AgCl Nanoparticles and their action

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Synthesis of Ag/AgCl Nanoparticles and their action on Human Serum albumin: A fluorescence study ⁎

Poonam Gawali , B.L. Jadhav Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz-East, Mumbai-400098, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Ag/AgCl NPs Mangroves Derris trifoliata Sonneratia alba Human Serum Albumin Fluorescence spectroscopy Antioxidant Anti-inflammatory

We report a one-step green synthesis of multifunctional silver nanoparticles AgNPs (Ag/AgCl NPs) using aqueous stem extracts of D. trifoliata and S. alba. Optimization results revealed that the maximum rate of synthesis could be achieved with 1.5 × 10−3 M and 2 × 10−3 M AgNO3 solution at room temperature in 4 h and 2 h respectively. The synthesized AgNPs were characterized by UV–vis spectroscopy, the bands were observed at 445 nm and 450 nm. High resolution transmission electron micrographs (HR-TEM) showed mostly spherical particles having diameters ranging from 20 to 60 nm. Energy dispersive x-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) confirmed elemental silver and chlorine, X-Ray diffraction (XRD) revealed the formation of face centered cubic. Interaction between human serum albumin (HSA) and AgNPs were studied at different temperatures. The Stern Volmer quenching constant, binding constant, number of binding sites thermodynamic parameters were calculated. Thermodynamic parameters (ΔG0, ΔH0 & ΔS0) hint that the binding process occurs spontaneously. Circular dichroism indicates that α helicity of HSA decreases due to the interaction with AgNPs. AgNPs showed potent antioxidant and anti-inflammatory activity. Our results reveal promising approach for alternative nano drug development.

1. Introduction Plants produce many diverse phytochemicals that act synergistically on targeted elements of the complex cellular biochemical pathway. Medicinal plants are the basis of wide varieties of biologically active compounds for many centuries and used broadly as crude material or as pure compounds for analyzing various disease conditions [1]. Throughout the world there are 77 mangrove plant species, the Indian mangroves consist of nearly 65 species belonging to 31 families [2]. Mangroves play a dynamic role in harsh infrequent disturbance (tsunamis), to chronic events (climate change) stabilizing environment of soft, low oxygen soils and varying salinity, mangroves acclimatize and adapt their leaves, stems, roots and their reproductive methods the degree of resilience of mangrove forests to large, and their role in coastal protection, and, they deliver habitat for a wide range of species, many of them arises only in the mangroves [3,4]. Derris trifoliata, a mangrove associate is a perennial climbing shrub which was previously reported to have various medicinal properties like antiplasmodial, larvicidal [5,6], antimicrobial [7], anticancer [8], antioxidant and anti-inflammatory [9]. Rotenoids, 7a-O-methylelliptonol, as an insecticide, vertebrate poison, and acaricide was isolated from the stem extracts of D. trifoliata. Flavanone, 4′,5,7-

⁎

trihydroxy-6,8-di-(2-hydroxy-3-methylbut-3-enyl)-flavanone, was isolated from the aerial parts of D. trifoliata, together with eleven known compounds; rotenone, tephrosin, 12a-hydroxyrotenone, deguelin, 6a,12a-dehydrorotenone, dehydrodeguelin, 7a-O-methyldeguelol, 7aO-methylelliptonol, 5,7,3′,4′-tetrahydroxy-6,8-diprenylisoflavone, daidzein and 4′-hydroxy-7-methoxyflavanone.2′, 4′-dihydroxy-4methoxy-3′-prenyl chalcone and leutolin [10–12]. Sonneratia alba, a true mangrove are used as traditional medicine for swellings and sprains, it has many commercial applications. The leaves, trunk and bark exhibited antioxidant properties, while the sepal shows antioxidant and anti-lipid peroxidation properties, antioxidant and antimicrobial activity [13]. Triterpenes and sterols are found in S. alba [14,15]. True mangrove presents a high level of inorganic ion accumulation for osmotic adjustment and displays high salt tolerance, optimal growth under moderate salinity. Many researchers have proved that true mangroves use inorganic salts (mainly Na and Cl) as energetically cheap osmolytes for osmotic adjustment. Mangrove associates did not show higher salt tolerance and grew best under freshwater [16]. Therefore salt concentration is more in true mangroves as compared to mangrove associate. Stems of mangroves are responsible for mechanical support for a growing plant and contain numerous secondary metabolites/phytochemicals. A bio-green method using plant

Corresponding author. E-mail addresses: [email protected] (P. Gawali), [email protected] (B.L. Jadhav).

https://doi.org/10.1016/j.procbio.2018.03.020 Received 1 November 2017; Received in revised form 4 March 2018; Accepted 22 March 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Gawali, P., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.03.020

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[24] revealed that incubation of nanoparticles with serum albumins is one of the abundant proteins bound to nanoparticle surface if in vivo experiments done then the binding between albumin and NPs will mediate NPs to navigate within body fluids and across membranes and be responsible for a plausible interpretation of many sites of accumulation and retention of NPs in different organs and tissues of the organism. Due to the redox potential of phytochemicals, antioxidant activity in plant extract is seen [25]. Non-steroidal Anti-inflammatory Drugs (NSAIDs) are among the most frequently used drugs worldwide they are reported to possess prevention of the denaturation of proteins, which act as antigens and lead to autoimmune diseases [26]. To develop a newer antioxidant and anti-inflammatory agents with sustained release and better efficacy is the use of green nanomedicine for drug development and delivery. Various nanoparticles systems are available in which silver nanoparticles are gaining more importance due to their diversified biological properties and potential applications widely used in the health industry, food storage, textile coatings, controlling pathogenic microbes and a number of environmental applications [27,28]. Silver has been used since ancient times for the treatment of wounds inflammation. Nowadays nanoparticles of silver have been developed for potent anti-inflammatory antioxidant and cancer treatments [29]. In this research, we have fabricated biosynthesized Ag/AgCl NPs from stems of mangroves (D. trifoliata and S. alba) as a reducing and stabilizing agent and thus it was very important to study their interaction with serum albumin. Besides this we investigated the comparison of its biomedical applications viz. antioxidant and anti-inflammatory activity using various assays. The data obtained from this work will form a basis for further research on more functional properties of serum proteins bound to Ag/AgCl NPs and various other applications in nanomedicine.

extracts has expanded the exceptional importance due to less time required for the synthesis of nanoparticles (NPs). While chemical synthesis of NPs has potential hazards which contain toxicities, plant-mediated synthesis of metal NPs is attaining impact in owing to its simplicity, rapid rate of synthesis of NPs of diverse morphologies and eco-friendliness [17]. Mangroves are biochemically unique with various abundant novel phytochemicals such as flavonoids, rotenoids, rich in polyphenols, phenolic glycosides and tannins have been isolated and characterized from mangroves and reported to possess different types of biological activities, these compounds and their derivatives are used in new drug discovery process which are responsible for the green nanomaterial synthesis the most exciting part of research in the field of nanotechnology for its cost-effectiveness and environmental nontoxic procedures [18]. D. trifoliata leaves and S. alba leaves synthesized AgNPs to reduced population of dengue vector and antimicrobial against several human pathogenic bacteria. [19,20]. Nanoparticles interactions with protein/biomolecules are of immense importance because proteins & nanoparticles are essential for biomedical applications used to develop diagnostic, sensors, targeted drug delivery and the growing biosafety concerns of Nanoparticles. Human Serum Albumin (HSA) is a water-soluble protein with 18 tyrosine residues and only one tryptophan, molecular mass of 66.5 kDa with 585 amino acids. Trp-214, which is situated in the physiologically important subdomain IIA which is synthesized in the liver as preproalbumin is in turn cleaved in the Golgi vesicles to produce secreted albumin which is found in human plasma. HSA was chosen because serum albumins are the most abundant proteins in plasma and have a wide range of physiological functions that binds to wide range of exogenous and endogenous compounds with the important involving transport of nutrition, drug efficacy and the delivery of fatty acids, bilirubin, steroids, etc. Adsorption of proteins and layering onto nanoparticle surface has been called the “protein corona” a competitive adsorption of proteins on the limited surface of nanoparticles containing the collective effects of incubation time period, concentration of protein and adsorption affinity between protein and nanoparticle surface is called “Vroman effect”. The study of interactions with HSA and nanoparticle, and the adsorption phenomenon with nanoparticles has significant relevance to the field of nanotechnology, pharmacology, biomaterials and nanomedicine [21]. Surface modification of biomolecules is a widely adopted technique for changing the surface of biomolecules to attain specific biocompatibility that is essential in different applications. The binding properties of HSA and AgNPs have been investigated in depth, these studies are widely used in vitro incubation of other nanoparticles with plasma proteins, and protein corona formed in vivo onto super para-magnetic nanoparticles [22,23]. Kreyling et al.

