Preparation, Characterization and Screening of Silver Nanoparticles Using Phenolic Rich Fractions of Amaranthus Gangeticus L. for its in-vitro Anti-Oxidant, Anti-Diabetic and Anti-Cancer Activities.

Introduction

In recent times, outstanding growth in research and applications has been observed in Nano science and` nanotechnology. Nanotechnology is bringing many advances in medical area especially for identification and treatment of disease. Now a days nano technology is gaining more popularity in drug delivery as well in in vitro and in-vivo diagnostics, nutraceuticals and production of improved biocompatible materials. Various metal nano particles have been proven for cancer treatment and also in biosensors. Amongst various metal nanoparticles, silver and gold nano particles are of prime importance for biomedical use.¹

The production of metal nanoparticles by synthetic method using chemicals will produce the toxic compounds because of the chemical encapsulation which will produce unwanted effects on human health. However, the green method of synthesizing nanoparticles is a better approach. A simple and eco-friendly green biosynthetic method which uses biological microorganisms, plants, or plant extracts are alternatives to chemical approaches.

Nowadays the diseases like diabetes and cancer are a major health problem in the world. Currently, chemical substances such as acarbose and doxorubicin are utilised for treatment of diabetes and cancer, respectively. However, all of these treatments are related with undesirable side effects like flatulence, stomach distention, and diarrhoea in case of diabetes and hair loss, low blood count in case of cancer leading to increase in the use of medicinal plants which are safe to use and less destructive to human body.²

By considering the above, this study gives the details on the synthesis of silver nanoparticles (AgNPs) using plants for its anti-cancer, anti-diabetic and anti-oxidant activity.

Materials and Methods

Collection of plant, extract preparation and phytochemical screening

The leaves of plant of Amaranthus gangeticus were collected from Tirunelveli district, Tamil Nadu and it was identified and authenticated by V. Chelladurai, Research officer – Botany, (Retired) Central Council for Research in Ayurveda & Siddha. The powdered leaf material (100gm) was extracted with 250 mL of solvents like Pet Ether (PEAT), chloroform (CEAT), ethanol (EEAT) using soxhlet apparatus at 60⁰C for 48 hours & water (AEAT) by cold maceration method. The phytochemical screening was carried out by standard protocols.^3,4

Total phenolic content determination

The total phenolic content of the various extracts of Amaranthus gangeticus was determined using the Folin

Ciocalteau reagent as mentioned by Singleton and

Rossi.⁵

Preparation of phenolic rich fraction from ethanolic extract of Amaranthus gangeticus

The ethanolic extract (179 g) was suspended in 525 mL water and subsequently fractionated with petroleum ether (Boiling range 30-60⁰C) and ethyl acetate. The later fraction was evaporated and dried in vacuum to yield a yellowish green extract, which was named the phenolic compounds – rich fraction from Amaranthus gangeticus.⁶ And total phenolic content has been determined using the Folin Ciocalteau reagent.

Preparation and characterization of AgNPs

90 mL of aqueous solution of 0.1M AgNO₃ was treated with 10 mL of phenolic rich fractions of Amaranthus gangeticus which was vigorously stirred manually and kept at room temperature. Reduction takes place rapidly and was completed in 10 min which was confirmed by its colour change from green to brown indicating the formation of silver nanoparticle.⁷ Further the solution was centrifuged at 20000 rpm for 30 min. The settled nanoparticles were collected and purified with water and dried using oven at 60 ⁰C for two hours which was utilised for further studies.⁸

The synthesized AgNPs using phenolic rich fraction of plant extract was monitored by various analytical techniques like UV–Visible Spectroscopy  (Shimadzu UV-2700), X-Ray Diffractometer (XRD), Scanning Electron Microscopy (SEM) (GEMINI 500 SEM machine), Transmission Electron Microscopy (TEM) (FEI TECNAI G2 TEM @200KV) and Selected

Area Electron Diffraction SAED pattern and Fourier-

Transform Infrared Spectroscopy FT-IR.⁹

In-vitro anti-oxidant activity of biosynthesized Ag-Nps

Free radical scavenging activity on 2, 2-diphenyl-2picrylhydrazyl (DPPH method):

To assess the scavenging ability on DPPH, phenolic rich fraction of A. gangeticus and synthesized AgNPs (10–100 μg/ mL) in water was mixed with 1 mL of methanol solution containing DPPH radicals (0.1mM). It was shaken vigorously and kept in the dark for 30 min. The absorbance at 517 nm against a blank was taken.¹⁰ The scavenging ability was determined using the following equation (1).

