Chromolaena odorata L (Asteraceae) is a bad invasive plant, found in the humid tropics and sub-tropics worldwide. It is used against dysentery, diarrhea, malaria, wound healing, headache and toothache in traditional medicine. In the present study, we investigated the antibacterial activities of different leaves extracts of Chromolaena odorata L. (cyclohexane, dichloromethane, ethyl acetate and butanol) against four clinical diarrheal strains (Klebsiella oxytoca, Salmonella enterica, Shigella sonnei and Vibrio cholera). We demonstrated that C. odorata leaves extracts show an antibacterial activity between 0.156 and 1.25 mg/mL. Bioassay-guided chromatography by bioautography with iodonitrotetrazolium-based colorimetric assay allowed the isolation and identification of two active compounds. After the combination of RP-HPLC, mass spectrometry analysis, 1D and 2D-NMR spectroscopy, we isolated and characterized two active molecules corresponding to 3’,4’,5,6,7-Pentamethoxyflavone (Sinensetin) and4’,5,6,7-Tetramethoxyflavone (Scutellareintetramethyl ether).
Intestinal infectious diseases are widespread around the world, especially in Africa where they constitute the second cause of mortality in children under five years [
According to the World Health Organization (WHO), 80% of the African population uses medicinal plants for their primary health needs and 40% of prescriptions in China are based on medicinal plants [
An ethnopharmacological survey in Benin allows identifying several plants used in traditional medicine for the treatment of intestinal infections. Among them, Chromolaena odorata (formerly Eupatorium odoratum) was less studied for antibacterial activities. In Vietnam, fresh leaves or their decoction are used throughout for the treatment of leech bite, soft tissue wounds, burning wounds, skin infection and dentoalveolitis [
In the present study, we focused on the antibacterial activities of Chromolaena odorata against four bacteria that causes intestinal tract infectious: Klebsiellaoxytoca Salmonella enterica, Shigella sonnei, and Vibrio cholerae. After determination of their minimal inhibitory concentration (MIC) and bioguided isolation, the antibacterial agents from C. odorata were characterized by mass spectrometry and NMR analysis.
Four strains (Klebsiellaoxytoca, Salmonella enterica, Shigella sonnei, and Vibrio cholera) were isolated from patients concerned by diarrhea at University hospital (CHRU) of Strasbourg (France) and obtained from the team of Dr. Gilles Prévost. Bacteria are cultivated in Mueller Hinton (MHB, Merck, Darmstadt, Germany).
Chromolaena odorata L. (Asteraceae) is collected during the rainy season in Abomey-Calavi (Benin). The taxonomic identification was obtained from a botanist and a voucher specimen was deposited at the National Herbarium of Abomey-Calavi in Benin (AA6390HNB).
Leaves were dried in the dark to avoid degradation of the active components and powdered with a blender. An aliquot (50 g) was sequentially extracted under agitation (IKA KS260 Basic, Staufen, Germany) at room temperature (20˚C) with cyclohexane (C6H12), dichloromethane (CH2Cl2), ethyl acetate (C4H8O2) and butanol (C4H10O). After the first extraction with C6H12 (2 × 150 mL, 1 h), the extract was filtered under vacuum and the filtrate was dried in a rotary evaporator (Stuart RE 300, Staffordshire, UK). The residue of this first extraction was dried under the laminar airflow chamber and used for successive similar extractions with the three others solvents.
Each extract (20 mg) was submitted to liquid chromatography on a Sephadex G-25 column (0.9 cm × 3.5 cm, GE Healthcare, SephadexTM G-25 Superfine, Uppsala, Sweden) and eluted with three successive solvents: solvent 1 (CHCl3/CH3OH/H2O; 60:30:4.5; v:v:v), solvent 2 (CHCl3/CH3OH; 2:1; v:v), solvent 3 (CHCl3/CH3OH/ H2O; 40:35:10; v:v:v). Elution was performed with 5 mL of solvent 1, 2.5 mL of solvent 2 and 2.5 mL of solvent 3. Each eluted fraction was dried under a stream of nitrogen using N-EVAP model 111 (Organomation Assoc. INC, Berlin South, USA).
