Calligonum polygonoides reduced nanosilver: A new generation of nanoproduct for medical applications

Journal Pre-proof Calligonum polygonoides reduced nanosilver; a new generation of nanoproduct for medical applications Naila Sher, Mushtaq Ahmad, Nadia Mushtaq

PII:

S1876-3820(19)31109-6

DOI:

https://doi.org/10.1016/j.eujim.2019.101042

Reference:

EUJIM 101042

To appear in:

European Journal of Integrative Medicine

Received Date:

16 October 2019

Revised Date:

18 December 2019

Accepted Date:

18 December 2019

Please cite this article as: Sher N, Ahmad M, Mushtaq N, Calligonum polygonoides reduced nanosilver; a new generation of nanoproduct for medical applications, European Journal of Integrative Medicine (2019), doi: https://doi.org/10.1016/j.eujim.2019.101042

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Calligonum polygonoides reduced nanosilver; a new generation of nanoproduct for medical applications

Naila Sher1 [email protected], Mushtaq Ahmad1* [email protected], Nadia Mushtaq2 [email protected] Department of Biotechnology, University of Science and Technology Bannu-KPK, Pakistan.

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Department of Biotechnology, University of Science and Technology Bannu-KPK, Pakistan.

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Department of Botany, University of Science and Technology Bannu-KPK, Pakistan.

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Abstract

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*Corresponding author: Mushtaq Ahmed (PhD), Department of Biotechnology, University of Science and Technology, Bannu-KPK, Pakistan., E:Mail:[email protected], Phone: +92928633425

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Introduction

Science and technology is always progressing and old technologies are replaced. Nanotechnology is one of the new growth areas that has emerged during this century. Nanotechnology deals with the synthesis of nanoparticles and nanomaterials of variable size, shape and its application in numerous fields. Biological methods are advantageous over chemical methods. The current study, has been designed to investigate the biological synthesis and pharmacological activities of silver nanoparticles (AgNPs) to demonstrate its

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utilization when used in combination with a natural medicine which has been used for the treatment of various diseases in the sub-continent. Methods In the current study, AgNPs were synthesized from 10 mM AgNO3 using the methanolic crude extract of the Calligonum polygonoides (CP) and was confirmed by UV visible and FTIR spectroscopy. The size and shape were determined by X-Rays Diffraction (XRD) and scanning electron microscope (SEM). In vitro antidiabetic, antifungal, antioxidants, cytotoxic and antibacterial potentials were determined by using the standard protocols. Results

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The AgNPs were monodispersed, spherical with an average size of 50 nm by SEM analysis. The average size 41.62 nm was calculated using three major peaks 380, 440 and 640 of XRD analysis. FT-IR spectra show the peaks for the functional groups involved in the synthesis and stability of the AgNPs.

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Conclusions

This study suggests that AgNPs showed enhanced properties when compared to the CP crude

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extract.

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activities; characterization

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Key words: Calligonum polygonoides; AgNPs synthesis; nanotechnology; biological

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Introduction

Silver nanoparticles (AgNPs) have become increasingly important due to their wide variety of applications [1]. Historically, silver has been an important material for domestic use [2]. Nanotechnology deals with application and synthesis of nanoparticles (generally range from 1-100 nm) of variable size and shapes. AgNPs are much consecrated amongst all-metal nanoparticles due to the surface plasmon resonance (SPR) (strong absorption in the visible region), which is determined by UV–visible spectrophotometer [3]. Chemical methods,

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incorporate the use of ethylene glycol [4] and sodium borohydride [5] in AgNPs synthesis. The chemicals used in these chemical methods are either toxic to the humans and environment or the processes are too costly to be realizable at an industrial scale. To overcome the complications resulting from the chemical methodology, there has been a search for a non-toxic, economical, reliable and “green” methodology for the synthesis of nanoparticles. The use of the plant extracts as capping, reducing and stabilizing agents has become specifically of interest in the synthesis of nanoparticles, due to the need for sterilizing and preserving the environment, during the process [6]. Silver is of more significance compared to other metals because of its vital role as an

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antimicrobial agent [7]. In small concentrations, silver has no diverse effect on human cells and can be considered as an environmentally friendly, antimicrobial agent. SEM analysis

confirms that AgNPs interact with the membrane of fungus which results in the formation of a hole in the membrane and finally cell death [8]. Diabetes mellitus is a major endocrine

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disorder characterized by chronic hyperglycemia. It mainly affects the humans due to defects in insulin secretion or resistance. α-amylase and α-glucosidase are the two major enzymes that play a vital role in the oligosaccharides and starch degradation to glucose and if these

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two enzymes are inhibited the absorption of the glucose in the intestine would be delayed. In diabetic patients, postprandial hyperglycemia occurs after a meal due to glucose absorption

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from the gastrointestinal tract. Preventing glucose uptake in the intestines and promoting glucose uptake in tissues can control the level of blood glucose in the case of postprandial hyperglycemia, which is common for people with diabetes. Green synthesis of nanoparticles

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from plants extracts can slow enzyme kinetics for catalytic activity, may provide better opportunities for manipulation, control over the crystal stability and growth [9]. Among the

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various methods, the biological and green methods are considerably preferred for the biosynthesis of AgNPs, using the plant extract that possesses phytochemicals with strong antioxidant properties [10]. Several studies have reported that AgNPs have significantly

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induced cell necrosis or apoptosis in many cell types [11]. C. polygonoides Linn. belongs to the family Polygonaceae. In Pakistan, Polygonoides are categorized by only one species from this genus. C. polygonoides (CP) which do not require any vegetation for their growth and grow well in sandy areas of the desert [12]. C. polygonoides flowers have high quantity of protein and possess tonic and digestive properties and are useful against asthma, cold, and cough [13]. C. polygonoides flowers are used for making delicious preparation by cooking in coconut oil or clarified butter [14] and as a food for camels in desert areas [15]. C.

