Antioxidant evaluation and bio-guided isolation from methanol leaf extract of Acalypha godse ffi ana

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Introduction
Medicinal plants are crucial in the fight against drug resistance, as they serve as foundations for chemical agents, lead drugs, and new drugs [1,2].In 2001, 25% of the world's bestselling medications were natural products or bioinspired medi-1 cations [3].This report was evident in that the bark of Cinchona is the source of malaria-attacking quinine; opium is a source of codeine, morphine, and paregoric (an anti-diarrhea agent), while morphine is still an acceptable pain-relieving medication to date [4].Aspirin is made from salicin, a glycoside that was separated from Salix alba Linn.Secondary metabolites from plants, such as flavonoids, alkaloids, terpenoids, and tannins, have antibacterial properties, making them a cost-effective alternative to synthetic antibiotics [5].Plant-derived metabolites, including flavonoids, alkaloids, terpenoids, and tannins, have antibacterial properties, suggesting they could be a costeffective and secure alternative to antibiotics in treating microbial diseases [6].Plant metabolites possess antibacterial properties, acting either bacteriostatic or bactericidal on bacterial strains [7].Novel antibiotic prototypes have been reported to be promising among higher plant sources [8].Plant phytochemicals, including phenolic compounds, nitrogen-containing compounds, and terpenes, are known to have antifungal properties.
The Euphorbiaceae plant Acalypha godesffiana MUELL ARG, also known as fire dragon, match-me-if-you-can, and Joseph's coat, is popular in the Euphorbiaceae family [9].It originated from the Pacific Islands and spread to the world, mostly to the tropics of Africa, Asia, and America.In Nigeria, the Yorubas' call it 'Jinwinini'; the Igbos call it "Ogbunizu".Traditional therapies make use of this plant due to its therapeutic properties, which are used to treat illnesses like fungal infections.The antimicrobial potentials of the leaf samples of the plants concerning solvents for extraction have been documented [10].The phytochemical composition of the plant's parts has been previously documented to include phenols, saponins, alkaloids, cardiac glycosides, terpenoids, and flavonoids [10][11][12].Acalypha species contain compounds like gallic acid, ethyl gallate, methyl gallate, ellagic acid, brevifolin carboxylate, 1, 2, 3-benzenetriol and kaempferol-3-O-α-L-rhamnoside [13,14].In this study, we investigated bioactive constituents, antioxidants, and antibacterial properties of partitioned extracts.This work produced compounds (1-3) from A. godesffiana for the first time.

Preparation of plant extract
The 1.5 kg ground sample of Acalypha godseffiana was macerated for 72 hours at room temperature in a shaker with 20 L of methanol.The solution was filtered, and 245.25 g of crude methanol extract was obtained by a rotary evaporator at 37 • C.

Solvent-solvent fractionation
After being suspended in 100 milliliters of water, the 245.25 grams of crude methanol extract were divided into four parts using n-hexane, dichloromethane, ethyl acetate, and finally nbutanol within that line, except for the extract of n-butanol, which was concentrated using a water bath at 90 • C. Each resultant fraction was independently concentrated by evaporation under a fume hood to give their respective fractions.

Diphenylpicrylhydrazyl (DPPH) radical scavenging
properties Previously established approach [15] was followed with minor adjustments to quantify the impact of each extract or fraction on DPPH radicals: Each extract or fraction was prepared in five different strengths (0.125-0.1 mg mL −1 ) and combined with a 1.0 mL methanol-based solution containing 0.135 mM DPPH radical.After shaking the reaction solution and letting it remain at room temperature under dark conditions for thirty minutes, the amount of light it absorbed was measured.with a spectrophotometer set at 517 nm.

