Antibacterial Effects of Chemical Constituents Isolated from the Roots of Cordia myxa (Boraginaceae) on Salmonella typhi (Enterobacteriaceae)

Effects of Chemical Constituents Isolated from the Roots of Cordia on Abstract: Cordia myxa is a Cameroonian pharmacopoeia traditional plant. It belongs to the Boraginaceae family, which different parts are used in the treatment of various bacterial infections. The phytochemical screening carried out on the Cordia myxa species indicates the presence of alkaloids, triterpenes, steroids, carbohydrates, flavonoids and saponins. Our investigations on the roots of this plant allowed us to isolate two triterpenes from the ethyl acetate extract, which crystallize in the Hex/AcEt [2/8] solvent system; (23R) campesta-9 (11), 24 (30) diene-18,23-diol 3-O-α-rhamnopyranoside (compound 1) and alphitolic acid (compound 2). The isolation and characterization of these compounds were carried out respectively by silica gel chromatography and by NMR (1D, 2D) spectral analysis. Compound 1and 2 were tested on the in vitro growth of E.coli and Salmonella typhi using the Muller-Hinton solid medium disc method. The results revealed that these compounds have a dose-dependent antibacterial activity on Salmonella typhi . However, compound 2 had a better antibacterial potential on the Salmonella strain (18.6±0.6mm, 30mg/ml) with the MIC=0.25mg/ml than the compound 1 (14.5±0.4mm, 30mg/ml,) with the MIC=0.44mg/ml. Few classes of antibiotics have been tested, to look for those with the best results and compare the different doses of our compounds. The two compounds did not have any effect on E. coli .


Introduction
Typhoid fever is a serious foodborne illness caused by Salmonella typhi, a bacterium found in faecally contaminated water and food. The disease is transmitted through contaminated water/food and occurs mainly in areas where hygiene conditions are poor [1]. The enterohaemorrhagic E.coli strains are responsable for a variety of infections ranging from watery diarrhea to haemorrhagic colitis and can develop into a haemolytic-ureenic syndrome in children, mainly under three years of age, or thrombotic microangiopathy in adults [2], they can acquire particular virulence factors and give rise to either extra-intestinal pathologies (meningitis, urinary tract infections) or intestinal pathologies [3,4]. Faced with the appearance of resistant forms of several bacteria to certain antibiotic, and the high cost of these products leading to the search for new active molecules with a broad spectrum of action has become a necessity [5]. One of the strategies for this research is to explore plants used in traditional medicine [6]. Cordia myxa is a plant from the tropical and subtropical region; it belongs to the Boraginaceae family. The phytochemical screening carried out on the Cordia myxa species indicates the presence of alkaloids, triterpenes, steroids, carbohydrates, flavonoids and saponins [7]. Traditionally, the different parts of this plant are used against urinary tract infections, antihelminthic, antidiuretic; their fruits have anti-inflammatory properties [8]. In Africa, the pulp of the fruit of Cordia myxa is also used to treat diarrhea, dysentery, tuberculosis and ulcers [9]. In Cameroon, Cordia myxa is used to treat diarrhea, stomachaches, fever and also urinary tract infections. Thus, the objective of this study is to isolate, characterize and evaluate the antimicrobial activity of secondary metabolites isolated from the root of Cordia myxa.

Plant Materials
The roots of Cordia myxa were collected in the northern region of Cameroon, in Mayo Louti division, in Kossel Danneel village. The plant was identified by the botanist Dr Froumsia Mouksia from the Department of Biological Sciences, University of Maroua, Maroua, Cameroon. One Voucher specimen (Nº 6410/HEFG) was deposited at the Herbarium of School for the Training of Specialists in Wildlife Management of Garoua, Garoua, Cameroon.

Extraction and Isolation
SOME 3.50 kg powders from the roots were subjected to successive extraction with the hexane and ethyl acetate, after total evaporation of the solvent, a black ethyl acetate extract was obtained with a mass of 75 g. This extract was submitted to the silica gel column chromatography (SiO 2 , 0.063-0.200) and the elution was carried out by gradient of increasing polarity of solvents (hexane, hexane-ethyl acetate). The elution of the extract with hexane/EtOAc [2: 8] polarity gradient yielded compounds 1 and 2.

