Isolation and Identification of Flavanone Derivative Eriodictyol from the Methanol Extract of Afzelia africana Bark and Its Antimicrobial and Antioxidant Activities

Background Afzelia africana is a plant species with well-documented ethnobotanical and medicinal properties. The plant is reported to have various secondary metabolites and had been applied for the treatment of various diseased conditions. Objectives The study objectives include fractionation, isolation, purification, and characterization of eriodictyol from the bark of A. africana, and the determination of its antimicrobial and antioxidant activities. Methodology. The series of methodologies that were employed include fractionations and purification (column chromatography), characterization (HPLC, LC-MS, IR, 1H, 13C, DEPT-135, HSQC, and HMBC), antimicrobial assays (microbroth dilution and checkerboard assay), and antioxidant activities assays (ABTS and DPPH scavenging capacity). Results The study reports the identification and characterization of eriodictyol from the bark of A. africana which exhibited potent antioxidant activities against ABTS and DPPH radicals with scavenging capacities (SC50) of 2.14 ± 0.05 and 2.51 ± 0.06 µg/mL, respectively. The compound exhibited its antimicrobial activity by reporting good bacteriostatic activities (MBC/MIC > 4) against Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), and fluconazole-resistant Candida albicans (CA2). Moreover, a broad spectrum of bactericidal effects (MBC/MIC ≤ 4) was reported against Streptococcus mutans (SM), Escherichia coli (EC), Bacillus subtilis (BS), Klebsiella pneumonia (KP), Pseudomonas aeruginosa (PA), Salmonella typhi (ST), and standard Candida albicans (CA1). The compound further exhibited synergistic effects against EC, KP, ST, and MRSA; ST; and CA2 when combined with ciprofloxacin, tetracycline, and nystatin, respectively. However, antagonistic effects were observed against PA and CA1 when combined with ciprofloxacin and ketoconazole, respectively. Conclusion The study reports for the first time the identification of eriodictyol from the bark of A. africana which exhibited significant antioxidant and antimicrobial properties.


Introduction
Te statistical data from the World Health Organization suggest that 80% of developing countries depend on plant medicines for the treatment of diseases and ailments through ethnobotany. As a result, the ethnobotanical study has provided a rich source of medicinal information from plants [1]. Te information has contributed to the incorporation of herbal medicine into the main health systems of various national health care systems [1,2]. Research beyond the ethnobotanical information has contributed to a deeper understanding of the phytochemistry, pharmacognosy, and ethnopharmacology of herbal medicines [3,4]. Consequently, strategies for the fractionation and purifcation of plant extracts based on biological activity have led to the isolation of bioactive compounds with subsequent application as drugs or lead agents for further drug development protocols [3,5,6].
Africa is endowed with a vast foral scope that has been utilized ethnobotanically for the treatment of diseases. One plant with enormous ethnomedicinal utilization is Afzelia africana, a plant species belonging to the Fabaceae family [7]. Te plant is commonly referred to as African mahogany with medium to large sizes and can grow up to 40 m [8]. Te medicinal prowess of A. africana has been reported through various ethnobotanical surveys. Te plant has been utilized ethnobotanically as an analgesic, anti-infammatory, antidiabetic, antimalarial, aphrodisiac, antimicrobial, emetic, emmenagogic, trypanocidal, and laxative agents [7,9]. Te antitrypanocidal, anthelmintic, antidiabetic, antimicrobial, and synergistic efects on standard antibacterial and fungal agents were also well-reported [10][11][12][13][14]. Moreover, the methanol extract of the plant was reported to have higher susceptibility against bacterial isolates such as Escherichia coli, Staphylococcus aureus, and Candida albicans [15,16]. Investigation of the phytochemical constituents of the plant extract revealed the presence of favonoids, tannins, saponins, alkaloids, and steroids [9,13]. Furthermore, the ethnobotanical usage of the plant as an antiplasmodial agent was authenticated against the 3D7 strain of Plasmodium falciparum [9,14]. Despite its rich medicinal potential, little or no compound has been isolated and linked to its numerous biological activities even though the plant is endowed with rich phytochemicals. A particular phytochemical class consistently identifed in A. africana is a favonoid. Flavonoid compounds are further classifed into several derivatives. Particularly, favanones are favonoid derivatives with the basic 2,3-dihydrofavone structure with reported numerous health benefts. Unlike their other favonoids counterpart, they lack a double bond at the C2-C3 portion of their chromene-4-one moiety [17]. Tey are biosynthesized and abundant in citrus fruits. Te main aglycone naringenin, hesperetin, and eriodictyol are, respectively, abundant in grapefruit, oranges, and lemons [18]. Tey are also abundantly available in various parts of cereals and other plant families such as Compositae, Leguminosae, and Rutaceae [19]. Te study sought to employ the principle of bioassay-guided fractionation studies to isolate, purify, and characterize a favanone compound from the bark of A. africana and to evaluate its antimicrobial and antioxidant activities.

