The biosynthesis of zinc oxide nanoparticles using aqueous leaf extracts of Cnidoscolus aconitifolius and their biological activities

ABSTRACT This study aims to biosynthesize zinc oxide nanoparticles using aqueous leaf extracts of Cnidoscolus aconitifolius and investigate their biological activities. UV–Vis Spectroscopy, FT-IR, SEM and TEM were used for characterization. The antioxidant properties were determined using H2O2, NO, ABTS, FRAP and DPPH scavenging assays. For anti-inflammatory activities, membrane stabilization, albumin denaturation and proteinase inhibitory activity were assayed. The antibacterial effect was tested using well diffusion method. Statistical analyses were done using ANOVA. Synthesized nanoparticles showed an intensity peak at 378 nm in UV–Vis Spectroscopy. FT-IR shows the presence of O-H stretching, C = C bending, O-H bending and C–N stretching functional groups of the stabilizing action of the plant extract on the surface of the nanoparticles. NPs were shown to be spherical by SEM analysis, and the sizes were 100 nm by TEM analysis. The antioxidant properties of ZnO NPs compared to ascorbic acid standard showed significant antioxidant potential in H2O2, NO, ABTS, FRAP and DPPH scavenging assays. ZnO NPs showed excellent anti-inflammatory activity with the synthesized ZnO NPs performing better in membrane stabilization. Clinical pathogens were inhibited by ZnO NPs when compared with Cefuroxime, a standard drug. These efficient biological activities could be utilized in several biological applications. GRAPHICAL ABSTRACT


Introduction
A nanoparticle, also known as an ultrafine particle, is a matter particle with a dimension of 1-100 nm (1, 2).In the fields of medicine, engineering, electronic pharmaceuticals, and industry (1), nanotechnology has played an important role as an accelerating technology that has revolutionized many scientific areas.Nanomaterials have a high volume/surface ratio, a surface tail or ability, multifunctionality, and high reactivity (3).Nanoparticles have piqued the interest of researchers due to their fascinating properties and applications that outperform their bulk counterparts (4).Spray pyrolysis, hydrothermal, sol-gel, microwave-assisted procedures, ultrasonic condition, chemical vapor deposition, and precipitation processes have all been developed for the manufacture of Zinc Nanoparticles.These preparations have a high energy requirement and include toxic and dangerous compounds, which may pose biological dangers.Biological techniques, on the other hand, are becoming popular since they are often safe, clean, one-step, and cost-efficient (1,3,4).Biosynthesis of nanoparticles is a method of producing nanoparticles that uses microorganisms and plants and has a wide range of applications.This method is eco-friendly, cost-effective, biocompatible, safe, and environmentally friendly (5).Applying this method enables large-scale production of nanomaterials free of impurities (6).Biosynthesized nanoparticles have higher catalytic activity and require fewer expensive and toxic chemicals (7).Zinc oxide nanoparticles have been reported to have a wide range of biological applications, including antibacterial (8), anticancer (9), anti-diabetic (10), drug delivery (11), antifungal (12) and agricultural properties (13).Different plant extracts, including those from Raphanus sativus var, Salvia officinalis, Eucalyptus globulus, Ocimum gratissimum, Vernonia amygdalina, Ocimum americanum, Laurus nobilis, Ocimum basilicum, Trifolium pratenese, Cassia fistula and others, have been used to carry out the green synthesis of ZNPs (1,5,9,12,(14)(15)(16).Flavones, terpenoids, polysaccharides, ketones, aldehydes, carboxylic acids, and amides are just a few of the phytochemicals found in plants that are involved in the bioreduction of nanoparticles.Numerous functional groups found in flavonoids enhance their capacity to reduce metal ions.When flavonoids undergo tautomeric changes from the enol-form to the keto-form, the reactive hydrogen atom is liberated.This is achieved by reducing metal ions into metal nanoparticles (17).
Generally, nanoparticles have found a wide range of applications, they have been used in wastewater treatment (18,19), treatment of cancer (20), bioimaging (21), soil stabilization (22), drug delivery systems (23), nano-fertilisers application (24) and biofuels production (25).Nanoparticles are being investigated as prospective strategies for the development of diagnostic biosensors and drug/gene delivery due to their new physicochemical and biological features (26).
Spinach tree (Cnidoscolus aconitifolius) is known locally as Chaya.It is a tropical perennial shrub in the Euphorbiaceae family.It is known as 'Iyana Ipaja' in South West Nigeria (27).It is also consumed by the people of south-eastern Nigeria, where it is known as 'hospital too far' (28).There have been numerous reports on the therapeutic effectiveness of Cnidoscolus aconitifolius (Spinach tree) in a variety of cases, including the ability to darken graying hair, strengthen finger nails, cure alcoholism, insomnia, vein infections, gout, scorpion stings, and brain function and memory recovery.Cnidoscolus aconitifolius has traditionally been used to treat stomach ailments, hypertension, kidney stones, hemorrhoids, eye defects, atherosclerosis, gallstones, and high cholesterol (27).The article is a report on the green synthesis of ZnO NPs using Cnidoscolus aconitifolius, since this plant extract has been reported to posses considerable bioactivity.Hence, owing to the ethnopharmacological potentials of the plant, we also investigate the antioxidant, anti-inflammatory and antibacterial activities of ZnO NPs from aqueous leaf extract of Cnidoscolus aconitifolius.

