Anti-inflammatory and Antinociceptive Activities of Aqueous and Ethanolic Extracts from Cereus jamacaru DC. (Cactaceae)

Abstract

Cereus jamacaru DC. (Cactaceae) is used in folk medicine for treating pain and inflammation. However, the therapeutic potential of C. jamacaru extracts has not been assessed experimentally. This study aimed to characterize the effect of both aqueous (CAECJ) and ethanolic (CEECJ) extracts of C. jamacaru in mouse models of pain and inflammation. CAECJ and CEECJ were chemically characterized by qualitative methods. Toxicity of extracts was evaluated in vitro against J774.A1 macrophages and in vivo by monitoring mice for 14 days following a single oral treatment at 2000 mg/kg. The antinociceptive activity of the extracts was assessed in models of pain in mice: acetic acid-induced writhing test, formalin test, hot plate test, and glutamate-induced nociception assay. The anti-inflammatory activity of the extracts was assessed following zymosan-induced peritonitis and in the model of arthritis induced by Complete Freund’s Adjuvant (CFA). Phytochemical analyses revealed alkaloids, condensed tannins, flavonoids, and anthraquinones in both extracts; saponins were present in CEECJ only. Neither extract was cytotoxic in vitro or induced toxicity in mice. Both extracts reduced nociception in all models of nociception without impairing motor function. Both extracts reduced leucocyte migration during experimental peritonitis and reduced paw edema during experimental arthritis. The effects of the extracts can be both due to anti-inflammatory and centrally-mediated mechanisms. This paper corroborates the traditional use of C. jamacaru extracts and demonstrates their therapeutic potential in the development of new analgesic and anti-inflammatory drugs with a good safety profile.

Keywords:
pain; inflammation; antinociceptive; anti-inflammatory; cladodes.

GRAPHICAL ABSTRACT

HIGHLIGHTS

C. jamacaru extracts contain alkaloids, tannins, flavonoids, anthraquinones, and saponins.

C. jamacaru extracts promote antinociceptive and anti-inflammatory effects in mice.

C. jamacaru extracts are not toxic in vitro or in vivo.

INTRODUCTION

Current pharmacological strategies used in pain management include non-steroidal anti-inflammatory drugs (NSAID) and opioid analgesics. NSAID, along with steroidal anti-inflammatories, are also used in the control of inflammatory conditions. Despite their therapeutic value, these drugs can induce toxicity and well-known side effects that can limit their clinical use [¹,²]. Because of these disadvantages, novel anti-inflammatory and analgesic compounds are being actively searched for. Many plant-based products are reported to have medicinal properties with few adverse effects [³]. The pharmacological study of medicinal plants greatly contributes to the dissemination of therapies based on natural products, especially those investigations that support folk medicine and the efficient use of natural resources aiming sustainable consumption [⁴-⁷].

Cereus jamacaru DC. (Cactaceae) is an herbaceous plant native to Brazil, where it is known as “mandacaru”. It occurs mainly in the Northeastern region of the country and composes the vegetation of the Caatinga biome [⁸]. In folk medicine, C. jamacaru is widely used in the form of tea, juice, and elixir for treating backache, inflammatory conditions, and infections [⁹-¹¹]. The major secondary metabolites described for C. jamacaru include tannins, alkaloids, anthraquinones, flavonoids, and polyphenols [¹²-¹⁴]. Some compounds that are known to promote anti-inflammatory and antinociceptive effects have been identified in C. jamacaru, including the polyphenols ellagic acid and caffeic acid [¹⁵-¹⁹] as well as the alkaloid hordenine [²⁰,²¹]. Moreover, the fact that this cactus species is widely consumed by wild animals suggests its low toxicity and safety [¹²,²⁰,²²].

Despite its traditional use in the treatment of inflammatory and painful conditions and the phytochemical data suggesting its analgesic potential, no studies have explored the antinociceptive and anti-inflammatory properties of C. jamacaru. Hence, the goal of this study was to characterize the effect of both aqueous and ethanolic extracts of C. jamacaru in mouse models of pain and inflammation.

MATERIAL AND METHODS

Plant material

C. jamacaru DC. cladodes were collected in Cachoeirinha (PE, Brazil; 08° 29' 11" S, 36° 13' 59" W) in July 2018 and correctly identified by Dr. Flávia de Barros Prado Moura. A plant specimen was deposited in the Arapiraca Herbarium of the Federal University of Alagoas, under the register code ARA000077.

