INTRODUCTION:

Silver nanoparticles are validated to be most potent as they hold excellent anti-microbial, anti-inflammatory, anti-cancer and anti-oxidant activities. In a biological system Reactive oxygen species generation during metabolism is a phenomenon that initiates oxidative stress¹. This leads to many diseases including Rheumatoid arthritis, cancer etc. Free radicals are easily scavenged by antioxidants preventing and repairing damages caused by reactive oxygen species thereby improving the immune defense. Rheumatoid arthritis is an autoimmune disease that results in a chronic disease in inflammatory disorder that causes pain swelling, stiffness and loss of function in joints². It occurs more frequently in women than in men and its prevalence depends upon age.

Butea monosperma and Cardiospermum halicacabum is a indigenous plants are widely used for the treatment of Rheumatism. Cardiospermum halicacabum family spindaceae also known as the ballon plant which are widely distributed in Asia and Africa. The whole plants has been used for the several countries in the treatment of Rheumatism, stiffness of limbs, snakebite its roots of nervous disease, swelling³. Butea monosperma and Cardiospermum halicacabum are used medicinally in different countries and are a source of many potent and powerful drugs.

Literature shows the structural elucidation and antioxidant activity of Butea monosperma and Cardiospermum halicacabum individually. None of the studies reported the synergistic effect of Butea monosperma and Cardiospermum halicacabum and its potential using silver nanoparticle.

The main objective of our study is to synthesis silver nanoparticle (NP) from aqueous extract combination of Cardiospermum halicacabum and Butea monosperma (CHBMC) and to analyze their applications. In our previous publication, we reported the synthesis of CHBMC silver nanoparticle and evaluation of its phytochemical constituents and antimicrobial activity⁴. In this investigation characterization of CHBMCSNP using UV-visible spectra, Fourier transform infrared spectra, Scanning electron microscopy, X-ray diffraction were carried out and evaluated for in-vitro antioxidant properties towards biomedical applications.

MATERIALS AND METHODS:

Extraction:

About each 15g of Butea monosperma and Cardiospermum halicacabum sample were weighted separately and transferred into 500ml beaker containing 300ml of water and boiled for 20 minutes. The extracts were then filtered thrice through whatmann No. filter paper to remove particulate matter and to get clear solution, stored in dark place and used for the further analysis.

Synthesis of silver nanoparticle:

Aqueous solution of 1mM silver nitrate was prepared and used for the synthesis of silver nanoparticles aqueous extract and silver nitrate added in 1:2 ratio kept at dark place for 72 hours. The leaf extract was added for reduction into silver ions. In the mean time, colour change of the mixture from green color to dark black observed.

Characterization of Silver Nanoparticles:

Ultra Violet Spectroscopy (UV):

The reduction of pure silver ion was monitored by the UV-Visible spectroscopy of the reaction medium after diluting a small aliquot of the sample into distilled water. UV visible spectrometer analysis was done by using Shimadzu 1800 spectrophotometer in 300 - 600nm range. The absorbance values were measured at required time period.

Fourier Transform Infrared Spectroscopy (FTIR):

FTIR measurement is undertaken in order to confirm the components and functional groups of the materials. The functional groups are responsible for the silver nanoparticle were analyzed using Shimadzu FTIR. The spectra were recorded at wave number in the range of 400 and 4000 cm^-1.

X-Ray Diffraction analysis (XRD):

The particle size and nature of silver nanoparticle were determined using XRD. This was carried out using Philips pw 1710 X-ray diffractometer model with 30 kv, 30mA with cuKa radians at 20 angle. The analyzed material is finely ground and average bulk composition is determined. The size of the particle on the silver nanoparticle was determined using Debye Scherrer's equation d=kλ/(β cosθ).

Scanning electron microscopy (SEM):

The film of the sample were prepared on the carbon coated grid by use dropping a very small amount of the sample and then the film on the grid were allowed to dry by putting it under an mercury vapor lamp for 5minutes for detecting the size and shape of silver nano particle using scanning electron microscopy. The electronic images were made on Hitachi - SEM analysis.

Antioxidant activity:

Nitric oxide radical scavenging activity, Super oxide free radical scavenging activity, Ferric Reducing Anti-oxidant Power Assay (FRAP), 2, 2- Diphenyl-1-picryhydrazyl Radical (DPPH) Inhibition assay were evaluated in this nanoparticles.

Statistical analysis:

The results are represented as mean ± standard deviation, n=5 and statistical significance between Nanoparticle and standard was analyzed using of One way ANOVA and the result were given as a mean ± standard Deviation (SD). A p value of < 0.05% was statistically significant.

RESULTS AND DISCUSSION:

The formation of silver nanoparticles was carried by aqueous extract of Cardiospermum halicacabum and Butea monosperma in combination (CHBMC). The extract was incubated for reduction of silver ions to silver nanoparticles as there was rapid reaction. The appearance of a brown color indicates the formation of colloidal silver nanoparticles. Intensity of color increased result from high nanoparticles formation.