Phosphomolybdenum-based total antioxidant capacity (TAC) method
To evaluate the TAC of extracts and fractions based on their capacity to convert Mo (VI) in an acidic medium to Mo (V), previously modified methodology [16] with the ability to transform Mo (VI) into Mo (V) was adopted.In 96-well plates, different quantities of a reagent solution, including ammonium molybdate, sodium phosphate, and sulfuric acid, were mixed with the extracts or reagent mixture, including ammonium molybdate, sulfuric acid, and sodium phosphate fractions.The blank was made by combining 300 µL of test reagent solution with 30 µL of methanol, with silymarin and gallic acid serving as standard controls.Using a microplate reader, measurements were made for the amount of Mo (VI) reduced to Mo (V) after being kept at a temperature (95 • C) for ninety minutes.+ )) The ABTS+ scavenging activities of extracts and fractions were assessed using a previously documented technique [17], with slight modifications as follows: The stock solution of ABTS radical was obtained by dissolving 7 mM ABTS and 38.4 mg of 2,2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) in 10 cm 3 of distilled water.In contrast, the potassium per sulfate solution was created by dissolving 6.62 mg K 2 S2O 8 .ABTS were combined in a one-to-one proportion with K 2 S2O 8 and left in the dark for a day.
The study used an ABTS reagent that was made by diluting the stock solution of ABTS radical cation using methanol to get an absorbance at 734 nm of 0.7 ± 0.005.The extract or fraction was added at different concentrations (0.125 to 0.1 mg mL −1 ) to an ABTS+ solution that had been diluted.After being vortexed, the mixture was left to react for seven minutes before measuring its absorbance with a Japanese-made BIORAD model 680 96well microplate reader at 734 nm.A control was created using a methanol solvent and ABTS solution alone, with silymarin and gallic acid as common antioxidants.

Ferric reducing antioxidant power assay (FRAP)
Acalypha godseffiana leaf extracts' and fractions' power to decrease ferric ions was ascertained by applying an established Müller et al. [18] method.After mixing 2.5 mL of phosphate buffer (20 Mm), 1% KFe(CN)6, and extract/fraction concentrations that range between 0.125 mg and 0.1 mg mL −1 , allow incubation at 50 • C for 30 minutes for the whole mixture.The mixture was added to 96-well plates along with 2.5 mL of a 10% weight/volume trichloroacetic acid solution and 0.1% (0.5 mL) weight/volume ferric chloride solution.The mixture is then left to stand for 10 minutes.Next, utilizing a microplate reader with 96 wells (BIO-RAD, model 680, Japan), the measurement of absorbance was at 700 nm.Common antioxidants, like silymarin and gallic acid, were used.

Scavenging activity of Hydroxyl radical (OH) by deoxyribose Method
The deoxyribose technique was utilized to evaluate the extracts and fractions' ability to scavenge hydroxyl radicals [19].The reaction mixture was combined with different extracts or fractions at varying concentrations (125 µL-1000 µL), containing FeCl 3 , EDTA, H 2 O 2 , and 2-deoxy-D-ribose.A 20-mM potassium phosphate buffer with a pH of 7.4 was utilized to prepare the reaction volume.Upon culture for thirty minutes at 37 • C, TBA (0.2 mL, 1% w/v) and TCA (0.2 mL, 15% w/v) had been incorporated into the solutions in 0.25 N HCl.After 30 minutes of heating in a bath of boiling water, the resulting mixture of the reaction was allowed to cool.A 96-well microplate scanner was used to quantify the absorbance of deoxyribose degradation at 532 nm, yielding a percentage inhibition percentage.The reaction volume was made using a buffer solution of potassium phosphate (20 mM, pH 7.4).After 30 minutes of incubation, 15% w/v and 0.2 mL of TCA at 37 • C, as well as 0.2 mL and 1% w/v of TBA, were introduced into the solutions in 0.25 N HCl.After 30 minutes of heating in a bath of boiling water, the blend used for the reaction was allowed to cool.A 96-well microplate scanner was used to quantify the absorbance of deoxyribose degradation at 532 nm, yielding a percentage inhibition percentage.

Metal chelating activity assay
With slight modifications, previously published methods [20] were used to assess the extracts' and fractions' ability to chelate ferrous ions or Fe 2+ .Each extract or fraction (40 µL) with variable concentrations that span between 0.125-0.1 mL was applied to a 0.2 mM iron (III) chloride (FeCl 3 ) solution (200 µL).The addition of 5 mM ferrozine (80 µL) after 30 seconds initiated the process.Divalent iron and ferrozine are combined to generate stable, highly soluble in-water magenta complex species.After 10 minutes at 562 nm and of room temperature incubation, the absorbance was calculated while utilizing a 96-well microplate scanner.

