Anti-inflammatory activity of essential oil from leaves of Myrciaria tenella and Calycorectes sellowianus

Abstract

The GC-MS analysis revealed that the leaf essential oils of Myrciaria tenella (DC.) Berg and Calycorectes sellowianus O. Berg (Myrtaceae) were composed of 34 and 37 compounds, respectively. The main constituents of M. tenella oil were β-caryophyllene (25.1%), and spathulenol (9.7%), while for C. sellowianus were guaiol (13.1%) and β-caryophyllene (8.6%). The anti-inflammatory effect of both essential oils was investigated in vitro and in vivo. Both oils reduced significantly (p < 0.005) the treated neutrophils chemotaxis with 93% and 91% inhibition for M. tenella and C. sellowianus, respectively. However, in the systemic treatment with the essential oils (50 mg/kg p.o.) only the M. tenella oil was able to significantly reduce the carrageenan-induced paw edema with a similar effect to that observed for indomethacin (10 mg/kg), the positive control.

Keywords::

• Myrciaria tenella
• Calycorectes sellowianus
• Myrtaceae
• essential oil
• anti-inflammatory activity

Introduction

The Myrtaceae is one of the most important families in Brazil, and it is the dominant woody family in the southeastern Atlantic rainforest (Tabarelli & Mantovani, 1999). The leaves of several species from this family have been used in folk medicine for their interesting bioactivities, such as antidiarrhetic, antibacterial, vermifuge and anti-inflammatory properties. These activities have been related to the presence of tannins and terpenoids (Schapoval et al., 1994; Slowing et al., 1994; Apel et al., 2002; Kim et al., 2003).

Myrciaria tenella (DC.) Berg (vernacular name: “cambium, camboí or camboizinho”) is a small tree with edible fruits, that grows up to 4-6 m high. It occurs in the forests from Amazon to Rio Grande do Sul in Brazil, spreading throughout Argentina, Paraguay and Peru. The phytochemical screening and mutagenic activity of M. tenella leaves has been previously reported (Alice et al., 1991; de Sa Ferreira & Vargas, 1999). The composition of the essential oil of leaves and thin branches of this species, growing in Para and Rio Grande do Sul, Brazil, was found to be rich in sesquiterpenes, with a predominance of β-caryophyllene for both collections (Apel, 2001; Andrade et al., 2006).

Calycorectes O. Berg is a genus that occurs from South America to Mexico and it is closely related to Eugenia Mich. ex L. Calycorectes sellowianus O. Berg is a small tree growing in the Brazilian coastal forests. The essential oil from two Calycorectes species, C. australis D. Legrand and C. psidiiflorus (O. Berg) Sobral, collected in Rio Grande do Sul (Brazil) has been previously characterized as rich in sesquiterpenes (Apel et al., 2006). However, to the best of our knowledge, this is the first report of chemical studies of C. sellowianus.

In this study the chemical composition profile of the essential oils obtained from Myrciaria tenella and Calycorectes sellowianus leaves were analyzed by gas chromatography coupled to mass spectrometry (GC-MS), and their anti-inflammatory effects was investigated in vitro, by the leukocyte migration method, and in vivo, with carrageenan-induced paw edema in rats.

Materials and methods

Plant material

Myrciaria tenella leaves were harvested in July 2004 at the Estação Ecológica and Experimental Fazenda Campininha (Mogi-Guaçu, São Paulo, Brazil). Calycorectes sellowianus leaves were collected in December 2004 at the Parque Estadual da Serra do Mar-Núcleo Caraguatatuba (São Paulo, Brazil). The voucher specimens (Moreno 54 and Cord 2976, respectively) were deposited in the Herbarium of the Instituto de Botânica of São Paulo.

Essential oil hydrodistillation

Leaf essential oils from Myrciaria tenella (156 g) and Calycorectes sellowianus (176 g) were obtained by hydrodistillation using a Clevenger-type apparatus, for 4 h. The yield (v/w) of essential oil was determined based on the dry leaf weight. The obtained oils were stored in sample vials and kept frozen at −5°C until analysis by gas chromatography (GC) and GC mass spectrometry (GC-MS).

