Antibacterial and Antioxidant Compounds from the Flower Extracts of Vernonia amygdalina

Vernonia amygdalina is traditionally used in Ethiopia to treat various diseases. This prompted us to isolate bioactive compounds from the flowers of this plant. The CHCl3 extract after silica gel column chromatography has led to the isolation of two compounds identified as tricosane (1) and vernolide (2), while the acetone extract furnished isorhamnetin (3) and luteolin (4). The acetone extract and isorhamnetin significantly scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by 91.6 and 94%, respectively. It was also shown that the acetone extract and isorhamnetin inhibited lipid peroxidation by 74 and 80%, respectively. The extracts and isolated compounds were also evaluated for their antibacterial activity with the CHCl3 extract and vernolide showing strong activity against S. aureus with an inhibition zone of 21 and 19 mm, respectively. On the other hand, the acetone extract and isorhamnetin were active against all bacterial pathogens tested. The work presented herein has demonstrated that vernolide and isorhamnetin had antibacterial activity. The antioxidant activity displayed by the flowers of V. amygdalina is accounted to the presence of isorhamnetin. Therefore, the biological activities displayed by the extracts and isolated compounds from this plant corroborate the traditional uses of this plant by the local people against various diseases.


Introduction
Vernonia amygdalina Del. is a soft woody shrub or tree belonging to the family Asteraceae and genus Vernonia [1]. e plant is predominantly found in Africa. It is a perennial plant characterized by its bitter sap from the leaf which has been widely explored for its medicinal use. is plant grows to 10 m tall with petiole leaf of about 6 mm in diameter and elliptic in shape [2]. In the Ethiopian highland, V. amygdalina has been classified by the farmer as a multipurpose fodder tree with high biomass yield, easy propagation, high adaptability, and high compatibility with other crops which do not compete with them for soil nutrients or moisture but instead help to improve the soil fertility and growth of perennial crops [3]. V. amygdalina, locally called ebicha in Afan Oromo and Grawa in Amharic (Ethiopia), is quite commonly used in Ethiopia in the preparation of local beer and also as fumigant. It is also used as fire wood [4].
e species is also used to promote wound healing [9] and to treat microbial infections [10]. e main bioactive constituents of the leaves were reported as sesquiterpene lactones [11]. Some of them include vernonioside A1, vernonioside A2, vernonioside B1, vernonioside B2 [12,13], vernodalin, vernolepin, vernomygdin, vernodalol, and vernodalinol [14,15]. Despite numerous report on the secondary metabolite profile of the leaves, to the best of our knowledge there is no prior scientific report on the chemical constituents of the flowers of this species. Furthermore, the antioxidant and antibacterial activities of the flower extracts and constituents of V. amygdalina have not been studied. Hence, in this paper, we report the results of our investigation of effects of the extracts and constituents of the flowers of V. amygdalina on E. coli, K. pneumonia, P. mirabilis, S. aureus, and S. bacillus. e results of the evaluation of the radical scavenging activities and antilipid peroxidation potentials of the flower extracts and constituents of V. amygdalina are also described herein.

Plant Material.
e flowers of V. amygdalina were collected on February 12, 2017 from Wonji district, which is located at 107 km Southeast of Addis Ababa, Ethiopia. Identification and authentication of the plant specimen was done at the National Herbarium of Addis Ababa University by Mr. Melaku Wondafrash and voucher specimen (AB002) was deposited ( Figure 1).
Likewise, the acetone extract (5 g) was adsorbed and fractionated over silica gel (180 g) column chromatography to furnish 15 fractions. In each case, a volume of 100 mL was collected.

Antibacterial Activities.
e antibacterial activities of the extracts and constituents of the flower of V. amygdalina were checked using agar well diffusion method [16] against five bacterial strains, three Gram negative (Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) and two Gram positive (Staphylococcus aureus and bacillus). Bacterial cultures were maintained on nutrient Muller-Hinton agar at 37°C, and the cultures were kept in appropriate media slants and stored at 4°C until used. Colonies of bacteria (24-hour old culture) were diluted by physiological normal saline (0.85%), to make a 0.1 McFarland standard suspension, and then the bacteria was inoculated into sterile Petri dishes of 60 mL of Muller-Hinton agar plates. e plates were shaken gently to allow evenly mixing of bacterial cells and agar. All samples were dissolved in methanol to furnish 10 mg/mL. From each sample, 100 µL of each concentration saturated with discs (6.00 mm diameter disc) was placed on plate and incubated at 37°C for 24 hours. e diameters of the inhibition zones were calculated. Clear inhibition zones formed around the discs indicated the presence of antibacterial activity [16]. Control wells containing neat solvents (negative control) and chloramphenicol (positive control) were also run parallel in the same plate.

