Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426

ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 411-426 http://www.ijcmas.com

Original Research Article Phytochemical analysis, in vitro evaluation of antioxidant and antimicrobial activity of methanolic leaf extract of Vernonia amygdalina (bitter leaf) against Staphylococcus aureus and Pseudomonas aeruginosa

Momoh Johnson1*, Odetunde Simeon Kolawole2 and Longe Adeteju Olufunmilayo1

1Department of Science Laboratory Technology (Biochemistry Unit), School of Pure and Applied Sciences, Lagos State Polytechnic, Ikorodu, Lagos, Nigeria 2Department of Science Laboratory Technology (Microbiology Unit), School of Pure and Applied Sciences, Lagos State Polytechnic, Ikorodu, Lagos, Nigeria

*Corresponding author

A B S T R A C T

The study was conducted to determine the phytochemical analysis, in vitro evaluation of antioxidant and antimicrobial activity of methanolic leaf extract of Vernonia amygdalina (bitter leaf) against Staphylococcus aureus and K e y w o r d s Pseudomonas aeruginosa. The qualitative phytochemicals in the extract of V.A include tannin, alkaloids, glycosides, anthraquinone, flavonoids, phlobatanin, Antimicrobial saponin etc. The amount of total proanthocyanidins, flavonoids, alkaloids, and total activity, phenols present in the extract are 0.09 ±0.03 mg quercetin/g of dry plant material, in vitro 1.1%, 9.3% and 163.24 mg PE / g DW respectively. The obtained values for - antioxidant, Carotene and lycopene were 0.49 ± 0.25 µg and 0.08 ±0.04 µg/g respectively. In minimum the in vitro antioxidant assay, V. amygdalina was found to have DPPH (95.65 inhibitory g/mL) and FRAP (0.708) scavenging activity. Antibacterial activity of the extract concentration, by disk diffusion method was characterized by inhibition zones of 20 ± 2.2 mm for minimum S. aureus and 23 ± 1.2 mm for P. aeruginosa. S. aureus was tested against 12 bacteriocidal standard antimicrobial agents. Of these, pefloxacin, gentamicin, ampiclox, concentration, roceptin, zinnacef, amoxicillin, septrin, erythromycin, augumentin and ampicilin Pseudomonas were resistance to the organism while ciprofloxacin and streptomycin were aeruginosa, sensitive. P. aeruginosa was sensitive to pefloxacin, gentamicin, amoxicillin, Staphylococcus ciprofloxacin, erythromycin, sparfloxacin and tarivid. Resistance to amplicox, aureus and roceptin, zinnacef, streptomycin, septrin, augumentin and ampicilin were also Vernonia observed with the same organism. The minimum inhibitory concentration (MIC) amygdalina for P. aeruginosa was 30 mg/ml while S. aureus has a value of 70 mg/ml. V. amydalina exhibited a minimum bacteriocidal concentration (MBC) of 60 mg/ml for P. aeruginosa and 140 mg/ml for S. aureus. The extract exhibited strong potency against these microorganisms with P. aeruginosa being the most susceptible. The results of this study support the use of Vernonia amygdalina as herbal remedies in Nigeria.

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Introduction chemical compounds such as resins, alkaloids, glycoside, saponins, lactose and Staphylococcus aureus is an important essential oils (Soraya, 2011). Many of these pathogen of humans and animals and is phytochemicals have beneficial effects on a implicated in a wide variety of infections. It long term human health and may be used to is a pathogen of greater concern because of effectively treat human disease (Lai and its virulence (Chambers, 2005), its ability to Roy, 2004). cause a diverse array of life threatening infections and its ability to adapt to different Oxidative stress leads to enhanced environmental conditions (Lowy, 2003). generation of reactive oxygen species (ROS) Pseudomonas aeruginosa is a gram and has been implicated in the etiology of negative, rod shaped, a sporogenous, and over one hundred human diseases including monoflagellated bacterium that has inflammation, metabolic disorders, cellular incredible nutritional versatility. aging, atherosclerosis, heart disease, stroke, diabetes mellitus, cancer, malaria, Antibiotics are naturally occurring or rheumatoid arthritis, HIV / AIDS, synthetic organic compounds which inhibit Alzheimer s disease, ulcerative colitis and or destroy selective bacteria, generally at Parkinsons disease (Smith et al., 2000; low concentrations (Brooks et al., 2007). In Olukemi et al., 2005 ; Hyun et al., 2006 and the last two decades, antibiotic resistance is Aliyu et al.,2008). Antioxidants are emerging as a serious problem worldwide substance or molecules that are capable of (Walsh, 2000 and Cohen, 2002). This has neutralizing the harmful effects of the ROS lead to the exploration for alternative, safe through the andogenous enzymatic defense and efficient antimicrobial compounds from system. The antioxidant effect of plant is natural resources like plants. The increasing mainly due to phenolic components like resistance of bacteria and fungi to currently flavonoids, phenolic acids and phenolic marketed antimicrobial agents is becoming a diterpenes (Shahidi et al., 1992). The world-wide medical problem (World Health antioxidants capacity of phenolic Organization, 2004). In recent years many compounds is mainly due to their redox bacteria have developed antimicrobial drug properties, which play an important role in resistance, these include but not limited to absorbing and neutralizing free radicals, Staphylococcus aureus and most of the quenching singlet and triplet oxygen and Enterobacteriaceae, such as Klebsiella decomposing peroxides (Osawa, 1994). pneumonia (WHO, 2004). Statistics indicate that more than 70% of the bacteria causing Vernonia amygdalina (V.A) is a shrub that infections are resistant to at least one of the grows predominantly in the tropical Africa. drugs most commonly used to treat them Leaves from this plant serve as food (WHO, 2004). vegetable and culinary herb in soup (Argheore et al., 1998). Vernonia Medicinal plants have formed the basis of amygdalina commonly known as bitter leaf health care throughout the world since the is a shrub or small tree of 2 5 m tall, earliest days of humanity and have remained belonging to the family Asteraceae. It has relevant in both developing and the petiolate leaves of about 6 mm diameter and developed nations of the world for various elliptic shape. The leaves are green with a chemotherapeutic purposes. Plants have characteristic odour and a bitter taste. The ability to synthesize a wide variety of taxonomic classification of Vernonia