Essential oils analysis

Analysis of the oil was performed using a Shimadzu GC-17A equipped with Shimadzu GC 10 software, using a DB-5 fused silica capillary column (25 m, 0.25 mm; film thickness 0.25 μm). The oven temperature was programmed from 60°C to 300°C at 3°C/min, with helium as carrier gas at an inlet pressure of 80 kPa (1 mL/min). Injector and detector temperatures were set at 220° and 250°C, respectively. The percentage compositions were obtained from electronic integration measurements using flame ionization detection (FID, 250°C).

The sample was analyzed by GC/MS in the same chromatographic conditions as described above, using a Shimadzu QP-5000-quadrupole MS system, operating with ionization energy of 70 eV and an interface temperature of 250°C.

Identification of the constituents

Identification of essential oils components was achieved based on the retention indices, determined relative to the retention times of a series of n-alkane standards (Sigma), by comparison of their mass spectral fragmentation patterns (National Institute of Standards and Technology (NIST) database), and with reference to published data (Apel, 2001; Apel et al., 2006; Adams, 2001).

Anti-inflammatory activity

Chemotactic migration

Chemotactic migration was measured by the method described previously by Zigmond and Hirsch (1973). Prior to the chemotactic assay, rat leukocytes were treated with 10 μg/mL of essential oil emulsion (essential oils were dispersed in Hanks buffer and Tween 80 (9:1 v/v) to a final volume of 1 mL) at 37°C for 1 h. Plasma collected from rats was incubated at 37°C for 30 min with 65 µg/mL of lipopolysaccharide (from Escherichia coli). The plasma was diluted in Hanks buffer at 20% concentration (v/v). Chemotactic migration of leukocytes through an 8 μm nitrocellulose filter towards the chemotactic stimulant (lipopolysaccharide treated plasma) was measured after incubation for 1 h at 37°C using the micrometer on the fine-focus knob of the microscope. The distance from the top of the filter to the farthest plane of focus still containing two cells in five microscopic fields allowed the evaluation of leukocyte migration. All experiments were carried out in duplicate.

Animals

Male Wistar rats (180–220 g) were obtained from the Breeding Laboratory, Universidade Federal do Rio Grande do Sul, Brazil. The animals were housed five per cage with free access to food and water, and were maintained on a 12 h light/dark cycle (lights on at 07:00 h), at a temperature of 23 ± 1°C.

Carrageenan-induced paw edema in rats

Anti-inflammatory activity was evaluated by the carrageenan-induced rat paw edema test, as described by Winter et al. (1962). A 9:1 (v/v) emulsion of the essential oils with Tween 80 in saline was administered orally 1 h before subplantar injection of carrageenan using a single dose of 50 mg/kg body weight for each sample. The control groups received equivalent volumes of the vehicle. Male Wistar rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and injected subplantarly into one of the hind paws with 0.1 mL of 0.5% λ-carrageenan type IV solution in isotonic saline (Sigma, St. Louis, MO). The contralateral paw was injected with 0.1 mL saline solution and used as control. Edema was measured using a digital plethysmometer Ugo Basile (model 7140, City of Comerio, Italy) at 1, 2, 3, and 4 h after carrageenan injection. Edema volume was expressed for each animal as the percentage change in rat paw volume after carrageenan injection, compared with placebo group. The activity was compared with the effect of indomethacin administration (10 mg/kg, p.o.; Sigma).

Statistical analysis

Data analysis results are expressed as the mean ± SEM, and were tested for significance using Student’s t-test. Probability values (P) of less than 0.05 were taken to indicate statistical significance.

Results and discussion

Chemical compositions of essential oil

The hydrodistillation of the leaves from Myrciaria tenella and Calycorectes sellowianus afforded pale yellow oils, with yields of 0.4% and 0.46% (v/w), respectively. The oils were analyzed by GC-MS, and the compounds identified in both oils are listed in Table 1. Thirty-four and 37 compounds were identified in the leaves, respectively, representing 92.1% and 87.8% of the total oil. Both oils presented a qualitatively similar composition; however, they differed quantitatively from each other. For both species, the chemical profile showed the predominance of sesquiterpenes (Figure 1). The main constituents of M. tenella oil were sesquiterpenes with caryophyllane and aromadendrane cluster, β-caryophyllene (25.1%), and spathulenol (9.7%). The same pattern has been previously observed for other M. tenella specimens, the oil from a specimen collect in Para showed the predominance of β-caryophyllene (32%) (Andrade et al., 2006), and in a specimen from southern Brazil the main constituents were bicyclogermacrene (12.3%), β-caryophyllene (8.2%) and globulol (9.1%) (Apel, 2001).