Preparation of the extracts

Aqueous and ethanolic extracts of C. jamacaru simulate traditional preparations used in folk medicine: juice and elixir, respectively. Cladode samples were weighed, cleaned, and grinded. To prepare the crude aqueous extract of C. jamacaru (CAECJ), half of the plant material was submitted to turbolysis following the proportion of 20 g of plant to 100 mL of distilled water. The extract was then filtered and stored in a refrigerator (2 to 8°C). To prepare the crude ethanolic extract of C. jamacaru (CEECJ), the other half of the plant material was dried at room temperature for 10 days. Next, it was submitted to maceration for 7 days following the proportion of 10 g of plant material to 100 mL of ethanol PA. The mixture was stirred twice a day. In the last day, the extract was filtered and the excess of solvent was removed with a rotatory evaporator. The extract was then placed in a desiccator for total removal of the solvent and stored at room temperature. For in vivo assays, doses of extracts are represented in milligrams of extract per kilogram of body weight. For CAECJ, doses were calculated based on the weight of raw plant material and dilutions were made in spring water. For CEECJ, doses were calculates based on the weight of the dry extract and dilutions were made in 5% Tween 80 in 0.9% saline.

Phytochemical screening of C. jamacaru extracts

CAECJ and CEECJ were screened for the presence of alkaloids, tannins, flavonoids, saponins, and anthraquinones as previously described [²³]. The procedures described in the following subsections were performed for both extracts, independently.

Alkaloids

1 g of extract was added to 2 mL of HCl (0.1 M), heated for 10 min, and allowed to cool to room temperature. The solution was filtered and equally divided into 3 tubes. A few drops of Dragendorff reagent were added to each tube. The formation of precipitate was considered a positive result for the presence of alkaloids.

Tannins

1 g of extract was diluted in 2 mL of methanol followed by the addition of 5 mL of distilled water. The solution was filtered and a few drops of ferric chloride (10% FeCl₃ in water) were added. Hydrolyzed tannins are detected by the solution turning blue, while condensed tannins are detected by the solution turning green.

Flavonoids

1 g of extract was diluted in 2 mL of a methanol-water solution (1:1). Magnesium chips were added to the solution, followed by the addition of concentrated hydrochloric acid. The presence of flavonoids was detected by the presence of red or light brown hues.

Saponins

1 g of extract was diluted in 2 mL of methanol. Then, 5 mL of boiling water was added. After cooled to room temperature, the tube was vigorously shaken for 15 s and then allowed to stand for 20 min. The presence of saponins was detected by the formation of stable foam.

Anthraquinones

1 g of extract was diluted in 2 mL of methanol. The solution was filtered and 2 mL of sulfuric acid was added. The solution was kept in a warm water bath for 1 min. After cooled to room temperature, a double liquid-liquid extraction was performed with 10 mL ethyl acetate. Reduced anthraquinones were detected by the presence of a yellow hue, while oxidized anthraquinones were detected by the presence of a red hue.

Drugs and reagents

The following substances were used: distilled water (Farmace, Brazil), ethanol (Sigma-Aldrich, USA), polyoxyethylenesorbitan monoleate (Tween 80®, Sigma-Aldrich, USA), acetic acid A.R. (Vetec, Brazil), glutamate (Sigma-Aldrich, USA), formaldehyde A.R (Vetec, Brazil), zymosan (Sigma-Aldrich, USA), complete Freund's adjuvant (Sigma-Aldrich, USA), dipyrone (Sigma-Aldrich, USA), indomethacin (Merck, Brazil), morphine (Cristália, Brazil), diazepam (Cristália, Brazil), dexamethasone (Fagron, Brazil), [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium] (MTT, Sigma-Aldrich, USA), Roswell Park Memorial Institute medium (RPMI, Sigma-Aldrich, USA), fetal bovine serum (Cutilab, Brazil), non-essential pyruvate (Sigma-Aldrich, USA) and glutamine (Sigma-Aldrich, USA) amino acids, dimethylsulfoxide (DMSO, Sigma-Aldrich, USA), and Triton™ X-100 (Neon, Brazil).

Cytotoxicity assay

The cytotoxicity of the extracts was determined on murine macrophages (J774.A1 cell line) obtained from the Cell Bank of Federal University of Rio de Janeiro (RJ, Brazil). This adherent-phenotype macrophage line was cultured in RPMI medium supplemented with fetal 10% bovine serum, pyruvate, glutamine and non-essential amino acids, at 37 °C, 95% humidity, and 5% CO₂ atmosphere. Briefly, cell suspensions containing 1.0 x 105 cells/mL were placed in a 96-well plate in triplicate and incubated at 37 °C for 1 h. Then, CAECJ and CEECJ were added at concentrations of 0.1, 1, 10, and 100 µg/mL. The cells were also cultured with medium only (basal growth control) or with 0.1% DMSO (vehicle control). 1% Triton™ X-100 was used as positive control to induce cell death. After 48 h, the cytotoxicity was evaluated by mitochondrial activity of the cells via MTT reduction and the absorbance was measured in a spectrophotometer at 530 nm. Results were expressed as percentual cell viability in relation to basal growth control [²⁴].

