# Synthesis and Characterization ofChrysophyllum albidum (African Star Apple) Plant-Mediated Zinc Oxide Nanostructures and Evaluation of Their Antioxidant, Anti-Inflammatory, and Antimicrobial Activity

Authors: Eric Kwabena Droepenu; Nancy Ama Na Jackson; Michael Odoi Kyene; Frederick Ayertey; Henry Brew-Daniels; Theophilus Mensah; Clement Adomako; Adwoa Padiki Nartey; Alexander Obiri Gyampoh; Linda Adutwumwaa Boamah
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

This study sought to evaluate the added advantage of mediating ZnO nanostructures with a medicinal plant. The synthesized ZnO nanocrystalline structures were confirmed by Fourier transform infrared spectrometer and characterized through scanning electron microscope, transmission electron microscope, ultraviolet–visible spectroscopy, and energy-dispersive X-ray spectrometer. The antioxidant, anti-inflammatory, and antimicrobial activities of the ZnO nanostructure mediated with methanol extracts of the leaf, fruit, and seed ofChrysophyllum albidum were then evaluated using DPPH assay, egg albumin denaturation assay, and agar well diffusion methods, respectively. All the characterization analyses revealed high-purity hexagonal-shaped ZnO nanoparticles which were agglomerated. The mean diameter of the particles determined were 11.3±1.7nm, 20.4±3.2nm, and 27.3±6.6nm for C. albidum seed, fruit, and leaf extract-mediated ZnO NPs, respectively. The EC50 values recorded for the antioxidant activity of the extract-mediated ZnO NPs were 0.507±0.015, 0.255±0.006, 0.193±0.003, and 0.004±0.000 mg/mL for leaf, fruit, seed, and ascorbic acid, respectively. From the antimicrobial analysis, C. albidum seed extract-mediated ZnO NPs recorded the highest zone of inhibition (24.33±0.47) against S. aureus whereas C. albidum leaf extract-mediated ZnO NPs gave the lowest zone of inhibition (13.00±1.41) against E. coli at a concentration of 50 mg/mL. Moreover, C. albidum fruit extract-mediated ZnO NPs presented the highest zone of inhibition (18.00±0.82) against the fungus (C. albicans) also at a concentration of 50 mg/mL. The IC50 values recorded for the anti-inflammatory activity of the extract-mediated ZnO NPs showed inhibition in the order fruit>seed>leaf. Meanwhile, extracts of the samples showed the presence of flavonoids, alkaloids, saponins, and glycosides as phytochemical constituents in the leaf, fruit, and seed samples. In conclusion, the synthesized ZnO NPs from the extracts of C. albidum displayed significant antioxidant, anti-inflammatory, and antimicrobial activities against some selected microbes and fungi.


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## Body

## 1. Introduction

Nanomedicine research is a rapidly growing area of nanotechnology where the focus is mainly on research and development of nanoparticles (NPs) for therapeutic applications [1]. Nanomedicines offer potential solutions for many of the current challenges in treating various kinds of diseases and other illnesses [2]. Although their functions are similar to many biological processes and cellular mechanisms, they are considered to have an extra promising approach due to the unique characteristics of the NPs involved, as well as molecular targeting as reported by Morigi et al. [3].NPs have emerged as novel materials attracting attention due to their unique properties which are strongly influenced by their morphological structure as reported by Gopalakrishnan et al. [4]. According to Sahayaraj and Rajesh [5], NPs can be used as antimicrobicides owing to their nanoscale size, surface-to-volume ratio, and strong affinity to target because they are attractive probes of biological markers. Zinc oxide is one of such NPs characterized with a wide band gap, high electrostatic charge, and high surface area-to-volume ratio leading to high surface reactivity, suitable for biomedical purposes [6, 7]. Das et al. [8] reported that the antioxidant properties in ZnO NPs enable them to scavenge free radicals generated in biosystems during their processes. Due to the low toxicity, heat resistance, and the essential mineral (Zn) that ZnO NPs offer to human cells, they are considered to be suitable antimicrobial agents as reported by Kasahun et al. [9].Basically, nanomaterials (NMs) can be classified into natural, anthropogenic, and engineered as documented in a review paper by Goswami et al. [10]. Natural NMs existed abundantly in the earth system through process (mechanical and biogeochemical) which are devoid of human activities and coexisted with living things as reported by Hochella et al. [11]. On the other hand, anthropogenic NMs are created by human action. However, engineered nanomaterials (ENMs) are thought to be synthesized by humans whose behaviours are different from their bulk counterparts as reported by Quigg et al. [12]. According to Verma et al. [13], NMs can be classified into organic (micelles, liposomes, chitosan, ferritin, and dendrimers) and inorganic NPs (metal NPs, semiconductor NPs, and magnetic NPs).Several approaches (physical, chemical, and biogenic or green) could be used in synthesizing ZnO NPs as reported by Jiang et al. [14]. As a new type of low-cost and low-toxicity NMs, ZnO NPs have piqued the interest of researchers in a variety of biomedical domains, including anticancer, antibacterial, antioxidant, antidiabetic, and anti-inflammatory activities, as well as drug delivery applications as investigated by Jiang et al. [14]. According to literature, the use of green synthesized NPs has been used as substitutes for chemically synthesized ones to help control chemical toxicity in the environment [15, 16]. Plant-mediated NPs result in better defined sizes and morphology as compared to other physicochemical methods as reported by Raveendran et al. [17].The last decades saw the exploit of plants, algae, bacteria, and other eukaryotic components as suitable bioreductants according to Ameta et al. [18]. It is reported that biochemical components of these materials serve as reducing and capping agents in biogenic processes as investigated by Makaro et al. [19]. According to literature, some bioactive components such as alkaloids, terpenoids, flavonoids, tannins, phenols, saponins, anthraquinone, steroids, amino acids, and glycosides account for the inhibition effects of pathogens in Refs. [20–22]. Moreover, Philippe et al. [23] reported that catechin and two dimeric procyanidins in C. perpulchrum were responsible for scavenging the free radicals produced by lipid peroxidation.In pharmaceutical research, nanotechnology of herbal medicine formulation not only improves drug absorption of poorly water-soluble phytochemicals but also improves drug therapeutic effectiveness. When compared to crude drugs, nanoparticle formulation is one of the innovative drug delivery technologies that has several advantages, including increased drug solubility, increased dissolving rate, improved bioavailability, and decreased dosage required for the same effects as reported by Yen et al. [24].Chrysophyllum albidum, a plant species of the Sapotaceae family with many other species, possesses ethnomedicinal values in Refs. [20, 25–29]. The ovoid to subglobose-shaped fruit is usually golden yellow in colour when ripe. Within the pulp (which maybe orange or pale yellow) are 3-5 hard dark-brown seeds covering inner, white-coloured cotyledons. The pulp may be used for jams or taken raw whereas the seeds are also used for traditional games or discarded as waste as reported by Amusa et al. [30].Although literature has reported on the strong medicinal potential of the various parts ofC. albidum extract, there is no available literature on green synthesis of C. albidum plant extract-mediated ZnO NPs. The authors report on the synthesis and characterization of nanocrystalline ZnO using methanolic leaf, fruit, and seed extracts of C. albidum. The study further evaluated their antioxidant, anti-inflammatory, and antimicrobial potential.


## 2. Materials and Methods

### 2.1. Collection and Identification of Plant

Healthy plant parts (fruits and leaves) ofC. albidum were collected from an uncultivated farmland located at Awukugua in the Akuapem North Municipality in the eastern region of Ghana on 22nd April 2021. The plant part samples were identified and authenticated by comparing with corresponding herbarium specimen. The samples were thoroughly washed with distilled water (DW) and dried (leaf and white cotyledon) at room temperature for two weeks to minimize the deterioration of biomolecules in the plant material as investigated by Chen et al. [31]. The dried leaf and white cotyledon samples were milled into fine and coarse powder, respectively, with a blender (FGR-350), stored in zip-lock plastic bags, and placed in a desiccator at room temperature. The ripe fruit was split open, the seeds taken out, and the pulp was shredded into pieces. The shredded pulp was then freeze-dried for further experiment.