Table 1.  Constituents of the essential oil from leaves of Calycorectes sellowianus and Myrciaria tenella (Myrtaceae).

RI	Constituents	C. sellowianus	M. tenella
Monoterpene hydrocarbons		0.4	2.1
895	α-Pinene	0.1	1.5
934	β-Pinene	-	0.6
987	Limonene	0.3	-
Sesquiterpene hydrocarbons		28.3	54.8
1297	δ-Elemene	1.9	-
1333	α-Copaene	0.4	3.2
1342	β-Boubornene	1.4	1.0
1350	β-Elemene	2.7	2.2
1376	β-Caryophyllene	8.6	25.1
1384	β-Gurjunene	0.4	-
1392	Aromadendrene	0.3	1.0
1397	α-Guaiene	0.3	-
1407	α-Humulene	1.4	4.2
1414	allo-Aromadendrene	0.5	1.6
1431	γ-Muurolene	2.8	1.5
1435	Germacrene D	0.7	0.7
1440	β-Selinene	1.0	2.8
1449	Viridiflorene	1.6	-
1450	α-Selinene	-	4.4
1455	α-Muurolene	0.7	0.6
1459	Germacrene A	-	0.4
1465	β-Bisabolene		3.1
1468	γ-Cadinene	0.8	0.6
1477	δ-Cadinene	1.6	1.8
1510	Germacrene B	0.7	-
Oxygenated sesquiterpenes		71.3	43.3
1504	Elemol	0.6	0.4
1514	E-nerolidol	0.4	-
1522	Ledol	0.8	0.3
1537	Spathulenol	6.6	9.7
1539	Caryophyllene oxide	4.4	3.4
1543	Globulol	4.1	5.9
1551	Guaiol	13.1	1.0
1560	Eudesmol**	-	2.5
1562	Humulene oxide I	3.1	-
1567	Humulene epoxide II	0.4	0.5
1574	1-epi-Cubenol	0.5	1.3
1581	Eudesmol**	0.6	1.5
1595	Caryophylladienol II	-	0.6
1593	iso-Spathulenol	7.3	0.7
1601	τ-cadinol	-	1.8
1604	τ-Muurolol	6.3	-
1608	α-Muurolol	2.1	0.6
1611	β-Eudesmol	1.0	-
1618	α-Cadinol	7.9	5.2
1631	Calamenenol**	-	0.5
1647	Khusinol	0.6	-
	Total	87.8	92.1

*RI, linear retention indices in a DB-5 column. **isomer not identified.

Figure 1.  Chemical profile of essential oils from Calycorectes sellowianus and Myrciaria tenella leaves, obtained by GC-MS in a DB-5 column.

C. sellowianus oil was characterized by the oxygenated sesquiterpene guaiol (13.1%) and sesquiterpene hydrocarbon β-caryophyllene (8.6%). To our knowledge, no previous chemical studies for this species are available. Two Calycoretes species, C. australis and C. psidiiflorus, had their essential oils analyzed and demonstrated to be also rich in sesquitepenes (Apel et al., 2006). However, in those species, the main components found were bicyclogermacrene (22.7% and 13.2%, respectively) and spathulenol (13.7% and 11.6%, respectively).

Anti-inflammatory activity of the essential oils from Myrciaria tenella and Calycorectes sellowianus

The anti-inflammatory activity of both essential oils was initially evaluated in vitro by the Boyden chamber assay (Zigmond & Hirsch, 1973). This method is based on a chamber containing two medium-filled compartments separated by a microporous membrane. In general, cells are placed in the upper compartment and are allowed to migrate through the pores of the membrane into the lower compartment, in which chemotactic agents are present. After an appropriate incubation time, the membrane between the two compartments is fixed and stained, and the number of cells that have migrated to the lower side of the membrane is determined. Treatment of polymorphonuclear neutrophils with 9 mg of M. tenella and C. sellowianus oils reduced significantly (P < 0.005) their migration towards the chamber filled with the lipopolysaccharide treated plasma in 93% and 91% of inhibition of the migration distance towards the lipopolysaccharide treated plasma compared with controls, respectively (Table 2). The essential oils inhibited the leukocyte migration in a rate comparable to that observed for the positive control, although the tested concentrations were a thousand times higher. This high concentration was employed because the samples were complex mixtures of terpenoids, not pure compounds.

