Green synthesis and Characterization of Silver Nanoparticles Synthesized Using Piper longum and its Antioxidant Activity

Background: Piper longum, a traditional plant used for regenerative medicine commonly known as the long pepper, is used to treat many pathological conditions. Piper longum is used as a source for obtaining the production of various nanoparticles and testing their efficacy. Piper longum has many beneficial activities, such as antifungal, anti-amoebic, anti-asthmatic, anti-cancer, anti-oxidant, antiinflammatory, antidepressants activities. Silver nanoparticles are widely and commonly used for its antimicrobial activity against various microorganisms. Our study aimed to synthesize Piper longum based silver nanoparticles and to investigate its anti-oxidant property. Original Research Article


INTRODUCTION
Piper longum, a traditional plant used for regenerative medicine commonly known as the long pepper, is used to treat many pathological conditions [1]. Various substitutes that are derived from Piper longum, one of the most important derivatives, is piperine. It's used to treat diarrhea, chronic bronchitis, stomach pain, asthma, hepatitis, and other respiratory insufficiencies. Advanced technology implying the use of nanoparticles in biomedical research, is gaining its agenda, incorporating almost all natural materials as sources [2].
Drug delivery is the most common use of nanoparticles in the current technological application.
Piper longum is used as a source for obtaining the production of various nanoparticles and testing their efficacy. Piper longum possesses various pharmacologically beneficial activities, such as insecticide and acaricidal, anti-fungal, anti-amoebic, anti-asthmatic, anti-cancer, antioxidant, anti-inflammatory, antidepressants, antiulcer activities on routine consumption [3][4][5]. The fruit of the Piper longum has hepatoprotective activity [6].
It is considered as an ayurvedic medicine providing enough treating ability to boost up the immune system to fight against various pathological disorders. Piperlongumine, sesamin, and sylvatin are important phytoconstituents of Piper longum [7]. The chemical nature of these constituents is the ability to produce antioxidant activity. Nanoparticles are minute particles that range from 1 to 100 nm on a nanometer scale. Different nanoparticles possess different properties; they vary in physical and chemical nature.
Nanotechnology is now widely used for synthesizing drugs in the pharmaceutical industry.
Formulation of nanoparticles, nanospheres, nano-capsules, nano-emulsion, and nanosuspensions are few applications of nanotechnology in the field of nanomedicine. The most common synthesized nanoparticles are plasmonic silver and gold because they possess unique physical and chemical properties when compared to other large surface areas and have strong electronic properties [8].
Nanoparticles are physically and chemically altered to have high potency to act upon the biological cell structure of the body [9,10]. Silver nanoparticles are widely and commonly used for the conduction of electricity [11]. They also have a localized resonance effect on the surface plasmon and have a wide range of antimicrobial activity against various microorganisms [12]. The synthesis of silver nanoparticles involves 3 complex methods, they include physical synthesis, chemical synthesis, and biological synthesis [13].
The most important method used to synthesize silver nanoparticles involves the chemical method because of the nature of the silver nanoparticles which have the ease to simply dissolve in the prepared aqueous solution [14]. Silver nanoparticles are commonly used in nanotechnology [15]. Antioxidant activity is generally stated as prevention of oxidation of various constituents of the body, thus substances that tend to possess antioxidant activity which will block the oxidation of protein, DNA, and lipids present in ourbody.
An antioxidant prevents the formation of free radicals and their storage in tissues thus helps to prevent tissue depletion [16,17]. Various naturally occurring plant sources and their products have antioxidant properties, which depresses and overcomes the oxidative stress caused by free radicals [18]. The beneficial activities of Piper longum include antioxidant properties which have the ability to treat chronic conditions.
The aim of this study is to green synthesize and characterize the silver nanoparticles using Piper longum plant extract and to observe its antioxidant capacity.

