Green Synthesis, Antioxidant and Antimicrobial Activity of Silver Nanoparticles Using Gnetum africanum Extracts

The synthesis and application of nanoparticles is an important area of research that is gaining attention recently. In this recent project, we report the synthesis of silver nanoparticles, AgNP using aqueous solution of silver nitrate and Gnetum africanum leaf extract (reducing agent). The synthesis of AgNP was achieved by mixing aqueous solution of silver nitrate (70ml, 15.75mM) with a solution of Gnetum africanum leaf extract 100 ml) in a reaction flask and allowed to stand for 24 hours in a dark cupboard. A color change from light brown to yellowish brown was observed which indicated that synthesis of silver nanoparticles took place. The presence of AgNP was ascertained using UVvis spectra analysis and absorption at 442 nm showed the presence of AgNP. The antioxidant assay of both the synthesized AgNP and the leaf extract was determined using DPPH. Antimicrobial activity was conducted using three different organisms which were Staphylococcus aureus, Escherichia coli and Pseudomonas respectively. The antioxidant results using DPPH scavenging ability of AgNp showed that at concentrations of 2mg/ml,1mg/ml and 0.1mg/ml, the percentage inhibition of DPPH by AgNp was 61.69, 53.06 and 38.31 respectively and that of Gnetum africanum leaf extract was 81.32, 78.49, and 58.29 respectively at the same concentrations using Original Research Article Uneze et al.; AJBGMB, 9(2): 1-7, 2021; Article no.AJBGMB.73507 2 Ascorbic acid as a standard. The antimicrobial activity of both the synthesized AgNps and Gnetum Africanum Leaf extract using one gram positive bacteria (Staphylococcus aureus) and two gram negative bacteria (Escherichia coli and Pseudomonas) revealed that the synthesized AgNps showed lesser activity than Gnetumafricanum leaf extract for both the gram positive bacteria (Staphylococcus aureus) and gram negative bacteria (Pseudomonas) and (Escherichia coli). From the above findings, it can be observed that Gnetum Africanum Leaf extract reduced Ag to Ag and also both the synthesized AgNps and the Gnetum Africanum Leaf extract showed reasonable antioxidant activity against DPPH and antimicrobial activity against the tested microorganisms. This implied that both samples have medicinal values.


INTRODUCTION
With antimicrobial resistance increasingly becoming a global concern, scientists have expanded the search for potentially new and safe antimicrobial agents to plants. There has been a steady increase in the use of plant remedies worldwide, including the search for new plant compounds that present potentials as sources of useful drugs for the treatment of diseases [1].
The Gnetum africanum plant popularly known as Afang and Gnetum africanum in Nigeria and Cameroon, is a member of Gnetaceae family, which has been used widely in treatment and curing of so many ailments and as food. It has been regarded as a medicinal plant, due to its natural content of active ingredients, with antiinflammatory and anti-carcinogenic properties and hence has, by virtue of its physico-chemical composition, tremendous health benefits [2].
The study of Nanoparticles has become fascinating because of their outstanding compositions in their size and shape, optically dependent, electrical, and magnetic properties. Also, is its importance in different areas of science such as chemical sciences, medical, environmental, physical sciences as well as engineering, pharmacological fields etc [3]. The use of AgNPs as antibacterial agents has a long history in the health industry [4]. The antipathogenic mechanisms of AgNPs although not fully understood have been explained to be by microbial damage, microbial sub-cellular structure damage, adhesion to microbial cells, ROS and free radical generation, microbial wall piercing and penetration inside cells and modulation and modification of microbial signaltransduction pathways [5].
In the Green synthesis of AgNPs, who synthesized silver nitrate with neem leaf extract, discovered the color change of the synthesized solution after the addition of the silver nitrate solution to the neem leaf extract, and observed the color change to brownish gray, which indicates the formation of AgNPs and reduction of Ag + to Ag 0 [6].
The objective of the study was to synthesize silver nanoparticles using Gnetum Africanum, leaf extracts and examine the antioxidant and antimicrobial activity of the synthesized silver nanoparticles.

Gnetum africanum Leaf (Sample) Collection
The fresh leaf sample was collected from Thinkers corner, Enugu State and was identified as Gnetum Africanum By a botanist.

Preparation of the Leaf Sample
The fresh leaves were washed thoroughly using distilled water and were air-dried for 2weeks. After drying, they were powdered using an electric blender. 90g of this powdered sample was weighed and mixed with 700ml of distilled water in a measuring cylinder. The mixture was heated using a Bunsen burner for 1hour 20 minutes in a fume hood, after which it was allowed to cool for 30 minutes. The mixture was filtered into a beaker, first using a muslin cloth, the filtrate of which was then filtered using a filter paper.

Synthesis of silver nanoparticles (AgNPs)
The 15.75mM silver nitrate (AgNO 3 ) solution was dissolved in 100ml of distilled water in the absence of light to avoid the decomposition of silver nitrate by light. 100 ml of the Gnetum africanum leaf extracts was measured into a conical flask. 70 ml of the above silver nitrate solution was mixed with 100 ml extracts solution. It was then placed in a dark cupboard for 24 hours for the synthesis of AgNPs to take place. It was then removed after 24 hours and concentrated using a water bath to obtain a semi-solid product.