### 2.2. Plant Extract Preparation

The solvent extraction method reported by Umaru et al. [32] was modified for this study. A mass of 10.0±0.1g of each sample (leaves, fruit, and seed) was soaked in 30 mL of methanol (1 : 3) at 28°C for 48 hours. The plant material was reextracted twice and filtered to obtain the crude. The methanol was evaporated by concentrating the crude extract under reduced pressure (200 mbar) and temperature (38°C) using a rotary evaporator (Heidolph Laborota 4000) at 40 rpm.


### 2.3. Phytochemical Screening

Preliminary phytochemical analysis of the three samples ofC. albidum was carried out using standard procedures as reported in Refs. [33–35].


### 2.4. Qualitative Test

#### 2.4.1. Test for Alkaloids

The appearance of a creamy white precipitate indicated the presence of alkaloids after 1 mL of 1% HCl was added to 3 mL of each extract and treated with drops of potassium mercuric iodide solution (Meyer’s reagent) in a test tube.


#### 2.4.2. Test for Tannins

Brownish green or blue-green colouration indicated the presence of tannins after a few drops of 0.1% ferric chloride was added to a filtrate of each sample obtained by boiling about 0.5 g of each powdered sample in 20 mL of deionised water in a test tube.


#### 2.4.3. Test for Saponins

The formation of an emulsion from the vigorous shaking of a froth mixture of each sample was obtained by boiling about 2 g of the powdered sample and 3 drops of olive oil.


#### 2.4.4. Test for Flavonoids

Red colouration of NH3 formed as layers after 2 mL of each extract was heated with 10 mL of ethyl acetate and cooled gives positivity of flavonoids.


#### 2.4.5. Test for Terpenoids

The appearance of a reddish-brown colour after 5 mL of each extract was mixed with 2 mL of chloroform and 3 mL of concentrated H2SO4 shows the presence of terpenoids.


#### 2.4.6. Test for Cardiac Glycosides

Brick-red precipitate formation after 10 mL of 50% H2SO4 was heated in boiling water for 5 min with 10 mL of Fehling’s solution indicates the presence of glycoside.


### 2.5. ZnO NP Synthesis

Nanocrystalline ZnO was synthesized by dissolving6.81±0.1 g of zinc acetate dihydrate Zn(CH3COO)2·2H2O with 50 mL ethanol (95%) in a 250 mL Schott bottle on an electrical stirring hotplate (Favorit) at 60°C. After dissolving the precursor, 15 mL of the methanolic plant extract was added dropwise and the mixture was maintained at the same temperature (60°C) with constant stirring for 1 h. The pH of the mixture was adjusted between 5 and 6 using 0.1 M each of KOH and NaOH solutions. Stabilization of the reaction mixture occurred at room temperature under 3 h. The cleaning and drying of the synthesized samples followed the method reported by Droepenu et al. [36]. The process was repeated for each plant extract to obtain individual nanocrystalline ZnO samples.


### 2.6. Characterization

The synthesized nanocrystalline ZnO samples were characterized using various spectroscopic and microscopic techniques.Spectral analysis of the samples was carried out with Fourier transform infrared (FTIR) spectrometer (Thermo Scientific Nicolet iS10, USA) at a range of 4000–400 cm-1 and resolution of 4 cm-1. The sample preparation used the method reported by Yang et al. [37] where potassium bromide (KBr) in a ratio of 1 : 19 was used to solidify the sample before analysis.Morphological analysis of the samples was determined using scanning electron microscopy (SU3500, Hitachi) with the spectral imaging system (Thermo Scientific NSS (EDS)) and detector tape (BSE-3D) attached to determine the elemental composition and purity of the samples. This was operated with acceleration voltage of 10.0 kV, working distance of 11.6 mm, and a pressure of 40 Pa. However, a transmission electron microscope (TEM) (JOEL 1230, Japan) was used in determining the particle shape of the as-prepared ZnO NP samples. Prior to analysis (SEM), the dry samples were mounted on an aluminium plate with the help of an adhesive membrane. In the case of TEM analysis, samples were diluted with ethanol and sonicated with ultrasonic cleaner (Elma, Germany) for 30 min. About 4μL of the sample was loaded onto a coated copper grid and observed under the microscope. Software (ImageJ) was used to determine the diameter of the particles.The optical property of the synthesized samples was evaluated using UV-visible spectrophotometer (UV-1800, SHIMADZU) with a spectrum range between 300 and 400 nm. Also, the energy band gap (E) was determined using the expression reported by Dharma and Pisal [38] in
(1)E=h×cλ,where λ is the wavelength in m, h is the Planck constant (6.626×10−34Js−1), and C is the speed of light (3.0×108ms−1; 1eV=1.6×10−19J).Sample preparation follows the method reported by Droepenu and Asare [39].


### 2.7. Antioxidant Activity Assay

This assessment was carried out using a DPPH assay to determine the radical scavenging activity of the plant-mediated ZnO NPs.The method was described previously by Ayertey et al. [40] with slight modifications. Different concentrations of the ZnO NPs ranging from 0.104 to 1.67 mg/mL were prepared in different test tubes. 3 mL of DPPH (2,2-diphenyl-1-picryl hydrazyl) with concentration 20 μg/mL was added to 1 mL of the extracts of the various concentrations and incubated at room temperature for 30 min. The absorbance was read at 517 nm against a methanol blank in triplicate using a UV-visible spectrophotometer (Biobase). The negative control was prepared by adding 3 mL of DPPH solution to 1 mL of methanol and treated under the same conditions as the samples. Ascorbic acid was used as a positive control. Percentage inhibition was calculated using
(2)%Inhibition=Ac−AsAc×100%,where Ac is the absorbance of control and As is the absorbance of the test sample.The effective concentration (EC) at 10, 40, and 90 was determined using GraphPad software, version 9.


### 2.8. Anti-Inflammatory Activity (Egg Albumin Denaturation Assay)

The method reported by Chandra et al. [41] was adopted for this study with slight modifications. Test samples consisted of 0.6 mL of fresh egg albumin, 3 mL of phosphate-buffered saline (PBS, pH 6.4), and 0.6 mL of varying concentrations of the sample so that the final concentrations became 1.67, 0.835, 0.4175, 0.2088, and 0.1044 mg/mL. Positive and negative control samples contained the same volume of egg albumin and PBS except that the same concentrations were done for the standard drug, diclofenac sodium, serving as the positive control and the vehicle as the negative control. The mixtures were incubated at 37±2°C for 10 min and then heated at 70°C for additional 20 min with a water bath. The absorbance of the cooled mixture was determined using a UV-visible spectrophotometer (Jenway, model 6715, USA) at a wavelength of 660 nm. Three replicate samples were used, and the percentage inhibition of protein denaturation is determined using
(3)%Inhibition=100×VtVc−1,where Vt is the absorbance of test sample and Vc is the absorbance of control.The IC50 values were determined using GraphPad prism software, version 9.


