Green synthesis of magnetite nanoparticles using calotropis procera leaf extract and evaluation of its antimicrobial activity

In this study, magnetite nanoparticles were successfully synthesized using Calotropis procera aqueous leaf extract. The effect of the whole extract and some of its phytochemicals on the particle size distribution was determined using Dynamic light scattering (DLS) while other characterizations such as UV–vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, x-ray diffraction (XRD) spectroscopy and Scanning electron microscopy (SEM) were carried out. The antimicrobial activity against selected microorganisms was also studied using the agar well diffusion method. The leaf extract showed the presence of phenols, flavonoids, saponins, alkaloids and tannins. The magnetite nanoparticle synthesized using the leaf extract (Calotropis procera/Fe3O4) had an average particle size of 11.1 nm with a PDI of 0.142 while the phenolic, flavonoid and saponin extracts of Calotropis procera leafs produced magnetites with average particle sizes of 62.83 nm, 68.02 nm and 134 nm respectively. The UV–vis spectra displayed the characteristic plasmon resonance peak at 420 nm, the FTIR plots highlighted important functional groups including peaks between 600 cm−1 and 400 cm−1 confirming the formation of iron oxide while the SEM micrographs showed the nanoparticles were mainly irregular in shape with areas of agglomeration. Calotropis procera/Fe3O4 displayed significant anti-microbial activity against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, Aspergillus niger and Fusarium oxysporum while no activity was recorded against Escherichia coli. The study indicated that Calotropis procera leaf extract was suitable for the green synthesis of magnetite with properties that suggest that it could be potentially applied as antimicrobial nanofillers.


Introduction
Magnetite nanoparticle (Fe 3 O 4 ) has gained a lot of interest due to its bio-compatibility, low toxicity, ease of preparation and biodegradability.Its unique magnetic property (ferromagnetism) has been well explored in different applications including biosensors, magnetic storage devices, magnetic resonance imaging and targeted drug delivery.Magnetites have also been utilized as nano-fillers to improve the properties of packaging films, however there are only a few of reports of this application [1].Their size, surface properties and shape vary based on the method of synthesis and intended application.Magnetites can be synthesized through chemical methods mainly co-precipitation, however this method is usually associated with high production cost, complicated procedure and the toxic chemicals.To this effect, green synthesis of magnetite has been explored as a viable, cheap and eco-friendly alternative [2].
Magnetites produced through green synthesis including the use of seeds, fruit peels, leaf extracts, fruits and even plant waste have been well reported as well as their effects on particle sizes, morphologies and magnetic properties [3].Generally the extract composition, solution pH and relative concentration of the iron salts are important during the synthesis of magnetite however, the phytochemicals present in these plant extracts are known to function as reducing, capping and stabilizing agents.A number of studies have highlighted the role of various phenolic and flavonoid compounds in nanoparticles synthesis while there are some reports of saponin involvement [4,5].
Calotropis procera (a type of milk weed) is a known medicinal plant that is widely spread in many regions around the world and has been reported to possess anticancer, anticoagulant, antioxidant, anti-inflammatory and antipyretic properties which has been attributed to the phytochemicals it contains [6].Aqueous extracts of Calotropis procera have been reported to be used in the synthesis of zinc oxide [7], silver nanoparticles [8], cerium oxide nanoparticles [9] and gold nanoparticles [10].With this plant widely available and the phytochemicals reportedly present, it could be explored for the synthesis of magnetites, hence the need to establish the phytochemicals present and their effects on important properties of magnetites.Generally nanoparticles must possess certain properties in order to be applied as fillers in packaging films, one of which is significant antimicrobial activity.The antimicrobial activity of magnetite synthesized using this plant against selected food spoilage microorganisms has also not been reported.
This present study therefore investigates the synthesis of magnetite nanoparticles using Calotropis procera leaf extract and the effect on some of its microstructural properties.The phytochemical composition of Calotropis procera was analysed and the individual effect of some of them on particle size distribution compared to the use of the whole leaf extract.The antimicrobial activity against selected gram positive bacteria, gram negative bacteria and fungi was also studied to evaluate its potential as an antimicrobial nano-filler.