Animals

Swiss mice of both sexes, 6-8 weeks of age, weighing 25-30 g, were obtained from the Animal Facilities of the Federal University of Alagoas (AL, Brazil). They were housed in single-sex cages under a 12 h light/dark cycle (dark phase: 7 pm to 7 am), at constant temperature (22 ± 2°C) with water and food ad libitum. The experiments were approved by the Ethics Committee for Animal Handling - UFAL (Protocol # 27/2018) and performed in accordance with the guidelines established by the Guide for the Care and Use of Laboratory Animals [²⁵].

Acute toxicity

Acute oral toxicity was evaluated in mice (n = 6 per group) according to the Guidelines for Testing Chemicals n° 420 of the Organization for Economic Co-operation and Development (OECD) [²⁶]. After acclimatization procedure, mice were orally treated with vehicle (control), CEECJ, and CAECJ at limit dose (2000 mg/kg). Then, mice were evaluated for signs of toxicity, according to parameters described by Malone [²⁷] every hour for the first 4 h and then daily for 14 days. Food and water intake, weight gain, and incidence of death were also verified daily.

Antinociceptive activity

Acetic acid-induced abdominal writhing test

This model was used as a screening assay to detect the antinociceptive effects of CAECJ and CEECJ. Dose-response curves were built to determine the doses to be used in the following tests. Abdominal writhing in mice was induced by intraperitoneal injection of acetic acid (0.6% solution, 0.1 mL/10 g of body weight). Mice (n = 6 per group) were orally treated with CAECJ (100, 150, and 300 mg/kg), CEECJ (100, 150, and 300 mg/kg), dipyrone (40 mg/kg), and vehicle (10 mL/kg) 40 minutes before intraperitoneal injections. Nociceptive responses were determined for 20 minutes after a latency period of 5 minutes. The observed behaviors included stretching of the hind limbs, contraction of abdominal muscles, and arching of the back [²⁸,²⁹].

Formalin-induced nociception

Mice (n = 6 per group) were orally treated with CAECJ (150 mg/kg), CEECJ (150 mg/kg), indomethacin (30 mg/kg), and vehicle (10 mL/kg), or intraperitoneally treated with morphine (5 mg/kg) 40 minutes before intraplantar injection of 2.5% formalin (20 μL) into the right hind paw. The nociceptive response was considered as the time that mice spent licking the injected paw. Following the intraplantar injection, the neurogenic phase (0 - 5 min) and the inflammatory phase (15 - 30 min) were evaluated [³⁰].

Hot plate test

The hot plate test was performed as previously described [³¹]. Mice (n = 6 per group) were orally treated with CAECJ (150 mg/kg), CEECJ (150 mg/kg), and vehicle (10 mL/kg), or intraperitoneally treated with morphine (5 mg/kg). Mice were placed on a hot plate at 54 ± 1°C. The reaction time until mice licked either fore paws or hind paws and/or jumped was recorded at 30, 60, 90, and 120 minutes. The cut-off time used to prevent skin damage was 15 seconds.

Glutamate-induced nociception assay

Mice (n = 6 per group) were orally treated with CAECJ (150 mg/kg), CEECJ (150 mg/kg), indomethacin (30 mg/kg), and vehicle (10 mL/kg), or intraperitoneally treated with morphine (5 mg/kg) 40 minutes before subcutaneous injection of glutamate (30 μmol/paw in 20 μL) in the dorsal surface of the hind paw. Nociceptive responses were recorded as the time mice spent licking the injected paw during the 15 minutes that followed glutamate administration [³²].

Motor performance in the rotarod test

Considering that the behaviors evaluated in the nociceptive tests depend on the integrity of mice’s motor function, the rotarod test was performed to exclude possible non-specific depressive or myorelaxant effects of CAECJ and CEECJ. The test consisted in placing each mouse on a rotating bar (16 rpm), 2.5 cm in diameter, raised 25 cm above ground. Mice need to keep moving forward to avoid falling from the bar. Mice were previously selected by being challenged to remain on the rotating bar for 240 seconds with a limit of up to three falls. On the following day, mice (n = 6 per group) were orally treated with vehicle (10 mL/kg), CAECJ (150 mg/kg), and CEECJ (150 mg/kg), or intraperitoneally treated with diazepam (5 mg/kg). After 40 min (for oral treatments) or 30 min (for the intraperitoneal treatment), each mouse was placed on the rotarod and the number of falls as well as fall latency time were evaluated [³³].

