Eco Friendly Synthesis, Characterization and Optimization of Silver Nanoparticles using Polyherbal Formulation and its Invitro Analysis for
Antidiabetic Study in L6 Cell Lines
Hemalakshmi.M1*,Dr.P. Chitra2
1,2 Sri Ramakrishna college of Arts and Science for Women, affiliated to Bharathiar University Coimbatore, TamilNadu
Email Id: 1[email protected] ABSTRACT
Green synthesis is the method of synthesising nanoparticles mediated through plant sources. For the synthesis of silver nanoparticles (AgNPs), an economically viable and “green" approach has been devised, which has been studied using UV-Visible absorption spectroscopy, FTIR, XRD, and SEM, as well as in vitro anti-diabetic efficacy to analyse the hypoglycaemic plants. The plants used for the present study includes a formulation of Mangifera indica, Vinca rosea and Allium sativum. These chosen plants showed higher a profile of efficient hypoglycaemic nature in many other studies. This polyherbal formulation is prepared with ethanolic extracts and further mixed with the silver nanoparticles. The colour shift and prominent peak in UV-spec analysis indicate that silver nanoparticles have been synthesised effectively. The FTIR research reveals the phytochemicals responsible for the reduction of silver and iron nanoparticles. The presence of silver and iron nanoparticles was crystalline, according to XRD measurements while the surface morphology and size were determined via SEM examination. The alpha-amylase inhibition assay and the percentage of inhibition with standard ascorbic acid were used to determine anti-diabetic efficacy using L6 cell lines. As a result, the study of green silver nanoparticle synthesis and its applications can be expanded to include a wide range of plant species to treat different ailments eliminating harmful side effects.
Keywords
Polyherbal formulation, Anti-diabetic assay, Molecular characterisation, Silver nanoparticles, Green synthesis, Anti-hyperglycaemic
INTRODUCTION
Diabetes mellitus is a chronic metabolic syndrome caused by total insulin shortage or reduced insulin activity. It primarily affects emerging nations such as India and China, with almost 500 million diabetics projected by 20251. Owing to the unwanted side effects faced in synthetic anti- diabetic drugs, alternative herbal based hypoglycaemic drugs can be used in the diabetes treatment. Studies also reveals that plants rich in polyphenols are potent antioxidant and hyperglycaemic agents that can act as better radical scavengers and prevent the diabetes linked complications. Usage of polyherbal formulation is encouraged in traditional medicinal system due to the synergistic activity and less side effects. Metal nanoparticles such as silver, gold, platinum, and copper have recently received a lot of attention since their properties differ dramatically from those of their bulk metal counterparts. Silver's antifungal, antioxidant, and anti-inflammatory properties make it a great choice for a variety of medical applications 2. Herbal formulation implies a dosage form composed of one or more herbs or herbs processed in defined amounts to provide particular dietary, cosmetic advantages intended for use in the diagnosis, mitigation or alteration of human or animal composition or physiology 3. A polyherbal formulation was synthesized, and Nano silver particles were manufactured in this work. The utilisation of more traditionally used medicinal plants to mediate the green synthesis of silver nanoparticles is examined. Parameters affecting the production of plant-mediated silver nanoparticles, as well as their characterization approaches and biological activities, are outlined and reviewed.
MATERIALS AND METHOD Preparation of polyherbal extract:
Preparation of extract is performed by 4g of coarsely ground air-dried material was weighed accurately and transferred into 250ml of glass tampered iodine flask. 100ml of specific solvent (water, ethanol and water respectively) was added in the flask. It was shaken and allowed to stand for 1 hour. Reflux condenser were attached to flask and boiled gently up to 24 complete cycles, then cooled to room temp and filtered. Filtrate was evaporated on a water bath and contents were dried for 6 hours in a hot air oven. The filtrate was then cooled in a desiccator for 30 minutes and weighed again without delay.
Synthesis of silver nanoparticles from polyherbal formulation
A 1mM aqueous solution of silver nitrate was made in an Erlenmeyer flask, and the pH was adjusted to 8. At room temperature, 1:4(v/v) extract was added to the silver nitrate solution. The first color shift was seen at 10-minute intervals up to 1 hour, followed by preliminary confirmation of synthesized AgNPs using a UV-vis spectrophotometer at 4-hour intervals starting at 4-hour and lasting up to 24 hours.
Optimization of pH and Temperature
Leaf extract (10%) was mixed with 1mM silver nitrate solution in 1:4, 1:3, 1:2, and 1:1(v/v) ratios to determine how varied mixing ratios of leaf extract and silver nitrate solution affect green synthesis of AgNPs. The leaf extract (10%) was mixed with a 1 mM silver nitrate solution with a pH range of 5 to 9 in a 1:4(v/v) mixing ratio to examine the influence of pH on AgNP production.