### 2.9. Antimicrobial Activity Assay

The agar well diffusion method reported by Holder and Boyce [42] was used to investigate the antimicrobial properties of the ZnO NPs against Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 19430), Staphylococcus aureus (ATCC 25923), and Candida albicans (ATCC 10231). Within 15 min after adjusting the turbidity of the inoculum suspension, 100 μL of the freshly cultured suspension was dispensed into the centre of the dried surface of Mueller-Hinton and Sabouraud 4% glucose agar plates for bacteria and fungi, respectively. Each plate was inoculated by streaking with a sterile swab over the entire sterile agar surface with the bacteria and fungi cells as described above. Following streaking, a sterilized cock borer of an internal diameter of 6 mm was used to punch holes in the medium, and 80 μL of the prepared concentrations (50.0, 25.0, 12.5, 6.25, and 3.25 mg/mL) of the ZnO NPs samples was dispensed into their respective labelled holes. Standard drugs, ciprofloxacin (15 μg/mL) for bacteria and fluconazole (150 μg/mL) for fungi, were used as positive controls and distilled water as negative control test. A triplicate of each plate was made and the procedure repeated for the other organisms. The plates were kept in the refrigerator for about 4 h for complete diffusion of the ZnO NP samples before incubating at 37°C for 24 h for bacteria and 48 h for C. albicans. After the incubation period, each zone of inhibition diameter was measured in millimeters (mm) with a sterilized ruler. Measured zone of inhibition for extracts less than 6 mm was considered as no antimicrobial activity observed and greater than 6 mm as sensitive or active against the test organism.


## 2.1. Collection and Identification of Plant

Healthy plant parts (fruits and leaves) ofC. albidum were collected from an uncultivated farmland located at Awukugua in the Akuapem North Municipality in the eastern region of Ghana on 22nd April 2021. The plant part samples were identified and authenticated by comparing with corresponding herbarium specimen. The samples were thoroughly washed with distilled water (DW) and dried (leaf and white cotyledon) at room temperature for two weeks to minimize the deterioration of biomolecules in the plant material as investigated by Chen et al. [31]. The dried leaf and white cotyledon samples were milled into fine and coarse powder, respectively, with a blender (FGR-350), stored in zip-lock plastic bags, and placed in a desiccator at room temperature. The ripe fruit was split open, the seeds taken out, and the pulp was shredded into pieces. The shredded pulp was then freeze-dried for further experiment.


## 2.2. Plant Extract Preparation

The solvent extraction method reported by Umaru et al. [32] was modified for this study. A mass of 10.0±0.1g of each sample (leaves, fruit, and seed) was soaked in 30 mL of methanol (1 : 3) at 28°C for 48 hours. The plant material was reextracted twice and filtered to obtain the crude. The methanol was evaporated by concentrating the crude extract under reduced pressure (200 mbar) and temperature (38°C) using a rotary evaporator (Heidolph Laborota 4000) at 40 rpm.


## 2.3. Phytochemical Screening

Preliminary phytochemical analysis of the three samples ofC. albidum was carried out using standard procedures as reported in Refs. [33–35].


## 2.4. Qualitative Test

### 2.4.1. Test for Alkaloids

The appearance of a creamy white precipitate indicated the presence of alkaloids after 1 mL of 1% HCl was added to 3 mL of each extract and treated with drops of potassium mercuric iodide solution (Meyer’s reagent) in a test tube.


### 2.4.2. Test for Tannins

Brownish green or blue-green colouration indicated the presence of tannins after a few drops of 0.1% ferric chloride was added to a filtrate of each sample obtained by boiling about 0.5 g of each powdered sample in 20 mL of deionised water in a test tube.


### 2.4.3. Test for Saponins

The formation of an emulsion from the vigorous shaking of a froth mixture of each sample was obtained by boiling about 2 g of the powdered sample and 3 drops of olive oil.


### 2.4.4. Test for Flavonoids

Red colouration of NH3 formed as layers after 2 mL of each extract was heated with 10 mL of ethyl acetate and cooled gives positivity of flavonoids.


### 2.4.5. Test for Terpenoids

The appearance of a reddish-brown colour after 5 mL of each extract was mixed with 2 mL of chloroform and 3 mL of concentrated H2SO4 shows the presence of terpenoids.


### 2.4.6. Test for Cardiac Glycosides

Brick-red precipitate formation after 10 mL of 50% H2SO4 was heated in boiling water for 5 min with 10 mL of Fehling’s solution indicates the presence of glycoside.


## 2.4.1. Test for Alkaloids

The appearance of a creamy white precipitate indicated the presence of alkaloids after 1 mL of 1% HCl was added to 3 mL of each extract and treated with drops of potassium mercuric iodide solution (Meyer’s reagent) in a test tube.


## 2.4.2. Test for Tannins

Brownish green or blue-green colouration indicated the presence of tannins after a few drops of 0.1% ferric chloride was added to a filtrate of each sample obtained by boiling about 0.5 g of each powdered sample in 20 mL of deionised water in a test tube.


## 2.4.3. Test for Saponins

The formation of an emulsion from the vigorous shaking of a froth mixture of each sample was obtained by boiling about 2 g of the powdered sample and 3 drops of olive oil.


## 2.4.4. Test for Flavonoids

Red colouration of NH3 formed as layers after 2 mL of each extract was heated with 10 mL of ethyl acetate and cooled gives positivity of flavonoids.


## 2.4.5. Test for Terpenoids

The appearance of a reddish-brown colour after 5 mL of each extract was mixed with 2 mL of chloroform and 3 mL of concentrated H2SO4 shows the presence of terpenoids.


## 2.4.6. Test for Cardiac Glycosides

Brick-red precipitate formation after 10 mL of 50% H2SO4 was heated in boiling water for 5 min with 10 mL of Fehling’s solution indicates the presence of glycoside.


## 2.5. ZnO NP Synthesis

Nanocrystalline ZnO was synthesized by dissolving6.81±0.1 g of zinc acetate dihydrate Zn(CH3COO)2·2H2O with 50 mL ethanol (95%) in a 250 mL Schott bottle on an electrical stirring hotplate (Favorit) at 60°C. After dissolving the precursor, 15 mL of the methanolic plant extract was added dropwise and the mixture was maintained at the same temperature (60°C) with constant stirring for 1 h. The pH of the mixture was adjusted between 5 and 6 using 0.1 M each of KOH and NaOH solutions. Stabilization of the reaction mixture occurred at room temperature under 3 h. The cleaning and drying of the synthesized samples followed the method reported by Droepenu et al. [36]. The process was repeated for each plant extract to obtain individual nanocrystalline ZnO samples.


## 2.6. Characterization

The synthesized nanocrystalline ZnO samples were characterized using various spectroscopic and microscopic techniques.Spectral analysis of the samples was carried out with Fourier transform infrared (FTIR) spectrometer (Thermo Scientific Nicolet iS10, USA) at a range of 4000–400 cm-1 and resolution of 4 cm-1. The sample preparation used the method reported by Yang et al. [37] where potassium bromide (KBr) in a ratio of 1 : 19 was used to solidify the sample before analysis.Morphological analysis of the samples was determined using scanning electron microscopy (SU3500, Hitachi) with the spectral imaging system (Thermo Scientific NSS (EDS)) and detector tape (BSE-3D) attached to determine the elemental composition and purity of the samples. This was operated with acceleration voltage of 10.0 kV, working distance of 11.6 mm, and a pressure of 40 Pa. However, a transmission electron microscope (TEM) (JOEL 1230, Japan) was used in determining the particle shape of the as-prepared ZnO NP samples. Prior to analysis (SEM), the dry samples were mounted on an aluminium plate with the help of an adhesive membrane. In the case of TEM analysis, samples were diluted with ethanol and sonicated with ultrasonic cleaner (Elma, Germany) for 30 min. About 4μL of the sample was loaded onto a coated copper grid and observed under the microscope. Software (ImageJ) was used to determine the diameter of the particles.The optical property of the synthesized samples was evaluated using UV-visible spectrophotometer (UV-1800, SHIMADZU) with a spectrum range between 300 and 400 nm. Also, the energy band gap (E) was determined using the expression reported by Dharma and Pisal [38] in
(1)E=h×cλ,where λ is the wavelength in m, h is the Planck constant (6.626×10−34Js−1), and C is the speed of light (3.0×108ms−1; 1eV=1.6×10−19J).Sample preparation follows the method reported by Droepenu and Asare [39].