Anti-inflammatory activity

Zymosan-induced peritonitis

The model of peritonitis [³⁴] was used to evaluate the acute anti-inflammatory potential of CAECJ and CEECJ. Dose-response curves were built to determine the doses to be used in the next test. Mice were orally pretreated with CAECJ (100, 150, and 300 mg/kg), CEECJ (100, 150, and 300 mg/kg), indomethacin (30 mg/kg), or vehicle (10 mL/kg). After 40 minutes, leukocyte migration was induced by intraperitoneal injection of zymosan (2 mg/mL, 250 µL/mouse). Six hours after zymosan injection, mice were euthanized and the peritoneal cavity was washed with cold PBS (3 mL), from which the exudate was retrieved. Naïve mice did not receive any treatment or zymosan injection prior to peritoneal washing. The number of recruited cells to the peritoneum was counted in a Neubauer chamber and the results were obtained using Equation 1:

(1) $$numberofcells/mL=\frac{totalcellcount\text{*}dilutionfactor\text{*}10000}{numberofquadrantscounted}$$

Arthritis induced by Complete Freund's Adjuvant (CFA)

Experimental arthritis was induced in mice as previously described [³⁵]. CFA (20 µL/paw) was injected into the right hind paw of mice. After 13 days, mice with pronounced arthritis were orally treated with CAECJ (150 mg/kg), CEECJ (150 mg/kg), dexamethasone (1 mg/kg), or vehicle (10 mL/kg) daily from the 14th to the 21st days after CFA injection. Naïve mice did not receive any treatments or CFA injections. Paw volume was measured on the 1st day followed by a daily assessment from the 14th to the 21st day. Paw edema was expressed by the mean difference between the right (arthritic) and left (without arthritis) paws using a digital plethysmometer (LE 7500; Panlab, Harvard Apparatus).

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM) and the statistical differences among groups were determined using one-way Analysis of Variance (ANOVA) followed by Dunnett’s post-hoc test. For repeated measures, two-way ANOVA followed by Bonferroni’s post-hoc test. Results were considered statistically significant when p < 0.05. Statistical analysis and creation of figures were performed using the software GraphPad Prism 8.0.

RESULTS

C. jamacaru extracts contain several classes of secondary metabolites

Qualitative screening of secondary metabolites showed the presence of alkaloids, condensed tannins, flavonoids, and anthraquinones in both CAECJ and CEECJ (data not shown). On the other hand, saponins were present in CEECJ, but not in CAECJ (data not shown).

Viability of murine macrophages is not affected by C. jamacaru extracts in vitro

Cell viability of J774.A1 murine macrophages (Figure 1) was not affected by incubation with CAECJ (0.1 - 100 μg/mL), CEECJ (0.1 - 100 μg/mL), or vehicle (0.1% DMSO). Cell viability was significantly lower in positive control wells treated with 1% Triton X-100 (p < 0.05). Since the extracts did not induce cytotoxicity at any of the tested concentrations, the study continued with in vivo assays.

Figure 1
Influence of C. jamacaru extracts on murine macrophages viability. J774.A1 macrophages were incubated with vehicle (VEH; 0.1% DMSO), crude aqueous extract of C. jamacaru (CAECJ; 0.1, 1, 10, and 100 µg/mL), crude ethanolic extract of C. jamacaru (CEECJ; 0.1, 1, 10, and 100 µg/mL), or 1% Triton X-100 (positive control; CTRL) for 48 hours. Cell viability in relation to basal growth control (axis of abscissa) was evaluated using the MTT assay. Results are expressed as mean ± SEM (n = 3). *** p < 0.001 compared to VEH as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

C. jamacaru extracts do not induce acute oral toxicity in mice

Oral administration of CAECJ (2000 mg/kg) and CEECJ (2000 mg/kg) did not induce visible manifestations of acute toxicity in mice during the two-week period of daily assessments. During the evaluations, no instances of drowsiness, salivation, tremor, restlessness, convulsion, piloerection, diarrhea, or death were registered (data not shown).

Acetic acid-induced nociception is partially prevented by C. jamacaru extracts

Intraperitoneal injection of 0.6% acetic acid induced nociceptive behaviors marked by lower body stretching and contraction of abdominal muscles (Figure 2). Oral treatments with CAECJ significantly reduced acetic acid-induced nociception at 150 mg/kg (53.4% reduction; p < 0.001) and at 300 mg/kg (50.6% reduction; p < 0.001) compared to vehicle-treated mice (40.6 ± 4.0 writhes). Similarly, oral treatments with CEECJ reduced acetic acid-induced nociception at 100 mg/kg (36.9% reduction; p < 0.05) and at 150 mg/kg (57% reduction; p < 0.001). The efficacy of the extracts was similar to that of the standard analgesic drug dipyrone (40 mg/kg), which reduced the nociceptive behaviors by 51.5% (p < 0.001). Conversely, CAECJ at 100 mg/kg and CEECJ at 300 mg/kg did not influence the nociceptive effect of acetic acid. Thus, the following assays were conducted using only the dose of 150 mg/kg for both extracts.

