Abstract: The water distilled oil obtained from rhizomes of Cochlospermum planchonii Hook.f.ex Planch (Apocynaceae) from Burkina Faso was examined by GC and GC/MS. Cochlospermum planchonii oil presents a particular chemical composition with a high rate of oxygenated components with predominance of ketones and esters (86.4%). The essential oil was tested against twelve strains of bacteria using a broth microdilution method. The results suggest that Cochlospermum planchonii essential oil has significant bactericidal activity.
INTRODUCTION
Cochlospermum planchonii Hook.f.ex Planch (Apocynaceae) is a West Africa species up to 0.5 and 1.5 m and growing from guinean region to Cameroon. In some African countries, it is a medicinal plant that rhizomes and leaves are used to treat many diseases: malaria, hepatitis, diabetes, infertility, touch, trypanosomiasis (Kone et al., 2002; Benoit-Vical et al., 2003; Anthony et al., 2005; Atawodi, 2005; Igoli et al., 2005; Pousset, 2006) and certain infections treated by traditional healers as diarrhoea, sexual transmissible infections (personal communication). Few studies concerning the chemical constituents were found (Adde-Mensah et al., 1985; Benoit-Vical et al., 1999). As far as our literature survey could as certain, rhizomes essential oil analysis and antibacterial properties of the plant have not previously been published. In this study we report volatile components and antibacterial activity of Cochlospermum planchonii (Co.p).
MATERIALS AND METHODS
Plant material: Samples of Cochlospermum planchonii Hook.f. Planch were collected in the rainy season (August, 2006) near the classed forest of Institute de Recherche en Biologie et Ecologie Tropicale de Saponé, 26 km south of Ouagadougou, Burkina Faso. Voucher specimens were kept in the herbarium of CRSBAN, University of Ouagadougou.
Extraction and analyses: The freshly comminuted rhizomes were subjected to hydrodistillation for 4 h with a clavenger-type apparatus. The essential oil was collected and dried, after decantation, over anhydrous sodium sulphate, then analysed by GC and GC/MS.
GC analyses were performed on a fused silica capillary column (30 mχ 0.25 mmχ 0.15 μm) coated with DB-1. The oven temperature was programmed from 60-220° C at 3° C min‾ 1; helium was used as a carrier gas at a flow rate of 1 mL min‾ 1.
GC/MS analyses were carried out on a Hewlett- Packard capillary GC-quadrupole MS system (model 5890) fitted with a fused silica column coated with DB-1 (25 mχ 0.23 mm) and using the same GC parameters. Helium was used as a carrier gas at a flow rate of 0.9 mL min‾ 1.
The volatile components were identified by comparison of their retention indices and their experimental mass spectra with those of reference compounds, further confirmation was done by referring to retention indices data generated from a series of alkanes: C9-C30 (Adams, 2001; Jennings and Shibamoto, 1980) (Table 1).
Bacterial strains: The micro organisms used were:
Reference bacterial strains: Bacillus cereus LMG13569, Enterococcus faecalis CIP103907, Escherichia coli CIP NCTC11609, Listeria innocua LMG1135668, Salmonella enterica CIP105150, Shigella dysenteria CIP5451, Staphylococcus aureus ATCC9244.
Hospital bacterial strains: Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes. They were kindly provided by the St. Camille Hospital of Ouagadougou, Burkina Faso.
Determination of the strains sensitivity: The tests were performed using Miller Hinton medium for bacteria strains using disk diffusion method following the National Committee for Clinical Laboratory Standards methods (Kiehlbauch Julia et al., 2000).
Overnight broth cultures of each strain were prepared in nutriment Broth (Diagnostic Pasteur, France). The final concentration of each inoculum was got making dilution of each strain in NaCl 9% solution. The turbidity of each innoculum was compared with McFarland 0.5 solution. The final concentration of each innoculum (approximatively 5.105 cfu mL‾ 1) was confirmed by viable count on Plate Count Agar (Merck, Germany). Three microliter of essential oil was put on every disk (8 mm diameter).