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polygonoides extract used is a therapeutic agent against various diseases [16]. In view the importance of nanoparticle, this research used as a novel approach in the green synthesis of AgNPs using C. polygonoide and to evaluate its biological activities. Materials and methodology Plant collection and C. polygonoides plants were collected from the area of Domel, District Bannu and were identified by Prof. Dr. Sultan Mehmood, Dean Faculty of Biological Sciences, UST Bannu. A voucher specimen was deposited (number1574). The collected plant samples were dried under shade at room temperature and ground mechanically up to approximately a mash size

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of 0.1 mm. About 600 gm of C. polygonoides powder was mixed with 3 L of the 80% methanol and kept on orbital shaker at 120 rpm for 12 h and then placed it at room

temperature for 7 days; After seven days the plant was extracted and filtered by using what man filter paper and concentrated with the help of the rotary evaporator; After the

then reserved at 4°C for more in-vitro studies.

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Synthesis of nanoparticles

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concentration, the extra methanol was evaporated at 37°C to obtain crude extract and was

AgNPs were synthesized from C. polygonoides, following the procedure of [17]. About 10

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mM (0.01 M) solution of silver nitrate was prepared in 50 mL deionize water. The 10 mM silver nitrate was further diluted to 10 times by mixing 1 mL silver nitrate and 9 mL

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deionized water. NaOH, ≥98% was used to adjust the pH 11. An aqueous solution of plant extracts was prepared by dissolving 1 gm of plant extract in 100 mL deionizes water. It is gently stirred on a magnetic stirrer for about 1 h. After dissolution, it was centrifuged at 6000

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rpm for 30 min. The supernatant was collected for activity and the pellets were discarded. The plant supernatant (50 mL) was mixed with the 10 fold dilute silver nitrate solution of pH

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11 (450mL+NaOH). The resulting solution became yellowish brown after 10 min of continuous stirring at pH 11 and at room temperature which indicated the formation of AgNPs by C. polygonoides. The solution was then stored at room temperature for 24 h for the complete settlement of nanoparticles and was then monitored using UV–Visible spectrophotometer. The colloidal suspension thus obtained was Centrifuged by cold centrifuge at -4°C at 13,000 rpm for 10 min and the pellet obtained after discarding supernants. The synthesized nanoparticles were lyophilized and recovered in powdered form which was further characterized and different activities were also determined.

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Factors affecting synthesis rate, size, and shape of AgNPs To study the effect of C. polygonoides extract on the synthesis of AgNPs concentration of the C. polygonoides extract was varied as 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL. In order to study the effect of the temperature AgNPs were synthesized at different temperature 25°C, 50°C, 75°C and 100°C. To study the effect of acidic and basic condition on the synthesis of the AgNPs pH of the reaction mixture was maintained from 2-12 respectively by using 0.2 M NaOH and 0.2 M HCl. To study the effect of the time on the completion of the reaction was monitored from 0 to 30 min after every 5 min interval and the last reaction was monitored after 24 hrs. The absorbance of the resulting solution was measured by using UV-visible

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spectrophotometer. Characterization of AgNPs

The reduction of the silver ion in the solution was determined using SHIMADZU UV

SPECTROPHOTOMETER (UV-1800). The purified AgNPs were examined for the presence

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of the various functional groups using FT-IR Shimadzu (IR Prestige-21) spectrometer

(Japan). FT-IR analysis was carried out through Shimadzu (IR Prestige-21) spectrometer

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(Japan). The prepared materials were fully dried in order to remove the traces of moisture before subjecting to FTIR analysis. Potassium bromide (KBr) powder was also dried 2-3

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times. For blank analysis, a pure KBr pellet was prepared and used. Than 5% solid solution of each sample with KBr in the form of thin pellet was prepared. The FT-IR spectra of the materials were obtained in the IR radiation range 400-4000 cm-1.The crystalline nature of the

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AgNPs was determined by JDX-3532 (JEOL JAPAN) X-ray diffractometer having a fixed radiation wavelength of λ-1.54 Aº. For structural analysis, the XRD patterns of the prepared materials were studied by JDX-3532 (JEOL JAPAN) X-ray diffractometer having a fixed

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radiation wavelength of λ-1.54 Aº. Each sample was sized to one square inch for XRD analysis. The samples were placed in a glass made holding system and then were transferred

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to the X-ray generating tube. The acceleration voltage of 35 kV and 20 mA current were applied while the degree of scanning was in the range from 10 to 50°. The diffracted intensities were recorded from 20° to 80°. The size and shape of nanoparticles of the prepared materials in the project was analyzed by using JEOL Scanning Electron Microscope Model JSM-5910 (Japan). Current-voltage applied was in the range from 5 kV-20 kV. Resolution of the microscopic lens varied from 10000X to 50000X. Appropriate amount of samples were taken and mounted on Aluminum stubs with conductive taps and then were subjected to SEM