## 2.7. Antioxidant Activity Assay

This assessment was carried out using a DPPH assay to determine the radical scavenging activity of the plant-mediated ZnO NPs.The method was described previously by Ayertey et al. [40] with slight modifications. Different concentrations of the ZnO NPs ranging from 0.104 to 1.67 mg/mL were prepared in different test tubes. 3 mL of DPPH (2,2-diphenyl-1-picryl hydrazyl) with concentration 20 μg/mL was added to 1 mL of the extracts of the various concentrations and incubated at room temperature for 30 min. The absorbance was read at 517 nm against a methanol blank in triplicate using a UV-visible spectrophotometer (Biobase). The negative control was prepared by adding 3 mL of DPPH solution to 1 mL of methanol and treated under the same conditions as the samples. Ascorbic acid was used as a positive control. Percentage inhibition was calculated using
(2)%Inhibition=Ac−AsAc×100%,where Ac is the absorbance of control and As is the absorbance of the test sample.The effective concentration (EC) at 10, 40, and 90 was determined using GraphPad software, version 9.


## 2.8. Anti-Inflammatory Activity (Egg Albumin Denaturation Assay)

The method reported by Chandra et al. [41] was adopted for this study with slight modifications. Test samples consisted of 0.6 mL of fresh egg albumin, 3 mL of phosphate-buffered saline (PBS, pH 6.4), and 0.6 mL of varying concentrations of the sample so that the final concentrations became 1.67, 0.835, 0.4175, 0.2088, and 0.1044 mg/mL. Positive and negative control samples contained the same volume of egg albumin and PBS except that the same concentrations were done for the standard drug, diclofenac sodium, serving as the positive control and the vehicle as the negative control. The mixtures were incubated at 37±2°C for 10 min and then heated at 70°C for additional 20 min with a water bath. The absorbance of the cooled mixture was determined using a UV-visible spectrophotometer (Jenway, model 6715, USA) at a wavelength of 660 nm. Three replicate samples were used, and the percentage inhibition of protein denaturation is determined using
(3)%Inhibition=100×VtVc−1,where Vt is the absorbance of test sample and Vc is the absorbance of control.The IC50 values were determined using GraphPad prism software, version 9.


## 2.9. Antimicrobial Activity Assay

The agar well diffusion method reported by Holder and Boyce [42] was used to investigate the antimicrobial properties of the ZnO NPs against Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 19430), Staphylococcus aureus (ATCC 25923), and Candida albicans (ATCC 10231). Within 15 min after adjusting the turbidity of the inoculum suspension, 100 μL of the freshly cultured suspension was dispensed into the centre of the dried surface of Mueller-Hinton and Sabouraud 4% glucose agar plates for bacteria and fungi, respectively. Each plate was inoculated by streaking with a sterile swab over the entire sterile agar surface with the bacteria and fungi cells as described above. Following streaking, a sterilized cock borer of an internal diameter of 6 mm was used to punch holes in the medium, and 80 μL of the prepared concentrations (50.0, 25.0, 12.5, 6.25, and 3.25 mg/mL) of the ZnO NPs samples was dispensed into their respective labelled holes. Standard drugs, ciprofloxacin (15 μg/mL) for bacteria and fluconazole (150 μg/mL) for fungi, were used as positive controls and distilled water as negative control test. A triplicate of each plate was made and the procedure repeated for the other organisms. The plates were kept in the refrigerator for about 4 h for complete diffusion of the ZnO NP samples before incubating at 37°C for 24 h for bacteria and 48 h for C. albicans. After the incubation period, each zone of inhibition diameter was measured in millimeters (mm) with a sterilized ruler. Measured zone of inhibition for extracts less than 6 mm was considered as no antimicrobial activity observed and greater than 6 mm as sensitive or active against the test organism.


## 3. Results and Discussion

Results of the phytochemical screening ofC. albidum plant parts (seed, fruit, and leaf) are illustrated in Table 1.Table 1
Phytochemical constituents ofC. albidum plant parts.
Phytochemical constituentsQualitative analysisLeafFruitSeedAlkaloids+++Flavonoids+++Phenols++−Tannins−++Saponins+++Cardiac glycoside++++: phytoconstituent detected; −: phytoconstituent not detected.The present study revealed the presence of medicinally active constituents in the different plant parts with phenol and tannins absent in seed and leaf, respectively.


### 3.1. FTIR Analysis

From the FTIR spectra (Figure1), a wide transmission band at 3266 cm-1 could be associated with O-H stretching vibration present in alcohols, flavonoids, and phenols. This confirms the study by Ibrahim et al. [43] who reports of a high concentration of alkaloid, flavonoid, and terpenoid in the fruit pulp and seed of C. albidum. However, Okoli and Okere [20] reported the absence of phenol in the seed of C. albidum. Meanwhile, these phytochemicals were reported to be present in most plant extracts that were utilized in plant-mediated ZnO NPs [44, 45]. Sharp peaks located at 1638 cm-1 and between 1044 and 1085 cm-1 could also be assigned to C=O and C-N stretching vibrations of proteins in the amide bands and aromatic rings of the phenols and polyphenols in the extracts as investigated by Karu et al. [46]. Similar bands were identified in C. fistula- and M. azedarach-mediated ZnO NPs as well as C. albidum-mediated Ag NPs as reported in literature [41, 47]. The peaks located between 550 and 580 cm-1 could probably be assigned to the stretching of Zn-O bonds in the prepared samples. These peak ranges were also assigned to Zn-O bonds as reported by Santhoshkumar et al. [48]. From the spectra analysis of the three (3) samples, slight shifts in peak positions and decreased intensities were observed which could be attributed to the different concentrations of the phytochemical constituents bound to the nanoparticles.Figure 1
FTIR spectra ofC. albidum (a) seed, (b) fruit, and (c) leaf extract-mediated ZnO NP samples.


### 3.2. SEM, TEM, and EDX Analysis

SEM micrographs of the different synthesized samples are illustrated in Figures2(a), 2(c), and 2(e) under various magnifications. The images revealed spherical-shaped particles which are clustered together in an agglomerated form as a result of the water content from the extracts used and the low temperature and drying conditions employed. According to a study by Wojnarowicz et al. [49], the presence of water molecules in the reaction mixture of the synthesis process affects the agglomeration state of NPs. The synthesized ZnO NPs were in the range of 10-40 nm as per the numbers (1-8) indicating the points in which the measurements were made in the SEM. On the other hand, TEM was employed in order to obtain high accuracy of the actual particle size and shape pattern. This is in fact one of the reliable techniques for NP characterization. Figures 2(b), 2(d), and 2(f) illustrate the TEM images with its particle distribution shown as histogram. The calculated mean diameter for C. albidum seed, fruit, and leaf extract-mediated ZnO NPs was 11.3±1.7 nm, 20.4±3.2 nm, and 27.3±6.6 nm, respectively. This clearly shows that samples synthesized from the seed were of the least particle size as compared to the leaf extract which gave a larger particle size. A similar morphology was reported by Ahmad et al. [50] using fruit extract of Ananas comosus. This result is almost similar to ZnO NPs synthesized using P. granatum reported by Sukri et al. [51].Figure 2
(a. c, e) SEM micrographs and (b, d, f) TEM images of seed, fruit, and leaf extract-mediated ZnO NPs usingC. albidum.
(a)(b)(c)(d)(e)(f)Elemental analysis was carried out on the samples to confirm the presence of zinc in the synthesized nanocrystalline samples using EDX. The spectrum of each sample as shown in Figure3 indicated two (2) characteristic peaks signifying O and Zn elements with percentage average mass of 18 and 82, respectively. Elemental oxygen gave a weak signal with its low percentage mass because of the X-ray emission of some phytochemical nutrients such as proteins, sugar, and vitamins present in the extract used as investigated by Satheshkumar et al. [52]. Table 2 gives the individual percentages of the different elements in the samples.Figure 3
(a–c) EDX spectra of seed, fruit, and leaf extract-mediated ZnO NPs usingC. albidum.
(a)(b)(c)Table 2
Elemental analysis ofC. albidum seed, fruit, and leaf extract-mediated ZnO NP samples.
ZnO NP sampleComponentMass %Atom %SeedO17.15±0.2045.82±0.53Zn82.85±0.6554.18±0.42FruitO18.48±0.2048.09±0.53Zn81.52±0.6351.91±0.40LeafO18.30±0.3147.79±0.80Zn81.70±0.9752.21±0.62