Figure 2
Influence of C. jamacaru extracts on acetic acid-induced nociception. Mice were orally treated with vehicle (VEH), dipyrone (DIP; 40 mg/kg), crude aqueous extract of C. jamacaru (CAECJ; 10, 150, and 300 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 10, 150, and 300 mg/kg). After 40 minutes, mice received intraperitoneal injections of 0.6% acetic acid and the nociceptive behaviors (axis of abscissa) were quantified for 20 minutes. Results are expressed as mean ± SEM (n = 6). * p < 0.05; *** p < 0.001 compared to VEH group as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

C. jamacaru extracts reduce both phases of the formalin test

Intraplantar injection of 2.5% formalin caused nociception, as shown by intense licking behavior in vehicle-treated mice in the first (85.4 ± 6.6 s) and second phases (265.0 ± 21.9 s) of the test (Figure 3). Both CAECJ (150 mg/kg) and CEECJ (150 mg/kg) significantly reduced the pain-like licking behaviors of mice in both phases of the formalin test. In the first phase, CAECJ and CEECJ reduced the licking time by 43% and 42.9%, respectively, in comparison to vehicle-treated mice (p < 0.01; Figure 3A). In the second phase, CAECJ and CEECJ reduced the licking time by 32.7% and 46.2%, respectively (p < 0.01; Figure 3B). Additionally, the reference opioid drug morphine (5 mg/kg) reduced the licking time in both phases (62.2% and 59%, respectively; p < 0.001). On the other hand, the reference anti-inflammatory drug indomethacin (30 mg/kg) reduced the licking time only in the second phase of the test (77.4%; p < 0.001).

Figure 3
Effects of C. jamacaru extracts in the formalin test. Mice were orally treated with vehicle (VEH), indomethacin (INDO; 30 mg/kg), crude aqueous extract of C. jamacaru (CAECJ; 150 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 150 mg/kg), or intraperitoneally treated with morphine (MOR; 5 mg/kg) 40 minutes before intraplantar injection of 2.5% formalin into the right hind paw. Time mice spent licking the injected paw (axis of abscissa) was quantified in the neurogenic phase (Panel A; 0 - 5 min) and in the inflammatory phase (Panel B; 15 - 30 min). Results are expressed as mean ± SEM (n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001 compared to VEH group as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

C. jamacaru extracts promote antinociception in the hot plate test

Vehicle-treated mice showed a stable latency time until reaction to the hot plate throughout the experimental period (3.16 ± 0.2 s), with no significant differences between time points (Figure 4). CAECJ (150 mg/kg) significantly increased latency time when compared to the vehicle group at 60 (p < 0.01), 90 (p < 0.01), and 120 minutes (p < 0.05). On the other hand, CEECJ (150 mg/kg) increased latency time only at 90 (p < 0.001) and 120 minutes (p < 0.01). As expected, the reference opioid drug morphine (5 mg/kg) also significantly prolonged latency time to response the thermal stimulus at 60 (p < 0.001), 90 (p < 0.001), and 120 minutes (p < 0.001).

Figure 4
Effects of C. jamacaru extracts in the hot plate test. Mice were orally treated with vehicle (VEH), crude aqueous extract of C. jamacaru (CAECJ; 150 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 150 mg/kg), or intraperitoneally treated with morphine (MOR; 5 mg/kg). Mice were placed on the hot plate at 54 ± 1°C and the latency time until reaction (axis of abscissa) was recorded at 30, 60, 90, and 120 minutes following treatments. Results are expressed as mean ± SEM (n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001 compared to VEH group as determined by two-way ANOVA followed by Bonferroni’s post-hoc test.

Glutamate-induced central nociception is reduced by C. jamacaru extracts

Because the results of the neurogenic phase of the formalin test and the hot plate test suggested that C. jamacaru extracts promote centrally-mediated antinociception, CAECJ (150 mg/kg) and CEECJ (150 mg/kg) were then tested against glutamate-induced nociception (Figure 5). Vehicle-treated mice showed intense pain-like paw licking behaviors (131.0 ± 12.8 s) that were significantly reduced by oral treatments with CAECJ (60.4%; p < 0.001) and CEECJ (47.2%; p < 0.01) or by intraperitoneal treatment with the reference opioid drug morphine (5 mg/kg) (93.4%; p < 0.001).

