Synergistic Antinociceptive Activity of Combined Aqueous Extracts of Artemisia Campestris and Artemisia Herba-Alba in Several Acute Pain Models
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SUPPLEMENTARY MATERIALS Synergistic antinociceptive activity of combined aqueous extracts of Artemisia campestris and Artemisia herba-alba in several acute pain models Imed Eddine Kadi, Mohamed Ouinten, Nadhir Gourine*, Mohamed Yousfi Laboratory of Fundamental Sciences. University of Amar Telidji, Road of Ghardaia, Po.Box 37G (03000), Laghouat, Algeria *Corresponding author at: Laboratoire LSF, B.P. 37G, Université Amar TÉLIDJI, Laghouat, (03000), Algeria. Tel.: +213 29 90 00 66 Fax: +213 29 90 00 66 E-mail address: [email protected] [email protected] Abstract: In this study, total phenolic and flavonoid contents, acute toxicity and the antinociceptive activity of Artemisia campestris and Artemisia herba-alba individually and in combination were investigated using multiple forms of pain in animals. Our results have been shown that plants are relatively safe without clinical signs of toxicity in animals. Thus, extracts were presented high levels in phenolic and flavonoid contents. Artemisia decoctions with 100, 200, 400 mg/kg b-w studied dose, clearly attenuate chemical and thermal noxious stimuli in writhing, formalin and hote plate tests, and significantly reduced paw edema in formalin test. Additionally, binary combination forms exhibited a great improvement in intensity and amplitude of antinociceptive activity in comparison with both plants used individually by a relative interference with opioid system. Our findings suggested the central and peripheral analgesic properties and confirmed the folkloric medicinal use of these plants in pain symptom treatment. Key words: Antinociceptive, Analgesic, Artemisia campestris, Artemisia herba-alba, Pain, Nociception. 1 1. Complementary introduction Somatic and mental health problems were often accompanied with a pain syndrome. It’s a major symptom in most chronic and acute diseases, especially those related to locomotor system (Dillworth et al. 2012, Marchand 2014). Pain is a complex phenomenon implicated neuro-physiological pathways and neuro-chemical mediators from peripheral nociceptors to higher centers in brain (Sinatra et al. 2009, Motoc et al. 2010). However, their adverse effects are unbearable in patients. It’s widely documented that NSAIDs generate cardiovascular effects, gastrointestinal ulcers and renal insufficiency (Higgs et al. 2013, Ymele et al. 2013). In addition, the prolonged use of opioids induces a tolerance and hyperalgesie. Which require us to research an alternative healthy and effective biopharmaceuticals. Natural products imposed themselves for a long time as an important and Inexhaustible origin in new therapeutics research. After the discovery of Artemisinin an anti-malarial agent from Artemisia annua, a great pharmacological interest was brought to Artemisia genus (Marchand 2014). Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures.(Ali et al. 2017) Grouping over 400 species with a wide distribution along the Mediterranean cost, Artemisia genus was considered to be a great source of biomolecules (Sainz et al. , Valant-Vetschera et al. 2003). In recent years (since 2010) the extracts of Artemisia plants were extensively studied, leading to isolation of 159 compounds with diverse structures from this genus (Zhang et al. 2017). The studies conducted in these plants (mostly based in vitro experiments) have revealed their potential in anti-inflammation, anti-cancer, antimalarial, antinociception and antimicrobial and antimicrobial. (Zhang, et al. 2017). More importantly, Hispidulin (40, 5, 7-trihydroxy-6- methoxyflavone) is a flavones derivative found in Artemisia species among other ones which have antioxidant, antifungal, anti-inflammatory, antimutagenic, and antineoplastic properties (Patel and Patel 2017). Artemisia herba-alba is a spontaneous aromatic plant frequently used in Algerian and Tunisian traditional medicine (Qnais et al. 2014, Aidi Wannes and Marzouk 2016, Najjaa et 2 al. 2017) to relieve gastric disorders with antispasmodic, hypoglycemiant, anti-inflammatory properties (Lee et al. 2012), vasorelaxant (Naoufel et al. 2016) and diabetes treatment (Aidi Wannes and Marzouk 2016, Telli et al. 2016, Bushara et al. 2017, El-Marasy et al. 2017). Similarly, the use of Artemisia campestris in Algerian folkloric medicine is recommended for its anti-Rheumatic, anti-inflammatory, diabetes treatment and anticancer activities (Naskar et al. 2013, Barkat et al. 2015, Aidi Wannes and Marzouk 2016). The meeting point of both previous plants