### 3.3. UV-Vis Analysis

The optical property of the three synthesizedC. albidum seed, fruit, and leaf extract-mediated ZnO NP samples is depicted in Figure 4.Figure 4
Optical absorption spectra ofC. albidum (a) seed, (b) fruit, and (c) leaf extract-mediated ZnO NP samples.
(a)(b)(c)From the spectra, the observed peaks gradually decreased from the seed sample (361.70 nm) to the leaf sample (359.90 nm). Similar green synthesis usingPseudomonas aeruginosa with particle size between 35 and 80 nm and absorption peak at 360 nm was reported by Singh et al. [53]. Besides, the greater the peak value, the smaller the particle size. From the observed peaks determined, it can be concluded that the energy band gap calculated using Equation (1) for the three (3) samples was within the same range of 3.4 eV. This result is in line with ZnO NPs synthesized with oleic acid as surfactants with energy band gaps of 3.4 and 3.39, respectively, as reported by Zare et al. [54].


### 3.4. Antioxidant Activity ofC. albidum-Mediated ZnO NPs

Figure5 reveals the inhibition of DPPH free radical by the synthesized ZnO NPs with reference to the standard ascorbic acid as a positive control.Figure 5
Inhibition of DPPH free radical by (a) reference ascorbic acid andC. albidum (b) fruit, (c) seed, and (d) leaf extract-mediated ZnO NPs. Data represents mean±SEM, n=3.
(a)(b)(c)(d)The study reports the scavenging activity of the extract-mediated ZnO NPs in the orderleaf<fruit<seed<ascorbicacid in terms of their EC50. The EC50 values recorded in Table 3 are 0.507±0.015, 0.255±0.006, 0.193±0.0003, and 0.004±0.000 mg/mL for leaf, fruit, seed, and ascorbic acid, respectively. The lower the EC50 value, the greater the antioxidant activity of the sample. The EC50 values recorded indicated that the synthesized ZnO NPs are promising antioxidant agents whose activity could possibly neutralize an oxidised system. From the data, it could be reported that particle size played a significant role in the activity. The smaller the size of the ZnO NP sample, the higher the antioxidant activity. When the antioxidant activity (EC50) of the different ZnO NP samples was compared to the activity of Cynometra cauliflora essential oil loaded-chitosan NPs and Albizia lebbeck stem bark extract-mediated ZnO NPs, the results showed that the current study recorded a similar activity in the range of 0.193-0.507 mg/mL as compared to 0.022-0.259 mg/mL by Samling et al. [55] and 0.457-0.666 mg/mL by Umar et al. [56]. According to the study by Umar et al., the particle size of Albizia lebbeck stem bark-mediated ZnO NPs is inversely proportional to the antioxidant activity. However, the synthesized Zn NPs recorded lower antioxidant activity when compared to green coffee-capped silver NPs with EC50 value of 0.02688 mg/mL by Kordy et al. [57].Table 3
Summary of the EC values of ascorbic acid andC. albidum fruit, seed, and leaf extract-mediated ZnO NP sample.
No.SampleEC10 (mg/mL)EC50 (mg/mL)EC90 (mg/mL)R21Ascorbic acid0.003±0.0000.004±0.0000.006±0.0000.99932ZnO NP-leaf0.104±0.0060.193±0.0030.366±0.0420.99263ZnO NP-fruit0.100±0.0030.255±0.0060.653±0.0440.89784ZnO NP-seed0.242±0.0110.507±0.0151.063±0.0300.9993Data is presented asmean±SEM (n=3).The dynamic ranges of these efficacies are depicted by the EC10 and EC90 values such that both the leaves (EC10=0.104±0.006mg/mL) and fruits (EC10=0.100±0.003mg/mL) require almost the same concentration to elicit the minimum antioxidant effects, but the seed (EC10=0.242±0.011mg/mL) requires about 2.4 more than the concentration of the leaves and fruits. However, the fruit requires a concentration fold of about 1.78 as the concentration of leaf to reach maximal antioxidant effect (EC90=0.653±0.044mg/mL for the leaf and 0.366±0.042mg/mL for fruits) while the seed (EC90=1.063±0.030mg/mL) requires about 3 times the concentration of the leaves to achieve the maximum effect. Even though similar doses are required for both leaf and fruit to achieve the minimum efficacy, their EC50 and EC90 vary. According to literature, the presence of phytochemicals such as phenolic acid and flavonoids in plant sample extracts could be responsible for greater antioxidant activity [58, 59]. However, two (2) isolated compounds in C. albidum (epicatechin and epigallocatechin) expressed higher antioxidant effect in a concentration-dependent manner as reported by Idowu et al. [60]. Other antioxidant studies on plant-mediated ZnO NPs were reported in literature [61, 62].


### 3.5. Anti-Inflammatory Activity

The anti-inflammatory activity of the extract-mediated ZnO NPs was analysed using egg albumin denaturation method, and results are shown in Figure6. The reported IC50 show inhibition in the order sodiumdiclofenac>fruit>seed>leaf as in Table 4.Figure 6
Inhibition of egg albumin denaturation by (a) reference standard sodium diclofenac and extract-mediated ZnO-NPs of the (b) seed, (c) fruit, and (d) leaf ofC. albidum. Data represents mean±SEM, n=3.
(a)(b)(c)(d)Table 4
Summary of the IC50 values of sodium diclofenac and C. albidum extract-mediated ZnO-NPs against egg albumin denaturation.
No.SampleIC50 (mg/mL)R2 value1Sodium diclofenac0.410±0.0000.99402ZnO-seed0.860±0.002∗∗∗abc0.98293ZnO-fruit0.714±0.002∗∗∗abc0.97714.ZnO-leaf0.933±0.000∗∗∗abc0.9884Data representsmean±SEM, n=3. ∗∗∗p≤0.0001 compared with the reference standard; a≤0.0001, b≤0.0001, and c≤0.0001 (ANOVA followed by Dunnett’s multiple comparison test).The inhibition of protein denaturation has long been used in most literatures to screen for potent anti-inflammatory agents. This is because protein denaturation involves an unpredictable mechanism that affects the tertiary and quaternary structures of functional proteins by destabilizing the electrostatic forces, disulfide linkages, hydrogen bonds, and hydrophobic interactions and further results in their modifications as reported by Sen et al. [63]. The process generates and accumulates autoantigens even at physiologic pH (6.2-6.5), which trigger the release of proinflammatory and inflammatory mediators to drive inflammation as implicated in most inflammatory conditions such as rheumatoid arthritis, cancer, and diabetes. This makes it necessary to pay attention to agents which inhibits protein denaturation since they could be potent anti-inflammatory agents as investigated by Sangeetha and Vidhya [64]. The egg albumin denaturation method employs a reliable protein denaturation technique and a cheaper alternative for screening natural products. The present study reports on the inhibition of protein denaturation by extract-mediated ZnO NPs. The result indicates that all the extract-mediated ZnO NPs have promising and comparable anti-inflammatory activity with reference to the standard NSAID sodium diclofenac, even though the ZnO NPs are crude extract-based. The anti-inflammatory activity could be explained by the different bioreductants in the methanolic extract of C. albidum and possibly their complexing zinc oxides. The current study can report that the absence of phenol and tannins in the seed and leaf extracts used in synthesizing the ZnO NPs could be responsible for the lower IC50 values recorded despite the effect of the particle sizes. These could also have an effect on the reduction effect of the Zn2+ ions to elemental Zn which could eventually affect the IC50 values.