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426 amygdalina is as follows: Kingdom: plantae, Biochemistry Laboratory, pulverised to Division: Angiosperms, Order : Asterales, coarse power using blender. Extraction was Family: Asteraceae, Genius: Vernonia, carried out by dispersing 200g of the Species: V. amygdalina, Botanical Name: grounded Vernonia amygdalina plant Vernonia amygdalina. It is commonly called material in 1L of 80% methanol and shaking Bitter leaf in English language, Onugbu was done with GFL shaker for 72 hours. in Igbo language, it is called Etidot , in This was followed by vacuum filtration and Efik, Ijaw and Ibibio, Ewuro in Yoruba concentrated by rotary evaporator at a language, Oriwo in Edo and Chusa-doki temperature not exceeding 40oC. The in Hausa (Egedigwe, 2010). In many parts concentrated extract was dried to complete of Nigeria, the plant has been domesticated. dryness in an aerated oven at 40oC for 48 Studies on the nutritional composition of the hours. The extract was latter stored in a bitter leaf are numerous (Nimenibo-Uadia, refrigerator at 4oC. 2003). V. amygdalina has been found to be rich in minerals, especially phosphorus, Qualitative phytochemical analysis of calcium, potassium, magnesium, zinc, iron Vernonia amygdalina and some vitamins like vitamin A, C and E. VA extracts have been shown to exhibit Phytochemical analysis for phytochemical profound ethnomedical and pharmacological constituents were carried out on the properties viz, anti-diabetic (Momoh et al., methanolic extract of Vernonia amygdalina 2014), antimalarial (Abort and Raserika, using standard phytochemical procedures by 2003), antihelminthic and antibiotic Sofowora (1993), Harborne (1973), Trease properties (Farombi, 2003). The present and Evans (1989). study investigated the in vitro antioxidant and antibacterial activity of methanolic leaf Quantitative phytochemical analysis of extract of Vernonia amygdalina against the methanolic leaf extract of Vernonia Staphylococcus aureus and Pseudomonas amygdalina aeruginosa Determination of total proanthocyanidins Materials and Methods content of the extract

Collection and identification of plant Determination of proanthocyanidin was extract based on the procedure reported by Sun et al., (1998). A volume of 0.5 ml of 0.1 mg/ml The leaves of Vernonia amygdalina were of extract solution was mixed with 3 ml of obtained from Ikorodu in Lagos State, 4% vanillin-methanol solution and 1.5 ml Nigeria. The plant was authenticated by Mrs hydrochloric acid; the mixture was allowed Shokefun a botanist from Science to stand for 15 min. The absorbance was Laboratory Technology Department measured at 500 nm. Extract samples were (Environmental biology Unit), Lagos State evaluated at a final concentration of 0.1 Polytechnic, Ikorodu. mg/ml. Total proanthocyanidin content were expressed as catechin equivalents (mg/g) Preparation of methanolic leaf extract of using the following equation based on the Vernonia amygdalina calibration curve: y = 0.5825x, R2 = 0.9277, where x was the The leaves of Vernonia amygdalina were absorbance and y is the catechin equivalent washed, air dried under shade in the (mg/g). 413