Antioxidant Assay
Antioxidant activity of both the synthesized AgNPs of the leaf extract of Gnetum africanum with AgNO 3 and the unsynthesized extract were determined by 2,2 diphenyl-1-picrylhydrazyl (DPPH) assay.
Stock solution of 0.07mmol/L of DPPH was prepared with methanol. 1.0ml of various concentrations (0.1, 0.5 and 1mg/ml) of the synthesized AgNPs of both the Synthesized and non-synthesized sample extract, were added to 6 different test tubes 3 for each. 1.0ml of the prepared solution of DPPH was added to each of the test tubes containing the sample. The mixture was shaken gently and placed in a dark room for 30minutes at room temperature. After 30minutes, it was removed from the dark room and the absorbance recorded at 517 nm against methanol control. Methanol was used as control and ascorbic acid was used as standard. % for DPPH radical scavenging activity was calculated with the formula: Where % inhibition -%DPPH radical scavenging

Antimicrobial Activity Determination
The antimicrobial activity of the synthesized AgNps and unsynthesized leaf extract of Gnetum africanum was tested against three different bacteria. The organisms are Staphylococcus aureus (gram positive),Escherichia coli (gram negative)and Pseudomonas(gram negative). Preparation of both the nutrient broth and media (the nutrient agar) was carried out according to the manufacturing guide.
A syringe was used to measure out 3ml of dimethyl sulfoxide (DMSO) used to dissolve the extract. A cock borer was then used to make three holes on each of the culture plates. 0.5ml of the dissolved extract was introduced carefully so as not to mistakenly touch the media, into the holes of the respective bacteria as labeled, one of each hole on the media was used as a control. It was incubated for 24 hours at 37 C.
The antimicrobial activity was determined using the zone of inhibition (mm) of the bacteria with ciprofloxacin and chloramphenicol used as standards.

Silver Reduction
According to (Kan-Senet al, 2003) explanation of synthesizes leaf extract, a yellowish-brown color change was observed after the aqueous Gnetum africanum leaf extract was added to the silver nitrate solution, which indicates the reduction of silver ion to silver nanoparticles. It was then preserved in a dark cupboard for 24hr. After 24hr the color further changed to deep brown which indicates that synthesis has occurred completely [8].

Antimicrobial Activity
Antimicrobial activity of synthesized AgNPs and the leaf extract of Gnetumafricanum showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas. The synthesized AgNPs showed the zone of inhibition while in the leaf extract of Telfairia Occidentalis Only Escherichia coli showed a zone of inhibition against the gram negative organisms used. Ciprofloxacin and Chloramphenicol were used as standard drugs for gram positive and gram negative organisms respectively [9].

DPPH scavenging property of synthesized
AgNPs of

Gnetum africanum
The antioxidant activity that was carried out for synthesized Gnetum africanum using silver nitrate and unsynthesized Gnetum africanum Leaf extract was determined by 2, 2diphenyl-1-1picrylhydrazyl (DPPH) assay. The graph of % radical scavenging activity is plotted against the three concentrations and was evaluated with ascorbic acid as the standard antioxidants. Methanol was used as the control, which has absorbance of 0.322%. DPPH radical scavenging activity was calculated with this formula [10].

DISCUSSION
The research findings showed that africanum leaf extract reduced Ag after addition of aqueous Gnetum africanum extract and it was confirmed after a color change of yellowish-brown was observed. After 24 hours of being stored in a dark cupboard, the color changed from yellow-brown to deep brown. The brown color was as a result of the surface Plasmon resonance which is a very important property of nanoparticles.

The synthesized silver nanoparticles and
Gnetum africanum leaf extract were tested against gram positive and gram negative microorganisms , in which the synthesized AgNps and extract of Gnetum Africanum showed maximum inhibition on the gram positive(Staphylococcus aureus microorganisms and minimum inhibition on t gram negative microorganism(E. coli and Pseudomonas). The extract of Africanum showed higher activity in the gram positive organism (S. aureus) and in one the of the gram negative organism(Pseudomonas low activity in one of the gram negativ organisms( E. coli), then the synthesized AgNps of Gnetum Africanum showed higher activity in the gram positive organism( S. aureus one of the gram negative organism ( it showed lower activity in one of the gram negative organisms (Pseudomonas) that both the synthesized AgNps and the extract of Gnetum Africanumhas more inhibitory activity on the gram positive organism than on the gram negative organism. This means that the Africanum leaf extract has more activity o the gram positive and gram negative organisms. This means that the Gnetum africanum The research findings showed that Gnetum leaf extract reduced Ag + to AgNPs Gnetum africanum leaf extract and it was confirmed after a color brown was observed. After in a dark cupboard, the brown to deep brown.