Dried fractions were solubilized with solvent 2 (30 - 50 µl) and loaded on glass plates, Kieselgel 60 F254 HPTLC (10 cm × 10 cm) or TLC (20 cm × 20 cm) (Merck, Darmstadt, Germany). The silica plates were developed in a pre-saturated vertical twin-through glass chamber 25 cm × 25 cm × 10 cm (Camag, Muttenz, Switzerland) containing 35 mL of solvent 4 ((C2H5)2O/ CH3OH/CH3COOH; 90:1:2; v:v:v) for monodimensional TLC. A two-dimensional thin-layer chromatography was performed for a better separation of different compounds present. For bidimensional TLC, plates were developed in solvent 5 (CHCl3/(CH3)2CO; 95:5; v:v) for the first dimension, dried and placed in solvent 4 (C2H5)2O/ CH3OH/CH3COOH; 90:1:2; v:v:v) for the second dimension. Compounds were detected under UV light (365 nm).
Antibacterial activity of plants extracts was determined by using a microdilution bioassay. Bacteria were cultured aerobically at 37˚C in a Mueller Hinton Broth (MHB, Merck, Darmstadt, Germany) with shaking at 300 rpm for 18 h in an incubator (Tritamax 1000, Heidolph, Germany). The following bacteria strains were tested: Klebsiella oxytoca, Salmonella enterica, Shigella sonnei, and Vibrio cholera. Bacterial activity was tested by measuring the inhibition of bacterial growth [
Isolation of the active components was obtained by using the bioautography procedure [12,13]. A culture of Salmonella enterica in MHB with 0.7% agar (Gibco, Paisley, Scotland) with absorbance of 0.01 at 620 nm was sprayed on HPTLC plates. Plates were incubated for 18 h at 37˚C and 100% relative humidity. The detection of antibacterial spot was obtained after spraying of plates with an aqueous solution of INT (1 mg/mL) and incubation for 1 h. Inhibition of bacterial growth was indicated by clear zones on the bidimensional plates. The inhibition zones were scraped on TLC plates similarly prepared. This material was used for a liquid column chromatography (1 cm × 0.2 cm) and the active compounds were eluted with methanol, dried by N-Evap, suspended in a mixture of isopropanol/H2O/trifluoroacetic acid (TFA) (30:69.9:0.1; v:v:v) and centrifuged at RT 15.000 rpm for 15 min.
In order to purify the active components, the supernatants were analyzed by HPLC using a DIONEX Ultimate 3000 system and a Vydac C18 218TP510 Grace column (particle size 3 - 20 µm and pore diameter 300 Å Davidson, USA). The elution was obtained at RT with a gradient (as indicated on the chromatogram) of Solvent A: Isopropanol/H2O/TFA (30:69.9:0.1; v:v:v) and Solvent B: Isopropanol/H2O/TFA (90:9.91:0.09; v:v:v) at a flow rate 1.5 mL/min. The detection was obtained at 254 nm.
Mass measurements were performed using MALDI-TOF (UltraflexTM MALDI-TOF/TOF, BrukerDaltonics, Bremen, Germany). This instrument was used with a positive reflectron and a maximum accelerating potential of 25 kV applied to the target and 26 kV applied to the reflectron. The value of the delayed extraction was optimized to 110 ns in order to obtain the best resolution of the peptides used for calibration. The laser used was a nitrogen laser (337 nm) operating at a frequency of 20 Hz. Data acquisition was performed in a mass range from 100 m/z to 500 m/z.
Extracts (0.5 µL) were mixed with matrix (0.5 µL) loaded on the target and dried at atmospheric pressure using the dried droplet method. In order to increase the sensivity of the technique, two matrices were used: a-cyano-4-hydroxy-cinnamic (CHCA) (BrukerDaltonics. Coventry, UK) and 2,5-dihydrobenzoic acid (DHB) (Sigma Aldrich, Steinheim, Germany). A saturated solution of CHCA matrix in H2O/ACN, 1:1 was diluted three times with H2O/ACN 50:50 acidified with 0.1% TFA. DHB matrix was prepared at a concentration of 10 mg/mL in H2O/ ACN, 1:1.