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analysis. Micrographs images of all the samples were obtained under suitable resolution and voltage. Antioxidant activity Ferric-reducing power assay The reducing power was determined according to the method of [18]. Extract solution (2 mL), phosphate buffer (2 mL, 0.2 M, pH 6.6) and potassium ferricyanide 2 mL (10 mg/mL) were mixed and then incubated at 50°C for 20 min. Trichloroacetic acid 2 mL (100 mg/mL) was added to the mixture. A volume of 2 mL from each of the above mixtures was mixed with 2 mL of deionizing water and 0.4 mL of 0.1% (w/v) ferric chloride in a test tube. After

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the 10-min reaction, the absorbance was measured at 700 nm. The percentage of the reducing power was calculated by using the formula; % Inhibition =

𝑨𝒄−𝑨𝒔 𝑨𝒄

× 𝟏𝟎𝟎

(i)

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Where Ac is the control absorbance and As is the sample absorbance

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Phosphomolybdate assay

The total antioxidant capacity (TAOC) was evaluated by the following procedure of [19]. An aliquot of 1 mL of concentration (1 mg/mL) was mixed with 9 mL of reagent solution (600

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mM sulphuric acid, 28mM sodium phosphate, and 4 mM Phosphomolybdate). The tubes were capped and incubated over a boiling water bath sustained at 95°C for 90 min. Once the sample cooled to room temperature the absorbance of the solution was measured at 695 nm

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against a blank. Ascorbic acid was used as a standard. The percentage of scavenging was calculated by using the formula (i).

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DPPH Activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging potential of the AgNPs was

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determined using the modified method of [20]. In this essay, first of all stock solution of the AgNPs was prepared by dissolving 1 mg/mL AgNPs in deionize water. After that from this stock solution, five sub solutions of (20, 40, 60, 80,100 µg/mL) were prepared by using the formula M1V1=M2V2. Standard ascorbic acid was also prepared in the same concentration as AgNPs. About 200 µL from different concentrations of AgNPs, CP extract and standard was mixed with 800 µL of DPPH (3 mg/50mL), incubated for 30 min at room temperature in

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dark and absorbance was recorded at 517 nm by spectrophotometer against deionize water as blank. The %age of scavenging was calculated using the formula (i). Hydrogen peroxide bioassay The H2O2 scavenging activity was analyzed by the following method of [21]. In brief, 200 µL from different concentrations (20,40,60,80,100 µg/mL) of AgNPs, CP extract and ascorbic acid (control) were mixed with 400 µL of 2 mM H2O2 solution and 400 µL of 50 mM phosphate buffer of PH =7.4. The mixture was incubated at 35°C for 20 min. The absorbance was measured at 610 nm against phosphate as blank. The %age of scavenging was calculated by using the formula (i).

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ABTS activity

The ABTS (2, 2 azobis, 3-ethylbenzothiozoline-6-sulphonic acid) radical cation scavenging activity was accomplished by the method of [22]. With slight modification. Equivalent

volumes of 7 mM ABTS solution and 2.45 mM K2(SO4) solution was prepared in deionized

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water. These two solutions were mixed and incubated in the dark overnight at room

temperature to yield a dark-colored solution comprising of ABTS•+ radicals. The optical

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density of the mixture was determined using a spectrophotometer and was brought to 0.700 (± 0.02) by the addition of more solvent. About 300 μL from different concentrations of

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AgNPs, CP extract, and standard (Ascorbic acid) were mixed with 3.0 mL of (K2(SO4) +ABTS) mixture. The absorbance was measured at 734 nm against water as blank. The experiment was completed in triplicate to eliminate errors. The percentage of ABTS

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scavenging was calculated by using the formula (i). Cytotoxic brine shrimp’s bioassay

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Cytotoxic effect of AgNPs was determined using brine shrimp lethality assay by following the modified method of [23]. The stock solution of AgNPs and CP extract were prepared at 1

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mg/mL concentration which fractioned into (25, 50, 75 and 100 µg/mL). Brine shrimps were hatched in two-compartment rectangular tray comprising sea salt saline. Eggs were scattered in the dark compartment of the tray and after 24 h of shrimps hatching larvae were collected by poster pipette from the lightened side. Solution (0.5 mL) from each concentration of AgNPs and CP extract was taken in the vial. The residue was determined in saline of 2 mL. Shrimps (n=12) were transmitted to each vial and raised the volume up to 5 mL and incubated at 25–28°C. After 24 h of incubation survivors were counted with help of 3x magnifying glass.

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The calculation was done by using Abbot’s formula; % Death =

𝑨𝒄−𝑨𝒔 𝑨𝒄

× 𝟏𝟎𝟎

Antifungal bio-assay Slightly modified method of [24] was followed for measuring the fungicidal capacities against three fungal strains A. niger, A. fumigates and A. flaves. About 6.2 gm savored dextrose agarose (SDA) was weighed and dissolved in 100 mL deionized water, autoclaved for 15 min at 121ºC and reserved for cooling at 40-50ºC. Brief 7 mL SDA along with 67 µL (25, 50, 75, 100 µg/mL) AgNPs dissolved in deionizing water and CP extract dissolved in

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methanol were transferred to each test tube. The negative control test tubes were treated only with DMSO and positive control received antifungal drug Terbinafine. All the tubes were

then placed in a slanted position at room temperature inside the laminar flow hood to solidify. The fungus strains were added to each tube and then placed in an incubator at 30ºC with open

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water up to 9 days and % inhibition was studied. Antibacterial activity of AgNPs

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The bactericidal activity of AgNPs and CP extract was determined by using the agar well diffusion method as described. Six bacterial strains, three Gram-negative P. vulgaris, E. coli

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and K. pneumonia and three Gram-positive M. luteus, S. aureus and S. epidermidis were used in this experiment. Bacterial cultures were refreshed in nutrients broth for 24 h at 37ºC. The antibacterial activity equipments i-e petri plates, media, tips, test tubes, cotton etc were