2. Experimental 2.1. Sample Collection D. trifoliata and S. alba were identified, authenticated by taxonomist and collected from pollution free zone located in Arabian Sea, Bhatye beach, Ratnagiri District, Maharashtra, India Fresh stems were separated, washed thoroughly, chopped, dried at 40° C and pulverized. 2.2. Extract preparation Stems of aqueous extracts (5% w/v) D. trifoliata (Fig. 1A) and S. alba

Fig. 1. Stems of Mangroves (A) Derris trifoliata (Mangrove associate) (B) Sonneratia alba (True mangrove). 2

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(Fig. 1B) were prepared. D. trifoliata stem aqueous extract (DS-AE) and S. alba stem aqueous extract (SS-AE) 5 g stem powder of both were soaked in 100 ml MilliQ water for 10 min. This mixture was boiled at 100 °C for 5 min and filtered through Whatman’s filter paper (42 ashless diameter 125 mm GE Healthcare Life Sciences) and filtrate is used for further study. The water used throughout the experiments was purified using Millipore Milli-Q (18.2 MΩ.cm at 25 °C) purification system.

2.4.2. Thermo gravimetric analysis (TGA) DS-AgNPs and SS-AgNPs were subjected to thermal gravimetric analysis (TGA) which were carried out under nitrogen atmosphere with a heating rate of 10 °C/min up to 800 °C on a Diamond model with Perkin Elmer USA apparatus. Since both the samples 1.5 × 10−3 M (DSAgNPs) and 2 × 10−3 M (SS-AgNPs) were prepared in aqueous solution for TGA analysis; samples were completely dried at Infra-Red lamp for 2 h on glass slides. After this both the samples were peeled for analysis and each sample of 2.245 mg (DS-AgNPs) and 6.247 mg (SS-AgNPs) were placed into experimental pan for the further measurement of weight loss.

2.3. Biosynthesis AgNPs were synthesized as per the Ghosh et al. [30] with slight modifications; silver nitrate (CAS Number: 7761-88-8) ACS reagent, ≥99.0% was purchased from Sigma-Aldrich (209139). AgNPs were synthesized between silver ions from AgNO3 and chloride ions from both the mangroves designated as D. trifoliata AgNPs (DS-AgNPs) and S. alba AgNPs (SS-AgNPs). DS-AgNPs: 142.5 ml aqueous solution of AgNO3 (1.5 × 10−3 M) was taken in 1000 ml conical flask, then 807.5 ml MilliQ water was added by 50 ml Hot (5% w/v DS-AE). SS-AgNPs: 190 ml aqueous solution of AgNO3 (2 × 10−3 M) was taken in 1000 ml conical flask, then 760 ml MilliQ water was added by 50 ml Hot (5% w/v SS-AE). Hot DS-AE and SS-AE were added drop by drop (1 ml/30 s) in the reaction flask under continuous stirring at 200 rpm, at ambient temperature to obtain clear solution and to avoid cloudy suspension and white precipitate. After synthesizing AgNPs they were subjected to initial characterization by UV–vis spectroscopy. Molar concentration of the AgNPs in the solution (assuming that the reduction was 100% complete) was calculated using Eq. (1)

C=

NTotal NVNA

2.4.3. Nanoparticle tracking analysis (NTA) NTA analyses were carried out for AgNPs using Nanosight UK-LM20 instrument. Metal particles were illuminated with laser light source of 640 nm diode with a temperature controller. Nearly perfect black background particles are enhanced and appear individually as point scatters which moves under Brownian motion. Brownian motion of the particles were tracked and video captured the settings for processing parameters were optimized in order to maximize the number of particles identified using the monochrome Marlin CCD camera (Allied Vision Technologies, Germany). Both DS-AgNPs and SS-AgNPs were sonicated for 10 min before analysis. 2.4.4. High resolution transmission electron microscopy (HR-TEM) Surface morphology and size of Ag/AgCl NPs were analyzed using high resolution transmission electron microscopy. The purified AgNPs were sonicated using Sonicator (Vibronics VS 80) for 10 min. A drop of both DS-AgNPs and SS-AgNPs were placed on carbon coated copper grids and dried under an Infra-Red lamp for 20–30 min and observation were performed on High Resolution Transmission Electron Microscopy (FEI Tecnai G2, F30 machine operated at accelerating voltage 300 kV).

(1)

Where N Total is the total number of Ag atoms added to the reaction solution. N is the number of Ag atoms present in each nanoparticle, V is the volume of the reaction solution in liters and NA is the Avogadro’s constant. N is calculated by considering spherical shape of the AgNPs (size of AgNPs were measured by TEM). Average number of silver atoms in each nanoparticle is 31 D3 where D is the average diameter of the particle [31]. By using the above equation the concentration of AgNPs was calculated to be in range of (1.9 × 10−8 M, 3.8 × 10−8 M, 5.7 × 10−8 M, 7.7 × 10−8 M, 9.6 × 10−8 M) and SS-AgNPs −8 −8 (2.5 × 10 M, 5 × 10 M, 7.5 × 10−8 M, 10 × 10−8 M, 12.5 × 10−8 M).

2.4.5. Energy-dispersive spectra (EDS) Energy-dispersive spectra for both DS-AgNPs and SS-AgNPs were taken using an energy dispersive spectrometer equipped with (EDS make FEI series Quanta 200 with EDS 3.0.13) at an energy range of 0–20 KeV. 2.4.6. X-ray photoelectron spectroscopy (XPS) The chemical bond states and elemental analysis of Ag/AgCl NPs were studied by an X-Ray Photoelectron spectroscopy (ULVAC-PHI Model PHI5000 Versa Probell) equipped with AlKα X-Ray of an energy of 1486.61 eV, pass energy 58.7 eV; step energy of 0.025 eV; pressure 5.8 × 10−8 Pa at 4.3 × 10−9 torr. Samples for XPS analysis were prepared by drying on slides and the observation were determined.

2.4. Characterization The AgNPs suspensions were centrifuged at 15,000 × g for 10 min. The supernatant was removed and Milli-Q water was added for further cleansing. The mixture was centrifuged at 15,000 × g for 10 min to confiscate any traces of unbound phytoconstituents. Reduction of the Ag+ ions was observed by measuring the ultraviolet-visible spectrum of the solution at regular intervals on Cary Varian 50 Bio UV–vis spectrometer (λ = 200–800 nm).

2.4.7. X-ray diffraction (XRD) Diffraction data for completely dried nanoparticle films on glass slides were recorded; crystalline nature of both AgNPs was recorded by X-ray diffraction (XRD Model recorded on Bruker D8 Discover with Cu Kα radiation (1.54 °A) step size 0.02 source operating at 40 kV and 40 mA). 2.5. AgNPs interaction studies with HSA

2.4.1. Fourier transform infrared spectroscopy (FTIR) Synthesized AgNPs were centrifuged thrice for better separation at 12,000 rpm for 10 min at room temperature, pellet obtained was redispersed in MilliQ water, The purified pellet was then dried and 2–3 drops of this colloidal solution of AgNPs were thoroughly mixed with potassium bromide (KBr) and exposed to an infrared source of 500–4000 cm−1 subjected to FTIR (3000 Hyperion Microscope with Vertex 80 FTIR System Bruker, Germany) using the pellet technique in diffuse reflection mode at a resolution of 4 cm−1. A similar process was carried out with aqueous stem extracts DS-AE and SS-AE to have a comparative study with their respective AgNPs.