The brown color was as a result of the surface Plasmon resonance which is a very important
The synthesized silver nanoparticles and xtract were tested against gram positive and gram negative microorganisms , in which the synthesized Gnetum Africanum showed maximum inhibition on the gram (Staphylococcus aureus) microorganisms and minimum inhibition on the E. coli and The extract of Gnetum showed higher activity in the gram ) and in one the of (Pseudomonas) and low activity in one of the gram negative ), then the synthesized AgNps showed higher activity in ( S. aureus) and in one of the gram negative organism (E.coli). Then it showed lower activity in one of the gram Pseudomonas). This means that both the synthesized AgNps and the extract has more inhibitory activity on the gram positive organism than on the gram negative organism. This means that theGnetum leaf extract has more activity on both the gram positive and gram negative organisms.
Gnetum africanum leaf extract can penetrate the high resistance cell wall of gram positive and gram negative organisms.
The antioxidant activity examined against DPPH was observed to have reasonable scavenging property for both the synthesized and africanum leaf extract, where the absorbance of the synthesize, Gnetum africanum acid are shown in the table in the previous chapter and the ascorbic acid was used as the standard. The antioxidant activity was observed to be high in the synthesized Gnetum africanum leaf extract at the concentration 0.1mg/ml, when compared with that of the ascorbic acid, and for the Gnetum africanumleaf extract was observed to be more active at the concentration of 0.1mg/ml which is a high concentration, when compared with the ascorbic acid. This result implies that for the synthesized AgNPs africanum leaf extract to be effective in drug formulation the concentration of the nanoparticles should not be so high and for the extract of Gnetum africanum nanoparticles to be effective it should be at the same concentration or higher.
DPPH is an antioxidant assay based on electron transfer, produces violet solution which when immersed in ethanol, the free radical which is stable at room temperature, is reduced to colorless in the presence of an antioxidant molecule. Antioxidant is a general term for any compound or molecule that can inhibit the activity of free radicals. Antioxidant is a general term for any compound or molecule that can inhibit the activity of free radicals. These free radicals lack completion of electrons thereby molecule to steal its electron from the molecule and in the process damage the molecule. Antioxidants neutralize the free radical by giving off its own electron, antioxidants acts as a natural or total switch off for the free radicals. The use DPPH assay provides a simple and well modified way to examine antioxidants by spectrophotometry. Antioxidants neutralize the free radical by giving off its own electron, therefore it means that the antioxidants acts as a natural or total switch off for the free radicals. The body or the cell generates free radicals from some environmental factors such as smoking of tobacco and cigarettes, excessive alcohol intake, air pollution, high intake of polyunsaturated fatty acids, ultraviolet rays etc. These en factors generate the R.O.S (reactive oxygen species). These R.O.S are a group of free radical ; Article no.AJBGMB.73507 extract can penetrate the high resistance cell wall of gram positive and gram negative The antioxidant activity examined against DPPH ved to have reasonable scavenging property for both the synthesized and Gnetum leaf extract, where the absorbance of Gnetum africanum and ascorbic acid are shown in the table in the previous chapter and the ascorbic acid was used as the standard. The antioxidant activity was observed Gnetum africanum leaf extract at the concentration 0.1mg/ml, when ed with that of the ascorbic acid, and for leaf extract was observed to be more active at the concentration of 0.1mg/ml which is a high concentration, when compared with the ascorbic acid. This result ed AgNPs Gnetum leaf extract to be effective in drug formulation the concentration of the nanoparticles should not be so high and for the nanoparticles to be effective it should be at the same concentration DPPH is an antioxidant assay based on electron transfer, produces violet solution which when immersed in ethanol, the free radical which is stable at room temperature, is reduced to colorless in the presence of an antioxidant a general term for any compound or molecule that can inhibit the activity of free radicals. Antioxidant is a general term for any compound or molecule that can inhibit the activity of free radicals. These free radicals lack completion of electrons thereby attacking a molecule to steal its electron from the molecule and in the process damage the molecule. Antioxidants neutralize the free radical by giving off its own electron, antioxidants acts as a natural or total switch off for the free radicals. The use of DPPH assay provides a simple and well modified way to examine antioxidants by spectrophotometry. Antioxidants neutralize the free radical by giving off its own electron, therefore it means that the antioxidants acts as a the free radicals. The body or the cell generates free radicals from some environmental factors such as smoking of tobacco and cigarettes, excessive alcohol intake, air pollution, high intake of polyunsaturated fatty acids, ultraviolet rays etc. These environmental factors generate the R.O.S (reactive oxygen species). These R.O.S are a group of free radical  ) 15mm 30mm -20

CONCLUSION
The emergence of AgNPs synthesis by biological methods has been a significant development in the field of nanoparticles. The interest for this type of synthesis has increased rapidly because these methods do not involve the use of hazardous or toxic chemicals. The use of plants, easily accessible factors, has led to the production of AgNPs through a simple, fast, cost effective and eco-friendly process. The synthesis of nanoparticles was attributed to the abundance of biomolecules in plant extract. The sample shown in the experiment was able to reduce silver ions to silver nanoparticles and exhibit great antibacterial and antioxidant properties. Silver nanoparticles synthesized from G.africanum can be incorporated into nanoparticles and applied as antibacterial agents and antioxidants [12].

CONSENT
It is not applicable.

ETHICAL APPROVAL
Animal Ethic committee approval has been taken to carry out this study.