Essential oil of Brachylaena hutchinsii Hutch. from Tanzania: composition and antimicrobial activity
Maria M. Oliva1 and Mirta S. Demo1
1Departamento de Microbiologia e Inmunología, Fac. de Cs. Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Argentina.
R. S. Malele2
2Faculty of Pharmacy, Muhimbili University College of Health Sciences,
P. O. Box 65013, Dar es Salaam. Tanzania
C. K. Mutayabarwa3
3Institute of Traditional Medicine, Muhimbili University College of Health Sciences,
P. O. Box 65001, Dar es Salaam. Tanzania.
Julius W. Mwangi* and Grace N. Thoithi4
4Faculty of Pharmacy, University of Nairobi, P.O. Box 19676, Kenya
Silvina M. Faillaci, Raquel L. Scrivanti, Abel G. Lopez and Julio A. Zygadlo5
5Instituto Multidisciplinario de Biologia Vegetal. Catedra de Quimica Organica. FCEFyN –UNC. Argentina.
* Address for correspondence
ABSTRACT: The hydrodistilled essential oil of fresh aerial parts of Brachylaena hutchinsii was analyzed by GC/MS. Thirteen compounds representing 94.7 % of the oil were identified. The main components of the oil were sesquiterpene hydrocarbons, caryophyllene (19.1 %), β-cubebene (15.5 %), cis-calamenene (10.5 %) and α-copaene (9.0 %). The oil exhibited antimicrobial activity which was as much as that of gentamycin against Proteus mirabilis. It also showed some activity against Bacillus cereus, Staphylococcus aureus, S. epidermidis, Micrococcus luteus and Enterococcus faecalis.
KEY WORD INDEX: Brachylaena hutchinsii, essential oil composition, sesquiterpenes, antimicrobial activity.
INTRODUCTION: Brachylaena hutchinsii Hutch. (B. huillensis O. Hoffm.), Compositae family, is a tree 10-18 m, evergreen; bark rough, grey, peeling longitudinally. Leaves are narrowly elliptic to slightly obovate, base cuneate or attenuate, apex acute or short-accuminate, margin entire or serrate in young plants, 3-12 by 1-4 cm,revolute, densely greywhite-tomentose beneath. Flowers white or greenish yellow, heads in 2-3 cm long erect axillary panicles. It grows in upland semidecidous forest and lowland dry forest or thicket [1]. It can be found in the north eastern part of Tanzania. The local people, the Sambaa, call it ‘Muhungwe’ or ‘Mhungwe’, while the Swahili name is ‘Muhuhu’ or ‘Mhuhu’. It is used for timber, woodcarving and firewood. Previous phytochemical reports on Brachylaena hutchisii were made [2]. However, There are not reports on the chemical composition and antimicrobial activity of the essential oil of Brachylaena hutchisii.
EXPERIMENTAL: Plant material and oil isolation. Leaves of Brachylaena hutchisii were collected on 10th March 1999 from Mombo, Korogwe district in the Tanga region of Tanzania. Voucher specimens are kept in the Herbarium of Institute of Traditional Medicine, Muhimbili University College of Health Sciences (Herbarium No. ITM
1426). Fresh leaves of Brachylaena hutchisii were hydrodistilled to yield 5.0 % oil. The oil obtained was dried over anhydrous sodium sulphate and stored in a refrigerator until analysis.
Gas Chromatography- The essential oils were analyzed with a Shimadzu GC-R1A gas chromatograph equipped with a fused silica column (30 m x 0.25 mm) coated with DB-5 (J&W). The temperature of the column was programmed from 60 0C to 240 0C at 4 0C/min. The injector and detector temperatures were at 250 0C. The gas carrier was helium, at a flow rate of 1 ml/min. Peak areas were measured by electronic integration. The relative amounts of the individual components are based on the peak areas obtained, without FID response factor correction. Programmed temperature retention indices of the compounds were determined relative to n-alkanes [3].
Gas Chromatography-Mass Spectrometry- GC-MS analyses were performed on a Perkin Elmer Q-910 using a 30 m x 0.25 mm capillary column coated with DB-5. The temperature of the column and the injector were the same as those of GC. The carrier gas was helium, at a flow rate of 1 ml/min. Mass spectra were recorded at 70 eV. The oil components were identified by comparison of their retention indices, mass spectra with those of authentic samples, by peak enrichment, with published data [4], mass spectra library of National Institute of Standards and Technology (NIST 3.0) and our mass spectra library which contains references mass spectra and retention indices of volatile compounds.
Antimicrobial assay- A collection of 8 microrganisms were used, including Gram-positive bacteria Bacillus cereus (from rice), Enterococcus faecalis (ATCC 29212), Micrococcus luteus (ATCC 9341), Staphylococcus aureus (ATCC 25212) and Staphylococcus epidermidis (from cow milk), Gram-negative strains Escherichia coli (from water), Klebsiella spp. (from bird food) and Proteus mirabilis (from human urine). All the samples of microorganisms were characterized at the Department of Microbiology, National University of Rio Cuarto, Argentina and voucher specimens were preserved. All the strains tested were maintained at 4 °C in Tripsein-Soy Agar and were subcultured every month. The fungus was stored at the same temperature as bacteria in Sabourand Agar and subcultured every month. The paper disc diffusion method was used to test antibacterial activity. It was performed using an 18 hr culture,
growth at 37 °C and adjusted to approximately 106 cfu/mL. The inoculum (200 μL) was spread over plates containing Mueller-Hinton agar and a paper filter disc (4 mm) impregnated with 10 μL of the oil was placed on the surface of the media. A gentamycin disc (Brittania Co.) containing 10 μg was used as a reference. The plates were left for 30 minutes at room temperature to allow the diffusion of the oil and then incubated at 37 °C for 24 hrs. After this the inhibition zone around the disc was measured with a calliper.
Antifungal experiments were carried out in the same way using Extracto de Malta Broth for the culture and Sabouraud Agar for the plates.
RESULTS AND DISCUSSION: Twenty-six constituents, representing 94.7 % of B. hutchinsii leaf oil were identified by GC and GC/MS. As can be seen from the results in Table 1, the oil is rich in caryophyllene (19.1 %), β-cubebene (15.5 %), cis calamenene (10.5 %) and α-copaene (9.0 %). Table 2 shows the oil is as active as gentamycin against Proteus mirabilis. It also showed some activity against Bacillus cereus, Staphylococcus aureus, S. epidermidis, Micrococcus luteus and Enterococcus faecalis.
However, it showed no activity against Escherichia coli, Klebsiela spp. and Candida albicans.
ACKNOWLEDGEMENTS: The authors thank the CONICET and SECyT-UNC for financial support.
REFERENCES
1. H. Beentje. Kenya trees, Shrubs and Lianas. National Museums of Kenya. Nairobi. 1994. Page 555.
2. P. C. Viera, M. Himejima and I. Kubo, Sesquiterpenoids from Brachylaena
Hutchinsii. J. Nat. Prod., 54, 416-420 (1991).

