Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

https://doi.org/10.1007/s00210-024-03264-8

RESEARCH

Carvacrol prevents D‑( +)‑galactose‑induced aging‑associated erectile

dysfunction by improving endothelial dysfunction and oxidative stress
in rats
Mathania Silva de Almeida Feitosa1 · Arthur José Pontes Oliveira de Almeida1 · Sabine Helena Dantas1 ·
Fátima de Lourdes Assunção Araújo de Azevedo1 · Javanyr Frederico de Souza Júnior1 ·
Tays Amanda Felisberto Gonçalves1 · Sonaly de Lima Silva1 · Evyllen Myllena Cardoso Soares1 ·
Hayaly Felinto Alves1 · Thais Trajano Lima1 · Larisse Virgolino da Silva Pontes1 · Ricardo Romão Guerra2 ·
Islania Giselia Albuquerque Araújo1 · Isac Almeida de Medeiros1

Received: 10 August 2023 / Accepted: 27 June 2024 / Published online: 5 July 2024
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024

Abstract
Aging is one of the risk factors involved in the development of erectile dysfunction (ED). Growing evidence suggests that
oxidative stress is the critical mediator of changes in endothelial function and penile vascular tone in the aging process. Thus,
reducing reactive oxygen species (ROS) levels may preserve the bioactivity of the penile vasculature. Antioxidant compounds,
such as carvacrol, limit the damage caused by ROS and, therefore, benefit the treatment of ED. Thus, this study aims to
evaluate the effects of carvacrol on ED using the D-( +)-galactose aging model. The animals were divided into five groups:
control, D-( +)-galactose 150 mg/kg, carvacrol 50 mg/kg or 100 mg/kg, and sildenafil 1.5 mg/kg treated daily for 8 weeks.
The physiological, functional, and morphological characteristics of aging-associated ED were evaluated after treatment with
carvacrol. Carvacrol prevented ED in a D-( +)-galactose-induced aging model by reducing hypercontractility, enhancing
endothelial dysfunction in the rat corpus cavernosum, and improving endothelial health of rat cavernous endothelial cells. In
addition, carvacrol prevented the destruction of erectile components essential for penile erection and promoted a reduction
of penile tissue senescence, probably through mechanisms that involve the harmful modulation of oxidative stress. Carvacrol
significantly improved the erectile function of rats in a D-( +)-galactose-induced aging model and has excellent potential as
a new therapeutic alternative in treating erectile dysfunction.

Keywords Sexual dysfunction · Accelerated aging · Reactive oxygen species · Monoterpene

Introduction (de Almeida Rezende et al. 2021, Crafa et al. 2023). The
high prevalence is related to the increase in life expectancy
Erectile dysfunction (ED) is a male sexual problem char- of the population; it is estimated that by 2025, ED will affect
acterized by the persistent or recurrent inability to obtain 322 million men (Kessler et al. 2019; Sin et al. 2021).
and/or maintain an erection sufficient for satisfactory sexual Several mechanisms involved in the aging process, such
intercourse (NIH 1993). The main risk factor associated with as endothelial dysfunction, increased contractility, decreased
ED is aging, affecting up to 90% of individuals over 70 years relaxation of the corpus cavernous (CC), and vascular senes-
cence, play a vital role in the development of ED (Aversa
* Isac Almeida de Medeiros et al. 2010; Echeverri Tirado et al. 2016). Furthermore,
[email protected] these mechanisms are tightly connected with the increased
1
reactive oxygen species (ROS) (de Almeida Rezende et al.
Departamento de Ciências Farmacêuticas, Centro de 2021).
Ciências da Saúde, Universidade Federal da Paraíba,
João Pessoa, Paraíba, Brazil Increased exposure to oxidative stress disrupts penile
2 hemodynamics, probably by depleting nitric oxide (NO)
Departamento de Ciências Veterinárias, Centro de Ciências
Agrárias, Universidade Federal da Paraíba, Areia, Paraíba, levels (Xu et al. 2022). NO may react with superoxide
Brazil anions ­(O2−), producing peroxynitrite, a highly cytotoxic

Vol.:(0123456789)

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10062 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

