E.P., C.M. and S.M.M.; validation, M.T. and
S.B.; formal analysis, C.M.; investigation, S.M.M., E.P., S.B., K.M. and I.P.; resources, A.G. and S.M.M.;
data curation, I.M., C.M., I.P. and A.M.D.; writing—original draft preparation, S.M.M., C.M., K.M.
and I.P.; writing—review and editing, C.M. and S.M.M.; visualization, F.T., M.D., M.M. and A.G.;
supervision, R.C.P. and M.T.; project administration, S.M.M. and I.P.; funding acquisition, S.M.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by The National Research and Development Projects for financing
excellence (PFE)-14/2022–2024 granted by the Romanian Ministry of Research and Innovation and
Internal Research contract, no. 6059, USAMV Cluj.
Institutional Review Board Statement: This experiment was approved by the Bioethics Committee
of the University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, no. 245/5 April
2021 and authorized by the Sanitary-Veterinary and Food Safety Directorate, Cluj-Napoca, by Project
Authorization no. 256/13 May 2021.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations

RA Rosmarinic acid
SAs Salvianolic acids
SyOf Comfrey concentrated extract
O/W Oil-in-water
SyOf10% 10% comfrey concentrated extract in oil-in-water cream
SyOf20% 20% comfrey concentrated extract in oil-in-water cream
Simple C Oil-in-water cream without SyOf
HaCaT Human keratinocytes cells
MH Mueller–Hinton agar
MICs Minimum inhibitory concentrations
MBC Minimum bactericidal concentration

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