Minimum inhibitory concentration (MIC) of extracts/fractions
The antibacterial potential of MeOH extract, HEX, DCM, EtOAc, BUOH, and ACF fractions was assessed using the minimum inhibitory concentration (MIC) in 96-well microtitre plates in cooperating two-fold serial dilutions of the fractions.The bacteria strains tested were Staphylococcus aureus and Pseudomonas aeurginosa, while the fungi strains were Candida.albicans, Trichophyton mucoides, and Cryptococcus.neoformans.The strains grew in a nutritional broth that had been autoclaved for 24 hours for fungus and 18-24 hours for bacteria, then corrected to the last level of density of 106 cfu/mL.The strains were cultivated to an ultimate density of 106 cfu/mL in autoclaved nutrient broth.After 24 hours of aerobic incubation (25 • C for fungus and 37 • C for bacteria), the plates were examined.
To quantify the amount of microbial growth, 40 microliters of 0.2 milligrams per milliliter of p-iodonitrotetrazolium solution (purity 97%, Sigma, South Africa) were introduced into every well.The wells were subsequently incubated for thirty minutes at 25 • C for fungi and 37 • C for bacteria.The organisms' activity converted the colorless tetrazolium salt to a reddish-brown result.Every treatment was performed in triplicate, and for a therapy to be deemed active, growth had to be completely suppressed at a particular fraction concentration [21].

Bioassay guided isolation of bioactive compounds
Further purification of the EtOAc fraction of MeOH extract-the most antifungal fraction-was achieved employing preparative thin-layered chromatography (PTLC) and frequent column chromatography.Fraction C from the first column was re-crystallized using hexane, chloroform, and ethyl acetate to afford compound 1.The third column's fraction, DB1L, was purified using preparative thin layer chromatography (PTLC) using a 5:4:1 ratio of chloroform, ethyl acetate, and formic acid to produce compounds 2 and 3.

Structural elucidation of the isolated compounds
Spectroscopic methods (NMR, ESI-MS, and FT-IR) were employed to elucidate the structure of the compounds.Utilizing as an internal standard tetramethylsilane (TMS) with CD 3 OD and CDCl 3 , at standard room temperature, a Bruker Avance II 600 MHz Fourier transform NMR spectrometer was used to obtain the nuclear magnetic resonance spectroscopic data.Water Synapt G2 mass spectrometry was used to record highresolution mass spectra.A Bruker FT-IR spectrometer was employed to obtain the infrared spectra of neatly isolated compounds.The melting point was determined using mercury in a glass thermometer and an electrothermal IA 9300 capillary melting point device.

Statistical analysis
The study used GraphPad Prism 5 for data analysis, including one-way ANOVA, Bonferroni tests, and student t-tests carried out at a p-value of less than 0.05.
Antioxidants are necessary to control oxidative reactions in human cells and, thus, prevent harm or even cell death.This investigation demonstrated that ABTS.+ had better scavenging properties by ACF (IC50, ACF = 0.46 mg mL −1 ) than did DPPH (IC50, ACF = 1.01 mg mL −1 ), corroborating previous research [4] that suggested certain compounds with ABTS.+ scavenging activity were unable to scavenge DPPH.Similar to what was discovered and described, certain compounds are moderate DPPH as well as ABTS.+scavengers.This supports the notion that the extracts and fractions have varying capacities for scavenging free radicals, a helpful strategy for addressing pathologic damage related to free radicals [22].A FRAP assay was also employed to assess the extracts' or fractions' antioxidant capacities.It has been previously established that the FRAP assay will detect most secondary metabolites that are redox-active substances [23].The analysis's findings proved compliant with the literature report of Sowndhararajan & Kang [24], showing that the extracts had moderate reducing capacity (FRAP), except the EtOAc fraction and ACF, which showed the best activity at the respective IC 50s of 1.26 mg mL −1 as well as 1.27 mg mL −1 .The phosphomolybdenum approach, which relies on a transformation of Mo (VI) to Mo (V) through antioxidant compounds as well as the generation of phosphate Mo (V) complexes with a green colour, was used to assess the TAC of the extracts or fractions.
According to this methodology, greater antioxidant activity is correlated with higher TAC values [25,26].This study's findings demonstrated that DCM fractions exhibited better activity while other fractions had comparable phosphomolybdenum reduction capacity to the standards.This study's TAC outcome is consistent with earlier research [27].The extracts' discovered antioxidant properties were linked to their redox features in controlling harmful reactive oxygen species (ROS) chain reactions and absorbing and eliminating free radicals that possess a preventative effect on the formation of malignancies.The high concentration of flavonoids, proanthocyanidins, and polyphenols found in A. godseffiana leaves may be the cause of the extracts' and fractions' potent antioxidant activity.