Table 2.  Chemotaxis inhibition of the essential oils of Myrciaria tenella and Calycorectes sellowianus treated cells.

Sample	Migrated distance (μm) ± MSE (% inhibition)
Control	119.0 ± 2.58
M. tenella (9 mg/mL)	8.0 ± 1.79* (93.3)
C. sellowianus (9 mg/mL)	10.28 ± 3.55* (91.4)
Genistein (108 µg/mL = 400 µM)**	9.9 ± 1.00 (91.7)

*Each value is the mean ± MSE. Student’s t-test: *P ≤ 0.005;**Positive control.

Since both essential oils demonstrated a similar the anti-inflammatory activity in the in vitro assay, they were further evaluated in vivo by the inhibition of carrageenan-induced paw edema. In this assay, there was a gradual increase in the edema paw volume in the control group from 1 to 3 h after the injection, decreasing later in the fourth hour. The results presented in Figure 2 indicated that, at the dose of 50 mg/kg, the essential oil of M. tenella was able to significantly reduce the carrageenan-induced paw edema in rats since the first hour, with inhibitions varying by 61.3%, 58.2%, 61.1%, and 61.9% during the 1, 2, 3, and 4 h after the treatment, respectively. This oil was as effective as the non-steroidal anti-inflammatory drug indomethacin used as the positive control at the dose of 10 mg/kg. On the other hand, the administration of C. sellowianus essential oil in the same concentration did not elicit any significant effect in reducing the paw edema in comparison to the control group.

Figure 2.  Effect of pre-treatment with the essential oil of Myrciaria tenella and Calycorectes sellowianus (50 mg/kg, p.o., 1 h beforehand) on carrageenan-induced paw edema in rats. Indomethacin (10 mg/kg, p.o., 1 h beforehand) was used as positive control. Each point represents the mean of seven animals, and vertical lines show the MSE (Mean Standard Error). *The significance levels in comparison to control values: P < 0.05 (Student’s t-test).

Some plant constituents, particularly terpenoids, have been reported to be useful in the management of inflammatory processes (Agarwal & Rangari, 2003; Esteves et al., 2005). Among the terpenoids, β-caryophyllene is a sesquiterpene present in a large number of natural products. e.g. clove oil, cinnamon leaves, and copaiba balsam, all of which have been used as natural remedies and as fragrances. This compound is also known to have antimicrobial (Alma et al., 2003; Lourens et al., 2004), antioxidant (Lourens et al., 2004, Singh et al., 2006), anticarcinogenic (Kubo et al., 1996) and skin-penetration-enhancing properties (Cornwell & Barry, 1994). Essential oils containing this compound as a major component showed marked anti-inflammatory activity against carrageenan-and prostaglandin E₁-induced edema in rats as well as anti-arthritic activity (Martin et al., 1993; Baricevic et al., 2001; Agarwal & Rangari, 2003). Oral administration of β-caryophyllene to rats significantly inhibited gastric mucosal injuries induced by acidic ethanol (Tamble et al., 1996) and DSS-induced experimental colitis, being useful in the prevention and treatment of colitis (Cho et al., 2007). Moreover, recent studies considering the anti-inflammatory activity for isolated terpenes demonstrated the effects of β-caryophyllene and its oxide, caryophyllene oxide, on the inhibition of nitric oxide (NO) and prostaglandin E (PGE) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages (Tung et al., 2008). Thus, the presence of β-caryophyllene might be responsible for the potent anti-inflammatory effect observed in the Myrciaria tenella leaf oil, as it was the major oil component and it has already been tested for this activity in a pure form. However, one must consider the synergism among the different oil constituents, which can also contribute to the observed effect.

Declaration of interest

The authors are grateful to (FAPESP) Fundação de Amparo a Pesquisa do Estado de São Paulo; and (CNPq) Conselho Nacional de Desenvolvimento Científico e Tecnológico for their financial support.