### 3.6. Antimicrobial Activity ofC. albidum-Mediated ZnO NPs

Table5 illustrates the antimicrobial activities of C. albidum fruit, seed, and leaf extract-mediated ZnO NPs using agar disc diffusion method against three bacterial strains (E. coli, S. aureus, and S. typhi) and a fungus (C. albicans). Generally, the zone of inhibition increased with increase in concentration of all the three plant extract-mediated ZnO NPs for the three bacterial strains and fungus. From Table 5, C. albidum seed extract-mediated ZnO NPs recorded the highest zone of inhibition (24.33±0.47mm) against S. aureus whereas C. albidum leaf extract-mediated ZnO NPs gave the lowest zone of inhibition (13.00±1.41mm) against E. coli both at a concentration of 50 mg/mL. Moreover, C. albidum fruit extract-mediated ZnO NPs presented the highest zone of inhibition (18.00±0.82mm) against the fungus (C. albicans) also at a concentration of 50 mg/mL. This result confirms the assertion that plant-mediated ZnO NPs are more effective on Gram-positive bacteria than Gram-negative based on their cell structure, metabolic activities, and degree of contact of the bacteria as reported in literature [65, 66]. Also, the data showed that antimicrobial activity increased with decreased particle size of the ZnO NP samples. The results obtained were in conformity with a study by Kasahun et al. [9].Table 5
Mean inhibition zone ofC. albidum-mediated ZnO NP samples from methanolic extracts of fruit, seed, and leaf against selected microbes (S. aureus (Gram +ve), E. coli (Gram −ve), A. typhi (Gram −ve), and C. albicans (fungi)).
Concentration (mg/mL)Mean inhibitionzone±SEM (mm)ZnO NP typeE. coli (Gram −ve)S. aureus (Gram +ve)S. typhi (Gram −ve)C. albicans (fungi)Control20.0±0.5821.3±0.3314.0±0.5818±0.583.125ZnO-fruit8.00±0.0012.33±0.478.00±0.009.33±0.47ZnO-seedZnO-leaf8.00±0.008.00±0.0014.33±0.4715.00±0.008.00±0.008.00±0.008.00±0.008.00±0.006.25ZnO-fruit8.00±0.0015.67±0.948.00±0.0013.00±0.82ZnO-seedZnO-leaf8.00±0.008.00±0.0017.00±1.4117.00±0.8210.67±0.949.00±0.009.00±0.0010.33±1.2512.5ZnO-fruit10.33±0.4718.33±0.949.00±0.0014.33±0.47ZnO-seedZnO-leaf10.23±0.458.00±0.0018.00±0.8218.67±0.4711.33±0.9410.00±0.0010.00±1.4111.67±0.9425ZnO-fruit11.67±0.4719.67±0.4711.67±0.4715.33±0.47ZnO-seedZnO-leaf12.33±0.478.00±0.0020.33±0.4720.33±0.4715.00±0.8212.67±0.4714.00±0.8215.33±0.9450ZnO-fruit15.33±0.4722.00±0.8215.33±0.4718.00±0.82ZnO-seedZnO-leaf16.33±1.2513.00±1.4124.33±0.4724.00±0.0016.67±1.2516.67±0.4717.33±1.2516.67±1.25Among the three plant extract-mediated ZnO NPs, the seed extract-mediated ZnO NPs showed the most significant antimicrobial activity against the three selected bacterial strains with its zone of inhibition in the range of 8.00-24.33 mm whereas fruit extract-mediated ZnO NPs gave efficient result onC. albicans with the inhibition zone in the range of 9.33-18.00 mm.According to Jan et al. [67], some species (H2O2, hydroxyl radicals, singlet oxygen, and Zn2+ ions) released on the surface of ZnO cause inhibition of bacterial growth. Reports showed that UV or visible light activates ZnO creating electron/hole (e-/h+) pairs. This initiation process results in the production of hydroxyl radicals (OH⋅) and superoxide radicals (O-2⋅), and finally, hydrogen peroxide (H2O2) kills the bacterial cell as illustrated in
(4)ZnO+hv⟶e−+h+H2O+h+⟶H++OH−⋅O2⟶O−2⋅+H+⟶OH2⋅OH2⋅+e−⟶HO2−+H2⟶H2O2The recombination of e-/h+ pair reduces the probability of reactive oxygen species (ROS) generation. It has been reported by Jayaseelan et al. [68] that efficient antibacterial activity depends on unique characteristics of ZnO NPs. The results from this study confirmed that ZnO NPs from C. albicans seed extract with the smallest particle size exhibited the highest antimicrobial activity against the selected microbes.


## 3.1. FTIR Analysis

From the FTIR spectra (Figure1), a wide transmission band at 3266 cm-1 could be associated with O-H stretching vibration present in alcohols, flavonoids, and phenols. This confirms the study by Ibrahim et al. [43] who reports of a high concentration of alkaloid, flavonoid, and terpenoid in the fruit pulp and seed of C. albidum. However, Okoli and Okere [20] reported the absence of phenol in the seed of C. albidum. Meanwhile, these phytochemicals were reported to be present in most plant extracts that were utilized in plant-mediated ZnO NPs [44, 45]. Sharp peaks located at 1638 cm-1 and between 1044 and 1085 cm-1 could also be assigned to C=O and C-N stretching vibrations of proteins in the amide bands and aromatic rings of the phenols and polyphenols in the extracts as investigated by Karu et al. [46]. Similar bands were identified in C. fistula- and M. azedarach-mediated ZnO NPs as well as C. albidum-mediated Ag NPs as reported in literature [41, 47]. The peaks located between 550 and 580 cm-1 could probably be assigned to the stretching of Zn-O bonds in the prepared samples. These peak ranges were also assigned to Zn-O bonds as reported by Santhoshkumar et al. [48]. From the spectra analysis of the three (3) samples, slight shifts in peak positions and decreased intensities were observed which could be attributed to the different concentrations of the phytochemical constituents bound to the nanoparticles.Figure 1
FTIR spectra ofC. albidum (a) seed, (b) fruit, and (c) leaf extract-mediated ZnO NP samples.


