Antimicrobial Activity of Chromolaena odorata Extracts against Bacterial Human Skin Infections

Leaf, stem and root of Chromolaena odorata were extracted by maceration extraction method using water, ethanol, methanol and hexane as solvents. The average percentage yield of leaf extracts in water (12.16 ± 0.13%), ethanol (8.42 ± 0.115%), methanol (10.45 ± 0.012%) and hexane (2.37 ± 0.215%) were significantly higher compared with stem and root extracts using the same solvents. All extracts were tested for antimicrobial activity against ten bacterial strains associated with human skin infections. Leaf extracts with ethanol, methanol and hexane solvents gave the best inhibitory activity against six gram-positive bacterial strains (Bacillus cereus TISTR 687, Enterococcus faecalis TISTR 379, Staphylococcus epidermidis TISTR 518, Staphylococcus aureus TISTR 1466, Streptococcus pyogenes ATCC 19615 and Propionibacterium acnes DMST 14916) and one gram-negative bacterial strain (Proteus vulgaris ATCC 13315). The hexane stem extract showed greater inhibitory activity against Pseudomonas aeruginosa ATCC 27853(15.3±0.5 mm), B. cereus TISTR 687(14.6±0.8 mm) and Klebsiella pneumoniae TISTR 1843 (14.0±1.0 mm), while hexane root extract showed high inhibitory activity against Enterococcus faecalis TISTR 379 (14.5±0.9 mm) and Kleb. pneumoniae TISTR 1843 (14.7±0.6 mm). The lowest minimum inhibitory concentration (MIC) of the ethanolic leaf extract was 0.81 mg/mL against Staph. aureus TISTR 1466, followed by methanolic and hexane leaf extracts with equal MIC of 1.62 mg/mL against both Staph. aureus TISTR 1466 and Strep. pyogenes ATCC 19615. The antimicrobial activity of the methanolic leaf extract of C. odorata was correlated with the amount of both total phenolic and flavonoid compounds. The results obtained suggest that the ethanolic, methanolic and hexane leaf extracts could be developed to treat bacterial skin infections. The hexane was sutiable solvent for extraction of the stem and root parts of C. odorata.


Introduction
Bacterial skin infection strains commonly infect patients in hospitals, leading to high nosocomial infection rates and use of antimicrobial drugs.The nosocomial pathogens that exhibit the greatest antimicrobial resistance are Staph.aureus (52.2%),E. coli (67.6%),Acinetobacter baumannii (100%) and Ps.aeruginosa (68.2%) (Ahoyo et al., 2014).According to the U.S. Food and Drug Administration, the bacterial pathogens that most commonly cause acute bacterial skin and skin structure infections (ABSSSI) are Strep.pyogenes and Staph.aureus, including methicillin-resistant Staph.aureus; less common causes are Streptococcus spp., Entc.faecalis and gram-negative bacteria.Furthermore, a report by the World Health Organization (WHO) in 2014 found antibacterial resistance in E. coli, Kleb. pneumoniae, Staph. aureus, Strep. pneumoniae, nontyphoidal Salmonella (NTS), Shigella spp.and Neisseria gonorrhoeae.
Nowadays, hospital-acquired infections (HAIs) are a major cause of patient morbidity and mortality, and are correlated with fomites.Fomites are reservoirs of pathogens which can infect patients by direct contact or indirectly from touching contaminated hospital environmental surfaces (Mangicaro, 2012).At a teaching hospital in northern Nigeria, pathogenic bacteria were found on 65.7% of the samples tested (e.g., stethoscopes, doorknobs), including Staph.aureus (21.7% frequency), Staph.epidermis (8.7%), Streptococcus spp.(8.7%), Bacillus spp.(13.0%),E. coli (26.1%),Pseudomonas spp.(8.7%) and Klebsiella spp.(13.0%) (Maryam et al., 2014).There has also been a rapid increase in the incidence of infections with drug-resistant microorganisms acquired by patients while in the hospital.Bacterial human skin infection strains are correlated with nosocomial infection, fomites and antimicrobial-resistant strains.Obtaining new antimicrobial agents from natural resources is necessary to develop alternative drugs for safe and cost-effective health care.
Previous studies on the antibacterial activity of this plant extract have been limited to clinical diarrheal strains such as B. cereus, E. coli, Kleb.oxytoca, Salmonella enterica, Salmonella Typhimurium, Shigella sonnei and Vibrio cholera, and skin infections with bacteria such as Staph.aureus and Staph.epidermidis (Vital & Rivera, 2009;Maji et al., 2010;Naidoo et al., 2011;Atindehou et al., 2013;Eze et al., 2013).A few reports have investigated the effect of C. odorata extract on bacterial human skin infection strains.
In this study, we investigated the antibacterial activity of C. odorata extracts against bacterial human skin infection strains.Various parts of C. odorata, i.e. leaf, stem and root, were extracted with water, ethanol, methanol and hexane.Antibiotics extracted from medicinal plants are an alternative method to reduce the antibiotic resistance of bacterial pathogens.

