© 2021 Journal of Pharmacy & Pharmacognosy Research, 9 (5), 677-684, 2021 ISSN 0719-4250 http://jppres.com/jppres Original Article Antispasmodic activity of Warionia saharae Benthem ex Benth. & Coss. on the rabbit and rat jejunums [Actividad antiespasmódica de Warionia saharae Benthem ex Benth. & Coss. sobre yeyunos de conejo y rata] Ouafa Amrani1, Mohamed Marghich1, Hanane Makrane2, Chakib Alem3, Mohammed Aziz1* 1Laboratory of Bioressources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences, Mohamed the First University, Boulevard Mohamed VI; BP: 717; 60000, Oujda, Morocco. 2Laboratoire de Biologie, Environnement et Développement Durable, Ecole Normale supérieure, Martil, Université Abdelmalek Essaidi, Tétouan, Morocco. 3Laboratory of Biochemistry, Department of Biology, Faculty of Sciences & Techniques, University of Moulay Ismail, Errachidia, Morocco. *E-mail: [email protected]; [email protected] Abstract Resumen Context: Warionia saharae Benthem ex Benth. & Coss has been widely Contexto: Warionia saharae Benthem ex Benth. & Coss se ha utilizado used for gastrointestinal problems in Morocco and Algeria. ampliamente para problemas gastrointestinales en Marruecos y Argelia. Aims: To explore the possible mechanism for its use to such ailments in Objetivos: Explorar el posible mecanismo de su utilización ante tales order to rationalize some of these folkloric uses. We investigated the dolencias con el fin de racionalizar algunos de estos usos folclóricos. aqueous extract of this plant (AqWs) for antispasmodic activity on Investigamos el extracto acuoso de esta planta (AqWs) para determinar isolated rat and rabbit jejunums. la actividad antiespasmódica en yeyunos aislados de ratas y conejos. Methods: To investigate the contractile activity of isolated jejunum Métodos: Para investigar la actividad contráctil de preparaciones aisladas preparations of rat and rabbit we had used in vitro techniques. Jejunums de yeyuno de rata y conejo utilizamos técnicas in vitro. Los yeyunos se were suspended in tissue baths filled with KHB culture medium (37°C) suspendieron en baños de tejido llenos de medio de cultivo KHB (37°C) and connected to force transducers. AqWs (0.1–3 mg/mL) was used y se conectaron a transductores de fuerza. Se usó AqWs (0,1-3 mg/mL) with or without carbachol (10-6 M), KCl (25 mM), yohimbine (10-5 M), con o sin carbacol (10-6 M), KCl (25 mM), yohimbina (10-5 M), prazosina prazosin (10-5 M), propranolol (10-5 M), hexamethonium (10-4 M), (10-5 M), propranolol (10-5 M), hexametonio (10-4 M), atropina (10-6 M), atropine (10-6 M), methylene blue (10-5 M), L-NAME (10-4 M) and azul de metileno (10-5 M), L-NAME (10-4 M) y nifedipina (10-6 M). -6 nifedipine (10 M). Resultados: AqWs a 0,1-3 mg/mL produjeron una relajación en las Results: AqWs at 0.1–3 mg/mL produced a relaxation on basal rabbit contracciones basales del conejo con un IC50 = 1,55 ± 0,06 mg/mL. Este contractions with an IC50 = 1.55 ± 0.06 mg/mL. This effect was reversible efecto fue reversible y no se vio afectado por el pretratamiento con los and was not affected by pretreatment with the inhibitors of α and β inhibidores de los receptores adrenérgicos α y β yohimbina, prazosina y adrenergic receptors yohimbine, prazosin and propranolol. The extract propranolol. El extracto tuvo una actividad antiespasmódica con una had an antispasmodic activity with an IC50 = 1.25 ± 0.02 mg/mL on rat CI50 = 1,25 ± 0,02 mg/mL en yeyuno de rata precontraído con medio rico jejunum precontracted with KCl rich medium (25 mM). This result en KCl (25 mM). Este resultado sugiere que el extracto tenía un efecto suggests that the extract had a Ca2+ antagonist effect. This was fortified antagonista de Ca2+. Esto se reforzó cuando el pretratamiento del when pretreatment of the intestine with the extract induced a rightward intestino con el extracto indujo un desplazamiento hacia la derecha en shift in the Ca2+ concentration-response curves. AqWs exhibited also las curvas de concentración-respuesta de Ca2+. Los AqW también antispasmodic effect on carbachol (10-6 M)-induced contractions of rat mostraron un efecto antiespasmódico sobre las contracciones del yeyuno -6 jejunums with an IC50 = 1.53 ± 0.04 mg/mL. The extract at 3 mg/mL on de rata inducidas por carbacol (10 M) con una CI50 = 1,53 ± 0,04 rat jejunums pre-incubated with hexamethonium (10-4 M), atropine (10-6 mg/mL. El extracto a 3 mg/mL en yeyuno de rata preincubado con M), and methylene blue (10-5 M), then contracted by KCl, decreased the hexametonio (10-4 M), atropina (10-6 M) y azul de metileno (10-5 M), maximum contraction but not totally like KCL alone without these luego contraído por KCl, disminuyó la contracción máxima pero no inhibitors. For the jejunum pre-incubated with L-NAME (10-4 M), the totalmente como KCL solo sin estos inhibidores. Para el yeyuno spasmolytic effect of AqWs was comparable with KCL. preincubado con L-NAME (10-4 M), el efecto espasmolítico