Preparation of leaf extracts
Distilled water was used to thoroughly rinsed off dust and dirt from fresh mature Cnidoscolus aconitifolius leaves.After the leaves had been finely chopped, 10 g of them were combined with 100 mL of distilled water to create a slurry.This was cooled to room temperature after being kept in a water bath at 60 ˚C for 10 min.The Whatman filter paper No. 1 was used to filter the aqueous plant extract (PE), and the filtrate was then kept at 4 °C for later use (14).The total phenolic content (TPC) of the leaf extract is expressed as microgram Tannic Acid Equivalent (mg TAE) per gram of aqueous leaf extract of C. aconitifolius.Quercetin Equivalents were used to express the total flavonoids content, which was determined using the aluminum chloride (AlCl 3 ) method (mg QE/g) (29).

Green synthesis of ZnO NPs
The ZnO NPs were biosynthesized using the method of Sabir et al. (30) as modified by Mfon et al. (15).To 30 mL leaf extract was added 70 mL of 0.5 M Zn (CH 3 COO) 2 and 30 mL of 0.2 M NaOH.The mixture was stirred for 2 h using a magnetic stirrer and the resulting precipitate was kept in a water bath at 50 ˚C for 9 h.The colloidal suspension was centrifuged for at 3000 rpm for 5 min and the supernatant discarded.The precipitate was washed twice with distilled water and then ovendried for 7 h.The product obtained was characterized using UV-Visible spectrophotometer (Shimadzu 3600 spectrophotometer Japan); FT-IR spectrophotometer (Shimadzu IR-Tracer 100 Japan); X-ray Diffractometer (PXRD) (SMART Bruker D8 Advance X-145 Ray Diffractometer; CuKα radiation (λ = 1.54 Å) Miniflex 600, Rigaku Corporation, Japan); Transmission electron microscope (FEI-Tecnai G2 20 S-TWIN Germany) and Scanning electron microscope (FE-SEM, Carl-Zeiss model ultra-microscope 55,151, Germany).
In vitro antioxidant assays 2, 2-Dipheny-l-picrylhydrazyl (DPPH) Radical Scavenging Assay followed the method described by Blois (31) and as modified by Marrassini et al. (32).The extent of inhibition of Ferric Reducing Ability (FRA) was described by Ferreira et al. (33).The ability of the ZnO NPs and PE to scavenge hydrogen peroxide was assessed as described by the method of Ruch et al. (34).The extent of H 2 O 2 scavenging of the nanoparticles and plant extracts was calculated according to Tantary et al. (35).The 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS *+ ) free radical scavenging activity was determined according to the method developed by Re et al. (36) as modified by Kumar et al., (16).The nitric oxide scavenging activity of the sample was measured spectrophotometrically according to the method described by Ewere et al. (37).The absorbance of the chromophore formed was measured at 546 nm (38).The percentage inhibition of nitric oxide radical formation was calculated (16):

In vitro anti-inflammatory activities
Membrane Stabilization Test was carried out using fresh Bovine blood (10 mL) and Diclofenac was taken as a standard drug.The Red Blood Cell (RBC) suspension was prepared following the method of Sadique et al. (39) as modified by Sakat et al. (40).The percentage of inhibition of the ZnO NPs was determined according to using the equation in Sakat et al. (40): The albumin denaturation assay was carried out using the method of Sakat et al. (40) and the percentage inhibition of protein denaturation calculated.The proteinase inhibitory activity test was performed according to the method of Oyedapo and Famurewa (41).The percentage of inhibition of proteinase inhibitory activity was calculated according to Sundar et al. (42).

