Phytochemical and Antinociceptive Properties of Matayba Elaeagnoides Radlk

Phytochemical and Antinociceptive Properties of Matayba elaeagnoides Radlk. Barks Michel Thomaz de Souzaa,b,Fa´tima de Campos Buzzia, Valdir Cechinel Filhoa, Sarai Hessa,b, Franco Delle Monachea, and Rivaldo Nieroa,*

a Programa de Mestrado em Cieˆncias Farmaceûticas e Nu´ cleo de Investigaço˜ es Quı´mico-Farmaceûticas (NIQFAR)/CCS, Universidade do Vale do Itajaı´ (UNIVALI), 88.302-202, Itajaı´, SC, Brazil. Fax: ++55(47)3341-7601. E-mail: [email protected] b Departamento de Farmaćia, Universidade do Contestado (UnC), 89.500-900, Campus Caçador, Caçador, SC, Brazil * Author for correspondence and reprint requests Z. Naturforsch. 62c, 550Ð554 (2007); received December 21, 2006/February 8, 2007 A mixture of triterpenes named lupeol (1), α-amyrin (2), -amyrin (3), and -sitosterol (4) has been isolated from the hexane fraction of Matayba elaeagnoides. In addition, scopoletin (5), umbelliferone (6), 3-O-d-glycopyranosyl-sitosterol (7) and betulin (8) were isolated from the chloroform fraction. All the structures were identified by spectroscopic techniques in accordance with literature data. The extracts (hydroalcoholic and methanolic) and some fractions (hexane, chloroform, ethyl acetate and butanol) exerted promising antinociceptive effects in mice. In addition, we have tested the pure compound betulin (8). When analyzed against induced pain using the writhing test (3Ð10 mg kgÐ1, i.p.), betulin showed a dose- Ð1 dependent effect with a calculated ID50 value of 7.74 (6.53Ð9.17) mg kg [17.5 (14.7Ð20.7) μmol kgÐ1] and a maximal inhibition (MI) of 58.3% in relation to the control group. When evaluated in the formalin test (3Ð10 mg kgÐ1, i.p.), this compound inhibited both phases of pain (neurogenic and inflammatory pain), with calculated ID50 values of 18.3 (17.7Ð18.9) and 8.3 (7.7Ð8.9) mg kgÐ1 [41.5 (38.4Ð42.7) and 18.8 (17.6Ð19.9) μmol kgÐ1] and maximal inhibition of 40.8 and 64.39% for the first and second phases, respectively. Using the same models, this compound was several times more active than two clinically used drugs, namely aspirin and paracetamol, suggesting that its main active principle is related to the antinociceptive effect found for the chloroform fraction of M. elaeagnoids barks. Key words: Matayba elaeagnoides, Antinociceptive, Betulin

Introduction et al., 2004). This study extends previous work carried out on this plant and describes the phyto- Sapindaceae consists of a large family of plants, chemical analysis and in vivo antinociceptive activ- with occurrence and distribution in distinct areas ity against two pain models in mice (acetic acid of the world, mainly in the tropical and subtropical and formalin) using the crude extract, fractions regions (Ferrucci, 2000; Guarim-Neto et al., 2000). and a pure compound isolated from M. elaeag- Several species have demonstrated different com- noides bark. pound classes such as saponins, terpenoids and fla- vonoids, which exhibit especially diuretic, stimu- lant, expectorant, anti-helmintic and anticancer Material and Methods properties (Arisawa et al., 1984; Mesquita et al., Plant material 2005). Matayba elaeagnoides, known as “camboata˜, camboata´ or cuvanta˜”, is frequently used in Barks of M. elaeagnoides (Sapindaceae) were folk medicine as an anti-inflammatory agent and collected in Cac¸ador Santa Catarina, Brazil in May to combat liver cancer (Lorenzi, 2000; Ribeiro, 2004 and identified by Dr. Ademir Reis (Depart- 2004). Recently, it was reported that it presents ment of Botany, Universidade Federal de Santa positive effects against inflammatory processes Catarina, Brazil). A voucher specimen was depos- and antiplasmodial activity in vitro against Plas- ited at Barbosa Rodrigues Herbarium (Itajaı´,SC, modium falciparum, associated with the presence Brazil) under number MTS 001. of matayosides AÐD (Mineo et al., 2004; Silva

0939Ð5075/2007/0700Ð0550 $ 06.00 ” 2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D M. T. de Souza et al. · Phytochemical and Antinociceptive Evaluation of Matayba elaeagnoides 551