Positive and negative growth controls were performed for every test. The plates were incubated aerobically at 30 or 37° C for 24 h. The bacterial sensitivity to the essential oil was assessed by measuring the diameter of inhibition zone. The inhibition zones were compared with that of tetracyclin (BIO-RAD Marnes-la coquette-France) and ticarcillin (BIO-RAD Marnes-la coquette-France).
Determination of antibacterial activity of Co.p. essential oil: A broth microdilution method was used to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) (Bassole et al., 2003). All tests were performed in Mueller-Hinton Broth (Becton Dickinson, USA).
RESULTS AND DISCUSSION
Chemical analyses: The yield of the essential oil of the fresh rhizomes was 0.12% (w/w). Its chemical composition was particular (Table 1). It exhibited a high rate of oxygenated components with predominance of ketones and esters (86.4%). The major constituents were: tetradecan-3-one (30.6%), tetradecen-3-one (15.3%), tetradecylacetate (15.0%), dodecylacetate (12.4%).
| Table 1: | Chemical composition of the essential oil of Cochlospermum planchonii Hook |
| aRetention indices on DB-1 column | |
| Table 2: | Diameter of inhibition zone (mm) of bacterial growth |
| Each value represents mean of three different observations; bTe: Tetracycline; Ti: Ticracilline, cnd: Not determined | |
The oil was characterized by the absence of monoterpenes and contained three minor sesquiterpenes: β-elemene (6.0%), β-selinene (1.9%), α-selinene (3.8%).
Antibacterial activity of essential oil: The results showed that almost of bacterial strains were sensitive to Co.p. (Table 2). Only Staphylococcus camorum LMG13567 was not sensible to Co.p. (zone of inhibition 9 mm). The best sensitivity to essential oil was, respectively obtained on Streptococcus pyogenes (41 mm), Escherichia coli CIP NCTC11602 (33 mm), Salmonella enterica CIP105150 (33 mm) and Listeria innocua LMG1135668 (30 mm). The other strains had sensitivities between 22-27 mm. Following the results in Table 2, the different strains were more sensitive to Co.p. than tetracycline. The most important information was that essential oil of Co.p. exhibited more activity on E. coli CIP NCTC11602 ( 33 mm) and S. pyogenes (41 mm) than tetracyclin (E. coli CIP NCTC11602, 22 mm; S. pyogenes 20 mm) and ticarcillin (E. coli CIP NCTC11602 , 8 mm; S. pyogenes 24.66 mm).
The MICs, MBCs of the Cochlospermum planchonii essential oil for the micro-organisms tested were consigned in Table 3.
| Table 3: | Minimum inhibitory concentration, minimum bactericidal concentration data (%v/v) obtained by microdilution method |
| Each value represents mean of three different observations | |
The essential oil failed to inhibit Staphylococcus camorum LMG 13567 and the Staphylococcus aureus obtained from hospital at the highest concentration (8%). Bacillus cereus, Escherichia coli, Listeria innocua, Streptococcus pyogenes were inhibited at the lowest MIC of 0.25%. The results of MBC demonstrated a bactericidal effect. The essential oil was bactericidal for Bacillus cereus, Enterococcus faecalis, Escherichia coli, Listeria innocua (reference strains), Streptococcus pyogenes (hospital strain). The most resistant strains with highest MBC (8% or more) were Shigella dysenteria and Staphylococcus camorum for reference strains and Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus for hospital strains. For Enterococcus faecalis and Staphylococcus aureus, strains isolated from hospital were found to be more resistant than reference strains to the essential oil action. Considering MICs and MBCs, no significant difference could be observed between Gram-negative and Gram-positive bacteria.
This study shows in vitro high and low antibacterial activities of the rhizomes essential oil of Cochlospermum planchonii. It was bactericidal for most of the reference strains and some hospital strains tested. These results indicate that the plant could be used as a potential remedy against diarrhoea and some sexual infections particularly in aromatherapy.