Determination of antibacterial activity
This test was carried out against a Gram-negative bacteria Escherichia coli and a Gram-positive bacterium was Bacillus cereus.The aqueous leaf extract of C. aconitifolius and ZnO NPs were tested using the well diffusion method according to Ahmed et al. (43).Cefuroxime was the standard antibiotic used as the positive control for the bacteria test organisms.The diameter zones of inhibition were determined.

Statistical analysis
Results were represented as Mean ± Standard Error of Mean (SEM).Statistical analyses were done using oneway analysis of variance (ANOVA) to compare the experimental groups followed by Tukey Multiple post-hoc test using GraphPad Prism 8 software (Version 8; GraphPad Software, La Jolla, California, USA).

Evaluated total phenolic and flavonoids contents
The result of TPC value and TFC content is shown in Table 1; the TPC value was 509.96 ± 0.44 mg TAE/g from the leaf extract of C. aconitifolius while the TFC value was 133.08 ± 0.83 mg QE/g.This showed the presence of both phenolic and flavonoids compounds in the aqueous extract responsible for the reduction of the metal, as capping agents for the stabilization of the NPs (44).

UV-Vis spectroscopy
The absorption spectrum showed a peak at 378 nm of the green synthesized ZnO NPs (Figure 1).The energy gap was calculated as 3.3 eV, which is consistent with previous research (45-47) using the following equation.
where Eg denotes the bulk band in eV.Lambda (λ) is the wavelength of peak absorbance in nanometers.UV-VIS absorption spectroscopy is a widely used technique to examine the optical properties of nanosized particles (48).Surface Plasmon Resonance (SPR) is responsible for the unique optical properties of ZnO NPs by factors such as particle size, particle shape, their distance from each other (concentration) and refractive index of the changes in the surrounding environment (49).The surface plasmon resonance (SPR) of ZnO NPS is between 310-380 nm (50).

Fourier transform infrared (FT-IR) spectroscopy of ZnO NPs
The IR spectra of the plant extract and the ZnO NPs (Figure 2(a,b)) have similar bands (Table 2) and are well supported by previous literature (14,43,51).The IR bands of ZnO NPs occurred around at 3358.X-ray diffraction (XRD) analysis of ZnO NPs XRD profile of the green synthesized ZnO NPs, was displayed in Figure 3 ZnO NPs.The image showed several Bragg peaks corresponding to the orientation of ZnO NPs. Green synthesized ZnO NPs show diffraction peaks at 2θ (in degrees) 31.21°,36.34°,39.70°and 47.01°of (100), ( 101), ( 110) and (112) planes respectively (Figure 3).These planes of hexagonal zinc oxide can be indexed to the wurtzite ZnO with high crystallinity (Joint Committee on Powder Diffraction Standard no.36-1451).A specific line broadening of diffraction peaks indicates that the synthesized materials are in the nanometer range and the manifestation of the peak at 2θ = 36.34°isthe confirmation of ZnO NPs (53).

TEM and SEM imaging of the ZnO NPs
The TEM micrographs of ZnO NPs are shown in Figure 4.
The size distribution of these ZnO NPs size was 200 nm.SEM micrograph displayed zinc oxide nanoparticles synthesized using C. aconitifolius to be spherical.The micrographs showed zinc oxide nanoparticles synthesized using C. aconitifolius in the scale bar of 0.1 µm and a distribution size of 100 nm (Figure 5).

In vitro antioxidant analysis of the ZnO NPs
DPPH radical scavenging assay of ZnO NPs Figure 6 showed in vitro DPPH activities of ZnO NPs and Tree spinach PE.DPPH is a stable synthetic free radical  that is easily reduced by antioxidants, either by accepting or donating electrons.The extent of discoloration shows the antioxidants' ability to scavenge free radicals, which is observed as a decrease in the absorbance intensity of DPPH at 517 nm.From the results obtained, ascorbic acid demonstrated the highest antioxidant activities, with ZnO NPs competing favorably with no significant difference at 40 µg/mL.A similar trend was reported by Naseer et al. (54), who synthesized zinc oxide (ZnO) nanoparticles (NPs) using leaf extracts of two medicinal plants Cassia fistula and Melia azadarach.

