Antinociceptive and hypothermic evaluation of the leaf essential oil and isolated terpenoids from Eugenia uniflova L. (Brazilian Pitanga).

Abstract

Eugenia uniflora L. (Myrtaceae), known as Brazilian cherry tree, is a fruity tree spread all over Brazil used in popular medicine to treat inflammations, rheumatic pain and fever, as hypoglycemic, diuretic and has been widely used in the cosmetics industry. The present study discusses the chemical composition, the antinociceptive and hypothermic profile of the essential oil of pitangueira leaves. The chemical composition was evaluated by GC-MS and the main constituent of the oil was characterized, after isolation, as a mixture of atractylone (1) and 3-furanoeudesmene (2). The essential oil, its pentane fraction and the isolated mixture of sesquiterpenes (1 and 2), given orally, significantly inhibited the acetic acid-induced abdominal constrictions, increased the latency time in hot plate test and showed a hypothermic effect. The results suggest that the responsible for the antinociceptive and hypothermic effect were the isolated furanosesquiterpenes. These findings provided additional pharmacological information and may contribute for the use of Brazilian cherry tree as a phytomedicine.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Eugenia uniflora L.; Essential oil; Antinociceptive; Hypothermic; Furanosesquiterpenes

Introduction

In the pharmaceutical industry essential oils are employed as medicines or their coadjutants. In Brazil, examples of success are the essential oil obtained from Cordia verbenacea (Boraginaceae), an anti-inflammatory medicine for topical use (Acheflan[R]) and the clove oil (Syzygium aromaticum, Myrtaceae), very used in dental care as a sealing component and as anti-septic for mouth hygiene. As coadjutant, the essential oils are used to promote medicine absorption, increasing penetration in the epidermis due to its lipophylic characteristics (Cal 2006). C. verbenacea anti-inflammatory activity was recently associated to the presence of two sesquiterpenes, [alpha]-humulene and (-)-trans-caryophyllene (Fernandes et al. 2007).

Eugenia uniflora L. (Myrtaceae), known as Brazilian cherry tree (or pitangueira), is a fruity tree spread all over Brazil. Their leaves are used in infusions or decoctions in popular medicine to treat inflammations, against rheumatic pains and fever, as hypoglycemiant, diuretic and to avoid stomach problems (Auricchio and Bacchi 2003). The hydroalcoholic extract from the leaves decrease the levels of the enzyme xanthine oxidase, supposed to be involved in the development of gout, besides showing vasorelaxing, antioxidant, antidiarrhoeal, hypoglycemic, hypotriglyceridemic and bactericide properties, this last one also observed in the essential oil (Ogunwande et al 2005).

Brazilian cherry tree leaves essential oil has been used by the Brazilian cosmetic industry due to its astringent properties, associated to its pleasant smell. Its main application is in shampoos, hair conditioners, face and bath soaps, body oils and perfumes.

Several works have described the chemical composition of the oil and the main compounds identified were seline-1,3,7(11)-trien-8-one and its oxide, germacrone, furanodiene and curzercne (Weyerstahl et al. 1988). There is a considerable variety in the concentration of these constituents when comparing essential oils from different origins and even a lack of these compounds. Brazilian cherry tree oils from the south of Brazil and Argentine have monoterpenes as major constituents (Henriques et al. 1993). Concentration changes of these metabolites seem to be due to chemotypes, geographical origin, seasonality and to the use of different oil extraction methods (Margis et al, 2002).

The Brazilian cherry tree is mentioned in the forth edition of the Brazilian Pharmacopoeia (2003) and the leaves are in the list of medicinal plants authorized by the Ministry of Health (ANVISA) to prepare infusions, as can be seen in the Brazilian Resolution number 267 (2005).

In view of that, the present study describes the evaluation of the antinociceptive and hypothermic profile of the oil obtained from Brazilian cherry tree leaves in order to confirm the pharmacological activities and contribute for the use of the essential oil of the leaves of E. uniflora as a phytomedicine.

Materials and methods

Plant material

Voucher specimen # RB 275960 was deposited in the Botanic Garden of Rio de Janeiro, RJ, Brazil.

Leaf harvesting

Brazilian cherry tree leaves were harvested from trees of the garden of the Chemistry Institute of the Federal University of Rio de Janeiro, in an area of 80 [m.sup.2] during one week, in February of 2006 in morning time (from 8 to 8:30h a.m.).

Essential oil extraction, isolation and identification

All solvents used were of analytical grade. The chemical composition of the crude essential oil, fractions and isolated products from Eugenia uniflora L. leaves was evaluated by GC-MS.

