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Received: 23 December 2024
Revised: 25 January 2025
Accepted: 27 January 2025
Published: 30 January 2025
Citation: Zapata-Morales, J.R.;
Alonso-Castro, A.J.; González-Rivera,
M.L.; González Prado, H.I.;
Barragán-Gálvez, J.C.;
Hernández-Flores, A.; Juárez-Vázquez,
M.d.C.; Domínguez, F.;
Carranza-Álvarez, C.; de Jesús
Pozos-Guillén, A.; et al. Synergistic
Interaction Between Justicia spicigera
Extract and Analgesics on the
Formalin Test in Rats. Pharmaceuticals
2025,18, 187. https://doi.org/
10.3390/ph18020187
Copyright: © 2025 by the authors.
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licenses/by/4.0/).
Article
Synergistic Interaction Between Justicia spicigera Extract and
Analgesics on the Formalin Test in Rats
Juan Ramón Zapata-Morales 1,*, Angel Josabad Alonso-Castro 1, María Leonor González-Rivera 1,
Hugo Israel González Prado 1, Juan Carlos Barragán-Gálvez 1, Araceli Hernández-Flores 1,
María del Carmen Juárez-Vázquez 1, Fabiola Domínguez 2, Candy Carranza-Álvarez 3,
Amaury de Jesús Pozos-Guillén 4, Juan F. López-Rodríguez 5, Patricia Aguirre-Bañuelos 6
and Marco Antonio Ramírez-Morales 1
1Department of Pharmacy, Natural and Exact Sciences Division, University of Guanajuato (UG),
Guanajuato 36050, Mexico; angeljosabad@ugto.mx (A.J.A.-C.); leonor.glez.rivera@outlook.com (M.L.G.-R.);
hi.gonzalezprado@ugto.mx (H.I.G.P.); jcbarragang@gmail.com (J.C.B.-G.); aracelihf@hotmail.com (A.H.-F.);
carmenjuarezv@gmail.com (M.d.C.J.-V.); marco.ramirezmo@hotmail.com (M.A.R.-M.)
2Eastern Biomedical Research Center of Mexican Social Security Institute (IMSS), Metepec 74360, Mexico;
irma.dominguez@imss.gob.mx
3School of Professional Studies Huasteca Zone, Autonomous University of San Luis Potosí,
Ciudad Valles 79060, Mexico; candy.carranza@uaslp.mx
4Basics Sciences Laboratory, School of Dentistry, Autonomous University of San Luis Potosi (UASLP),
San Luis Potosí 78290, Mexico; apozos@uaslp.mx
5Animal Laboratory, School of Medicine Autonomous University of San Luis Potosi,
San Luis Potosi 78290, Mexico; francisco.lopez@uaslp.mx
6Faculty of Chemical Sciences, Autonomous University of San Luis Potosí, Manuel Nava Martínez 6 Avenue,
San Luis Potosí 78210, Mexico; paguirreb@uaslp.mx
*Correspondence: mzrj@hotmail.com or juan.zapata@ugto.mx; Tel.: +52-473-732006 (ext. 1420)
Abstract: Background: Combining antinociceptive drugs with different mechanisms of ac-
tion can reduce the doses and the adverse effects, with a possible increase in the antinocicep-
tive effect. This work evaluated the antinociceptive effect of the combination of an ethanol
extract of Justicia spicigera (JSE) with naproxen (NPX) or tramadol (TML) using the formalin
test in rats. Methods: Rats received JSE (30–200 mg/kg p.o.), NPX
(50–300 mg/kg p.o.),
or
TML (5–50 mg/kg p.o.) 60 min before paw administration with formalin (5%). Different pro-
portions of the combination between NPX and JSE, as well as TML and JSE, were used in the
formalin test to obtain the dose–response curve of each drug and the experimental effective
dose 50 (ED
50
). The levels of IL-1
β
and COX2 were assessed using a Western blot analysis
as a possible mechanism of action for the combination of JSE and analgesics. A pharmacoki-
netic study was conducted to evaluate the effect of JSE on the pharmacokinetic parameters
of NPX. Results: The ED
50
values for the proportions NPX:JSE were
107.09 mg/kg (1:1),
102.44 mg/kg (3:1), and 73.82 mg/kg (1:3). The ED
50
values for the proportions TML:JSE
were 66 mg/kg (1:1), 29.5 mg/kg (1:3), and 78 mg/kg (3:1). The combination
NPX:JSE (1:3)
showed the best synergistic interaction index (0.501). The pharmacokinetic study revealed
that there were no significant changes in the pharmacokinetic parameters of NPX admin-
istered individually and the combination NPX:JSE.
Conclusions:
In this preclinical study,
the combination NPX:JSE showed antinociceptive effects by decreasing the levels of COX2
and IL-1βwithout affecting NPX’s pharmacokinetics.
Keywords: Justicia spicigera; naproxen; tramadol; formalin test; isobolographic analysis;
synergism
Pharmaceuticals 2025,18, 187 https://doi.org/10.3390/ph18020187
Pharmaceuticals 2025,18, 187 2 of 17
1. Introduction
Since pre-Hispanic times, Mexican traditional medicine has used Justicia spicigera, also
known as “muitle” or “muicle”, as an empirical treatment for pain, superficial wounds or
injuries, fever, inflammation, and liver infections [1,2].
Numerous studies have demonstrated the
in vitro
and
in vivo
effectiveness of Justicia
spicigera in treating anemia and cell damage [
3
,
4
], infections and parasites [
5
], high blood
pressure [
4
], and inflammation [
6
,
7
]; boosting the immune system; and fighting tumors [
8
].
In addition, Zapata-Morales et al. [
9
] showed that an ethanol extract from Justicia spicigera
leaves (JSE) exerted antinociceptive activity in the acetic acid writhing test, the formalin
test in both phases, and the tail-flick test. The antinociceptive effects of JSE (200 mg/kg p.o.)
were like those of naproxen (150 mg/kg p.o.) in the acetic acid writhing test and phase 2 of
the formalin test.
The treatment of pain involves the use of different drugs, such as non-steroidal anti-
inflammatory analgesics (NSAIDs) and opioids, among others, according to the degree
and location of pain [
10
]. Another alternative includes a combination of two or more
types of treatments. Combining two painkillers with different mechanisms of action could
potentially address the issues related to their effectiveness, duration of action, and tolerance.
In some cases, it might even make the analgesics work better together [
11
]. Combining
analgesics with different types of activities can reduce the doses of the drugs, leading to
a reduction in the number of prescribed drugs, a decrease in the possibility of adverse
effects, and potentially an increase in the pharmacological effect. A combination of drugs
may extend the duration of the therapeutic effect without exacerbating the adverse effects
compared to individual drug administration.
