Composition and Antibacterial Activity of the Essential Oils from Zanthoxylum rhoifolium

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The essential oils from the aerial parts of leaves, fruits and flowers of Zanthoxylum rhoifolium of Southern Brazil (Rio Grande do Sul), were analysed by GC, GC/MS, and chiral phase gas chromatography (CPGC). Forty-eight compounds were identified from the essential oils. The major constituents of the essential oil of the leaves were germacrene D (34 %) and bicyclogermacrene (23 %) and of the fruits, menth-2-en-1-ol (46.2 %), β-myrcene (30.2 %), (-)-linalool (15 %) and (-)-α-terpineol (8.45 %). β-Myrcene (65 %) and menth-2-en-1-ol (5.4 %) dominate the essential oil of the flowers. The oils of the leaves and fruits were bioactive with antibacterial activity against Staphylococcus aureus (Gram positive), and Klebsiella pneumoniae and Salmonella setubal bacteria (Gram negative) microorganisms, while the essential oil of the flowers was inactive.

Zanthoxylum rhoifolium (Rutaceae), locally called ”mamica-de-porca”, ”mamica-de-cadela”, ”juvevê” is a plant that grows in South America (Brazil, Uruguay, Paraguay and Argentina). It has been used in Brazilian folk medicine against a variety of diseases. As a continuation of our chemical studies on Rutaceae plants [1], especially our research on the essential oils of Rio Grande do Sul aromatic plants [2], [3], we now report on the volatile constituents of leaves, fruits and flowers of Z. rhoifolium. The oils were analysed by means of GC and GC/MS. The identification of the chemical constituents was based on comparison of their relative retention times and mass spectra with those obtained from authentic samples and/or the Wiley and NBS/NIST libraries and those published by Adams [4].

The oils of Z. rhoifolium were obtained by hydrodistillation in 0.57 %, 1.5 % and 0.45 % yield, for leaves (sample A), fruits (sample B) and flowers (sample C), respectively. The quantitative analysis for each sample showed twelve main compounds (&γ> 1 %) for sample A, four compounds for sample B and six compounds for sample C. The oils displayed some qualitative and quantitative differences (Table [1]). The oil of the leaves (sample A) was richer in sesquiterpenes, while the oils of the fruits and flowers were richer in monoterpenes. The configurations of the chiral monoterpenes constituents (linalool, α-terpineol and limonene) were analysed on a dual column system [5], using two 25 m fused silica columns coated with modified cyclodextrins (2,6-Bu₂ - 3-Pe-γ-CD and 6-Me-2,3-Pe₂-β-CD) as the chiral stationary phase.

The antimicrobial activities were evaluated by means of direct bioautography in a TLC bioassay [6], [7]. Table [2] shows the results obtained with three Gram-positive: Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, and with three Gram-negative bacteria: Klebsiella pneumoniae, Salmonella setubal and Escherichia coli. The antibacterial studies showed that the oils of leaves and fruits were active against the three Gram (±) species, while the oil of flowers was inactive against the six bacteria at the highest sample amount tested (50 μg).

Materials and Methods

Plant material: The aerial parts of leaves and flowers of Zanthoxylum rhoifolium were collected in November 2001 and fruits were collected in January 2002, in the town of Jaguari, Rio Grande do Sul, Brazil, and identified by Prof. Renato Záchia. The voucher specimen (HDFI 194) is deposited in the Herbarium of the Federal University of Santa Maria.

Chemical analysis: Fresh plant leaves (sample A), fruits (sample B) and flowers (sample C) were subjected to hydrodistillation for 4 h using a modified Clevenger-type apparatus, followed by exhaustive extraction of the distillate with diethyl ether. After removal of the solvent, the yields of the crude oils were 0.57 %, 1.5 % and 0.45 % (v/w) for samples A, B and C, respectively.

Sample A: d ^{20 :} 0.67 g mL^-1; n ^{20 :} 1.4854; [α]²⁵ _D= + 3.27 (c 0.09, CHCl₃).

Sample B: d ^{20 :} 0.67 g mL^-1; n ^{20 :} 1.4758; [α]²⁵ _D= + 3.55 (c 0.1, CHCl₃).

Sample C: d ^{20 :} 0.9 g mL^-1; n ^{20 :} 1.4780; [α]²⁵ _D= + 17.8 (c 0.1, CHCl₃).

Gas chromatographic (GC) analyses: The essential oils were submitted to GC analysis in a Varian 3800 Gas Chromatograph equipped with a capillary fused silica column (25 m × 0.25 mm) coated with SE-54. The GC conditions used were: carrier gas H₂ (1 mL min^-1); on column injector 220 °C; FID 280 °C; column temperature 50 °C to 250 °C at 4 °C/min.