At ambient temperature and at higher temperatures ranging from 50°C to 80°C, the aqueous extract (10%) and 1mM silver nitrate solution were allowed to interact at a mixing ratio of 1:4 (v/v) to analyze the effects of temperature on the synthesis of AgNPs.
Characterization of green synthesized silver nanoparticles FTIR Spectroscopy
The functional groups capping in silver nanoparticles were identified using FTIR spectroscopic analysis of the produced nanoparticles. After centrifuging the colloidal solution for 10 minutes at 8000 rpm, the pellet was washed three times with Milli Q water. Before FTIR analysis, the resultant suspension was fully dried in a freeze drier (Perkin Elmer Spectrum-one, USA). The materials were dried before being combined with KBr powder and pelletized for FTIR; with wavelengths ranging from 4000 cm1 to 400 cm1 acquired using Perkin Elmer.
X-ray diffraction
X-ray diffraction analysis was used to determine the crystalline AgNPs. An apparatus running at 30 Kv, 40 mA, with Cu K radians at two angles was used to deposit crystalline dry powder silver nanoparticles onto glass slides. Cu K radiation with a wavelength of 1.5406 A° and a nickel monochromator were used to capture the pattern. Scanning was done in a two-degree range, from 10 to 90 degrees. Scherer's equation was used to calculate the size of the nanoparticles. D = 0.94 λ / (β cos θ) Where D is the average crystal size, λ is the X-ray wave length (λ=1.5406 A°), θ is Bragg's angle (2θ), β the full width at half maximum (FWHM) in radians.
Scanning electron microscopy
Dry nanoparticle samples were obtained by dropping a drop onto a carbon-coated grid and allowing it to dry before measuring using a Hitachi S-3400 N.
Invitro antidiabetic assay
Alpha-amylase is an enzyme that aids in the breakdown of big, insoluble starch molecules into smaller, more absorbable units. Thus, the anti-diabetic effect of Mangifera indica, Vinca rosea and Allium sativum leaf extracts was determined using the Amylase Inhibition test and the Glucose uptake by yeast cell technique.
α- Amylase Inhibition assay
The anti-diabetic effect of the plant extract was determined using an in vitro -amylase inhibition assay. The amylase enzyme solution for the experiment was prepared by dissolving amylase at a concentration of 0.5 mg/ml in 20 mM phosphate buffer (pH 6.9). The extracts were mixed with 1.0ml of enzyme solution and incubated at 25°C for 10 minutes at varied concentrations (0.1, 0.2, 0.3, 0.4, and 0.5g/ml). After incubation, the mixture was given 1.0 ml of 0.5 percent starch solution and incubated for another 10 minutes at 25 ° C. After that, the reaction was stopped by adding 2.0 mL of di nitro salicylic acid (DNS, colour reagent) and heated the solution for 5 minutes in a boiling water bath. After cooling, the absorbance at 565 nm was measured colorimetrically. Metformin was utilised as the assay's standard. The measurements were obtained in triplicates. Using the formula provided, the inhibition percentage was computed.
%inhibition = (Absorbance of Control – Absorbance Sample/Absorbance control) X 100 Glucose Uptake by Yeast Cells Method:
The glucose absorption by yeast cells methodology was used to test the extracts' anti-diabetic activity in vivo. After treating the commercial baker's yeast with distilled water, it was centrifuged again (3,000g, 5min) until clear supernatant fluids were recovered. Plant extracts in various concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 g/ml) were added to 1.0ml of glucose solution (5,10, and 25 mM) and incubated at 37°C for 10 minutes. The reaction was begun by adding 100 l of yeast suspension, followed by an incubation period of 60 minutes at 37°C. After 60 minutes, the tubes were centrifuged (2500g, 5 min) and the amount of glucose in the supernatant was calculated. The standard was Metronidazole. The readings were taken as Triplicates.
The percentage increase in glucose uptake by yeast cells were calculated by the formula:
%inhibition = (Absorbance of Control – Absorbance Sample/Absorbance control) X 100
RESULTS AND DISCUSSION Optimization of mixing ratio
For the efficient green production of silver nanoparticles, the effects of varied mixing ratios have been improved (AgNPs) 4. The optical density readings were observed to increase as the mixing ratio was increased, indicating that AgNP production was progressing (Figure 1). Among the various mixing ratios, 1:4 (polyherbal extract and silver nitrate solution) was shown to be the most effective for green AgNP production.
