VALIDATION OF ETHNOPHARMACOLOGICAL USE AS ANTI-INFLAMMATORY OF A DECOCTION FROM ANNONA MURICATA LEAVES

M.A


Introduction
Inflammation is a complex process that results into up-regulation of inflammatory mediator as prostaglandins, cytokines, chemokines, oxidants such as nitric oxide and superoxide and granular lytic enzymes by macrophages and neutrophils, the main phagocytic cells (Moncada et al., 1991). It is a beneficial host-response, but upon long persistence, it may result into chronic conditions such as cancer (Aggarwal et al., 2006), cardiovascular diseases (Libby, 2006), diabetes (Wellen & Hotamisligil, 2005), pulmonary disorders (Sevenoaks & Stockley, 2006), neurological diseases (Yong et al., 2001) and arthritis (Goldring & Otero, 2011). Non-steroideal anti-inflammatory drugs (NSAIDs) are commonly used to treat the symptoms of acute or chronic conditions such as pain, fever and inflammation. However, the extensive use of these drugs is associated with several side-effects. Due to such reasons, there has been a growing interest in the use of natural compounds and medicinal plants (Zeng et al., 2012).
Annona species (Annonaceae) have been used as natural remedy for a variety of illnesses, the literature is rich in studies showing the many and important pharmacological activities of this genus of medicinal plants. Annona muricata L. is a broadleaved, flowering, evergreen tree commonly known as soursop, graviola, guanábana or corossol among its many popular names. It is found wild and cultivated from sea level to 1,000 m elevation in the tropical areas of America, Asia, and Africa (Lim, 2012).
Phytochemical investigations of the leaves from Annona muricata revealed as the main components: alkaloids (Leboeuf et al., 1982), phenolic compounds, terpenoids, essential oils (Thang et al., 2013;Kossouoh et al., 2007) and acetogenins. These latter active compounds have been studied further, as they have demonstrated antitumor properties, presenting selective toxicity against various types of cancer cells and against different multidrug-resistant cancer cell lines (Mishra et al. 2013; Barbalho et al. 2012;Lim, 2012;Lima, 2008). In the consulted bibliography, pharmacological studies are found supporting the popular use of Annona muricata in the original countries, especially related to the antitumor, anti-inflammatory, antidiabetic, antiviral, anti-bacterial, insecticidal and antiparasitic activities (Mishra et

Reagents
All the reagents were purchased from Sigma Aldrich Chem. (St. Louis, MO, USA). Stock solutions of the extracts were prepared in dimethyl sulfoxide (DMSO) and later dissolved in ethanol.

Plant Material and Extract Preparation
Young leaves of Annona muricata were collected in July 2004 in "Roca del Mar" area (Santo Domingo, Dominican Republic) and were identified by the botanist A. Veloz in the National Botanic Garden of Santo Domingo (JBSD). A voucher specimen was deposited in the JBSD and kept under reference Tramil-Gef Project 3142. Decoction of the leaves was prepared using water according to the Spanish Farmacopeia method (Real Farmacopea Española, 2011). The aqueous decoction was concentrated in a rotary evaporator.

Phytochemical Screening
Phytochemical screening of the aqueous extract was performed using conventional protocol. The presence of alkaloids with Mayer, Dragendorff and Bouchardat´s reagents, flavonoids with the use of Mg and HCl, tannins with 1% gelatin and 10% NaCl and FeCl 3 solutions, cyanogenic glycosides with picrate paper, the phenolic acids were identified by TLC, anthraquinones with the Borntranger´s reaction, saponins with the aphrometric index and phenylpropanoids with Amow reagent (Evans, 2006).

Animals
Forty-eight male Swiss albino mice of five-weeks-old, weighting 25-30 g were used for the experiments. They were randomly placed in cages (6 mice/cage) kept in a room at 22 ± 2°C, humidity 60 ± 5% and ad libitum feeding of a standard laboratory diet and tap water before use. All animal care and experimental procedures complied with the Guidelines of the European Union regarding animal experimentation (Directive of the European Council 86/609/EC) and followed a protocol observed and approved by the Animal Ethics Committee of the University of Seville.

Toxicity and Viability Cell Acute Oral Toxicity Study
The acute oral toxicity study was conducted using test guideline on acute oral toxicity test 423 according to OECD (2001). Six male Swiss albino mice of five-weeks-old per group fasted overnight, but allowed free access to water ad libitum were randomly assigned into the following four groups: Control group received distilled water and three groups received the extract at the doses of 250, 500 and 1000 mg/kg. Mice were not fed for 3 h following the administration. The signs of toxic effects and/or mortality were observed 3 h after administration and then, for the next 48 h.