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Flavonoid content determination 500 l of the F-C reagent. After shaking, the mixture was incubated for 3 min at room One hundred millilitres of 80% aqueous temperature. Then 2000 l of 20 % Na2CO3 methanol was used to repeatedly extract 1 g solution was added. The volume obtained of the defatted sample at room temperature. was mixed vigorously, and held for 60 min The solution was then filtered through in the dark at ambient temperature. The Whatman filter paper. The filtrate was absorbance of the solution was then evaporated to dryness in a crucible over a measured at 650 nm against a blank in a water bath and weighed to a constant weight spectrophotometer. The sample was using the method of Oseni et al (2014). analysed in triplicate and the average content was noted for each measurement. Alkaloid content determination The total phenolic content, expressed as mg of pyrrocatechol equivalents (PE) per g of To about 1 g of the defatted sample of dry weight of plant material (mg PE / g Vernonia amygdalina, 80 ml of 10% acetic DW), was calculated through the calibration acid in ethanol was added. The beaker was curve obtained using the equation given covered and then allowed to stand for 4 bellow: hours. The suspension was then filtered and the extract concentrated on a water bath to Absorbance = 0.0828 x C, R2 = 0.9993 one-quarter of the original volume. Concentrated ammonium hydroxide was where C was the concentration in mg/l. added drop wise to the extract until the precipitation was completed. The precipitate Determination of -Carotene and was collected and washed with dilute lycopene content of the extract ammonium hydroxide and then filtered to obtain the alkaloid residue. This was dried -Carotene and lycopene were determined and weighed using method of Oseni et al by the method of Barros et al (2007). The (2014). dried extract of Vernonia amygdalina (100 mg) was vigorously shaken with acetone- Total phenolic content of the extract hexane mixture (4:6, 10 mL) for 5 min and filtered through a disposable filter (0.45 m, The quantitative determination of total Millipore). The absorbance of the filtrate phenolic content using Folin-Ciocalteu (F- was measured at 453, 505, and 663 nm. C) reagent involves oxidation in alkaline Contents of -carotene and lycopene were solution of phenols by the yellow calculated according to the following molybdotungstophosphoric heteropolyanion equations: reagent and colorimetric measurement of the resultant molybdotungstophosphate blue -carotene (mg/100 mL) = 0.216 (A663) according to the method of Singleton and 0.304 (A505) + 0.452 (A453); Rosi (1965) and modified by Dogÿan et al., (2005). The polyphenol fraction (extract lycopene (mg/100 mL) = 0.0458 (A663) + corresponding to 1 g of dry plant material) 0.372 (A505) 0.0806 (A453). was dissolved in 5 ml of double distilled water. An aliquot of 100 l of this solution The assays were carried out in triplicates, was diluted with double distilled water to 3 the results were mean ± SD and expressed as ml. Afterward, the obtained solution was g of carotenoid/g of the extract. added to 300 l of double distilled water and 414

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In-vitro plant antioxidant assay of (2.5 ml, 1% w/v ). After incubation at 50°C Vernonia amygdalina for 20 min, 2.5 ml of trichloroacetic acid (10 % w/v) was then added to the mixture DPPH Radical Scavenging Activity Assay. followed by centrifuging at 3000 rpm for 10 min. Consequently, 5 ml of the upper layer 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free was mixed with 2.5 ml of distilled water and radical scavenging activity was measured 0.5 ml of ferric chloride (0.1 % w/v). After according to the method of Barros et al 30 min of incubation at room temperature in (2007) with slight modification on the basis the dark, absorbance of the resulting of the method of Blois (1958). Different solution was measured at 700 nm using a concentrations of ethanol dilutions of spectrophotometer. The ferric reducing samples were mixed with 2.0 volume of 6.5 power capacities of the plant extracts and × 10 5M solution of DPPH. The resulting standard antioxidants were expressed solutions were thoroughly mixed and graphically by plotting absorbance against absorbance was measured at 517 nm after concentration. Samples for the assay were keeping the tubes in dark for 30 minutes. prepared in triplicate The scavenging activity was determined by comparing the absorbance with that of Preparation of methanolic extract control containing equal volumes of DPPH impregnated paper discs solution and ethanol. The radical scavenging activity was obtained by the following The methanolic extract impregnated paper equation: discs were prepared as described by Ekundayo and Ezeogu (2006). Whatman Radical scavening activity (%) = No. 1 filter paper was cut into discs of 6 mm Acontrol Asample d i a m e t e r u s i n g a n o f f i c e p e r f o r ator. The Acontrol × 100 discs were placed in glass Petri dish and sterilized in hot air oven at 160°C for 1 The IC50 was defined as the concentration hour. Each disc was impregnated with 20 l (in g/mL) of the extract required to deplete portion of stock solution of the methanolic the amount of DPPH radical by 50%. Gallic extract of Vernonia amygdalina (100 acid (GA) and butylated hydroxytoluene mg/ml). The discs were dried in an incubator (BHT) were used as positive control at 35-37°C for 2 hours.

Ferric reducing antioxidant power assay Test organisms (FRAP) The two bacterial strains used in this The reducing power assay was conducted as investigation were obtained from previously described by Wang et al. (2008) Microbiology Department, University of and Oyaizu (1986) with ascorbic acid (AA) Lagos, Nigeria. The organisms are and tert-butyl-4-hydroxyanisole (BHA) Staphylococcus aureus, a Gram-positive being used as the positive controls. In brief, bacteria and Pseudomonas aeruginosa a 2.5 ml of individual deionized water diluted Gram-negative bacteria. The Vernonia amygdalina extract (ranged from microorganisms were maintained at 4°C on 0.1 to 1 mg/ml) was sequentially mixed with Nutrient Agar slant in the Department of equal volume of phosphate buffer (2.5 ml, Science Laboratory Technology 0.2 M, pH 6.6) and potassium ferricyanide (Microbiology Unit), School of Pure and