FRAP assay of ZnO NPs
Figure 7 showed in vitro ferric reducing power ability of ZnO NPs and Tree spinach From the results obtained, ZnO NPs and Tree spinach PE at concentrations between 100 and 200 µg/mL have no significant difference in the antioxidant activity as compared with the Ascorbic acid standard, Tree spinach PE showed better antioxidant activities than ZnO NPs when compared with the ascorbic acid standard at higher concentrations of 250 µg/mL to 300 µg/mL.Similar observations were also made by Reddy and Mandal ( 14) with synthesized ZnO NPs using Eucalyptus globulus.
Hydrogen peroxide scavenging effect of ZnO NPs           From the results obtained, ZnO NPs and Tree spinach PE demonstrated Nitric oxide scavenging antioxidant activities at all the concentrations used.However, both showed low antioxidant activities when compared with the ascorbic acid standard.However, there was no significant correlation between the flavonoid and phenolic content of the plant to the scavenging activity of the nanoparticles.These, therefore, suggests that the metals present in the nanoparticles participate in scavenging free radical in a given biological system (50).

Membrane stabilization test of ZnO NPs
The effects of zinc nanoparticle and the plant extract were as shown in Figure 11.From the results, the zinc oxide nanoparticle and Tree spinach PE extract exhibited significant bovine erythrocyte membrane stabilizing activity at the various concentrations tested as compared with the positive control.The maximum RBC stability was 82.59 ± 0.00, 72.88 ± 0.00, and 69.91 ± 0.01%, for ZnO NPs, Tree spinach extract and diclofenac, respectively.At lower concentrations, the Tree spinach extract exhibited a higher and slight biphasic mode of protection.On the other hand, the zinc nanoparticle exhibited a steady, dose-dependent and monophasic mode of protection on the bovine erythrocyte; with maximum stability of 82.59 ± 0.00% as against 69.91 ± 0.01% for diclofenac.In the comparison of the nanoparticles in this study, ZnO NPs had inhibited the release of RBC lysosomal to various levels and effectively induced the hemolysis of anti-inflammatory effect; this is in agreement with the study carried out by Agarwal and Shanmugam (50).

Albumin denaturation assay of ZnO NPs
The percentage inhibition of heat-induced albumin denaturation of the crude extract of Tree spinach and its ZnO NP as compared with diclofenac drug is shown in Figure 12.The maximum percentage inhibitions were found to be 82.81 ± 0.10 and 65.97 ± 0.01% for crude extract and ZnO NPs.These values were lower than the standard (95.20 ± 0.04%) at 0.35 mg/mL.From the results, the crude extract and standard drug   protected the BSA better than the ZnO NPs.Zinc oxide nanoparticles have been shown in the past to have potent anti-inflammatory activity (50).Agarwal and Shanmugam (50) reported ZnO NPs with a size of 40-50 nm to heat-induced albumin denaturation.

Proteinase inhibitory activity of ZnO NPs
The ability of the crude extract and ZnO NPs to inhibit tryptic activity against the hydrolysis of BSA is shown in Figure 13.ZnO NPs inhibited the trypsin activity maximally at 10 μg/mL with a maximum % inhibition of 95.93 ± 0.03.The effect of ZnO NPs was significantly better than the crude extract, whose effect was concentration-dependent.

Antibacterial activity of ZnO NPs
The minimum zone of inhibition for ZnO NPs against Gram-positive bacteria Bacillus cereus was found to be 20 and 27 mm at 50 and 100 µg.Similarly, that of Tree spinach PE was found to be 20.7 mm and 23.3 mm at 50 and 100 µg, while that of the standard was found to be 28 mm.Comparing the results obtained at high concentrations of the extract ZnO NPs showed a better zone of inhibition to Tree spinach PE.Though the standard cefpirome (CPR) still maintains a significant higher zone of inhibition to the test samples.
The test samples ZnO NPs and Tree spinach PE also showed significant inhibition on gram-negative bacteria E. coli.The minimum zone of inhibition for ZnO NPs was found to be 28.0 and 24.3 mm at 50 and 100 µg, respectively, that of Tree spinach PE was found to be 24 mm and 21 at 50 and 100 µg, respectively.CPR standard was found to have 28 mm against the Gram-negative  bacteria.Figure 14 shows a chart of the minimum zone of inhibition against Gram-positive bacteria Bacillus cereus and Gram-negative bacteria E. coli as against the test samples.The antibacterial effect of the ZnO NPs samples is mainly due to the combination of various factors, such as reactive oxygen species (ROS) generation and the release of Zn 2 ions.The generated ROS can penetrate the cell membrane and kills the bacteria.Similar observations were made by Elshama et al. (57).This report is also in agreement with Kumar et al. (16), who reported the antimicrobial activity of zinc oxide nanoparticles synthesized using aqueous plant extract of Ocimum americanum, results showed significant inhibition against test pathogens.ZnO NPs inhibited E. coli and Bacillus cereus.