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autoclaved for 15 min at 121ºC and were cooled up to 60ºC. To each Petri plate, 30 mL media was transferred and allowed for overnight solidification. The 24 h old cultures were swabbed in the nutrients agar plates by using sterile cotton swab aseptically. About 67 µL

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from the four different concentrations of AgNPs and CP (25, 50, 75 and 100 µg/mL) were loaded in the labeled wells. Standard levofloxacin and DMSO were also loaded into the

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wells. The plates were incubated at 37ºC for 24 h. Results were obtained by measuring the zone of inhibition around each well and expressed in millimeter. Antidiabetic activity Inhibition of β–glucosidase enzyme In-vitro β–glucosidase inhibitory activity of AgNPs and CP extract was determined by following the method described by [25]. About 290 mM solution of β-D glucopyranoside (pNPGlc) was prepared in 20 mM citrate buffer of pH 5.6. The stock solution of AgNPs, CP

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and standard (tagipmet) was prepared at 1 mg/mL concentration which fractioned into (200,400,600, and 800 µg/mL). About 200 µL was taken from each concentration of AgNPs, CP and standard (tagipmet) to be mixed with 980 µL of β -D glucopyranoside. Now it was incubated for 5 min at 37ºC. The reaction was initiated by adding 20 µL of a β-glucosidase enzyme (IU/mL) to each test tube followed by incubation for 40 min at 35ºC. Then the reaction was terminated by the addition of 200 µL of 6 N-HCl and the absorbance was calculated at 405 nm against water as a blank. The experiment was repeated twice. The percentage inhibition was calculated by using the formula (i). α-amylase assay

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In-vitro α-amylase inhibitory activity of AgNPs and CP extract was determined by following the method described by Malik [26]. To obtain starch solution (0.1% w/v) 0.1 gm potato

starch was dissolved in 100 mL sodium acetate buffer (16 mM). The enzyme solution was

prepared by mixing 300 µL of α-amylase from stock (250 units/mL) with 700 µL deionizes

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water. A mixture of Sodium potassium tartrate and 3, 5 dinitro salicylic acid (96 mM) was

used as calorimetric reagent. The stock solution of AgNPs, CP and standard (tagipmet) was

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prepared at 1 mg/mL concentration which fractioned into (25, 50, 75 and 100 µg/mL). About 250 µl from the four different concentrations of AgNPs, CP and standard tagipmet was mixed

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with 250 µL potato starch solution and 250 µL alpha-amylase and incubate for 5 min at 25°C (room temperature). After 5 min 250 µL from the combine solution of Sodium potassium tartrate and 3, 5 dinitro salicylic acid was added to each concentration. The reaction is

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detected at 450 nm. Deionized water was taken as a reference.Tagipmet served as a positive control. The experiment was repeated twice. The percentage inhibition was calculated by

Results

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using the formula (i).

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Synthesis of AgNPs

UV-Visible spectrophotometric analysis of AgNPs In the aqueous AgNPs exhibited yellowish-brown color due to the SPR. After mixing the plant extract with AgNO3 the color of the solution start to change from the milky to yellowish indicated the bioreduction of the Ag+ (Fig. 1). Factors affecting the synthesis of AgNPs

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The UV-vis spectra of AgNPs aqueous medium was noted at the range of 300 to 800 nm wavelength for 0.2-1 mL plant concentration which indicated absorbance at 403,405,405, 405 and 414 nm respectively (Fig. 2.A). Fig. 2.B indicated that a sharp band appeared at 414 nm for the colloidal suspension at 25°C (room temperature) at higher absorption 2.1583. With an increase in the temperature the sharp band became broader and broader. A final broad band appeared at 410 nm at 100°C and at lower absorption 1.5833. Fig. 2.C indicated the effect of reaction time on the AgNPs synthesis by UV-vis spectroscopy at the range between 350 to 800 nm wavelengths. Absorption of 409 nm appeared after 5 min

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of the stirring. Due to the continuous formation of the AgNPs, the absorption peak quickly increases with the passage of time. A clear sharp peak 414 nm at high absorbance 2.375 obtained after 24 hrs of the reaction.

In the current project effect of the pH on the AgNPs synthesis was carried out using a UV-vis spectrophotometer. Even after 24 hours of incubation, no band was observed in the range 420

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to 450 nm. Different peaks i-e 413,420, 417,410, 414 and 413 nm was observed as the pH increase from 7 to 12 (Fig. 2.D).

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Analysis through scanning electron microscopy (SEM)

Fig. 3.A indicated high density, monodispersed and spherical shape AgNPs. The average

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particle size was reported by Nano Measurer software is 50 nm. Nano Measurer software varied from 0-1000 nm (1µm).The particle size calculated for 1 mL sample by marking 20 particles which reported that 80% AgNPs are in the average size 50 nm (0.05µm) Fig. 3.B.