Bacterial Strains
The microorganisms used in this study are Gram negative bacteria Escherichia coli and Salmonella typhi (Enterobacteriaceae). These are clinical isolates provided by the "Centre Pasteur", Yaounde, Cameroon. The biochemical and serological tests were used for confirmation of both bacterial strains.

Study of Antibacterial Activity
First, the discs are prepared and then the different concentrations of compounds 1 and 2 (60mg/ml, 30mg/ml, 15mg/ml). These compounds were dissolved in methanol before preparing the concentrations. These solutions are coated and sterilized in an autoclave (121°C for 15mn) [1]. Blotting paper discs of 6mm diameter are impregnated with these different concentrations and even methanol (negative control disc). All the discs prepared are dried in an oven at 37°C. Some classes of antibiotics are used to search for the best reference antibiotics [1].

Agar Diffusion Method
The antibacterial activity of the different compounds was evaluated by the agar diffusion method as described by Bauer and collaborators [10] and taken up by Barry and collaborators [11]. From 18 to 24-hour young colonies, a bacterial suspension is made in sterile distilled water for each strain. The turbidity of this suspension was adjusted to 0.5Mc Farland and then diluted to 1/100. An estimated inoculum of 10 6 colony-forming units per milliliter (cfu/ml) was then obtained. This inoculum was inoculated by flooding on petri dishes containing Muller-Hinton agar [12]. The discs impregnated with the different concentrations of compounds 1and 2, methanol and antibiotics were then delicately deposited on the surface of the agar. The petri dishes were first left for 1 hour at room temperature for pre-diffusion of the substances, before being incubated at 37°C in an oven for 24 hours [13]. Antibacterial activity was determined by measuring the diameter of the inhibition zone around each disc [14]. The minimum inhibition concentration (MIC) was determined by the dilution method described by Haddouchi and his collaborators [5].

Identification of the Compounds
Compounds 1 and 2 are all white powders. Compounds 1 and 2 crystallize in the Hex/AcEt [2/8] solvent system. These compounds are all soluble in methanol. The Libermann-Buchard test of componds 1 and 2 is positive. This information confirms that the compounds belong to the triterpene family. The RMN spectrum proton of Compound 1 reveal six (06) methyl between δ 0.67-1.74ppm; five (05) singlets at δ 0.81ppm (3H,s, Me-19); 0.97ppm (3H,s, Me-21); 1.09ppm (3H, s, Me-26); 1.07ppm (3H,s, Me-27); 0.67ppm (3H,s, Me-28); a broad singlet at δ 5.34ppm which corresponds to a proton of the double bond; two (02) singlets at δ 5.06ppm and 4.88ppm, which are the protons of methylene at the C-30 position. These informations confirm the basic structure of the genin. The proton at δ 3.07ppm (H-3), which is a deflated proton, and the presence of an anomeric proton at δ 4.82ppm, suggest that compound 1 has a sugar. Its COSY spectrum shows the follow correlations: the anomeric proton correlates with the signal at δ 3.94ppm (H 2' ), and which correlates with the proton at δ 3.74ppm (H 3 '). The proton H 3 'correlates with the signal at δ 3.43ppm (H 4' ) which correlates with the signal at δ 3.80ppm (H 5' ). The proton H 5' correlates with the methyl proton at δ 1.29ppm (H 6' ). The spectral analysis of the 13 C NMR of compound 1 confirms the presence of a steroid. Its 13 C-NMR exhibit the signals at δ 160.5ppm (C-24); 105.6ppm (C-30); 124.4ppm (C-11) and 50.6ppm (C-18). These chemical shifts of the carbon confirm the basic structure of the genin.