Figure 5
Influence of C. jamacaru extracts on glutamate-induced nociception. Mice were orally treated with vehicle (VEH), crude aqueous extract of C. jamacaru (CAECJ; 150 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 150 mg/kg), or intraperitoneally treated with morphine (MOR; 5 mg/kg) 40 minutes before subcutaneous injection of glutamate (30 μmol/paw in 20 μL) in the dorsal paw surface. Time mice spent licking the injected paw (axis of abscissa) was quantified for 15 minutes. Results are expressed as mean ± SEM (n = 6). ** p < 0.01; *** p < 0.001 compared to VEH group as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

Motor function of mice is not impaired by C. jamacaru extracts

Since the experiments aiming to evaluate nociception rely on the integrity of mice’s motor capacities, impairment of motor function was assessed in the rotarod test. As shown in Table 1, oral treatments with CAECJ (150 mg/kg) and CEECJ (150 mg/kg) did not affect the number of falls nor fall latency in comparison with vehicle-treated mice. On the other hand, intraperitoneal treatment with the anxiolytic-sedative drug diazepam (5 mg/kg) significantly increased the number of falls (p < 0.001) and decreased fall latency (p < 0.001) when compared to vehicle-treated mice.

Table 1
Influence of C. jamacaru extracts on mice’s motor function. Mice were orally treated with vehicle (VEH), crude aqueous extract of C. jamacaru (CAECJ; 150 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 150 mg/kg), or intraperitoneally treated with diazepam (DZP; 5 mg/kg) and then challenged to remain on the rotating rod. Results are expressed as mean ± SEM (n = 6). ^*** p < 0.001 compared to VEH group as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

Treatment	Number of falls	Fall latency (s)
VEH	0.0 ± 0.0	240 ± 0.0
CAECJ	0.0 ± 0.0	240 ± 0.0
CEECJ	0.0 ± 0.0	240 ± 0.0
DZP	11.40 ± 1.89***	23.27 ± 5.22***

C. jamacaru extracts reduce leucocyte migration induced by experimental peritonitis

Vehicle-treated mice induced with zymosan showed a significant increase in the number of cells present in peritoneal washing compared to the Naïve group (p < 0.001), confirming the development of experimental peritonitis (Figure 6). CAECJ significantly inhibited cell recruitment at 100 mg/kg (61.2%; p < 0.01), 150 mg/kg (66.6%; p < 0.01), and 300 mg/kg (66%; p < 0.01) compared to vehicle-treated mice. CEECJ also inhibited zymosan-induced leukocyte migration at 100 mg/kg (73.5%; p < 0.001), 150 mg/kg (59.5%; p < 0.01), and 300 mg/kg (47.1%; p < 0.05). The reference anti-inflammatory drug indomethacin (30 mg/kg) significantly decreased cell recruitment by 46% (p < 0.05).

Figure 6
Influence of C. jamacaru extracts on leucocyte migration during zymosan-induced experimental peritonitis. Mice were orally pretreated with vehicle (VEH), indomethacin (INDO; 30 mg/kg) crude aqueous extract of C. jamacaru (CAECJ; 100, 150, and 300 mg/kg), and crude ethanolic extract of C. jamacaru (CEECJ; 100, 150, and 300 mg/kg) and then intraperitoneally injected with zymosan (500 µg) after 40 minutes. Naïve mice did not receive any pretreatments or zymosan injection. After 6 hours, the peritoneal cavity was washed with cold PBS (3 mL) and leukocyte migration was determined by counting the number of cells (axis of abscissa). Results are expressed as mean ± SEM (n = 6). ### p < 0.001 compared to Naïve group and * p < 0.05; ** p < 0.01; *** p < 0.001 compared to VEH group as determined by one-way ANOVA followed by Dunnett’s post-hoc test.

C. jamacaru extracts promote anti-inflammatory effects during experimental arthritis

The results gathered so far were used for selecting the dose of C. jamacaru extracts to be tested in the CFA model of arthritis. Since this model has an important inflammatory component while also causing hypernociception, we selected a dose that promoted both anti-inflammatory and antinociceptive activities. Thus, the effect of oral daily treatments with CAECJ (150 mg/kg) and CEECJ (150 mg/kg) was tested in this model of chronic inflammation (Figure 7). Intraplantar injections of CFA triggered an inflammatory response marked by local edema, as shown by the significant increase in paw volume of vehicle-treated mice compared to the Naïve group (p < 0.001). Paw edema was persistent throughout the entire experimental period (1st and then 14th to 21st day after CFA injection). Both CAECJ (150 mg/kg) and CEECJ (150 mg/kg) as well as the reference drug dexamethasone (1 mg/kg) significantly reduced paw edema from the 15th to the 21st day following the induction of the model (p < 0.05).

Figure 7
Antiedematogenic effect of C. jamacaru extracts in the CFA model of arthritis. Experimental arthritis was induced by intraplantar injection of CFA (20 µL). From the 14th to the 21st day following CFA injections, mice were orally treated with CAECJ (150 mg/kg), CEECJ (150 mg/kg), dexamethasone (1 mg/kg), or vehicle (10 mL/kg) daily. Naïve mice did not receive any treatments or CFA injections. Paw volume was measured daily and edema was expressed as variation in paw volume (axis of abscissa) between the right (arthritic) and left (without arthritis) paws. Results are expressed as mean ± SEM (n = 6). ### p < 0.001 compared to Naïve group and * p < 0.05; ** p < 0.01; *** p < 0.001 compared to VEH group as determined by two-way ANOVA followed by Bonferroni’s post-hoc test.