Extraction preparation
The aqueous extract of Calotropis procera, was prepared as described by Dhar et al [11].The leaves were then soaked in deionized water, washed thoroughly and then dried under the Sun for 10 days.The dried leafs were then milled to powder and stored in airtight bags.Ten grams of the leaf powder added to a round bottom flask containing 100 ml of deionized water and heated at 70 °C for 1 h.After the solution has cooled adequately it was then filtered using Whatmann filter paper.The Calotropis procera leaf extract solution was then stored at 4 °C for further use.

Phytochemical analysis of leaf extracts
The aqueous Calotropis procera leaf extract was screened for phytochemicals mainly phenols, flavonoids, alkaloids, tannins, glucosides and saponins.The total phenol content of the leaf extract was determined using Folin-Ciocalteu's method described by Amrulloh, et al [12].The total flavonoid content of the aqueous leaf extract was determined using the aluminum chloride method described by Chang et al [13].Total alkaloid, cardiac glucoside and saponin of the aqueous leaf extract was determined using ethanol extraction method described by Oloye [14].Tannin content of the crude extract was determined using the method described in AOAC [15].

Synthesis of calotropis procera/Fe 3 O 4
The method described by Yew et al [16] was used for the synthesis of magnetite with some modifications.The synthesis started with the dissolution 6.25 g of FeCl 3 and 3.12 g of FeCl 2 (2:1 molar ratio) in 50 ml of deionized water.The solution was then heating at 80 °C with continuous stirring for 10 min.Ten milli-litres (10 ml) of Calotropis procera aqueous extract was added, the mixture was stirred thoroughly for another 10 min and then 1.0 M sodium hydroxide was added in a drop wise manner till a pH of 11 was obtained.The solution was further stirred for 1 h and allowed to cool to complete the synthesis of magnetites.The precipitate was separated using a permanent magnet and washed several times with deionized water to avoid impurities.Finally the magnetite obtained designated (Calotropis procera/Fe 3 O 4 ) was oven-dried in an oven 70 °C for 24 h and then stored in air tight bags for further characterisation.

Effect of phytochemicals on particle size distribution
The effects of phenol, flavonoid and saponin extracts from the Calotropis procera leaf on particle size distribution was studied.The extraction of phenols was done using methods described by Liu et al [17], flavonoids using methods described by Latif et al [18] and saponins using methods described by Pn et al [19].After this, the phytochemical extracts were used (in place of the aqueous leaf extract) for the synthesis of magnetites as described in 2.4.

Characterisation of calotropis procera/Fe 3 O 4
The UV-vis absorption spectrum for the nanoparticles was carried out using the Ultravoilet Visible spectrophotometer (Shimadzu UV-1800) within 200-800 nm.The nanoparticles sizes were determined by dynamic light scattering (DLS) using a Zetasizer 3000 (Malvern Instruments, UK).The different functional groups present in the nanoparticles were evaluated using FTIR-8400S Fourier Infrared Spectrophotometer.The samples were pre-dried and ground with KBr pellets, the transmittance recorded within the range of 400-4,000 cm −1 [10].The morphology of the synthesized magnetites were studied using images from Scanning Electron Microscope (model: PRO:X: 80007334 Phenom World, MVE01570775) with secondary electron detectors at an operating voltage of 30 kV while the x-ray diffraction patterns were evaluated using an x-ray diffractometer (Thermoscientific model: ARL XTRA, 197492086).