Phytochemical analysis Writhing test Air-dried material from M. elaeagnoides (2.6 kg, The abdominal constriction, induced by intra- bark) was cut into small pieces and macerated peritoneal injection of acetic acid (0.6%), was car- with methanol at room temperature for 10 d. ried out according to the procedures described After filtration, the solvent was removed by rotary previously by Collier et al. (1968) and Souza et al. evaporation under reduced pressure, yielding the (1998) with minor modifications. The animals were respective methanolic extract (ME). Additionally, pretreated with extract, fractions or compound 8 a hydroalcoholic extract (HE) was prepared by (3Ð10 mg kgÐ1, i.p.) 30 min before the acetic acid maceration with an ethanol/water mixture (1:1) injection. Control animals received a similar vol- and stored until pharmacological test. The metha- ume of 0.9% (10 ml kgÐ1, i.p.). All experiments nolic extract (220.0 g) was then suspended in were carried out at (23 ð 2) ∞C. After challenge, MeOH/water (9:1) and successively partitioned pairs of mice were placed in separate boxes and with hexane, chloroform, ethyl acetate and bu- the number of constrictions of the abdominal mus- tanol, furnishing the respective fractions. The hex- cles and stretching were counted cumulatively ane fraction (6.85 g) was chromatographed on a over a period of 20 min. Antinociceptive activity silica gel column eluted with a hexane/acetone was expressed as the reduction of the number of mixture with increasing polarity. The chloroform abdominal constrictions comparing control and (1.2 g) and ethyl acetate (3.6 g) fractions were pre-treated animals. chromatographed separately on a silica gel column eluted with a mixture of chloroform/methanol Formalin-induced nociception with increasing polarity. Similar fractions, which The procedure used was essentially similar to showed positive reaction with anisaldehyde/sulfu- that previously described (Hunskaar and Hole, ric acid reagents and short-wave UV light, were 1987). Animals from the same strain were used combined and re-chromatographed as described and 20 μl of 2.5% formalin solution (0.92% form- above, yielding a mixture of terpenes, steroids and aldehyde), made up in a phosphate-buffered solu- coumarins. The compounds were identified based α tion (137.0 mm NaCl, 2.7 mm KCl and 10 mm phos- on their spectral data as lupeol (1), -amyrin (2), phate buffer), were injected intraplantarly in the -amyrin (3), -sitosterol (4), scopoletin (5), um- right hindpaw. After injection, the time spent lick- belliferone (6), 3 -O-d-glycopyranosyl-sitosterol ing the injected paw was measured and considered (7) and betulin (8). The purity of all isolated sub- as indicative of pain. The initial nociceptive scores stances was examined by thin layer chromatogra- normally peaked 5 min after formalin injection phy (TLC) using Merck silica gel pre-coated alu- μ (first phase) and 15Ð30 min after formalin injec- minum plates, with a layer thickness of 200 m, tion (second phase), representing the neurogenic with several solvent systems of different polarities. and inflammatory pain, respectively. In an attempt to investigate the possible antinociceptive action Animals 3Ð10 mg kgÐ1, i.p., of compound 8 were used. Swiss mice (25Ð35 g), housed at (22 ð 2) ∞C under a 12 h light/12 h dark cycle and with access to Statistical analysis food and water ad libitum, were acclimatized to The results are represented as mean ð S.E.M., the laboratory for at least 1 h before testing. For except for the ID50 values (i.e., the dose that re- each experiment, one group of 6Ð8 animals was duced responses by 50% in relation to control val- used. The experiments reported here were carried ues), and presented as geometric mean and their out in accordance with the current ethical and care respective 95% confidence limit. The ID50 values guidelines for the care of laboratory animals and were determined by linear regression GraphPad. the investigation of experimental pain in conscious Statistical significance between groups was calcu- animals (Zimmermann, 1983). The experiments lated by means of analysis of variance followed were approved by the local Ethics Committee of by Newman-Kuels’ multiple comparison tests. P- this institution (number 412/06-UNIVALI). The values less than 0.05 were considered as indicative number of animals and intensities of noxious stim- of significance. uli used were the minimum necessary to demon- strate consistent effects of the drug treatments. 552 M. T. de Souza et al. · Phytochemical and Antinociceptive Evaluation of Matayba elaeagnoides

Results and Discussion they were separately chromatographed on a silica Recently, many plants have received special at- gel column eluted with a hexane/acetone gradient, tention as sources of new antinociceptive agents monitored by TLC, yielding lupeol (1), α-amyrin (Calixto et al., 1998; Patocˇka, 2003; Oliveira et al., (2), -amyrin (3), -sitosterol (4), scopoletin (5), 2005; Lima-Junior et al., 2006; Abdel, 2006). De- umbelliferone (6), 3-O-d-glycopyranosyl-sito- spite its common use in folk medicine to treat vari- sterol (7) and betulin (8), which were directly com- ous pathologies associated with dolorous proc- pared with authentic samples and spectroscopic esses, it was observed a lack of literature on data (IR, 1H NMR and 13C NMR). Although the experimental studies concerning the analgesic ethyl fraction presented promising antinociceptive properties of M. elaeagnoides. On the other hand, activity (Table I), we obtained a limited quantity some plants belonging to the Matayba genus have of material for separation and purification of other exhibited important antiplasmodial activity (Mes- active principles. However, the chromatographic quita et al., 2005). profile by TLC demonstrated the strong presence Table I shows that hydroalcoholic and methano- of phenolic compounds when revealed with spe- lic extracts and different fractions caused the pro- cific reagents, whose studies will be continued for nounced effect when analyzed against the writhing further publications. model in mice. As can be observed, hydroalcoholic It is well documented that 1Ð7 present distinct and methanolic extracts as well as hexane, chloro- pharmacological properties particularly anti-in- form, ethyl acetate and butanolic fractions showed flammatory and analgesic effects and for this rea- antinociceptive activity, with MI values of 71.1, son, they were not included in this study (Santos 54.6, 46.4, 46.4, 66.0 and 25.7%, respectively, at et al., 1995; Delira et al., 2003; Patocˇka, 2003; Ot- 10 mg kgÐ1, i.p. Considering that hexane and chlo- uki et al., 2005; Oliveira et al., 2005; Lima-Junior roform fractions demonstrated a better chromato- et al., 2006; Reyes et al., 2006; Meotti et al., 2006). graphic profile and a good antinociceptive effect, However, the antinociceptive profile of 8, to the