2.5.1. Fluorescence quenching measurement Fluorescence spectra were recorded on Varian, Cary Eclipse fluorescence spectrometer using excitation wavelength which was set at 295 nm to excite the tryptophan residue of HSA and emission was measured in the scan range of 280–600 nm, with fixed excitation and emission slit width of 5 nm each, voltage 550 mV. For interaction of AgNPs-HSA complex, HSA (purchased from sigma Aldrich) stock of 2 mg/ml solution dissolved in 1X PBS 0.01 M (phosphate buffered saline) pH 7.4 with DS-AgNPs (1.5 × 10−3 M) and SS-AgNPs (2 × 10−3 M). Three sets of fluorescence measurements were recorded 3

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Fig. 2. Visual observation of color change from (A) DS-AE yellow to (B) DS-AgNPs reddish brown and (C) SS-AE dark yellow to (D) SSAgNPs reddish brown after bioreduction by stem extract of mangroves at room temperature. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.5.3. Dynamic light scattering (DLS) and zeta (ζ) potential Average size, conductivity and zeta potential were performed to analyze DS-AgNPs, SS-AgNPs and AgNPs-HSA complex using Zetasizer (Nanoseries Nano-ZS90, Malvern Instruments). The AgNPs were filtered with the syringe having pore size 0.22 μm to remove the dust that might interfere with the results. All measurements were performed in triplicate and carried out at 25 °C. 2.5.4. Circular dichroism (CD) spectroscopy The CD spectra were recorded using JASCO, J-815-150S spectrometer. The quartz cuvette had pathlength of 1 cm. With bandwidth of 10 nm the measurements were taken in the UV region of wavelength range 190–260 nm. 2.6. In vitro antioxidant assay Samples of standard, test extracts and AgNPs were evaluated for Free radical, hydrogen peroxide percent scavenging and reducing power assay of DS-AE, SS-AE and DS-AgNPs and SS-AgNPs of both the mangrove plants were carried out in triplicate. Ascorbic acid (AA) was used as positive control.

Fig. 3. UV–vis absorption spectra of (A) DS-AE aqueous extract of D. trifoliata stem (B) Plasmon resonance of silver nanoparticles DS-AgNPs and (C) SS-AE aqueous extract of S. alba stem (D) Plasmon resonance of silver nanoparticles SS-AgNPs.

2.6.1. DPPH free radical scavenging assay 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging potential were analyzed [33]. Samples of different concentrations (20, 40, 60, 80, and 100 μg/ml) were taken and 1 ml of freshly prepared DPPH (200 μM dissolved in ethanol) was added and vortexes thoroughly. The control contained all reagents except samples. Finally, for 30 min the solutions were incubated in dark. The absorbance of stable DPPH was measured at 517 nm. % Inhibition was calculated using following Eq. (2).

at different temperature in kelvin 298, 306, 313 and 318 for different concentrations of DS-AgNPs (1.9 × 10−8 M, 3.8 × 10−8 M, −8 −8 −8 5.7 × 10 M, 7.7 × 10 M, 9.6 × 10 M) and SS-AgNPs (2.5 × 10−8 M, 5 × 10−8 M, 7.5 × 10−8 M, 10 × 10−8 M, 12.5 × 10−8 M). The interaction between AgNPs and HSA were analyzed after 30 min of incubation period and readings recorded with mirror coated quartz cuvette [32].

2.5.2. UV–visible absorption studies UV–visible absorption studies of pure HSA (2 mg/ml), AgNPs-HSA complex against different concentration were recorded at pH 7.4 adjusted by PBS Buffer with UV–visible spectrometer Shimadzu, UV-1800. The quartz cuvette with 1 cm path length was used. The solutions for the measurements were prepared in Milli-Q ultra-pure water.

% inhibition =

Acontrol − Asample Acontrol

× 100

(2)

2.6.2. Hydrogen peroxide (H2O2) assay H2O2 were estimated according to the method reported by Ruch 4

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Fig. 4. Fourier transform infrared (FTIR) absorption spectral analysis (A) DS-AE before bioreduction (B) DS-AgNPs after complete bioreduction and (C) SS-AE before bioreduction (D) SS-AgNPs after complete bioreduction.

Fig. 6. Nanoparticle tracking analysis graph Particle Size/Concentration (A) DS-AgNPs (B) SS-AgNPs.

H2O2. The control solution having all the reagents except sample. The percentage of inhibition for H2O2 scavenging activity was calculated using Eq. (2).

2.6.3. Reducing power assay The reducing power was determined by Oyaizu’s method [35] with slight modification Samples of different concentrations (20, 40, 60, 80 and 100 μg/ml) were mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and then cooled. Afterwards, 2.5 ml 10% Trichloroacetic acid (TCA) was added to the above-mentioned solution and centrifuged at 3000 rpm for 8 min. The supernatant was collected and mixed with equal amount of Millipore Milli-Q water. Finally, 1 ml 0.1% ferric chloride was added to the upper layer and the absorbance was measured spectrophotometrically at 700 nm.

2.7. In-vitro anti-inflammatory assay Fig. 5. TGA of (A) DS-AgNPs and (B) SS-AgNPs.

Human red blood cells (HRBC) membrane stabilization, proteinase and protein denaturation inhibition assay were carried out in triplicate. For all the in vitro anti-inflammatory bioassay samples contained the stock solution (10,000 μg/ml). Diclofenac sodium (DS) injection IP (I.M/intragluteal/I.V) 25 mg/ml as a positive control, DS-AE, SS-AE, DS-AgNPs and SS-AgNPs were prepared in ethanol. From this (20, 40, 60, 80, 100 μg/ml) concentration were made and used for all the bioassay.

et al. [34] with minor modification as follows. Hydrogen peroxide solution (30% w/v Thomas Baker SD Fine Chem, Mumbai) was prepared in 1 M phosphate buffer (pH 7.4). Samples of different concentrations (20, 40, 60, 80, and 100 μg/ml) were added to 60 μl hydrogen peroxide solution. An absorbance of H2O2 at 230 nm was determined after 10 mins against a blank solution containing phosphate buffer without 5

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Table 1 Binding parameters (Stern-Volmer quenching constants, binding constant and binding sites with thermodynamic parameters of AgNPs interacted with HSA). Sr. No.

Temperature in (K)

AgNPs –HSA complex

No of Binding sites (n)

Binding constant K (L mol−1)

Stern Volmer constant Ksv (x108 M)

Biomolecule Quenching Constant Kq (M−1 S−1)

1.

298

2.

308

3.

313

4.

318

DS-AgNPs SS-AgNPs DS-AgNPs SS-AgNPs DS-AgNPs SS-AgNPs DS-AgNPs SS-AgNPs

1.60 1.35 1.67 1.40 1.69 1.46 1.63 1.49

1.72 1.71 1.64 1.61 1.52 1.52 1.36 1.42

0.4 0.6 0.5 0.7 0.6 1.0 0.8 1.1

0.8 1.2 1.0 1.4 1.2 2.0 1.6 2.2

± ± ± ± ± ± ± ±

0.03 0.02 0.09 0.07 0.08 0.10 0.05 0.08

± ± ± ± ± ± ± ±

0.45 0.37 0.47 0.39 0.47 0.40 0.45 0.41

± ± ± ± ± ± ± ±

0.17 0.20 0.21 0.24 0.25 0.31 0.35 0.34

± ± ± ± ± ± ± ±

0.17 × 1016 0.20 × 1016 0.21 × 1016 0.24 × 1016 0.25 × 1016 0.31 × 1016 0.35 × 1016 0.34 × 1016

Free Energy ΔG° (kJ/mol) −34.69 −162.96 −35.89 −168.46 −36.48 −171.21 −37.08 −173.96

Table 2 DLS at RT Size (nm) and Zeta potential (mV) of AgNPs interacted with HSA (DS-AgNPs and SS-AgNPs Stock Solutions: Bold). Sr.No.

AgNPs

Concentration (Molar)

DLS Size (nm)

Poly dispersive index (PDI)

Zeta potential (mV)

Conductivity (mS/cm)

DS-AgNPs (Stock Solution) 1. DS-AgNPs and HSA complex 2. 3. 4. 5.

1.5 × 10−3 M 1.9 × 10−8 M 3.8 × 10−8 M 5.7 × 10−8 M 7.7 × 10−8 M 9.6 × 10−8 M

73.06 ± 41.08 308.5 ± 21.53 297.3 ± 26.32 247.3 ± 35.43 198.0 ± 53.20 172.1 ± 83.35

0.230 0.554 0.512 0.428 0.392 0.396

−13.6 −9.81 1.34 −10.2 −10.9 −10.0

0.242 15.1 15.4 14.5 15.8 13.8

SS-AgNPs (Stock Solution) 1. SS-AgNPs and HSA complex 2. 3. 4. 5.