%Scavenging activity =

                                          [Abs (control) - Abs (AgNPs / Extract / standard)] / Abs (control)

Hydrogen peroxide scavenging activity:

The H₂O₂ scavenging activity was assayed, in brief. 0.1 mL of different concentrations (10, 20, 40, 60, 80 and 100 μg/ mL) of phenolic rich fraction of A. gangeticus and AgNPs and ascorbic acid (control) were mixed with 0.6 mL of 50 mM H₂O₂ solution and kept at room temperature (26 ± 2 °C, 10 mins incubation). The absorbance was measured at 230 nm.¹¹ The percentage of H₂O₂ scavenging was calculated using Eq. (1)

Hydroxyl radical scavenging activity:

Exactly, 0.2 mL of different concentrations (10, 20, 40, 60, 80 and 100 μg/ mL) of phenolic rich fraction of A. gangeticus and synthesized AgNPs and ascorbic acid (control) were added with 1.0 mL of EDTA solution and added with 1.0 mL of DMSO (0.85%) in 0.1 M phosphate buffer (pH 7.4). Then the above content was maintained in a water bath at 90°C (15 min) and followed by 1.0 mL of ice-cold 17.5% trichloroacetic acid, 3.0 mL of Nash reagent (75 g of ammonium acetate, 3.0 mL of glacial acetic acid and 2.0 mL of acetyl acetone in 1.0 L of water) was added to all the test tubes and subjected to incubation (15 min) for color development which was observed at 412 nm.¹² The hydroxyl radical scavenging activity was determined by using Eq. (1)

In-vitro anti-diabetic activity of biosynthesized AgNps

Alpha-amylase inhibitory activity:

50 µL of phosphate buffer (100 mM, pH = 6.9), 20 µL alpha-amylase (2 U/mL) were prepared and added in 96-well plate and 20 µL of test solutions in different concentrations (500, 400, 300, 200 and 100 µg/mL) were pre-incubated at 37⁰C for 20 min. Thereafter, 20 µL of 1% soluble starch (phosphate buffer (100 mM), pH 6.9) was added as a substrate and incubated again at 37⁰C for 30 min. The DNS (3,5-dinitrosalicylic acid) color reagent (100 µL) was added which was boiled for 10 min. The absorbance of the resulting mixture was measured at 540 nm using a plate reader.¹³ Acarbose at various concentrations (500, 400, 300, 200 and 100 µg/ mL) was used as a standard. The percentage inhibition was calculated. 

% Inhibitory activity =

   (1-A (absorbance test substance)) / B (absorbance of control) X100

Alpha-glucosidase inhibitory assay:

In 96-well plate ,5 μL test samples (phenolic rich fraction of ethanolic extract of plant and their corresponding silver nanoparticles) at different concentration (500, 400, 300, 200 and 100 µg/mL) and 20 μL of 1.0 U/mL alpha-glucosidase solution followed by 60 μL of 67 mM potassium phosphate buffer (pH 6.8) were added. After 5 min of incubation, 10 μL of 10 mM ρ-nitrophenylα-D-glucoside solution (PNP-GLUC) was added which was again incubated for 20 mins (37°C). After incubation, 25 μL of Na₂CO₃ (100 mM) solution was added and absorbance was measured at 405 nm. Blank was taken using without test samples. Positive control was the sample extract. Each test was taken thrice and calculations were made.¹⁴

Glucose diffusion inhibition

In a dialysis tube (6 cm×15 mm), 6 mL (50 g/L) of phenolic rich fraction of ethanolic extract of the plant and their corresponding silver nanoparticles and 2 mL of 0.15 M NaCl containing 1.65 mM D-glucose were added. The dialysis tube was sealed at both sides and kept in a centrifuge tube containing 0.15 M NaCl (45 mL). After occasional shaking of the tubes, they were incubated at 37°C (3 h). Glucose concentration in the dialysis tube was observed and control tests were conducted in the absence of samples. The movement of glucose into the external solution was monitored at set time intervals by glucose oxidase kit method. Each test was repeated thrice.¹⁵