Extraction

The essential oil was extracted by hydrodistillation using a modified Clevenger apparatus adapted to a condenser at 5 (+ or -2)[degrees]C. The fresh leaves (150 g) were cut with scissors into small parts and put in a 2000 ml reaction flask with 1000 ml of distilled water. After 3 hours of extraction the organic phase was separated and dried over [Na sub 2][SO sub 4]. The distillations were performed in triplicate.

GC-MS and GC-FID

GC-MS equipment and ionization mode were the same as described in Melo et al. (2007). An HP-1 GC capillary column (30 m x 0.25 mm x 0.25 [micro]m, Agilent., PA, USA) was used to inject 2[micro]l of a solution of 2mg/ml of sample in[ CH.sub.2][C1.sub.2]. The oven temperature program was from 80[drgrees] C (4min) to 240 [degrees]C at 2[degrees]C/min and from 240[degrees]C to 270[degrees]C (5min) at 5[degrees]C/min, injector (spiritless, 0.5 min), and transfer line temperature were hold at 270 and 290[degrees]C, respectively. He (l.0ml/min) was used as carrier gas. Reference literature (Adams 1995) and standards were co-injected in order to identify the chemical constituents. The same temperature program was used for CG-FID.

The specific rotation of Eugenia uniflora essential oils obtained from fresh leaves was analyzed in a JASCO apparatus (UK). The oils were dissolved in [CH.sub.2][CI.sub.2] (10%, w/v) using a 3.5 cm x 50 mm cell at 25 [degrees]C. Each oil was analyzed ten times.

Preparation of E. uniflora essential oil fractions

The essential oil was submitted to an open silica gel column chromatography impregnated with a KOH methanolic solution as described in detail by Pinto et at (2000) for Copaifera cearensis.

Isolation and characterization of the major compounds

The essential oil obtained from the E. uniflora leaves was kept in the freezer for one week, at--8 [degrees]C. The white crystals obtained were filtered and washed with cold methanol (0 [degrees]C). GC-MS showed one peak with IRL of 1467 calculated in relation to a linear n-hydrocarbon mixture. The major compounds, the furanosesquiter-penes atractylone (1) and 3-furanoeudesmene (2) were identified by (1) H NMR, (l3) C NMR and MS.

Pharmacological activities

Experiments were carried out in albino Swiss mice (20-25g) of both sexes from LASSBio (Faculty of Pharmacy, UFRJ, Brazil) breeding unit. All animals were kept in standardized conditions, maintained only with water ad libitum for 12 hours before the experiment. Animal experiments were performed according to the "Principles of Laboratory Animal Care and Use in Research" (Colegio Brasileiro de Experimentacao Animal-COBEA/Instituto de Biofisica Carlos Chagas Fil-ho- [IBCCF sup o], Brazil), based on international guidelines for the care and use of laboratory animals and ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann 1983).

The crude essential oil, fractions and isolated products were administered orally (0.1 ml/20 g) as a suspension in tween 80:ethanol:[H sub 2 O] (1:1:10) (vehicle). The crude essential oil and the pentane fraction were administered at the doses of 50, 100, 200 e 500 mg/kg. The dichloromethane and methanol fractions were administered at a dose of 200 mg/kg and the major sesquiterpenes at l00 mg/kg. Indomethacin (l0 mg/kg) and morphine (5 mg/kg) were used as positive controls when appropriate.

Acetic acid-induced abdominal constriction in mice

The peripheric antinociceptive activity was determined in vivo using the mouse abdominal constriction test induced by acetic acid 0.6% (0.l ml/10g; i.p.) (Ribeiro et al. 1998). Essential oil and fractions were administered one hour before the noxious stimulus. Ten minutes after i.p. injection of the acetic acid the number of constrictions per animal was recorded for 20 minutes. Control animals received an equal volume of vehicle.

Hot plate test

Central antinociceptive activity was investigated using the hot plate test as described earlier (Kuraishi et al. 1983). In these experiments, the hot plate apparatus (Ugo Basile, Model-DS 37) was maintained at 56[+ or -]1 [degrees]C. Mice were placed on the heated surface and the time between placement and the first sign of paw licking or jumping was recorded as latency. Latencies were recorded at 0, 30, 60, 90 and 120 min after oral administration. The basal latencies were found to be 5-7 s. A cut-off time of 30 s was followed to prevent any injury to the paws.