Antimicrobial activities
The outcomes of the antibacterial examination showed that the fraction of ethyl acetate demonstrated the greatest inhibition against both harmful bacterial strains, S. aureus and P. aeruginosa, with MIC values of 0.469 mg/mL.Furthermore, with MICs (0.156 mg mL −1 and 0.313 mg mL −1 ) for S. aureus as well as P. aeruginosa, respectively, ACF from the ethyl acetate fraction was found to have greater potential against the bacteria tested than both the fractions from methanol and ethyl acetate extracts.
Table 2 presents the specific results in detail.The EtOAc fraction demonstrated robust activity against the three pathogenic fungi tested, namely Candida albicans, Candida mucoides, and Candida neoformans, possessing a uniform MIC of 0.059 mg mL −1 superior to the standard antifungal drug, nystatin, which had MICs (0.500 mg mL −1 , 0.250 mg mL −1 0.250 mg mL −1 ) for Candida mucoides, Candida albicans, and Candida neoformans, respectively.Conversely, ketoconazole demonstrated MICs of 0.250 mg mL −1 for each of the three (3) tested fungal strains.Compared to the conventional medications, MeOH extract and ACF displayed much superior activity, with MIC values of 0.020 mg mL −1 for Candida albicans, Candida neoformans, and T. mucoides, and as indicated in Table 2, this activity may be related to synergistic effects.
Saponins exhibit properties that include antiinflammatory, antimelanogenic, and antispasmodic activities, along with cancer-inhibiting potentials [28].Saponins, alkaloids, and polyphenolic metabolites are perhaps accountable for the antibacterial characteristics of the A. godseffiana plant.Flavonoids are reported to be produced by plants to defend themselves against microbial infections [29].According to in vitro investigations, flavonoids have been shown to exhibit broad-spectrum antibacterial capabilities [30].This activity may be related to flavonoids' ability to bind with soluble and extracellular both bacteria's cell walls and proteins.The lipophilicity of flavonoids correlates positively to their ability to disrupt the microbial membrane, which enhances their microbial potential [31].
This study also revealed that, possessing a MIC (0.059 mg mL −1 ), the EtOAc fraction outperformed the MeOH extract, which had a MIC (0.098 mg mL −1 ) against the fungal strains Trichophyton mucoides, Candida albicans, and Cryptococcus neoformans.This may be due to the nature of the phytochemicals that are more expressive in EtOAc solvent fractions as compared to MeOH solvent fractions.The antifungal result further revealed that ACF showed better antifungal potency against C. neoformans and C. albicans (MIC: 0.02 mg mL −1 ; Table 2), indicating that the extracts express their antifungal potentials less when together; this may arise from antagonistic effects of various components of the extract or fractions.