product (Shannon et al. 2022). In addition, the uncoupling 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (x-gal),
of endothelial nitric oxide synthase (El-Far et al. 2022) con- glutaraldehyde, OCT (Optimal Cutting Temperature) Com-
tributes to the greater production of ROS (de Almeida et al. pound (Tissue Plus®), fluorescence mounting medium
2020). With reduced or insufficient production of NO, char- (DAKO®), dihydroethidium (DHE), Dulbecco’s modified
acteristic of endothelial dysfunction, it is unable to compen- Eagle’s medium (DMEM), trypsin, penicillin and streptomy-
sate for cavernous compliance impaired by the aging process cin and endothelial growth factor were obtained from Sigma-
(Helmy and Senbel 2012). Therefore, reducing ROS levels Aldrich (Brazil). Ketamine and xylazine were acquired from
may be necessary for preserving endothelial function and Syntec (Brazil). Heparin (Hepamax-s®) was purchased
penile vasculature (Wang et al. 2020). from Blau Farmacêutica S.A. (Brazil); formaldehyde 10%
Previous studies have shown that essential oils can reduce from Medi Química Indústria Farmacêutica Ltda (Brazil);
oxidative damage and prevent changes related to the patho- hematoxylin–eosin from Química Especializada Erich Ltda
physiology of ED (Pavan et al. 2015; Sharifi-Rad, Varoni (Brazil); sildenafil from Roval Pharmacy (Brazil); fetal
et al. 2018). In this context, essential oils have been the sub- bovine serum (FBS) from Induslab (Brazil); and 4-amino-5-
ject of scientific interest, with extensive screening indicating methylamino-2′,7′-difluororescein diacetate (DAF-FM DA)
that many plant extracts and their isolated components have from Thermo Fisher (Brazil). The carbogen mixture (95%
beneficial activities for the cardiovascular system (Costa ­O2 and 5% ­CO2) was acquired by White Martins (Brazil).
et al. 2019; Ramsey et al. 2020). We highlight carvacrol for The carvacrol was obtained commercially from Sigma-
being described as a potent antioxidant, anti-inflammatory, Aldrich (Brazil), whose density is 0.976 g/ml at 20 °C with a
vasorelaxant, and antiapoptotic (Dantas et al. 2015; Guan content of 98%. The carvacrol was solubilized in Kolliphor®
et al. 2019; Hou et al. 2019; Singh et al. 2023). In addition, EL and diluted daily in physiological saline solution (NaCl
carvacrol seems to regulate senescence markers negatively 0.9%) to prepare the desired doses. The D-( +)-galactose and
(El-Far et al. 2022). sildenafil were dissolved in a physiological saline solution.
Carvacrol (2-methyl-5-isopropylphenol) is a phenolic
compound found in oils of oregano (Origanum vulgare,
Origanum dictamnus, Origanum majorana), thyme (Thym- Animals
bra capitate, Thymus vulgaris, Thymus zygis, Thymus
serpyllum), pepper (Lepidium favum), and wild bergamot Eight-week-old male Wistar rats were purchased from the
(Citrus aurantium bergamia) (Gholami-Ahangaran et al. animal production unit of the Instituto de Pesquisa em Fár-
2022). The high antioxidant activity of carvacrol is due to macos e Medicamentos (IPeFarM) of the Universidade
the hydroxyl group (OH), which is covalently linked to the Federal da Paraíba (UFPB). The experimental animals were
aromatic ring (Shoorei et al. 2019). Moreover, carvacrol housed under conditions at 22 ± 1 °C with 12-h light–dark
can improve the activity of antioxidant enzymes, such as cycles and free access to water and food (Nuvilab CR-1,
superoxide dismutase, catalase, and glutathione peroxi- Quimtia®). The physical and mental health of the rats was
dase (Imran et al. 2022). In addition, carvacrol negatively monitored every day. All animal procedures were conducted
modulates the expression of the pro-oxidant nicotinamide in accordance with the Health Guide for the Care and Use of
adenine dinucleotide phosphate (NADPH) oxidase (Sharifi- Laboratory Animals, submitted and previously approved by
Rad, Varoni et al. 2018). Together, these mechanisms can the UFPB animal ethics committee (Ethic-approval number:
limit the damage caused by ROS and, therefore, benefit the 9706070319).
treatment of ED associated with aging (Gholami-Ahangaran
et al. 2022). Based on these findings, the present research
provides subsidies to evaluate the possible beneficial actions Experimental protocol
of carvacrol in diseases whose biological effects have not
yet been studied, as is the case of erectile dysfunction in a The rats were divided randomly into five groups: the con-
D-( +)-galactose-induced aging model. trol group (CTL), which received physiological saline solu-
tion (NaCl 0.9%) intraperitoneally (Suntres, Coccimiglio
et al.), and the D-( +)-galactose group (DGAL), which
Materials and methods received D-( +)-galactose 150 mg/kg via ip, the carvacrol
groups, which received both D-( +)-galactose 150 mg/kg
Chemicals ip and carvacrol 50 mg/kg (DGAL + CVC50) or 100 mg/
kg (DGAL + CVC100) by oral gavage, and the sildenafil
D-( +)-galactose (purity ≥ 98%), Kolliphor® EL, group (DGAL + SD1.5) which received both D-( +)-galac-
dimethyl sulfoxide (DMSO), phenylephrine (Phe), tose 150 mg/kg ip and sildenafil 1.5 mg/kg by oral gavage.
acetylcholine (ACh), sodium nitroprusside (SPN), Administrations were performed daily for 8 weeks.

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Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073 10063