Extraction of Plant Material.
Te bark of the plant material was air-dried under shade and pulverized. 500 g of the pulverized plant material was macerated in 90% methanol for three days with periodic agitation, followed by fltration. Te fltered extract was then concentrated and evaporated to dryness using the rotary evaporator (BUCHI Rotavapor R-114) at 50°C. Te percentage yield of the extract was determined to be 12% w/w (60 g) and kept at 2°C until further use.

Structural Elucidation.
Te structure of the isolated compound was characterized by employing analytical techniques such as HPLC, FTIR, LC-MS, MS, and NMR.

HPLC Condition for Purity Determination.
Te HPLC chromatogram was determined on the PerkinElmer (PE) series 200 (Kyoto, Japan) liquid chromatography using the BDS Hypersil C18 reversed-phase column (250 × 4.6 mm, 5 μm). Te isolated compound was solubilized in 5.0 mL of methanol and 20 μL injected into the HPLC using methanolwater-formic acid (40 : 60 : 1) as the mobile phase. Te compound was detected at a fow rate and wavelength of 1 mL/min and 254 nm, respectively, using the diode array UV detector.

FTIR Spectroscopy.
Te IR spectrum of the compound was determined using the FTIR (820IA single beam laser Shimadzu infrared spectrophotometer, (Tokyo, Japan)) within the wavenumber regions of 4,000 cm − 1 -400 cm − 1 .

LC-MS.
Te LC-MS of the compound was determined using the LC system (Terkinelmer Agilent infnity 1,290 LC coupled to the 6,420 triple quadrupole MS (Melbourne, Australia)). Te solvent system consisted of methanol-water (50 : 50) at a fow rate of 1.5 mL/min.