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Fourier transforms infrared spectrometer (FT-IR) analysis of AgNPs FTIR analysis gives various peaks which are corresponded to different functional groups involved in the bio-reduction of Ag+ ion and also act as a capping agent for AgNPs. The

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observed FTIR peaks were then compared to standard values in the FTIR chart to detect the exact functional groups involved in the bioreduction process. Fig. 4 showed peaks at 700,

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832, 993, 1333, 1620, 1730 and 2921 cm-1, these peaks correspond to different functional groups. The peak at 2921 in FT-IR indicates that there must O-H group of aromatic phenolic compounds; peak at1730 cm-1 corresponded to C=O functional group of sterols; 1620 cm-1 band corresponds to C-N and C-C revealed the existence of proteins. All other peaks correspond to different vibrating stretching of functional groups. X-Ray Diffraction analysis (XRD)

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Fig. 5 indicated 4 Bragg reflections at position 38°, 44°, 64°, and 78°; these have corresponded to the planes (1 1 1), (2 0 0), (2 2 0) and (3 1 1) correspondingly which can be indexed conferring to the face of face-centered cubic crystal structure of Ag+. The interplanar spacing (d calculated) and Miller constants values were calculated, using the Debye Scherrer equation (dhkl ꞊π/2sinθhkl and a0=dhkl(h2+k2+l2)1/2) respectively (Table. 1). The average crystalline size is calculated, using the Debye Scherrer formula (D = kλ/βcosθ). Where D is the average crystalline size of the nanoparticles, k is a geometric factor (0.9), λ is the wavelength of the X-ray radiation source and β is the angular FWHM (full-width at half maximum) of the XRD peak at the diffraction angle θ. The average size 41.62 nm calculated

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using three major peaks 380, 440 and 640 of XRD analysis. In-Vitro antioxidant capacity of AgNPs and CP

In the present study, reducing the power of AgNPs and CP decreases with increasing dilution. Significant scavenging activity was shown by different concentrations of AgNPs and CP with increasing concentration (20, 40, 60, 80 and 100 µg/mL). It is cleared from the Fig. 6.A that

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AgNPs and CP showed appreciable results 97% and 47% at 100 µg/mL respectively.

In this study, various concentrations (20-100 µg/mL) of green synthesised AgNPs and CP

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were tested as a scavenger against free radicals phosoho molybdate (Fig. 6.B). The phosoho molybdate percent scavenging potential of AgNPs is appreciable as compared to standard

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ascorbic acid and CP increase with increasing concentration 44-68%, 9-22% and 12-27% respectively.

DPPH reducing capacity of AgNPs and CP was measured by color changes. DPPH

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scavenging assay exhibited by AgNPs shows effective inhibition compared to CP extract. Fig. 6.C indicated that at high concentration 100 µg/mL the AgNPs, CP and ascorbic showed high scavenging capacity 93%, 35% and 78% respectively then decrease with an increase in

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dilution (100-20 µg/mL).

In this study, different concentration (20-100 µg/mL) of AgNPs, CP and ascorbic acid were

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tested against hydrogen peroxide. Fig. 6.D showed that at high concentration 100 µg/mL the inhibition was found to be 88, 51 and 81% for AgNPs, CP and ascorbic acid respectively and then decrease with a decrease in concentration of AgNPs and ascorbic acid. In the present project AgNPs and CP were perceived against ABTS in comparison with a standard (ascorbic acid). The results showed (Fig. 6.E) that at high concentration 100 µg/mL AgNPs and CP d offered 79 and 55% inhibition respectively. In-vitro cytotoxic investigation of brine shrimps

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In this study, the cytotoxicity of AgNPs and CP was determined against brine shrimps. The cytotoxic effect of AgNPs and CP increased with increasing their concentration from 25 to 100 (µg/mL). Fig. 7 revealed that at a lower concentration of 25 µg/mL AgNPs and CP revealed 47% and 42% death and 53 % and 58% survival. While at higher concentration 100 µg/mL 77 and 60 % death and 23 and 40 % survival was observed respectively. In-vitro antifungal and antimicrobial activities In the present study, it has been proved that various concentrations of AgNPs and CP 25-100 (µg/mL) exhibited the strong inhibition of fungal strains and proved significant antifungal sources (Table. 2). The results showed a higher % inhibitions of AgNPs (% inhibition 36-61

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mm) compared to CP extract (% inhibition 30-53 mm). DMSO served as a negative control. No fungal growth was observed in positive control terbinafine (Relonchem) test tubes.

In this study, the antibacterial activity of synthesised AgNPs and CP at various doses 25-100 (µg/mL) was determined by the agar diffusion method in comparison with standard

levofloxacin (GEOFMAN). In the present study, AgNPs (zone of inhibition 4-25 mm)

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exhibited higher antimicrobial activity in terms of inhibition zone offered by CP extract (zone of inhibition 3-21 mm). These results are appreciable and indicate that AgNPs and CP were

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active in all cases. DMSO served as negative control and levofloxacin (1000 µg/10mL) as a positive control (Table. 3).

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Inhibition of β-glucosidase and α-amylase

In this study, in-vitro inhibition of different concentrations of AgNPs and CP 200-800 (µg/mL) were tested against the β-glucosidase enzyme. Fig. 8 indicated that AgNPs, CP and

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tagipmet revealed 21-79%, 15-68% and 30-888% inhibition with IC50 545 µg/mL, 700 µg/mL and 340 μg/mL respectively (Fig. 9.A-9.C). AgNPs and CP was also tested against α-amylase enzyme in comparison with standard

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tagipmet at five different concentrations 20,40,60,80 and 100 (μg/mL). Fig 10 indicated that AgNPs, CP and tagipmet revealed maximum %age of inhibition (18-66%), (12-60%) and

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(36-80%) with IC50 73 µg/mL, 87 µg/mL and 45 respectively (Fig. 11.A-11.C). Discussion

Synthesis of AgNPs UV-Visible spectrophotometric analysis of AgNPs In the aqueous medium AgNPs exhibited yellowish-brown color due to the SPR. The SPR of the AgNPs obtained corresponded to the 414 nm (Fig. 1). Factors affecting the synthesis of AgNPs

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The surface plasmon resonance bands in the UV-visible spectra of AgNPs aqueous solution were noted for different volumes of CP extracts (Fig. 2.A). At a lower concentration of extract a broad-band appeared because the phytochemicals i-e phenols, saponins, flavonoids, tannins, proteins, and alkaloids, etc diminishes [27]. Fig. 2.B indicated the effect of the temperature on the synthesis of the AgNPs. At 25ºC a sharp band 414 nm appeared but this band became broader with an increase in the temperature, and a final broad band 410 appeared at 100ºC. From the temperature study, it was observed that an optimum temperature is required for the synthesis of the AgNPs due to the instability of the formed nanoparticles. Similar results were reported by [28].

