Chemical composition and cholinesterase inhibition of essential oils of three chemotypes from Croton zehntneri

 

Hélcio S. Santos^a*, Elaine F. Furtado^a, Luciana M. Bertini^b, Paulo N. Bandeira^a, Maria R. J. Ribeiro Albuquerque^a, Jane E. S. Alencar Menezes^b, Maria Teresa S. Trevisan^b, Telma L. G. Lemos^b

 

^a Centro de Ciências Exatas e Tecnologia, Universidade Estadual Vale do Acaraú, Sobral–CE, Brazil. *Corresponding Author: Tel. 55–88–36774243, Fax. 55–88–36116342, Email: helciodossantos@gmail.com

^b Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60451–970 Fortaleza–CE, Brazil.

^c Departamento de Química, Universidade Estadual do Ceará, Itapipoca–CE, Brazil.

 

Received November 2009.
Accepted April 2010.

 

ABSTRACT

Croton zehntneri Pax. & K. Hoffm. (Euphorbiaceae) is an aromatic plant native of the northeaster region of Brazil; and is popularly known as "canela de cunhã". The chemical composition of the essential oils from leaves, stalks and roots from three chemotypes of C. zehntneri obtained by hydrodistillation were analyzes by GC–MS. E–anethole, was the main component in the essential oils of all plants parts of che–motype 1. While the phenylpropanoids stragole, eugenol, Z–methyl isoeugenol and E–methyl isoeugenol were the major component in the essential oils of chemotypes 2 and 3, respectively. On TLC the essential oils and the major compounds showed an acetylcholine esterase inhibitory effect.

Key words: Croton zehntneri, Euphorbiaceae, essential oils, phenylpropanoids, chemotypes, cholinesterase inhibition.

 

RESUMEN

Croton zehntneri Pax. & K. Hoffm. (Euphorbiaceae) es una planta aromática nativa de la región nororiental de Brasil, y es popularmente conocido como "canela de cunhã". La composición química de los aceites esenciales de hojas, tallos y raíces de três quimiotipos de C. zehntneri obtenido mediante hidrodestilación fueron examinados por GC–MS. E–anetol, fue principal componente en los aceites esenciales de todas las partes de la planta de quimiotipo 1. Si bien la fenilpropanóides estragol, eugenol, Z–metil isoeugenol and E–metil isoeugenol fueron el componente principal en los aceites esenciales de quimiotipos 2 e 3, respectivamente. El TLC estos aceites esenciales y los principales compuestos mostraron una acetilcolina esterase efecto inhibitorio.

 

