Antimicrobial properties of the methanol leaf extract of V ernonia glaberrima Welw. Ex O. Hoffm (Asteraceae)

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INTRODUCTION
The World Health Organization (WHO) has long recognized antimicrobial resistance (AMR) as a growing global health threat, and one of the important global strategies is a continuous search for effective antimicrobial agents (WHO, 2012).AMR is driven by both appropriate and inappropriate use of anti-infective medicines for human, animal health and food production, together with inadequate measures to control the spread of infections (Goosens et al., 2005;Mathew et al., 2007;Orzech and Nichter, 2008;WHO, 2014).More than 2 million people become infected with bacteria that are resistant to antibiotics each year in the United States out of which about 23,000 die as a direct result of these infections, while many more people die from other complications related to antibiotic resistance (Centers for Disease Control and Prevention CDC, 2013).Although, the data for developing countries such as Nigeria is not very comprehensive, the mortality figures are expectedly higher (WHO, 2014).
Theoretically, bacteria will continue to develop resistance once exposed to any antimicrobial agent, thereby imposing the need for a permanent search and development of new drugs (Silver and Bostian, 1993).Natural products have been the most significant source of drugs and drug leads in history (Newman and Cragg, 2007).The emergence of multidrug resistance in human and animal pathogenic bacteria as well as undesirable side-effects of certain antibiotics has triggered immense interest in the search for new antimicrobial drugs of plant origin (Ahmed and Beg, 2001).V. glaberrima Welw.Ex O. Hoffm (Asteraceae), Shìwaákár-ján-gágári (Hausa language -N.Nigeria) is an erect shrub, 2 m high, found on hillside grassland in Guinea to Northern Nigeria, Western Cameroon and Central Africa to Angola (Burkill, 1997).It was reported to be used against malaria, migraine, psoric and dysmenorrhoea (Burkill, 1997).The leaves decoction is also employed traditionally in Nassarawa State, Northern Nigeria for treating pain, inflammation, vertigo and microbial infections (Personal communication).Preliminary phytochemical screening, acute toxicity studies and antidiabetic property of the plant have been reported (Abdullahi et al., 2015).
The aim of this study was to evaluate the antimicrobial properties of V. glaberrima using agar diffusion and broth dilution methods.

Collection and Identification of Plant material
The whole plant material of V. glaberrima was collected from Nasarawa State 8°32'N 8°81'E, Northern-Nigeria in June, 2012 during the rainy season.It was authenticated by Mallam U. S. Gallah of the herbarium section of Biological Sciences Department, Ahmadu Bello University, Zaria.A voucher specimen (No. 899) was deposited at the herbarium for future reference.

Preparation of extract
The leaves were removed, shade dried, pulverized, labelled, and stored at room temperature in an air-tight container prior to extraction.The Powdered leaves (2500 g) were extracted with 70% methanol using maceration method for 10 days with occasional shaking.The extract was evaporated in-vacuo using rotary evaporator at 40°C to obtain 400 g of a gummy greenish product (16.0%w/w)subsequently referred to as the crude methanol leaf extract VGLE.

Test organisms
Clinical isolates of the test organisms were obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital Zaria, Nigeria.All bacterial cultures were checked for purity and maintained in a blood agar slant while the fungi were maintained on a slant of Sabraud dextrose agar (SDA).

Susceptibility test
Antimicrobial activity of the methanol leaf extract of V. glaberrima was determined through susceptibility test using agar diffusion method (Sardari et al., 1998).The stock concentration of the extract (40 mg/ml) was prepared by dissolving 0.4 g of the extract in 10 ml dimethyl sulfoxide (DMSO).Mueller Hinton agar and the growth medium, was prepared according to manufacturer's instructions, and sterilized for 15 min at 121°C.Nutrient broth and sabraud dextrose broth were used for antibacterial and antifungal evaluations respectively.The test organisms were inoculated and incubated for 24 h for bacteria and 48 h for fungi.The solidified sterile medium contained in petri dish was seeded with 0.1 ml standard inoculum of the test microbe at 45°C.Wells were bored into the solidified inoculated nutrient agar plates using cork borer of 6 mm diameter.The wells were filled with 0.1 ml (400 µg) DMSO solution of the extract.Discs containing blank extraction solvents served as control.The reference antibacterial drug Sparfloxacin (5µg) and antifungal drug Fluconazole (5µg) were used as a positive control.The extract and standard drugs were allowed to diffuse into the agar and incubated overnight.All incubations were done at 37 and 25°C for bacteria and fungi respectively.At the end of incubation period, diameter of inhibition zone was measured using transparent ruler and recorded.The zones of inhibition of microbial growth were tested in duplicates and the mean of the results was recorded in millimeters (mm).

Minimum inhibitory concentration (MIC)
The MIC of the extract was carried out using Broth dilution method (Volekobia et al., 2001).Mueller Hinton broth was prepared of which 10 ml was dispensed into test tubes, sterilized at 121°C for 15 min, and allowed to cool; Mc-Farland's standard turbidity scale number 0.5 was prepared.Dilution of the organism suspension was done continuously using sterile normal saline until the turbidity matched that of Mc-Farland's scale by visual comparison.At that point, the concentration of the test microbe was about 1.5 x 10 8 cfu/ml.Two-fold serial dilution of the extract in the sterile broth was made to obtain the concentrations of 40, 20, 10, 5 and 2.5 mg/ml, respectively.0.1ml of the standard inoculum of the test microbe was then inoculated into the different concentrations of the extract in the broth.The tubes were incubated at 37°C for 24 h and 25°C for 48 h for bacteria and fungi respectively after which the plates were observed for turbidity (growth).The MIC was defined as the lowest concentration of the extract inhibiting the visible growth of each micro-organism.