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The effect of the time on the completion of the reaction of the synthesis of the AgNPs was also studied. Absorption at 409 nm appeared after 5 min of the stirring. The absorption peak rapidly increased with an increase in the reaction time due to the continuous formation of the AgNPs. A clear sharp peak of 414 nm obtained after 24 h of the reaction (Fig. 2.C). From the time study, it was observed that an optimum time is required for the completion of the

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reaction.

pH is another important factor affecting the synthesis of the AgNPs. At pH 2 to 6 no

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absorption peak was observed in the range of 420-450 for all the samples even after 24 h of the reaction. However, absorption bands at about 413, 420, 417,410, 414 and 413 nm were

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observed as the pH increase from 7 to 12, indicating the formation of the AgNPs (Fig. 2.D). From the pH study, it was observed that the absorption peak intensity increased gradually with an increase in the pH. The synthesis of the AgNPs was repressed by acidic conditions

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and improved by basic conditions [27].

Analysis through scanning electron microscopy (SEM) In the present study, the SEM image shows that AgNPs are nearly uniformly distributed in

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the matrix. It showed a high percentage of silver signals in the image. Monodispersed spherical AgNPs were formed on the surface of BPE derived biological materials as indicated

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in Fig. 3.A. The average particle size was calculated by Nano Measurer software. The particle size was calculated by marking 20 particles and the result reported that 80% AgNPs are in the average size of 50 nm (0.05 µm) Fig. 3.B. The size of the nanoparticles mainly depends on the temperature, time, pH, and nature of the plant extract and concentration [29]. Fourier transforms infrared spectrometer (FT-IR) analysis of AgNPs FTIR measurements were carried out in order to identify the presence of various functional groups in biomolecules responsible for the reduction of Ag+ and capping/stabilization of

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AgNPs. The observed intense bands were compared with standard values to identify the functional groups. Fig. 4 showed peaks at 700, 832, 993, 1333, 1620, 1730 and 2921 cm-1, these peaks correspond to different functional groups present in the biological material synthesized. The functional groups adsorbed on the AgNPs were identified by FTIR analysis. [30]. The peak at 2921 in FT-IR indicates that there must C-H stretching of an aromatic compound. The band at 1730 cm-1 was assigned for C-C stretching (non-conjugated). The band at 1620 cm-1 in the spectra corresponds to C-N and C-C stretching indicating the presence of proteins. All other peaks correspond to different vibrating stretching of functional groups [31]. A study carried out by [32] supports our results.

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X-Ray Diffraction analysis (XRD) For structural analysis, the XRD patterns of the prepared materials were studied. Four strong Bragg reflections at 38°, 44°, 64°, and 78° resemble to the planes of (1 1 1), (2 0 0), (2 2 0) and (3 1 1) respectively which can be indexed according to the face of face-centered cubic

crystal structure of silver (Fig. 5). The calculated interplaner spacing at d111, d200, d220, and

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d311 and miller constants are shown in the table 1. The calculated average crystallite of the AgNPs is 41.62 nm for three major peaks 38°, 44° and 64° of XRD. Similar results also

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reported by [33].

In-Vitro antioxidant capacity of AgNPs and CP

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In Fig. 6.A AgNPs and CP extract shows appreciable results 97% and 47% respectively at high concentration 100µg/mL. In the present study, the reducing power of AgNPs was better than the CP extract and standard ascorbic acid [19, 34]. The results of the present study, were

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also supported by [35].

In this study, AgNPs, CP extract, and standard ascorbic acid offered the inhibition in a dosedependent manner varies from 44-68%, 9-22% and 12-27% respectively (Fig. 6.B). In the

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current study, Phosphomolybdate scavenging activity of AgNPs is appreciable as compared to CP extract correlated with the work of [36].

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DPPH also known as 1, 1-diphenyl-2-picrylhydrazyl is an organic compound and is used as a free radical to check the free radical scavenging efficiency of the desired substance. The DPPH scavenging activity showed that at high concentrations 100 µg/mL the AgNPs, CP extract and ascorbic showed a high scavenging capacity of 93%, 35% and 78% respectively (Fig. 6.C). AgNPs showed the enhanced scavenging activity compared to the CP extract [37]. The color of the solution changes as we add the AgNPs which indicated that the AgNPs donate electron to DPPH due to which DPPH become stable [38]. This efficiency of the

14

biosynthesized AgNPs is due to the presence of the different phytochemicals in the extract [34]. Hydrogen peroxide is an inorganic compound also known as dioxide or oxidant is unstable species and causes huge damage to cell membranes in the living system. In this study, the hydrogen peroxide activity of different concentrations of AgNPs and CP extract 20-100 (µg/mL) was quantified spectrophotometrically using ascorbic acid as a standard (Fig. 6.D). At a high concentration, the inhibition was found to be 88, 51 and 81% for AgNPs CP extract and ascorbic acid respectively. [35] has also reported the enhanced antioxidant and biological activities of AgNPs of Iresine herbstii. The results of this study, are in good accordance with

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an earlier report of [39]. ABTS is a potent free radical which shows maximum absorbance at 734 nm but this

absorbance decreases as we increase the concentration of the AgNPs [22]. In this study, the ability of the biosynthesized AgNPs and CP extract to scavenge the ABTS free radical was

examined which decreased with increase in the dilution (Fig. 6.E). The ABTS activity results

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showed the effective free radical % scavenging potential of AgNPs and CP extract as 79 and 55% respectively. The results of the present study have also been supported by [40].