ESI-MS.
Te ESI-MS (positive ion mode) was obtained on an LC system coupled to an Agilent single quadrupole mass-selective detector (Terkinelmer Agilent infnity 1,290 LC, 6,420 triple quadrupole MS (Melbourne, Australia)). Te MS was generated at the following conditions: fragmentor voltage = 100 V, capillary voltage = 2500 V, nebulizer pressure = 30 psi, drying gas temperature = 350°C, and mass range = 100-600 Da. Te fragmented ions were separated according to their mass to charge ratios (m/z).
2.5.5. NMR Spectroscopy. Te 1 H and 13 C NMR spectra of the compound were determined at 500 and 125 MHz, respectively, using the Bruker 500 MHz NMR facility. Te 2-D spectra that were also performed included DEPT-135, HSQC, and HMBC.
2.6. Antioxidant Activity. Two main assays, the ABTS and DPPH free radicals scavenging activity, were used for the determination of the antioxidant activity of the plant compound.
2.6.1. ABTS Radical Scavenging Activity. Te ABTS free radical scavenging capacity of the compound was examined with reference to a previous procedure by Re et al. and Sebastian et al. [21,22] with minor optimization. Te activity was performed by mixing the ABTS (10 mL) and 2.4 mM potassium persulphate (10 mL) to produce the ABTS free radical. Te resulting solution was diluted with 50 mL of methanol to generate the working stock solution. A 0.150 mL of the stock solution was mixed with 0.050 mL of the prepared compound with concentrations (C) of 100.00, 50.00, 25.00, 12.50, 6.25, and 3.13 µg/mL. Te resulting mixtures were vortexed and incubated at 30°C for 30 min. Te absorbances of the mixtures were measured with the UV spectrophotometer (Jenway, Bibby Scientifc Ltd, Staf, UK) at 734 nm for control ABTS (Ao) and the test samples (A). Te experimental protocol was repeated for ascorbic acid with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, and 3.13 µg/mL. Te experimental procedure was performed in triplicate. Te percentage (%) scavenging activity was evaluated by employing the relation as follows: A graph of the % scavenging activity against the logarithm of the concentration [Log C] was plotted. Te ffty percent scavenging capacity ((SC 50 ), µg/mL) was deduced from the regression equation [Y � mx + C] from the graphs by evaluating the relation at Y � 50: where m and C are the slope and intercept from the regression equation.
A plot of the % scavenging activity versus the logarithm of [Log C] was performed and the SC 50 (µg/mL) was deduced from the regression equation [Y � mx + C] from the graphs by evaluating the relation at Y � 50: where m and C are the slope and intercept from the regression equation.

Antimicrobial Activity.
Te antimicrobial activity of the isolated compound was accessed by evaluating the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) by employing the micro broth dilution assay. Te synergistic efect was determined by evaluating the fractional inhibitory concentration (FIC) index using the checkerboard assay.
Te interaction was considered synergistic if the FIC index was ≤0.5; partial synergistic if the FIC index was >0.5 and <1; additive if FIC index was � 1; indiference if the FIC index was >1 and ≤4; and antagonistic if the FIC Index was >4.0.

Statistical Analysis of Data.
All the data generated from the experiments were analyzed with the GraphPad Prism software package 5.00 for Windows (San Diego, California, USA). Te data were further subjected to one-way ANOVA analysis (P < 0.01). Te antioxidant data were reported as the mean ± SD.  In view of the analysis of all the spectral data, the isolated compound was identifed as eriodictyol (5,7-dihydroxy-3′,4′-dihydroxyfavanone) and is being reported for the frst time from the bark of A. africana.

