Synthesis and antimicrobial activities of a metallic oxide nanoparticle complex of Moringa oleifera leaves extracts against selected microorganisms

This research work aimed at synthesizing and investigating the antimicrobial activities of a metallic oxide nanoparticle complex of Moringa oleifera leaves extracts against some microorganisms. Moringa oleifera leaves were washed, dried and blended. They were extracted with distilled water and ethanol using standard methods. The nanoparticle was synthesized by coordinating with manganese oxide. The physicochemical properties were determined following standard procedures. The phytochemical screening was carried out by standard methods. The antibacterial activities were done using agar well diffusion method. Antifungal activity was carried out following the plate technique. The leaves extract had a 75% yield and melting point of 116 °C while the nanoparticle had a yield of 60% and melted at 78 °C with pH of 3.46. The molar conductance of the nanoparticle revealed at 10.6 Ωcmmol. The ethanolic extract of the leaves showed the presence of alkaloids, tannin, steroids and saponins. The ethanolic extract of M. oleifera exhibited the highest antibacterial activity of 33.05±0.10 mm against Bacillus subtilis while its antifungal activity revealed the highest inhibition of 48.40±0.53 mm at 30 mg/mL against Aspergillus niger. Staphylococcus aureus had a zone of inhibition of 19.00±0.16 using the aqueous extract. The ethanolic extract of M. oleifera nanoparticles showed antibacterial and antifungal activity against B. megaterium and A. niger with a zone of inhibition of 49.21±0.32 mm and 50.35±0.29 mm respectively while the aqueous extract showed antibacterial activity against S. aureus with a zone of 26.00±0.38mm. As it was concluded ethanolic extract in both leaves extract and its nanoparticle, possessed higher antibacterial and antifungal activities than the aqueous extract.


Introduction
Nano-techonology has enhanced our daily lives quietly and significantly with the help of researchers globally (Wong et al., 2001). Different nanoparticles have been studied due to nano-dimension, nanostructured materials which are analysed by irregular electronics and mechanical properties. Coordination and characterization of some nanoscale materials are important to scientific studies which grow rapidly as a

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Notulae Scientia Biologicae result of their usefulness for producing new products for virtually all areas of human activity including medicine and communication. Nanotechnology has great a concern in enhancing the treatment of different disorders and diagnostics. (Cagin et al., 1999). The metallic nanoparticles have been produced through the borohydride reduction method at a determined temperature (Jana and Gearheart, 2001). From previous research, studies on nanoscale material based on some selected metals such as copper, cobalt, nickel and iron have been done due to their physicochemical properties and potential use of technology for keeping information.
Moringa oleifera leaves are used as a local source for a target in the development of biomaterials, bionanotechnology and engineering. Importantly, Moringa oleifera leaves powder constitute high concentration of some organic materials MaryKensa and Neelamegam (2014). Moringa is a local plant in Africa and Asia which are mostly grown in North Western India. It is the sole genus in the family of Moringaceae. It has about thirteen species from tropical and subtropical climates within the range of tiny herbs to massive trees (Azwanida, 2015).
Several works have been performed on the beneficial effects in humans. It is well known as having a large number of bioactive compounds. The part used most is the leaves which are very rich in vitamins, polyphenols, flavonoids, alkaloids, glucosinolates, tannins, saponins e.t.c. The number of bioactive compounds helps to describe the pharmacological properties of Moringa oleifera leaves. Researchers have proven the pharmacological properties of the plant. Few years back, synthesis of different forms of active antimalarial substances from the leaves have been on the increase (Yakubu et al., 2006).
M. oleifera plant has recently generated great attention as potential therapeutic agents against different diseases like those involving radical damage. These polyphenolic compounds, ubiquitous in higher plants, are commonly major dietary constituents. The biological and medicinal properties of flavonoids have been reviewed extensively, with wealth of data on their activity as reducing agents, hydrogen-donating antioxidant, and singlet oxygen quenchers (MaryKensa and Neelamegam, 2014).
M. oleifera are natural chelators and its metal complexes have been shown to possess high cytotoxic activity (Ramakrishna and Pragna (2011) which has great benefits in curing various diseases. It has also been shown that synthesis of metals with bioactive ligands can enhanced the pharmaceutical activity of the drugs and help to decrease their toxicity effects (Enemose et al., 2014). From previous work, Doddanna et al., 2013) showed the activity of some plant extracts against Candida albicans. It was observed that the results obtained revealed that the ethanolic extract of curry leaves are effective against Candida albicans with 24.05±0.07 after 48 hours. According to Alzoreky and Nakahara (2003) and Castro et al. (2008), the antimicrobial activities of some parts of plants has been carried out. These include crude extracts of cinnamon, curry and ginger mustard, and other herbs which possessed antimicrobial properties against gram-positive and gram-negative organisms. It has also been observed that the extracts obtained from Chinese chives and cassia can actively help to reduce the rate at which Escherichia coli and other organisms develop during storage of meat, juices, and milk (Mau et al., 2001).