MS analyses were performed using an Agilent Technologies 78901A (Santa Clara, CA, USA) gas chromatograph equipped with a split/splitless programmed temperature injector and an Agilent J&W DB5MS fused silica column (30 m by 0.25 mm; film thickness, 0.25 µm) connected to a JEOLAccut of JMS-T100GCV spectrometer (ChemSW, Inc., Fairfield, CA, USA). The mass spectrometer was operated in electron impact mode at 70 eV, and spectra were recovered over a mass range from m/z 35 to 700 with a cycle time of 0.4 scans/s. The oven temperature was programmed to increase from 80˚C to 300˚C at a rate of 10˚C/min, and then kept isothermal for 8 min. The other conditions were as follows: helium split, 1:10; constant flow rate, 1 mL/min; transfer line temperature, 280˚C and MS source temperature, 250˚C. Samples were injected with an auto-injector at 280˚C using the split mode with a ratio of 1:5.
The two isolated compounds were dissolved in CDCl3 and analyzed using a BRUKER Avance III 500 MHz spectrometer equipped with CapNMRTMProtasis 5 µL Probe Head (Savoy, IL, USA).
Chromolaenaodorata selection was based on an ethnopharmacological survey conducted among traditional healers of Benin. 14 plants were selected and tested in a preliminary study and C. odorata was selected as one of the most active plant. C. odorata leaves extracts were prepared and antibacterial activity extracts was determined by using iodonitrotetrazolium salt. Results demonstrate that C. odorata display an antibacterial activity ranging from 0.156 to 1.25 mg/mL against Klebsiellaoxytoca, Salmonella enterica, Shigella sonnei and Vibrio cholerae (
After purification of the extracts on a Sephadex G-25, TLC was performed (
The inhibition zones named Cp 1 and Cp 2 (
The molecular formula of Cp 1 and Cp 2 was determined as C20H20O7 at m/z 372 and C19H18O6 at m/z 342
respectively. Structural determination of the compounds by NMR enabled us to identify two flavonoids. The complete assignments of 1H and 13C NMR spectra were achieved with the help of COSY, NOESY and HSQC.
Compound 1: d 7.49 (1H, dd, J = 17, J = 4 HZ, Hc), 7.31 (1H, d, J = 4 HZ, He), 6.95 (1H, d, J = 17 HZ, Hd), 6.78 (1H, s, Ha), 6.60 (1H, s, Hb), 3.97 (6H, s, 7-OCH3, 5-OCH3), 3.96 (3H, s, 3’-OCH3), 3.94 (3H, s, 4’-OCH3), 3.90 (3H, s, 6-OCH3).
Compound 2: d 7.82 (2H, d, J = 18 HZ, Hc), 7.00 (2H, d, J = 18 HZ, Hd), 6.79 (1H, s, Ha), 6.68 (1H, s, Hb), 3.97 (6H, s, 7-OCH3, 5-OCH3), 3.90 (3H, s, 6-OCH3), 3.87 (3H, s, 4’-OCH3).
Cp 1 has been identified as 3’,4’,5,6,7-Pentamethoxyflavone or Sinensetin and Cp 2 as 4’,5,6,7-Tetramethoxyflavone or Scutellareintetramethyl ether (
The synthetic molecules corresponding to our natural isolated compounds were purchased from Extrasynthese (Genay, France) and their NMR spectra were compared showing similar profiles between the two series of analysis.
Recent data demonstrated that the bacteria used in this study were resistant to antibiotics. In 2011, Younes re-
ported that K. oxytoca isolated from Scottish patients display a resistance to cefotaxime and ceftazidime. This resistance is due to the expression of cefotaximase by the bacteria [
Some studies demonstrated that C. odorata leaves extracts display antibacterial activities against Pseudomonas aeruginosa, Streptococcus faecalis [
The two compounds identified in this study belong to the flavonoid family. Flavonoids are polyphenolic compounds mostly present in vegetables. Some flavonoids isolated from medicinal plants show important biological activities such as antiallergic, antiinflammatory, antiviral, anticancer, antioxidant. Interestingly flavonoids exert anti-inflammatory activities in bowel inflammation disease [
To conclude, this study describes for the first time antibacterial activity of C. odorata against four diarrheagenic pathogens strains and validates somehow traditional use of this plant in the treatment of intestinal infectious diseases. Bioguided isolation by chemical and biological methods allowed us to characterize two flavonoids, sinensetin and scutellareintetramethyl ether as responsible for these antibacterial activities of Chromolaena odorata.
We are grateful to the Embassy of France in Benin, the Benin government (ARHES project) and the Faculty of Odontology of the University of Strasbourg for their financial supports (M.A.). We thank Drs Céline Marban and Loïc Jerry for critical reading of the manuscript.