Anti-inflammatory Activity: Carrageenan-induced Mouse Hind Paw Edema
Edema was induced on the right hind paw of the mouse by subplantar injection of 0.1 ml of a solution of carrageenan, 1% w/v in saline solution (Garcia et al., 2004). A single dose by oral gavage of the aqueous extract, 250 or 500 mg/kg, was administered 1 h before the injection of carrageenan. The control group received only saline solution. Ibuprofen at 50 mg/kg was used as a reference drug. Paw volumes was measured by means of plethysmometer (LETICA-7500) before administering carrageenan (V 0 ), at 1, 3 and 5 h later (V t ). The percentages of inhibition were calculated for each group and each measurement, comparing with the control group, using de following ratio:

TPA-Induced Mouse Ear Edema
Each mouse received 2.5 µg tetradecanoylphorbol acetate (TPA) as phlogistic agent dissolved in 20 µl acetone (Xian et al., 2011; Fernandez-Arche et al., 2010). Extracts were applied topically, at doses of 2.5 and 5 mg/ear dissolved in water, immediately after application of TPA in the right ear. Ibuprofen (0.05 mg/ear) was used as a reference compound. Inflammation was allowed to develop for 4 h and the swelling was assessed as the increase of weight of the right ear against the left ear (received only the vehicle).

Myeloperoxidase Assay (MPO)
The method of Bradley (Bradley et al., 1982) was followed, modified for lecturing in a microplate reader. MPO activity was determined on the supernatants from the homogenates of the ear biopsies prepared as it is described (Xian et al., 2011).

MPO Release by A-23187-Stimulated Rat Neutrophils
Rat neutrophils were obtained as described in Moroney et al. (1988). The cells were resuspended in complete Hank Balance Salt Solution at 0.5×10 6 cells/ml containing 1.26 mM Ca 2+ and 0.9 mM Mg 2+ . Cells were pre-incubated at 37°C for 10 min with 50, 100, 200 µg/ml of AMAEL doses. After this, calcium ionophore A23187 (1 μM) was added. The cells were pelleted by centrifugation at 2500 g for 10 min at 4°C, and the supernatants were assayed for the MPO activity (Bradley et al., 1982).

Nitric Oxide Assay
Nitrite, as index of nitric oxide (NO) generation, was determined in culture supernatants by Griess reagent (Green et al., 1982).

Statistical Analysis
All results were expressed as mean ± SEM. Statistically significant differences were evaluated by analysis of variance (ANOVA) followed by Dunnett's test. P value less than 0.05 was considered significant.

Phytochemical Screening
Aqueous extraction yield was 24%. AMAEL showed the presence of phenol compounds (tannins, flavonoids and phenolic acids) and alkaloids in the qualitative phytochemical screening.

Toxicity and Viability Cell
A single dose of 250, 500 and 1000 mg/kg did not indicate modification of behavior in Swiss Albino mice. No mortality was recorded during the study. After sacrifice on the 2 nd day, macroscopic pathology observations, revealed no visible lesions in any animal. The animals did not show any stereotypical symptoms associated with toxicity at these doses. AMAEL did not induced toxicity in the murine peritoneal macrophage when assessed by mitochondrial reduction of MTT after 24 h of treatment. Viability of cells treated with the extract was 100.3 ± 0.6%, 99.8 ± 0.7% and 100.8 ± 1.0 at concentrations 500, 250 and 100 µg/ml respectively (data not shown).

Anti-inflammatory Activity Carrageenan-induced Edema Model
The results of this test are reported in Table 1. AMAEL exerted a significant edema reduction from the first hour and remained along in the time compared to the untreated control group (p<0.001). Interestingly, a single dose of 500 mg/kg by oral gavage was found to be slightly more effective in reducing the paw edema as the standard Ibuprofen (50 mg/kg). Table 2 shows the results of the topical effect of AMAEL on the TPA-induced edema in mouse ear. The edema was significantly and dose-dependently reduced (56 and 78% at doses of 2.5 and 5 mg per ear respectively).

Myeloperoxidase Activity (MPO) in Inflammed Tissue
The effect of AMAEL on MPO activity in inflamed tissue is depicted in Fig. 1. The topical administration significantly reduced MPO overproduction in a dose-dependent manner. The highest inhibition was achieved at 5 mg/ear (92.5% ± 1.83, p<0.001).

MPO Release by Rat Neutrophils
MPO assay in A23187-stimulated neutrophils was performed to assess possible in vitro effects of AMAEL on activated neutrophils. Fig. 2 shows that Annona muricata L. at 200 µg/ml caused an inhibition of the A23187-induced MPO expression (81.98% ± 1.01, p<0.001). At doses of 100 and 200µg/ml, the effect was higher than that produced by caffeic acid, the reference compound (59.66% ± 2.27).