Minimum bactericidal concentration/Minimum Fungicidal Concentration (MBC/MFC)
The MBC/MFC was carried out to determine whether there is complete death of test microbes or just growth inhibition.Mueller Hinton agar broth was prepared, sterilized at 121°C for 15 mins, and transferred into sterile petri dishes to cool and solidify.The contents of the MIC in the serial dilution were sub-cultured into the prepared medium and incubated at 37°C for 24 h; the plates were observed for colony growth; the MBC/MFC was the plate with lowest concentration of the extract in serial dilution without colony growth (Volekobia et al., 2001).

RESULTS AND DISCUSSION
The results of susceptibility test, MIC and MBC/MFC of the methanol leaf extract of V. glaberrima are shown in Tables 1, 2 and 3, respectively.The methanol leaf extract of the plant exhibited antimicrobial activity against the susceptible organisms tested.Susceptibility test result showed inhibition range of 18 to 29 mm against Methicillin resistance S. aureus, Vancomycin resistant Enterococci, S. aureus, H. pylori, P. fluorescence and the anti-fungi, Candida stellatoidea; The crude extract of the plant can be said to have a good broad spectrum of activity at the concentrations tested with mean zone of inhibition diameter > 18 mm (Tania et al., 2000).No activity was observed against L. monocytogenes, C. fetus, P. vulgaris and Candida.tropicalis.
The standard antibacterial drug, Sparfloxacin had inhibitory activity against all the organisms except H. pylori, P. flourescens, C. tropicalis and C. stellatoidea while Fluconazole, the standard anti-fungal drug showed activity only on the two fungi species tested C. tropicalis and C. stellatoidea.S. aureus was the most sensitive organism (29 mm) and C. stellatoidea was the least (18 mm).The MIC and the Minimum Bactericidal/Fungicidal Concentration (MBC) ranges for the extract were 5 to 20 mg/ml and 10 to 40 mg/ml, respectively.The low MIC value suggests that the extract has good antimicrobial activity.The highest bactericidal activity was recorded on S. aureus at 10 mg/ml while C. stellatoidea has the least activity at 40 mg/ml.S. aureus is the most dangerous of all the many common staphylococcal bacteria causing skin infections, pneumonia, breast infections, endocarditis, and other fatal infections in humans (Baorto et al., 1994).The extract also exhibited inhibitory effect on Methicillinresistant S. aureus (MRSA) which is a bacterium that is resistant to many antibiotics causing life-threatening bloodstream infections, pneumonia, skin and surgical site infections (Bush, 1989;CDCP, 2015).
Helicobacter pylori has been implicated in peptic and stomach ulcer (NLM, 2014) as there is a strong association between infection with H. pylori and gastric ulcers (Kosunen et al., 2005;Kusters et al., 2006), and antibacterial drugs are included in the prescribed gastric and peptic ulcer treatment regimen (Sung et al., 1995).The ability of the crude extract of V. glaberrima to inhibit this pathogenic microbe suggests its potential relevance in the management of peptic and gastric ulcers.P. fluorescens is an unusual cause of disease in humans, and usually affects patients with compromised immune systems (Gershman et al., 2008) Sparfloxacin, the standard drug is a broad spectrum antibiotic with better activity on gram positive bacteria; it exhibited high activity on the tested gram positive bacteria (MRSA, VRE, L. monocytogenes, and S. aureus) but was not active against the gram negative bacteria, H. pylori and P. flourescens.It is noteworthy that the extract inhibited the growth of H. pylori and P. flourescens, the only bacteria that were resistant to Sparfloxacin.
The ability of the extract to exert its antimicrobial effects may be attributed individually or collectively to the presence of flavonoids, alkaloids, saponins, tannins, steroids and terpenes detected in the plant (Abdullahi et al., 2015).Other Vernonia species such as V. amygdalina have been reported to possess antimicrobial activity (Ibrahim et al., 2008) though with varying degrees; the result of this work for example, showed V. glaberrima exhibited better inhibition of S. aureus (5mg/ml) than 12.5 mg/ml by V. amygdalina (Ibrahim et al., 2008).
These observed activities will give impetus for the isolation and characterization of bioactive constituents responsible for the observed activity, thereby aiding the attempt to combat these pathogens which are causative agents of serious and life threatening infections.

Conclusion
The results of this study suggest that the methanol leaf extract of V. glaberrima contains bioactive principles that have good antibacterial and antifungal activity, validating the antimicrobial use of the plant in traditional medicine.Further work aimed at the isolation of the bioactive principles is being pursued.

Table 1 .
Susceptibility test (Zone of Inhibition) of Methanol Leaf Extract of V. glaberrima.: mean zone of inhibition measured in millimeter (mm), -= activity not detected, VG= V. glaberrima extract. Key

Table 2 .
Minimum inhibitory concentration of methanol leaf extract of V. glaberrima against the test organisms.

Table 3 .
Minimum bactericidal/fungicidal concentration of methanol leaf extract of V. glaberrima against the test organisms.