2 × 10−3 M 2.5 × 10−8 M 5 × 10−8 M 7.5 × 10−8 M 10 × 10−8 M 12.5 × 10−8 M

40.58 ± 35.20 163.14 ± 28.60 124.8 ± 33.47 114.2 ± 40.85 111.2 ± 30.44 103.7 ± 40.83

0.540 0.347 0.328 0.320 0.284 0.216

−15.7 −10.9 −12.3 −11.8 −11.0 −9.70

0.331 15.9 14.4 14.9 14.6 14.5

3. Results and Discussions

2.7.1. HRBC membrane stabilization Human RBC membrane stabilization bioassay was standardized as per [36]. 4.5 ml reaction mixture consisted with 2 ml hyposaline (0.25% NaCl), 1 ml 0.15 M phosphate buffer (pH 7.4) and 1 ml samples (20, 40, 60, 80, 100 μg/ml of final volume) in normal saline, 0.5 ml 10% human RBC in normal saline was added. For control tests, 1 ml isosaline was used instead of test solution while product control tests lacked red blood cells. For 30 min the mixtures were incubated at 56 °C. The tubes were cooled under tap water for 15 min, centrifuged and the absorbance of the supernatants was read at 560 nm. % Inhibition was calculated as per Eq. (2).

3.1. Characterization of AgNPs 3.1.1. UV–visible spectroscopy Color changes of the DS-AE to DS-AgNPs and SS-AE to SS-AgNPs from light yellow to reddish brown to dark brown were observed after 4 h and 2 h respectively (Fig. 2) when silver salt (AgNO3) was added to aqueous stem extracts of mangroves. Color changes of the solutions are due to secondary metabolites such as alkaloids, flavonoids, saponins present in plant extract which acts as a reducing agent that reduced silver ions (Ag+) to a silver atom (Ag0). The UV–visible profile of mangrove stem aqueous extracts and its synthesized silver nanoparticles was studied at the wavelength of 200–800 nm (Fig. 3). For DSAE, one major peak was recorded at 270 nm with absorbance values of 2.88, the spectra for phenolic compounds (tannins and flavonoids) typically lie in the range of 230–290 nm (Fig. 3A). SS-AE no such peak in that range (Fig. 3B). DS-AgNPs showed maximum absorption at 445 nm while, SS-AgNPs displayed maximum absorption at 450 nm (Fig. 3C and D). It is well known that the size of AgNPs influences their brown color, because of the excitation of surface plasmon resonance (SPR) phenomenon [39]. Similarly, SPR value confirms synthesis of AgNPs in various other mangrove species, Avicennia marina [40], Rhizophora lamarckii [41], R.mucronata [42,43], R. apiculata [44]. These reports are in accordance with our present reports. The role of temperature on the reaction rate where we found that at room temperature (25 °C) was the best optimized experimental condition, for stable DS-AgNPs and SSAgNPs.

2.7.2. Proteinase inhibitory bioassays Proteinase inhibitory bioassay is followed as per Kritika et al. [37] with modifications. Initially, a concentration of enzyme trypsin 0.06 mg/ml, 2% casein and 5% trichloroacetic acid at pH 7.6 was optimized. 3 ml reaction mixture contained 100 μl trypsin, 350 μl 25 mM Tris-HCl buffer (pH 7.4) and 50 μl samples were incubated at 37 °C for 5 min. After this 500 μl 2% w/v casein was added and incubated at 37 °C for 20 min. 2 ml 5% trichloroacetic acid (TCA) was added to terminate the reaction. Cloudy suspension was centrifuged at 5000 rpm for 10 min. An absorbance of the supernatant was observed at 280 nm. A control where buffer compensated the volume is measured. Percentage inhibition at the different concentration of the samples was calculated as per Eq. (2).

2.7.3. Protein denaturation inhibition bioassays In-vitro protein denaturation inhibition bioassay was optimized using DS according to Sakat et al. [38]. In 3 ml reaction mixture 50 μl samples and 450 μl, 5% w/v BSA was added. Tubes were incubated at 37 °C for 20 min and then heated at 57 °C for 3 min. Then cooled, 2.5 ml phosphate buffer saline (pH 6.3) was added to each tube and absorbance was read at 660 nm. Percentage protein denaturation inhibition at the different concentration of samples was calculated as per Eq. (2).

3.1.2. FTIR spectroscopy FTIR analysis before and after reduction of plant extract and NPs was carried out to identify the possible interaction between AgNO3 and bioactive molecules present in extract which might have been involve in the synthesis and stabilization of NPs. The results suggest that the biological molecules could possibly perform a function for the formation and stabilization of AgNPs in an aqueous medium. Before 6

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Fig. 7. HR-TEM micrograph showing size at 25 °C of DS-AgNPs (A) Scale bar corresponds to 100 nm (B) 50 nm scale with spherical diameter (C) 20 nm scale (D) 5 nm scale (E) scale bar corresponds to 2 nm (F) shows the SAED pattern of DS-AgNPs.

2923.66 cm−1, 2427.25 cm−1 were responsible for Methylene CeH asym/sym stretch. We could spot peaks for SS-AE 1730.88 cm−1 and bioreduced SS-AgNPs at 1741.07 cm−1 as aldehyde and ester group. The peaks signifies DS-AE at 1624.14 cm−1 which is bioreduced to 1628.03 cm−1, SS-AE at 1621.24 cm−1 and bioreduced to 1627.49 cm−1 corresponds to alkenyl C]C stretch, NeH Primary amine and NeH bend. Further, DS-AE showed phenol/tertiary alcohol OH bend at 1404.81 cm−1 and SS-AE showed carbonate ion at 1446.77 cm−1. A prominent sharp peak of 1384.31 cm−1 in DS-AgNPs and 1384.18 cm−1 was seen in SS-AgNPs that were assigned to Methyl-

bioreduction D. trifoliata and S. alba stem extract showed hydroxyl group in alcoholic and phenolic compound (Fig. 4A & C) which is supported by the presence of a strong peak at approximately 3383.51 and 3408.22 cm−1. The sharp peak represented as OeH bond which was not seen in the after bioreduction 3423.04 and 3424.19 cm−1 respectively (Fig. 4B & D); specifies that the alcohol containing multiple hydroxyl groups are mainly responsible for reduction of Ag+ into silver nanoparticles this may lead to the disappearance of the OeH bond [30]. The peak observed of extracts DS-AE at 2931.12 cm−1 and SS-AE at 2935.38 cm−1 and DS-AgNPs was 2924.66 cm−1 and SS-AgNPs at

7

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Fig. 8. HR-TEM micrograph showing size at 25 °C of SS-AgNPs (A) Scale bar corresponds to 100 nm (B) 50 nm scale with spherical diameter (C) 20 nm scale (D) 5 nm scale (E) scale bar corresponds to 2 nm (F) shows the SAED pattern of SS-AgNPs.

CH3 group (Fig. 4B and D). Additional 1368.96 cm−1and 1327.46 cm−1 peaks were ascribed to SS-AE. An aromatic phosphate (PeOeC Stretch) was seen only in SS-AE (1228.14 cm−1) and SS-AgNPs (1207.37 and 1227.55 cm−1). A peak of DS-AE was 1076.38 cm−1 which was bioreduced to 1027.25 cm−1 and of SS-AE was 1076.57 cm−1 bioreduced to 1024.61 cm−1 are attributed to CN stretch. CeH Aromatic and phosphate stretch of peaks 923.99, 871.31 cm−1 shown in DS-AE and 882.71 cm−1 exhibited in SS-AE. The peaks specified above are well discussed with functional groups [45]. FTIR results showed the binding efficiency of several functional groups like alcohols, carboxylic acids, amides, amines, esters and ethers. These groups are proved to have certain reducing agents such as proteins with amide groups and with major phytochemical classes (flavonoids, triterpenoids and

polyphenols) with metal to form AgNPs.