Antimicrobial Activity.
Te antimicrobial activity of the isolated compound was assessed against 10 diferent microbes by determining its bacteriostatic and bactericidal efects (MICs, MBCs, and MBC/MIC ratios) over a concentration range of 100.0− 0.1 mg/mL. Te results of the antimicrobial assay are shown in Table 3 as follows.
According to Mogana et al., compounds with MIC ≤ 2 mg/mL and MIC > 2-8 mg/mL have been found to exhibit some degree of good and weaker inhibitory or bacteriostatic efects, respectively [26]. Based on this classifcation, the compound was found to possess various degrees of bacteriostatic efects (MIC ≤ 8 mg/mL) against all the microbial strains under consideration. Te highest level of inhibitory efects was reported against SM, SA, MRSA, BS, PA, ST, and CA1 (MIC ≤ 2 mg/mL) whilst the weakest (2 mg/mL ≥ MIC ≤ 8 mg/mL) activity was reported against EC, CA2, and KP. Furthermore, the compound exhibited a broad spectrum of bactericidal efects against all the microbial strains at concentrations ranging from 1. Te antimicrobial results further confrm similar experiments reported for various extracts of the plant as well as the isolated compound. For instance, the leaves, barks, roots, and seeds of A. africana have registered a broad spectrum of biological activities such as antimicrobial, antiplasmodial, antitrypanocidal, antioxidant, anti-infammatory, and analgesic properties [9][10][11][12]38]. Specifc authentication of the antimicrobial activities of A. africana was confrmed for the methanol extract of the bark with higher susceptibility for Staphylococcus aureus and Bacillus subtilis. Te extract was however reported to be inactive against Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi [10]. Agbelusi et al. investigated the synergistic efects of chewing stick extracts with standard antibiotics which were confrmed against the drug-resistant strain of some bacterial isolates [11]. Moreover, the isolate was also reported to exhibit antimicrobial activities against SA, CA, and EC [39,40]. Te series of reported activities further corroborate those reported for the compound in this study. Te antimicrobial activities of the isolated compound and its favonoid classes have been associated with the presence of α-β unsaturated carbonyl and multiple phenolic groups in their structures. Te structural motifs in these compounds are known to enhance their antimicrobial actions by either interfering or inhibiting bacterial cell wall and protein synthesis at the 30S subunit of ribosomes. In fact, the motifs in these phytochemicals are known to enhance bacterial cell wall permeability for the expression of their antimicrobial activities [41,42]. Furthermore, favonoids exhibit their antibacterial action by causing cell lyses of pathogenic bacteria [40,41]. Te isolated compound (eriodictyol) and its favonoid families such as naringenin have been identifed as inhibitors of β-ketoacyl acyl carrier protein synthase III (KAS III), a key protein involved in the initiation of fatty acid synthesis in bacterial. Te utilization of the compound would therefore serve as a key approach to overcoming antibiotic drug resistance associated with the methicillin-resistant bacteria Staphylococcus aureus and other antibacterial resistance [41,43].

Antibacterial Synergistic Activity.
In view of the series of bactericidal activities reported by the compound, further tests to determine its synergistic activity with standard antibacterial agents such as ciprofoxacin and tetracycline were performed (Table 4).
Te data suggest that the compound reported synergistic activity against EC, KP, ST, and MRSA; partial synergism against SA; antagonism against PA; and no synergistic efect (indiferent) against SM and BS when combined with ciprofoxacin. Te compound further showed synergistic activity with tetracycline against ST, and partial synergism against EC, KP, SA, SM, MRSA, BS, and PA. Te mechanistic account for the reported synergistic efects could be linked to Evidence-Based Complementary and Alternative Medicine its favanone phytochemical class, and its accompanying structural motifs. Te phytochemical class is known to exhibit synergistic efects with several antibiotic agents against several bacterial strains [44,45]. For instance, their combination with ciprofoxacin and tetracyclines is known to exhibit synergistic efects against S. aureus, its methicillinresistant strains, and several other bacterial strains [44,45]. Te results, therefore, corroborate the synergistic efects reported by the compound against S. aureus, methicillinresistant S. aureus, and the other microbial strains in this study. Tey exhibit their synergistic efects by facilitating the interaction of the antibiotic with its target site in the pathogen. Te mechanisms of action have been established to proceed through the major facilitator superfamily (MSF) and the multidrug resistant (MDR) efux pump pathway. Tey are also known to enhance the activities of antibiotic interaction by disintegrating the oily outer membrane of bacteria, leading to membrane permeability for the expression of antibiotic activity [44][45][46].

Antifungal Synergistic Activity.
Te antifungal synergistic activity of the isolated compound when combined with fuconazole, ketoconazole, and nystatin was determined with the checkerboard assay (Table 5). Te interpretation of the synergistic activity was based on the criteria adopted by Yang    Evidence-Based Complementary and Alternative Medicine (eriodictyol) reporting synergistic and partial activity against CA2 and CA1, respectively, when combined with fuconazole. Moreover, when the compound was combined with ketoconazole, antagonistic, and additive were reported against CA1 and CA2, respectively. Finally, synergistic and indiferent activity were reported against CA2 and CA1, respectively, when combined with nystatin. Te myriad of synergistic activities reported for the compound further re-enforces the vast antimicrobial activities reported for A. africana against C. albicans strains. Te identifed compound and its favonoid derivatives had been reported against C. albicans. Tey exhibited their synergistic efects by decreasing the ability of cells to efux out the antibiotics [47]. Te efects lead to the permeability, bioavailability, and efectiveness of antibiotics at their target sites [47]. Te favonoid compounds were also known to perform their synergistic efects by inhibiting cell-cell adhesion communication in the fungal strain, leading to the disruption of gene expression responsible for bioflm formation in the fungal strain [48].