Determination of body weight and blood glucose mixture (95%O2 and 5%CO2), as previously described (Clau-
dino et al. 2010). Voltage changes were measured using iso-
The body weight of the animals was assessed during treat- metric transducers (MLT020, ADInstruments, Australia) and
ment. Measurements were performed individually, three recorded in a PowerLab® data acquisition system (ML870/P,
times a week, always before administration. Also, blood LabChart version 7.0, ADInstruments, Australia).
glucose was evaluated at the end of treatment. Blood was The contractile response was investigated through
collected from the caudal end and introduced into a strip increasing and cumulative addition of Phe (10 nM–300 μM)
attached to an Accuchek Guide glucometer (Roche 1, Bra- and via electrical field stimulation (EFS). EFS was applied
zil). Body weight values were expressed as weekly average between two electrodes (Stimulating Electrode Double
in grams and blood glucose in mg/dL. Ring MLA0302/8, ADInstruments) connected to a stimula-
tor. The CC was stimulated for 10 s at different frequencies
Measurement of erectile responses — ICP/MAP (1, 2, 4, 8, and 16 Hz), with 50V electrical pulses of 1-ms
(intracavernous pressure/mean arterial pressure) duration. In addition, cumulative concentration–response
ratio curves for Ach (1 nM–10 μM) and SNP (100 pM–100 μM)
were obtained in cavernosal strips precontracted with Phe
The erectile response was investigated using the ICP/MAP (10 μM) to investigate the relaxing response.
ratio, as described previously (Kim et al. 2019; de Almeida
Rezende et al. 2021). In short, the rats were anesthetized ROS quantification
using ketamine (75 mg/kg) and xylazine (10 mg/kg), ip, after
8 weeks. The rat was then placed on a surgical table, and the As previously described (de Almeida Rezende et al. 2021),
right carotid artery was cannulated to continuously measure oxidative fluorescent dye DHE was used to demonstrate
mean arterial pressure (MAP). Next, the penis was denuded levels of ROS production. Briefly, the CC isolated was cut
of the skin and a 30G gauge needle connected to a polyethyl- into 8 μm thickness sections at − 20 °C. Next, the sections
ene catheter (10 mm) was inserted into one crura of the penis were fixed on glass slides and incubated with 5 µM DHE
to measure the intracavernosal pressure (ICP). Both of the in the dark at 37 °C for 30 min. The fluorescence inten-
catheters were filled with heparinized saline (200 IU/mL) sity of DHE staining was observed using a Fluorescence
and connected to a computerized system with pressure trans- Eclipse Ti-U Nikon® microscope (Japan) with appropriate
ducers (Disposable BP Transducer, MLT0699, ADInstru- excitation/emission filters. Data were quantified using the
ments) coupled to the PowerLab® data acquisition system NIS-element® software, normalized by the CTL group, and
(LabChart® software, version 8.1; ADInstruments, USA). expressed as percentage fluorescence.
The cavernous nerve was identified on the posterolateral
aspect of the prostate, and a bronze bipolar stimulator (Ani- Morphometric measurement
mal Nerve Stimulating Electrode, MLA0320, ADInstru-
ments, USA) was positioned, performing a unilateral electri- In order to perform histological analysis, hematoxylin–eosin
cal stimulation of 16 Hz with a pulse duration of 1 ms (ms) (HE) staining was used, as previously described (Corrêa
at 6 V (V) for 60 s (s). Two cavernous nerve stimulations et al. 2017). For that, sections of the mid-transversal part of
were performed with a rest period of at least 5 min between the penis were fixed in buffered formaldehyde (10%), incor-
each stimulation. The maximum value of the ICP/MAP dur- porated into paraffin blocks, and cut 8 μm thickness sections.
ing cavernous nerve electrical stimulation was used to char- Then, morphometric areas were measured using the “poly-
acterize the erectile response. gon area” function of the Olympus CellSens Dimension
Program, and the images were obtained using an Olympus
Corpus cavernosum reactivity BX-60 microscope and an Olympus camera coupled with
the Olympus CellSens Dimension digital image capture
After euthanasia, the penis was carefully isolated and imme- program (UK).
diately placed in a Krebs–Ringer nutrient solution (pH
7.4) to dissection and remove two strips of CC (10 mm). Histochemical detection of senescence associated
The composition of Krebs–Ringer solution was as follows β‑galactosidase (SA‑β‑galactosidase)
(Shoorei, Khaki et al.): NaCl 118.0; KCl 4.7; ­CaCl2 2.50;
­KH2PO4 1.20; ­MgSO4 1.17; ­NaHCO3 25.00; and glucose Analysis of SA-β-galactosidase was adapted as previously
5.60 (Claudino et al. 2010). The strips were suspended verti- described (Chang et al. 2017). Briefly, the isolated penile
cally in organ-bath chambers (Panlab Multi Chamber Organ segment was cut into 5-μm-thick sections at − 20 °C and
Baths, ADInstruments, Australia) containing Krebs–Ringer then fixed on glass slides with a formaldehyde (2%) and
solution at 37 °C, continuously bubbled with a carbogenic glutaraldehyde (0.2%) solution for 5 min. Tissues were

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10064 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

incubated with the x-gal staining solution (x-gal 1 mg/mL; 7.0. For the statistical analysis of the concentration–response
citrate–phosphate buffer 40 mM, pH 6.0; NaCl 150 mM; curves, the maximum effect (Emax) values were calculated
­MgCl2 2 mM; C ­ 6N6FeK4 5 mM; C­ 6N6FeK3 5 mM) in the from the non-linear regression of the responses obtained.
dark at 37 °C for 18 h and taken immediately for analy- One-way or two-way analysis of variance (ANOVA) with
sis under a microscope (Nikon Eclipse Ti-E, Nikon, Japan) Bonferroni post-test was performed. Statistical significance
(Silva et al. 2017). Data were quantified using the NIS-ele- was set at a p-value of less than 0.05.
ment® software normalized by the CTL group.