3.1.3. TGA analysis TGA is a technique that measures the weight changes as a function of temperature; it provides information of thermal behavior of decomposition of samples. TGA analysis of both DS-AgNPs and SS-AgNPs are shown in Fig. 5A & B which indicated the relationship between weight loss and temperature in the TGA curve. The initial weight loss from 50 °C to 120 °C is ascribed to vaporization or loss of absorbed H2O. The amount of the end product of weight loss for DS-AgNPs that were around 22.108% and 24.648% for SS-AgNPs. In view of these TGA results, samples were in accordance with XRD results [46]. The phytochemicals are responsible for the reduction thermally low stable 8

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SS-AgNPs was found to be d = 0.27 nm corresponding to the (200) lattice plane of the face centered cubic (FCC) structure of AgCl [51,52,53]. HR-TEM analysis clearly proves the co-presence of Ag/ AgCl, in Figs. 7F & 8F represents the SAED pattern of Ag/AgCl NPs which supports the unit cell. Compact and closed interface interaction of Ag/AgCl NPs could be seen from HR-TEM images. Stems of true mangroves S. alba has high salinity (NaCl salts in abundance) occurrence of salt glands and an ability to maintain water uptake in saline conditions as compared to mangrove associate D. trifoliata. Mangroves exert tight control over salt concentrations in their stem tissues by decoupling water uptake from ion uptake. Moreover, nature of capping agents present in the stem extracts gave shapes of AgNPs [54,55] 3.1.6. EDS spectroscopy Occurrence of metallic silver and chlorine was identified by energy dispersive x-ray spectroscopy. The results were confirmed with the presence of significant amounts of silver and chlorine. Optical absorption peak of both the DS-AgNPs (Fig. 9A) and SS-AgNPs (Fig. 9B) were detected at about 3 KeV, which is characteristic of silver atoms and peak of 2.6 KeV attributed to chlorine. In DS-AgNPs, % weight of AgL (AgLα, AgLβ, and AgLβ2) was 82.99% and ClK (ClKα, ClKβ1) was 17.01%. Whereas in SS-AgNPs, % weight of AgL (AgLα, AgLβ and AgLβ2) was 78.79% and ClK (ClKα, ClKβ1) was 21.21%. As per the earlier reports representative spot EDS profile confirms the presence of silver and chlorine [56]. 3.1.7. XPS spectroscopy Chemical environment and Surface oxidation state of Ag/AgCl nanoparticles were determined from XPS measurements. Figs. 10C & 11 C showed wide scan spectrum for both DS-AgNPs and SS-AgNPs which specifies presence of Ag, Cl, C, and O. DS-AgNPs centered at 366.18, 368.10, 369.56, 372.18 eV, 371.25 and 373.00 eV for Ag at 3d core levels which could be ascribed to Ag3d5/2 and Ag3d3/2 (Fig. 10A) and Cl 2p centered at 196.29 and 197.87 eV represents Cl present in Ag and AgCl NPs. Fig. 11(A) showed XPS data of SS-AgNPs, where Ag3d5/2 centered at 367.2 and 373.2 eV which represents Ag/AgCl. The Cl species displays binding energies of Cl 2p3 and Cl 2p1 at about 197.61 and 199.3 eV, respectively as shown in Fig 11B. These are in agreement with the presence of FCC structure obtained by XRD and EDS results [57,58].

Fig. 9. EDX analysis of (A) DS-AgNPs and (B) SS-AgNPs.

compound that gets extracted in water [47,48] 3.1.4. NTA analysis Particle size and concentration is measured and analyzed by NTA, the trajectories of particles undergoing Brownian motion are tracked by illuminating the particles from a 90° angle by an intense laser beam it is possible to visualize particles considerably smaller than the diffraction limit. Where the mean size, size variation which were marked in the histogram and concentration of DS-AgNPs was observed as 20 nm and 2.96 × 108 particles/ml (Fig. 6A) and SS-AgNPs indicated 25 nm and 2.19 × 108 particles/ml (Fig. 6B) respectively [49]. The main central advantage that NTA has over the other measurement technique is that it is not biased towards larger particles or aggregates. The software is based on the tracking of single particles, whereas DLS systems place a strong bias on the largest particles present in the sample. NTA results are in accordance with [50].

3.1.8. XRD analysis X-Ray diffraction pattern were recorded to estimate the crystal structural phase of DS-AgNPs as shown in (Fig. 12A). The Bragg reflections with 2θ values of 38.23, 44.43, 64.68, and 77.66 correspond to the (111), (200), (220) and (311) planes. The data was matched with the standard cubic Ag° (Joint Committee on Powder Diffraction Standards – JCPDS file no. 00-001-1167). Similarly, the X-Ray diffraction pattern of SS-AgNPs was shown in (Fig. 12B). The Bragg reflections with 2θ values of 38.20 and 77.40 correspond to (111) and (311). The resultant data was matched with the database (JCPDS file no. 01-0870717) for both the nanoparticles composition. According to Gnanadesigan et al. [43] these values are comparable with Avicennia marina nanoparticles where XRD analysis showed 2θ intense values (38.11 and 70.57) within the ranges of Bragg’s reflection, confirming a face-centered cubic (FCC) crystal lattice structure for the silver nanoparticles. Additional intense peaks of D. trifoliata nanoparticles are 27.88, 32.30, 46.32, 54.89, 54.94 and 57.55 attributed to planes (111), (200), (222), (220), (311) of cubic phase of AgCl crystal matched with (JCPDS file no. 00-031-1238). Similarly, S. alba nanoparticles showed intense peaks 27.89, 32.32, 46.32, 54.92 and 57.58 indicates lattice planes (111), (220), (311), (222), and (311) of cubic phase of AgCl crystal matched with (JCPDS file no. 00-031-1238). However, mangroves have various salt tolerance mechanisms that vary with species as they can exclude salt, accumulate salt, and/or excrete salt. Salinity is one of the outstanding environmental features of mangrove ecosystems where S. alba

3.1.5. HR-TEM analysis The shape and size of the biosynthesized AgNPs were analyzed with HR-TEM. Image morphologies show that both DS-AgNPs and SS-AgNPs are well dispersed and displayed with Poly Dispersity Index (PDI) as 0.230 and 0.540 respectively as shown in Table 2. Spherical nanoparticles were formed in SS-AgNPs (Fig. 8A), as well as in DS-AgNPs (Fig. 7A). The HR-TEM images (Figs. 7B & 8B) at 50 nm scale and 20 nm scale (Figs. 7C & 8C) which corresponded to reveal mostly spherical shape with varying sizes from 20 to 40 nm. Average particle size of 24.49 ± 4.69 nm for DS-AgNPs and 25.74 ± 3.62 nm for SS-AgNPs. As shown in Figs. 7E and 8E the arranged lattice fringes with d- spacing d = 0.23 nm corresponds to (111) for DS-AgNPs and SS-AgNPs were clear crystallographic planes of Ag. Lattice spacing of the DS-AgNPs and 9

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Fig. 10. XPS Survey spectra of Ag/AgCl NPs of DS-AgNPs (A) Ag3d (B) Cl 2p (C) Wide scan.

3.2. Fluorescence spectroscopy: Human Serum Albumin bound with AgNPs

can grow in 100% seawater. There are many reports indicating the importance of salinity for mangrove species as well as evidence that various mangroves may have diverse tolerances and salinity. All true mangroves indicated significantly higher Cl and Na contents than mangrove associates [15,16]. The occurrence of these external peaks did not alter the Bragg reflection peaks dedicated to silver. X-ray diffraction is a powerful tool used to estimate the crystalline size and lattice strain using the Debye–Scherrer’s formula given in the following equation

D=

Kλ nm β cosθ

3.2.1. Fluorescence quenching of human serum albumin in presence of AgNPs Fluorescence spectroscopy is a most appropriate method to study the interaction between HSA and AgNPs, the fluorescence spectra were recorded with AgNPs and for the native HSA. To evaluate the interaction between them; HSA was incubated with different concentration of DS-AgNPs and SS-AgNPs. Valuable feature of intrinsic fluorescence of protein is the abundant sensitivity of tryptophan to its environment. Changes in emission spectra of tryptophan are common in response to protein conformational transition, denaturation, subunit association or substrate binding [59]. Hence, the intrinsic fluorescence of protein (quenching) can offer considerable information about their structure and dynamics that is considered for the study of protein folding and its association. The effect of AgNPs on tryptophan residues revealed in the (Fig. 13A and B) noticeably specifies that the HSA has strong emission band at 343 nm when excited with 295 nm wavelength. Fluorescence band intensity decreases gradually with increase in the concentration of DS-AgNPs & SS-AgNPs. However, this change in fluorescence characteristic of the HSA indicates the binding between AgNPs and HSA to form an assured complex.

(3)

Where D is the crystalline size; K = 0.9 is the shape factor, The values for the coefficient “K” depends on factors such as the geometry of the crystallites, here it is 0.9 for cubic system, λ is the X-Ray wavelength (λ = 0.154 nm for Cu Kα); β is the full width at half the maximum (FWHM) of Braggs peak (in radians), θ is the Bragg angle. From Eq. (3) the crystalline size is calculated.

