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EurAsian Journal of BioSciences Eurasia J Biosci 13, 2249-2260 (2019)

Synthesis, characteristics and biological activities of silver nanoparticles from Euphorbia dracunculoides

Umbreen Khattak 1, Rehman Ullah 2, Shafqat Ali Khan 1, Saiqa Afriq Jan 1, Abdur Rauf 3, Mohamed Fawzy Ramadan 4* 1 Department of Botany Faculty of Biological Sciences, Islamia College, Peshawar, PAKISTAN 2 Department of Botany, Faculty of Biological Sciences, University of Peshawar, PAKISTAN 3 Department of Chemistry, University of Swabi, Anbar, KPK, PAKISTAN 4 Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Zagazig, EGYPT *Corresponding author: mframadan@zu.edu.eg

Abstract The present study was conducted to synthesize and characterize silver nanoparticles (AgNPs) from the ethanol extract of Euphorbia dracunculoides (EEE). AgNPs were synthesized by mixing the EEE solution with silver nitrate solution at different ratios (1:1, 1:2, 1:3, 1:4, v/v). The structure and characteristics of synthesized AgNPs were studied by SEM (Scanning electron microscopy), Energy dispersive X- rays (EDX), X-ray diffraction (XRD) and FTIR. The EEE and synthesized AgNPs were subjected to investigate antioxidant, phytotoxic, cytotoxic and analgesic activities. Change in color of the solution to dark brown was the indicator of AgNPs synthesis in the EEE solution. UV-Vis spectroscopy showed maximum absorbency at 1:1 in the range of 400-500. EDX profile showed strong signal silver atom. XRD analysis indicated the crystalline shape of face-centered cubic AgNPs. FTIR analysis identified molecules responsible for the reduction of silver ion and capping. The peak (1396 cm-1) present in EEE disappears in the AgNPs indicated that -C-H bending (alkane) bound to synthesized AgNPs. SEM showed that the shape of AgNPs was spherical and its size ranged from 14.0-45.6 nm. The antiradical activity of EEE and AgNPs showed minimum absorbency 34.2% and 71.4% at 1000 µg/mL, respectively. Brine shrimp cytotoxicity assay of EEE and AgNPs showed 100% lethality at 1000 µg/mL. Phytotoxicity of EEE showed 40.2% inhibition of fronds while AgNPs showed 69.0% inhibition of fronds at 1000 µg/mL. The analgesic effect was dose-dependent. EEE and AgNPs showed maximum MLT 19.4 (129.4%) and 19.22 (135.7%) at 300 µg/mL, respectively. It could be concluded that EEE and synthesized AgNPs had potent pharmacological traits. Keywords: nanotechnology, scanning electron microscopy, energy dispersive X- rays, X-ray diffraction (XRD), FTIR Khattak U, Ullah R, Khan SA, Jan SA, Rauf A, Ramadan MF (2019) Synthesis, characteristics and biological activities of silver nanoparticles from Euphorbia dracunculoides. Eurasia J Biosci 13: 2249-2260. © 2019 Khattak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License.

INTRODUCTION The nanotechnology field possesses a large part of

the research area of the advanced material sciences. Nanoparticles (NPs) show particular characteristics of morphology and sizes, therefore exhibit new or improved properties when compared to the bulk materials. Nanotechnology is a field that is growing day-by-day, making the potential for the welfare of human beings. Nano-materials and NPs provide various applications that are increasing rapidly (Jain et al. 2019). NPs are synthesized by both physical and chemical methods. These methods are costly and use harmful chemicals, which have negative effects; therefore using a green synthesis of inorganic NPs is safer (Agrawal et al. 2014). Green synthesis of noble metals is important because they are environment-friendly and are beneficial to human health (Jae and Beom 2009). Biological synthesis of NPs is of great interest due to the

rising need to decrease toxicity, increase renewable resources, and provide environment-friendly solvents. These have captured the attention of major corporations in the last few decades (Gericke and Pinches 2006, Kamranifar et al. 2018). Silver nanoparticles (AgNPs) are synthesized by chemical, physical and biological methods. The biological methods are very useful because it provides natural capping agents and there is no need to use high pressure, temperature, toxic chemicals, and excessive energy. Bio-methods are also an inexpensive, simple and environment-friendly process. Using plant extracts to synthesize NPs is relatively cheaper than NPs synthesis by microorganisms (Naghizadeh et al. 2016). They also

Received: September 2019 Accepted: October 2019 Printed: December 2019

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have significant uses in the field of high sensitivity biomolecular detection, therapeutics, diagnostics, catalysis, microelectronics and have used as an antimicrobial agent (Singh et al. 2010).

Euphorbia dracunculoides (family Euphorbiaceae) is called dragon spurge in English. E. dracunculoides has many medicinal uses. Plant leaves are used for the treatment of epilepsy and snakebite (Sharma et al. 2010). Its fruits are used for the removal of warts from skin (Rahman et al. 2004). Lice are removed from the body of cattle by applying the decoction of the whole plant (Sikarwar et al. 1994). Local practitioners used it for purgative and diuretic purposes. From the aerial parts of the E. dracunculoides, 19 diterpenoids were isolated (Wang et al. 2015).

The present research was carried out to synthesize and characterize AgNPs prepared from E. dracunculoides ethanol extract (EEE). Biological and pharmacological traits including antioxidant, cytotoxic, phytotoxic and analgesic traits of synthesized AgNPs and EEE were also investigated.