No study has examined the combination of Justicia spicigera with NSAIDs or opioids.
Therefore, the present study aimed to evaluate the pharmacodynamic interaction between
the ethanolic extract of Justicia spicigera and naproxen or tramadol, two of the most widely
used analgesics in the world. The molecular mechanism of the antinociceptive effect of the
combination of JSE and naproxen was evaluated. In addition, this study assessed the effect
of JSE on the pharmacokinetics of naproxen.
2. Results
2.1. Kaempferitrin (KM) Quantitation
KM is the active compound in JSE. The retention time of KM was 10.48 min, and the
content of KM in JSE was 42 mg/g (Figure 1).
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 2 of 17
Keywords: Justicia spicigera; naproxen; tramadol; formalin test; isobolographic analysis;
synergism
1. Introduction
Since pre-Hispanic times, Mexican traditional medicine has used Justicia spicigera,
also known as “muitle” or “muicle”, as an empirical treatment for pain, superficial
wounds or injuries, fever, inflammation, and liver infections [1,2].
Numerous studies have demonstrated the in vitro and in vivo effectiveness of Justicia
spicigera in treating anemia and cell damage [3,4], infections and parasites [5], high blood
pressure [4], and inflammation [6,7]; boosting the immune system; and fighting tumors
[8]. In addition, Zapata-Morales et al. [9] showed that an ethanol extract from Justicia spic-
igera leaves (JSE) exerted antinociceptive activity in the acetic acid writhing test, the for-
malin test in both phases, and the tail-flick test. The antinociceptive effects of JSE (200
mg/kg p.o.) were like those of naproxen (150 mg/kg p.o.) in the acetic acid writhing test
and phase 2 of the formalin test.
The treatment of pain involves the use of different drugs, such as non-steroidal anti-
inflammatory analgesics (NSAIDs) and opioids, among others, according to the degree
and location of pain [10]. Another alternative includes a combination of two or more types
of treatments. Combining two painkillers with different mechanisms of action could po-
tentially address the issues related to their effectiveness, duration of action, and tolerance.
In some cases, it might even make the analgesics work beer together [11]. Combining
analgesics with different types of activities can reduce the doses of the drugs, leading to a
reduction in the number of prescribed drugs, a decrease in the possibility of adverse ef-
fects, and potentially an increase in the pharmacological effect. A combination of drugs
may extend the duration of the therapeutic effect without exacerbating the adverse effects
compared to individual drug administration.
No study has examined the combination of Justicia spicigera with NSAIDs or opioids.
Therefore, the present study aimed to evaluate the pharmacodynamic interaction between
the ethanolic extract of Justicia spicigera and naproxen or tramadol, two of the most widely
used analgesics in the world. The molecular mechanism of the antinociceptive effect of
the combination of JSE and naproxen was evaluated. In addition, this study assessed the
effect of JSE on the pharmacokinetics of naproxen.
2. Results
2.1. Kaempferitrin (KM) Quantitation
KM is the active compound in JSE. The retention time of KM was 10.48 min, and the
content of KM in JSE was 42 mg/g (Figure 1).
Figure 1. Chromatogram of the Justicia spicigera extract (JSE) showing the detection of kaempferitrin.
2.2. Antinociceptive Activity of JSE, NPX, and TML
JSE, NPX, and TML showed a dose-dependent antinociceptive activity (Figure 2).
The highest doses tested, 200 mg/kg JSE, 300 mg/kg NPX, and 50 mg/kg TML, exhib-
ited antinociceptive activities of 59.87%, 54.29%, and 89%, respectively (Figure 2). The
ED50 values were 121.15 mg/kg (JSE), 226.21 mg/kg (NPX), and 14.58 mg/kg (TML).
Pharmaceuticals 2025,18, 187 3 of 17
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 3 of 17
Figure 1. Chromatogram of the Justicia spicigera extract (JSE) showing the detection of
kaempferitrin.
2.2. Antinociceptive Activity of JSE, NPX, and TML
JSE, NPX, and TML showed a dose-dependent antinociceptive activity (Figure 2). The
highest doses tested, 200 mg/kg JSE, 300 mg/kg NPX, and 50 mg/kg TML, exhibited an-
tinociceptive activities of 59.87%, 54.29%, and 89%, respectively (Figure 2). The ED50 val-
ues were 121.15 mg/kg (JSE), 226.21 mg/kg (NPX), and 14.58 mg/kg (TML).
Figure 2. The formalin test in the rats demonstrated the antinociceptive effect of naproxen (A), the
Justicia spicigera extract (B), and tramadol (C). The vertical bars represent the mean ± S.E.M. for each
experimental group (n = 6). * p < 0.05 versus vehicle group. ANOVA followed by Dunne’s test.
Figure 2. The formalin test in the rats demonstrated the antinociceptive effect of naproxen (A), the
Justicia spicigera extract (B), and tramadol (C). The vertical bars represent the mean
±
S.E.M. for each
experimental group (n = 6). * p< 0.05 versus vehicle group. ANOVA followed by Dunnett’s test.
2.3. Antinociceptive Activity of JSE, NPX, and TML
The combination NPX-JSE and its three proportions (1:1, 1:3, and 3:1) showed antinoci-
ception in a dose-dependent manner (Figure 3). The highest values of antinociception were
recorded as 55.44% (173.68 mg/kg p.o. 1:1 NPX-JSE), 68.67%
(147.42 mg/kg p.o. 1:3 NPX-JSE)
,
and 72.54% (199.94 mg/kg p.o. 3:1 NPX-JSE). The ED
50
values for the proportions NPX:JSE
were 107.09 mg/kg (1:1), 73.82 mg/kg (1:3), and 102.44 mg/kg (3:1).
Pharmaceuticals 2025,18, 187 4 of 17
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 4 of 17
2.3. Antinociceptive Activity of JSE, NPX, and TML
The combination NPX-JSE and its three proportions (1:1, 1:3, and 3:1) showed antino-
ciception in a dose-dependent manner (Figure 3). The highest values of antinociception
were recorded as 55.44% (173.68 mg/kg p.o. 1:1 NPX-JSE), 68.67% (147.42 mg/kg p.o. 1:3
NPX-JSE), and 72.54% (199.94 mg/kg p.o. 3:1 NPX-JSE). The ED50 values for the propor-
tions NPX:JSE were 107.09 mg/kg (1:1), 73.82 mg/kg (1:3), and 102.44 mg/kg (3:1).