ε=

β cosθ 4

(4)

From Eq. (4), the microstrain (ε) – induced broadening in the powders are due to crystal imperfection. The average calculated crystallite size of both DS-AgNPs and SS-AgNPs were 40 nm whereas, average lattice strain of DS-AgNPs (0.3) and SS-AgNPs (0.27).

3.2.2. Binding mechanism between HSA and silver nanoparticles At different temperatures, fluorescence-quenching data were 10

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Fig. 11. XPS Survey spectra of Ag/AgCl NPs of SS-AgNPs (A) Ag3d (B) Cl 2p (C) Wide scan.

interaction between both AgNPs with HSA.

evaluated by Stern-Volmer’s equation

Fo/ F = 1 + kqτo [Q] = 1 + Ksv [Q]

(5) 3.2.3. Number of binding sites (n) and binding constant (K) HSA fluorescence quenching also delivers the information on the number of binding sites (n) and the binding constant (K) as per Eq. (6),

Where Ksv represents the Stern-Volmer quenching constant, [Q] is the concentration of AgNPs, Kq denotes quenching rate constant of the biological macromolecules, τ0 signifies average life time of the molecules without any quencher, F0 is the fluorescent intensity of the fluorescent dye without the quencher and F is fluorescent intensity of fluorescent dye with the quencher indicates the steady-state fluorescence in the absence and presence of AgNPs. The fluorophore fluorescence intensity can be quenched by ground state quencher fluorophore reactions (static quenching) and excited state quencher fluorophore reactions (dynamic quenching) [60]. Quenching actions can be differentiated by evaluating the fluorescence quenching at different temperatures. The Stern-Volmer plot F0/F vs. [Q], after linear regression of this plot, the slope yields the Stern-Volmer constant (Ksv) (Fig. 14A and B). The biomolecular quenching constant (Kq) was calculated by considering τ0 for HSA = 5 × 10−9 S for Trp residues [21]. Kq increases with the temperature and displays k values, at 298 K, 308 K, 313 K and 318 K respectively. Results revealed Kq which increases for DS-AgNPs from 0.8 × 1016 to 1.6 × 1016 and for SS-AgNPs from 1.2 × 1016 to 2.2 × 1016 is due to binding interaction as shown in Table 1 and not because of diffusion control [61]. Therefore, it states that the static quenching and formation of a ground state complex between HSA: DSAgNPS and HSA: SS-AgNPs. Hence, confirming that the binding

log [(F0 − F)/ F] = logK + nlog [Q]

(6)

The values of n and k were obtained from the slope and Y-axis intercept. These values are summarized in Table 1 and in Fig 15A and B, DS-AgNPs and SS-AgNPs both have displayed n and K values which decreased with the rise in temperature and may partly decompose at higher temperature [62]. These results suggest the formation of an unstable complex of HSA with AgNPs and the values of n approximately equal to 1 represents the existence of 1 binding site in HSA for both AgNPs [63].

3.2.4. Binding forces amongst AgNPs-HSA complexes Formation of hydrogen bonds or by Vander Waals interaction, hydrophobic and hydrophilic, electrostatic interaction etc. is because biological molecules bind with inorganic molecules [64]. The thermodynamic parameters & evaluation of change in entropy (ΔS°) and enthalpy (ΔH°) of binding reaction enables to interpret the binding mode. These parameters can be evaluated by Vont Hoff plot and as mentioned in Eq. (7). 11

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Fig. 12. XRD pattern of Ag/AgCl NPs (A) DS-AgNPs (AgCl Chlorargyrite JCPDS no: 00-031-1238 and Ag JCPDS no: 00-001-1167) (B) SS-AgNPs (AgCl Chlorargyrite JCPDS no: 00-031-1238 and Ag Silver 3C JCPDS no: 01-0870717).

lnK = −

ΔH° ΔS° + RT R

(7)

Fig. 13. Fluorescence spectroscopy at RT (A) DS-AgNPs and (B) SS-AgNPs.

Here K is the binding constant at the corresponding temperature and R is the gas constant. ΔH° and ΔS° are calculated from the linear Vont Hoff plots. The Gibbs free energy (ΔG°) is assessed from Eq. (8).

ΔG° = ΔH° − TΔS°

16A and B the results showed that the absorption wavelength of HSA and both AgNPs-HSA at different temperatures. The absorbance spectrum at approximately 279 nm escalates gradually from curve to curve with rise in temperature as well as increase in DS-AgNPs & SS-AgNPs concentration respectively. These results specified that the HSA molecule gets adsorbed on the surface of AgNPs and increases the intensities with incubation time and formation of ground state complex of AgNPsHSA complex. According to Kiselev et al; [70] demonstrated the calculation the number of HSA molecules bound per nanoparticle with diameter of HSA (8.5 nm) using small angle neutron scattering. Therefore approximately 27 molecules of HSA will be accommodated per DS-AgNPs and SS-AgNPs assuming monolayer formation Mariam et al. [71].

(8)

Various temperatures (298 K, 308 K, 313 K, and 318 K) of binding constant were studied to calculate the thermodynamic functions involved in the binding process for DS-AgNPs & SS-AgNPs. The plot of ln (K) vs 1/T were fitted linearly to attain the value of ΔS° and ΔH° from the intercept and slope. According to Marium et al. [63] AgNPs-BSA complex interaction showed ΔH° 37.712 kJ/mol−1 and ΔS° 396.8 J mol−1 K−1 at 277 K, 301 K, 310 K, and 315 K temperature respectively. Our results of DS-AgNPs:HSA complex showed ΔH° (1070.24 kJ/mol−1) and ΔS° (0.120 J mol−1 K−1) and SS-AgNPs:HSA ΔH° (933.88 kJ/mol−1) and ΔS° (0.55 J mol−1 K−1) at 298 K, 308 K, 313 K and 318 K temperature. As per the previous findings [65,66] the positive ΔH° & ΔS° value is associated with hydrophobic interaction. The negative ΔH° and ΔS° values are associated with hydrogen bonding and Van der Waals interaction in low dielectric medium. Firstly, very low positive or negative ΔH° and possible ΔS° values are characterized by electrostatic interaction [67]. The Gibbs free energy (ΔG°) are negative (Table 1) which confirms that the process includes hydrogen bonding and Vander Waals interactions. It suggests that the binding process of AgNPs to the surface of HSA is spontaneous [68].

3.2.6. DLS analysis and zeta (ζ) potential Dynamic light scattering technique relies on Rayleigh scattering from the suspended nanoparticles that undergo Brownian motion. The hydrodynamic diameter of Ag/AgCl NPs is measured and is usually larger because it includes waters of hydration. The DLS size distribution of DS-AgNPs was 75.93 nm and SS-AgNPs was 40.58 nm respectively, while the polydispersity index (PDI) showed 0.230 and 0.540. Conductivity was found to be 0.242mS/cm and 0.284mS/cm respectively. The stability of the colloidal system and the surface charge of AgNPs were determined by the magnitude and measurements of the zeta (ζ) potential of the biosynthesized DS-AgNPs were −13.6 mV and SS-AgNPs that was found to be −15.7 mV. This result signifies that the surface of the nanoparticles is negatively charged and dispersed in the medium and the negative value confirms the repulsion among the

3.2.5. Adsorption studies of AgNPs-HSA complex UV–vis absorption measurement is applicable to explore the structural change and to know the AgNPs-HSA complex formation [69]. The λmax of uninteracted HSA was 278 nm, where absorption spectra of HSA in presence and absence of DS-AgNPs & SS-AgNPs were recorded. Fig 12

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Fig. 14. Stern Volmer plot for (A) DS-AgNPs-HSA Complex (B) SS-AgNPs-HSA Complex.

Fig. 15. Plots of the AgNPs quenching effect on HSA fluorescence at different temperature (A) DS-AgNPs-HSA Complex (B) SS-AgNPs-HSA Complex.

particles and proves that they are very stable. Whereas it is essential to mention that DS-AgNPs showed lower potential due to little agglomeration of the particles as seen in HR-TEM images, while SS-AgNPs has shown higher (ζ) potential because of uniform distribution of particles. Results displayed in Table 2 showed changes in hydrodynamic diameter and agglomeration kinetics in AgNPs-HSA complex, the DLS size decreased with increased concentration of both AgNPs and stability conditions depend on both the characteristics of AgNPs and the ratio between the concentrations in the reaction mixture. These results further acclaims that the stability of AgNPs is mediated by steric repulsion provided by proteins (which stay in their native state).

after the addition of AgNPs respectively. The decrease in intensity indicates decrease in α-helical content, which is due to the binding of AgNPs with amino acid residues (Fig 17A and B). Proteins need to be in their correct 3-dimensional form for enzymatic activity, decrease in αhelix can lead to loss in enzymatic activity of HSA [72,73].