Antioxidant Assay.
e antioxidant activities of the extracts and constituents were studied using DPPH and ferric thiocyanate methods.

DPPH Radical Scavenging Assay.
e radical scavenging assay of the extract and constituents of the flowers of V. amygdalina was assessed using DPPH according to the following procedure [17]: the CHCl 3 extract was dissolved in methanol to afford 1 mg/mL. It was serially diluted in methanol to give concentration of 500, 250, 125, and 62.5 µg/mL. To 1 mL of each concentration, 4 mL DPPH  (0.04% DPPH in MeOH) was added to make 100, 50, 25, and 12.5 µg/mL solutions. is was repeated for the acetone extract and pure constituents. en, all the samples prepared were incubated in an oven at 37°C for 30 min and then absorbance was recorded at 517 nm using a UV-Vis spectrophotometer. e experiments were performed in triplicates.
e percentage inhibition was calculated using the following formula: where A control is the absorbance of DPPH solution and A extract is the absorbance of the test sample (DPPH solution plus sample).

Ferric iocyanate Method.
e antioxidant potential of the extracts and constituents of the flowers of V. amygdalina was also assessed according to the method of Nagatsu [18]. Each 0.1 mg of the extracts and pure compounds of V. amygdalina, 100 µL of linoleic acid, EtOH (5 mL), and phosphate buffer (5 mL, 0.05 M, pH � 7) in water were separately added into a vial and incubated at 40°C in an oven. After 24 h, 0.1 mL from each were taken and added into a vial containing 75% aqueous EtOH (7 mL), 30% of NH 4 SCN (0.15 mL), and 0.15 mL of 0.02 M FeCl 2 in 3.5% HCl. Each was then subjected to UV-Vis spectrophotometery to record the absorbance at 500 nm. Absorbance of the blank and ascorbic acid were done in the same fashion. e experiments were performed in triplicates. e percentage inhibition using ferric thiocyanate method is calculated according to the following formula: where As is the absorbance of the sample and Ab is the absorbance of the blank [19].

Results and Discussion
Four compounds were isolated and characterized from the flower extracts of V. amygdalina (Figure 2). e NMR spectral data generated for compound 1 agreed well with tricosane ( Figure 2). is compound has not yet been reported from the genus.
Compound 2 (77 mg) was obtained as yellow solid from the chloroform extract of the flower of V. amygdalina. It was eluted with hexane : ethyl acetate (1 : 9). e 1 H-NMR spectral data of compound 2 (Supplementary Material 1) showed the presence of exomethylene δ-lactone at δ 6.3 (1H, d) and δ 5.9 (1H, d). Other diastereotopic methylene protons were evident at δ 6.1 (1H, d) and 5.7 (1H, d). Another olefinic methine proton is observed at δ 5.5 (1H, d). e signal at δ 5.2 (1H, t) is due to methine proton on oxygenated carbon. Methylene protons on oxygenated carbons are evident at δ 4.6 and 3.7 (2H, d, H-15). e signal at δ 4.5 (1H) indicated that the presence of methine proton on carbon bearing two oxygens. Moreover, the spectral data of this compound showed one methyl group on quaternary carbon at δ 1.9 (3H, s). e 13 C-NMR spectral data (Supplementary Material 2) displayed the presence of nineteen carbon resonances which agreed well with literature reported from vernolide [20] whose structure is shown in Figure 2. e structure was further confirmed with extensive 2D-NMR including COSY, HSQC, and HMBC (Supplementary Materials 4-6).
Compound 3 (70 mg) was isolated from the acetone extract of the flower of V. amygdalina. It was eluted using . e carbon resonance at δ 60.0 is due to the presence of the methoxy group. Based on the above spectral data, compound 3 was identified as isorhamnetin whose structure is shown in Figure 2.