Plant Extraction
Leaf, stem and root parts were collected from C. odorata plants in Amnat Charoen province, Thailand.Each plant part was washed, dried in sunlight, and then placed in a hot-air oven with temperature controlled at 60 o C. Dried plant parts were then collected, blended and weighed before extraction.A 3×4 factorial design was employed on the C. odorata extracts, giving an output of 12 experiments.Twenty g of dried plant part powder was soaked in a 500 mL conical flask containing 200 mL of water, 95% ethanol, methanol or hexane solvents, and shaken on a vertical shaker at 150 rpm for 24 h.The extract solutions were filtered through Whatman filter paper No. 1.The filtrates were concentrated by evaporation (R-200 rotary evaporator; Büchi, Flawil, Switzerland) and frozen in a freeze dryer (CoolSafe; Labogene, Lynge, Denmark).Crude extract powder was stored at 4 o C until used.The percentage yield of crude extract powder was calculated by the following equation:

Determination of Total Phenols and Total Flavonoids by HPLC-DAD Method
The content of total phenols and flavonoids was analyzed by high-performance liquid chromatography (HPLC) (LC-20AD; Shimadzu, Kyoto, Japan) using a UV-vis diode array detector (DAD) recording at 280 and 360 nm (SPD-M20A, variable wavelength; Shimadzu) (Peñarrieta, Alvarado, Bergenstahl & Akesson, 2007).The conditions of phytochemical analysis were set as follows: mobile phase (methanol: 1% acetic acid), retention time (20 min), and flow rate (0.8% mL/min).The analytical column used in this study was an Intersil ® ODS-3, size 4.6×150 mm (GL Sciences, Tokyo, Japan).Gallic acid and Trolox were used as standards for phenolic contents, and rutin and quercetin for flavonoid contents.

Pathogenic Bacteria
The

Determination of Inhibition Zone of Crude Extracts by Agar Well Diffusion
The inhibition zone was determined using agar well diffusion method.Twenty-eight mL of trypticase soy agar (TSA) was poured into individual Petri dishes until the media became solid.One loop of each strain taken from each working stock was incubated on a TSA plate with 5 mL of trypticase soy broth (TAB) at 35 ± 2 o C for 18-24 h, except Entc.faecalis TISTR 379 was incubated under anaerobic conditions.Bacterial strain cultures were adjusted to obtain a final concentration of 10 8 CFU/mL using a 0.5 McFarland standard.One hundred μl of the optimum cell concentration was then swabbed on a TSA plate by three-way method.Wells (7 mm diameter) were cut into the agar plates using a sterile cork borer (No. 3).About 100 μl of extract from each plant part was poured into individual wells.Stock solutions of the extracts were prepared at a final concentration of 100 mg/mL.DMSO (dimethyl sulfoxide, 10%) was used as a negative control.The agar well plate was then incubated for 18-24 h at 35 ± 2 o C. Antimicrobial activity was measured in terms of the diameter of the inhibition zone (mm).A value of the inhibition zone of ≥ 10 mm was selected to study the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

Determination of Bacterial Susceptibility to Extracts by MIC and MBC
The MIC and MBC were determined by two-fold serial dilution method using a macro-broth dilution technique modified from Schwalbe, Steele-Moore & Goodwin (2007).A stock solution (100 mg/mL) of each extract was serially two-fold diluted in a tube with 1 mL TSB to obtain concentrations ranging from 0 mg/mL to 100 mg/mL.The zero concentration (0 mg/mL) tube served as a growth control.The inoculum size was prepared by diluting cell cultures in TSB to obtain a cell concentration of 10 8 CFU/mL using 0.5 McFarland standard and then diluting with TSB to obtain a cell concentration of 10 6 CFU/mL.Then 0.1 mL of each standard inoculum was added to the diluted extracts.Cultivation was carried out at 35 ± 2 o C for 18-20 h.The reduction of bacterial growth in each tube, containing various extract concentrations, was determined using a standard plate count compared with the growth control tube.MIC was defined as 80% inhibition in growth of the initial bacterial inoculum when compared with the control.MBC was defined the lowest concentration of a plant extract that killed the majority (99.9%) of the initial bacterial inoculum.