## 3.2. SEM, TEM, and EDX Analysis

SEM micrographs of the different synthesized samples are illustrated in Figures2(a), 2(c), and 2(e) under various magnifications. The images revealed spherical-shaped particles which are clustered together in an agglomerated form as a result of the water content from the extracts used and the low temperature and drying conditions employed. According to a study by Wojnarowicz et al. [49], the presence of water molecules in the reaction mixture of the synthesis process affects the agglomeration state of NPs. The synthesized ZnO NPs were in the range of 10-40 nm as per the numbers (1-8) indicating the points in which the measurements were made in the SEM. On the other hand, TEM was employed in order to obtain high accuracy of the actual particle size and shape pattern. This is in fact one of the reliable techniques for NP characterization. Figures 2(b), 2(d), and 2(f) illustrate the TEM images with its particle distribution shown as histogram. The calculated mean diameter for C. albidum seed, fruit, and leaf extract-mediated ZnO NPs was 11.3±1.7 nm, 20.4±3.2 nm, and 27.3±6.6 nm, respectively. This clearly shows that samples synthesized from the seed were of the least particle size as compared to the leaf extract which gave a larger particle size. A similar morphology was reported by Ahmad et al. [50] using fruit extract of Ananas comosus. This result is almost similar to ZnO NPs synthesized using P. granatum reported by Sukri et al. [51].Figure 2
(a. c, e) SEM micrographs and (b, d, f) TEM images of seed, fruit, and leaf extract-mediated ZnO NPs usingC. albidum.
(a)(b)(c)(d)(e)(f)Elemental analysis was carried out on the samples to confirm the presence of zinc in the synthesized nanocrystalline samples using EDX. The spectrum of each sample as shown in Figure3 indicated two (2) characteristic peaks signifying O and Zn elements with percentage average mass of 18 and 82, respectively. Elemental oxygen gave a weak signal with its low percentage mass because of the X-ray emission of some phytochemical nutrients such as proteins, sugar, and vitamins present in the extract used as investigated by Satheshkumar et al. [52]. Table 2 gives the individual percentages of the different elements in the samples.Figure 3
(a–c) EDX spectra of seed, fruit, and leaf extract-mediated ZnO NPs usingC. albidum.
(a)(b)(c)Table 2
Elemental analysis ofC. albidum seed, fruit, and leaf extract-mediated ZnO NP samples.
ZnO NP sampleComponentMass %Atom %SeedO17.15±0.2045.82±0.53Zn82.85±0.6554.18±0.42FruitO18.48±0.2048.09±0.53Zn81.52±0.6351.91±0.40LeafO18.30±0.3147.79±0.80Zn81.70±0.9752.21±0.62


## 3.3. UV-Vis Analysis

The optical property of the three synthesizedC. albidum seed, fruit, and leaf extract-mediated ZnO NP samples is depicted in Figure 4.Figure 4
Optical absorption spectra ofC. albidum (a) seed, (b) fruit, and (c) leaf extract-mediated ZnO NP samples.
(a)(b)(c)From the spectra, the observed peaks gradually decreased from the seed sample (361.70 nm) to the leaf sample (359.90 nm). Similar green synthesis usingPseudomonas aeruginosa with particle size between 35 and 80 nm and absorption peak at 360 nm was reported by Singh et al. [53]. Besides, the greater the peak value, the smaller the particle size. From the observed peaks determined, it can be concluded that the energy band gap calculated using Equation (1) for the three (3) samples was within the same range of 3.4 eV. This result is in line with ZnO NPs synthesized with oleic acid as surfactants with energy band gaps of 3.4 and 3.39, respectively, as reported by Zare et al. [54].


## 3.4. Antioxidant Activity ofC. albidum-Mediated ZnO NPs

Figure5 reveals the inhibition of DPPH free radical by the synthesized ZnO NPs with reference to the standard ascorbic acid as a positive control.Figure 5
Inhibition of DPPH free radical by (a) reference ascorbic acid andC. albidum (b) fruit, (c) seed, and (d) leaf extract-mediated ZnO NPs. Data represents mean±SEM, n=3.
(a)(b)(c)(d)The study reports the scavenging activity of the extract-mediated ZnO NPs in the orderleaf<fruit<seed<ascorbicacid in terms of their EC50. The EC50 values recorded in Table 3 are 0.507±0.015, 0.255±0.006, 0.193±0.0003, and 0.004±0.000 mg/mL for leaf, fruit, seed, and ascorbic acid, respectively. The lower the EC50 value, the greater the antioxidant activity of the sample. The EC50 values recorded indicated that the synthesized ZnO NPs are promising antioxidant agents whose activity could possibly neutralize an oxidised system. From the data, it could be reported that particle size played a significant role in the activity. The smaller the size of the ZnO NP sample, the higher the antioxidant activity. When the antioxidant activity (EC50) of the different ZnO NP samples was compared to the activity of Cynometra cauliflora essential oil loaded-chitosan NPs and Albizia lebbeck stem bark extract-mediated ZnO NPs, the results showed that the current study recorded a similar activity in the range of 0.193-0.507 mg/mL as compared to 0.022-0.259 mg/mL by Samling et al. [55] and 0.457-0.666 mg/mL by Umar et al. [56]. According to the study by Umar et al., the particle size of Albizia lebbeck stem bark-mediated ZnO NPs is inversely proportional to the antioxidant activity. However, the synthesized Zn NPs recorded lower antioxidant activity when compared to green coffee-capped silver NPs with EC50 value of 0.02688 mg/mL by Kordy et al. [57].Table 3
Summary of the EC values of ascorbic acid andC. albidum fruit, seed, and leaf extract-mediated ZnO NP sample.
No.SampleEC10 (mg/mL)EC50 (mg/mL)EC90 (mg/mL)R21Ascorbic acid0.003±0.0000.004±0.0000.006±0.0000.99932ZnO NP-leaf0.104±0.0060.193±0.0030.366±0.0420.99263ZnO NP-fruit0.100±0.0030.255±0.0060.653±0.0440.89784ZnO NP-seed0.242±0.0110.507±0.0151.063±0.0300.9993Data is presented asmean±SEM (n=3).The dynamic ranges of these efficacies are depicted by the EC10 and EC90 values such that both the leaves (EC10=0.104±0.006mg/mL) and fruits (EC10=0.100±0.003mg/mL) require almost the same concentration to elicit the minimum antioxidant effects, but the seed (EC10=0.242±0.011mg/mL) requires about 2.4 more than the concentration of the leaves and fruits. However, the fruit requires a concentration fold of about 1.78 as the concentration of leaf to reach maximal antioxidant effect (EC90=0.653±0.044mg/mL for the leaf and 0.366±0.042mg/mL for fruits) while the seed (EC90=1.063±0.030mg/mL) requires about 3 times the concentration of the leaves to achieve the maximum effect. Even though similar doses are required for both leaf and fruit to achieve the minimum efficacy, their EC50 and EC90 vary. According to literature, the presence of phytochemicals such as phenolic acid and flavonoids in plant sample extracts could be responsible for greater antioxidant activity [58, 59]. However, two (2) isolated compounds in C. albidum (epicatechin and epigallocatechin) expressed higher antioxidant effect in a concentration-dependent manner as reported by Idowu et al. [60]. Other antioxidant studies on plant-mediated ZnO NPs were reported in literature [61, 62].