Table I. Antinociceptive activity of extracts and fractions of M. elaeagnoides against acetic acid-induced abdominal constrictions in mice, in comparison with aspirin and paracetamol (10 mg kgÐ1, i.p.).

Treatment MI (%)

Methanolic extract 54.6 ð 2.8 Hydroalcoholic extract 71.1 ð 1.9 Hexane fraction 46.4 ð 1.5 Ethyl acetate fraction 66.0 ð 0.6 Chloroform fraction 46.4 ð 0.4 Butanolic fraction 25.7 ð 2.4 Fig. 1. Effect of compound 8 (3Ð10 mg kgÐ1, i.p.) against Paracetamola 38.0 ð 1.0 acetic acid-induced abdominal constrictions in mice. Aspirina 35.0 ð 2.0 Each column represents mean ð S.E.M. of six experiments. Significance levels when compared with the con- a Bresciani et al. (2003). trol group: **p Ͻ 0.01; *p Ͻ 0.05.

Fig. 2. Effect of compound 8 (3Ð10 mg kgÐ1, i.p.) against formalin-induced pain in mice; (A) first phase (0Ð5 min); (B) second phase (15Ð30 min). Each column represents mean ð S.E.M. of six experiments. Significance levels when compared with the control group: **p Ͻ 0.01; *p Ͻ 0.05. M. T. de Souza et al. · Phytochemical and Antinociceptive Evaluation of Matayba elaeagnoides 553 best of our knowledge, is being evaluated for the first time. Figs. 1 and 2 show the result of 8, analyzed against writhing and formalin models. As can be noted, in the writhing test, 8 caused signifi- cant dose-dependent inhibition, with a calculated Ð1 ID50 value of 7.74 (6.53Ð9.17) mg kg [17.5 (14.7Ð20.7) μmol kgÐ1] and a maximum inhibition of 58.33% (Fig. 1). Although the writhing test is a non-specific model, it is widely used in the search for synthetic and natural compounds because it in- volves local peritoneal receptors, which are gener- Fig. 3. Effect of anti-inflammatory activity of compound ally associated with prostanoids, increasing the lev- 8 in formation of oedema in the formalin test. Each column represents mean ð S.E.M. of six experiments. Sig- els of PGE2 and PGF2a in peritoneal fluids nificance levels when compared with the control group: (Vongtau et al., 2004). **p Ͻ 0.01; *p Ͻ 0.05. When analyzed in the formalin test, 8 caused dose-dependent inhibition in both phases of pain (Hunskaar et al., 1985; Souza et al., 1998). By an- by the systemic route (Fig. 2). The calculated ID50 values were 18.3 (17.7Ð18.9) and 8.3 (7.7Ð8.9) mg tagonizing the formalin-induced hindpaw oedema kgÐ1 [41.5 (38.4Ð42.7) and 18.8 (17.6Ð19.9) μmol was observed suggesting an associated anti-inflam- kgÐ1] with maximal inhibition of 40.8 and 64.39% matory effect (Fig. 3). The factor by which this for the first and second phases, respectively. When compound inhibits such an inflammatory oedema compared with aspirin and paracetamol [123.0 induced by irritants could be the modulation of (77.0Ð209.0) and 120.0 (90.0Ð161.0) μmol kgÐ1, the liberation or blocking of B2 and prostaglandin respectively], two reference drugs, compound 8 receptor systems (Correˆa and Calixto, 1993). was about 6-fold more active. It is interesting to For the first time, the extract, fractions and pure note that the reference drugs practically prevented compounds obtained from M. elaeagnoides that only the inflammatory effects (second phase). produced antinociception against the acetic acid- However, 8 also inhibited the neurogenic pain induced visceral nociceptive response were distin- (first phase), being about 2-fold more active com- guished, supporting the ethnomedical use of this pared to the inflammatory pain (second phase), plant. Experimental studies are currently in suggesting that other mechanisms could be in- progress in order to determine the other active volved. The effect produced in the first phase may principles present in this plant. be due to immediate and direct effects on sensory receptors, bradykinin receptors or the glutama- Acknowledgements tergic way whereas for the last phase the antinoci- The authors are grateful to Dr. Ademir Reis for ceptive effect is related to the inflammatory re- the botanical identification, to CNPq, FAPESC- sponses induced by the arachidonic acid cascade SC, and UNIVALI/Brazil for financial support.

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