Statistical Analysis
Data were subjected to one-way analysis of variance (ANOVA) using SPSS Statistics 17.0 (SPSS, USA).The different treatments were analyzed using Duncan's multiple range test method at a confidence level of 95%.

Extraction Yield
Twelve samples of C. odorata extracts were obtained from leaf, stem and root plant parts, and were used for water, ethanol, methanol and hexane extractions, as shown in Superscript letters within a column indicate significant (p<0.05)differences of means within solvents.Subscript letters within a column indicate significant (p<0.05)differences of means within plant parts.Statistics analyzed using Duncan's multiple range test.

Content of Total Phenols and Flavonoids
Evaluation of total phenolic and flavonoid contents was determined for three plant parts and four solvents, as shown in Table 2.The maximum content of total phenols was found in the ethanol leaf extract, followed by water, methanol and hexane.For the stem, water extract contained the highest total phenols.The highest flavonoid contents were found in the ethanol leaf extract, compared with other plant parts and solvents.The ethanol leaf extract also had the highest total phenolic and flavonoid contents, followed by water and methanol; hexane leaf extract only contained phenols.

Antibacterial Activity
Leaf extracts in ethanol, methanol and hexane exhibited strong inhibitory effects against B. cereus TISTR 687, Entc.faecalis TISTR 379, Staph.epidermidis TISTR 518, Staph.aureus TISTR 1466, Strep.pyogenes ATCC 19615 and Prop.acnes DMST 14916, with inhibition zones in the ranges of 14.6 ± 0.6 to 20.0 ± 0.5, 14.7 ± 1.5 to 17.0 ± 1.0, 11.2 ± 1.4 to 14.7 ± 1.5, 12.0 ± 0.5 to 20.3 ± 0.6, 15.7 ± 0.6 to 20.3 ± 2.9, and 9.7 ± 0.6 to 14.2 ± 0.8 mm, respectively.In gram-negative bacteria, the ethanol, methanol and hexane leaf extracts affected Ps. aeruginosa ATCC 27853, with inhibition zones ranging from 12.5 ± 0.3 to 15.3 ± 0.5 mm.Pr. vulgaris ATCC 13315 was inhibited by the leaf extracts in all solvents.Methanol and hexane leaf extracts also showed antimicrobial activity against Kleb.pneumoniaeTISTR 1843, while the water, ethanol and methanol extracts only had an effect against E. coli TISTR 780.None of the plant extracts were effective against Entb.aerogenes ATCC 13048.Hexane extract of the stem of C. odorata showed inhibitory activity against B. cereus TISTR 687, Entc.faecalis TISTR 379, Staph.epidermidis TISTR 518, Strep.pyogenes ATCC 19615, Kleb.pneumoniae TISTR 1843, Pr. vulgaris ATCC 13315 and Ps.aeruginosa ATCC 27853, while the root extract also exhibited inhibitory activity against all of the above except for B. cereus TISTR 687 and Pr.vulgaris ATCC 13315.Furthermore, the ethanolic stem and water root extracts showed inhibitory activity against Strep.pyogenes ATCC 19615 and E. coli TISTR 780, respectively (Table 3, 4 and 5 and Figures 1 and 2  The ethanolic leaf extract was found to be more active against Staph.aureus TISTR 1466, with a MIC of 0.81 mg/mL (MBC = 1.62 mg/mL), followed by methanolic and hexane leaf extracts, which exhibited inhibitory activity against Staph.aureus TISTR 1466 and Strep.pyogenes ATCC 19615, with an MIC equal to 1.65 mg/mL.The hexane root extract had the lowest activity, while hexane stem extract showed inhibitory activity against B. cereus TISTR 687 (MIC = 6.25 mg/mL) and Pr.vulgaris ATCC 13315 (MIC = 6.25 mg/mL).C. odorata extracts were most active against Staph.aureus TISTR 1466, followed by Strep.pyogenes ATCC 19615, Staph.epidermidis TISTR 518 and Prop.acnes DMST 14916; while for gram-negative bacteria, C. odorata extracts showed inhibitory activity against only Pr. vulgaris ATCC 13315 (Table 6, 7 and 8).Previous reports found that methanol leaf extract from C. odorata obtained in Nigeria gave a MIC value against Staph.aureus in a range of 0.125-2.0mg/mL, a