Nitrite Production by LPS-Stimulated Murine Peritoneal Macrophages
The in vitro effect of AMAEL on the release of inflammatory mediators such as nitric oxide by LPS-stimulated murine peritoneal macrophages is depicted in Fig. 3. Co-incubation with Annona muricata significantly reduced nitrite overproduction in a dose-dependent manner. The highest inhibition was achieved at 500 µg/ml (73.18% ± 2.36, p<.001). AMAEL at 100, 250 and 500 µg/ml did not decrease nitrite production in vitro NO scavenging test, using sodium nitroprusside as a NO donator, indicating this effect was not a consequence of a direct scavenging of this radical (data not shown).

Discussion
Based on the need to validate phytopharmaceutical products that allow the use of local resources in basic health systems, we have shown in this study the anti-inflammatory activity of AMAEL against two inflammation animal models and in two different cell systems, macrophages and neutrophils, the major inflammatory cells. We used the aqueous leaf extract because this is the preparation equivalent to the use in folk medicine. In the qualitative phytochemical screening we have detected phenols compounds, alkaloids having sparingly but we have not found acetogenins in detectable amounts, perhaps because of its poor water solubility (Le Ven et al., 2012). The lack of toxicity showed in the acute mice toxicity and in the MTT cell viability assays, could likely be to a poor presence in the aqueous extract of acetogenins, described by some authors as cytotoxic compounds (De Pedro et (Table 1) and the presence of phenol compounds (tannins, flavonoids and phenolic acids) could explain this anti-inflammatory activity and the possible synergistic effect among these compounds. This evidence suggests that the anti-inflammatory actions of the aqueous extract may be related to inhibition of one or more signaling intracellular pathways involved with the inflammatory mediators. On the TPA-induced edema, the application of AMAEL produced a reduction of about 78% compared with the control group (Table 2). This positive response on the TPA test would indicate that the inhibition of edema could be essentially due to protein Kinase C (PKC) inhibition (Nishizuka et al., 1988). TPA stimulates the actions of PKC in a manner similar to that of endogeneous diacylglycerol, liberated from membrane phospholipids (Marucha et el., 1991).
It is generally accepted that tissue injury associated with inflammation is attributed to infiltration of neutrophils and macrophages followed by the release of proinflammatory mediators such as eicosanoids, toxic radical species and lytic enzymes. Accordingly, inhibition of the function of the macrophages and neutrophils participates on the mechanism of action of a number of anti-inflammatory drugs. AMAEL significantly inhibited some of the functions of these cells, which may be implicated in the anti-inflammatory action detected in the in vivo assays. In this sense, the MPO activity, as an indicator of the migration of neutrophils to the inflamed tissue, was calculated and the reduction in the activity of this enzyme in both, ear homogenates and activated rat neutrophils, were highly significant (Fig.1, Fig.2, respectively). On the other hand, the inflammatory macrophages constantly express inducible nitric oxide synthase (iNOS) producing an increase amount of NO, which plays a multifaceted role in inflammation ranging from an increase in vascular permeability and edema formation to tissue cytotoxicity (Moncada et al., 1991). AMAEL exerted significant and dose-dependent inhibition of LPS-stimulated NO production in macrophages without any evidence of cytotoxic effect (Fig.3). The mechanism by which the extract inhibits NO seems to involve attenuation in induction or activity of iNOS, because of that, it was checked that AMAEL did not directly scavenged the NO radicals produced by the NO donor as the sodium nitroprusside. NO inhibition results in suppression of the inflammatory response in different models including carrageenan induced paw edema (Salvemini et al., 1996) thus NO inhibition may be implicated in AMAEL associated reduction for this inflammation model assayed.

Conclusion
The results obtained in this study demonstrated the significant anti-inflammatory activity of the decoction of Annona muricata L. leaves that may be related to inhibition of one or more signaling intracellular pathways involved with the inflammatory mediators. This fact validates the ethnomedicinal use of this plant in different pathologies related to inflammation and suggests that this species could be a valuable source of phytodrugs. It is important to note that plant extracts, like AMAEL, usually constitute a range of substances in different concentrations that are capable of establishing a synergism for certain biological effects. The efficacy and safety presented by AMAEL leads to our research group to make advancements to identify the active principles responsible for its anti-inflammatory effects and their possible synergistic activity. production Ethnopharmacological resources as Annona muricata, with a large scientific literature that validates its popular uses, represent a potential source of effective, safe, low-cost and alternatives tools to primary health care.