Preparation of Plant Extract
Commercially available dry powder of Piper longum was used for this experiment. This experiment was an intro vitro study which was conducted in Saveetha Dental College, Chennai, Tamil nadu. The experiment was carried out by dissolving 1g of Piper longum powder in 100ml of water. The moisture was then boiled in a heating mantle at 70 degrees Celsius for up to 10 minutes. The boiled mixture was then filtered using a Whattman number 1 filter paper to obtain the plant extract. Then 40 ml of plant extract was measured using a measuring cylinder and the mixture was added to 60ml of 1 mM silver nitrate (0.0169 g) dissolved in 60ml distilled water. The lab technician reconfirmed the synthesized Piper longum silver nanoparticles to avoid sampling error for further analysis. The further analysis was that the formation of brownish-red color observed was evident, resulting in the synthetization of silver nanoparticles.

Antioxidant Activity
Piper longum was the plant used to determine the antioxidant activity by taking only 5 samples in distinct test tubes and adding 10μL, 20μL, 30μL, 40μL, and 50μL of the solution respectively using a micropipette. The micropipette usage was dealt with care to avoid methodology error. Random sampling method was performed to eliminate sampling bias and the validation of the procedure was done by principal investigator and by experts in nanotechnology. Other plants and activities were not tested in this present study.
To check the antioxidant activity, a DPPH assay was used to test the antioxidant activity of biogenic synthesized silver nitrate nanoparticles. Diverse concentrations (2-10μg/ml) of Piper longum leaf extract interceded silver nitrate nanoparticle was mixed with 1 ml of 0.1 mMDPPH in methanol and 450µl of 50mM of tris HCl buffer(pH7.4)and incubated for30 minutes. Later, the reduction in the quantity of DPPH free radicals was assessed dependent on the absorbance at 517 nm. BHT was employed as control. The percentage of inhibition was determined from the following equation: The percentage of Inhibition was then calculated and correlated with the standard vitamin C concentration values which were not performed. And the antioxidant activity was correlated using Spearman's correlation in SPSS software version 23.0, the correlation graph was then represented and the p value was less than 0.05 for the nonparametric correlation analysis proving statistical significance.

RESULTS
The optical observation of the experiment was determined using a UV-visible spectrum providing the characterization of the prepared silver particles. The formation and synthesis of silver nanoparticles were determined by the brownish-yellow discoloration shown (Fig. 1). The corresponding formation of silver nanoparticles with peaks at 517nm in UV spectrophotometer graph (Fig.  4). The presence of the surface plasmon resonance was the reason for the color change of the solution. Silver nanoparticles were formed by the reaction between the plant extract and the silver ions.
Only potential plants were able to synthesize nanoparticles thus the change in color of the solution acted as a primary identification tool. The DPPH assay performed to test the antioxidant activity of the synthesized Piper longum silver nanoparticles was shown (Fig. 2).
The percentage of inhibition of the synthesized Piper longum silver nanoparticles was noted to be 61.2%, 64.5%, 66.1%, 67.4% and 69.3% at 10μL, 20μL, 30μL, 40μL and 50μL respectively and whereas the values of the standard vitamin C solution are 76.5%, 78.8%, 84.9%, 89.2% and 92.3% at 10μL, 20μL, 30μL, 40μL and 50μL respectively ( Table 1). The correlation analysis performed using SPSS version 23.0 and was shown in (Fig. 5). There was a positive correlation between the concentration of the sample prepared and percentage of inhibition.
Thus, the antioxidant activity was increasing with increased concentration of the synthesized silver nanoparticles (Fig. 3).