Isolation and culture of rat cavernous endothelial Results

cells (RCECs)
Evaluation of physical characteristics, body weight,
After euthanasia, the penis was carefully isolated and trans- and glycemic levels
ferred into sterile vials containing Hank’s solution (Ivanov
et al. 2018) (NaCl 137; KCl 5.4; C ­ aCl2 1.3; ­Na2HPO4 0.25; The animals presented differences in appearance at the end
­KH2PO4 0.44; ­MgSO4 1.0; ­NaHCO3 4.2; and glucose 5.5) of each treatment (Fig. 1a–d). The rats in the CTL group
and was washed two times in phosphate-buffered saline. The had smooth hair, healthy-looking, shiny, and uniform colors.
glans penis, urethra, and dorsal neurovascular bundle were However, the animals in the DGAL group had curly, coarse,
removed, and only the corpus cavernosum tissue was used and opaque hair with darker regions, plus severe hair loss
for primary endothelial cell culture. The corpus cavernosum (Fig. 1a–b). Carvacrol (50 or 100 mg/kg) promoted improve-
tissue was cut into 4–6 fragments (3–6 mm) and placed in ment in appearance compared to animals in the DGAL
a cell culture bottle containing DMEM supplemented with group, similar to the CTL group (Fig. 1a–d). The animals in
20% FBS and 1% of penicillin and streptomycin. Endothelial the experimental groups did not show significant differences
growth factor (50 ng/mL) was added to DMEM to induce in body weight (n = 5; p > 0.05) (Fig. 1e) and glycemic levels
sprouting of cavernous tissue endothelial cells. The cells (n = 6; p > 0.05) (Table 1).
were maintained in an incubator with 5% C ­ O2 and a tem-
perature of 37 °C. After cells were 80% confluent, they were
trypsinized and resuspended in DMEM 10% FBS for pas- Carvacrol prevents erectile dysfunction in rats
saging and cultivation. Cells at passages between 2 and 4 in a D‑( +)‑galactose‑induced aging model
were used for experiments. The cells were plated at a con-
centration of 5 × ­104 cells/well, and the percentage of FBS The erectile function of groups was assessed. The ani-
contained in the DMEM was reduced from 10 to 2% during mals in the DGAL group exhibited a significant decrease
incubation. (0.470 ± 0.007; p < 0.0001) in ICP/MAP when compared to
the CTL group (0.733 ± 0.040). Conversely, carvacrol (50
Quantification of NO in RCEC or 100 mg/kg) and sildenafil showed a significant increase
(0.645 ± 0.040, p = 0.0024; 0.761 ± 0.025, p < 0.0001;
As previously described (Gu et al. 2022), a NO-sensitive 0.852 ± 0.012, respectively; p < 0.0001) in ICP/MAP when
fluorescent dye DAF-FM DA was used to demonstrate lev- compared to the DGAL group (0.470 ± 0.007) (Fig. 2).
els of NO. Briefly, RCECs were incubated with 20 mg/mL
of D-( +)-galactose to induce premature senescence (Wang Carvacrol reduces hypercontractility and prevents
et al. 2022), and carvacrol ­(10−8 and ­10−7 M) for 48 h in endothelial dysfunction in the corpus cavernosum
5% ­CO2 at 37 °C. Next, 2 μM DAF-FM DA was added for in rats
30 min in the dark at 37 °C. All protocols were performed
in triplicate. The fluorescence intensity of DAF-FM DA The addition of Phe (10 nM–300 μM) for the DGAL
staining was observed using a Fluorescence Eclipse Ti-U group promoted a significant increase in contrac-
Nikon® microscope (Japan) coupled to the NIS Elements tile response (Emax = 171.95 ± 19.24%, p = 0.0003;
software with appropriate excitation/emission filters. Data pD2 = 4.93 ± 0.09; n = 4) as compared to the CTL group
were quantified using the ImageJ® software, normalized by (Emax = 100.00 ± 9.44%; pD2 = 4.853 ± 0.08; n = 4), without
the CTL group, and expressed as percentage fluorescence. statistical differences in potency (p > 0.05) (Fig. 3a). In addi-
tion, the groups DGAL + CVC50 (Emax = 119.51 ± 20.74%,
Data analysis p = 0.0198; pD2 = 5.28 ± 0.17; n = 4) and DGAL + CVC100
(Emax = 59.75 ± 5.76%, p < 0.0001; pD = 25.08 ± 0.16;
All results were presented as mean ± standard error of the n = 4) showed a significant reduction in the contractile
mean (SEM) and analyzed using GraphPad Prism version response induced by PHE when compared with the DGAL

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Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073 10065

Fig. 1  Physical appearance at 8 weeks of treatment for CTL (a), ment. The data were expressed as mean ± SEM (n = 5). The data were
DGAL (b), DGAL + CVC50 (c), and DGAL + CVC100 (d) groups, analyzed using the two-way ANOVA statistical test, followed by the
and average body weight during treatment (e) at 8 weeks of treat- Bonferroni post-test

Table 1  Glycemic levels after 8 weeks of treatment p = 0.0011; 16 Hz: 90.53 ± 25.10%, p < 0.0001, n = 5)
Experimental groups Glycemic levels SEM
showed a significant reduction in the contractile response
(mg/dL) induced by EFS at frequencies of 8 and 16 Hz when com-
pared with the DGAL group (Fig. 3b).
CTL 121.2 4.094 The relaxation response induced by the addition of ACh
DGAL 118.8 5.735 (1 nM–10 μM) was significantly lower in the DGAL group
DGAL + CVC50 120.0 5.978
(Emax = 65.38 ± 7.77%; n = 4; p = 0.0105) when com-
DGAL + CVC100 125.0 6.723 pared to the CTL group (Emax = 101.52 ± 2.65%; n = 4)
The data were expressed as mean ± SEM (n = 6). The data were ana- (Fig. 3c). A similar effect was observed with the animals in
lyzed using the one-way, followed by the Bonferroni post-test the DGAL + CVC50 group (Emax = 77.07 ± 6.35%; n = 5;
p = 0.0012), which also showed a reduction in ACh-induced
relaxation when compared to the CTL group (p < 0.05)
group without statistical differences in potency (p > 0.05) (Fig. 3c). Furthermore, the animals in the DGAL + CVC100
(Fig. 3a). group (Emax = 104.04 ± 3.75%; n = 3; p = 0.0009) showed a
The EFS (1, 2, 4, 8, and 16 Hz) in the DGAL group (1 Hz: significant increase in the relaxing response induced by ACh
44.34 ± 12.56; 2 Hz: 69.50 ± 16.49; 4 Hz: 104.67 ± 22.63; when compared to the DGAL group, similar (p > 0.05) to the
8 Hz: 163.19 ± 22.23, p = 0.0009; 16 Hz: 228.51 ± 17.79%, CTL group (Fig. 3c).
p < 0.0001; n = 5) promoted hypercontractility at all fre- The relaxation response induced by the addition of
quencies tested, as compared to the CTL group (1 Hz: SNP (100 pM–100 μM) did not result in a significant dif-
13.60 ± 6.15; 2 Hz: 21.20 ± 9.37; 4 Hz: 39.30 ± 15.85; 8 Hz: ference in maximum effect (p > 0.05). However, there
65.50 ± 20.56; 16 Hz: 93.60 ± 15.68%, n = 5) (Fig. 3B). The was a significant reduction in the potency for the DGAL
animals in the DGAL + CVC50 (1 Hz: 29.51 ± 9.55; 2 Hz: group (Emax = 94.77 ± 10.93%; pD2 = 6.45 ± 0.22,
41.20 ± 11.37; 4 Hz: 57.24 ± 9.41; 8 Hz: 69.41 ± 18,45, p = 0.0008; n = 5) as compared to the CTL group
p = 0.0007; 16 Hz: 85.74 ± 26.01%, p < 0.0001, n = 5) (Emax = 114.07 ± 4.44%; pD2 = 7.70 ± 0.16; n = 4)
and DGAL + CVC100 groups (1 Hz: 24.31 ± 8.24; 2 Hz: (Fig. 3d). The animals in the groups DGAL + CVC50
43.99 ± 17.53; 4 Hz: 59.03 ± 20.04; 8 Hz: 72.18 ± 19.74, and DGAL + CVC100 (Emax = 112.98 ± 2.74%,

