Biosynthesis of Copper Nanoparticles using Mucuna Pruriens and its Antioxidant and Antidiabetic Activity

Introduction: The field of Nanotechnology has gained importance since last century. Nanoparticles can be used in medicine due to its increased interaction with microbes and has less side effects than drugs. Antioxidant compounds scavenge free radicals and inhibit the oxidative mechanisms that lead to degenerative diseases. There is a growing number of diabetes patients all over the world. Wide varieties of synthetic drugs are being used for the treatment of Type 2 diabetes mellitus, most of them possess side effects in the long run such as hepatotoxicity, abdominal pain, flatulence and diarrhea. Therefore, there is a need for a search of an alternate antidiabetic agent Aim: The aim of the study is to synthesize Copper nanoparticles from Mucuna pruriens and to evaluate its antioxidant and antidiabetic activity. Original Research Article Ali et al.; JPRI, 33(47B): 621-629, 2021; Article no.JPRI.74402 622 Materials and methods: Plant extract of Mucuna pruriens was prepared and filtered by Whatman No 1 filter paper. Copper sulphate was added to the plant extract and kept in a magnetic stirrer for nanoparticle synthesis. The synthesized nanoparticle was preliminarily analysed using UV visible spectroscopy. Finally the left over solution was taken to calculate antioxidant activity and antidiabetic activity. Results: Antioxidant activity was calculated by DPPH method and the percentage of inhibition of copper nanoparticles synthesised from Mucuna pruriens was 58.5% for 10μL, 59.6% for 20μL, 67.5% for 30μL, 71.4% for 40μL and 72.3% for 50μL. Antidiabetic activity was calculated by alphaamylase inhibitory assay and the percentage of inhibition of copper nanoparticles synthesised from Mucuna pruriens was 66% for 10μL, 69% for 20μL, 73% for 30μL, 79% for 40μL and 80% for 50μL. Conclusion: We can conclude that copper nanoparticles synthesised from Mucuna pruriens are a potent antioxidant and antidiabetic agent. Since it shows a good activity in free radical scavenging, copper nanoparticles can be used in a clinical therapeutic application and also in the management of type 2 diabetes mellitus.


INTRODUCTION
The field of Nanotechnology has gained importance since last century. Nanotechnology has given particles of various nanoscale levels. These nanoparticles are within the size of 1 to 100 nanometers in diameter [1]. Nanoparticles can be used in medicine due to its increased interaction with microbes and has less side effects than drugs [2,3]. Nanoscience and nanotechnology has become a priority field of research for today's researchers around the world [1,4,5]. Copper nanoparticles have important applications in diverse fields such as catalysis, water treatment, solar cells and a significant role in advanced electronic circuits due to its good electrical conductivity. It is also used as a disinfectant due to antibacterial properties and has wide use in pharmaceutical and health care [6,7]. Biological synthesis of copper nanoparticles are ecological, economical and easily scalable when compared to physical and chemical methods [8].
The use of Mucuna pruriens seed extract for the synthesis of copper nanoparticles has been investigated for the first time. Mucuna pruriens is a herb which belongs to the family fabaceae and is used for the management of nervous disorders, male infertility, as an antidepressant and also for treating Parkinson's disease [9]. Among the various wild legumes, the velvet bean Mucuna pruriens is found throughout the world's tropical and subtropical regions.
Mucuna pruriens is a viable source of dietary proteins due to its high protein concentration and hence it is a good source of food. It is a popular Indian medicinal plant and used in Ayurveda as a powerful aphrodisiac used for treating nervous disorders and arthritis. The main phenolic compound of Mucuna pruriens seeds is the L-Dopa, this substance is used as a first line treatment for Parkinson's disease [10]. L-Dopa obtained from Mucuna pruriens have less or no side effects when compared with the synthetic L-Dopa administered to Parkinson's patients [11].
Oxidative stress is one of the important risk factors in the pathogenesis of many chronic diseases [12][13][14]. Free radicals and other reactive oxygen species are recognized as agents responsible for human aging [15]. An antioxidant is a substance that inhibits oxidative damage to a target molecule [16]. The characteristic property of an antioxidant is its ability to trap free radicals. Antioxidant compounds scavenge free radicals and inhibit the oxidative mechanisms that lead to degenerative diseases [17,18]. Diabetes Mellitus is a metabolic syndrome, associated with severe physiological imbalances. It is characterized by chronic hyperglycemia that leads to multiple biochemical impairments and oxidative stress. There is a growing number of diabetes patients all over the world. Wide varieties of synthetic drugs are being used for the treatment of Type 2 diabetes mellitus , most of them possess side effects in the long run such as hepatotoxicity, abdominal pain, flatulence and diarrhea. Therefore, there is a need for a search of an alternate antidiabetic agent [19]. Evidence has shown that copper nanoparticles synthesised from medicinal plant Dioscorea bulbifera tuber extract showed antioxidant and antidiabetic activity [20].Our team has extensive knowledge and research experience that has translate into high quality publications [21][22][23][24][25][26][27][28][29][30][31][32], [33][34][35][36][37]. [38] [39] [40]. [41][42][43][44][45] The aim of the study is to evaluate the antioxidant and antidiabetic activity of Copper nanoparticles synthesised from Mucuna pruriens which have not yet been investigated.