INTRODUCTION

Croton is an extensive genus comprising around 1,300 species from Euphorbia–ceae family. This genus with wide range of bioactive compounds have been found to exert vasorelaxant activity (Baccelli et al., 2007). Popular uses include treatment of cancer, constipation, diabetes, digestive problems, dysentery, external wounds, fever, hypercholesterolemia, hypertension, inflammation, intestinal worms, malaria, pain, ulcers, and weight–loss (Salatino et al., 2007). Previous phytochemical investigations show that this genus possesses alkaloids (Murillo et al., 2001; Araujo–Junior et al., 2004), flavonoids (Peres et al., 1997; Maciel et al., 2000; Graikou et al., 2004), triterpenoids and steroids (Peres et al., 1998; Guadarrama et al., 2004), and a large number of diterpenoids (McChesney and Silveira, 1990; El Mekkawy et al., 2000; Barbosa et al., 2003; Giang et al., 2004; Santos et al., 2008; Santos et al., 2009). C. zehntneri (Euphorbiaceae) is an aromatic plant native in northeastern Brazil; and popularly known as "canela de cunhã". The specie is used in traditional medicine as sedative, appetite stimulating, antianorexigen and for the relief of gastrointestinal disturbances (Oliveira et al., 2001). The essential oil also acts as intestinal muscle relaxant (Coelho–de Souza et al., 1997, 1998), depressor central effect (Lazarini et al. , 2000) and antinociceptive (Oliveira et al., 2001). The literature reports chemical composition and larvicidal activity of the essential oil of leaves, stalk and inflorescences of chemotype 1 from C. zehntneri (Santos et al., 2007). Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognite impairment and personality changes. Inhibition of acetylcho–linesterase (AchE) serves as a strategy for the treatment of AD, senile dementia and Parknson's disease (Anonymous, 2000). Synthetic medicines as tacrine, donepezil, and the natural product–based rivastigmine used for treatment of cognitive dysfunction and memory loss associated with AD have their adverse effects including gastrointestinal disturbances and problems associated with bioavailability (Schulz, 2003), which necessitates finding better AChE inhibitors from natural resources. In recent years, essential oils and their monoterpene constituents have received much attention for their effects on AChE, e.g. essential oils from Melissa offcinalis, Rosmarinus officinalis and Salvia lavandulaefolia and monoterpenes such as geraniol, 3–carene, α–caryophyllene, limonene, sabinene, 1,8–cineole, α and β–pinene, γ–terpinene, bornyl acetate, geraniol, linalool, camphor, bor–neol, have been reported to inhibit AChE in vitro (Howes et al., 2003; Perry et al., 2003). The literature reports the antioxidant activities of the essential oil of leaves of chemotype 1 from C. zehntneri (Morais et al. , 2006), which is associated with the activation of lipoxygenases, which catalyse the formation of hydroperoxides of poly–unsaturated fatty acids (PUFAs); a hydroperoxide radical may react with fatty acids to produce dioxoenes, which are regarded as plant defence compounds (Spiteller, 1993). The antioxidant effects in essential oil may therefore have relevance in mammals, particularly in disorders involving oxidative stress such as AD. Besides the fact that phenylpropanoid E–anethole, the main component in all oils of chemotype 1 from C. zehntneri have been reported to inhibition AChE (Menichini et al., 2009). This work we report an evaluation of the cholinesterase inhibition effect, as well the chemical composition of essential oils from three chemotypes of C. zehntneri.

 

MATERIALS AND METHODS

Plant material

Leaves, stalks and roots of three chemo–types from C. zehntneri were collected in april and may 2008, in Ubajara and Croatá da Serra, Ceará State, Brazil. The plant material was identified by Dr. Edson Paula Nunes at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil, where the vouchers specimens (No. 42389, 42774 and 43048) was deposited.

Extraction of the essential oils

The fresh leaves (954 g, 327 g and 1000 g), stalks (1500 g, 1015 g and 700 g) and roots (198 g, 174 g and 186 g) of chemotypes 1, 2 and 3 from C. zehntneri were subjected to hydrodistillation in a Clevenger–type apparatus for 2 hours to afford leaves (0.80%, 0.90% and 0.84%), stalks (0.30%, 0.27% and 0.29%) and roots (0.60%, 0.06% and 0.14%) of pale yellow oils, respectively. The yields (w/w) were calculated based on the fresh weight of the plant materials. The isolated oils, after drying over anhydrous sodium sulfate and filtered, were stored in sealed glass vials and maintained under refrigeration before analysis.

Gas Chromatography–Mass Spectrometry

GC–MS for the analysis of the volatile constituents was carried out on a Hewlett–Packard Model 5971 GC/MS using a non–polar DB–5 fused silica capillary column (30 m x 0.25 mm i.d., 0.25 μm film thickness); carrier gas helium, flow rate 1 ml/min and with split mode. The injector temperature and detector temperature were 250 °C and 200 °C, respectively. The column temperature was programmed from 35 °C to 180 °C at 4 °C/min and then 180 °C to 250 °C at 10 °C/min. Mass spectra were recorded from 30 – 450 m/z. Individual components were identified by matching their 70 eV mass spectra with those of the spectrometer data base using the Wiley L–built library and two other computer libraries MS searches using retention indices as a preselection routine (Alencar et al., 1990), well as by visual comparison of the fragmentation pattern with those reported in the literature (Adams, 2001).

Measure the activity of acetylcholinesterase Solutions patterns

The following solutions were prepared: (1) 50 mM Tris–HCl, pH 8; (2) 1 mM 5,5'–dithiobis–(2–nitrobenzoic acid) (DTNB or Ellman's reagent) and (3) 1 mM acetylthiocoline iodide (ACTI). Lyophilized enzyme AChE was dissolved in buffer solution (1) to make 1000 U / mL stock solution, and further diluted with buffer solution (1) to get 3 U / mL enzyme (Rhee et al., 2001).