Anti-microbial activity
Six microbial strains were used to screen the antimicrobial activity of Calotropis procera/Fe 3 O 4 ; two grampositive (Staphylococcus aureus and Bacillus subtilis), two gram-negative (Escherichia coli and Klebsiella pneumoniae) bacterial isolates and two fungal isolates (Aspergillus niger and fusarium oxysporum).They were isolated deteriorated fruits and identified by microbiology department, Centre for Genetic Engineering and Biotechnology (CGEB), Federal University of Technology, Minna, Niger State, Nigeria after morphological and biochemical tests.Agar well diffusion method as described by Thukkaram et al [20] was used to screen the antimicrobial activity of Calotropis procera/Fe 3 O 4 .One ml of freshly prepared pure cultures of each microorganism was spread on nutrient agar (NA) plates and incubated at 37 °C for 30 min.Wells (diameter of 6 mm) were made using sterile cork borers and were filled with Calotropis procera/Fe 3 O 4 (100 mg ml −1 ) and then incubated at 37 °C for 24 h.The antimicrobial activity was examined by measuring the zone of inhibition that appeared after the incubation period.The analysis was done in triplicates, the results were presented as means ± SE of the mean and data considered significant at P < 0.05.
The phytochemical content of Calotropis procera observed in this study agrees with other reports which showed the presence of phenols, alkaloids, tannins, flavonoids and saponins [6].Similar values for the total content of some of the phytochemicals have also been reported; Banerjee et al [21] reported a total phenol content of 40.7 (mg GAE g −1 ) while Najar et al [22] recorded a total flavonoid content of 36.75 (mg GAE g −1 ).Manal & Amal [23] however reported higher values, with a total phenol and flavonoid content being 56.3 (mg GAE g −1 ) and 41.4 (mg QE g −1 ) respectively.Variation in the phytochemical content could be attributed to agro-climatic factors in the environment where the plant was collected from.
The result therefore establishes that Calotropis procera contains important phytochemicals that can be involved in the reduction, stabilization and capping processes during magnetite synthesis [24].

UV-visible spectroscopy
The UV-Vis spectra of Calotropis procera and Calotropis procera/Fe 3 O 4 was shown in figure 1; Two peaks were observed for Calotropis procera at 307 nm and 350 nm while a surface resonance peak at 420 nm was observed for Calotropis procera/Fe 3 O 4 .The formation of peaks between 400 nm and 450 nm has been reported in most studies on the synthesis of magnetites and is attributed to the direct formation of iron oxides.A similar study showed absorption peaks at 415 nm when seaweed was used for the synthesis magnetite [25], while another report showed resonance band peak at 417 nm [26].The presence of the peak at 420 nm therefore confirms the synthesis of magnetite.

Particle size of calotropis procera/Fe 3 O 4
From figure 2, an average particle size of 11.1 nm was obtained while the poly-dispersity index (PDI) was 0.143 as determined by DLS.The PDI measures the size distribution of magnetite nanoparticles hence the value in this study indicates that the sizes of the synthesized magnetite was relatively uniform.Acceptable PDI range depends on the applications, however smaller particle sizes and PDI values of 0.2 or below are usually recommended for nanoparticles intended for polymer based applications [27].The result in this study is similar to the sizes reported using leaf extracts of some plants for the synthesis of magnetite.Average particle sizes of 18.1 nm, 20 nm, 14.7 nm and 17.72 nm were reported in magnetite synthesized using Sargassum muticum seaweed [25], Uncaria tomentosa leaves [28], Seaweed Kappaphycus alvarezii [16] and Lathyrus sativus peel extracts [11] respectively.PDI values of 0.24 and 0.229 were observed in some reports involving green synthesis of magnetites [29,30].
The results showed that calotropis procera leaf extract was effective in reducing salts to iron oxide nanoparticles with relatively small particle sizes and low PDI suggesting that they could be suitable as nanofillers in packaging films.