Extract Preparation
In the present study, 1gm of powder of Mucuna pruriens seed was added in 100 ml of distilled water and boiled for 10-15 minutes at 70 degree celsius. After boiling, the plant extract was filtered by Whatman No 1 filter paper. 60 ml of 20 milli molar copper sulphate was prepared in 250 ml of conical flask, 40 ml of filtered plant extract was mixed to it and kept in a magnetic stirrer for nanoparticle synthesis (figure1). The synthesized nanoparticle was preliminarily analysed using a UV visible spectrophotometer (figure 2). Prior to the final step the nanoparticle solution was centrifuged at 8000 rpm to prepare nanoparticle pellet powder, it was dried in a hot air oven at 80 degree celsius. The dried powder was sent for characterisation. Finally the left over solution was taken to calculate antioxidant and antidiabetic activity. All the results were taken photographs and recorded in the excel sheets.

Antioxidant Assay-DPPH Method
DPPH assay was used to test the antioxidant activity of biogenic synthesized copper nanoparticles. Diverse concentrations (2-10 μg/ml) of Mucuna pruriens plant extract interceded copper nanoparticles were mixed with 1 ml of 0.1 mM DPPH in methanol and 450 µl of 50 mM Tris HCl buffer (pH 7.4) and incubated for 30 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.
% inhibition= Absorbance of control-Absorbance of test sample × 100 Absorbance of control

Antidiabetic Assay-Alpha-Amylase Inhibitory Assay
The in-vitro antidiabetic assay was performed using Alpha-amylase inhibitory assay. The amount of maltose liberated during the experiment was used to determine alphaamylase inhibition. The method described by Bhutkar and Bhise was used. (Bhutkar and Bhise, 2012). Different concentration of nanoparticles (10, 20, 30, 40, 50 mU/L) was preincubated with 100 mU/L of alpha-amylase solution ( 1% w/v) was further added to it and the mixture was incubated at room temperature for 10 minutes. 100 mU/L of 96 mM (3,5-Dinitrosalicylic acid solution) DNSA reagent was added to it to stop the reaction and the solution was heated in a water bath for 5 minutes. A control was maintained in which an equal amount of enzyme extract was replaced by a sodium phosphate buffer kept at a pH of 6.9. Reading was measured at 540 nm. The experiment was performed in triplicate. Acarbose was used as a positive control.
% inhibition was calculated using the formulae: % inhibition = C-T × 100 C Where, C= control, T= test sample. From the results, we can conclude that copper nanoparticles inhibit the function of alphaamylase enzyme. Inhibition of this enzyme has a therapeutic effect on diabetes mellitus by controlling the level of glucose in the blood [46].Hyperglycemia due to chronic diabetes generates reactive oxygen species leading to oxidative stress which have a crucial role in lipid peroxidation and membrane damage. Preventing oxidative damage with free radical scavengers and inhibiting digestive enzymes such as αamylase and α-glucosidase are the two important therapeutic strategies for prevention of diabetes.

RESULTS
Previous research works have reported on the various activities exhibited by the nanoparticles synthesised from natural sources such as cytotoxic, antimicrobial activity [47][48][49][50][51]. The antifungal effect of copper nanoparticles isolated from white fish (Rutilus frisii kutum) eggs were tested against the fungus Saprolegnia sp.
Copper nanoparticles at a concentration of 10 ppm have been found to have antifungal effects on Saprolegnia species. Antifungal activity of copper nanoparticles was found to be positively correlated with their concentration and exposure time. This makes them a good alternative to malachite green which is also carcinogenic.
[52]. A study on copper nanoparticles synthesised using medicinal plants such as Gnidia glauca and Plumbago zeylanica were then tested for anti-diabetic activity by inhibiting porcine pancreatic -amylase and -glucosidase. CuNPs were able to inhibit porcine pancreatic α-amylase by 30 to 50 %, while α-glucosidase was inhibited by 70 to 88 %. This research demonstrates that phytogenic CuNPs synthesised with G. glauca and P. zeylanica can be used to develop antidiabetic nanomedicines [53]. In our study, antidiabetic activity was checked for the copper nanoparticles synthesised from Mucuna pruriens by alpha amylase inhibitory assay and it showed good antidiabetic activity.
In future, copper nanoparticles synthesised from Mucuna pruriens can be assessed for its anticancer, antiinflammatory, antifungal and antibacterial activity and clinical trials can be carried out. The study's limitation was that it was a preliminary study and conducted in vitro, so it cannot be assumed that the results of antidiabetic and antioxidant activity could be translated into clinical effectiveness.

CONCLUSION
The copper nanoparticles biosynthesised from seed extract of Mucuna pruriens have good antioxidant and antidiabetic activity. We can conclude that copper nanoparticles are a potent antioxidant and antidiabetic agent. Since it shows a good activity in free radical scavenging, copper nanoparticles can be used in a clinical therapeutic application and also in the management of type 2 diabetes mellitus.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.