Antibacterial Activity.
e antibacterial activities of the hexane, chloroform, acetone extract, and pure constituents of the flowers of V. amygdalina were investigated using agar well diffusion method, against the selected human pathogens such as E. coli, K. pneumoniae, P. mirabilis, S. aureus, and S. bacillus. e results are presented in Table 1. e extracts and isolated compounds showed considerable difference in antibacterial activities against all selected bacteria's with zone of inhibition ranging from 6 to 21 mm (Table 1). e acetone and chloroform extracts of the flowers of V. amygdalina demonstrated pronounceable activity against S. aureus (Figure 3) compared to chloramphenicol used as a standard antibiotic. is is in conformity with the result reported for the leaf extract of V. amygdalina against S. aureus [21]. e activity displayed by the chloroform extract is likely accounted to the presence of vernolide which showed an inhibition zone of about 19 mm.
is is significantly higher than the activity displayed by chloramphenicol. Vernolide exhibited relatively better activity against Gram-positive bacteria with maximum inhibition zone (19 mm) observed for S. aureus. is is in close agreement with the literature reported for same compound [20]. e zone of inhibition displayed by the constituents of the flowers of V. amygdalina is depicted in Figure 3. e chloroform extract was found insensitive against E. coli, K. pneumonia, P. mirabilis, and S. bacillus. On the other hand, the acetone extract displayed broad range of antibacterial activities against all five strains tested in this study. Similar effects have been reported for the leaves extract of V. amygdalina against E. coli [22]. e wide zone of inhibition of the acetone extract of V. amygdalina showed that it had great potential as a remedy for infections/diseases caused by bacterial pathogens. Isorhamnetin isolated in this work from this plant showed modest activity with inhibition zone ranging from 9 to 14 mm against all bacterial strains.
e work presented herein has demonstrated that the acetone extract, vernolide, and isorhamnetin identified in the flowers of V. amygdalina had antibacterial activity.
ough their degree varies, the extracts and pure constituents of the flowers of V. amygdalina are able to reduce the stable DPPH radical to the yellow-colored diphenylpicrylhydrazicine indicating their potential as radical scavengers.
e acetone extract reduced DPPH radicals significantly as compared to the chloroform extract, with   Table 2). is is evident from the low IC50 (42 µg/mL) value observed for the acetone extract. At the same concentration standard, ascorbic acid scavenged the DPPH radical by 97.7%. Plant phenolics are a major group of compounds acting as primary antioxidants or free radical scavengers [23]. erefore, the observed high free radical scavenging activity of the acetone extract is accounted to the presence of two natural antioxidants, isorhamnetin and luteolin, identified in this study from the flowers of this species. e radical scavenging potential of isorhamnetin was found to be 94%, which is comparable to ascorbic acid used as the positive control. Both isorhamnetin and luteolin are flavonoids with free hydroxyl group which can donate hydrogen and electron and hence responsible for the radical scavenging activities of the flower of V. amygdalina. is agrees well with the literature reported for the antioxidant activity of luteolin [23].

Ferric iocyanate Method.
e degree of lipid peroxidation which was evaluated using ferric thiocyanate method can be used to measure the antioxidant potential of compounds or extracts. Table 3 shows the results of the antilipid peroxide formation of the flower extracts and constituents of V. amygdalina.
As depicted in Table 3, the acetone extract and isorhamnetin inhibit peroxide formation by 74 and 80%, respectively, demonstrating their potential in preventing the formation lipid peroxides. e results were turned out to be comparable with ascorbic acid with inhibition of 83%. On the other hand, the chloroform extract and vernolide were shown to have low ability of inhibiting peroxide formation compared with the natural antioxidant. is indicates that the antioxidant compound of the flowers of V. amygdalina resides in the acetone extract with the main active ingredient found to be isorhamnetin.

Conclusion
In conclusion, the work presented herein has demonstrated that the acetone extract and vernolide had strong antibacterial activity compared to chloramphenicol. e antioxidant activities displayed by the acetone extract and isorhamnetin were significant compared with ascorbic acid indicating the potential of the flowers of this species as natural antioxidants. erefore, biological activities displayed by the flower extracts and constituents of the flowers of V. amygdalina corroborate the traditional uses of this plant against various ailments including bacteria.

Conflicts of Interest
e authors declare that there are no conflicts of interest.