Analgesic and Anti-Arthritic Potential of Methanolic Extract and Palmatine Obtained from Annona squamosa Leaves

Abstract

Background/Objectives: Annona squamosa is used in folk medicine to treat pain and arthritis. Palmatine is an alkaloid isolated from several plants, including A. squamosa leaves. The aim of the present study was to investigate the analgesic, anti-arthritic, and anti-inflammatory potential of the methanolic extract of A. squamosa (EMAS) and palmatine. Methods: The chemical profile of EMAS was evaluated by ultra high-performance liquid chromatography with electrospray ionization coupled to mass spectrometry (UHPLC-ESI/MS). EMAS and palmatine were evaluated in carrageenan-induced pleurisy, zymosan-induced joint inflammation, formalin-induced nociception, and tumor necrosis factor (TNF)-induced mechanical hyperalgesia in experimental models in mice. A cytotoxicity test of EMAS and palmatine was performed using a methylthiazolidiphenyl-tetrazolium (MTT) bromide assay. Results: The analysis of the chemical profile of the extract showed the presence of palmatine, liriodenine, and anonaine. Oral administration of EMAS and palmatine significantly reduced leukocyte migration and oxide nitric production in the carrageenan-induced pleurisy model. EMAS and palmatine reduced mechanical hyperalgesia, leukocyte migration, and edema formation in the joint inflammation induced by zymosan. In the formalin test, palmatine was effective against the second-phase nociceptive response, mechanical hyperalgesia, and cold allodynia. In addition, palmatine reduced mechanical hyperalgesia induced by TNF. EMAS and palmatine did not demonstrate cytotoxicity. Conclusions: The present study showed that A. squamosa and palmatine are analgesic and anti-inflammatory agents, and that the anti-hyperalgesic properties of palmatine may involve the TNF pathway. Palmatine may be one of the compounds responsible for the anti-hyperalgesic and/or anti-arthritic properties of this medicinal plant.

Keywords: Annona; alkaloids; anti-hyperalgesic; arthritis; efficiency; inflammation.

Figure 1

UHPLC-MS chromatograms. (A) From the methanolic extract of the leaves of A. squamosa (EMAS) with some identified alkaloids. (B) From the standard samples of alkaloids used as a standard.

Figure 2

Effects of oral administration of EMAS and palmatine on leukocyte migration (A,B) and nitric oxide production (C,D) in pleurisy model induced by carrageenan. Animals received, by gavage, EMAS (30, 100, 300 mg/kg), palmatine (0.3, 1, 3 mg/kg), vehicle (naïve and control groups), or prednisolone (PRED, 3 mg/kg), and, after 1 h, intrathoracic injection of carrageenan was given to groups, except naïve. Each bar represents mean ± SEM of 6 animals. Letters a, b and c indicate significant differences among groups according to Tukey’s multiple comparison test.

Figure 3

Effects of oral administration of EMAS and palmatine on mechanical hyperalgesia (A,B), knee edema (C,D), and on leukocyte migration (E) caused by zymosan-induced joint inflammation in female mice. Animals received, by gavage, EMAS (100 mg/kg), palmatine (1 mg/kg), saline solution (control group), or the reference drug prednisolone (PRED, 3 mg/kg) and, after 1 h, 200 μg of zymosan was injected into left knee. After induction of inflammation, mechanical hyperalgesia and edema were measured four (A,C) and six (B,D) hours later, respectively. After that, animals were euthanized and joint exudate was used to assess leukocyte migration (E). Each bar represents mean ± SEM of 6 animals. Letters a, b, and c indicate significant differences among groups according to Tukey’s multiple comparison test.

Figure 4

Effects of oral administration of EMAS and palmatine on formalin test. One hour before applying 30 μL of 1% formalin, animals were orally treated with EMAS (100 mg/kg), palmatine (PALM 1 mg/kg), or vehicle (control group). First phase represents neurogenic phase (A), while second phase is inflammatory phase (B). Paw withdrawal threshold (Von Frey test) (C) and cold sensitivity (D) were performed immediately after formalin test. Formalin-induced edema was measured one hour after formalin application (E). Each bar represents mean ± SEM of 5 animals. Letters a and b indicate significant differences between groups according to Tukey’s multiple comparison test.

Figure 5

The effects of the intraplantar administration of palmatine on TNF-induced mechanical hyperalgesia. One hour before the injection of 20 μL of TNF into the right hind paw, the animals were intraplantar treated with saline solution or palmatine (PALM 3 and 30 µg/paw) in the same region as the inducing agent. Three and four hours after the TNF injection, mechanical hyperalgesia (Von Frey test) was analyzed. In addition, the basal response to mechanical stimulus was performed before treating the animals. Each bar represents the mean ± SEM of 5 animals. The letters a and b indicate significant differences among groups according to Tukey’s multiple comparison test.