Effect of phytochemicals on particle size distribution
The individual effect of the some phytochemicals extracted from Calotropis procera leafs on magnetite synthesis and particle size distribution was described in figure 3. The results show that the magnetite synthesized from phenol extract produced an average particle size of 62.83 nm with a PDI of 0.241 while that of the flavonoid extract was 68.02 nm with PDI of 0.186.This indicates that although the flavonoid synthesized magnetite had slightly higher average particle size, it had a lower PDI suggesting a more controlled nanoparticle synthesis and uniform size distribution.The saponin extract however, had average particle size of above nano-size (134 nm) and a PDI of 0.323.
Generally a number of mechanism of synthesis of nanoparticles by different phenolic compounds and flavonoids have been proposed.The use of individual phytochemicals tend to produce highly stable nanoparticles when compared to the use of organic acids and it has also been suggested that the ability to reduce iron salts to iron oxide nanoparticles can be affected by the number of hydroxyl groups present in these phytochemicals [31].
There are reports on the direct use of the phytochemicals obtained from the extracts for nanoparticle synthesis.The synthesis of iron oxide nanoparticles using phenolic extracts of different plants has been reported and the study highlighted that different types and structures of phenolic compounds influenced particle size distribution [32].Flavonoid (quercetin) isolated from Combretum ovalifolium was used in the synthesis of zinc oxide nanoparticles with an average particle size of 31.24nm [33] while flavonoid extracts from citrus plants was used in the synthesis of silver nanoparticles with particle sizes between 5-80 nm [34].Overall the results showed that although the phenol and flavonoid extract of Calotropis procera was capable of synthesizing magnetite individually, synthesis using the whole leaf extract had much lower particle size (11.1 nm) and PDI.This suggests the combined effect of the different phytochemicals in the reduction and stabilization process, however further studies would be required to establish the specific phenolic or flavonoids compounds present in Calotropis procera that could be involved in magnetite synthesis as well as their possible mechanism of action.

Fourier-transform infrared (FT-IR) spectroscopy
The FTIR spectra of as shown in figure 4 highlights the different functional groups present in both calotropis procera leaf extract and Calotropis procera/Fe 3 O 4 .In figure 4(a), peaks were observed at 3434 cm −1 , 2908 cm −1 , 1627 cm −1 , 1108 cm −1 which can be attributed to N-H, −CH, C=O, C-O, C-N functional groups of the phytochemicals present in Calotropis procera.In figure 4(b), distinct absorption peaks were observed at 3434 cm −1 , 2908 cm −1 , 1627 cm −1 , 1108 cm −1 , 568 cm −1 and 435 cm −1 .The peak at 3434 cm −1 can be attributed to the stretching of the phenolic OH groups from the leaf extract.The peak at 2908 cm −1 corresponds to CH stretching while the peak at 1627 cm −1 corresponds to the carbonyl group C=O of a carboxylic acid possibly from the phenolic or flavonoid compounds present.The peak at 1108 cm −1 can be ascribed to the stretching frequency of the phenolic C-O group.The peaks in this result are similar to other reports of biosynthesis of magnetites [25,28].The peaks after 600 cm −1 (at 568 cm −1 and 435 cm −1 ) corresponds to the stretching vibration mode of Fe-O present and confirms the synthesis of Fe 3 O 4 [11].This results also suggests that the functional groups present due to the surface properties Calotropis procera/Fe 3 O 4 could improve its compatibility when applied as nanofillers, however this will depend on the packaging polymer of interest.