de AqWs fue Conclusions: These results suggest that the myorelaxant and comparable con KCL. antispasmodic effects are mediated possibly via Ca2+ antagonist, Conclusiones: Estos resultados sugieren que los efectos miorrelajantes y anticholinergic, and guanylate cyclase mechanisms but not by antiespasmódicos están mediados posiblemente por mecanismos adrenergic and nitric oxide pathway. antagonistas de Ca2+, anticolinérgicos y guanilato ciclasa, pero no por la vía adrenérgica y del óxido nítrico. Keywords: anticholinergic; antispasmodic; calcium antagonist; guanylate Palabras Clave: anticolinérgico; antiespasmódico; calcio antagonista; cyclase; jejunum; Warionia saharae. guanilato ciclasa; Warionia saharae; yeyuno. ARTICLE INFO AUTHOR INFO Received: February 25, 2021. ORCID: 0000-0002-3006-2523 (OA) Received in revised form: April 30, 2021. Accepted: April 30, 2021. Available Online: May 9, 2021. _____________________________________ Amrani et al. Antispasmodic activity of Warionia saharae INTRODUCTION NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2 and glucose 10; calcium free high The genus Warionia (Compositae) is represented K+ KHB ([K+] = 75 mM): NaCl 48, KCl 75, CaCl2 0, by only one species named Warionia saharae Ben- MgSO4 1.2, NaHCO3 25, KH2PO4 1.2 and glucose them ex Benth. & Coss. For Katinas et al. (2008), 10; and calcium free KHB (in mM): NaCl 121.7, the palynological and morphological proof places KCl 4.7, CaCl2 0, MgSO4 1.2, NaHCO3 25, KH2PO4 this plant between the tribes Cichorieae and Cardue- 1.2 and glucose 10. All components of these solu- ae, and it a shrub endemic in Morocco and Algeria. tions were purchased from Sigma-Aldrich, USA, In Morocco, it is known locally by the vernacular made up in distilled water. These solutions were name ‘‘Afessas’’. The leaves are used in the tradi- employed for incubation jejunum strip and main- tional remedy of Morocco to treat gastrointestinal tained at pH 7.4. Throughout the long-term exper- disorders, inflammatory diseases and against epi- iments, while being heated to 37°C and bubbled leptic crisis (Bellakhdar, 1997). In previous studies, with 95% O2 + 5% CO2. researchers wrought on different extracts of W. saharae have shown that it has antioxidant (Sek- Plant material koum et al., 2011), anti-inflammatory, antibacteri- al, and cytotoxic activities against a cancer cell line The crude extract of W. saharae (AqWs) was ob- (Hilmi, 2002a; Znini et al., 2011). tained by following the traditional use of Moroc- can population. Therefore, air-dried aerial part of Despite its wide use in the treatment of diges- this plant was infused in boiling distilled water for tive tract diseases in the southeastern of Morocco, 30 min, after filtration, the filtrate was concentrat- to our best knowledge, no study on W. saharae an- ed by rotary evaporator, and then dried at 37°C in tispasmodic activity was conducted before. There- an oven. Finally, the resulting crude aqueous ex- fore, and like in developing country, this disease is tract was recovered and preserved at -20°C until a major health care problem. This study aimed to use. The specie was collected in Errachidia region obtain for the first-time information on the anti- (31°55′53″ N, 4°25′35″ W) identified by a Prof. Mo- spasmodic effect of this plant on rodent isolated stafa Elachouri and a specimen was deposited in jejunum. the herbarium of Faculty of Sciences, University Mohamed first, Oujda, Morocco, under number MATERIAL AND METHODS HUPOM 450. Chemicals Animals Carbamylcholine chloride (carbachol, CCh), New-Zeeland rabbits (1.5-2 kg) and Wistar rats calcium chloride (CaCl2), propranolol, prazosin, (200-350 g) of both sexes were used in these exper- yohimbine, L-NAME and hexamethonium were iments. They were placed under standard condi- purchased from Sigma Chemical Co (Sigma- tions in the animal house of Faculty of Sciences, Aldrich, USA). Atropine from Research Biochemi- Oujda, Morocco, with free access to drinking water cal Incorporated, USA; papaverine hydrochloride ad libitum. Maintained at controlled lighting (12 h– (Pap) from Fluka, India; and verapamil hydrochlo- 12 h light-darkness cycle), humidity, and tempera- ride (Vrp) from Tocris, USA were also used. All ture. All animals were starved 18 h prior the ex- chemical products used were solubilized in dis- periments. The experiments were conducted ethi- tilled water. cally in accordance with the internationally ac- Incubation media cepted guidelines for the care and use of laborato- ry animals published by the United States Nation- The solutions used in this study were normal al Institutes of Health (NIH, 1985). Krebs-Henseleit buffer (KHB) solution (in mM): http://jppres.com/jppres J Pharm Pharmacogn Res (2021) 9(5): 678 Amrani et al. Antispasmodic activity of Warionia saharae Collection and preparation of animal isolated precise concentration of AqWs 5 min before jejunum strips adding of carbachol.