Ultra Violet Spectroscopy (UV):

The UV-Vis spectroscopy could be used to examine size and shape-controlled nanoparticles in aqueous extract suspensions. In order to verify the synthesis of silver nano particles, the test samples were subjected to UV-Vis spectrophotometric analysis (Fig.1). This analysis showed the sharp absorbance at round 400-450nm. This was specific for silver nano particles. The UV-visible absorption band in the current visible light region was 430nm. The reduction was ascribed to the phenolics, terpenoids, and flavonoids compounds present in the extract⁴. Our result is closely related to Anandalakshmi et al., 2016⁵.

Fig.1: UV –Visible spectra of Cardiospermum halicacabum and Butea monosperma in combination of silver nanoparticle

Fourier Transform - Infrared (FT-IR) Spectroscopic analysis:

To investigate the functional groups of CHBMCSNP, a FT-IR study was carried out spectra is shown in fig.2. This silver nanoparticles shows a number of absorption at 3363.86 cm-1 corresponds to stretching of -N-H bond amino group and indicative of bonded hydroxyl (OH) group which coincides with Awwad and Salem⁶. The absorption peak 2924.09cm-1 could be assigned to -CH stretching vibration of -CH and -CH2 functional groups. Nearly similar peak (2913 cm^-1 and 2916.13 cm^-1) was absorbed in Awwad and Salem, 2012 ⁶ and Shyam sundar et al.⁷. The peak 1651 and 1527.6 cm that corresponds to the bending vibrations of amide I and Amide II of proteins respectively while their corresponding stretching of -NH bond of amino groups and indicative of bonded hydroxyl group were seen at 3363.86 and 2924.09 which coincides with the results of Awwad and Salem⁶. The two bands observed at 1381.03 and 1033 cm^-1 can be assigned to the C-N stretching vibrations of aromatic and aliphatic amine which is coincides with Vanmathi Selvi⁸. The peak near 833 cm⁻¹ assigned to C=CH2 and the peaks near 677 cm⁻¹ and 651.96 cm⁻¹ assigned to CH out of plane bending vibrations are substituted ethylene systems –CH=CH (cis) which is also coincides with Priya et al.⁹. The two bands observed at 1381.03 and 1033 cm^-1 can be assigned to the C-N stretching vibrations of aromatic and aliphatic amine which is coincides with Malarvizhi et al.¹⁰. These observations indicate the presence of and binding of proteins with silver nanoparticles which can lead to their possible stabilization. These bands denote stretching vibrational bands responsible for compounds like flavonoids and terpenoids and may be responsible for efficient capping and stabilizing of obtained silver nanoparticles. FTIR study indicates that the carboxyl (-C=O), hydroxyl (-OH) and amine (N-H) groups of CCHBM leaves extract are mainly involved in reduction of Ag+ to Ag nanoparticles.

Scanning Electron Microscopy (SEM):

SEM analysis shows high density silver nanoparticles synthesized by CHBMCSNP fig.3. It was shown that relatively spherical or cubidal and uniform silver nanoparticles were formed with diameter of 11 to 13.81nm which is coincides with Priya et al., 2011⁹.

X-Ray Diffraction analysis (XRD):

To confirm the crystalline nature of silver nanoparticles further studies were done by X-ray diffraction (fig.4). This figure illustrates peaks at 36.250, 41.27, 61.53, 73.47 and 83.61 corresponding to the lattice planes ((111), (200), (220), (311)) of face-centered cubic (FCC) structure of CHBMCSNP. The average gained n size of the silver nanoparticles formed in the bioreduction process is determined using scherrs formula d = (0.9 * 180) / Cos and is estimated to be 5.2nm. Similar pattern of XRD for silver nanoparticles has been reported⁸.

Figure 4: XRD diffractogram of Cardiospermum halicacabum and Butea monosperma in combination of silver nanoparticle

The XRD pattern at were in accordance with the reported pattern which confirmed that the green synthesized silver nanoparticles were nanocrystalline with focus crystal structure respectively. Tirupathy et al., 2010¹¹ observed various additional peaks in the diffractogram of silver nanoparticles prepared from Azadirachta indica leaf extract. These peaks were attributed to the presence of some bioorganic compounds/protein (s) existing in the neem leaf broth.