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10066 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

Fig. 2  Original record (a)

and statistical graph (b) of
the ICP/MAP in response to
electrical stimulation (16 Hz,
6 V, 1 ms for 60 s) of the
cavernous nerve in CTL,
DGAL, DGAL + CVC50
and DGAL + CVC100, and
DGAL + SD1.5 animals at
8 weeks. The results are
expressed as mean ± SEM
(n = 4). The data were analyzed
using the one-way ANOVA
statistical test, followed by the
Bonferroni post-test. #, p < 0.05
vs. CTL. *, p < 0.05 vs. DGAL.
ES, electrical stimulation

pD2 = 6.14 ± 0.15, n = 5; Emax = 98.07 ± 17.13%, revealed a significant decrease in the CC by total area when
pD2 = 5.47 ± 0.23, n = 5, respectively) did not demonstrate compared to the CTL group (4.90 × ­106 ± 1.19 × ­105 μm2,
significant improvements in maximal effect (p > 0.05) and n = 5) (Fig. 5).
potency (p > 0.05) when compared to the DGAL group The animals in t he DGAL + CVC100 g roup
(Fig. 3d). (5.49 × ­106 ± 1.19 × ­105 μm2, n = 5, p < 0.0001) showed a
significant increase in the total area of the CC when com-
Carvacrol reduces the levels of superoxide anions pared to the DGAL group, similar (p > 0.05) to the CTL
in the corpus cavernosum isolated from rats group. However, the animals in the group DGAL + CVC50
(4.16 × ­1 0 6 ± 4.14 × ­1 0 4 μm 2, n = 5) showed no changes
Superoxide anions measurements were performed in the (p > 0.05) in the total area of the CC when compared to
CC isolated from rats. The animals in the DGAL group the DGAL group (Fig. 5).
(213.92 ± 14.10%, n = 5, p < 0.0001) presented a significant
increase in fluorescent intensity when compared to the CTL
group (100.00 ± 10.63%, n = 5) (Fig. 4). Carvacrol induces a decrease in SA‑β‑galactosidase
The animals in the groups DGAL + CVC50 and activity in the corpus cavernosum isolated from rats
DGAL + CVC100 (140.92 ± 10.93%, n = 5, p = 0.0005;
139.78 ± 3.79%, n = 5, p = 0.0004, respectively) showed a The SA-β-galactosidase activity of animals in the DGAL
significant reduction in the intensity of fluorescence emit- group (196.592 ± 5.00%, n = 5, p < 0.0001) revealed a
ted by the DHE probe when compared to the DGAL group, significant increase when compared to the CTL group
similar (p > 0.05) to the CTL group (Fig. 4). (100.00 ± 10.97%, n = 5) (Fig. 6).
The animals in the groups DGAL + CVC50 and
Carvacrol induced an increase in the total corpus DGAL + CVC100 (141.994 ± 6.272%, n = 5, p = 0.0009;
cavernosum area isolated from rats 133.42 ± 9.99%, n = 5, p = 0.0002, respectively) showed a
significant reduction in SA-β-galactosidase activity when
The histomorphometry analysis of animals in the DGAL compared to the DGAL group, similar (p > 0.05) to the
group (4.34 × ­1 0 6 ± 8.28 × ­1 0 4 μm 2, n = 5, p = 0.0026) CTL group (Fig. 6).