Figure 1: Optimization of mixing ratio for green synthesized Optimization of pH
When the pH range was changed from acidic to basic, both the pace of reaction and the optical density value increased (Figure 2). pH 8 was found to be the best for synthesis of silver nanoparticles when compared to acidic pH, where optical density values fell (AgNPs) 5.
Figure 2: Optimization of pH for green synthesized AgNPs Optimization of temperature
Given the energy required to maintain the higher temperature ranges, room temperature was found to be optimum/best for effective AgNPs production 6. The optimum temperature was found to be 80ºc in the present study.
Figure 3: Optimization of temperature for green synthesized AgNPs
X-Ray diffraction analysis
XRD pattern examination validated the crystalline nature of AgNPs, as shown in Fig.4. The XRD spectrum showed four distinct diffraction peaks at 38.28o, 44.33o, 64.33o, and 77.53o, which corresponded to lattice plane values indexed at (111), (200), (220), and (311) planes of face centred cubic (fcc) silver with a lattice parameter that was in good agreement with the fcc structure reference. The Debye Scherrer's equation was used to calculate the mean size of AgNPs based on the breadth of the Bragg's reflection 7. As a result, the size of AgNPs generated at normal temperature is expected to range between 10 and 80 nanometres. The XRD patterns obtained are consistent with earlier research. The unassigned peaks (*) were detected because the bio-organic phase crystallised on the surface of the AgNPs 8. According to the broad bottom of the peak, AgNPs were smaller. The crystallite size was calculated using the Debye Scherrer formula from the reflection's line broadening spectrum: D = 0.94 k / cos D = 0.94 k / cos D = 0.94 k / cos D = 0.94 k / cos D = 0.94 k / cos D = 0.94 The X-ray wavelength is k, the full width at half maximum is b, and the diffraction angle is y. The average crystallite size is D, the X-ray wavelength is k, the full width at half maximum is b, and the diffraction angle is y.
Figure 4: X-Ray diffraction analysis of green synthesizezd AgNPs Scanning Electron Microscopy analysis
The SEM image offered more information on the shape and size of the created AgNPs. AgNPs were deposited on a carbon-coated copper grid. AgNPs were discovered to be widely diffused due to their spherical shape 9. In this study, little AgNPs were discovered attaching to the surface of very large macromolecules.
Figure 5: SEM photograph of green synthesized AgNPs
Fourier Transform Infrared Spectroscopy analysis
The probable biomolecules in leaf extract that are responsible for capping, which leads to efficient AgNP stabilisation, were identified using FTIR measurements. Plant metabolites have two peaks that correspond to C=C stretching vibrations from aromatic rings at 1595cm-1 and 1496cm-1, as well as stretching vibrations of OH bonds at 3186cm-1 (alcohols and phenols) (alcohols and phenols). C-O stretching caused by functional groups of proteins and metabolites covering AgNPs causes peaking at 1065cm-1. The absorption band of 1601-1595cm-1 change after bio-reduction, showing the creation of green AgNPs capped with bio-molecules. It shows that the extract's water-soluble components played a complex function in the bio-reduction of precursors and the formation of AgNP 10.
Figure 6: FTIR spectrum of green synthesized AgNPs Glucose uptake assay
The rate of glucose absorption increased in nanoparticles treated cells in a dose-dependent manner. The results were statistically significant when compared to the control group. In the presence of glucose, PHF was found to increase basal glucose absorption (**P>0.001). 2- deoxyglucose-6-phosphate is the standard here (2DG6P). The rate is increased mainly due to the action of GLUT4 transporter and the anti-hyperglycaemic activity 11. The translocation of GLUT4 transporter within cells were able to show the cell’s ability to increase the uptake of glucose (Figure 7).
Table 1: Standard 2DG6P Std. 2DG6P conc. µM RLU
0.00 260.00
0.39 449.00
0.78 695.00
1.56 1154.00
3.13 2474.00
6.25 6323.00
12.50 12152.00
25.00 22812.00
Figure 7: Estimation of rate of glucose uptake in Insulin treated cells
The reason for employing L6 myotubes in glucose transport investigations is widely understood.
The effects of free and nano DDA on 2-deoxy-D-[1-3H] glucose absorption was studied to see if they had anti-diabetic properties12. Both free DDA and nano-DDA increased glucose absorption in a dose-dependent manner. Both free DDA and nano DDA increased glucose absorption in a dose-dependent manner, confirming its utility (p 70 percent glucose uptake compared to control).
DDA's anti-diabetic effectiveness was improved after it was nano encapsulated (Figure 8).
Following with gliclazide-loaded alginate-methyl cellulose mucoadhesive microcapsules had a similar effect13.