Figure 3. The formalin test demonstrated the antinociceptive effect of the Naproxen (NPX)–Justicia
spicigera extract (JSE) combination at ratios of (A) 1:1 (NPX 113.1 mg/k +60.58 mg/kg = 173.68
mg/kg), (B) 1:3 (NPX 56.6 mg/kg + 90.9 mg/kg = 147.42 mg/kg), and (C) 3:1 (NPX 169.7 mg/kg + 30.3
mg/kg = 199.94 mg/kg). The vertical bars represent the mean ± S.E.M. for each experimental group
(n = 6). * p < 0.05 versus the vehicle group. ANOVA followed by the Dunne’s test.
A)
B)
C)
Figure 3. The formalin test demonstrated the antinociceptive effect of the Naproxen (NPX)–Justicia spi-
cigera extract (JSE) combination at ratios of
(A) 1:1 (NPX 113.1 mg/k +60.58 mg/kg = 173.68 mg/kg),
(B) 1:3 (NPX 56.6 mg/kg + 90.9 mg/kg = 147.42 mg/kg), and (C) 3:1 (NPX 169.7 mg/kg + 30.3 mg/kg
= 199.94 mg/kg). The vertical bars represent the mean
±
S.E.M. for each experimental group (n = 6).
*p< 0.05 versus the vehicle group. ANOVA followed by the Dunnett’s test.
The combination of TML-JSE and its proportions exhibited dose-dependent antinoci-
ceptive activity (Figure 4). The highest doses registered the maximum antinociceptive effect
as follows: 51.68% (67.86 mg/kg p.o. 1:1 TML-JSE), 73.54%
(94.51 mg/kg p.o. 1:3 TML-JSE),
and 70.79% (41.22 mg/kg p.o. 3:1 TML-JSE) (Figure 4). The ED
50
values for the pro-
portions TML:JSE were 66 mg/kg (1:1), 39.23 mg/kg (1:3), and 14.79 mg/kg (3:1). The
proportions 3:1 (NPX-JSE) (Figure 3) and 1:3 (TML:JSE) (Figure 4) registered the highest
antinociceptive effects.
Pharmaceuticals 2025,18, 187 5 of 17
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 5 of 17
The combination of TML-JSE and its proportions exhibited dose-dependent antino-
ciceptive activity (Figure 4). The highest doses registered the maximum antinociceptive
effect as follows: 51.68% (67.86 mg/kg p.o. 1:1 TML-JSE), 73.54% (94.51 mg/kg p.o. 1:3
TML-JSE), and 70.79% (41.22 mg/kg p.o. 3:1 TML-JSE) (Figure 4). The ED50 values for the
proportions TML:JSE were 66 mg/kg (1:1), 39.23 mg/kg (1:3), and 14.79 mg/kg (3:1). The
proportions 3:1 (NPX-JSE) (Figure 3) and 1:3 (TML:JSE) (Figure 4) registered the highest
antinociceptive effects.
Figure 4. Antinociceptive effect of the tramadol (TML)–Justicia spicigera extract (JSE) combination:
(A) 1:1 (TML 7.29 mg/kg + 60.58 mg/kg = 67.86 mg/kg), (B) 1:3 (TML 3.6 mg/kg + 90.9 mg/kg = 94.51
mg/kg), and (C) 3:1 (TML 10.9 mg/kg + 30.3 mg/kg = 41.22 mg/kg) ratios in the formalin test. The
vertical bars represent the mean ± S.E.M. for each experimental group (n = 6). * p < 0.05 versus the
vehicle group. ANOVA followed by the Dunne’s test.
A)
B)
C)
Figure 4. Antinociceptive effect of the tramadol (TML)–Justicia spicigera extract (JSE) combination:
(A) 1:1 (TML 7.29 mg/kg + 60.58 mg/kg = 67.86 mg/kg), (B) 1:3 (TML 3.6 mg/kg + 90.9 mg/kg =
94.51 mg/kg), and (C) 3:1 (TML 10.9 mg/kg + 30.3 mg/kg = 41.22 mg/kg) ratios in the formalin test.
The vertical bars represent the mean
±
S.E.M. for each experimental group (n = 6). * p< 0.05 versus
the vehicle group. ANOVA followed by the Dunnett’s test.
2.4. Isobolographic Analysis
In the isobolographic analysis, the combination of NPX-JSE and its three proportions
demonstrated a synergistic type of interaction, with the antinociceptive effect graphically
placed below the line of additivity (Figure 5). The values of
γ
were 0.617 (1:1 NPX:JSE),
0.512 (3:1 NPX:JSE), and 0.501 (1:3 NPX:JSE) (Table 1). The values of the interaction index
were below one, representing a synergistic effect in the pharmacological combination
NPX:JSE (Table 1). Similarly, the combination TML:JSE and its three proportions showed
Pharmaceuticals 2025,18, 187 6 of 17
synergism (Figure 6). The values of
γ
were 0.973 (1:1 TML:JSE), 0.359 (3:1 TML:JSE), and
0.415 (1:3 TML:JSE) (Table 2).
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 6 of 17
2.4. Isobolographic Analysis
In the isobolographic analysis, the combination of NPX-JSE and its three proportions
demonstrated a synergistic type of interaction, with the antinociceptive effect graphically
placed below the line of additivity (Figure 5). The values of γ were 0.617 (1:1 NPX:JSE),
0.512 (3:1 NPX:JSE), and 0.501 (1:3 NPX:JSE) (Table 1). The values of the interaction index
were below one, representing a synergistic effect in the pharmacological combination
NPX:JSE (Table 1). Similarly, the combination TML:JSE and its three proportions showed
synergism (Figure 6). The values of γ were 0.973 (1:1 TML:JSE), 0.359 (3:1 TML:JSE), and
0.415 (1:3 TML:JSE) (Table 2).
(A)
(B)
(C)
Figure 5. The isobolograms illustrate the experimental interaction between naproxen (NPX) and the
Justicia spicigera extract (JSE) at the following ratios: (A) 1:1, (B) 3:1, and (C) 1:3 in the formalin test.
T: theoretical additive ED
50
; and E: experimental ED
50
. The points represent the mean
±
S.E.M. for
each experimental group (n = 6).
Pharmaceuticals 2025,18, 187 7 of 17
Table 1. Theoretical (Z
add
) and experimental (Z
exp
) ED
50
values and interaction index for the different
ratios for the combinations of NPX:JSE.
Proportion
NPX1:JSE1 NPX3:JSE1 NPX1:JSE3
Zadd (mg/kg) 173.68 ±17.49 199.94 ±13.55 147.42 ±24.11
Zexp (mg/kg) 107.09 ±0.01 * 102.44 ±0.09 * 73.82 ±0.10 *
Interaction Index 0.617 0.512 0.501
*p< 0.05 Z
add
versus Z
exp
, by Student’s t-test. The values were expressed as the mean
±
standard error of the
mean (SEM).