DISCUSSION

This is the first experimental study to demonstrate the antinociceptive and anti-inflammatory activities of both aqueous and ethanolic extracts of Cereus jamacaru DC. in murine models of pain and inflammation. Our research corroborates the traditional use of this plant for treating painful and inflammatory conditions, a common practice in the Northeastern Brazilian folk medicine [⁹,¹⁰]. In addition to confirming the therapeutic potential of CAECJ and CEECJ, this paper provided evidence that supports the safety of C. jamacaru extracts, as neither induced noticeable toxic effects in mice nor were they cytotoxic for murine macrophages in vitro.

Prior to pharmacological characterization in models of pain and inflammation, CAECJ and CEECJ were screened for safety both in vitro and in vivo. The extracts were not cytotoxic for J774.A1 murine macrophages, which agrees with a previous in vitro study showing that the hydroalcoholic extract from C. jamacaru is not cytotoxic against lymphocytes [³⁶]. We then proceeded to perform an assay for detecting acute oral toxicity in mice; neither of the extracts at the dose of 2000 mg/kg induced signs of systemic toxicity. Treatments did not impair the general behavior or mobility of mice, nor did they alter food and water intake or body weight during the 14 days of evaluation (data not shown). No deaths were registered during the 14 days following oral treatments. According to the guidelines established by OECD [²⁶], we concluded that both CAECJ and CEECJ can be considered low toxicity xenobiotics, so their pharmacological characterization could continue.

The antinociceptive and anti-inflammatory activities of CAECJ and CEECJ were assessed by using classical animal models of pain and inflammation often employed in the process of drug discovery [³⁷]. The maximum dose used during the pharmacological characterization was 300 mg/kg for both extracts, which corresponds to 15% of the limit dose tested for acute toxicity (2000 mg/kg). The antinociceptive effect of the extracts was initially tested in the acetic acid-induced abdominal writhing assay, which has been largely used as a screening test for detecting the analgesic potential of new compounds [²⁹]. The intraperitoneal injection of acetic acid leads to the release of endogenous mediators that promote peripheral sensitization and nociception in mice [³⁸]. These mediators include biogenic amines (e.g., histamine and serotonin) and prostaglandins as a result of the induction of cyclooxygenase (COX) enzymes [²⁸,³⁸]. Both CAECJ and CEECJ reduced the abdominal writhes induced by acetic acid; the antinociceptive effect of the extracts could be due to the inhibition of the aforementioned nociceptive mediators.

In the writhing test, the maximum effective dose for both extracts was 150 mg/kg. Therefore, following the ethical procedures established by OECD [²⁶], this dose was selected and used in all the subsequent nociceptive assays. Interestingly, CEECJ at 300 mg/kg did not promote antinociception in the writhing test, demonstrating that this effect was not dose-dependent. This apparent inconsistency can be explained by the fact that plant extracts are complex mixtures containing different compounds that can interfere with each other’s pharmacological actions [³⁹]. In fact, it is not uncommon that studies that characterize crude plant extracts report similar results, as demonstrated for Allium macrostemon (Alliaceae) [⁴⁰] and Heteropterys brachiata (Malpighiaceae) [⁴¹].

To further characterize the antinociceptive activity of CAECJ and CEECJ, we performed other behavioral assays that can provide insights into the antinociceptive mechanisms of the extracts. Formalin-induced nociception is known to be driven by a biphasic mechanism; the neurogenic phase is caused by the direct activation of nociceptors and can be inhibited by opioid drugs, whereas the inflammatory phase results from the release of inflammatory mediators and is inhibited by anti-inflammatory drugs such as NSAID [⁴²,⁴³]. The extracts of C. jamacaru reduced the pain-like licking behavior of mice in both phases of the test, suggesting that CAECJ and CEECJ have the potential to promote both centrally-mediated antinociception and anti-inflammatory effects. Therefore, both actions were investigated in the following steps of this study.

Two assays for evaluating centrally-mediated antinociception were performed: the hot plate test, a thermal pain model that requires a complex and integrated response that involves supraspinal regions [³¹]; and glutamate-induced nociception, that involves the activation of NMDA and non-NMDA receptors in the central nervous system and depends on the activation of the L-arginine-nitric oxide pathway [³²]. Both CAECJ and CEECJ increased the latency time of mice on the hot plate and reduced the pain-like licking behavior caused by intraplantar glutamate injections, supporting the hypothesis that the extracts promote antinociception by central mechanisms. Centrally-mediated antinociception has been reported for other species of Cactaceae, including Opuntia dillenii [⁴⁴] and Pereskia bleo [⁴⁵]. Importantly, neither CAECJ nor CEECJ caused motor impairment in mice in the rotarod test. These results validate the antinociceptive effect of C. jamacaru extracts, since they indicate that changes in behavior caused by CAECJ and CEECJ in the nociceptive tests were not due to non-specific depressant or muscle relaxing effects.