Figure 8: Estimation of rate of glucose uptake in cells treated with various concentrations of test samples (AB- represents polyherbal extracts and AgNPs; C - represents AgNPs)
CONCLUSION
The silver nanoparticles were synthesized using the polyherbal formulation through green synthesis that is economical and eco-friendly methodology. Capping agents play important roles in determining the final characteristics of the metal NPs. Further the characterization of synthesized nanoparticles was carried out using UV analysis, FTIR, XRD and SEM analysis. The anti-diabetic potential had higher potential with standard drug. The natural products/nanoparticles combination is a new important dimension in the area of drug discovery and the use of natural product compounds, such as procyanidins, that have well-established pharmacological profiles will improve the future usage of the metal nanoparticles in the field of biomedical applications.
CONFLICT OF INTEREST Declared none.
REFERENCES
[1] Stump CS, Henriksen EJ, Wei Y, Sowers JR. The metabolic syndrome: role of skeletal muscle metabolism. Ann Med. 2006;38(6):389–402.
[2] Babu, S.; Kumar, B.; Kumar, K. Environment friendly approach for size controllable synthesis of biocompatible silver nanoparticles using diastase. Environ. Toxicol.
Pharmacol. 2017, 49, 131–136. 5.
[3] Lee, J.; Park, E.Y.; Lee, J. Non-toxic nanoparticles from phytochemicals: Preparation and biomedical application. Bioprocess. Biosyst. Eng. 2013, 37, 983–989.
[4] Xia, D.-L.; Wang, Y.-F.; Bao, N.; He, H.; Li, X.-D.; Chen, Y.-P.; Gu, H.-Y. Influence of reducing agents on biosafety and biocompatibility of gold nanoparticles. Appl. Biochem.
Biotechnol. 2014, 174, 2458–2470.
[5] Okaiyeto, K.; Hoppe, H.; Okoh, A.I. Plant-Based Synthesis of Silver Nanoparticles Using Aqueous Leaf Extract of Salvia officinalis: Characterization and its Antiplasmodial Activity. J. Clust. Sci. 2020, 38, 15-23.
[6] Saratale, R.G.; Saratale, G.D.; Shin, H.S.; Jacob, J.M.; Pugazhendhi, A.; Bhaisare, M.;
Kumar, G. New insights on the green synthesis of metallic nanoparticles using plant and waste biomaterials: Current knowledge, their agricultural and environmental applications.
Environ. Sci. Pollut. Res. 2017, 25, 1–20.
[7] Benelli, G.; Kadaikunnan, S.; Alharbi, N.S.; Govindarajan, M. Biophysical characterization of Acacia caesia-fabricated silver nanoparticles: Effectiveness on mosquito vectors of public health relevance and impact on non-target aquatic biocontrol agents. Environ. Sci.
Pollut. Res. 2017, 25, 10228–10242.
[8] Nath, D.; Banerjee, P. Green nanotechnology—A new hope for medical biology. Environ.
Toxicol. Pharmacol. 2013, 36, 997–1014.
[9] Ovais, M.; Khalil, A.T.; Islam, N.U.; Ahmad, I.; Ayaz, M.; Saravanan, M.; Shinwari, Z.K.;
Mukherjee, S. Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles. Appl. Microbiol. Biotechnol. 2018, 102, 6799–6814.
[10] Chahardoli, A.; Karimi, N.; Fattahi, A. Biosynthesis, Characterization, Antimicrobial and Cytotoxic Effects of Silver Nanoparticles Using Nigella arvensis Seed Extract. Iran. J.
Pharm. Res. 2017, 16, 1167–1175.
[11] Arassu, R.R.T.; Nambikkairaj, B. Pelargonium graveolens plant leaf essential oil mediated green synthesis of Silver Nano particles and its antifungal activity against human pathogenic fungi. J. Pharm. Phytochem. 2018, 7, 1778–1784.
[12] Ranoszek-Soliwoda, K.; Tomaszewska, E.; Małek, K.; Celichowski, G.; Orlowski, P.;
Krzyzowska, M.; Grobelny, J. The synthesis of monodisperse silver nanoparticles with plant extracts. Colloids Surf. B Biointerfaces 2019, 177, 19–24.
[13] Nayaka, S.; Bhat, M.P.; Chakraborty, B.; Pallavi, S.S.; Airodagi, D.; Muthuraj, R.;
Halaswamy, H.M.; Dhanyakumara, S.B.; Shashiraj, K.N.; Kupaneshi, K.N.S.A.C. Seed Extract-mediated Synthesis of Silver Nanoparticles from Putranjiva roxburghii Wall., Phytochemical Characterization, Antibacterial Activity and Anticancer Activity Against MCF-7 Cell Line. Indian J. Pharm. Sci. 2020, 82, 260–269.
