1. INTRODUCTION:

Nanoscience makes use of materials and systems applicable on a nanometer scale. Nanoscale world is far more different that the microscopic world that we are accustomed to. With the decrease in length scale of material, importance of surface area increases and it leads to development of quantum effects which contribute to changes in properties of material¹. Nanoparticles are solid in nature and can modify the physico-chemical properties compared to bulk material. The chemical methods of synthesis have low productivity thus new biological methods have been synthesized². Silver nanoparticles (Ag NPs) have high stability and low sintering temperature which makes it more valuable for research. Silver ion is highly toxic to bacteria but has a low toxicity towards animal cells. The bacteria fail to develop an immunity towards silver thus silver nanoparticles are synthesized as they provide large surface to volume ratio along with anti-bacterial properties³.

Copper nanoparticles (CuNPs) have a low cost preparation and have good thermal and electrical conductivity. They have a strong catalytic activity and thus they achieve a higher reaction yield and shorter reaction time. These exhibit potential antibacterial/ antifungal properties that are not observed in commercially available copper⁴.

Diabetes is a chronic disease characterized by persistent high blood sugar and effects a wide population across the world. Hyperglycemia is caused due to insulin resistance or the inability to make insulin⁵. Nanoscience is a focal point in diabetes research. Nanoparticles have proven to be of great importance in treatment and management of diabetes⁶.

Citrus limonum (lemon) is found to be rich in phytoconstituents. The essential oils of lemon exhibit strong antioxidant activities⁷. These not only provide an ample supply of vita minutes, pectins and minerals but also contain phytochemicals including phytophenolics (flavanones, flavones, phenolic acids, etc) which provide us better health due to their anti-inflammatory, anti-microbial and strong anti-oxidant activity⁸. Citrus flavonoids consist of an important series of flavonoids, Naringin and its aglycone naringenin. These display strong antioxidant and anti-inflammatory activities. Lemon pulp has a low glycemic index and is known to lower the glycemic index of other foods⁹.

We are working on an approach to find anti-diabetic activity of lemon pulp through green synthesis of silver and copper nanoparticles from lemon pulp extract since it is a highly consumed fruit.

2. MATERIALS AND METHODS:

2.1 Preparation of lemon pulp extract:

Fresh lemons were purchased from a local grocery shop in Vellore, India. The lemon pulp extract was prepared by cutting the lemons into small parts and discarding the seeds from it. Peeled off the lemon and discarded the peels. Weighed 20g of the obtained pulp and crushed it using mortal pester. Immersed the crushed pulp in 100 ml distilled water and kept in water bath at 80°C for 45 minutes. Cooled the solution at room temperature and filtered it using Whatman filter paper and used the filtrate for further processes. Stored it at 4°C for further use.

2.2 Biosynthesis of silver nanoparticles:

AgNO₃was used for synthesis of silver nanoparticles (AgNPs). The synthesis was carried out at three concentrations 1:9, 1:7, 1:5. 20ml of extract was added to 180ml, 140ml and 100ml of 0.1M of aqueous AgNO₃ ¹⁰_.The solution was stirred and heated at 80°C for 5 hours and color change was observed (Fig 1).

Fig 1 Lemon pulp extract, synthesis of AgNPs (from left to right)

The solution was centrifuged for 15 minutes at 5000rpm for 2-3 times. Discarded the supernatant and rinsed the precipitate with distilled water for 2-3 times. Immersed the precipitate in small amount of distilled water and poured it in a dish. Covered the dish using aluminum foil, made holes in foil for air to pass and placed the dishes in hot air oven at 80°C for 9-10 hours. After the content has dried, scraped off the dish using a needle and collected the powdered content in a vial.

2.3 Biosynthesis of copper nanoparticles:

CuSO₄.5H₂0 was used for synthesis of copper nanoparticles. 100ml of three concentrations, 1mM, 10mM and 100mM were prepared in Erlenmeyer flask. 30ml of lemon pulp extract was added to all 3 solutions and was kept at 100°C for 15 minutes. After cooling, the pH of solution was adjusted to 11 using 1N NaOH and was further stirred at 100°C for 30 minutes and color change was observed (Fig 2)¹¹.

Fig 2. Lemon pulp extract in 100mM copper sulphate solution.

