Antinociceptive effect of triterpene acetyl aleuritolic acid isolated from Croton zehntneri in adult zebrafish (Danio rerio)

https://doi.org/10.1016/j.bbrc.2020.11.056Get rights and content

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  • Triterpene acetyl aleuritolic acid showed no acute toxicity.

  • Triterpene presented analgesia without causing a sedative effect.

  • Triterpene as an inhibitor of corneal nociception involving the TRPV1 channel.

Abstract

Croton zehntneri is a plant known as canelinha de cunhã, prevalent in the northeast region of Brazil. Many constituents of the vegetable have already been studied, and their pharmacological properties have been proven, but this is the first study to analyze the antinociceptive effect in adult zebrafish (ZFa) of the triterpene acetyl aleuritolic acid (AAA) isolated from the stem bark. The animals (ZFa; n = 6/group) were treated intraperitoneally (ip; 20 μL) with AAA (0.1 or 0.3 or 1.0 mg/mL) or vehicle (0.9% saline; 20 μL), and submitted to the locomotor activity test, as well as 96 h acute toxicity. Other groups (n = 6/each) received the same treatments and underwent acute nociception tests (formalin, cinnamaldehyde, glutamate, acid saline, capsaicin, and hypertonic saline). Possible neuromodulation mechanisms were evaluated. AAA (0.1 or 0.3 or 1.0 mg/mL) reduced the nociceptive behavior induced by acid saline and capsaicin, as well as inhibited corneal nociception induced by hypertonic saline, both without altering the animals’ locomotor system and without toxicity. These analgesic effects of AAA were significantly (p > 0.05) similar to those of morphine, used as a positive control. The antinociceptive effect of AAA was inhibited by methylene blue, ketamine, camphor, ruthenium red, amiloride, and mefenamic acid. The antinociceptive effect of AAA on the cornea of animals was inhibited by capsazepine. Therefore, AAA showed pharmacological potential for the treatment of acute pain, and this effect is modulated by cGMP, NMDA receptors, transient receptor potential channels (TRPs), ASICs and has pharmacological potential for the treatment of corneal pain modulated by the TRPV1 channel.

Introduction

Pain is a physiological response generated after mechanical tissue damage and inflammation, which can be stimulated by generalized factors being the primary indicator of tissue damage [1]. In order for the painful stimulus to process, it is necessary for nociceptors to be activated by mechanical, physical, including thermal (temperature 34 to 43 °C) or chemical stimuli, through mediation by nociceptive agents such as: formalin, capsaicin, glutamate, acid saline, and solution hypertonic saline [2,3].

Tactile and painful sensations in the cornea are captured by innervations that arise from the first branch of the trigeminal nerve [4]. The debilitating discomfort of chronic corneal neuropathic pain has an immensely negative impact on patients’ quality of life, causing impaired functioning and inability to perform activities of daily living [5], which promotes the investigation of drugs that inhibit or sensitize endings corneal nerves in the transduction of nociceptive potential [6].

The antinociceptive effect of triterpenes in animal models has been reported [[7], [8], [9], [10], [11], [12]]. AAA is a triterpene of molecular formula C32H50O4 that was isolated from the stem bark of Croton zehntneri, a plant native to Northeast Brazil known as “canelinha de cunhã” [13]. AAA has been identified to have moderate cytotoxic effect [14], anti-inflammatory and analgesic effect in rodents [15].

Zebrafish (D. rerio) has the cellular components necessary to respond to various harmful stimuli [16], as it expresses genes involved in pain neuromodulation such as NMDA, TRPV1, TRPV2, TRPM3, TRPM8, TRPA1, and ASICs [[17], [18], [19], [20]]. Research has used zebrafish to mimic nociceptive behavior and thereby identify new biologically active compounds [2,7,21,22]. In this perspective and based on the reported data, the antinociceptive effect of AAA isolated from the bark of the stem of C. zehntneri and possible mechanisms of action in adult zebrafish (Danio rerio) were investigated.

Section snippets

Triterpene acetyl aleuritolic acid

The triterpene acetyl aleuritolic acid (Fig. 1) was extracted from the stem of C. zehtneri. The plant material was collected in March 2006, in Tianguá, Ceará, Brazil, identified and cataloged under no. 42,389 at Herbário Prisco Bezerra. The characterizations and vibrational properties of the triterpene were carried out by Melo et al. [13].

Drugs and reagents

The drugs and reagents used in the experiments were: formaldehyde, acetic acid (Dinâmica, Brazil), sodium chloride (NaCl; Synth), saline solution (0.9%;

96 h open field test and acute toxicity

No sedation and/or locomotor dysfunction was observed in animals treated with AAA or vehicle (0.9% saline; 20 μL; i.p.). The mobility of animals treated with AAA varied between 86.6 and 93%, with no difference between the Naive group and vehicle (p > 0.05). Animal mortality was also not recorded after 96 h (LC50 > 1.0 mg/mL).

Antinociceptive activity

The highest concentration of AAA [(1.0 mg/mL; 20 μL; i.p.) ∗p < 0.05 vs. control] inhibited neurogenic formalin-induced nociception and acid-saline-induced nociception, as

Discussion

Although the antinociceptive potential of AAA isolated from C. cajucara has been reported [15], it is believed that this is the first study to ascertain the analgesic capacity of AAA isolated from C. zehntneri against various nociceptive stimuli in ZFa. In this study, we performed nociceptive models induced by chemical products, and identified the antinociceptive potential of the triterpene AAA through the modulation of receptors in adult zebrafish. There was no toxicity or any change in the

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

We would also like to thank FUNCAP, CAPES, and CNPq for the support and scholarships.

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