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 7 of 17
Figure 5. The isobolograms illustrate the experimental interaction between naproxen (NPX) and the
Justicia spicigera extract (JSE) at the following ratios: (A) 1:1, (B) 3:1, and (C) 1:3 in the formalin test.
T: theoretical additive ED50; and E: experimental ED50. The points represent the mean ± S.E.M. for
each experimental group (n = 6).
Table 1. Theoretical (Zadd) and experimental (Zexp) ED50 values and interaction index for the different
ratios for the combinations of NPX:JSE.
Proportion
NPX1:JSE1 NPX3:JSE1 NPX1:JSE3
Zadd (mg/kg) 173.68 ± 17.49 199.94 ± 13.55 147.42 ± 24.11
Zexp (mg/kg) 107.09 ± 0.01 * 102.44 ± 0.09 * 73.82 ± 0.10 *
Interaction Index 0.617 0.512 0.501
* p < 0.05 Zadd versus Zexp, by Student's t-test. The values were expressed as the mean ± standard
error of the mean (SEM).
A)
B)
C)
Figure 6. The isobolograms illustrate the experimental interaction between tramadol (TML) and the
Justicia spicigera extract (JSE) at the following ratios: (A) 1:1, (B) 3:1, and (C) 1:3 in the formalin test.
T: theoretical additive ED
50
; and E: experimental ED
50
. The points represent the mean
±
S.E.M. for
each experimental group (n = 6).
Pharmaceuticals 2025,18, 187 8 of 17
Table 2. Theoretical (Z
add
) and experimental (Z
exp
) ED
50
values and interaction index for the different
ratios for the combinations of TML:JSE.
Proportion
TML1:JSE1 TML3:JSE1 TML1:JSE3
Zadd (mg/kg) 67.87 ±15.97 41.22 ±8.31 94.51 ±23.85
Zexp (mg/kg) 66 ±0.02 14.79 ±0.10 * 39.23 ±0.03 *
Interaction Index 0.973 0.359 0.415
*p< 0.05 Z
add
versus Z
exp
, by Student’s t-test. The values were expressed as the mean
±
standard error of the
mean (SEM).
2.5. Western Blot Analysis
The combination of 1:3 NPX:JSE demonstrated the best synergistic effect, exhibited a
low ED
50
value of 73.82 mg/kg, and accounted for the use of NSAID. Therefore, this work
conducted further experiments with this combination to evaluate its molecular mechanism
of action. The expression of COX2 was detected in the vehicle group, and it was slightly
decreased in the NPX group (Figure 7A, Lane 2–3 from the upper blot). JSE decreased
(p< 0.05)
the COX2 expression in the rats using the formalin test (Figure 7B). However, the
combination NPX:JSE showed a higher reduction (p< 0.05) in COX2 expression compared
to JSE alone (Figure 7B). Only the group receiving a saline solution exhibited the pro-
inflammatory cytokine IL-1. The groups treated with NPX, JSE, or the combination NPX:JSE
did not exhibit this cytokine (Figure 7A,C).
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 8 of 17
Figure 6. The isobolograms illustrate the experimental interaction between tramadol (TML) and the
Justicia spicigera extract (JSE) at the following ratios: (A) 1:1, (B) 3:1, and (C) 1:3 in the formalin test.
T: theoretical additive ED50; and E: experimental ED50. The points represent the mean ± S.E.M. for
each experimental group (n = 6).
Table 2. Theoretical (Zadd) and experimental (Zexp) ED50 values and interaction index for the different
ratios for the combinations of TML:JSE.
Proportion
TML1:JSE1 TML3:JSE1 TML1:JSE3
Zadd (mg/kg) 67.87 ± 15.97 41.22 ± 8.31 94.51 ± 23.85
Zexp (mg/kg) 66 ± 0.02 14.79 ± 0.10 * 39.23 ± 0.03 *
Interaction Index 0.973 0.359 0.415
* p < 0.05 Zadd versus Zexp, by Student's t-test. The values were expressed as the mean ± standard
error of the mean (SEM).
2.5. Western Blot Analysis
The combination of 1:3 NPX:JSE demonstrated the best synergistic effect, exhibited a
low ED50 value of 73.82 mg/kg, and accounted for the use of NSAID. Therefore, this work
conducted further experiments with this combination to evaluate its molecular mecha-
nism of action. The expression of COX2 was detected in the vehicle group, and it was
slightly decreased in the NPX group (Figure 7A, Lane 2–3 from the upper blot). JSE de-
creased (p < 0.05) the COX2 expression in the rats using the formalin test (Figure 7B). How-
ever, the combination NPX:JSE showed a higher reduction (p < 0.05) in COX2 expression
compared to JSE alone (Figure 7B). Only the group receiving a saline solution exhibited
the pro-inflammatory cytokine IL-1. The groups treated with NPX, JSE, or the combina-
tion NPX:JSE did not exhibit this cytokine (Figure 7A,C).
Figure 7. Expression of COX2 and pro-IL-1β in the formalin-induced pain model. As shown in Fig-
ure (A), COX2 and pro-IL-1β levels were measured in tissues from the right hind paw that had been
treated with a vehicle (V), naproxen (NPX), and naproxen mixed with the Justicia spicigera extracts
Figure 7. Expression of COX2 and pro-IL-1
β
in the formalin-induced pain model. As shown in
Figure (A), COX2 and pro-IL-1
β
levels were measured in tissues from the right hind paw that had
been treated with a vehicle (V), naproxen (NPX), and naproxen mixed with the Justicia spicigera
extracts (NPX + JSE) ED
50
= 73.82 mg/kg. The left hind paw not treated with formalin and drugs is
denoted with basal, and the saline solution was the vehicle. The COX2 (B) and pro-IL-1
β
(C) protein
expression underwent a densitometry analysis, n = 6. The data are presented as the mean
±
standard
error of the mean (S.E.M) ((*p< 0.05; **p< 0.01; *** p< 0.001 compared to the vehicle group).
Pharmaceuticals 2025,18, 187 9 of 17
2.6. Myeloperoxidase (MPO) Activity
The effect of the combination of 1:3 NPX:JSE on myeloperoxidase activity was eval-
uated. The activity of MPO in the vehicle group was higher than the left paw or basal
without formalin (p< 0.01). The right paws of the rats pretreated with formalin—NPX,
JSE, or a combination of NPX and JSE—decreased the MPO activity (p< 0.05) by 42.73,
37.94, and 58%, respectively. This effect was comparable to the MPO levels in the left
paw (Figure 8).