Considering that the results of the formalin test indicated that C. jamacaru extracts effectively reduced inflammatory nociception, we then tested the effects of CAECJ and CEECJ in murine models of inflammation. To investigate whether the extracts modulate the inflammatory response, CAECJ and CEECJ were tested against zymosan-induced leukocyte migration. Intraperitoneal injection of zymosan causes an inflammatory reaction marked by leukocyte influx and increased expression of the inducible nitric oxide synthase (iNOS) and COX-2, with consequent release of nitric oxide and prostaglandins [⁴⁶]. Both C. jamacaru extracts reduced cell migration induced by zymosan in the peritoneal cavity, corroborating their anti-inflammatory potential. Similarly, it has been demonstrated that the juice of Opuntia ficus-indica (Cactaceae) reduces the release of pro-inflammatory mediators and, consequently, reduces mucosa damage during intestinal inflammation [⁴⁷].

The anti-inflammatory action of CAECJ and CEECJ was also tested in the mouse model of arthritis induced by CFA. Rheumatoid arthritis is a chronic autoimmune disease characterized by persistent inflammation of the joints accompanied by leucocyte infiltration, increased levels of cytokines, chemokines, and C-reactive protein, as well as intense pain [⁴⁸,⁴⁹]. The pharmacological treatment of rheumatoid arthritis is based on the use of anti-inflammatory and immunosuppressive drugs that cause deleterious side effects [⁵⁰], which justifies the need for new treatments that are safe and effective. Daily treatments with both CAECJ and CEECJ significantly reduced paw edema throughout the experimental period, demonstrating the potential of the extracts in the treatment of persistent joint inflammation. Accordingly, other authors have reported that extracts of Opuntia dillenii and Opuntia ficus-indica promote therapeutic effects in rodent models of arthritis [⁴⁴,⁵¹].

Although investigating the mechanisms of the antinociceptive and anti-inflammatory effects of C. jamacaru extracts was beyond the scope of this study, the phytochemical screening of CAECJ and CEECJ provided valuable clues about the chemical classes of compounds that could contribute to these effects. The presence of alkaloids, condensed tannins, flavonoids, and anthraquinones was detected in both extracts, whereas saponins were only found in CEECJ. The qualitative chemical characterization of the extracts used in our research agrees with previous studies that explored the composition of C. jamacaru extracts and reported the presence of similar chemical classes [¹²,¹³,⁵²,⁵³]. Several studies have demonstrated the antinociceptive and/or anti-inflammatory activity of alkaloids, anthraquinones, saponins, and polyphenols (especially tannins and flavonoids) [⁵⁴-⁵⁸]. Therefore, it is possible that these classes of compounds contribute to the antinociceptive and anti-inflammatory effects of CAECJ and CEECJ, either by acting on their own or synergically with other compounds.

CONCLUSION

This study provides pharmacological evidence that supports the traditional use of the cladodes of Cereus jamacaru in the treatment of inflammatory and painful conditions. We demonstrated that both the aqueous and the ethanolic extracts of C. jamacaru promote antinociception in murine models of pain; our results suggest that this effect is both due to anti-inflammatory and centrally-mediated mechanisms. Additionally, both CAECJ and CEECJ reduced signs of acute and chronic inflammation in mouse models. The extracts did not induce signs of toxicity, sedation, or motor impairment in mice. Therefore, this paper demonstrates that C. jamacaru extracts have therapeutic potential in the development of new analgesic and anti-inflammatory drugs with a good safety profile. Future studies should aim to elucidate the mechanisms of action and fractions of the extracts responsible for the antinociceptive and anti-inflammatory effects described herein.

Acknowledgments:

The authors thank their colleagues from Federal University of Alagoas for their constructive criticism and assistance during the development this project.

• Funding: This research was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant numbers 479822/2013-1, 485854/2011-2, 404344/2012-7; the Instituto Nacional de Ciência e Tecnologia de Fármacos e Medicamentos (INCT-INOFAR), grant number 573.564/2008-6; the Fundação de Amparo à Pesquisa do Estado de Alagoas (FAPEAL), grant number Pronem 20110722-006-0018-0010; and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) along with the Financiadora de Estudos e Projetos - Brasil (FINEP) with the joint grant of student fellowship.

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Editor-in-Chief: Paulo Vitor Farago

Associate Editor: Paulo Vitor Farago

Publication Dates

• Publication in this collection
  09 Sept 2024
• Date of issue
  2024

History

• Received
  11 Mar 2024
• Accepted
  21 Mar 2024