3.3. Antioxidant activity After characterization of AgNPs were tested for antioxidant and anti-inflammatory activity. In antioxidant assay, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) scavenging activity and reducing power was quantified spectrophotometrically using ascorbic acid as a standard. Table 4A shows the obtained antioxidant results. Ascorbic acid was used as a positive control. DPPH scavenging activities have shown dose dependent antioxidant potential in both the plants the IC50 values of SS-AgNPs as 46.01, and DS-AgNPs was 60.88 as compared with AA. SS-AgNPs were slightly superior to DS-AgNPs but lower than standard AA (40.66). These results indicate lower activity in aqueous extracts of DS-AE and SS-AE than standard AA. However, the antioxidant potential of aqueous extract of both the plants enhanced after synthesizing nanoparticles. The DPPH antioxidant potential of SSAgNPs at highest concentration 100 μg/ml (90.62 ± 0.00) was almost similar to standard AA at 100 μg/ml (93.99 ± 0.02). The result of similar magnitude was observed at 100 μg/ml for both DS-AgNPs (83.15 ± 0.03) and SS-AgNPs (95.13 ± 0.02) over standard AA (95.80 ± 0.01) for H2O2 assay and SS-AgNPs showed more potent activity with IC50 value of 43.21–45.53 of AA. Here SS-AgNPs activity showed slightly higher and are almost equivalent to AA. The reducing ability of a compound usually depends on the presence of reductant that showed antioxidant activity by breaking the free radical chain through

3.2.7. CD spectroscopy The CD spectrum obtains the information on conformational changes in the secondary structure of HSA protein. The CD results were expressed in terms of mean residue ellipticity (MRE) in degree cm2 dmol−1 according to Eq. (9)

MRE =

Intensity of CD (m deg) at 208 nm Cp nl

(9)

Where Cp = molar concentration of the protein, n = number of amino acid residues and l = pathlength in cm. The percentage of α helicity was calculated using Eq. (10)

∝ −Helix (%) =

−MRE208 − 4000 X100 33,000 − 4000

(10)

Where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm and 33,000 is the MRE value of the pure α-helix at 208 nm. As shown in Table 3, pure HSA the α-helicity was about ∼57.35% this type of CD result indicated a decrease in the α-helical structure 13

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Fig. 17. Circular dichroism spectra of HSA in absence and in presence of AgNPs at different concentrations (A) DS-AgNPs (B) SS-AgNPs.

3.4. Anti-inflammatory activity In anti-inflammatory assay, membrane stabilization involves the process in which the integrity of the erythrocyte membrane and lysosomal membrane is maintained by anti-inflammatory drugs by stabilizing the membrane. Table 4B results revealed anti-inflammatory activities. Percent membrane stabilization results in have shown dose dependent activity in both plants. While SS-AgNPs recorded better potential than DS-AE and SS-AE as well as DS-AgNPs. Percentage stabilization was (79.35 ± 0.04) for DS-AgNPs and (83.33 ± 0.04) for SS-AgNPs compared with DS (93.70 ± 0.01). IC50 value of SS-AgNPs was 58.40 that were compared to standard 52.98. Lower the IC50 higher is the activity. In determining trypsin’s inhibitory activity, using trypsin enzyme and casein as substrate. Accurate concentration of both enzyme (0.06 mg/ml) and (2%) casein substrate has shown the optimum concentration dependent proteolysis activity. At highest concentration 100 μg/ml (80.39 ± 0.06) inhibition was recorded in DS-AgNPs whereas (88.89 ± 0.01) for SS-AgNPs and (89.54 ± 0.02) for SS-AE as compared to (92.48 ± 0.03) for DS. An IC50 value of SS-AgNPs (58.41) was almost comparable to the IC50 value of DS (50.42). AgNPs to inhibit protein denaturation was studied where SS-AE showed better activity than D. trifoliata. SS-AgNPs showed better IC50 value (58.86) than DS-AgNPs (61.69) as compared to standard DS IC50 value (43.85). From the results of in-vitro anti-inflammatory assay reveals that the maximum inhibition of membrane stabilization, trypsinase activity and protein denaturation in SS-AgNPs has showed best activity than DSAgNPs as well as DS-AE and SS-AE extracts. The present study might be useful for the development of newer and more potent natural antioxidant and anti-inflammatory. Flavonoids present in S. alba and D. trifoliata stems are major antioxidants and anti-inflammatory agents, some of them act as phospholipase inhibition and some have been described as TNF-α inhibition in different inflammatory conditions. The

Fig. 16. UV–vis spectroscopy at RT (A) DS-AgNPs-HSA Complex (B) SS-AgNPsHSA Complex.

Table 3 Percentage of α-helix in HSA and in presence of different concentration of AgNPs in PBS buffer pH 7.4 at 25 °C. Sr.No.

HSA-AgNPs

Concentration

% α-helix

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

HSA HSA+DS-AgNPs HSA+DS-AgNPs HSA+DS-AgNPs HSA+DS-AgNPs HSA+DS-AgNPs HSA HSA+SS-AgNPs HSA+SS-AgNPs HSA+SS-AgNPs HSA+SS-AgNPs HSA+SS-AgNPs

Control 1.9 × 10−8 M 3.8 × 10−8 M 5.7 × 10−8 M 7.7 × 10−8 M 9.6 × 10−8 M Control 2.5 × 10−8 M 5 × 10−8 M 7.5 × 10−8 M 10 × 10−8 M 12.5 × 10−8 M

57.35 55.65 54.83 53.87 52.3 48.92 53.87 51.34 50.55 48.92 47.57 47.22

donation of a hydrogen atom. Reducing ability of DS-AgNPs, SS-AgNPs, DS-AE, SS-AE compared to standard AA. SS-AgNPs at 100 μg/ml showed (1.24 ± 0.31) and DS-AgNPs showed (1.15 ± 0.13) which exhibited comparatively almost similar to AA 100 μg/ml (1.12 ± 0.09) but due to presence of phytoconstituents in SS-AgNPs, at 100 μg/ml activity showed slightly more compared to AA whereas DS-AgNPs was almost similar to AA. These results were correlated with biosynthesized AgNPs of Helicteres isora [74] and Iresine herbstii [75].

14

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Table 4A Antioxidant Activity. Sr. No.

Concentration (μg/mL)

DPPH Assay % inhibition

IC50

Hydrogen peroxide Assay % inhibition

IC50

Reducing Power Assay

1. AA

20 40 60 80 100

30.08 50.45 67.57 87.69 93.99

± ± ± ± ±

0.05 0.06 0.06 0.06 0.02

40.66

29.73 40.24 61.86 87.09 95.80

± ± ± ± ±

0.03 0.01 0.03 0.03 0.01

45.53

0.30 0.48 0.71 0.90 1.12

± ± ± ± ±

0.02 0.03 0.01 0.03 0.09

2. DS-AE

20 40 60 80 100

11.55 24.09 32.67 53.80 68.65

± ± ± ± ±

0.01 0.02 0.07 0.06 0.08

76.46

14.09 23.71 34.36 43.64 60.14

± ± ± ± ±

0.05 0.03 0.03 0.02 0.02

86.44

0.12 0.30 0.40 0.65 0.91

± ± ± ± ±

0.00 0.01 0.01 0.05 0.03

3. DS-AgNPs

20 40 60 80 100

27.78 32.35 45.42 62.75 78.76

± ± ± ± ±

0.04 0.01 0.02 0.02 0.02

60.88

22.47 42.70 61.80 73.41 83.15

± ± ± ± ±

0.01 0.02 0.03 0.03 0.03

51.18

0.13 0.22 0.51 0.74 1.15

± ± ± ± ±

0.02 0.03 0.06 0.04 0.13

4. SS-AE

20 40 60 80 100

12.73 29.39 32.12 43.64 60.00

± ± ± ± ±

0.05 0.02 0.05 0.03 0.05

86.52

16.84 27.15 39.86 54.98 63.92

± ± ± ± ±

0.01 0.02 0.04 0.01 0.03

75.44

0.21 0.36 0.55 0.76 0.98

± ± ± ± ±

0.02 0.05 0.05 0.01 0.02

5. SS-AgNPs

20 40 60 80 100

28.83 44.72 61.25 80.22 90.62

± ± ± ± ±

0.01 0.10 0.05 0.03 0.00

46.01

25.47 49.44 68.54 84.64 95.13

± ± ± ± ±

0.02 0.02 0.03 0.02 0.02

43.21

0.26 0.40 0.62 0.87 1.24

± ± ± ± ±

0.02 0.02 0.01 0.02 0.31

Table 4B Anti-inflammatory Activity. Sr. No.