Hypothermic activity

Rectal temperature (TR) was measured by inserting a lubricated thermistor probe (external diameter: 3 mm) 2.8 cm into the rectum of the animal. The probe was linked to a digital device, which displayed the temperature at the tip of the probe with a 0.1 [degrees]C precision. The values displayed were manually recorded. Animals were orally administered immediately after measurement of their basal rectal temperature. T R changes were recorded every hour up to 3h, and expressed as the difference from the basal value (Souza et al. 2002).

Statistical analysis

Results are expressed as mean [+ or -] SEM of "n" animals per group and the activities expressed as percentage of inhibition when compared with the vehicle control group. All data were statistically analyzed by the Student's t test or ANOVA One-way (Dunnett's post-test) for a significance level of *p<0.05. When appropriated, the [ED sub 50] values (i.e. the dose able to elicit 50% of the maximum effect observed) were determined by non-linear regression using GraphPad Prism software.

Results and discussion

The essential oils from Brazilian cherry tree leaves were analyzed by GC-MS with the aid of standards co-injection and retention index calculations. Accordingly to previous studies on the chemical composition of E. uniflora oil, sesquiterpenes were the major compounds (Weyerstahl et al. 1988).

The MS fragmentation pattern of the chemical constituents, their molecular weight range, linear retention index (LRI) and standards co-injection permitted the identification of three main classes of sesquiterpenes in the oil: hydrocarbon, monohydroxylated and furanoid derivatives. Most of the hydrocarbons were characterized by the molecular ions at m/z 204 e 202 followed by methyl loss, besides the characteristic sesquiterpene fragments. For the monohydroxylated compounds, molecular ions in the range of m/z 222 to 220 (when visible) were typical and ions rearrangement due to the water loss, fragmentation due to methyl loss and both were also observed. For the furanoids, the molecular ions at m/z 218 e 216, together with ion at m/z 108 due to the retro-Diels Alder rearrangement of furan rings with neighbor methylene groups were used for this class characterization. Minor peaks due to di e tri-oxygenated sesquiterpenes were observed after 42 min, with molecular ions in the range of m/z 230 to 250. This classification allowed the inclusion of up to 95% of the substances detected in GC-MS.

By GC-MS, the major constituent in the oils was a furanosesquiterpene with LRI of 1467. The other sesquiterpenes observed in great amount in the fresh leaves oils were spathulenol, [beta]-elemene, [lambda]-elemene, globulol, ledene and [beta]-caryophyllene.

In order to obtain fractions enriched with the main classes of sesquiterpenes searching for compounds responsible for the pharmacological properties, Brazilian cherry essential oil was submitted to an open column chromatography by two methodologies. First, it was eluted with pentane, dicloromethane and methanol in a silicagel column, which failed to concentrate on the sesquiterpenoids. Based on our previous successful experience with KOH impregnated silica gel column chromatography applied to copaiba oils (that also have furanoterpenes) (Pinto et al. 2000; Gomes et al. 2008), Brazilian cherry oil was submitted to this methodology and showed that the most apolar fraction (obtained with pentane) gave hydrocarbon sesquiterpenes and furano-sesquiterpenes in 56% (w/w). Monohydroxysesquiter-penes were enriched in the dichloromethane fraction (26% w/w) and, after neutralization of the potassium methanolic fraction with aqueous HCl, monohydroxy-and most oxidated sesquiterpenes were concentrated (8% w/w), summing up to 89% of the oil. Diazomethane/ethyl ether derivatization did not suggest acidic compounds in the oil.

To identify the major furanoid compound, the essential oil and the pentane fraction were kept in the freezer from which the white crystals obtained were recrystalized with cold methanol. By GC-FTD the substance showed a LRI of 1467 and a peak purity of 95%. [1.sup.H] and [13.sup.CNMR] analyses showed the presence of two compounds (1 e 2), in proportion of 2:1, that were identified as atractylone (1) and 3-furanoeudesmene (2) (Fig. 1), according to Adams (1995), Bagal et al. (2004), Gavagnin et al. (2003) and Blay et al. (1996). Copies of the original spectra can be obtained from the author for correspondence. The physical and chemical data agree with these reported in the literature.

[FIGURE 1 OMITTED]

Atractylone is found in rhizomes like Atractylodes, which species A. macrocephala is used in Chinese traditional medicine for stomach problems as antiinflammatory and anti-cancer (Yosioka et al. 1976; Guo et al. 2006). Its chemical structure had been characterized both in natural and synthetic origin material (Minato and Nagasaki 1965). The furanoeudesmane (2) was already isolated from the Antarctic gorgonian Dasystenella acanthine (Gavagnin et al. 2003).