In-vitro anti-cancer activity of biosynthesized AgNps MTT assay:

Cytotoxicity of samples were examined by using MTT assay. In brief, HeLA cells obtained from NCCS, Pune, India, were plated in 96-well plates (1 x 10⁴ cells/well). Cells were exposed to 2, 5,10, 25, 50 and 100 µg/mL phenolic rich fraction of ethanolic extract of the plant and their corresponding silver nanoparticles for 48 hrs at 37°C in a 5% CO₂ atmosphere. MTT reagent was added in the wells, and plates were subjected to incubation (4 hrs). Then the mixture was taken out and added with 100 µL/well DMSO, mixed many times by pipetting up and down. The absorbance was measured at 570 nm.¹⁶

Morphological analysis:

The changes in the morphology were observed under the microscope to determine the alterations induced

by different samples in HeLa cells treated with 1 µg/ ml to 100 µg/ml of different samples. The cell images are grabbed at 20x by using the phase contrast inverted microscope.

Results

The plant of Amaranthus gangeticus is shown in figure 1. The leaf shows the presence of glycosides, alkaloids, carbohydrates, proteins, amino acids, phenolic compounds, flavonoids, steroids, tannins. The total phenolic content (Gallic acid equivalents, mg/g) in the Chloroform, Ethanolic and Aqueous extracts were 45.6 + 1.33, 105.6 + 1.10, 99.2 + 0.95 mg of Gallic Acid Equivalent (GAE) /g of extract, respectively.

Preparation of phenolic rich fraction and its total phenolic content determination

The phenolic compounds – rich fraction from Amaranthus gangeticus (47.256 g) was prepared and its Total phenolic content was calculated as 104.4 mg of Gallic Acid Equivalent (GAE) /g.

Synthesis of silver nanoparticles Phenolic rich fraction of ethanolic extract of Amaranthus gangeticus mediated silver nanoparticles were synthesized (Figure 2). The colorless silver nitrate solution changed dark brown colour after the addition of green color of phenolic rich fraction which indicated that silver ions were reduced to silver nanoparticles.¹⁷

Characterization of biosynthesized AgNPs

After visual confirmation by detecting a colour change in the biosynthesis of AgNPs, the samples were exposed to spectral analysis. In this study, Amaranthus gangeticus silver nanoparticles showed the SPR peak from 400 – 450 nm as shown in Figure 3. Broadening of peak indicated that the particles were polydispersed.¹⁸ The biosynthesized AgNPs XRD pattern is shown in figure 4. It showed three well-resolved diffraction peaks at as shown in Figure 6.  Further, FT-IR analysis were carried out for the plant extract and AgNPs as shown in Figure 7 (a&b). In plant extract, the peaks were observed at 3370.14 cm^-1, 1625.88 cm^-1, 1397.69 cm^-1, 1347 cm^-1, 1237 cm^-1, 1053.97 cm^-1 (Figure 7(a)) and at 3412.92 cm^-1, 3241.92 cm^-1, 2362.52 cm^-1,1700.76 cm^-1, 1696.48 cm¹, 1499.66 cm^-1, 1372.02 cm^-1, 1181.62 cm^-1 (Figure 7(b)) for the AgNPs. In the plant extract, the peak was broad and blends, but after encapsulation of nanoparticles, the peak was narrow and sharper. The absorption peak at 3370.14 cm^-1 was observed in control extract, which was due to OH stretching vibration, 1625.88 cm^-1 was due to C=O stretching, 1397.69 cm^-1 and 1347 cm^-1 was due to C-H stretching of aromatic ring, 1237 cm^-1 and 1053.97 cm^-1 was for CO stretching which indicates that control extract may have the phenolic substances. These structural changes indicated that the reduction and stabilization of silver nanoparticles proceed via the coordination between the phenolic substances of the plant extracts and silver ions. The FTIR studies have confirmed the fact that the phenolic group has the stronger ability to bind metal indicating that the phenolic constituents could possibly form a layer covering the metal nanoparticles (i.e., capping of silver nanoparticles) to prevent agglomeration and thereby stabilizes the medium.