Concentration (μg/mL)

Membrane Stabilization Assay % stability

IC50

Proteinase Inhibitory % inhibition

IC50

Protein denaturation % inhibition

IC50

1. DS

20 40 60 80 100

23.33 41.48 52.96 69.63 93.70

± ± ± ± ±

0.01 0.03 0.03 0.03 0.01

52.98

16.34 29.41 75.82 85.95 92.48

± ± ± ± ±

0.04 0.01 0.02 0.01 0.03

50.42

28.32 50.18 67.03 73.12 92.11

± ± ± ± ±

0.02 0.02 0.01 0.03 0.01

43.88

2. DS-AE

20 40 60 80 100

14.86 24.64 41.67 60.51 71.38

± ± ± ± ±

0.02 0.02 0.03 0.01 0.02

69.92

13.07 29.08 44.77 56.54 66.34

± ± ± ± ±

0.03 0.06 0.05 0.04 0.04

72.00

15.41 30.47 43.73 56.99 68.10

± ± ± ± ±

0.02 0.04 0.03 0.03 0.02

70.70

3. DS-AgNPs

20 40 60 80 100

17.75 26.45 44.57 62.68 79.35

± ± ± ± ±

0.04 0.03 0.01 0.01 0.04

60.63

14.38 25.16 55.88 69.28 80.39

± ± ± ± ±

0.04 0.06 0.03 0.06 0.06

61.11

19.71 31.18 47.67 63.08 81.72

± ± ± ± ±

0.03 0.04 0.06 0.04 0.07

61.69

4. SS-AE

20 40 60 80 100

14.49 24.28 40.58 48.19 69.57

± ± ± ± ±

0.03 0.02 0.04 0.01 0.02

75.78

10.13 25.16 46.41 67.32 89.54

± ± ± ± ±

0.02 0.01 0.03 0.02 0.02

62.27

17.20 32.26 38.35 59.50 69.89

± ± ± ± ±

0.05 0.02 0.03 0.04 0.04

69.89

5. SS-AgNPs

20 40 60 80 100

21.74 35.51 49.28 66.30 83.33

± ± ± ± ±

0.02 0.01 0.02 0.03 0.04

58.40

16.01 36.27 51.31 64.38 88.89

± ± ± ± ±

0.05 0.01 0.02 0.04 0.01

58.41

20.79 32.97 53.05 62.37 85.30

± ± ± ± ±

0.06 0.02 0.02 0.03 0.02

58.86

Note: IC50 – Concentration of the aqueous extract, ethanol extract and synthesized nanoparticles causing 50% scavenging ability. AA: Ascorbic Acid; DS: Diclofenac sodium; DS-AE: D. trifoliata stem aqueous extract; DS-AgNPs: D. trifoliata Ag nanoparticles synthesis; SS-AE: S. alba stem aqueous extract; SS-AgNPs: S. alba Ag nanoparticles synthesis

using Membrane stabilization, Proteinase inhibitory and Protein denaturation assays (Table 5B). Antioxidant and Anti-inflammatory activities of all groups were comparable to positive control (Ascorbic acid and Diclofenac sodium) that was not significant. Both the activities of AgNPs were better than AE groups in DPPH, H2O2, Reducing power, Membrane stabilization, Protein inhibitory and Protein denaturation

statistical analysis results showed that in Shapiro wilk test, to test normality data which was found to be normally distributed, therefore we performed one-way ANOVA with post hoc-tukey’s test where, p < 0.05 is considered significant. We evaluated antioxidant activity of D. trifoliata and S. alba using DPPH, H2O2 and Reducing power assays (Table 5A) and Anti-inflammatory activity of D. trifoliata and S. alba 15

Process Biochemistry xxx (xxxx) xxx–xxx

P. Gawali, B.L. Jadhav

Table 5A Antioxidant activity of one-way ANOVA and Post hoc tukey’s test. Sr. No. 1. 2. 3.

Assay DPPH Hydrogen Peroxide Reducing Power

Standard Ascorbic Acid 66 ± 26 66 ± 26 0.70 ± 0.32

DS-AE

DS-AgNPs

38 ± 22 35 ± 17 0.47 ± 0.30

#

49 ± 21 56 ± 24# 0.55 ± 0.41#

SS-AE

SS-AgNPs

35 ± 17 40 ± 19 0.57 ± 0.30

61 ± 25# 64 ± 28# 0.67 ± 0.38#

For Table 5A: Values represents Mean ± SD (p < 0.05) DS-AgNPs and SS-AgNPs. # p < 0.05 as compared to DS-AE and SS-AE. Table 5B Anti-inflammatory activity of one-way ANOVA and Post hoc tukey’s test. Sr. No.

Assay

Standard Diclofenac Sodium

DS-AE

DS-AgNPs

SS-AE

SS-AgNPs

1. 2. 3.

Membrane stabilization Proteinase Inhibitory Protein denaturation

56 ± 27 53 ± 28 62 ± 24

42 ± 23 48 ± 22 42 ± 21

46 ± 25* 41 ± 25* 48 ± 24*

39 ± 21 45 ± 29 43 ± 21

51 ± 24* 51 ± 27* 50 ± 25*

For Table 5B: Values represents Mean ± SD (p < 0.05) DS-AgNPs and SS-AgNPs. * p < 0.05 as compared to DS-AE and SS-AE.

Professor, Department of Pharmacology and Therapeutics at Seth GSMC & KEM Hospital, Parel Mumbai) for statistics analysis.

assays. Thus, the anti-oxidant and anti-inflammatory properties from true mangroves differ from mangrove associates physiologically and ecologically in their ability to survive.

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Biosynthesized AgNPs was confirmed using different characterization techniques such as FTIR, TGA, DLS, NTA, HR-TEM, EDS, XPS and XRD results revealed Ag/AgCl NPs. Fluorescence spectroscopy showed both AgNPs-HSA fluorophore interaction in colloidal solution, which however gets destabilized at a higher temperature. The fluorescence quenching data were analyzed by Stern-Volmer equation that endorsed us to obtain the number of binding sites and binding constant at varying temperature. The results of binding studies reveal that the adsorption of HSA on both AgNPs surface tends to form corona like structure, the value of ‘n’ ≈1 indicates the existence of a single binding site in AgNPsHSA complex. The calculated negative values of enthalpy and entropy change depicts that the interaction is mainly driven by thermodynamic parameters (ΔG°, ΔH° and ΔS°) suggests binding which occurs spontaneously in both AgNPs involving hydrogen bond and Vander Waals forces. UV–vis spectroscopy revealed the formation of aggregates of HSA-AgNPs complex to induce conformational modification. DLS size decreases when the concentration of AgNPs: HSA complex increases. In addition, circular dichroism spectra clarified that α- helicity of the HSA decreases due to its interaction with both AgNPs. The antioxidant and anti-inflammatory activities were studied for both the stems of aqueous extracts of mangrove and their AgNPs; from overall results of biomedical applications it can be concluded that stems of a true mangrove S. alba, have shown better antioxidant and anti-inflammatory activities than stems of a mangrove associate D. trifoliata. Conflict of interest We have no conflict of interest. Acknowledgements The work was financially supported by University Grants Commission (UGC), New Delhi, India [F1-17.1/2016-17/RGNF-201517-SC-MAH-25252/(SA-III/Website)] to the first author. We thank Department of Life Sciences, Department of Chemistry, Department of Biophysics, National Centre for Nanosciences and Nanotechnology, University of Mumbai; Sophisticated Analytical Instrument Facility (SAIF) and Indian Nanoelectronics User Program (INUP)- IIT Bombay for accessing Instrument facility and Dr. Sanket B. Raut (Assistant 16

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