The infusion of E. uniflora L. leaves is used in popular medicine as anti-inflammatory, against rheumatic pain and fever among other activities. Schapoval et al. (1994) described the anti-inflammatory activity for infusions and decocts of fresh and dry leaves but they did not observe an antinociceptive effect even at a dose of 300 mg/kg.

The antinociceptive and hypothermic activities of E. uniflora L. leaves essential oil have never been measured. The antinociceptive activity in mice constrictions test is illustrated in Fig. 2A. Considering the effect showed for the essential oil, inhibiting the constrictions by 48% orally at a dose of 200 mg/kg, the crude oil was fractionated and tested for antinociceptive activity. The pentane fraction was the only to shown an important and significant antinociceptive effect, inhibiting the constrictions by 70% at the same dose (Fig. 2A). The antinociceptive effect for both essential oil and pentane fraction was dose-dependent and the [ED.sub.50] values were 218.6 mg/kg and 176.7 mg/kg, respectively. The pentane fraction showed to be more potent than the crude oil.

[FIGURE 2 OMITTED]

As the pentane fraction was the most active, we decided to identify and to isolate its major constituents, in view to determine which compounds were responsible for the observed effects. From the results, we can infer that the antinociceptive effect was associated to the furanosesquiterpenes (1) and (2), mostly found in the pentane fraction, that were able to inhibit the constrictions at the same magnitude (70%) of the pentane fraction with half the dose, showing to be much more active when comparing the effects observed for the same dose employed (l00mg/kg) (Fig. 2B). It is the first time that an analgesic effect is reported to atraclylone (1) and to 3-furanoeudesmene (2).

Dolara et al. (1996) described the in vivo analgesic effect on the hot plate and abdominal constrictions tests for two furanosesquiterpenes isolated from mirra (C molmol), curzerene and furanodiene that was mediated by the interaction with opioid receptors. In view of that, the effect of the essential oil (200mg/kg), the pentane fraction (200 mg/kg) and the mixture of the furanosesquiterpenes (1) and (2) (100 mg/kg) from Brazilian cherry tree was evaluated in the hot plate test (Fig. 3). They all increased significantly the latency time in the hot plate. As well as the observed in the abdominal constriction test, the isolated products were more effective at a half of the dose of the oil and fraction, increasing the latency from 30min, suggesting that the major sesquiterpenes were also the responsible for the central antinociceptive activity observed.

[FIGURE 3 OMITTED]

Infusion of leaves of E uniflora was usually used against fever and there are no reports confirming this activity. In this study we evaluated the ability of the essential oil and the mixture of the furanosesquiterpenes (1) and (2) to diminish the body temperature. Both presented a hypothermic effect, lowering significantly the normal body temperature up to three hours later (Fig. 4). These results reinforce the effect of the furanosesquiterpenes (1) and (2) in the central nervous system, suggesting that induced-hypothermia involves centrally mediated mechanisms that must be further investigate and in some ways confirm its popular use. Dypirone also presented a hypothermic and anti-pyretic effects probably by different mechanisms of action that are not fully understood (Souza et al. 2002).

[FIGURE 4 OMITTED]

This paper reports for the first time the antinociceptive and hypothermic activities of the essential oil of the leaves of Brazilian cherry tree or pitangueira and of the furanosesquiterpenes, atractylone (1) and 3-furanoeu-desmane (2) that are orally effective. These findings provide additional pharmacological evidences for the popular use of the leaves of Brazilian cherry tree and may contribute for its use as phytomedicine.

Acknowledgments

The authors would like to thank International Flavors and Fragrances-IFF for providing sesquiterpenes for co-injection and also Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Br), CNPq (Br) and CAPES (Br) for financial support and fellowships.

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Ana Carolina L. Amorim (a), Cleverton Kleiton F. Lima (b), Ana Maria C. Hovell (a), Ana Luisa P. Miranda (b), *, Claudia M. Rezende (a)

(a) Instituto de Quimica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, Sala 626-A, Cidade Universitaria, Itha do Fundao, 21945-970 Rio de Janeiro, RJ, Brazil

(b) Laboratorio de Avaliacao e Sintese de Substancias Bioativas (LASSBio), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Bloco Blss, Sala 22, Cidade Universitaria, Itha do Fundao, 21941-902 Rio de Janeiro, RJ, Brazil

* Corresponding author. Tel./fax: [phone].

E-mail address: [e-mail] (A.L.P. Miranda).

0944-7113/$-see front matter [c] 2009 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2009.03.009