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Applied Science, Lagos State Polytechnic, Antibiotic susceptibility testing Ikorodu, Lagos, Nigeria and fresh subcultures were made before use. Susceptibility of organisms to different antibiotics were tested using disk diffusion Inoculum preparation method on freshly prepared Mueller Hinton agar and standardized by the method of A loopful of isolated colonies was National Committee for Clinical Laboratory inoculated into 4 ml of peptone water, Standard using some selected antibiotics incubated at 37°C for 4 hours. This actively namely: Amoxicillin (30ug), Ciprofloxacin growing bacterial suspension was then (5ug), Gentamicin (10ug), Ampicillin adjusted with peptone water so as to obtain a (25ug), Streptomycin (25ug) etc. For each turbidity visually comparable to that of 0.5 combination of the antibiotics and the McFarland standard prepared by mixing 0.5 bacterial strains, the experiment was ml of 1.75% (w/v) barium chloride performed in triplicate. The bacteria with a clear zone of inhibition of more than 17 mm dehydrate (BaCl2. 2H2O) with 99.5 ml of were considered to be sensitive 1% (v/v) tetraoxosulphate (vi) acid (H2SO4). This turbidity is equivalent to approximately108 colony forming units per Minimum inhibitory concentration (MIC) ml (CFU/ml). MIC is defined as the lowest extract concentration that inhibited the growth of Determination of antibacterial activity by the test organisms as indicated by absence of disk diffusion method visible turbidity in the tube compared with the control tubes. The minimum inhibitory Sensitivity of Staphylococcus aureus and concentration (MIC) of the extracts was Pseudomonas aeruginosa strains to determined using the tube serial dilution methanolic leaf extracts of Vernonia method. Extract (100 mg/ml) was dissolved Amygdalina were measured in terms of zone in water and diluted with Nutrient broth in of inhibition using disk diffusion method as two fold serial dilutions in test tubes. An described by Kirby-Bauer diffusion overnight broth culture of the test organism technique (Cheesbrough, 2002). Here the was adjusted to McFarland turbidity discs were soaked in the leaf extract as standard and 50 l (0.05 ml) of the cell described above before use. suspension added to each of the tubes. The tubes were incubated aerobically at 37°C for The plates containing Muller Hinton were 18 hours. smear with 0.1ml of the inoculums using swab stick. This was followed by mounting Minimum bactericidal concentration of the impregnated paper disc. The (MBC) inoculated Staphylococcus aureus and Pseudomonas aeruginosa isolates were The MBC of the methanolic leaf extract of incubated at 37°C for 24 hours and the Vernonia amygdalina was prepared by diameter of any resultant zone of inhibition modification of the method of Spencer and was measured. For each combination of Spencer (2004). Here 0.1ml, aliquots of extract and the bacterial strains, the samples taken from the non-turbid tubes of experiment was performed in triplicate. The the MIC assay tube was sub-cultured onto bacteria with a clear zone of inhibition of Mueller Hinton agar plates. The resulting more than 17 mm were considered to be plates were then incubated aerobically at sensitive. 416

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37°C for 24 hrs. The lowest concentration of lower when compared to the standard the extract at which no colonies of compounds used in this study. Staphylococcus aureus and Pseudomonas aeruginosa were seen was taken as the Determination of -Carotene and MBC. The results were compared with that lycopene of control using sterilized distilled water. The experiment was performed in triplicate. -Carotene and lycopene were found in The MBC was taken as the concentration of small amounts. The obtained values for - the extract that did not show any growth on Carotene and lycopene were 0.49 ± 0.25 µg a new set of agar plates. and 0.08 ±0.04 µg/g respectively.

Data analysis Figure I below shows the DPPH radical scavenging activities of Vernonia All analyses were carried out in triplicate amygdalina. Figure 2 below shows the and results expressed as mean ± SEM. The reducing power activities of Vernonia data analysis was done using the Graph Pad amygdalina. prism computer software version 6. Student s -test and one-way analysis of Vernonia amygdalina is widely consumed as variance (ANOVA) were used for food and used in traditional medicine comparison. A P-value < 0.05 was practice because of its immense medicinal considered significant. The IC50 values were properties which exert bacteriostatic and calculated using linear regression graphs. bacteriocidal effect on some bacteria. The Result and Discussion qualitative phytochemical screening revealed the presence of various secondary Qualitative phytochemical analysis of the metabolites in the extracts examples are: methanolic leaf extract of Vernonia tannins, phlobatanin, flavonoids, alkaloids, amygdalina glycosides, terpenoids, saponin, polyphenol, cardenolide etc (Table 1). A study The results obtained from the conducted by Oseni et al (2014) on phytochemicals analysis of Vernonia Vernonia amygdalina (bitter leaves) amygdalina leaf extract showed the presence revealed that the plant contained flavonoids, of some secondary metabolite like tannins, saponins, tannins, glycosides, phenolic, Phlobatanin, alkaloids, saponins, flavonoids, terpenes, anthracenosides, reducing sugar anthraquinone, glycoside, polyphenols, and alkaloids. Flavonoids have been terpenoid etc (Table I). The presence of reported to be synthesized by plants in these secondary metabolites in this extract response to microbial infections and are may be responsible for the anti-microbial good antibacterial agents and tannins have activity of the extract. been demonstrated to have antibacterial Quantitative analysis of the activities (Akiyama et al., 2001). phytochemical constituents of the Some of the previously isolated constituents methanolic leaf extract of Vernonia found in Vernonia amygdalina Del. include: amygdalina sesquiterpene lactones (Cimanga et al., The results of table 2 below revealed the 2004; Izevbigie, 2003 and Igile et al., 1995), level of phenolic compounds in Vernonia flavonoids like luteolin, luteolin 7-O- amygdalina. The values are significantly glucosides and luteolin 7-O-glucuronide (Igile et al., 1994), steroid glycosides (Igile 417