Spectroscopic and spectrometric analysis of the isolated compounds
From the ACF fraction, three chemicals were later separated, and their structures were clarified.Compound 1 was obtained from the recrystallization of fraction C in the first column.This was isolated as an off-white crystal (3.0 mg), with a melting point of -238 o C and a R f value of 0.37 (Hexane: EtOAc 4:1).The HR-ESI-MS [M-H] = 153.0188(Calc.153.0266) corresponds to a molecular formula of C 7 H 6 O 4 (Figure 1) .The IR absorption bands show the presence of OH (3188 cm −1 ) confirming the hydroxyl group, C=C (1598 cm −1 ) of the aromatic ring, and C=O (1672 cm −1 ) for carboxylic acid (Figure 1).A tri-substituted benzene ring with two proton signals at δH 6.80 (1H, d, J = 12 Hz) and δH 7.42 (1H, d, J = 18 Hz) was identified in the 1H NMR spectra (CD 3 OD, 600 MHz) (Figure 1).
3,5-dihydroxybenzoic acid, also known as α-resorcylic acid, is being reported here for the very first time, as far as we know, from Acalypha godseffiana.It is a polyphenolic acid that may present some natural products that may have healing benefits [32].It demonstrates the moderate antioxidant and scavenging potential of free radicals and additionally inhibits lipolysis in adipocytes [33].
The proton signal at δ H 3.68 (2H, m) germinal to the ester carbonyl group was allocated to a methylene group.The methine proton signal was seen at δ H 1.47 (1H, m), and there were four methylene groups with signals at δ H 1.31, 1.40, and 1.71 (2H, m).Compound 2's 13C-NMR spectrum showed the anticipated 12-carbon resonance of the molecule (Figure 1).DEPT-135 and HSQC spectra confirmed two quaternary carbons, including the ester carbonyl group at δ 170.4,three methine signals ascribed to aromatic rings at δ 132.9, 132.0, and 128.0, five methylene carbons, including an oxygen-bearing group at δ 170.4 (C4), and two terminal methyl groups (Figures 1, 2 and supplementary data).Compound 2's identity as di-(2ethylhexyl) phthalate (Figure 1) was verified by comparing the 13 C-NMR and 1H values matching those documented in Nair et al. [33] and utilizing the COSY and HMBC signals, which correlate to the report of Nair et al. [33].The well-known synthetic plasticizer di-(2-ethylhexyl) phthalate (DEPH) is obtained from a variety of plant species, particularly Alchornea cordifolia and Aloe vera [34].Hence, it may have taxonomic significance.The antiviral, anti-tumor, and antioxidant activities of DEHP have been documented [34].The antimicrobial properties of DEHP were also reported in Abri & Maleki [35].
Further purification of DB 1 L using PTLC offered compound 3 as a crystalline colorless powder with a melting 6 point of -237 o C and a molecular formula deduced as C 7 H 6 O 5 through HR-ESI-MS [M-H] =169.0167(Calc.169.0215).The IR absorption bands show a very broad signal typical of OH at 3199 cm −1 , a C=O sharp stretching band at 1672 cm −1 , and a C=C aromatic vibration (1598-1437 cm −1 ) typical of the benzene ring. 13 1, 2 and supplementary data) to be gallic acid [36,37] previously reported in the literature.
3, 4, 5-trihydroxy benzoic acid, occasionally referred to as gallic acid, is a phenolic acid and has been isolated from species of Acalypha [13].Gallic acid possesses a broad range of actions, according to descriptions, like strong antioxidants, scavenging, and antifungal activities of free radicals.The activity of the polyphenolic compounds is influenced by the number and configuration of hydroxyl groups, as evidenced by the fact that, due to the presence of a pair of hydroxyl groups bonded in a meta position to one another in 3,5-dihydroxybenzoic acid (αresorcylic acid), gallic acid has higher biological activities [32].One of Toona sinensis' main ingredients, gallic acid, has been suggested as a possible medication agent to treat prostate cancer [38].

Conclusion
This study investigates the antioxidant and antimicrobial properties of Acalypha godesffiana, a plant used in conventional medicine for fungal-related illnesses.The extracts were purified and characterized using chromatographic and spectroscopic techniques.Three biologically active compounds were identified, including 3, 5-dihdroxylbenzoic acid (1), 3, 4, 5trihydroxybenzoic acid (2), and Di-(2-ethylhexyl) phthalate (3).The extract and fractions showed varying scavenging capacities on different anti-oxidative models.The study validates the antifungal potentials of A. godseffiana leaves.The phytochemicals and antioxidant activities revealed in this study can be employed for human health benefits.This study's biological activities support the plant's use in folk medicine as a potent antifungal therapy for fungal skin infections.Many of the biological activities reported in the literature for the compounds correlate well with the activities revealed in this study, suggesting that the three isolated compounds may be responsible for the potent antifungal capacity exhibited by the EtOAc fraction and column fraction (ACF) of Acalypha godseffiana methanol leaf extract from this study.Further investigations are needed to fully explore the biological potentials of Acalypha godseffiana.

Figure 2 .
Figure 2. (a) Proton NMR spectrum of compound 1 (b) Proton NMR spectrum of compound 2 (C) HR-ESI-MS spectrum of compound 3; detailed spectroscopic data of compounds 1-3 have been presented in the supplementary document.

Table 1 .
IC 50 of leaf extract and fractions of A. godseffiana antioxidant potentials.