Antioxidant Activity:

O₂ is an element indispensable for life oxidation processes are very important to living organisms. Most of the potentially harmful effects of O₂ are due to the formation of reactive oxygen species (ROS). The uncontrolled production of ROS and unbalanced mechanism of antioxidant protection result in the onset of many diseases and accelerate ageing ROS include superoxide anion radical hydroxyl radical, H₂O, singlet O₂. There is a balance between the generation of ROS and inactivation of ROS by antioxidant system in organisms¹². Antioxidant are compounds that inhibit or delay the oxidant of other molecules by inhibiting the initiation (or) propagation of oxidizing chain reaction (or) suppressing formation of free radicals by binding metal ions, reducing H₂O₂ and quenching superoxide and singlet O₂ free radicals present in the human organisms causes oxidative damage to different molecules neurodegenerative diseases, inflammation, viral infections, autoimmune pathologies and digestive system disorders such as gastro intestinal inflammation, gastric ulcer¹³. Many phytochemicals possess antioxidant activity and reduce the risk of many diseases. As reported in our earlier work, phytoconstituents in CHBMCSNP contributes directly to antioxidative action. Superoxide is a biologically important as it can form singlet oxygen and hydroxyl radical. In vitro superoxide radical scavenging is measured by riboflavin /light /NBT (Nitro Blue Tetrazolium) reduction. This method is based on generation of superoxide anion radical by auto oxidation of riboflavin in the presence of light. Over production of superoxide anion radical contributes to redox imbalance and associated with harmful physiological consequences. Superoxide anion are generated by the oxidation of NADH and assayed by the reduction of NBT resulting in the formation of blue Formosan that can be measured at 560nm¹⁴. From our results, it is confirmed CHBMCSNP is a potential free radical scavenger with effective inhibition activity (Table 1). Result of NO radical scavenging assay reveals a maximum inhibition of 63.2±4.32% was shown in Table 1. Oxygen reacts with excess nitric oxide to generate nitrite and peroxy nitrite anion, which act as radicals. Nitric oxide is an important chemical mediator generated by endothelial cells, macrophages, neurons and involved in the regulation of various physiological process including neurotransmission, vascular homeostasis, antimicrobial and antitumor activities. Excess concentration of nitric oxide is implicated in the cytotoxic effects observed in various disorders like AIDS, cancer, alzheimer’s and Rheumatoid arthritis. Similar finding was observed in silver nanoparticles of Aegle marmelos leaf extract¹⁵. The high scavenging activity may also help to arrest the excess generation of NO that results in adverse effects on human health. Reductive ability was investigated from the ability of the extract to perform Fe3+ to Fe2+ transformation. The reducing capacity of compound may serve as significant indicator of its potential antioxidant activity. Increased absorbance of reaction mixture indicated increased reduction power which is observed in our results. The reducing power was consistently lower for CHBMCSNP in comparison with their DPPH and NO scavenging activities. DPPH assay shows 50 % inhibition in this sample. Similar results were seen in aqueous Alangium salvifolium seed extract¹⁶. As shown in table 1 reducing powers of CHBMCSNP reached peak at 61.8±1.92 that might be attributed either to capping agents such as flavonoids described in FTIR (Fig.2) or due to the contribution of silver nanoparticles on account of their large surface to volume ratios. A similar finding reported in the characterization of antioxidant functions of flavonoids and proanthocyaanins in marratian black teas, which stated that when samples react with FRAP solution a dark colour will appear which corresponds to the Ferrous tripyridyl complex. Ferrous tripyridyl triazine complexes are the product from the0020reaction in which the samples had the ability to reduce Fe3⁺ reduced to Fe2⁺¹⁷.

Table 1: Determination of antioxidant activity in CHBMCSNP

Assay	CHBMCSNP (% of inhibition)	Ascorbic acid (% of inhibition)
Superoxide radical scavenging activity	50.8±10.06	66.8±2.4
Nitric oxide radical scavenging assay	63.2±4.32	69.2±1.92
FRAP reducing activity	61.8±1.92	67 ±1.58
DPPH assay	50.0±10.1	66.0±2.3

CHBMCSNP is Cardiospermum halicacabum and Butea monosperma in combination of silver nanoparticle, Data values are represented by mean ± SD, n=5, P < 0.05 significant, SD is standard deviation for n=5 observations

CONCLUSIONS:

On the basis of the present results and available reports, it can be finally concluded that silver nanoparticles of CHBMC can be used as an effective treatment for ROS involved diseases such as rheumatoid arthritis. Their antioxidant activity may be responsible for their usefulness in the management and treatment of various diseases. Further research involves the isolation of individual components and formulation of a potent antimicrobial and antioxidant drug from CHBMCSNP.

ACKNOWLEDGEMENTS:

We acknowledge the Kalasalingm University, Krishnankoil, Srivilliputhur and SFR College, Sivakasi, Tamil Nadu, India for SEM, XRD, FTIR studies. The authors are thankful to the V.V. Vanniaperumal College for Women management, Virudhunagar, Tamilnadu, India for utilizing the lab facilities.

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Received on 09.12.2019 Modified on 25.02.2020

Research J. Pharm. and Tech. 2020; 13(12):6218-6223.

DOI: 10.5958/0974-360X.2020.01084.7