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Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073 10067

Fig. 3  Concentration–response
curves for Phe (a), EFS (b),
ACh (c), and SNP (d) in the
corpus cavernosum isolated
from rats at 8 weeks of treat-
ment. The results are expressed
as mean ± SEM (n = 4). The
data were analyzed using the
two-way ANOVA statistical
test, followed by the Bonferroni
post-test. #, p < 0.05 vs. CTL. *,
p < 0.05 vs DGAL

Fig. 4  Representative image (a)

and quantitative analysis (b) of
the superoxide anions produc-
tion as measured by fluorescent
intensity emitted by the DHE
probe in the corpus cavernosum
isolated from rats. Data are
expressed as mean values of
the percentage of fluorescence
relative to the control ± SEM
(n = 5). The data were analyzed
using the one-way ANOVA
statistical test, followed by the
Bonferroni post-test. #, p < 0.05
vs. CTL. *, p < 0.05 vs. DGAL.
Scale bars, 100 μm

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10068 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

Fig. 5  Photomicrographs (a)

and histomorphometric analysis
(b) of the total corpus caver-
nosum area isolated from rats.
Data are expressed as mean
values ± SEM (n = 5). The
data were analyzed using the
one-way ANOVA statistical
test, followed by the Bonferroni
post-test. #, p < 0.05 vs. CTL. *,
p < 0.05 vs. DGAL. Scale bars,
200 μm

Fig. 6  Representative image

(a) and quantitative analysis
(b) of SA-β-galactosidase
activity in the corpus caver-
nosum isolated from rats. The
data are expressed in mean
percentage values normalized
by the CTL ± SEM (n = 5). The
data were analyzed using the
one-way ANOVA statistical
test, followed by the Bonferroni
post-test. #, p < 0.05 vs. CTL. *,
p < 0.05 vs. DGAL. Scale bars,
100 μm

Carvacrol induced an increase in nitric oxide levels n = 6). Treatment with carvacrol 0.01 µM (127.914 ± 3.572,
in RCECs n = 6, p < 0.0001) and 0.1 µM (129.910 ± 2.015, n = 6,
p < 0.0001) promoted a significant increase in the fluores-
NO levels in the DGAL group showed a significant reduction cence intensity emitted by DAF-FM DA probe when com-
in the fluorescence intensity emitted by the DAF-FM DA pared to the CTL group (100.00 ± 1.806, n = 6) (Fig. 7).
probe (83.056 ± 2.081%, n = 6, p < 0.0001) when compared
to the CTL group (100.00 ± 1.806, n = 6). However, the
DGAL + CVC 0.01 µM (142.002 ± 1.863, n = 6, p < 0.0001) Discussion
and DGAL + CVC 0.1 µM (146.089 ± 2.098, n = 6,
p < 0.0001) groups showed a significant increase in fluo- While current strategies for the pharmacological treat-
rescence intensity emitted by the DAF-FM DA probe when ment of ED may be effective, they are far from perfect and
compared to the DGAL 20 mg/mL group (0.874 ± 0.015, remain unable to meet the growing medical needs of the

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Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073 10069

Fig. 7  Representative image

(a) and quantitative analysis
(b) of the nitric oxide produc-
tion as measured by fluorescent
intensity emitted by the DAF-
FM DA probe in the RCEC.
The data are expressed in mean
percentage values normalized
by the CTL ± SEM (n = 6). The
data were analyzed using the
one-way ANOVA statistical
test, followed by the Dunnett’s
post-test. #, p < 0.05 vs. CTL. *,
p < 0.05 vs. DGAL. Scale bars,
100 μm

aging population (Chung 2019). Nevertheless, studies show actions in diseases whose biological effects have not yet
that first-line therapy for ED (phosphodiesterase-5 inhibi- been studied, as is the case of ED associated with aging
tors) has a rate of effectiveness and safety in over 80% of induced by D-( +)- galactose.
patients (Madeira et al. 2021; Raheem et al. 2021). In addi- Given the promising effects of carvacrol, it was inves-
tion, several alternative or complementary therapies are tigated whether it would promote a beneficial action on
being investigated in this condition as new treatment options the erectile function of rats in a D-galactose-induced
that could potentially cure or attenuate ED progression (Sin aging model. It is known that chronic administration of
et al. 2021). D-( +)-galactose for 8 weeks mimics the natural aging pro-
Natural products have been highlighted as a source of cess and promotes erectile dysfunction in rats (Azman et al.
structurally diverse compounds that may represent good 2021; Rinkūnienė et al. 2021). Excess D-( +)-galactose gen-
starting points for developing alternative therapies for ED erates a disturbance in its metabolism, producing decreased
(Pavan et al. 2015; Sin et al. 2021). In this context, essential total antioxidant capacity, leading to tissue damage, inflam-
oils have been the subject of scientific interest, with exten- mation, and increased apoptosis via oxidative metabolism
sive screening indicating that many of these plant extracts (Ni et al. 2018; Jing et al. 2019; Zhao et al. 2020). Thus,
and their isolated components have beneficial activities for oxidative stress may decrease the endothelium-dependent
the cardiovascular system, mainly because they have anti- vasodilatory of the corpus cavernosum, favoring the devel-
oxidant properties (Aebisher et al. 2021; Alves-Silva et al. opment of ED (Helmy and Senbel 2012).
2021). Among the most active natural antioxidants found in The hypothesis that carvacrol could prevent erectile
essential oils, carvacrol stands out (Costa et al. 2019). dysfunction in aged animals was investigated. The 50 or
Carvacrol has many benefits for clinical applications, 100 mg/kg doses were used, corresponding to approximately
such as antioxidant, antiapoptotic, anticancer, and anti- 6 and 12% of the carvacrol oral LD50, respectively (Suntres
inflammatory (Zhao et al. 2020). Furthermore, carvacrol is et al. 2015). In addition, a group of animals received 1.5 mg/
believed to produce these effects through its action in oppo- kg of sildenafil, the standard gold drug used in the treatment
sition to the effects of ROS; therefore, they may be indicated of ED (Li et al. 2017).
for the protection and prevention of oxidative damage to The animal’s physical appearance was analyzed. After the
tissues (Samarghandian et al. 2016). Together, these proper- treatment, the DGAL group showed signs of aging, such as
ties provide support for evaluating their possible beneficial severe hair loss and frizzy and opaque hairs with pigmented