it’s their extensively use in the management of several forms of abdominal (Silva et al. 2002), gastric (Wagner and Ulrich-Merzenich 2009) and Rheumatic (Motoc, et al. 2010) pain paradigms. The strategy of natural products combination is an effective approach to optimize and improve pharmacological and therapeutic properties of phytopharmaceuticals in order to treat multifactorial diseases (Wagner and Ulrich-Merzenich 2009). It has been frequently reported that herbal extracts in combination improve the bioavailability (Yang et al. 2014), neutralize the side effects of ingredients and optimize their pharmacokinetics and pharmacodynamics properties (Williamson 2001, Wagner and Ulrich-Merzenich 2009). 2. General literature background Antinociceptive (Qnais, et al. 2014) and anti-inflammatory (Khlifi et al. 2013) activities of A. herba-alba, and anti-inflammatory properties in vivo for A. campestris (Fotso et al. 2014, Ghlissi et al. 2016) were previously confirmed. Moreover, it’s widely documented that phenolic compounds, in particular flavonoids plays an effective role in nociceptive response management (Mothana et al. 2012, Higgs, et al. 2013, Wang et al. 2014, Rauf et al. 2016). Detailed studies reported the phenolic molecular profile of these Artemisia plants; flavonols and flavonones are the main categories (Sefi et al. 2010, Megdiche-Ksouri et al. 2015, Dif et al. 2016, Bourgou et al. 2017), with phenolic acids, coumarine and its derivative in A. campestris (Ferchichi et al. 2006, Dib et al. 2016). In addition, sesquiterpene lactones, flavonoids from flavone and flavonol glucosides to highly methylated flavonoids and phenolic acids such as chlorogenic acid with their derivatives have been identified in A. herba-alba (Mohamed et al. 2010, Bourgou, et al. 2017, Peron et al. 2017). Furthermore, Hispidulin (40, 5, 7-trihydroxy-6-methoxyflavone) is a flavone derivative found in both Artemisia herba-alba and campestris exhibited antioxidant, antifungal, anti- inflammatory, antimutagenic, and antineoplastic properties (Patel and Patel 2017). Some very 3 recent publications reported the antioxidant and the biological activities of the total phenolic compounds of Algerian A. herba alba and A. campestris plants using several solvents extractions: organic (methanolic, ethanolic, hexanic) or aqueous, (Dif, et al. 2016, Belkacem et al. 2017, Dib et al. 2017, Salhi, Rahmani, et al. 2017, Salhi, Saghir, et al. 2017). This variability in the qualitative and quantitative levels on molecular profile stimulated us to mix extracts from these plants to hone their analgesic properties. Acetic acid, Formalin and Hot-plate tests using mice and rats were applied in this study. Writhing test is a non-selective classical method to evaluate the antinociceptive responses (Silva et al. 2015). Acetic acid injected in animals abdomen was induced an irritation process (Zakaria et al. 2008), accompanied with an increase in endogenous mediator levels in abdominal cavity such as histamine, serotonin, bradykinin, substance P and prostaglandin (Reanmongkol et al. 2009), especially PGE2 and PGF2 (Ymele, et al. 2013). Thus, the release of pro-inflammatory cytokines IL1, IL6, IL8 and TNFα (Silva, et al. 2015) with increasing in capillary permeability. All the preceding molecules were participated in the stimulation of peripheral and central chemo-sensitive receptors (Bukhari et al. 2010). Aqueous extracts gavages individually and in combination, showed a remarkable antinociceptive effect in writhing test by immediate effects on prostaglandin actions, or indirectly by interference with acid arachidonic metabolism and inhibiting of cyclooxygenase and lipooxygenase functions (Zakaria, et al. 2008, Fotso, et al. 2014). Intra-plantar injection of formalin in rats was generated a moderate continuous pain sensation (Reanmongkol, et al. 2009) distributed in two forms, chronologically and neuo- physiologically different (Nonato et al. 2011). Neurologic pains (Early phase 0-10 min) were created by a direct stimulation of sensory neurons such as C fibers, and inflammatory pains (Late phase 15-60 min) were discussed by increasing in inflammatory mediators such as prostaglandins, serotonin, histamine and bradykinine (Bukhari, et al. 2010). A quiescent- interval period separates previous phases (Silva, et al. 2015). Peripheral analgesic drugs such as NSAIDs, Diclofenac and aspirin are known as inhibitors of inflammatory nociception, whereas opioids central drugs can attenuate both forms of pain (Ymele, et al. 2013). Hot-plate test is a selective model to appreciate the central analgesic effects by direct stimulation of nociceptors C and Aδ (Reanmongkol,