Centrifuged the solution for 10 minutes at 5000rpm for 2 times. Discarded the supernatant and rinsed the precipitate with distilled water for 2-3 times. Immersed the precipitate in small amount of distilled water and poured it in a dish. Covered the dish using aluminum foil, made holes in foil for air to pass and placed the dishes in hot air oven at 80°C for 9-10hours. After the content has dried, scraped off the dish using a needle and collected the powdered content in a vial.

2.4 Characterization of biosynthesized silver and copper nanoparticles:

The green synthesized silver and copper nanoparticles were characterized using UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR) and Zeta potential analysis.

2.4.1 UV- Vis spectroscopy:

UV-Vis spectroscopy was used to monitor the color changes in the solution while stirring. For AGNO₃solutions, the readings were taken at every 30 minutes in the wavelength range 400-500nm while for copper, readings were taken in the wavelength range 400-700nm.

2.4.2 Fourier Transform Infrared Spectroscopy (FTIR):

FTIR spectroscopy was performed of obtained powdered content of silver and copper nanoparticles. It was used to identify possible functional groups present.

2.5 Zeta potential and Size analysis:

Stability is an essential parameter for using the synthesized nanoparticles for further perspectives. Zeta potential analysis was performed along with size analysis. For Zeta potential, synthesized nanoparticles were mixed in distilled water in 1:10 dilution ratio and was sonicated until the particles dissolved. The solution was further filtered through Whatman filter paper and analyzed through zeta potential analyzer.

2.6 Assessment of Antibacterial activity:

The antibacterial activity of the obtained powder content of copper and silver nanoparticles was tested for staphylococcus spp., and E.coli were used as model strains for agar-gel diffusion inhibition test¹².

Bacteria culture of staphylococcus spp., and E.coli was grown overnight and used. Mueller Hinton agar plates were prepared with each bacterial suspension by for both silver and copper and 0.1mg of AgNPs and CuNPs were dissolved in 1ml distilled water¹³. Three wells of approximately 6mm diameter were punched on the agar plates using well borer. The control well was in the centre with distilled water in it¹⁴. For the other two wells, one had 20µl AGNPs solution/20µl CuNPs solution and last one had antibiotic gentamycin respectively. The plates were incubated for 24hrs at 37°C.

2.7 Assessment of anti-diabetic activity:

2.7.1 Inhibition assay for alpha amylase activity (DNSA):

Three concentration of lemon pulp extract were prepared which were 25mg/ml, 50mg/ml and 100mg/ml. 500µl of plant extract was mixed with 500µl sodium phosphate buffer (pH 7 with 0.006M sodium chloride) containing α-amylase (0.5mg/ml) and was incubated at 25°C for 10 minutes. 1% of starch solution in sodium phosphate buffer was added to each tube and was again incubated at 25°C for 10minutes. 1ml of DNSA reagent was added for stopping the reaction and was incubated for 5 minutes in boiling water bath. The solution was further diluted by adding 10ml distilled water and absorbance was observed at 540nm¹⁵.

% inhibition = [(A_CONTROL - A_SAMPLE)/A_CONTROL] *100

2.7.2 Glucose diffusion inhibitory study:

This was performed using dialysis method. Glucose solution (0.22mM in 0.15M sodium chloride) and 2ml lemon pulp extract was added to dialysis membrane and was tied at both ends¹⁶. 40ml of 0.15M sodium chloride and 10ml of distilled water was mixed in a beaker. Dialysis membrane was placed in the beaker. Prepared control by adding 0.22M glucose solution in 0.15M sodium chloride and 1ml distilled water. The beakers were placed on a rotary shaker. Movement of glucose was observed every 30 minutes. Replicated the procedure 3 times for 3 hours each.

3. RESULTS:

3.1 Characterization of biosynthesized silver and copper nanoparticles:

3.1.1 UV- Vis spectroscopy:

UV-Vis spectroscopy has been used to detect the presence of silver nanoparticles during synthesis. Reduction of Ag+ to metallic Ag has been confirmed in the absorbance range 400nm to 450nm. Hence we carried our research in the range 400nm to 500nm.

Formation of AgNPs was observed at different time intervals. With increase in time interval, increase in intensity of absorbance was observed. Best results were obtained at concentration 1:7, 1:5 for AgNps at 4^th hour between 440 to 470nm (Fig. 3, 4).