DISCUSSION
The present study investigated the optical observation which was the initial and novel technique that revealed the characterization of the synthesized silver nanoparticles as it presented a result of the UV-visible absorption spectra. The brownish-yellow discolorationindicated the formation of the silver nanoparticles. The silver nanoparticle formation showed significant color change because of the occurrence of the surface plasmon resonance [19][20]. As the color change served as a primary tool, this was useful in the detection of the potential plants for the synthesis of silver nanoparticles.
The color change in the prepared solution was correlated with its UV spectrophotometer graph, in which the peak of the synthesized Piper longum solution with silver nanoparticles production is 517 nm. Similar studies showed that the prepared nanoparticles accompanied with the respective natural plant components used in assessing its antioxidant activity [21][22].
The production of the chosen nanoparticleswere formed at the range of 400-520 nm in a UV visible spectrophotometer graph [23]. So compatible values of the UV spectrophotometer graph revealed the silver nanoparticle produced in 48 hrs.
2,2-diphenyl-1-picrylhydrazyl is an organic chemical compound which stands as the full form of DPPH, it is an assay performed to determine the antioxidant that is the free radical scavenging activity of a substance [24]. So, the DPPH assay was performed to observe the antioxidant activity of the synthesized silver nanoparticles in this present study. A bar graph comparison represented the percentage of inhibition between the silver nanoparticles synthesized and the standard solution and a line graph version of the already displayed bar graph of the results were elicited.
In comparison to various works conducted by different authors, the study conducted by S.N Kharat etal., [25] the DPPH assay scavenging ability was in a purely dose-dependent manner, the scavenging property at 50μL was 15.23% which was the lowest concentration whereas when compared to the present study the antioxidant activity was 61.2% at the lowest concentration that is at 10μL which represents a correlation analysis.
The work conducted by Rajeshkumar [19] also concluded that the free radical scavenging activity was concentration dependent as well. Keshari et al., [26] concluded in study that AgNPs had the scavenging property of more than 29% stating its high free radical scavenging activity. Comparing works of DPPH radical scavenging with the work done by Zdenka et al., [27] stated that the activity was more than 89% at the highest concentration being 50μL.
Opposing work conducted by Chaudhary SK et al., [28] withoutthe involvement of nanoparticles showed that there was potential antioxidant activity exhibited by the synthesized Piper longum extract. The Piper species were accountable and known for their reduction of oxidative stress which made them a good source of an antioxidant substance. They were successfully used to treat Alzheimer's disease and Parkinson's disease which are common disorders affecting the central nervous system [29].
Generally the silver nanoparticles were remarkable for its significant antioxidant activity [30], they also showed numerous beneficial activities such as antimicrobial [31-32] and antiinflammatory [33], so silver nanoparticles were most commonly used metal nanoparticles in nanomedicine [34]. So silver nanoparticles played a crucial role in nanomedicine involving nano technological advances and also for biomedicine using genetic engineering [35]. The use of silver nanoparticles made the present study economical, less cost effective, and easily available [36].
But not so on the bright side, recent researchers had found that silver nanoparticles consumption had caused some adverse effects on human metabolism. The toxic adverse effects included suppressing activity of the cell growth, division and multiplication [37], which was found to be in a pure concentration consumption and exposure manner.
The present study, was based on the silver nanoparticles having the property of possessing equally accessible antioxidant activity when compared to the standard scavenging property of the vitamin C solution [38]. Vitamin C was chosen for this present study from the other two free radical scavenging vitamins that are A and E because of the slightly higher values of the antioxidant activities shown by them on various biomedical facilities [39].
Vitamin C free radical scavenging property is due to its capacity to donate an electron which provided the basic properties of a vitamin which was potent enough to show strong antioxidant activity. Limitations of the present study, were less sample size taken for the study, different plants could be taken as they all showed different antioxidant capacity and other beneficial properties. Only antioxidant activity of the Piper longum plant was assessed, and the specification in using silver nanoparticles. Value changes may be formed due to errors in experiment performance.
According to the present study conducted, the free radical scavenging property of the prepared Piper longum plant extract silver nanoparticles was from an average range of 60% to 70% of the antioxidant activity. The maximum antioxidant activity was shown at the highest concentration that is at 50μL it showed nearly 70% of inhibition. Thus, the prepared Piper longum silver nanoparticles had good antioxidant activity and can be potentially used in nanomedicine to treat various biological conditions.

CONCLUSION
The Piper longum mediated silver nanoparticles showed remarkable andconsiderable antioxidant activity when compared with the standard values of vitamin C. The potential free radical scavenging property of the plant leaves extract mediated silver nanoparticles, was applicable in nanomedicine through the help of advanced technologies to support the medical voided fields. The properties of the synthesized nanoparticles were further implied in nanotechnology which may be useful for other sourcing fields. Further research studies were required to assess the full potential of the plant mediated nanoparticles.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable. Prevalence of Acb and non-Acbcomplex in