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426 et al.,1995; Jisaka et al., 1992 and Jisaka et popular methods utilized in screening for al., 1993), and vernonioside A, B, A1, A2, anti-oxidative activity. DPPH is a free A3, B2, B3 and A4 (Igile et al., 1995; Jisaka radical compounds and has been widely et al., 1992; Jisaka et al., 1993 and used to test the free radical scavenging Ohigashi et al., 1991). ability of various samples. The DPPH radical is employed as a substrate to The amount of total proanthocyanidins, evaluate antioxidant activity. IC50 expresses flavonoids, alkaloids, and total phenols the amount of antioxidant required to present in the extract of V.A are 0.09 ± 0.03 decrease the DPPH radical concentration by mg quercetin/g of dry plant material, 1.1%, 50% (Chanda et al., 2011). Basically, a 9.3% and 163.24 mg PE / g DW respectively higher DPPH radical scavenging activity is (Table 2). Strong antioxidant activities have associated with a lower IC50 value. IC50 been reported for flavonoids from V.A and, value was determined from plotted graph of its saponins have been reported to elicit scavenging activity against the different antitumoral activities in leukemia cells concentrations of V. amygdalina extracts, (Jisaka et al., 1993). Antimicrobial activities gallic acid (GA) and butylated in plants have been attributed to the hydroxytoluene (BHT). The highest percent presence of alkaloids, saponins, tannins, DPPH radical-scavenging activities were flavonoids and terpenes (Odugbemi, 2006). observed in the GA, followed by BHT whilst the extract showed lowest scavenging The plant has been used as remedies for activity. At a concentration range of 10 50 many infectious diseases, plants with g/mL, the IC50 of GA was 32.20 g/mL that secondary metabolites such as flavonoids, of BHT was 48.72 g/mL and V. alkaloids, saponins, terpenoids, amygdalina has a value of 95.65 g/mL sesquiterpene lactones and steroids have (Figure 1). been found to have antimicrobial properties in vitro ( Cartagena et al., 2008, Cowan, This result suggested that the extract can 2002 and Sibanda and Okoh, 2007).These scavenger DPPH radicals. The reducing phytochemicals were all found to be present capacity of an extract may be an important in the methanolic leaf extract of V. indicator of its potential antioxidative amygdalina. -Carotene and lycopene were activity (Chanda et al., 2011). A higher found in small amounts. The obtained values absorbance indicates a higher ferric reducing for -Carotene and lycopene were 0.49 ± power. Increase absorbance indicates an 0.25 µg and 0.08±0.04 µg/g respectively. increase in reductive ability. Figure II The presence of these compounds in the presents the dose dependent ferric reducing extract indicates the anti-oxidant potential of powers of the extracts of V. amygdalina, the plant. BHA, and AA respectively. The extract of V.A has a reducing power value of 0.708 Oxidative stress which is caused by compared to the standard BHA and AA with insufficient capacity of biological system to reducing power of 1.36 and 0.9 respectively. neutralize excessive free radical product, has This is an indication that the extract has a been associated with all kind of human ferric reductive ability. diseases and aging (Jensen et al., 2008). In this study, V. amygdalina extract possessed Resistance to antimicrobials is highly in vitro antioxidant activity when tested with prevalent in bacterial isolates worldwide, DPPH radical scavenging assay. DPPH free particularly in developing countries. radical scavenging ability is one of the most 418

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Table.1 The qualitative phytochemical constituents of the methanolic leaf extract of Vernonia amygdalina

Phytoconstituent Qualitative abundance Tannins + Phlobatanin + Flavonoids ++ Alkaloids +++ Glycosides ++ Cyanogenic glycosides ++ Anthraquinone ++ Terpenoids + Saponin ++ Polyphenols ++ Cardenolide + + (Present in low concentration), ++ (present in moderate concentration) and .+++ (present in high concentration).

Table.2 Quantitative analysis of the phytochemical constituents of the methanolic leaf extract of Vernonia amygdalina

Phenolics Vernonia amygdalina extract Total proanthocyanidinsb 0.09±0.03 Flavonoids 1.1% Alkaloids 9.3% Total phenola 163.24

Table.3 Zones of inhibition indicating the antimicrobial activity of the methanolic leaf extract of Vernonia amygdalina on the tested organisms

Test organisms Gram Conc. of the methanolic Diameter of zones of property extract of VA inhibition (mm) S. aureus +ve 100 mg/ml 20 ± 2.2 P. aeruginosa -ve 100 mg/ml 23 ± 1.2 y t i v i t c

a 100 g GA n i

g 80 BHT n e

v V. amygdalina a

c 60 s l a c

i 40 d a r

e 20 e r f

H 0 P 10 20 50 60 P

D Concentration (µg/ml) % Figure.I DPPH radicals scavenging activities of the extracts of V. amygdalina, GA and BHT at different concentrations. Each value represents mean ± SD (n =6).

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Table.4 Antimicrobial susceptibility pattern of standard antibiotics against Staphylococcus aureus

Antibiotic Concentration Diameter of zone of Interpretation sensitive disc ( g ) inhibition (mm) Pefloxacin 10 14.8 R Gentamicin 10 12.3 R Ampiclox 30 10.6 R Rocephin 25 9.2 R Zinnacef 20 8.1 R Amoxycilin 30 15.1 R Ciprofloxacin 10 22.2 S Streptomycin 30 18.3 S Septrin 30 12.0 R Erythromycin 10 17.2 R Augmentin 30 14.6 R Ampicilin 10 16.1 R KEY: S = Sensitive (zone of bacterial inhibition is 18 mm) and R = Resistance (zone of bacterial inhibition is < 18 mm).