Fig 3 UV-Vis analysis of AgNPs at 1:7 concentration

Fig 4 UV-Vis analysis of AgNPs at 1:5 concentration

Formation of CuNPs was carried out for 1 hour. Change in color was observed which indicated formation of CuNPs. Best results were obtained at 10mM concentration (Fig 5). While at 100mM concentration, no dignified peak was obtained (Fig 6) thus no further characteristics were performed for this concentration.

Fig 5 UV-Vis analysis of CuNPs at 10mM concentration

Fig 6 UV-Vis analysis of CuNPs at 100mM concentration

3.1.2 Fourier Transform Infrared Spectroscopy (FTIR):

The FTIR analysis of pulp extract of Citrus Limon for production/synthesis of silver nanoparticles exhibits absorbance peak in the range of 3250.05 cm^-1to 624.64 cm^-1 (Fig. 7) Peaks. The FTIR analysis shows the presence of alkaloids due to N-H stretching, polyphenols and flavonoids due to O-H stretching and terpenes due to C-H groups. The test plant consists of several functional groups which may include aldehydes, amines, alcohol, phenol, aromatics, disulfides, carboxylic acids, quinines, phosphates, cyanates, sulfates and halogen compounds. FT-IR spectrophotometer study predicted the presence of the groups such as O-H, N-H, C-H, C=C, nitrates and silicates stretching. Presence of the characteristic functional groups of alcohols, phenols, amines, terpenoids, tannins, carboxylic acids, hydroxyl group and glycosides are responsible for the antidiabetic and antibacterial property¹⁷.

Fig 7 FTIR analysis of Silver nanoparticles (1:7 conc)

Fig 8 FTIR analysis of Copper nanoparticles (10 mM)

The FTIR analysis of pulp extract of Citrus Limon for synthesis of copper nanoparticles displays a absorbance peak between the range of 3226.91 to 451.34 (Fig 8). The FTIR analysis exhibits the presence of polyphenols and flavonoids due to O-H stretching, alkaloids due to N-H stretching, and terpenes due to C-H groups. The test plant contains several functional groups which may include phenol, alcohol, ester, aldehyde, secondary amines, open chain imino, open chain azo, organic nitrates, disulphide, peroxides, carboxylic acid, sulfates, phosphates, silicates, Epoxy, oxirane and halogen compounds. Presence of the characteristic functional groups of alcohols, phenols, amines, terpenoids, tannins, carboxylic acids, hydroxyl group and glycosides are responsible for the antidiabetic and antibacterial property.

3.2 Zeta potential and size analysis:

For stable nanoparticles, the Zeta potential values should be between the ranges -25mV to 25mV. The Zeta potential values of synthesized Ag Nps resulted in -18.4mV and for CuNps the obtained Zeta potential was -24.1mV (Fig 9 and Fig 10), thus the negative zeta potential value confirms the strong repellant force among the synthesized nanoparticles. The obtained negative value shows that the obtained particles were stable. On performing the size analysis, the results were 15.3nm for AgNps and 0.5nm for CuNps (Fig 11 and Fig 12). Thus it was confirmed that the particles synthesized are in nanometer range.

Fig 9 Zeta potential analysis of AgNps

Fig 10 Zeta potential analysis of CuNps

The shift in zeta-potential was attributed to an increase in contribution of the signal from extraneous particulate matter¹⁸.

Fig 11 Size analysis of AgNps

Fig 12 Size analysis of CuNps.

3.3 Assessment of Antibacterial activity:

The diameter of the zone of inhibition represents the effectiveness of the sample used. Larger the diameter, greater is the sensitivity of the bacterium towards the sample. We used antibiotic gentamycin and compared the results with already obtained zone size of gentamycin in earlier researches¹⁹

It was inferred that at 1:5 concentration of AgNps, maximum inhibition zone was observed (Table 1) while for CuNps, at 10mM good inhibition zone was observed while at 100mM it was comparatively very less (Table 2). We can conclude that AgNps synthesized at 1:5 concentration are more appropriate.

Table 1: Inhibition zone of AgNPs at different concentrations.