MIC value which closely matched our results (Eze et al., 2013;Stanley et al., 2014).However, our results indicated that the methanolic leaf extract of C. odorata (6.25 mg/mL) had a better MIC against Staph.epidermidis TISTR 518 than the methanolic leaf extract from C. odorata grown in Durban, South Africa (8.0 mg/mL) (Naidoo et al., 2011).Furthermore, the results obtained suggest that the extracts of C. odorata exhibited better antibacterial activity against gram-positive bacteria than gram-negative bacteria.The outer phospholipid membrane of gram-negative bacteria has lipopolysaccharide (LPS) components, making it impermeable to lipophilic solutes.The cell wall of gram-positive bacteria has only an outer peptidoglycan layer, which is not an effective permeability barrier (Vital & Rivera, 2009).Our results indicated that water and methanol showed the highest percentage yield, followed by ethanol and hexane, when using a 1:10 (w/v) ratio of dried plant material and solvent.The effects on antibacterial activity were associated with the plant part and extraction solvent.The leaf was absorbed by polar organic solvents more than the other plant parts.These results were similar to the report of Stanley et al. (2014) but different from Mondal et al. (2012).The hexane extract of C. odorata gave the lowest yield which similar to the reported of Kusuma et al. (2014).Hexane is a low-polarity solvent, which can dissolve and separate the non-polar secondary compounds from the plants such as tannins, terpenes and quinines (Lavanya & Brahmaprakash, 2011;Delahaye et al., 2009).The extraction efficiency from plants is related to solvent type, plant material to solvent ratio, time of extraction, and particle sizes of the plant (Tiwari, Kumar, M. Kaur, G. Kaur, & H. Kaur, 2011).The rate of extraction can be increased by increasing the surface area and using a sample to solvent ratio of 1:10 (w/v) (Tiwari et al., 2011).
Total phenolic contents were found in almost all previous reports on C. odorata leaf extracts.Interestingly, in several studies no flavonoid contents were discovered during phytochemical screening (Anyasor et al., 2011;Danlami et al., 2013).However, Mondal et al. (2012) reported on the flavonoid contents found in C. odorata leaf extract but not the total phenolic contents.In the present study, only ethanol leaf extract showed higher flavonoid content (rutin) than total phenol contents.
Our results indicated that the water, ethanol and methanol leaf extracts of C. odorata possessed antibacterial activity, as they exhibited the highest total phenol and flavonoid contents (Table 4).Both ethanol and methanol solvents could extract antimicrobial compounds (flavonoids, flavones, polyphenols, terpenes and tannins) from the leaf of C. odorata (Akinmoladun et al., 2007;Tiwari et al., 2011;Mondal et al., 2012;Vijayaraghavan, Ali, & Maruthi, 2013;Stanley et al., 2014).Normally, the polarity of a solvent plays a key role in increasing phenolic solubility (Naczk & Shahidi, 2006).The polar secondary compounds from medical plants contain alkaloids, flavonoid and some phenols (Delahaye et al., 2009).In many plants, the types of flavonoids and phenols that show antimicrobial activity are chrysin, quercetin and rutin, and catechols, epicatechin and cinnamic acid (Tiwari et al., 2011).The antimicrobial compounds of C. odorata (flavonoids) are effective at inhibiting the growth of pathogens by binding to the bacterial cell wall, leading to inhibition of cell wall biosynthesis (Anyasor et al., 2011;Lavanya & Brahmaprakash, 2011).In terms of antibacterial activity, the results indicated that the organic solvents ethanol, methanol and hexane were the most suitable extraction solvents.Highly polar organic solvents such as ethanol and methanol are typically used for extraction of bioactive compounds from plants.