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 9 of 17
(NPX + JSE) ED50 = 73.82 mg/kg. The left hind paw not treated with formalin and drugs is denoted
with basal, and the saline solution was the vehicle. The COX2 (B) and pro-IL-1β (C) protein expres-
sion underwent a densitometry analysis, n = 6. The data are presented as the mean ± standard error
of the mean (S.E.M) ((*p < 0.05; **p < 0.01; *** p < 0.001 compared to the vehicle group ).
2.6. Myeloperoxidase (MPO) Activity
The effect of the combination of 1:3 NPX:JSE on myeloperoxidase activity was evalu-
ated. The activity of MPO in the vehicle group was higher than the left paw or basal with-
out formalin (p < 0.01). The right paws of the rats pretreated with formalin—NPX, JSE, or
a combination of NPX and JSE—decreased the MPO activity (p < 0.05) by 42.73, 37.94, and
58%, respectively. This effect was comparable to the MPO levels in the left paw (Figure 8).
Figure 8. Myeloperoxidase activity in the formalin-induced pain model. The right hind paw was
pretreated with the vehicle (V), naproxen (NPX), and naproxen with the Justicia spicigera extract
(NPX + JSE) plus formalin. The left hind paw was not treated with formalin (basal). The bars repre-
sent the mean values (±SEM). n = 6, ** p < 0.01, and *** p < 0.0001 compared to the vehicle group.
2.7. Effect of JSE on the Pharmacokinetics of NPX
Figure 9 indicates the chromatogram showing the retention times for NPX (3.007
min) and dicloxacillin (4.1 min). Figure 10 depicts the mean arterial plasma concentration–
time profiles of naproxen after its oral administration to the rats with or without the sim-
ultaneous oral administration of JSE. Table 3 presents the main pharmacokinetic (PK) pa-
rameters of naproxen when administered alone or with JSE, along with the statistical test
results. As shown in Table 3, after oral administration, NPX was absorbed and reached a
maximum concentration of 68.6 ± 11.73 µg/mL at 1.25 h. The combination of JSE and NPX
reached a maximum concentration of 55.15 ± 2.39 µg/mL at 1.17 h. There were no signif-
icant changes in the pharmacokinetic parameters of NPX administered individually and
the combination NPX:JSE.
Figure 8. Myeloperoxidase activity in the formalin-induced pain model. The right hind paw was
pretreated with the vehicle (V), naproxen (NPX), and naproxen with the Justicia spicigera extract
(NPX + JSE)
plus formalin. The left hind paw was not treated with formalin (basal). The bars
represent the mean values (
±
SEM). n = 6, ** p< 0.01, and *** p< 0.0001 compared to the vehicle
group.
2.7. Effect of JSE on the Pharmacokinetics of NPX
Figure 9indicates the chromatogram showing the retention times for NPX (3.007 min)
and dicloxacillin (4.1 min). Figure 10 depicts the mean arterial plasma concentration–time
profiles of naproxen after its oral administration to the rats with or without the simultaneous
oral administration of JSE. Table 3presents the main pharmacokinetic (PK) parameters of
naproxen when administered alone or with JSE, along with the statistical test results. As
shown in Table 3, after oral administration, NPX was absorbed and reached a maximum
concentration of 68.6
±
11.73
µ
g/mL at 1.25 h. The combination of JSE and NPX reached
a maximum concentration of 55.15
±
2.39
µ
g/mL at 1.17 h. There were no significant
changes in the pharmacokinetic parameters of NPX administered individually and the
combination NPX:JSE.
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 10 of 17
Figure 9. Chromatogram showing the retention times for NPX (3.007 min) and dicloxacillin (4.1
min).
Figure 10. Mean arterial plasma concentration–time profiles of naproxen (NPX) after oral admin-
istration at a dose of 30 mg/kg (●; n = 6) or naproxen at a dose of 30 mg/kg with JSE at a dose of 60
mg/kg (○; n = 6). The bars represent standard deviations.
Table 3. Mean (±standard deviation) pharmacokinetic parameters of naproxen after single oral ad-
ministration at a dose of 30 mg/kg to the rats with 60 mg/kg of JSE.
Parameter NPX NPX + JSE
Tmax (h) 1.25 ± 0.9874 1.16 ± 0.41
t1/2 (h) 17.05 ± 7.46 10.78 ± 8.41
Cmax (ug/mL) 68.70 ± 11.72 55.15 ± 5.86
AUC 0-t (µg/mL×h) 650.52 ± 149.01 529 ± 67.54
AUC 0-inf_obs (µg/mL×h) 1038.77 ± 208.75 718.65 ± 222.93
3. Discussion
Balanced analgesia proposes a new pharmacological alternative for the treatment of
pain. Drugs such as naproxen have been combined with other analgesics, i.e., paraceta-
mol, to promote changes to individual treatment and provide various benefits, mainly at
the therapeutic level [11].
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
0 5 10 15 20 25
Naproxen in plasma (µg/mL)
Time (h)
NPX
NPX-JSE
Figure 9. Chromatogram showing the retention times for NPX (3.007 min) and dicloxacillin (4.1 min).
Pharmaceuticals 2025,18, 187 10 of 17
Pharmaceuticals 2025, 18, x FOR PEER REVIEW 10 of 17
Figure 9. Chromatogram showing the retention times for NPX (3.007 min) and dicloxacillin (4.1
min).
Figure 10. Mean arterial plasma concentration–time profiles of naproxen (NPX) after oral admin-
istration at a dose of 30 mg/kg (●; n = 6) or naproxen at a dose of 30 mg/kg with JSE at a dose of 60
mg/kg (○; n = 6). The bars represent standard deviations.
Table 3. Mean (±standard deviation) pharmacokinetic parameters of naproxen after single oral ad-
ministration at a dose of 30 mg/kg to the rats with 60 mg/kg of JSE.
Parameter NPX NPX + JSE
Tmax (h) 1.25 ± 0.9874 1.16 ± 0.41
t1/2 (h) 17.05 ± 7.46 10.78 ± 8.41
Cmax (ug/mL) 68.70 ± 11.72 55.15 ± 5.86
AUC 0-t (µg/mL×h) 650.52 ± 149.01 529 ± 67.54
AUC 0-inf_obs (µg/mL×h) 1038.77 ± 208.75 718.65 ± 222.93
3. Discussion
Balanced analgesia proposes a new pharmacological alternative for the treatment of
pain. Drugs such as naproxen have been combined with other analgesics, i.e., paraceta-
mol, to promote changes to individual treatment and provide various benefits, mainly at
the therapeutic level [11].