Test on Layer Chromatography (TLC) Samples (1.5 – 2.5 uL) were applied in CCD, DC–Alufolien, Silicagel 60 F254, 0.2 mm Merck. Spatter to the plate with solutions (2) + (3), leaving over 3 min. After drying, spraying is the enzyme 3 U/mL and in 10 minutes, appeared to be yellow. Where there was inhibition of the enzyme, there is a white halos. In about 20 to 30 minutes, the color disappeared (Rhee et al., 2001).

 

RESULTS AND DISCUSSION

The essential oils extracted from leaves, stalks and roots of three chemotypes from C. zehntneri were analysed by CG/MS and the constituents identified and quantified are summarized in Table 1. A total of 32 compounds were identified in the six sample oils and they are arranged in order of elution on a DB–5 column. As can be seen, the oils analysed were characterized by a high amount of phenylpropanoids (Figure 1). Sixteen constituents were identified in the oil from leaves (98.7%), stalks (94.4%) and roots (99.8%) from chemotype 1, the phenylpropanoid E–anethole 1 was the main component in all these oils. In the essential oils from chemotype 2 were identified twenty two constituents were identified in the oil from leaves (99.1%), stalks (97,4%) and roots (99.7%) the main component in the oil from leaves and stalks was eugenol 2, while Z–methyl isoeugenol 4 was the major constituent in the roots. In addition, fourteen constituents from leaves (92.2%), stalks (94.0%) and roots (99.6%) from chemotype 3, the main component in leaves was stragole 3, while Z–methyl isoeugenol 4 and E–methyl isoeugenol 5 were the major in stalks and roots, respectively. The finding of inhibition of AChE is possible following the methodology of Elmann, adapted by Rhee for thin layer chromatography (TLC). In this test, is used the reagent 5,5'–dithiobis–2–nitrobenzoic acid (DTNB) and acetylthiocholine iodide (ATCI) in buffer, and subsequently applies to AChE enzyme (3 U / ml). A preview of inhibition is made through observation of white halos. The essential oils of leaves, stalks and roots from three chemotypes of C. zehntneri was guided by bio–enzyme inhibition. The presence of essential oils downloads is confirmed by the appearance of white halos (Elmann et al., 1961, Rhee et al., 2001). On TLC the essential oils from leaves, stalks and roots and major constituent E–Anethtole of chemotype 1 from C. zehntneri showed an acetylcholine esterase inhibitory effect (Table 2). This result is in agreement with the literature, since E–anethole has been reported to inhibition AChE (Menichini et al., 2009). The essential oils from stalks and roots of chemotypes 2 and 3 showed an acetylcholine esterase inhibitory effect, respectively. The major constituent eugenol of the essential oils from leaves and stalks of chemotype 2 showed an anti–cholinesterase activity. While stragole the major constituent of the essential oils from leaves of chemotype 3 was not active (Table 2). These results can be explained by the presence in these oils of constituents β–pinene, 1,8–cineole, camphor and borneol, which have been reported to inhibit AChE in vitro (Howes et al., 2003; Perry et al., 2003), besides the fact that the monoterpenoids and phenylpropanoids presence in these oils may act synergistically to inhibit AChE (Savelev et al., 2003). The results clearly indicate that some structural features are important for biological activity. The presence of a conjugated double bond and a hydroxyl group seem to be important for the activity test, since the E–anethole and eugenol containing these structural features have shown activity, while the stragole which has no such characteristics was not active. The anti–cholinesterase activity of this compounds can be explained by hydro–phobic interactions between hydrophobic active site of AChE and hydrocarbon skeleton of the phenylpropanoids (Mukherjee et al., 2007).

 

CONCLUSION

Acetylcholinesterase (AchE) inhibitors have therapeutic applications in Alzheimer's disease (AD). On TLC the essential oils from three chemotypes of C. zehntneri. showed an anti–cholinesterase effect. The results from the present study demonstrate these essential oils are the promising source of cholinesterase inhibitors natural agents.

 

ACKNOWLEDGEMENTS

We thank the Brazilian funding agency FUN–CAP, CNPq and Governo do Estado do Ceará for fellowships and financial support.

 

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