Effect of C. Odorata Extracts on Antibacterial Activity
Our results were similar to previous findings on the effect of C. odorata extracts against pathogens.Leaf extracts demonstrated the best antimicrobial activity, followed by stem and root extracts.The best extraction solvent for antibacterial activity was methanol, followed by ethanol and hexane.(Vital & Rivera, 2009;Maji et al., 2010;Naidoo et al., 2011;Mondal et al., 2012;Eze et al., 2013;Stanley et al., 2014).Furthermore, it was found that the gram-negative bacteria Entc.aerogenes was not inhibited by water, ethanol, methanol or hexane leaf extracts, results which are in accordance with the leaf extract from C. odorata in Durban, South Africa (Naidoo et al., 2011).E. coli and Klebsiella pneumoniae were inhibited by leaf extracts with acetone and benzene from C. odorata in India but not by water leaf extract (Maji et al., 2010).The previous report found that the hexane leaf extacts of Callistemon viminalis leave showed more potent inhibition against skin pathogen (Staph.aureus, Strep.pyogenes and the enteric B. cereus) than intestinal pathogen (Shigella sonnei, Salmonella enteritidis and E. coli) (Delahaye et al., 2009).The factors affecting the quality of plant extracts are the nature of the plant material (season, topography and climate) and its origin (locations) (Tiwari et al., 2011).The type of solvent used in the extraction procedure plays an important role in the diversity of compounds in the plant extracts (Tiwari et al., 2011).
C. odorata stem and root extracts in hexane showed better antibacterial activity compared with other solvents.Venkata et al. (2012) reported that extracts from the root of C. odorata, using ethyl alcohol, methanol, chloroform, and methanol:chloroform:water in a ratio of 12:5:3 as solvents, exhibited activity against the same pathogen strains as in our results.

Conclusions
In comparing the antibacterial activities of different plant parts and extraction solvents, it was found that the most successful extraction procedure for C. odorata leaf was by using ethanol and methanol solvents and maceration extraction method. ).

Table 1 .
Leaf extracts gave significantly higher percentage yields than stem and root extracts for all extraction solvents.The water and methanol solvents showed significantly higher yields compared with the other extraction solvents for the plant leaf and stem, but not the root.The lowest percentage yield was obtained from C. odorata root extracted with hexane solvent.Furthermore, hexane solvent gave a significantly lower percentage yield for all plant parts compared with water, ethanol and methanol solvents.

Table 1 .
Extraction yields of leaf, stem and root of C. odorata with four different solvents

Table 2 .
Phytochemical analysis of leaf, stem and root of C. odorata extracted using different solvents

Table 4 .
Antibacterial activity (zone of inhibition, mm) of C. odorata stem extract

Table 5 .
Antibacterial activity (zone of inhibition, mm) of C. odorata root extract

Table 6 .
Antibacterial activity (MIC and MBC values, mg/mL) of C. odorata leaf extract

Table 7 .
Antibacterial activity (MIC and MBC values, mg/mL) of C. odorata stem extract

Table 8 .
Antibacterial activity (MIC and MBC values, mg/mL) of C. odorata root extract The ethanolic and methanolic leaf extracts gave high extraction yields and high contents of both total phenols and flavonoids.Ethanolic and methanolic leaf extracts also exhibited good antibacterial activity against the gram-positive bacteria Staph.aureusTISTR1466, Strep.pyogenesATCC19615 and Staph.epidermidisTISTR518 and the gram-negative bacteria Pr. vulgaris ATCC 13315.C. odorata extracts were also more active against gram-positive bacteria than gram-negative bacteria.The antimicrobial activity of C. odorata extracts was correlated with the amount of both total phenol and flavonoid compounds.For stem and root parts, hexane solvent was more suitable for extraction of antibacterial compounds than other solvents.This is the first report to demonstrate the antibacterial activity of ethanolic and methanolic leaf extracts of C. odorata against Strep.pyogenesATCC19615 and Prop.acnesDMST 14916, as well as the inhibitory activity of stem extract of C. odorata against the bacterial skin infection(B.cereusTISTR 687, Pr. vulgaris ATCC 13315, Entc.faecalisTISTR 379 and Strep.pyogenesATCC 19615).The results obtained suggest that the ethanolic and methanolic leaf extracts could be used as a potential natural source for drug development to treat bacterial skin infections.