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
0 5 10 15 20 25
Naproxen in plasma (µg/mL)
Time (h)
NPX
NPX-JSE
Figure 10. Mean arterial plasma concentration–time profiles of naproxen (NPX) after oral admin-
istration at a dose of 30 mg/kg (
•
; n = 6) or naproxen at a dose of 30 mg/kg with JSE at a dose
of 60 mg/kg (#; n = 6). The bars represent standard deviations.
Table 3. Mean (
±
standard deviation) pharmacokinetic parameters of naproxen after single oral
administration at a dose of 30 mg/kg to the rats with 60 mg/kg of JSE.
Parameter NPX NPX + JSE
Tmax (h) 1.25 ±0.9874 1.16 ±0.41
t1/2 (h) 17.05 ±7.46 10.78 ±8.41
Cmax (ug/mL) 68.70 ±11.72 55.15 ±5.86
AUC 0-t (µg/mL ×h) 650.52 ±149.01 529 ±67.54
AUC 0-inf_obs (µg/mL×h) 1038.77 ±208.75 718.65 ±222.93
3. Discussion
Balanced analgesia proposes a new pharmacological alternative for the treatment of
pain. Drugs such as naproxen have been combined with other analgesics, i.e., paracetamol,
to promote changes to individual treatment and provide various benefits, mainly at the
therapeutic level [11].
Previous studies of Justicia spicigera have found the presence of kaempferol, kaempferol-3,
7-bisrhamnósido (kaempferitrin), cryptoxanthin, allantoin, and
β-glucosyl-o-sitosterol [12,13].
This work found kaempferitrin content similar to the previous study [
9
]. An ethanol extract of
Justicia spicigera exerted antinociceptive activity in different murine models [
9
]. However, the
antinociceptive mechanism of Justicia spicigera remains undefined, underscoring the importance
of evaluating its mechanism of action and its combination with analgesics in future studies.
This study used the formalin test, a pain model that involves two phases of
pain: the first phase corresponds to neurogenic pain, which includes substance P and
bradykinin [14,15],
and the second phase corresponds to inflammatory pain, which is
characterized by the appearance of inflammation mediators like histamine, serotonin,
prostaglandins, and even bradykinin itself [
16
,
17
]. The individual administration of JSE,
NPX, and TML showed antinociceptive activity in a dose-dependent manner. The antinoci-
ceptive effect of these treatments was observed in the following order: TML > JSE > NPX.
The combination of JSE with NPX and TML in three different proportions (1:1, 3:1,
and 3:1) showed an increase in antinociception in the formalin test. The combination
also demonstrated the existence of synergism between both treatments when combined,
Pharmaceuticals 2025,18, 187 11 of 17
confirmed by the interaction index of less than one and in the isobolographic analysis. The
proportions 3:1 (NPX-JSE) and 1:3 (TML:JSE) registered the highest antinociceptive effects,
whereas the combination 3:1 (NPX-JSE) showed the highest synergism. Further studies
were carried out with this combination. Synergism occurs because the drugs’ mechanisms
of action differ, causing antinociception. It is known that naproxen works by blocking
cyclooxygenase (COX-1 and COX-2) [
18
]. This stops the production of prostaglandins from
arachidonic acid, which is directly connected to controlling pain. Tramadol’s antinocicep-
tive activity stems from its affinity for the opioid and the neuropeptide substance P receptor,
as well as the participation of K+ channels and the nitric oxide synthase [19,20].
Canonically, pro-IL-1
β
is converted to the mature IL-1
β
form mediated by caspase-1
cleavage after inflammasome formation [
21
]. IL-1
β
has been described as capable of induc-
ing COX2 expression through IL-1
β
receptors and protein kinase C (PKC) signaling [
22
].
COX2, for its part, induces the production of prostaglandin E2 (PGE2), which plays a role
in the generation of inflammatory and neuropathic pain [
23
]. Myeloperoxidase, an enzyme
present in primary azurophilic granules of neutrophils, is another inflammatory media-
tor. MPO catalyzes the formation of reactive oxygen intermediates such as hypochlorous
(HOCl), hypobromous, and hypothiocyanous acids [
24
]. This work detected the expression
of pro-IL-1
β
(~30 kDa) in the vehicle group where there is inflammation and pain, and the
levels of this cytokine were reduced with the treatment of NPX, JSE, and the combination
NPX:JSE. The levels of COX2 (~70 kDa) decreased with the treatment of NPX and JSE and
markedly reduced with the combination NPX:JSE. Additionally, the MPO activity was
lower in the treated groups with NPX, JSE, and the combination NPX:JSE compared to the
vehicle group (formalin without treatment). This study found that JSE reduces pain by
lowering the levels of COX-2 and IL-1
β
and the MPO activity in the formalin test in rats.
The combination NPX:JSE exerts its antinociceptive actions by markedly decreasing the
MPO activity and the COX2 levels, and reducing the IL-1βlevels.
In this study, KM was the main compound found in JSE. The main compound in a rich
flavonoid extract of lotus (Nelumbo nucifera) leaves was KM. The mice that received this
plant extract daily for 14 days at a dose of 100 mg/kg p.o. experienced a 74% reduction in
ethanol-induced stomach damage. This plant extract increased the levels of nitric oxide
and IFN-gamma and lowered the levels of IL-6, IL-12, and TNF-alpha in gastric tissue [
25
].
These results indicated that KM is participating in the gastroprotective actions of lotus
extract. Therefore, KM is unlikely to produce gastric damage in rodents.
The pharmacokinetic parameters of NPX found in this work agree with previous re-
ports in rats [
26
]. CYP2A1 and CYP2C9 enzymes metabolize NPX [
27
,
28
]. JSE did not affect
the pharmacokinetic parameters of NPX. Therefore, JSE did not affect the bioavailability
(absorption, distribution, or elimination) of NPX. NSAIDs like naproxen present the risk of
inducing bleeding. The coadministration of JSE and NPX decreases the possibility of this
side effect.
4. Materials and Methods
4.1. Reagents and Drugs
Tris, sodium chloride, Tween-20, non-fat dry milk, sodium dodecyl sulfate (SDS),
naproxen sodium (NPX), tramadol (TML), and acrylamide were from Sigma-Aldrich
(St. Louis, MO, USA).
Anti-COX2 (PA5-17614), anti-beta actin (BA3R), and HRP-conjugated
anti-rabbit (#31460) antibodies were purchased from Thermo Scientific (Rockford, IL, USA),
whereas anti-IL-1β(D6D6T) was from Cell Signaling Technology (Danvers, MA, USA).