Antioxidant Activity.
Te antioxidant activity of the compound was determined against the ABTS-and DPPHfree radicals scavenging with a concentration range of (100.00− 3.13 µg/mL). Te study shows signifcant free radical scavenging activity for the isolated compound against the ABTS and DPPH radicals with SC 50 (µg/mL) values and ascorbic acid indicated in Table 6 and Figure, SI 10.
Te study further reveals the isolated compound indicating superior activity against standard ascorbic acid. Te compound belonging to the favonoid and phenolic phytochemical class further confrms the vast antioxidant activities reported for such a class of compounds [49]. Te antioxidant activities of A. africana and its fractions had been reported for various parts of the plant but no compound was assigned to their reported activities [38]. Te isolated compound, therefore, gives further credence to the utilization of A. africana in managing medical conditions related to oxidative stress. Te antioxidant activities of the identifed compound and its favonoid derivatives have been linked to the presence of multiple phenolic groups in their structures. Te phenolic groups in their structures perform their antioxidant activities by acting as pro-oxidants by chelating metals and other reactive radicals [49]. Such phytochemicals are good hydrogen donors and normally react with reactive oxygen and nitrogen radicals. Te resultant efect could terminate the generation of free radicals and consequently break the cycle of the generation of new ones [26,50]. Several mechanistic pathways have been reported for the numerous medicinal potentials of the compound (eriodictyol). In fact, the compound had been reported to protect the LPS-triggered oxidative stress, neuroinfammation, and synaptic dysfunction through the MAPK NF-κB mediated by ROS, Sirt1, and Nrf2/Keap1 signal pathways. Te reported property confrms the antioxidant activity reported in this study and could be applied as a nutraceutical for managing oxidative-related neurodegenerative ailments and diseases [18]. Furthermore, the Fc″RI-mediated human basophilic KU812F cells antiallergic activity of the 7-O-β-d-glucuronide derivative of the compound (eriodictyol) and its chromene moiety, 5,7-dihydroxy-4-chromene have been investigated. Te study concluded that the compounds can downregulate protein and messenger RNA (mRNA) expression of Fc″RI on the cell surface implicating its potential for the treatment of allergic disorders [51]. In other studies, eriodictyol was identifed as a novel insulin secretagogue through the stimulation of the cAMP/PKA signaling pathway at higher glucose concentration and hence could be applied as prophylaxis for treating drug-induced hypoglycemia [52]. Moreover, the anti-infammatory, antioxidant, antimicrobial, immunomodulatory, cytoprotective efects on the kidney, hepatoprotective, neuroprotective, analgesic, antipyretic, anticancer, and antiallergenic were well documented [41,[51][52][53][54][55][56][57]. Tese numerous pharmacological activities reported for the plant could contribute to the vast medicinal potentials reported for A. africana.

Conclusion
A favanone derivative, eriodictyol ((S)-2-(3ʹ,4ʹ-dihydroxyphenyl)-5,7-dihydroxychroman-4-one), was isolated for the frst time from the bark of A. africana. Te compound exhibited signifcant antioxidant activity against ABTS and DPPH radicals and a broad spectrum of antimicrobial and synergistic efects against several bacterial and fungal strains. Te study gives scientifc credence to the numerous ethnobotanical and medicinal properties of A. africana.

Data Availability
All the data used to support the fndings of the study are included within the article.