Table.5 Antimicrobial susceptibility pattern of standard antibiotics against Pseudomonas aeruginosa

Antibiotic Concentration Diameter of Interpretation sensitive disc ( g ) zone of inhibition (mm) Pefloxacin 10 17.6 S Gentamicin 10 18.1 S Ampliclox 30 12.2 R Rocephin 25 17.2 R Zinnacef 20 5.6 R Amoxycillin 30 20.1 S Ciprofloxacin 10 18.1 S Streptomycin 30 15.3 R Septrin 30 15.2 R Erythromycin 10 18.1 S Augmentin 30 9.3 R Ampicilin 10 15.2 R Sparfloxacin 10 18.1 S Tarivid 10 20.2 S KEY: S = Sensitive (zone of bacterial inhibition is 18 mm) and R = Resistance (zone of bacterial inhibition is < 18 mm).

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Table.6 MIC and MBC determination of Vernonia amygdalina against Staphylococcus aureus and Pseudomonas aeruginosa

Test organisms MIC MBC Staphylococcus aureus 70 mg/dl 140 mg/ml Pseudomonas aeruginosa 30 mg/dl 60 mg/dl

2.5 BHA m

n 2.0 AA 0 0

7 V. amygdalina t

a 1.5 e c

n 1.0 a b r o

s 0.5 b a 0.0 0.2 0.4 0.6 0.8 Concentration (mg/ml)

Figure.II Reducing power of BHA, AA and V. amygdalina at different concentrations. Each value represents mean ± SD (n =6).

Figure.3 Agar plate showing zones of inhibition of the methanolic leaf extract of Vernonia amygdalina against Staphylococcus aureus.

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Figure.4 Sensitivity and resistance of Staphylococcus aureus strain to multi drug sensitive antibiotic disc

Figure.5 Sensitivity and resistance of Pseudomonas aeruginosa strain to multi drug sensitive antibiotic disc

The mean diameters of zones of inhibition strains to standard multi drug antibiotic for the methanolic leaf extract were 20 ± 2.2 sensitivity discs. mm against S. aureus and 23 ± 1.2 mm against P. aeruginosa (table 3).The activity Twelve different antimicrobial standard of the extract significantly (P<0.05) showed antibiotic agents were used to test the antimicrobial activities against all the tested susceptibility against S. aureus. The organisms used. The extract exhibited strong organism was resistant to the first line potency against the micro-organisms with P. antibiotics that are commonly prescribed by aeruginosa being the most susceptible. physicians. The organism (S. aureus) was Therefore, it appears that the methanolic leaf found to be resistance to pefloxacin, extract of V. amygdalina could present an gentamicin, ampiclox, roceptin, zinnacef, important treatment option of infections amoxicillin, septrin, erythromycin, caused by these organisms (S. aureus and P. augumentin and ampicilin. It was also aeruginosa). Figure III shows the zone of observed that the bacterium was too inhibition of V.A against S. aureus. The sensitive to ciprofloxacin and streptomycin clear zone of inhibition is an indication that (table 4). In table 5, fourteen antibiotic the extract has antimicrobial activities. sensitive discs were used to test the Figure IV and V show the sensitivity and susceptibility against P. aeruginosa. The resistance of S. aureus and P. aeruginosa organism was sensitive to pefloxacin,

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426 gentamicin, amoxicillin, ciprofloxacin, assistance when carrying out this research erythromycin, sparfloxacin and tarivid. P. work. aeruginosa was resistance to amplicox, roceptin, zinnacef, streptomycin, septrin, References augumentin and ampicilin. Abort, A.O., Raserika, B.H 2003. In vivo The zone of inhibition that is 18mm is antimalarial activity of Vernonia considered to be sensitive while those that amygdalina. British Journal of are < 18mm are considered to be resistance. Biomedical Science; 60:89-91. Low MIC indicates the minimum inhibitory Akiyama, H., Fujii, K., Yamasaki, O., Oono concentration required to inhibits the growth T., Iwatsuki, K. 2001. Antibacterial of the test organism, which translate to high Action of Several Tannins against potency of the extract. The MIC of 30 Staphylococcus aureus, J. mg/ml was exhibited by the extract on P. Antimicrob. Chemother. 48(4):487- aeruginosa while 70 mg/ml was observed in 491. S. aureus. Similar report revealed the Aliyu, A.B., Musa, A.M., Abdullahi, M.S. antibacterial potency of active leaf extract of and Oyewale, A.O. 2008. V. amygdalina with MICs of 22.5 to 26.0 Phytochemical and antibacterial mg/ml and 19.8 to 26.4 mg/ml for the Gram- properties of Ludwigia suffruticosa negative and Gram positive isolates tested (Willd.) Oliv. ex. O. Ktze (Iwalokun et al., 2003). V. amydalina (Onagraceae). Int. Jor. P. App. Scs., exhibited a minimum bacteriocidal 2 (4), 1 5. concentration (MBC) of 60 mg/ml for P. Argheore, E.M., Makkar, H.P.S., Becker, K. aeruginosa and 140 mg/ml for S. aureus. 1998. Feed value of some browse The extract exhibited strong potency against plants from central zone of Delta these microorganisms with P. aeruginosa State of Nigeria. Trop Sci, 38:97 being the most susceptible. The results of 104. this study support the use of the extract as Barros, L., Ferreira, M.J., Queiros, B., herbal remedies in Nigeria. Ferreira, I.C.F.R., Baptista, P. 2007. Total phenols, ascorbic acid, b- In conclusion, this study showed that carotene and lycopene in Portuguese methanolic leaves extract of Vernonia wild edible mushrooms and their amygdalina exerted significant (P<0.05) antioxidant activities. Food Chem. antimicrobial activities against 100, 413 419. Staphylococcus aureus and Pseudomonas Blois, M. S. 1958. Antioxidant aeruginosa and might be source of active determinations by the use of a stable antimicrobial agents for the development of free radical, Nature, vol. 181, no. drugs for the treatment of these 4617, pp. 1199 1200. microorganisms. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A. 2007. The growth and Acknowledgments death of microorganism. In Jawetz, Melinick and Adelberg s Medical The authors are grateful to Hassan Ameenat Microbiology 24th ed Mcgraw-Hill Olayinka, Adeleke Wakilat Romoke and Toronto, 57p. Okajare Oluwasegun Michael for their