Pharmaceuticals 2025,18, 187 12 of 17
4.2. Plant Material and Extraction
Samples of Justicia spicigera were collected in Mexico City, México (21
◦
58
′
43.90
′′
N and
98
◦
58
′
31.44
′′
W) in October 2019, identified (voucher number FEZA 4656) by a specialist,
and preserved at the herbarium School of Higher Studies of Zaragoza, UNAM. Dried leaves
of Justicia spicigera (40 g) were extracted with 1 L of ethanol using a Soxtherm apparatus
(Soxtherm automatic, Gerhadt, Germany) for 3 h. The Justicia spicigera extract (JSE) was
filtered, evaporated, and then lyophilized (Free Zone 2.5, Labconco, MO, USA). The yield
of JSE was 3.4%.
4.3. Chemical Standardization of Plant Extract
The quantitation of kaempferitrin, an active compound in JSE, was carried out accord-
ing to the protocol described by Alonso-Castro et al. [
8
] using an HPLC method in a Waters
2795 chromatograph (Waters Corp., Milford, MA, USA) instrument.
4.4. Animals
Male Wistar rats aged 6–8 weeks (weight range 200–250 g) were from the University
of Guanajuato animal facility and were housed in isolated cages at 24
◦
C under a light-dark
cycle of 12:12. The animals were supplied with food and water ad libitum. Each animal
was used only once and euthanized at the end of the test by an overdose of anesthetic. This
study conducted all the experiments in accordance with the NIH Guide for Treatment and
Care for Laboratory Animals [
29
] and the Official Mexican Norm NOM
062-ZOO-1999
,
which provide technical specifications for the production, care, and use of laboratory
animals. The University of Guanajuato’s ethical committee for research (CIBIUG-P30-2019)
revised and approved the protocol of this study. This research also followed the Guidelines
on Ethical Standards for Investigations of Experimental Pain in Animals [30].
4.5. Antinociceptive Assay
4.5.1. Design
Different groups of rats (n = 6) were used to characterize the dose–response curve of
each drug. Justicia spicigera extract (JSE) at 30, 100, 150, and 200 mg/kg; naproxen (NPX) at
50, 100, 200, and 300 mg/kg; or tramadol (TML) at 5, 12.5, 25, and 50 mg/kg were orally
administered 60 min before the administration with formalin. The drug treatments were
dissolved in saline solution as a vehicle. The control group received a saline solution. Once
the dose–response curve of each drug was obtained, an experimental effective dose (ED
50
,
the dose that produces a response of 50% of the maximum response) value was determined
for each compound, and the NPX-JSE and TML-JSE combinations were assessed in the
second phase of the formalin test using three different proportions (1:1, 3:1, and 1:3).
4.5.2. Formalin Test
The formalin-induced nociception test was performed as described previously [
31
].
The rats were placed in transparent plastic cylindrical chambers, positioning a mirror at
a 45
◦
angle to prevent any unobstructed view of their paws. The animals were injected
into the dorsal surface of the hind paw with 50
µ
L of dilute formalin (5%) and observed
for nociceptive behavior immediately after injection. Nociceptive behavior was quantified
as the number of paw flinches during 1 min periods every 5 min up to 60 min after
injection. Time courses of antinociceptive response to individual drugs and combinations
were constructed by plotting the mean number of paw flinches as a function of time.
Dose–response
data were quantified as the percentage of reduction in paw flinches relative
to the control total in the second phase of the formalin test (15–60 min), where JSE showed
Pharmaceuticals 2025,18, 187 13 of 17
antinociceptive effects in the previous study [
9
]. The percentage of antinociception was
calculated according to the following equation:
% Antinociception = ((Control-Drug)/Control)
×
100, where “Control” and “Drug”
refer to the number of paw flinches elicited by formalin plus saline and formalin plus drug,
respectively. At the end of the experiment, an anesthetic overdose euthanized the rats.
4.6. Western Blot
The rats from the formalin-induced pain model were euthanized to obtain the right
and left hind paws. Approximately 120 mg of tissue were sectioned and placed in a
tube containing zirconium beads and 0.6 mL of immunoprecipitation (IP) lysis buffer
(Thermo Scientific) supplemented with protease inhibitor. The BeadBug
TM
system ho-
mogenized the tube containing the sample through three cycles of 35 s and 2 min in ice.
The homogenate was centrifuged at 16,000
×
gfor 10 min at 4
◦
C, and the supernatant
was recovered for measuring the protein content using the Bradford assay. Then, 30 µg of
protein per lane was loaded and resolved in 10% SDS-PAGE gel and electro-transferred
onto polyvinylidene difluoride (PVDF) membranes, which were blocked with 10% of
non-fat dry milk in Tris-buffered saline buffer with 0.1% Tween-20 (TBST) and incubated
with the respective primary antibody (1:500 for COX2, 1:200 for IL-1
β
, and 1:1500 for
β
-actin) overnight
at 4 ◦C.
The membranes were washed with TBST and then incubated
with
1:3000 HRP-conjugated
anti-rabbit secondary antibody for 1 h at room temperature.
The membranes were visualized in x-ray film using an ultra-sensitive enhanced chemilu-
minescent (ECL) kit (SuperSignal West Femto from Thermo Scientific). The intensity of
the bands on the blots was quantified using the ImageJ software version 1.54j [
32
] and
normalized for beta-actin values as a loading control for the densitometry analysis.
4.7. Bradford and Myeloperoxidase (MPO) Assays
The quantitation of proteins in the tissue from the rats’ paws in the formalin
test and MPO as activity was realized following the same methodology described by
Barragan-Galvez et al. [33].
4.8. Pharmacokinetic Study
4.8.1. Catheterization of the Femoral Artery
The rats received sodic pentobarbital (50 mg/kg i.p.), and the catheterization was
performed in accordance with the protocol of Jespersen et al. [
34
] with minor modifications.
The surgical regions (back of the neck and inner leg) were shaved and disinfected using
iodopovidone and 70% ethanol. Each rat was lying prone (on its stomach). Then, a
horizontal incision (3 mm) was made on the back of the neck at the level of the shoulder
blades with scissors, followed by another incision made in the inguinal area (15 mm)
along the natural angle of the hind leg, placing each rat onto its back (supine position).
We performed the dissection to separate the connective tissue, and then introduced the
catheter into the inguinal area at the back of the neck using a disinfected linear guide of
steel. The femoral artery was separated from the nervous and femoral veins. This artery
was cannulated for blood sampling with a PE-10 catheter rinsed with heparin in a saline
solution (125 U/mL) [
34
]. The wounds were sutured, and the catheter was secured with
a high-tension spring located in the back of the neck region. Each animal was placed
in an acrylic cage with free access to water and a standard diet to aid in their recovery
from surgery.