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In-vitro cytotoxic investigation of brine shrimps

Shrimps cytotoxic activities of any plant extract determine their different pharmacological

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properties. In this study, results of the cytotoxic furnished by the biosynthesized AgNPs and CP extract at different concentration 25-100 (µg/mL) were appreciable which increased in a dose-dependent manner (Fig. 7). Cytotoxic activity of the AgNPs increased with increase of

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concentration and showed enhanced cytotoxic activity compared to CP extract [41]. Due to the cytotoxic effect of the AgNPs, they can be used as a source of anticancer drugs [42]. AgNPs are effective against cancers cells [43].

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In-vitro antifungal and antimicrobial activities In the present study, the green synthesized AgNPs and CP extract were tested against three

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fungus strains A. niger, A. fumigatus and A. flavus (Table. 2). By measuring % inhibition, it was found that the higher % inhibitions of AgNPs (% inhibition 36-61 mm) were observed compared to CP extract (% inhibition 30-53 mm). AgNPs have a good antifungal effect compared to CP extract and other chemical drugs which increase with the increase in the concentration [8]. AgNPs can be used as potent antifungal agents and new drugs for therapy of Human infections [44]. The growth inhibition of various fungus strains by AgNPs at difference concentration was best supported by [45].

15

Silver has been used as an antimicrobial agent since Roman times but due to advance in new technologies, AgNPs are generated which have also the strong power of antimicrobial properties. In this study, AgNPs (zone of inhibition 4-25 mm) exhibited higher antimicrobial activity in terms of inhibition zone when compared with the CP extract alone (zone of inhibition 3-21 mm). A study carried out by [46] reported the enhanced antimicrobial activity of AgNPs using Piper longum compared to the extract to support our results. [47] has reported the antimicrobial activities of AgNPs and gold nanoparticles. AgNPs exhibited good antimicrobial activity against both Gram positive and Gram -negative bacteria (Table. 3). The maximum zone of inhibition was studied against Gram-negative compared to Gram positive due to the difference in the cell wall between Gram-positive and Gram-negative bacteria [48].

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AgNPs inhibit bacterial growth by releasing Ag+ ions which interfere with the DNA and suppress respiratory enzyme and ETC component in the bacterial cell [49]. Inhibition of β-glucosidase and α-amylase

In this study, in-vitro inhibition of AgNPs, CP extract and standard drug tagipmet were tested

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against β-glucosidase enzyme and α-amylase. Fig. 8 indicated that against β-glucosidase AgNPs, CP extract and tagipmet revealed 21-79%, 15-68% and 30-888% inhibition

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respectively. IC50 value of AgNPs, CP methanolic extract and tagipmet was found up to 545 µg/mL, 700 µg/mL and 340 μg/mL respectively (Fig. 9.A-9.C). The lower IC50 value

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suggests, the antidiabetic nature of AgNPs compared to CP methanolic extract. In this present study, AgNPs, CP extract and tagipmet revealed %age of inhibition (18-66%), (12-60%) and (36-80%) respectively (Fig. 10). IC50 value of AgNPs, CP methanolic extract

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and tagipmet against α-amylase was found up to 73 µg/mL, 87 µg/mL and 45 μg/mL respectively (Fig. 11.A-11.C). In this present high percent, inhibition was reported against αamylase compared to β–glucosidase. The inhibition of the α-amylase and β–glucosidase

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interrupt the carbohydrate degradation, which results in the decrease absorption of glucose as a result of postprandial blood glucose level elevation [50]. In the present study, AgNPs

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exhibited excellent antidiabetic activity compared to CP extract similar results were also reported by [51].

Scope and limitation of the study. This study, focused mainly on the determination of the biological property of the CP reduced nanosilver. Amongst all other methods the use of the plant extract as a bioreductant in AgNPs synthesis has achieved distinctive attention, due to preservative and sterilizing environment during the process. Among all other noble metals silver (Ag) is excellent because it is used as a health preservative medicine traditionally.

16

Nanotechnology deals with the creation of useful materials, devices and systems using the particles of nanometer length scale and exploitation of novel properties. Silver is nontoxic, safe inorganic and less expensive compared to gold. AgNPs have large surface area and small size 1-100 nm in size. The limitation of green synthesised AgNPs is obtaining small size of nanoparticle. Collection and preparation of materials is done for preliminary test of CP for AgNPs synthesis. Conclusion Our results demonstrated that the green synthesis of AgNPs was carried successfully by using Calligonum polygonoides extract. The morphology of the AgNPs depends on the extract

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concentration, temperature, reaction time and pH of the medium. Furthermore, these

nanoparticles were evaluated for their biological activities and showed higher antidiabetic, antifungal, cytotoxic, antioxidant and antimicrobial activities as compared to CP extract. AgNPs improved the biological activities of the phytochemicals.