In-vitro anti-oxidant activity

The anti-oxidant potential of biosynthesized AgNPs were examined by DPPH free radical scavenging, Hydrogen peroxide scavenging and Hydroxyl radical scavenging assays and graphically the results were shown in Figure 8 (a-c). IC50 values for AgNPs and phenolic rich fraction of A. gangeticus were tabulated in Table 1.

In-vitro anti-diabetic activity of biosynthesized Ag-Nps Alpha-amylase and α-glucosidase inhibitory activity:

As the results showed, the α-amylase inhibitory activities of all the samples were varied IC₅₀ values from 78.23+ 0.92 to 372.31+ 1.09 μg/mL and showed the α- glucosidase inhibitory activity with varied IC₅₀ values from 82.32 + 1.52 to 445.7 + 1.09 μg/mL. Concentrationdependent inhibition was observed.

Figure 9 (a & b) shows the α-amylase and α- glucosidase inhibitory activity of the AgNPs and plant extract.

Glucose diffusion potential of AgNPs:

The effect of phenolic rich fraction of A. gangeticus, AgNPs as anti-diabetic agents has been studied. The effects of phenolic rich fraction of A. gangeticus, AgNPs on glucose diffusion inhibition were summarized in Table 2. At the end of 27 hours, glucose movement of control (without plant extract) in the external solution had reached a plateau with a mean glucose concentration above 300 mg/dL (311.2+2.72). It was evident from the table that the AgNps were potent inhibitors of glucose diffusion.

In-vitro anti-cancer activity

 MTT assay:

The cytotoxic effect of AgNPs and phenolic rich fraction was studied by MTT assay. The percentage growth inhibition was increasing with increasing concentration of test compounds which is graphically represented in Figure 10 (a & b).

Morphological analysis

Morphological changes of HeLa cells treated with Phenolic Rich Fraction of A. gangeticus and AgNPs are shown in Figure 11 & 12.

Discussion

During the past several years, production of metallic nanoparticles using low-cost biological resources such as plants, algae, fungi and bacteria were reported.

This study gives the details on the synthesis of silver NPs using plants to exhibit the potent anti-cancer, antidiabetic and anti-oxidant activity. The anti-oxidant activity of biosynthesized AgNPs were examined by DPPH free radical scavenging, Hydrogen peroxide scavenging and Hydroxyl radical scavenging assays. The DPPH free radical scavenging activity of AgNPs at five different concentrations (10–100 μg/mL) was found in the extent of  24.65%– 84.95% (Figure 8a) whereas that of Hydrogen peroxide scavenging assay was in the extent of 21.38%–59.45% at the same concentration (Figure 8b). The Hydroxyl radical scavenging activity of AgNPs at a concentration of 10–100 μg/mL ranged from 22.42% to 88.72%. In-vitro anti-diabetic activity of biosynthesized Ag-Nps was also studied. Among the samples, the silver nanoparticles showed the highest alpha–amylase enzyme inhibition activity with an IC₅₀ value of 91.82+ 0.09 and alpha–glucosidase enzyme inhibition activity with an IC₅₀ value of 69.23+ 0.75. The cytotoxicity study of test samples of various concentrations (2, 5, 10, 25, 50 & 100 µg/mL) was also done by MTT assay method using HeLa cell lines. The IC₅₀ value of the phenolic rich fraction of ethanolic extract of A. gangeticus and their silver nanoparticles were 49.82 µg/mL and 8.93 µg/mL. The percentage growth inhibition depends on the increasing concentration of test compounds.

Conclusion

A simple one-pot green synthesis of stable AgNPs were prepared by the phenolic rich fraction of Amaranthus gangeticus L. (Leaves) at room temperature. It is an ecofriendly, rapid green approach which is low cost and a better way for the preparation of silver nanoparticles. In this study, AgNPs exhibited excellent anti-oxidant, anti-diabetic and anti-cancer potential as compared to phenolic rich fraction of A. gangeticus. Thus, A. gangeticus mediated AgNPs could serve as a viable source for natural anti-diabetic and anti-cancer drugs in the pharmaceutical industry.