423

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426

Cartagena, E., Montanaro, S., Bardon, A. University of Agriculture Umudike, 2008. Rev. Latinoamer, Quim. 36, Nigeria. 43. Ekundayo, E.O., Ezeogu, L.I. 2006. Chambers, H.F. 2005. Community- Evaluation of Antimicrobial associated MRSA-resistance and activities of extracts of five plants virulence converge. New Engl. J. used in Traditional Medicine in Med. 352: 1485-1487. Nigeria. Int. J. Trop. Med. 2: 93-96. Chanda, S., Dave, R., and Kaneria, M. Farombi, E. O. 2003. African indigenous 2011. In vitro antioxidant property of plants with chemotherapeutic some Indian medicinal plants. potentials and biotechnological Research Journal of Medicinal approach to the production of Plant, 5(2):169-179. bioactive prophylactic agents. http://dx.doi.org/10.3923/rjmp.2011. African Journal of Biotechnology, 2: 169.179 662 667. Cheesbrough, M. 2002. District Laboratory Harborne, J.B.1973. Phytochemical practice in Tropical Countries Part 2: methods. Chapman and Hall Ltd., Cambridge University Press. UK pp. London. pp 49-188. 136-142. Hyun, D., Jo, D., and Emerson, S. 2006. Cimanga, R.K., Tona, L., Mesia, K., Calorie restriction up-regulates the Musuamba, C.T., De Bruyne, T., plasma membrane redox system in Apers, S., Hernan, N., Miert, V.S., brain cells and suppresses oxidative Pieters, L., Totte, J., Vlietink, stress during aging. The Proceedings A.J.2004. In vitro antiplasmodia of the National Academy of acivity of extravts and fractions of Sciences.,103: 19908 19912. seven medicinal plants used in the Igile, G.O., Oleszek, W., Jurzysta, M., democratic republic of Congo. J. Burda, S., Fafunso, M., Fasanmade, Ethnopharmacol. 93: 27-32. A.A. 1994. Flavonoids from Cohen, M.L. 2002. Changing patterns of Vernonia amygdalina and their infectious disease. Nature 406: 762- antioxidant activities. J. Agric. Food 767. Chem. 42: 2445-2448. Cowan, M.M. 2002. Clin. Microbiol. Rev. Igile, G.O.,Oleszek, W., Burda, S., Jurzysta, 12, 564. M. 1995. Nutritional assessment of Dogÿan, S., Turan, Y., Ertuerk, H., and Vernonia amygdalina leaves in Arslan, D. 2005. Characterisation growing mice. J. Agr. Food Chem. and purification of polyphénols 43: 2162-2166. oxidase from artichoke(Cyanara Iwalokun, B.A., Bamiro, S.B., Durojaiye, ScolymusL). Journal of Agricultural O.O. 2003. West Afr. J. Pharmacol. Food Chemistry, 53, 776 785. Drug Res. 19, 9. Egedigwe, C. A. 2010. "Effects of Dietary Izevbigie, E.B. 2003. Discovery of water- Incorporation of Vernonia soluble anticancer Agents (Edotides) amygdalina and Vernonia colorata from a vegetable found in Benin on Blood Lipid Profile and Relative City, Nigeria. Exp. Biol. Med. 228: Organ Weights in Albino Rats", 293-298. M.Sc. Dissertaton, Department of Jensen, G.S., Wu, X., Patterson, K.M., Biochemistry, Michael Opara Barnes, J., Carter, S.G., Scherwitz, L., Beaman, R., Endres, J.R.,