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10070 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

regions. In a study by Zhao et al. (2018), these character- 2021). After the treatment period, in response to Phe and
istics were observed in mice treated with D-( +)-galactose EFS, the animals in the DGAL group demonstrated hyper-
for 8 weeks. Carvacrol (50 and 100 mg/kg) demonstrated contractility compared to the CTL group. This effect may
apparent improvements when compared to the animals of the be related to the upregulation of the contractile pathways of
DGAL group, preventing changes in the physical appearance the CC, autonomic neuropathy (caused by exacerbation of
of these animals. sympathetic activity), and/or increased noradrenergic recep-
The body weight of the animals was monitored during tor sensitivity (Andersson 2011). Carvacrol (50 or 100 mg/
treatment. The animals from all experimental groups showed kg) promoted a reduction in the contractile response when
a similar gradual increase in their body weights, demonstrat- compared to the DGAL group, suggesting that the negative
ing that treatments do not interfere with the body weight modulation of α1-adrenergic receptors or a decrease in adr-
of these animals, as previously observed by Cardoso et al. energic discharge may be involved in the improvement of the
(2015). There was also no significant change in the glycemic erectile function of the animals.
levels of the animals, demonstrating that none of the treat- As NO is considered the central mediator of penile erec-
ments interfered with glucose metabolism. tion, changes in its synthesis or bioavailability may favor the
After the treatment period, the most used method for state of cavernous contraction and, consequently, the devel-
in vivo evaluation of erectile function in rats, the ICP/MAP opment of ED (Silva et al. 2016). Therefore, we evaluated
ratio, was assessed (McMurray et al. 2006). Electrical stimu- whether the treatments would affect NO production through
lation of the cavernous nerve promotes nitrergic discharge, the action of ACh on endothelial cells. In animals from the
inducing relaxation of the CC with consequent elevation of DGAL group, the relaxation of the CC induced by ACh was
ICP (Li et al. 2011). The ICP/MAP ratio in the DGAL group significantly attenuated compared to the CTL group. This
was reduced significantly compared to the CTL group, dem- effect is attributed to endothelial dysfunction and, conse-
onstrating that the D-( +)-galactose-induced-aging model quently, to a decrease in NO bioavailability that impairs
effectively promoted ED. Similar results were observed the relaxation of the CC (Andersson 2011). Furthermore,
in a study that showed that aged rats (physiological aging) Lafuente-Sanchis Triguero demonstrated that reducing
and rats treated for 8 weeks with D-( +)-galactose showed a endothelium-dependent vasodilation in response to ACh in
decrease in the ICP/MAP ratio (Helmy and Senbel 2012; de aged animals might be related to endothelial dysfunction in
Almeida Rezende et al. 2021). Carvacrol (50 or 100 mg/kg) the cavernous trabeculae of these animals (Lafuente-Sanchis
showed a significant increase in ICP/MAP compared to the et al. 2014). Carvacrol (100 mg/kg) significantly improved
DGAL group, suggesting prevention of ED since ICP/MAP the ACh-mediated relaxation of the CC compared to the
was similar to the CTL and DGAL + SD1.5 groups. The ICP/ DGAL group, probably by preventing endothelial dysfunc-
MAP increases are associated with increased cGMP levels, tion and increasing NO bioavailability by endothelial cells.
probably due to phosphodiesterase 5 (PDE-5) inhibition in Endothelial dysfunction is commonly accompanied by
the CC, consequently leading to the trabeculae’s smooth a decrease in NO production and sensitivity, resulting in
muscle relaxation, resulting in penile erection (Irfan et al. an imbalance in vascular homeostasis (Crafa et al. 2023).
2020). The ability to produce NO served as a marker for healthy
Given these findings, the next step was to assess whether endothelial cells. In RCEC, in the DGAL group, NO pro-
alterations in the contractile and relaxing reactivity of the duction was significantly attenuated compared to the CTL
CC were involved in this process (Irfan et al. 2020). These group. Consistent with our studies, data reveal a reduction
results are essential since the erectile function is a hemody- in NO bioavailability in a senescence model in endothelial
namic process that depends on the relaxation of the smooth cells and this effect is associated with changes that impair
muscle of the CC (Irfan et al. 2020). endothelial function (Hwang et al. 2022). However, carvac-
Therefore, knowing that noradrenergic discharge and rol increased NO production in the presence and absence of
stimulation of α-adrenergic receptors favor increases in CC D-( +)-galactose, suggesting that it can improve endothe-
smooth muscle tone and consequently impair the state of lial health, and consequently increase NO bioavailability in
erection, the response of the CC in contractile reactivity RCEC cells. Nanomolar concentrations of NO have been
was evaluated using Phe (selective α1-adrenergic recep- reported to possess anti-inflammatory and antioxidant activi-
tor agonist) and EFS curves (Aydın et al. 2018; Lim et al. ties (Gu et al. 2022).
2020). EFS is a technique in which electrical stimulation is In addition to evaluating endothelium-dependent relaxa-
applied to the isolated corpus cavernosum to evaluate adr- tion, we investigated whether there is any impairment in
energic, cholinergic, and non-adrenergic non-cholinergic pathways directly involved in the relaxation of the CC. The
events (Britto-Júnior et al. 2021). However, in the absence SNP (NO donor) was used, whose induced relaxation did not
of blockages, the noradrenergic discharge stands out, caus- present statistical differences between groups in the maxi-
ing a contraction of the cavernous tissue (Britto-Júnior et al. mum response (Silva et al. 2016). However, SNP promoted