Materials and Methods
Preparation of aqueous and ethanolic extract of Moringa oleifera leaves Procedure followed by Bamigboye and Ahmed (2019) was adopted for this research work. Fresh Moringa oleifera leaves were collected from the environment of University of Ilorin, Nigeria. The leaves were freed from twigs and extraneous matter. In order to remove the dirt of foreign matter, the samples were thoroughly washed with distilled water and dried. After drying, the leaves were grinded into a fine powder with the use of mortar and pestle. About 10 g of the powder was then weighed and soaked into a separate round bottom flask of distilled water and ethanol. Each of the contained flasks was heated for 30 minutes at 100 °C and 78 °C respectively. They were incubated at 37 °C and 130 rpm in a shaking incubator (Kahlil et al., 2013).
The extracts were separated by filtration with the use of Whatman No. 1 filter and concentrated using rotary evaporator.

Synthesis of Moringa oleifera leaves-manganese oxide nanoparticles
The procedure of Pirtarighat et al. (2019) was adopted. Briefly, an aqueous solution of manganese oxide (MnO) (1 mM) was prepared and mixed in different beakers with aqueous and ethanolic extracts of M. oleifera leaves respectively. Each mixed solution was introduced into a shaker at a determined temperature for about 3 hours. It was decanted and dried. The product was kept in a sample bottle for further analysis.
Determination of phytochemical constituents of leaves extracts of M. oleifera The phytochemical constituents of M. oleifera leaves extract were determined by the procedures adapted by Ahmed et al. (2013). They include steroids, alkaloids, saponins and tannins.
Test for alkaloids: In the leaves extract, about 5 percent drop of 1 mL of leaves extract was added to 2 mL of 1% HCl for 20 minutes. The mixed extract was left to cool. After it was filtered, 2 drops of picric acid were introduced into it. A form of cream precipitate was obtained. This revealed the presence of an alkaloid in the leaves extract.
Test for saponins: 10 mL of distilled water was measured and added to the aqueous and ethanolic extracts in each test tube. Frothing was introduced to a few drops of olive oil. A form of foam showed the presence of saponin.
Test for steroids: About two drops of hydrogen tetraoxosulphate (VI) acid were added to about 1 mL of the leaf's extracts. A form of red colour was observed revealing the presence of steroid in the leaves extract.
Test for tannins: Bromine water (7 mL) was introduced to the ethanolic and aqueous leaves extract. The bromine water was decolorized, revealing the presence of tannins.
Collection and maintenance of test microorganisms The Bacterial isolates include Escherichia coli, Bacillus megaterium, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis, and fungal isolates used were Aspergillus niger, Candida albicans, and Penicillium chrysogenum. They were collected from the Department of Microbiology, Lagos State University, Lagos State, Nigeria. They were aseptically sub-cultured in appropriate agar slants and maintained in the refrigerator at temperature of 4 °C until further use.

Antimicrobial activities
Antibacterial activity of the leaves extract and nanoparticles were done using agar well diffusion method as adopted by Daoud et al. (2015) and Bamigboye and Ahmed (2019). About 1 mL of bacterial culture was pipetted into the centre of the prepared sterile Petri dish. Nutrient agar was then introduced into the Petri dish which contained the inoculum. Upon solidification, holes of 5 mm were drilled with the use of a sterile cork borer into the agar plates. About 0.1 mL of ethanolic and aqueous extracts were introduced into each hole. The agar plates could stay for about 30 minutes in order to allow the extracts diffuse well into the agar. Thereafter, the plates were incubated at 37 °C for 24 hours. Zone of inhibition were determined with use of a metre rule.
Procedure followed by Ahmed et al. (2013) was followed in carrying out the antifungal activity.
Poisoned plate technique was used for the antifungal activity. Mycelial plugs from the margins of each plate was removed with the use of a cork borer. The nanoparticle was affixed at the middle of the solidified sterile potato dextrose agar plate. The agar plates were incubated at 28±2 °C for about 72 hours. The growth was determined and recorded. Both the antibacterial and antifungal activities were carried out for the leaves extracts and its nanoparticles.

Statistical analysis
The results were statistically analysed by expressing as mean and standard deviation of triplicates readings using ANOVA of SPSS statistical package of Minitab software 2011 version. Values were considered significant at p<0.05

Results
The physicochemical properties of Moringa oleifera leaves extract and its nanoparticle is presented in Table 1. The melting points of the leaves extract and its nanoparticle are 116 and 78 °C respectively. The phytochemical screening of the aqueous and ethanolic leaves extracts of M. oleifera revealed the presence of alkaloid, saponin, steroid and tannin in the ethanolic extracts while they were absent in the aqueous extract ( Table 2). The antibacterial and antifungal activities of the leaves extracts indicated that the ethanolic extract exhibited a better activity than the aqueous extract as presented in Tables Tables 5 and 6. According to the data obtained, the ethanolic leaves extract of M. oleifera nanoparticles exhibited highest antibacterial activity against B. megaterium with a zone of 49.00±0.32 mm at 30 mg/mL ( Table 5). The highest antifungal activity of the ethanolic leaves extract was shown against Aspergillus niger with a zone of inhibition of 50.00±0.29mm while the highest activity for the aqueous leaves nanoparticles was found to be 29.00±0.39 b mm against Penicillium chrysogenum as presented in Table 6.     Values are means ± standard deviation of three replicates. Values in the same column with different superscript are significantly different at P< 0.05.