## 3.5. Anti-Inflammatory Activity

The anti-inflammatory activity of the extract-mediated ZnO NPs was analysed using egg albumin denaturation method, and results are shown in Figure6. The reported IC50 show inhibition in the order sodiumdiclofenac>fruit>seed>leaf as in Table 4.Figure 6
Inhibition of egg albumin denaturation by (a) reference standard sodium diclofenac and extract-mediated ZnO-NPs of the (b) seed, (c) fruit, and (d) leaf ofC. albidum. Data represents mean±SEM, n=3.
(a)(b)(c)(d)Table 4
Summary of the IC50 values of sodium diclofenac and C. albidum extract-mediated ZnO-NPs against egg albumin denaturation.
No.SampleIC50 (mg/mL)R2 value1Sodium diclofenac0.410±0.0000.99402ZnO-seed0.860±0.002∗∗∗abc0.98293ZnO-fruit0.714±0.002∗∗∗abc0.97714.ZnO-leaf0.933±0.000∗∗∗abc0.9884Data representsmean±SEM, n=3. ∗∗∗p≤0.0001 compared with the reference standard; a≤0.0001, b≤0.0001, and c≤0.0001 (ANOVA followed by Dunnett’s multiple comparison test).The inhibition of protein denaturation has long been used in most literatures to screen for potent anti-inflammatory agents. This is because protein denaturation involves an unpredictable mechanism that affects the tertiary and quaternary structures of functional proteins by destabilizing the electrostatic forces, disulfide linkages, hydrogen bonds, and hydrophobic interactions and further results in their modifications as reported by Sen et al. [63]. The process generates and accumulates autoantigens even at physiologic pH (6.2-6.5), which trigger the release of proinflammatory and inflammatory mediators to drive inflammation as implicated in most inflammatory conditions such as rheumatoid arthritis, cancer, and diabetes. This makes it necessary to pay attention to agents which inhibits protein denaturation since they could be potent anti-inflammatory agents as investigated by Sangeetha and Vidhya [64]. The egg albumin denaturation method employs a reliable protein denaturation technique and a cheaper alternative for screening natural products. The present study reports on the inhibition of protein denaturation by extract-mediated ZnO NPs. The result indicates that all the extract-mediated ZnO NPs have promising and comparable anti-inflammatory activity with reference to the standard NSAID sodium diclofenac, even though the ZnO NPs are crude extract-based. The anti-inflammatory activity could be explained by the different bioreductants in the methanolic extract of C. albidum and possibly their complexing zinc oxides. The current study can report that the absence of phenol and tannins in the seed and leaf extracts used in synthesizing the ZnO NPs could be responsible for the lower IC50 values recorded despite the effect of the particle sizes. These could also have an effect on the reduction effect of the Zn2+ ions to elemental Zn which could eventually affect the IC50 values.


## 3.6. Antimicrobial Activity ofC. albidum-Mediated ZnO NPs

Table5 illustrates the antimicrobial activities of C. albidum fruit, seed, and leaf extract-mediated ZnO NPs using agar disc diffusion method against three bacterial strains (E. coli, S. aureus, and S. typhi) and a fungus (C. albicans). Generally, the zone of inhibition increased with increase in concentration of all the three plant extract-mediated ZnO NPs for the three bacterial strains and fungus. From Table 5, C. albidum seed extract-mediated ZnO NPs recorded the highest zone of inhibition (24.33±0.47mm) against S. aureus whereas C. albidum leaf extract-mediated ZnO NPs gave the lowest zone of inhibition (13.00±1.41mm) against E. coli both at a concentration of 50 mg/mL. Moreover, C. albidum fruit extract-mediated ZnO NPs presented the highest zone of inhibition (18.00±0.82mm) against the fungus (C. albicans) also at a concentration of 50 mg/mL. This result confirms the assertion that plant-mediated ZnO NPs are more effective on Gram-positive bacteria than Gram-negative based on their cell structure, metabolic activities, and degree of contact of the bacteria as reported in literature [65, 66]. Also, the data showed that antimicrobial activity increased with decreased particle size of the ZnO NP samples. The results obtained were in conformity with a study by Kasahun et al. [9].Table 5
Mean inhibition zone ofC. albidum-mediated ZnO NP samples from methanolic extracts of fruit, seed, and leaf against selected microbes (S. aureus (Gram +ve), E. coli (Gram −ve), A. typhi (Gram −ve), and C. albicans (fungi)).
Concentration (mg/mL)Mean inhibitionzone±SEM (mm)ZnO NP typeE. coli (Gram −ve)S. aureus (Gram +ve)S. typhi (Gram −ve)C. albicans (fungi)Control20.0±0.5821.3±0.3314.0±0.5818±0.583.125ZnO-fruit8.00±0.0012.33±0.478.00±0.009.33±0.47ZnO-seedZnO-leaf8.00±0.008.00±0.0014.33±0.4715.00±0.008.00±0.008.00±0.008.00±0.008.00±0.006.25ZnO-fruit8.00±0.0015.67±0.948.00±0.0013.00±0.82ZnO-seedZnO-leaf8.00±0.008.00±0.0017.00±1.4117.00±0.8210.67±0.949.00±0.009.00±0.0010.33±1.2512.5ZnO-fruit10.33±0.4718.33±0.949.00±0.0014.33±0.47ZnO-seedZnO-leaf10.23±0.458.00±0.0018.00±0.8218.67±0.4711.33±0.9410.00±0.0010.00±1.4111.67±0.9425ZnO-fruit11.67±0.4719.67±0.4711.67±0.4715.33±0.47ZnO-seedZnO-leaf12.33±0.478.00±0.0020.33±0.4720.33±0.4715.00±0.8212.67±0.4714.00±0.8215.33±0.9450ZnO-fruit15.33±0.4722.00±0.8215.33±0.4718.00±0.82ZnO-seedZnO-leaf16.33±1.2513.00±1.4124.33±0.4724.00±0.0016.67±1.2516.67±0.4717.33±1.2516.67±1.25Among the three plant extract-mediated ZnO NPs, the seed extract-mediated ZnO NPs showed the most significant antimicrobial activity against the three selected bacterial strains with its zone of inhibition in the range of 8.00-24.33 mm whereas fruit extract-mediated ZnO NPs gave efficient result onC. albicans with the inhibition zone in the range of 9.33-18.00 mm.According to Jan et al. [67], some species (H2O2, hydroxyl radicals, singlet oxygen, and Zn2+ ions) released on the surface of ZnO cause inhibition of bacterial growth. Reports showed that UV or visible light activates ZnO creating electron/hole (e-/h+) pairs. This initiation process results in the production of hydroxyl radicals (OH⋅) and superoxide radicals (O-2⋅), and finally, hydrogen peroxide (H2O2) kills the bacterial cell as illustrated in
(4)ZnO+hv⟶e−+h+H2O+h+⟶H++OH−⋅O2⟶O−2⋅+H+⟶OH2⋅OH2⋅+e−⟶HO2−+H2⟶H2O2The recombination of e-/h+ pair reduces the probability of reactive oxygen species (ROS) generation. It has been reported by Jayaseelan et al. [68] that efficient antibacterial activity depends on unique characteristics of ZnO NPs. The results from this study confirmed that ZnO NPs from C. albicans seed extract with the smallest particle size exhibited the highest antimicrobial activity against the selected microbes.


## 4. Conclusion

The current work, green synthesis of ZnO nanoparticles fromC. albidum, is a green approach, which is inexpensive, nontoxic, and eco-friendly. All characterization techniques revealed high-purity hexagonal-shaped ZnO NPs which are agglomerated. The presence of bioreductants such as alkaloids, flavonoids, terpenoids, and saponins seems to play an important role in the synthesis and stabilization of the ZnO NPs. From the microscopy analysis, samples synthesized from the seed recorded the least mean particle size with diameter of 11.3±1.7 nm as compared to the fruit and leaf extracts which gave larger mean particle sizes with diameter 20.4±3.2 nm and 27.3±6.6 nm, respectively. From the analysed results, C. albidum-mediated ZnO NPs from the seed extract with small particle size recorded the highest antioxidant and antimicrobial activities as compared to the larger-sized particles of fruit and leaf extract ZnO NPs. However, the presence of bioreductants played a more significant role than the particle size in the anti-inflammatory activities in this study. The results showed that C. albidum-mediated ZnO NPs from the fruit extract recorded the least inhibition as compared to the seed and leaf extract samples. In conclusion, the presence of the different phytochemical constituents in the extracts serves as reductants to reduce the Zn2+ ions to ZnO NPs through the green route adopted. Therefore, these effects eventually displayed a significant antioxidant, anti-inflammatory, and antimicrobial activities against some selected microbes and fungi. These biogenic synthesized ZnO NPs could be an excellent source for antimicrobial drugs.



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*Source: 5508224-2022-06-13.xml*