424

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426

Schauss, A.G. 2008. In vitro and in 10th information supplement vivo antioxidant and anti- approved standard, M100 - S10, inflammatory capacities of an Wayne P.A. antioxidant-rich fruit and berry juice Nimenibo-Uadia, R. 2003. Effect of blend. Results of a pilot and Vernonia amygdalina in alloxan randomized, double-blinded, induced diabetic albino rats. J. Med. placebo-controlled crossover study. Lab. Sci., 12(1): 25-31. J. Agric. Food Chem. 56: 8326-8333. Odugbemi, T. 2006. Outline of Medicinal Jisaka, M., Ohigashi, H., Takagaki, T., Plants in Nigeria.1st ed. Lagos: Nozaki, H., Tada, T., Hirota, M., University of Lagos Press. Irie, R., Huffman, M.A., Nishida, T., Ohigashi, H., Jisaka, M., Takagaki, T., Kaji, M., Koshimizu, K. 1992. Bitter Nozaki, H., Tada, T., Huffman, steroids glucosides, vernoniosides M.A., Nishida, T., Kaji, M., A1, A2, and A3 and related B1 from Koshimizu, K. 1991. Bitter principle a possible medicinal plant, Vernonia and a related steroid glucoside from amygdalina used by wild Vernonia amygdalina, a possible chimpanzees. Tetrahedron. 48: 625- medicinal plant for wild 632. chimpanzees. Agr. Biol. Chem. 55: Jisaka, M., Ohigashi, H., Takegawa, K., 1201-1203. Hirota, M., Irie, R., Huffman, M.A., Olukemi, O.A., Olukemi, I.O., Sofidiya, Koshimizu, K. 1993. Steroid M.O., Aniunoh, O.A., Lawal, B.M., glucosides from Vernonia and Tade, I.O. 2005. Antioxidants amygdalina, a possible chimpanzee activity of Nigerian Dietary spices. plant. Phytochemistry. 34: 409-413. Electr. J. Environ. Agric. Food Lai, P. K., and Roy, J. 2004. Antimicrobial Chem., 496: 1086 1093. and chemopreventive properties of Osawa, T. 1994. Novel natural antioxidants herbs and spices. Journal of Current for utilization in food and biological Medicinal Chemistry, 11(11): 1451- systems. In I. Uritani, V.V. Garcia, 60. Lowy, F.D. 2003. Antimicrobial and E.M. Mendoza (Eds.), resistance: the example of Postharvest Biochemistry of Plant Staphylococcus aureus. J. Clin. Food-Materials in the Tropics. Invest. 111:1265-1273. Tokyo, Japan: Japan Scientific Momoh, J., Akoro,S.M., and Godonu, K.G. Societies Press., 241 251. 2014. "Hypoglycemic and Oseni, .L. A., Abugri. J., Sumabe, .B. K., Hepatoprotective Effects of and Onilimor, P. J. 2014. Leaf Vernonia Amygdalina (Bitter Leaf) Extracts of Vernonia amygdalina and Its Effect on Some Biochemical Del. From Northern Ghana Contain Parameters in Alloxan-induced Bioactive Agents that Inhibit the Diabetic Male Albino Rats" Science Growth of Some Beta-lactamase Journal of Biochemistry, Volume Producing Bacteria in vitro. British 2014, Article ID sjbt-194, 7 Pages, Journal of Pharmaceutical Research, 2014, doi: 10.7237/sjbt/194. 4(2): 192-202, 2014 National Committee for Clinical Laboratory Oyaizu, M. 1986. Studies on products of Standards (NCCLS), 2000. browning reactions-antioxidative Performance standard for activities of products of browning antimicrobial susceptibility testing,

425

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426

reaction prepared from glucosamine. Wang, H., Gao, X.D., Zhou, G.C., Cai, L., Jap. J. Nutr. 44: 307 315. and Yao, W.B. 2008. In vitro and in Shahidi, F., Janitha, P. K., and vivo antioxidant activity of aqueous Wanasundara, P. D. 1992. Phenolic extract from Choerospondias antioxidants. Critical Rev. Food Sci. axillaris fruit. Food Chemistry., 106: Nutr., 32(1): 67 103. 888 895. Sibanda, T., Okoh, A.I., 2007. Afr. J. WHO., 2004. World Health Organization, Biotechnol. 6, 2886. author. The World Health Report: Singleton, V. L., and Rossi, J. A. 1965. The problem of antibiotic resistance. Colorimetry of total phenolics with Geneva. phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture., 16: 144 158. Smith, M., Perry, G., and Raina, A. 2000. Oxidative stress in Alzheimer s disease. Biochimica et Biophysica Acta, 1502: 139 144. Sofowora, A., 1993. Medicinal plants and traditional medicines in Africa Spectrum Book Ltd. Ibadan, Nigeria. p 289. Soroya, A. S. 2011. Herbalism, phytochemistry and ethnopharmacology. Science Publishers, Enfield, New Hampshire. Retrieved from www.scipub.net/cited 15/05/2014 Spencer, A.L.R., Spencer, J.F.T. 2004. Public Health Microbiology: Methods and Protocols. Human Press Inc. New Jersey. pp. 325-327. Sun, J.S., Tsuang, Y.W., Chen, I.J., Huang, W.C., Hang, Y.S., Lu, F.J. 1998. An ultraweak chemiluminescence study on oxidative stress in rabbits following acute thermal injury. Burns., 24:225-231. Trease, G.E., Evans, W.C. 1986. Pharmacognsy. 11th edition. London: Brailliar Tiridel Can Macmillian Publishers., 60 75. Walsh, C., 2000. Molecular mechanisms that confer antibacterial drug resistance. Nature 406:775-781.

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