Conclusions
Green synthesis of nanoparticles using plants has been regarded as a cheap, eco-friendly and effective method for the delivery of drugs.Metal oxide nanoparticles exhibit remarkable biological applications and are of great interest for both research and technological development (58).In this study, ZnO NPs were synthesized using an aqueous plant extract of C. aconitifolius, Zinc acetate and NaOH as precursor materials.NaOH has been reported by Koutu et al. (59) to help in reducing the size of crystallite size as well as a co-precipitating agent in the synthesis of ZnO NPs characterized via several electron microscopy techniques.The study revealed ZnO NPs to be a better antioxidant in H 2 O 2 and NO scavenging assays.ZnO NPs inhibited albumin denaturation and trypsin at the lowest concentration used better than diclofenac.Furthermore, the ZnO NPs act as a potent bactericidal agent against E. coli and B. cereus.A literature survey revealed that C. aconitifolius leaves process a vast range of biological activities.This study has shown that zinc oxide nanoparticles synthesized via green synthesis from C. aconitifolius leave extract possess antioxidant, antiinflammatory and antibacterial potentials.These biological and pharmacological activities could be exploited further for drug designs and deliveries.

Figure 8 showed
Figure 8 showed in vitro hydrogen peroxide radical scavenging (H) activities of ZnO NPs and Tree spinach
Figure 9 showed in vitro ABTS radical scavenging activities of ZnO NPs and Tree spinach PE.From the results

Figure 6 .
Figure 6.Profile of DPPH assay.The data represent an average of three determinations ± SEM and subjected to one-way analysis of variance (ANOVA) for comparing the means of all groups.* indicates values that are significant at (p < .05)when compared to control Ascorbic acid.α indicates values that are significant at (p < .05)when compared to Tree spinach Plant Extract.β indicates values that are significant at (p < .05)when compared with ZnO NPs.

Figure 8 .
Figure 8. Profile of hydrogen peroxide scavenging effect.The data represent an average of three determinations ± SEM and subjected to one-way analysis of variance (ANOVA) for comparing the means of all groups.*indicates values that are significant at (p < .05)when compared to control Ascorbic acid.α indicates values that are significant at (p < .05)when compared to Tree spinach Plant Extract.β indicates values that are significant at (p < .05)when compared with ZnO NPs.

Figure 9 .
Figure 9.A profile of in vitro ABTS radical scavenging activities.

Figure 10 showed
Figure 10 showed in vitro NO activities of ZnO NPs and Tree spinach PE.Different concentrations of extracts fractions were compared with ascorbic acid standard.From the results obtained, ZnO NPs and Tree spinach PE demonstrated Nitric oxide scavenging antioxidant activities at all the concentrations used.However, both showed low antioxidant activities when compared with the ascorbic acid standard.However, there was no significant correlation between the flavonoid and phenolic content of the plant to the scavenging activity of the nanoparticles.These, therefore, suggests that the metals present in the nanoparticles participate in scavenging free radical in a given biological system(50).

Figure 10 .
Figure 10.A bar chart showing in vitro nitric oxide activities.Data represent an average of three determinations ± SEM and subjected to one-way analysis of variance (ANOVA) for comparing the means of all groups.* indicates values that are significant at (p < .05)when compared to control Ascorbic acid.α indicates values that are significant at (p < .05)when compared to Tree spinach Plant Extract.β indicates values that are significant at (p < .05)when compared with ZnO NPs.

Figure 11 .
Figure 11.Profile showing effect of ZnO NPs, Spinach Tree PE and Diclofenac on the Bovine RBC membrane stabilization.

Figure 12 .
Figure 12.The albumin denaturation assay of ZnO NPs, Spinach Tree PE and Diclofenac.Data represent an average of three determinations ± SEM and subjected to one-way analysis of variance (ANOVA) for comparing the means of all groups.* indicates values that are significant at (p < .05)when compared to control Ascorbic acid.α indicates values that are significant at (p < .05)when compared to Tree spinach Plant Extract.β indicates values that are significant at (p < .05)when compared with ZnO NPs.

Figure 13 .
Figure 13.A profile of effects of the crude extract and ZnO NPs on trypsin activity (in vitro proteinase inhibitory activity).

Table 1 .
Total phenolic content and flavonoids in aqueous extract of C. aconitifolius.
Note: The results are shown for n = 3 determinations as the average ± standard error of mean (SEM).QE: Quarcetin Equivalents; TAE: Tannic acid equivalents.