Scanning electron microscopy (SEM)
Figure 6 shows the SEM images of calotropis procera/Fe 3 O 4 .The micrographs showed mainly irregular shaped particles with areas of uniform dispersion and areas of particle aggregation.The average particle size as determined by Image J software was 13.1 nm slightly higher than obtained through the DLS.It has been suggested that these areas of aggregation can be attributed to the interaction between the formed nanoparticles and some of the phytochemicals involved in its synthesis [11] or could be due steric magnetic interactions between individual nanoparticles [35].Different studies however report various morphologies including cubic, spherical and irregular clusters of magnetites synthesized using green extracts however this morphological variation can be attributed to the method of synthesis [36].This particle aggregration observed could however limit its ability to disperse uniformly when applied as nanofillers.
3.8.Antimicrobial activity of calotropis procera/Fe 3 O 4 Table 2 describes the antimicrobial activity of Calotropis procera/Fe 3 O 4 against isolated microorganisms.It can be observed that antimicrobial activity ranged between 7.1 mm to 22.5 mm (zone of inhibition) against K. pneumonia, S. aureus, B.subtilis, A.niger, F.oxysporum while no activity was observed against E. coli.The result in this study also showed the antimicrobial activity against gram positive bacteria (S. aureus and B. subtilis) was significantly (P 0.05) higher than other microorganisms tested while the activity against fungal isolates was significantly (P 0.05) higher than the gram negative bacteria.
The higher activity against gram positive bacteria can be attributed to the absence of an outer lipid membrane which plays an important role of providing an extra layer of protection in gram negative bacteria, as compared to gram positive bacteria.Overall the antimicrobial activity of iron oxide nanoparticles and sensitivity to different microbes vary in different reports; this could be due to the method of synthesis, functionalization, particle size, magnetite concentration, magnetic property and the microbial strain tested [37].Generally the antimicrobial activity of magnetites has been attributed to the electrostatic attraction between magnetite and the negatively charge microbial membrane which subsequently leads to membrane disruption by the reactive oxygen species (ROS) generated [38].The interaction between magnetite and microorganism hence plays a key role in its antimicrobial activity.While some reports indicate antimicrobial activity of magnetite against E. coli, other studies show no significant effect on E. coli or a surface charge dependent effect which could be attributed to variation in E. coli strains [39][40][41].E. coli resistance to magnetite has been studied through genomic analysis and the involvement certain genes (rpoA and rpoC) in this resistance was highlighted [42].Strain identification of E. coli and subsequent antimicrobial activity of magnetite against other E. coli strains will be required to further understand the antimicrobial resistance observed in this study.
The antimicrobial activity of magnetites against ten pathogenic bacteria was studied by Behera et al [43]; while no activity was observed against Shigella flexneri and pseudomonas aeruginosa, significant antimicrobial activity was recorded against staphylococcus aureus, staphylococcus epidermidis bacillus subtilis and E. coli and a host of others.The result also showed significantly higher antimicrobial activity against gram positive bacteria similar to the observation in this study.Thukkaram et al [20] also observed similar higher antimicrobial activity against S. aureus when compared to other microorgamisms.Magnetite synthesized using Glycosmis mauritiana leaf extract was also reported to have antimicrobial activity values (zone of inhibition) of 16 mm and 19 mm against S. aureus and B. subtilis repectively [44].
This result indicates that Calotropis procera/Fe 3 O 4 possesses significant antimicrobial activity hence can be applied as an nanofiller in packaging films.The reported lack of antimicrobial activity against E. coli could be limiting however, the effect of magnetite on different strains of E. coli and a growth kinetic study of each microbial strain in the presence of magnetite might be required to fully establish the pattern of antimicrobial activity.
Data were expressed as mean of three replicates ±SEM.Values with different lower case superscript alphabets across the row are significantly different (P 0.05).

Conclusion
The study demonstrated that Calotropis procera leaf extract was effective in the synthesis of magnetites.The average particle size and the PDI of magnetite synthesized using individual phenol and flavonoid extracts of Calotropis procera leaf were significantly higher than when the whole leaf extract was used highlighting the combined effect of all the phytochemicals in acting as reducing, stabilizing, and capping agents.Other characterisations such as UV, FTIR spectra and XRD confirmed the synthesis of magnetites with properties comparable to other available reports involving green synthesis.The magnetite nanoparticles displayed antimicrobial activity against most of the microorganisms tested with E. coli being the exception.Overall the study suggests that Calotropis procera leaf can be recommended as a viable source of extract material for the synthesis of magnetite nanoparticles for different applications including as nanofillers in packaging films.Further studies on the phenolic and flavonoid compounds present in Calotropis procera leaf and the effect of magnetites on different microbial strain types was however recommended.