Phytothérapie © Lavoisier SAS 2014 DOI 10.1007/s10298-014-0875-1
Article original Pharmacognosie
Phytochemical Screening, Immunomodulatory and Anti-inflammatory Activities of the Phenolic Fraction of Aphloia theiformis (Vahl) Benn. Plant Used in Comorian Traditional Medicine S. Hsoidrou1, D. Bousta1, M. Lachkar2, S.O. Said Hassane3, A. El Youbi-Hamsas1, L.E.L. Mansouri1, J. Benjilali1, H. El-Hajaji2, A. Farah1 1
PAMSN, National Institute of Medicinal and Aromatic Plants, Taounate, University Sidi-Mohamed-Ben-Abdellah, Fez, Morocco LIMOM, Faculty of Sciences Dhar el Mehraz, University Sidi Mohamed Ben Abdellah, Fez, Morocco 3 Faculty of Sciences and technology, University of Comoros, BP 2585, Moroni, Comoros 2
Correspondance :
[email protected]
Abstract: Aphloia theiformis (Vahl) Benn. is used as ethnical medicine for diverse diseases in the Indian Ocean islands and the East Africa region. This study investigated the potential immunomodulatory effect of the phenolic fraction of A. theiformis leaves and their anti-inflammatory potent. Phenolic fraction from the leaves of A. theiformis was obtained by methanol extract, and recovered by an aqueous solution of 5% HCl, then filtered. We investigated the potential immunomodulatory effect in rat, by flow cytometer. Anti-inflammatory activity was also investigated in carrageenan-induced paw oedema rat model. Results demonstrated an immunosuppressive effect on lymphocyte for all doses administered. However, we noted an interesting immunostimulant effect on monocytes and granulocytes number at high doses. Then, we noted an important anti-inflammatory-like effect for these doses in vivo. This work suggests the eventual use of this plant in the treatment of some inflammatory symptoms and immune dysfunctions. Keywords: Aphloia theiformis (Vahl) Benn. – Immunomodulatory – Anti-inflammatory – Phytochemical screening – Phenolic fraction
Caractérisation phytochimique, activités immunomodulatrice et anti-inflammatoire de la fraction phénolique d’Aphloia theiformis (Vahl) Benn., plante utilisée dans la médecine traditionnelle comorienne Résumé : Extrêmement répandu dans les îles de l’océan Indien, Aphloia theiformis est très utilisée contre diverses maladies. Dans cette étude, nous nous sommes penchés sur les possibles effets immunomodulateur et anti-inflammatoire de la fraction phénolique d’Aphloia theiformis. Cette fraction phénolique est obtenue à partir de l’extrait méthanolique des feuilles d’Aphloia theiformis après récupération par une solution d’HCl à
5 %. L’activité immunomodulatrice a été réalisée par cytométrie en flux, et l’activité anti-inflammatoire par le biais du modèle d’œdème de la patte induit par la carragénine chez le rat. Pour ce qui est de l’activité immunomodulatrice, les résultats ont montré un effet suppressif sur les lymphocytes pour toutes les doses testées. Cependant, pour les doses élevées, on a noté un intéressant effet immunostimulant pour les sous-populations monocytaires et granulocytaires, suggérant ainsi un potentiel effet anti-inflammatoire qui a été démontré in vivo. Ces résultats ouvrent donc la voie à une utilisation potentielle de cette plante dans le traitement des symptômes inflammatoires et certains dysfonctionnements de la réponse immunitaire. Mots clés : Aphloia theiformis (Vahl) Benn. – Effet immunomodulateur – Effet anti-inflammatoire – Criblage phytochimique – Fraction phénolique
Introduction Approximately 80% of the world’s population uses plants for their primary care [1]. Plants are recognized for their ability to produce secondary metabolites, and mankind has used many species for centuries to treat a variety of diseases [2]. Many natural plant products and synthetic compounds have demonstrated their capacity to modulate immune responses [3,4]. Immune system plays a vital role as the main line of defense. Its efficiency and integrity are important in chemotherapeutic interventions for treatment of many diseases [5]. In Comoros, traditional medicine is considered as an integral part of ancient traditions, but they are few written documentation. For the recovery and diversification of medicinal plants used in Comoros, we focused our research
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on Aphloia theiformis (Vahl) Benn, a plant known locally as the “Mfandrabo”, the leaves of which are widely used alone or in combination for the preparation of tea. Previously included in the Flacourtiaceae family, Aphloia theiformis (Vahl) Benn is currently the only representative of the Aphloiaceae family [6]. In Comoros, the A. theiformis leaves are used for treating dizziness and leucorrhoea [7]. In Mauritius, they are also used as a febrifuge [8]. In Madagascar, the bark of this species is used as emetic [9]; its young leaves are demonstrated effective against haematuria and the old ones against rheumatisms [10]. In terms of chemical composition, the literature reports the presence of saponins and xanthone in A. theiformis leaves [6,8]. In this study, we aimed to (i) determine the acute toxicity threshold of aqueous extract of phenolic fraction of A. theiformis, (ii) to investigate some pharmacological activities such as the potential anti-inflammatory-like profile and immunomodulatory properties in the rat and (iii) to measure the percentage of some chemical components like total phenols and flavonoïds.
Materials and methods Plant material Leaves of A. theiformis were collected in September 2009 from the Hantsongoma forest (950 m altitude, Oichili province, NE of Ngazidja, Comoros) and identified by Professor Jean-Noël LABAT from Muséum National d’Histoire Naturelle (Paris, France). A Voucher specimen (AND501) was deposited in the Herbarium of Botanic Department from Faculty of Sciences and Technology, University of Comoros. Extract preparation Dried and ground leaves of A. theiformis were successively extracted with hexane, ethyl acetate, dichloromethane and methanol in soxhlet. Phenolic fraction was obtained according the method described by Maiza-Benabdesselam et al., [11], with minor modifications. Methanol extract was concentrated and recovered by an aqueous solution of 5% HCl, and then filtered. The residue was dried and stored for different tests.