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Aims of the study

The aims of the present study, were to analyze CP for the green synthesis of AgNPs.

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Green synthetic systems use environmentally friendly agents such as sugars, plant extracts, bacteria and fungi to form and stabilize AgNPs. It is important to measure nanosilver size, surface charge, concentration, shape, crystal structure, surface chemistry, and surface

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transformation in AgNPs synthesis.

Conflict of Interest

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The authors do not have any conflict of interest regarding this article and its publication.

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Acknowledgments

The author wishes to thanks the Higher Education Commission of Pakistan (HEC-PAK) for financial support of project No: 20-5082/NRPU/R&D/HEC.

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[47] M. Zargar, A.A. Hamid, F.A. Bakar, M.N. Shamsudin, K. Shameli, F. Jahanshiri, F. Farahani, Green synthesis and antibacterial effect of silver nanoparticles using Vitex negundo L, Molecules (Basel, Switzerland) 8(16) (2011 ) 6667-6676. [48] K. Chaloupka, Y. Malam, A.M. Seifalian, Nanosilver as a new generation of nanoproduct in biomedical applications, Trends biotechnol 28(11) (2010) 580-8. [49] Y. Li, P. Leung, L. Yao, Q.W. Song, E. Newton, Antimicrobial effect of surgical masks coated with nanoparticles, Hospital Infection Society 62(1) (2006) 58-63. [50] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Green synthesis of silver nanoparticles using latex of Jatropha curcas, Colloids and surfaces A: physicochemical and

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engineering aspects 339(1–3) (2009) 134-139. [51] K.M. Vishnu, S. Murugesan, Biogenic silver nanoparticles by Halymenia poryphyroides and its in vitro anti-diabetic efficacy, Journal of chemical and pharmaceutical research 5(12)

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(2013) 1001-1008.

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Legends of the figures

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Fig. 1. The UV–vis absorption spectrum of AgNPs

23

B

D

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C

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A

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Fig. (2.A, 2.B, 2.C and 2.D). UV–spectra of AgNPs with different volume of extract, temperature, time and pH

24

B

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A

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Fig. (3.A and 3.B). SEM analysis and SEM demonstrated the size of AgNPs

100

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95

85

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% Trasmittance

90

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80

2921

1730

1620

993

75 70 65 500

700 832

1000

1333

1500

2000

2500

3000

3500

4000

Wavelenght (cm-1) Fig. 4. FTIR analysis of AgNPs

25

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A

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Fig. 5. X-ray diffraction pattern analysis of AgNPs

26

D

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C

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E

Fig. (6.A, 6.B, 6.C, 6.D and 6.E). In-vitro reducing power, Phosoho molybdate, DPPH, Hydrogen peroxide (H2O2) and ABTS %scavenging assessment of silver nanoparticles (AgNPs) and C. polygonoides (CP).

27

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Fig. 7. Total Cytotoxic measurements of silver nanoparticles (AgNPs) and

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polygonoides (CP).

Fig. 8. In-vitro β-glucosidase inhibitory capability of silver nanoparticles (AgNPs) and C. polygonoides (CP).

28

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C

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B

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A

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Fig. (9.A, 9.B and 9.C). A plot of the percentage residual activity of β-glucosidase in the absence and presence of AgNPs, C. polygonoides and standard antidiabetic drug tagipmet versus various concentrations of AgNPs

29

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\

polygonoides

B

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A

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Fig. 10. α-amylase Inhibitory activity of silver nanoparticles (AgNPs) and

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C

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Fig. (11.A, 11.B and 11.C). A plot of the percentage residual activity of α-amylase in the absence and presence of AgNPs, C. polygonoides and standard antidiabetic drug tagipmet

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versus various concentrations of AgNPs

31

Legends of the tables

Table.1. Interplaner spacing and lattice constant 2θ Value 38

Element Ag

plane 111

Interplaner spacing (d) 2.3650 Å

Lattice constants (a0) 4.0963 Å

2

44

Ag

200

2.055 Å

4.1109 Å

3

64

Ag

220

1.4530 Å

4

78

Ag

311

1.2235 Å

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S. No 1

4.1097 Å

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4.0578 Å

Table.2. Antifungal activity of Silver nanoparticles (AgNPs) and C. polygonoides

strains 25 µg/mL

Zone of inhibition (mm) 75 µg/mL 100 µg/mL

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S. no

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(CP).

50 µg/mL

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AgNPs CP AgNPs CP AgNPs

CP

AgNPs CP terbinafine

DMSO

A. niger

41.5

30

45.5

35

46.5

37

53

41

90

10

2

A.fumigatus

61

45

57.5

47

53

50.2

60.5

53

90

nill

3

A. flavus

36

35

40.5

38

48

41

84

nill

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1

33

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38.5

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Table.3. Antibacterial activities of silver nanoparticles (AgNPs) and C. polygonoides (CP).

strains 25 µg/mL

50 µg/mL

Zone of inhibition (mm) 75 µg/mL 100 µg/mL

AgNPs CP AgNPs CP AgNPs

CP

P. vulgaris

19

12

21

15

20

15

2

S. epidermidis

9

8

20

12

19

13

3

S. aureus

15

9

16

10

19

13

4

M. luteus

15

7

21

10

20

5

K. pneumonia

16

3

19

7

6

E. coli

19

9

4

12

re

12

Levofloxacin

DMSO

25

21

30

nill

20

15

39

nill

18

16

29

nill

19

16

30

nill

-p

1

AgNPs CP

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S. no

9

23

12

28

nill

10

13

20

15

40

nill

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19

33