3. A. A. Craveiro, F. J. A. Matos and J. W. Alencar, Kovat's indices as preselection routine in Mass Spectra Library Search of volatiles. J. Nat. Prod., 47, 890-892 (1984).


4. R. P. Adams. Identification of Essential Oil Components by Gas Chromatogrphy and Mass Spectroscopy. Allured Publ. Corp., Carol Stream, IL. (1995).

Table 1. Chemical composition of the essential oil of B. hutchisii leaves Compounds Retention Index Peak area (relative %) Methods of identification
α-thujene 931 0.5 MS-CO
α-pinene 939 0.2 MS-CO
sabinene 976 0.2 MS-CO
β-pinene 980 0.2 MS-CO
myrcene 991 0.4 MS-CO
carene-3- 1011 0.3 MS
δ-elemene 1339 0.1 MS
α-cubebene 1352 0.1 MS
α-ylangene 1373 5.2 MS
α-copaene 1376 9.0 MS
β-bourbonene 1385 0.2 MS
β-cubebene 1390 15.5 MS
β-caryophyllene 1418 19.1 MS-CO
α-cadinene 1450 0.3 MS
germacrene D 1480 0.1 MS
α-muurolene 1499 8.0 MS
γ-cadinene 1513 3.5 MS
cis-calamenene 1521 10.5 MS
δ-cadinene 1524 8.5 MS
α-calacorene 1542 6.0 MS
β-calacorene 1556 1.5 MS
spathulenol 1576 1.2 MS-CO
β-oplopenone 1606 1.5 MS
β-eudesmol 1649 0.9 MS
cadalene 1678 1.5 MS
α-santalol 1684 0.2 MS


Источник: East and Central African Journal of Pharmaceutical Sciences Vol.6(3) 2003, с. 61-63