Plant material
Madeng’ai samples were collected from Grassland above 800 meters in Tongdao County, Huaihua City, Hunan Province, and identified by the Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine. Collected plant materials were powdered and stored at 4℃ until further analysis.
Experimental animals
Preparation of the extract (medicine suspension)
Weigh 10g powder of the medicine (Madeng’ai), add 50ml autoclaved Milli-Q water, then autoclave it and get the pasty medicine suspension (0.2 g/ml).
Microorganisms
Six bacterial strains were provided by the First Affiliated Hospital of Chongqing Medical University, China. The strains used are Staphylococcus aureus (S. aureus, ATCC25923), Escherichia coli (E. coli, ATCC259220), Enterococcus faecalis (E.faecalis, ATCC29212), Pseudomonas aeruginosa (PAE, ATCC2785), as well as Klebsiella pneumoniae (K.pneumoniae) and Acinetobacter baumannii (A.baumannii). The latter two were isolated from the patient’s sputum, and their drug susceptibility tests (Minimum inhibitory concentration, MIC) were non-multiple antimicrobial resistant.
Minimum inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) of Madeng’ai extracts for antimicrobial testing was determined by the LB agar plate. The solvent without extracts was served as the negative control, and the others in different extract concentrations were the experimental groups. Inhibition of organism growth in the plates containing test crude extracts was judged by comparing growth in blank control plates. The MIC values were determined as the lowest concentration of extracts inhibiting visible growth of each organism on the agar plate. All the samples were tested in triplicate.
In vitro antimicrobial activity
Preparation of test solution
Madeng’ai powder 0mg, 100mg, 200mg, 300mg, 400mg, and 500mg were added into six flasks containing 100ml LB culture respectively, and high temperature autoclaved to prepare extract medicine suspension concentration at 0 mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, and 5 mg/ml. Then, 100 μl aliquots of culture containing MRSA or E.coli were added into six 100ml flasks, respectively, and shaking cultured at 37℃, 220 round/min for 2-3 hours until OD600nm = 0.5. Finally, 100 μl aliquots from six kinds of culture containing MRSA or E.coli at different medicine suspension concentration (0 mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, and 5 mg/ml) were pick up and spread on normal LB agar to culture overnight to get the optimum concentration.
Microorganisms for animals
Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAE) were cultured in LB broth at 37℃, 220 round/min for 2-3 hours until OD600nm = 0.5.
Animal model
All animal experiments were performed under the experimental animal use guidelines of the National Institutes of Health. All procedures for the mouse experiments were approved by the Ethics Committee of Animal Experiments of Chongqing Medical University. All mice were housed in laminar flow cabinets under specific pathogen-free conditions at room temperature with a 12 h light/dark cycle, with food and water available ad libitum in the Experimental Animal Centre, Chongqing Medical University. 12 female BABL/c mice (5-6 weeks old) were randomly divided into 4 groups (MRSA-medicine group, MRSA-PBS group, PAE-medicine group, PAE-PBS group). The back of the mice was unhaired and cut a 0.5cm diameter wound on the mice’s surface with a sterilized surgical scissor, then subcutaneously 100μL MRSA/PAE suspension was injected in the wound area. After 48h, the wound surface was swab with extracts (Madeng’ai, medicine suspension) by the cotton swab twice a day for the MRSA/PAE-medicine groups; similarly, PBS was swab as control twice a day for the MRSA/PAE-PBS groups.
In vivo antimicrobial activity
Changes of mice weight and wound recovery condition
Mice body weights and wound recovery conditions were recorded daily for 10 days after swabbing the extracts (Madeng’ai, medicine suspension) and control (PBS) s treatment.
Bacteria growth condition and histological analysis after treatment
After 10-day treatment with extracts (Madeng’ai, medicine suspension) and PBS, 75% ethanol was used to sterilize wound surfaces (recovered or unrecovered) of the mice. The wound surfaces were then cut down with the sterilized surgical scissor, swabbed on the LB agar plates for bacteria culture, Then, wound surfaces were cut into 4 μm tissue slices and subjected to hematoxylin and eosin (H&E) staining for histological analysis.
Statistical analysis
All experimental data are expressed as the means and standard error of the mean (SEM).