Phytochemical screening Flavonoids, tannins, alkaloids, saponosids and total phenolic constituents were determined according the work of Hajaji et al., [12]. Total flavonoids contents were determined by spectrophotometer by using method described in the work of Hadj Salema et al., [13] with some modifications.
Animals Male Wistar Rats (100-150g) were obtained from the center ECWP “Emirates Wildlife Propagation Center” (Missour, Morocco). They were kept in well-ventilated environment, had a free access to water and food ad libitum and were housed in a quiet room under a “12-h light: 12-h dark” cycle for 2 weeks before experimentations.
Acute toxicity and median lethal dose (LD50) The method described by Randhawa, using methods of Miller, Tainter and Ghosh [14–16] was used in this study in the determination of LD50. A. theiformis has been studied by “i.p” at 200, 300, 600 and 1200 mg/kg b.w. The animals were observed for obvious toxic symptoms and mortality in each group during 5 days.
Flow cytometer The animals are anesthetized by “ip” with sodium pentobarbital at the dose of 30 mg/kg, 24 h, after treatment with the phenolic fraction of A. theiformis. The blood is collected in heparinized tubes, and then, subjected to analysis by FCM (flow cytometer Epics-XL MCL type). FCM was used to evaluate the proportion of leukocyte sub-populations. This is based on cell morphology, size and structure.
Anti-inflammatory activity For anti-inflammatory properties we used the method described by Winter et al, [17]. Four groups each containing five animals per group were used in the study. Control group received normal saline solution by “ip”. The other groups received indomethacine (2 mg/kg), phenolic fraction of A. theiformis (100 and 200 mg/kg). 30 min after administration, 0.1 ml of Carrageenan was injected into the plantar region of the right hand paw by intradermal injection. The paw size was measured before carrageenan injection and every hour, after 3 hours of injection, for a period of 6 hours. This measure was carried out by wrapping a piece of cotton thread round the paw and measuring the circumference with a meter rule. The percentage inhibition was calculated by: S t −S 0 Þ treated ¼ 100 % inhibition ¼ ðS t −S 0 Þ control−ð ðS t −S 0 Þcontrol St = the mean paw size for each group after carrageenan treatment. S0 = the mean paw size obtained for each group before carrageenan injection.
Statistical analysis Data were expressed as the mean ± SEM. Comparisons of means were performed by using the t-test of Student. The level of statistical significance was set at p < 0.05.
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ment of monocytes and granulocyte sub-populations at doses of 100 and 200 mg/kg b.w. compared to control and to the prednisolone groups. This result suggested a probable anti-inflammatory effect of the phenolic fraction of A. theiformis. A similar result was observed in indomethacine group especially on granulocyte subpopulation. The relative percentage was augmented at 788% at dose of 200 mg/kg, b.w. of the phenolic fraction of A. theiformis compared to control group.
Results Phytochemical screening As shown in Table 1, phytochemical screening of residue of A. theiformis revealed an abundance of flavonoids and tannins. However, alkaloids and saponosids were not detected. The total phenolic contents (mg/l) was determined from regression calibrate curve (Y = 0, 0005x + 0, 131; R2: 0, 9392) and expressed in gallic acid equivalent (GAE). The result was calculated at 442 mg /l GAE. The total flavonoid content was also determined from regression calibrate curve (Y= 0.0224x + 0.6183; R2= 0.8673) and expressed in quercetin equivalent (QE). The result was calculated at 104 mg /l QE.
Anti-inflammatory activity The anti-inflammatory potent of the phenolic fraction of A. theiformis was evaluated by carrageenan-induced paw edema model in rat. Results showed an anti-inflammatory activity for all doses (Fig. 3), but the most important activity was observed at 200 mg/kg, b.w. We noted an important efficiency of A. theiformis at a dose of 200 mg/kg, b.w., five hours after Carrageenan injection and the percentage of edema inhibition was 93% compared to indomethacine
Acute toxicity and median lethal dose (DL50) The results revealed that the doses of 200mg/kg, b.w. of A. theiformis extract did not produce any acute toxic effect or death in the two tested groups of rats (N=6). However for the ranging doses of 300, 600 and 1200 mg/kg, b.w., animals showed some side effects like fatigue, decreased weight and ultimately death (Table 2). The median lethal dose was estimated at 501 mg/kg, b.w. (Fig. 1).
Discussion Our study was conducted to identify the major phenolic coumpounds contained in leaves of A. theiformis from Comoros and their anti-inflammatory and immunomodulatory properties. Phytochemical screening showed the abundant presence of flavonoids and tannins (Table 1). It’s also reported that A. theiformis from Reunion islands contains flavonoids and tannins presence in [18]. In their study Gopalsamy et al., [19] reported the presence of saponins in the leaves of A. theiformis, but thes compounds are absent in our phenolic fraction. This absence can be explained by the use of HCl solution in our extraction method. For acute toxicity, the doses of 200, 300, 600 and 1200 mg/kg b.w. were tested; the lethal median dose was established at 501 mg/kg b.w (Fig. 1).
Immunomodulatory effect The immunomodulatory effect of A. theiformis extract was studied by evaluation of relative percent of leukocyte subpopulations in flow cytometer (Fig. 2). We noted an immunosuppressive effect of lymphocytes subpopulation at all doses tested of A. theiformis. The dose of 15 mg/kg, b.w. (8%) was the most active compared to the other doses and to control group (P