4.8.2. Experimental Design
Twelve male Wistar rats were maintained under the same laboratory conditions de-
scribed above. After 3 days of acclimatization, the rats (308.7
±
42.25 g) were randomly
Pharmaceuticals 2025,18, 187 14 of 17
distributed into two groups (NPX and NPX-JSE). The randomization of groups was per-
formed using the GraphPad QuickCalcs software V5 (San Diego, CA, USA). The animals
were catheterized by the femoral artery according to the previously described protocol.
Twenty-four hours after the anesthesia and a 12 h fast, the rats received a single oral dose
of NPX (30 mg/kg) or NPX (30 mg/kg)-JSE (60 mg/kg). The selection of NPX doses was
guided by previous studies [
9
,
26
], and this work determined the ED50 of JSE to achieve
antinociceptive effects. Five hundred microliter blood samples were obtained at 30, 60, 90,
120, 180, 240, 360, 480, 600, and 1440 min after drug administration [
35
]. The blood volume
drawn was replaced with the same saline solution volume to avoid reducing the circulating
volume [
35
], and the catheter was maintained in sodic heparin (125 U/mL) [
34
]. Finally,
the plasma was obtained by centrifuging blood samples at 11,400 rpm at 4
◦
C for 10 min
(MicroCL 21 Centrifuge, Thermo Fisher, Rockford, IL, USA).
4.8.3. HPLC Analysis
Our laboratory developed a method to determine the quantification of NPX in the rat
plasma using reversed-phase high-performance (HPLC). Ten microliters of dicloxacillin
(1000
µ
g/mL) were mixed with 100
µ
L plasma samples as an internal standard (IS). Af-
terward, two hundred microliters of acetonitrile were added to eliminate the plasma
proteins. This mixture was shaken in a vortex for 1 min and then centrifuged for 5 min at
100,000 rpm (HSIANGTAI, New Taipei City, Taiwan, China). The samples were filtered
and analyzed by HPLC at room temperature using a C18 column (4.6
×
250 mm, 5
µ
m,
Agilent,
Palo Alto,
CA). The mobile phase was a solution of acetonitrile and phosphoric
acid (1.46 mM pH 3) 30:70 v/v. It had a constant flow rate of 1.2 mL/min and a pressure
of 2000 ±24 psi (1500 HPLC pump
, Waters, Milford, MA, USA). A UV/Visible Detector
(2489 HPLC, Waters, Milford, MA, USA) was used to measure NPX at 225 nm.
4.9. Data Analysis and Statistics
The results are shown as the mean
±
SEM for 6 animals per group. The dose–response
data were analyzed using a one-way analysis of variance followed by Dunnett’s test.
The possible synergism between JSE-NPX and JSE-TML was evaluated by constructing
isobolograms using the individual ED
50
values. The theoretical additive ED
50
and the
experimentally derived ED
50
values were compared using the Student’s t-test. An experi-
mental ED
50
value significantly lower than the theoretical additive ED
50
value indicated a
synergistic interaction between JSE-NPX and JSE-TML. The isobolographic analysis was
used to characterize drug interactions. The isobologram was constructed by connecting
the significantly lower than theoretical additive ED
50
value of naproxen plotted on the
abscissa with the ED
50
value of JSE plotted on the ordinate to obtain the additive line.
For the mixture of JSE and naproxen, the ED
50
value was determined by the linear re-
gression analysis of the log dose–response curve (six animals with at least four doses)
and compared using a t-test to a theoretical additive ED
50
value [
36
,
37
]. The interaction
index (
γ
) was obtained by dividing the ED
50
value of the experimental combination by the
ED
50
value of the theoretical combination. This index showed the difference between the
theoretical and experimental ED
50
values for each combination. The
γ
indicates the portion
of the
ED50 value
of individual drugs that accounts for the corresponding
ED50 value
in
the combination. Values of approximately 1 correspond to an additive interaction; values
greater than
1 imply
an antagonistic interaction; and values less than 1 indicate a synergistic
interaction. Statistical significance was considered when p< 0.05.
For the pharmacokinetic study, the NPX plasma concentrations were plotted against
time after the drug administration. These curves were constructed using the GraphPad
V5 software. The pharmacokinetic parameters of naproxen were determined by a non-
Pharmaceuticals 2025,18, 187 15 of 17
compartmental method using the Excel add-on PKSolver [
37
]. The pharmacokinetics
parameters were expressed as the mean ±standard deviation (SD).
5. Conclusions
The combination NPX:JSE (3:1) showed antinociceptive effects by decreasing the levels
of COX2, IL-1β, and MPO without affecting NPX’s pharmacokinetics.
Author Contributions: Conceptualization, J.R.Z.-M., A.J.A.-C. and M.L.G.-R.; methodology,
J.R.Z.-M.,
A.J.A.-C., M.L.G.-R., J.C.B.-G. and A.H.-F.; formal analysis, J.R.Z.-M., A.J.A.-C.,
M.L.G.-R.,
J.C.B.-G.
and A.H.-F.; investigation, H.I.G.P., M.L.G.-R., A.d.J.P.-G., J.F.L.-R., P.A.-B., J.C.B.-G., M.d.C.J.-V., F.D.,
C.C.-Á., M.A.R.-M. and A.H.-F.; writing—review and editing J.R.Z.-M., A.J.A.-C., M.L.G.-R. and
J.C.B.-G.; supervision, J.R.Z.-M. and A.J.A.-C.; project administration, J.R.Z.-M. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance with the
NIH Guide
for Treatment and Care for Laboratory Animals [
28
] and the Official Mexican Norm
NOM 062-ZOO-1999
(technical instructions for the production, care, and use of laboratory animals).
The protocol for the use of animals was approved by the Research Bioethics Committee of the
University of Guanajuato (CIBIUG-P30-2019). The animals were sacrificed in a CO2chamber.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained in this manuscript.
Acknowledgments: This work was partially supported by the Investigadores Jovenes program from
SICES 90/2016 (provided to AJAC) and the Consejo Nacional de Ciencia y Tecnologia (CONACyT;
Ciencia Basica-CB-2015-01-257872; provided to JRZM). We thank M. E. Labrada-García for the
initial biological assays with Tramadol. M.L.G.R (CVU 705887), M.C.J.V (CVU 545284), and J.C.B.G
(CVU 489981) received a postdoctoral fellow from CONACYT.
Conflicts of Interest: The authors declare no conflicts of interest.
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