Results and Discussion on Antimicrobial Activity
The evaluation of the antimicrobial activities of compounds 1 and 2 was carried out by the Muller-Hinton agar diffusion method, as proposed in the recommendations of the Antibiogram committee of the French Society of Microbiology [20]. The antibacterial ability was classified according to the DIZ (diameters of the inhibition zone) as follows: not sensitive (DIZ < 8.0mm) moderately sensitive (8.0 < DIZ <14.0mm); sensitive (14.0 < DIZ < 20.0), and extremely sensitive (DIZ > 20.0mm) [21,22]. The results obtained on the antibacterial activity of our compounds varied significantly depending on the concentration on Salmonella typhi. For the compound 2, the highest concentration (30mg/ml) at an inhibition zone diameter of 18.6±0.6mm (Table 3 and figure 2), at this dose was sensitive because it is between (14.0<DIZ<20.0mm), we can say that, at this concentration, compound 2 has the same effect as the antibiotic CAZ10 (Ceftazidim) which belongs to the β-lactam class, the diameter of its inhibition zone was 19.2±0.2 mm, it was also sensitive because it belongs to the same range. Compound 2 showed the sensitive effect at a concentration of 15mg/ml, as its inhibition zone was 16.5±0.7mm (14.0<DIZ<20.0mm) ( Table 3 and figure 2). Our results are similar to that of Xiao and his collaborators, who tested dihydromyricetin onSsalmonella typhi, at a dose of 11.34mg/ml, the diameter of the inhibition zone obtained is 13.1±0.5mm, this is a moderately sensitive effect (8.0<DIZ<14.0mm) [22]. ND=not detected, DIZ=diameter of the inhibition zone, MIC=minimum inhibitory concentration. Values represent means of three independent replicates ± standard deviation (SD) At these two concentrations of the compound 2, the larger effect on Salmonella typhi than the reference antibiotic C30 chloramphenicol was shown. Chloramphenicol had a moderate sensitive effect because its inhibition zone diameter was 10.2±0.1mm (8.0<DIZ<14.0mm) ( Figure 2 and Table 3), as of compound 2 at a dose of 7.7mg/ml (8.6±0.6mm).   Similarly, for compound 1, at doses of 30mg/ml and 15mg/ml we observe respectively the diameter of the inhibition zone, 14.5±0.4 and 8.3±0.5mm. For the 30mg/ml dose, it has the sensitive effect because its inhibition zone belongs to the interval of 14.0<DIZ<20.0mm and it has the moderately sensitive effect (8.0<DIZ<14.0mm) for 15mg/ml dose (Table 4 and figure 3) [21].
These different maximum concentrations of compound 1 had the same effect as the reference antibiotic C30. The lowest concentration of compound 1 (7.7mg/ml), inhibits Salmonella typhi at an inhibition diameter of (7.4±0.6mm), it is not a sensitive effect. Among the antibiotics belonging to the β-lactam class, the best that has had an effect on Salmonella typhi is CXM30 (cefuroxim), followed by CAZ (ceftazidim). Their inhibition diameter were respectively, 20.9±0.1mm and 19.2±0.2mm, CXM30 had an extremely sensitive effect (DIZ>20.0mm), but CAZ10 had a sensitive effect (14.0<DIZ<20.0mm) ( Table 5 figure 3). The compounds 1 and 2, had no effect on E. coli. Among the reference antibiotics belonging to the Aminoside and phenicol classes, CN10 (Gentamicin) had an extremely sensitive effect (DIZ>20.0mm) with an inhibition diameter of 23.1±0.1mm and TOB10 (Tobramycin) with an inhibition diameter of 18.9±0.4mm, these references antibiotics were sensitive on Salmonella typhi ( Table 6 figure 4). Antimicrobial substances are defined as substances used to kill or inhibit the growth of microorganisms, including antibiotics and other antibacterial agents [23]. Salmonella typhi and E. coli are both gram (-) bacteria, the effect of compound 1 and 2 on Salmonnella typhi was dose-dependent, their MIC were respectively 0.25mg/ml and 0.44mg/ml. Our two compounds did not have any effect on E. coli. The process of the high resistance of E.coli to our compounds could probably be attributed to their outer membranes, which surround the cell wall and limit the diffusion of hydrophobic compounds by the covering lipopolysaccharides [23].

Conclusion
The investigations on the roots of Cordia myxa leaded to isolation of two triterpenes from the ethyl acetate extract, the (23R) campesta-9 (11), 24 (30) diene-18,23-diol 3-O-αrhamnopyranoside and the alphitolic acid-The effect of these compounds on Salmonnella typhi was dose-dependent, their MIC were respectively 0.25mg/ml for (23R) campesta-9 (11), 24 (30) diene-18,23-diol 3-O-α-rhamnopyranoside and 0.44mg/ml for 2α,3β-dihydroxybetulinic acid. The two compounds did not have any effect on E. coli. Therefore, it is suggested that Cordia myxa could be a potential source of natural antimicrobial that could have great importance as therapeutic agent in the treatment of Salmonella typhi infections. Further research on others parts of the plant is recommended for characterization of important active constituents, responsible for antibacterial activity.