Strain	1:7 Conc	1:5 Conc	Reference Gentamycin
*E.coli*	8.1	14.8	21.4
*Staphylococcus*	6.2	13.2	18.0

Table 2: Inhibition zone of CuNPs at different concentrations

Strain

10mM Conc

1mM Conc

Reference

Gentamycin

E.coli

9.4

13.8

22.3

Staphylococcus

7.2

8.5

19.3

3.3 Assessment of anti-diabetic activity:

3.3.1 Inhibition assay for alpha amylase activity (DNSA):

The obtained results of DNSA analysis are given in table. Lemon pulp extract showed elevated inhibition with increase in concentration. Maximum inhibition of α-amylase was 83.7% and was observed at 250mg/ml while least was 45.2% at 25mg/ml (Table 3).

Table 3% Inhibition of α-amylase enzyme brought about by aqueous extracts of varying concentrations of lemon pulp.

Concentration	Control %	% Inhibition of α-amylase
25mg/ml	0	45.2%
50mg/ml	0	49.4%
100mg/ml	0	62.1%
250mg/ml	0	83.7%

Thus it can be inferred that with increasing lemon pulp concentration, inhibition of enzyme also increases. Thus lemon pulp can be a good source to control breakdown of food into glucose.

3.3.2 Glucose diffusion inhibitory study:

On performing dialysis, inhibition of glucose was observed. Dialysis was carried out for 180 minutes. 3 sets were carried out and average %inhibition was recorded for each time period. It has been recorded that maximum inhibition is observed at 120minutes for all samples. As compared to all the samples, AgNps showed maximum inhibition followed by CuNps. Dialysis was carried out for concentrations 50mg/ml and 250mg/ml and AgNps of prepared from extract concentration 1:5 a CuNps prepared from 10mM concentration of copper sulphate. (Table 4).

Table 4 Effect of aqueous extracts of lemon pulp on diffusion of glucose out of a dialysis membrane over 180 minutes

Concentration	Control Mean ± SEM	Sample Mean± SEM	Relative movement at 210nm
30 minutes
50 mg/ml	0.006±0.0003	0.0049±0.0017	82.3%
250 mg/ml	0.006±0.0003	0.0042±0.0015	70.65%
CuNps	0.006±0.0003	0.0038±0.0014	64.75%
60 minutes
50 mg/ml	0.04± 0.0014	0.0297±0.001	74.45%
250 mg/ml	0.04± 0.0014	0.0244±0.0241	61.11%
CuNps	0.04± 0.0014	0.0225±0.0063	56.35%
90 minutes
50 mg/ml	0.112±0.0042	0.0589±0.0041	52.6%
250 mg/ml	0.112±0.0042	0.0637±0.0065	56.9%
CuNps	0.112±0.0042	0.0503±0.0041	44.98%
120 minutes
50 mg/ml	0.135±0.0054	0.0623±0.0025	46.16%
250 mg/ml	0.135±0.0054	0.0529±0.0024	39.21%
CuNps	0.135±0.0054	0.0447±0.001	33.15%
150 minutes
25 mg/ml	0.156±0.0063	0.0816±0.021	52.31%
250 mg/ml	0.156±0.0063	0.1190±0.002	76.32%
CuNps	0.156±0.0063	0.0838±0.003	53.74%
180 minutes
25 mg/ml	0.175±0.0076	0.1317±0.003	75.314%
250 mg/ml	0.175±0.0076	0.1519±0.004	86.8%
CuNps	0.175±0.0076	0.1246±0.025	71.2%

Relative movement of glucose was initially high. After an hour we observed a slower relative movement of glucose. While it elevated after 150 minutes. Thus maximum inhibition was observed from 120 minutes to 150 minutes.

4. CONCLUSION:

Our study has demonstrated that lemon pulp also shows anti-diabetic activity. Silver and copper nanoparticles synthesized from the pulp will be an easy source to use it as an antidiabetic agent for the treatment and for preparation of medicines. The synthesis of particles is simple and leads to production of stable nanoparticles. Any industrial product can be made for treatment of diabetes using this method. Stability of both metal particles was observed to be optimum while some changes in parameters can be done while synthesizing silver nanoparticles for enhancing the stability.

The particles synthesized from lemon pulp have good antibacterial properties and in all aspects have the means to control glucose transfer. Thus we can infer this that specifically the pulp part of lemon can be used in treatment of diabetes and medicines made from it can be used against bacterial infections.

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Received on 31.05.2020 Modified on 27.04.2022

Research J. Pharm. and Tech 2023; 16(11):5101-5106.

DOI: 10.52711/0974-360X.2023.00827