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Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073 10071

a reduction in the potency of the relaxation response of the mimicked animal aging conditions. Similar results were
DGAL group compared to the CTL group, suggesting that demonstrated in cardiac tissue from animals that received
the functionality of the smooth muscle cells of the CC was D-( +)-galactose treatment for 8 weeks (Chang et al. 2017).
altered by the treatment with D-( +)-galactose and the car- Carvacrol (50 or 100 mg/kg) decreased SA-β-
vacrol was not able to improve this parameter, indicating galactosidase activity in cavernous tissues compared to the
that its beneficial effect on erectile function does not involve DGAL group, suggesting that carvacrol is able to reduce
alteration in the functionality of smooth muscle cells in the penile tissue senescence. Consistent with our findings,
CC. Chang et al. (2017) revealed that a substance character-
Studies demonstrate that ROS contribute to endothe- ized as anti-aging decreased SA-β-galactosidase activity
lial dysfunction in aging-associated ED and that the in cardiac tissues of aged rats induced by D-( +)-galactose
D-( +)-galactose model increases ROS production, which compared to the young animals.
may be the main cause of age-related ED (Angulo et al. In conclusion, daily administration of carvacrol for
2019; Jing et al. 2019). However, we cannot rule out other 8 weeks prevented ED in a D-( +)-galactose-induced
conditions, such as atherosclerosis, hypertension, and diabe- aging model by reducing hypercontractility and improving
tes, that may participate in the advancement of ED in aging endothelial dysfunction in isolated rats’ CC. In addition,
(Kloner and Speakman 2002). Thus, we evaluated whether carvacrol prevents the destruction of erectile components
ROS would be involved through the fluorescence intensity and promotes a reduction in SA-β-galactosidase, probably
of the DHE probe. Animals from the DGAL group showed through mechanisms that involve the harmful modulation
a significant increase in fluorescence intensity compared to of oxidative stress.
the CTL group, suggesting an increase in superoxide anion Thus, carvacrol demonstrated significant effects on
levels in the cavernous tissues. Consistent with our find- rat erectile function and has excellent potential as a new
ings, Gur et al. (2008) revealed increased superoxide anion therapeutic alternative in the treatment of ED in its vari-
levels in smooth muscle and endothelium of the corpora ous degrees (mild, moderate, or severe). However, further
cavernosa of aged rats compared to young animals. Car- studies will be needed to elucidate the participation of
vacrol (50 or 100 mg/kg) reduced the levels of superoxide signaling pathways in the mechanism of action induced
anion produced by D-( +)-galactose, and this decrease may by this substance.
be related to its antioxidant activity through the action of
Acknowledgements The authors thank Coordenação de Aper-
enzymatic and non-enzymatic antioxidants and negative feiçoamento de Pessoal de Nível Superior (CAPES), Conselho
modulation of NADPH oxidase (Sharifi-Rad, Varoni et al. Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and
2018). Notably, reducing oxidative stress by carvacrol may Instituto de Desenvolvimento da Paraíba (IDEP/PB) for all the support
improve contractility and endothelial function, as observed provided for the development of this research.
in the results described above. Authors contributions Conceptualization: M.S.A.F., A.J.P.O.A.,
In addition to functional abnormalities, age-related ED S.H.D. I.A.M.; formal analysis: M.S.A.F., I.G.A.A.; funding acquisi-
is associated with structural changes resulting in the loss tion: I.A.M; investigation: M.S.A.F., A.J.P.O.A., S.H.D., F.L.A.A.A.,
of erectile components (Cho et al. 2018). At the end of the J.F.S.J., T.A.F.G., S.L.S., E.M.C.S., H.F.A., T.T.L., R.R.G.; method-
ology M.S.A.F., A.J.P.O.A., S.H.D., F.L.A.A.A., J.F.S.J., T.A.F.G.,
treatment, there was a decrease in the total area of the CC of S.L.S., E.M.C.S., H.F.A., T.T.L., L.V.S.P., R.R.G.; project administra-
the animals in the DGAL group compared to the CTL group, tion M.S.A.F., I.A.M.; resources I.A.M; supervision, I.G.A.A., I.A.M.;
suggesting the loss of essential erectile components for erec- validation M.S.A.F., I.G.A.A., I.A.M.; visualization M.S.A.F.; writing
tion. Similar data are observed in a diabetes-induced ED - original draft M.S.A.F., A.J.P.O.A., L.V.S.P., I.A.M.; writing – review
and editing M.S.A.F., I.A.M.. All authors have approved the final ver-
model (Tao et al. 2017). Carvacrol (100 mg/kg) protected sion of the manuscript. The authors declare that all data were generated
the CC structure compared to the DGAL group, maintaining in-house and that no paper mill was used.
the erectile structures in normal conditions.
In addition to these changes, the accumulation of Funding This work was supported by the CAPES (process number
88887507825/202000) and CNPq (process number 311711/2018–9).
senescent cells in tissues is an organizational character-
istic of aging, which the detection of SA-β-galactosidase Data availability The data presented in this study are available on
activity can confirm. Since it was first reported, SA-β- request from the corresponding authors.
galactosidase activity has been a widely used biomarker
for senescence due to the simplicity of the assay method Declarations
and its apparent specificity for senescent cells (Lee et al. Ethical approval All animal procedures were submitted and previously
2006). The animals in the DGAL group showed an increase approved by the UFPB animal ethics committee (Ethic-approval num-
in SA-β-galactosidase activity in cavernous tissues com- ber: 9706070319).
pared to the CTL group, confirming that D-( +)-galactose
Competing interests The authors declare no competing interests.

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10072 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) 397:10061–10073

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