Discussion
The physicochemical properties of the leaves extract and its nanoparticles are presented in Table 1. According to the data, it was observed that the nanoparticles possessed low pH of 3.46 and 3.05 in aqueous and ethanolic extract respectively which is synthesized in slightly acidic medium. The optimum pH for the synthesis of nanoparticles is 6 which is strongly dependent on the feature of the central metal ion. It was also observed that the pH formation of nanoparticles is less than 3 due to the presence of flavonoid in an undissociated form according to Maitera et al. (2018). The nanoparticle is non-electrolytic in nature which revealed conductivity at 10.18 due to its large surface area. The melting point of the nanoparticle is very low due to the environmental condition Mabhiza et al. (2016).
The phytochemical screening of the ethanolic leaves extract of Moringa oleifera showed the presence of alkaloids, saponins, steroids and tannins. The presence of the phytochemicals in the ethanolic extract is majorly responsible for its antimicrobial activity which might be due to the chemical constituents (secondary metabolites) that is being produced during the process of normal plants metabolism according to Oda et al. (2000). Based on previous work, it has been observed that ethanol can be used for extraction in most herbal medicinal plants in order to yield a pure and active compound Pal et al. (2007). It has been documented that 80% of leaf extracts are used for treatment by fighting against diseases worldwide Blanco et al. (2001). According to Mabhiza et al. (2016) antibacterial studies of alkaloid extracts from Callistemon citrinus and Vernonia adoensis against S. aureus and P. aeruginosa possessed the antibacterial activity as well as the inhibition of ATP-dependent transports of compounds through the microbial cell membranes. According to Ahmed et al. (2009), it has been observed that alcohol can be used for extraction in some medicinal plant in order to yield a good pure and active compound. Ethanol acts as the control which enhance the activities of the extracts. Based on the result obtained, it was observed that ethanolic extract was found to be more potent than the aqueous extract against the selected tested organisms. This might be as a result of a good solubility of the active composition of the leaves in ethanol than that of water Ezeifeka et al. (2004). This is in good agreement with the findings of Airaodion et al. (2019) who discussed the antibacterial activity of ethanolic extract and aqueous extracts of medicinal plant of Vernonia amygdalina leaves against some selected Gram (+) and Gram (-) bacteria. Based on the data obtained, the zone of inhibition of the bacterial growth in ethanolic extract exhibited highest antibacterial activity. This is also in good agreement with the previous studies Aruljothi et al. (2014). From previous research, ethanol has been observed to be more useful in the extraction of leaves which is a good solvent for extraction and not toxic for human consumption Banskota et al. (2001).
Ethanol is known to have higher polarity and has the ability to dissolve different types of compounds from plant materials inserted in them. Karaman et al. (2003) and Wei et al. (2008) also reported that ethanol and methanol are the most useful solvents for extraction of antibacterial compounds. According to Jayaraman et al.
(2008) solvents such as ethanol and methanol can be used for extraction of antibacterial and antifungal compounds from M. citrifolia. There is proof from previous work that ethanol is more suitable than other solvents most especially water in the process of extracting the constituents of medicinal plants Ahmed et al. (2009) Cowan (1999 and Emad et al. (2009). According to the result obtained, antibacterial and antifungal activities indicated that the ethanolic (extracts) nanoparticle are more active than aqueous extract against the microorganisms. Based on the data obtained in the antifungal activity of the leaves extract nanoparticles, there was remarkable activity against the organisms which might be as a result of the coordination of the manganese oxides in the compound. The activity of plant nanoparticles is as a result of their high surface-to-volume ratio which allows them to be more active according to Prabhu and Poulose (2012). The compounds have the ability to cross the membranes of microbial cells by gaining access to target cells. The increase in antimicrobial studies might be as a result of the presence of phytochemicals in the leaves and their tendency to penetrate through the cell wall and cytoplasmic membranes of the organisms, thereby allowing the growth of inhibition zone in the organisms which agrees with Barnabas and Nagarajan (1988).

Conclusions
M. oleifera leaves extract has been synthesized with Manganese oxide nanoparticle possessing antibacterial and antifungal activities. It has been observed that the ethanolic extracts for both the leaves and its nanoparticle were found to exhibit higher activities than the aqueous extracts.

Authors' Contributions
Both authors read and approved the final manuscript.