Gas chromatography-mass spectrometry (GC-MS): Analyses were performed on a Varian-3800 Saturn system operating in the EI mode at 70 eV, equipped with a CP-SIL cross-linked capillary column (30 m × 0.25 mm). The temperatures of the column and the injector were the same as those from GC. The identification of the components of the oils was based on comparison of the retention times and Kovats retention indexes on both columns and mass spectra with those of NBS/NIST Libraries and those described by Adams [4].

Chiral phase gas chromatography (CPGC): Three of the chiral monoterpenes constituents (linalool, α-terpeneol and limonene) of Z. rhoifolium oils were identified by peak enrichment by enantioselective capillary GC with two fused capillary columns, 25 m × 0.25 mm, coated with heptakis(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin and octakis(3-O-methyl-2,6-di-O-butyryl)-γ-cyclodextrin, each diluted with the polysiloxane OV-1701, using a Varian-3800, equipped with a flame ionization detector (FID), and hydrogen as the carrier gas. All runs were performed with a temperature program from 35 °C for 15 min. and 35 °C to180 °C at 3 °C/min.

Antibacterial activity: The antibacterial activity of the oils of samples A, B and C against three Gram-positive bacteria: Staphylococcus aureus (ATCC 6538p), Staphylococcus epidermidis (ATCC 12 228), Micrococcus luteus (ATCC 9341) the three Gram-negative bacteria: Klebsiella pneumoniae (ATCC 10 031), Salmonella setubal (ATCC 19 196) and Escherichia coli (ATCC 11 103), were determined using the bioautographic technique [6], [7]. The microorganisms used in the antibacterial assay have been maintained at the Chemistry Department of the University of Santa Maria, RS, Brazil. For the antimicrobial assay, 50.0, 25.0, 12.5, 6.25, 3.12, 1.06 μg of oils were applied to pre-coated TLC plates. The inoculum was prepared by culturing each organism in tryptone soya agar (TSA, Oxoid) at 37 °C to a turbidity equivalent to McFarland 0.5 standard (1.5 × 10⁸ CFU/mL). One microliter of each diluted inoculum (10⁴ - 10⁶ CFU) was applied onto Mueller-Hinton Agar medium (MHA-DIFCO), and distributed over TLC plates (5 × 5 cm). After solidification of the media, the TLC plates were incubated overnight at 37 °C [8]. Subsequently, bioautograms were stained with an aqueous solution of 2,3,5-triphenyltetrazolium chloride (TCC, 1 mg/mL) and incubated at 37 °C for 1 h. Amoxicillin was used as positive control.

Acknowledgements

The authors are grateful to IPT (Instituto de Pesquisas Tecnológicas), of Blumenau-SC for mass spectral data.

References

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Prof. Dr. Ademir Farias Morel

Universidade Federal de Santa Maria

Departamento de Química

Camobi

Santa Maria RS

Brazil CEP: 97105-900

Email: afmorel@base.ufsm.br

References

• 1 Morel A F, Moura N F, Ribeiro H B, Machado E CSM, Ethur E M, Zanatta N. Benzophenanthridine alkaloids from Zanthoxylum rhoifolium .  Phytochemistry. 1997;  46 1443-6
  CrossrefPubMedSearch in Google Scholar
• 2 Morel A F, Moreira J S, Pereira C S, Ethur E M, Machado E C, König W A. Volatile constituents composition of Blepharocalix salicifolius leaf oil.  Journal of Essential Oil Research.. 1999;  11 45-8
  PubMedSearch in Google Scholar
• 3 Morel A F, Flach A, Gregel B, Simionatto E, Silva U F, Zanatta N, Linares C EB, Alves S H. Chemical analysis and antifungal activities of the essential oil of Calea clematidea (Asteraceae).  Planta Medica. 2002;  68 836-8
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Adams R P. Identification of essential oil components by gas chromatography/mass spectroscopy. Allured Publishing Co Carol Stream, Illinois; 1995
  Search in Google Scholar
• 5 König W A, Icheln D, Runge T. Enantiomeric composition of the chiral constituents in essential oils. Part I. Monoterpene hydrocarbons.  Journal of High Resolution Chromatography. 1992;  15 184-90
  CrossrefPubMedSearch in Google Scholar
• 6 Homans A L, Fuchs A. Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances.  Journal of Chromatography. 1970;  51 327-9
  CrossrefPubMedSearch in Google Scholar
• 7 Hamburger M O, Cordell A G. Direct bioautography TLC assay for compounds possessing antibacterial activity.  Journal of Natural Products. 1987;  50 19-22
  CrossrefPubMedSearch in Google Scholar
• 8 Rahalison L, Hamburger M, Hostettmann K, Monod M, Frenk E. A bioautographic agar overlay method for the detection of antifungal compounds from higher plants.  Phytochemical Analysis. 1991;  2 199-203
  PubMedSearch in Google Scholar

Prof. Dr. Ademir Farias Morel

Universidade Federal de Santa Maria

Departamento de Química

Camobi

Santa Maria RS

Brazil CEP: 97105-900

Email: afmorel@base.ufsm.br