MATERIALS AND METHODS E. dracunculoides Plant About 10 Kg of E. dracunculoides whole plant was

collected from the southern districts of Khyber Pakhtunkhwa (Pakistan). A specimen was mounted in kept on a herbarium sheet provide voucher number Khattak-UOP-Bot-2016, and kept in the Department of Botany, University of Peshawar (Pakistan). Plants were cleaned, jumbled thoroughly, dried in shade then as electric grinder was used to thrash dried plants into powder.

Preparation of E. dracunculoides Ethanol Extract (EEE)

Five hundred g of E. dracunculoides powder was dissolved in 3 L of ethanol and placed at room temperature with continuous shaking for 7 days. After 7 days, the extract was filtered off through Whatman No. 1 filter paper. The filtrate was evaporated using a rotary evaporator under reduced pressure below 50°C. To obtain EEE, the saturated or thick filtrates were set to air dry for entire dissipation of ethanol. EEE was stored in a refrigerator at 4°C and used for further research work.

Synthesis of AgNPs About 0.2 g of EEE was dissolved in 100 mL of

distilled water while 0.017 g of silver nitrate was dissolved in 100 mL distilled water, then the EEE solution was mixed with silver nitrate solution to make up 200 mL. For the green synthesis of AgNPs, different ratios (1:1, 1:2, 1:3, 1:4, v/v) of EEE solution and silver nitrate solution were mixed with a magnetic stirrer for 30 min with continuous stirring and occasional heating (45 ±5oC) till the characteristic color was obtained by the reduction of silver ions through phytometabolites indicating the formation of AgNPs. The synthesized

AgNPs showed maximum absorbency at 1:1. The sample was heat-dried to obtain AgNPs in solid form which is used for further studies. Synthesized AgNPs were investigated for structure and average size by SEM (Scanning electron microscopy), Energy dispersive X- rays (EDX), X-ray diffraction (XRD) and FTIR analysis (Roy and Das 2015).

Characteristics of Synthesized AgNPs UV-Vis spectroscopy The synthesized AgNPs were studied by UV-Vis

spectroscopy analysis. UV-Vis spectroscopy was conducted for observing the synthesis of AgNPs using UV-1602 Double Beam UV-Vis spectrophotometer with a resolution of 1 nm. The surface plasmon resonance showed the wavelength ranging from 300 to 800 nm for the synthesized AgNPs (Dipankar and Murugan 2012).

FTIR spectroscopy The synthesized AgNPs were mixed with KBr pellet.

The KBr was pressed by Hydraulic Pellet Press to prepare a sample pellet. Samples were subjected to FTIR spectroscopy using PerkinElmer spectrometer FTIR SPECTRUM ONE at a resolution of 4 cm−1 in the range of 4000-0 cm−1. EEE was also subjected to FTIR spectroscopy using the same procedure (Ranjitham et al. 2013).

X-Ray Diffraction (XRD) XRD pattern of synthesized AgNPs was analyzed

using JEOL JDX 3532 X-ray diffractometer. The diffraction pattern of NPs was recorded by nickel monochromator filtering the wave at a tube voltage of 40 kV and tube current of 30 mA with Cu-Kα radiation (λ = 1.5406 Å). The average diameter of NPs was calculated from the line width of the maximum intensity reflection peak. The sizes of AgNPs were calculated through the Scherrer’s equation.

D=Kλ/(β1/2cosθ) where “D” represents the average crystalline domain size perpendicular to the reflecting planes, λ is the X-ray wavelength (λ=1.5406 Å), K (0.89) is the Scherrer’s coefficient, θ is Bragg’s angle representing diffraction angle and β is the full width at half maximum (FWHM) in radians (Dipankar and Murugan 2012).

Scanning Electron Microscopy (SEM) The synthesized AgNPs were coated on a carbon

tape and subjected to silver coating with auto fine coater (Spi-module sputter coater) then analyzed for morphological features with SEM using FE-SEM (JSM-5910-JEOL, Japan) (Ranjitham et al. 2013).

Energy Dispersive X-Ray Spectroscopy (EDS) For the elemental composition, synthesized AgNPs

suspension in deionized water was centrifuged at 10,000 rpm for 20 min, oven-dried at 50oC and the powdered mass was used for EDX analysis using Oxford Inca 200 SEM instrument equipped with a Thermo EDX attachment (Ranjitham et al. 2013).

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Biological Activities of Synthesized AgNPs and EEE

The following bioassays were performed on EEE and synthesized AgNPs.

Antioxidant efficacy For determining antioxidant activity, the procedure of

Lalitha et al. (2013) was used. 1, 1- diphenyl-2-picryl hydrazyl (DPPH·) was used to measure EEE and synthesized AgNPs radical scavenging activity. DPPH· solution (0.1 Mm) was prepared in ethanol. Different concentrations (10, 100 and 1000 µg/mL) of EEE and synthesized AgNPs were tested. One mL of DPPH· solution was mixed with 3 mL of EEE or AgNPs at different concentrations. The dilution method was used to make the different concentrations. After shaking the mixture thoroughly, it was kept at room temperature for 30 min before being incubated in the dark. A spectrophotometer (UV-VIS Shimadzu, Japan) was used to measure the absorbance at 517 nm after 30 min, 45 min, and 60 min. Ascorbic acid was used as the standard compound.

Phytotoxic activity Limna minor was used as a test species to conduct

the phytotoxic activity using the method of Atta-ur-Rhman et al. (2001). The stock solution was prepared by adding 15 mg of EEE or AgNPs in 1.5 mL of ethanol solvent (70%). For each concentration, three sterilized Petri dishes were used in the form of replicates. 10, 100 and 1000 µg/mL were taken from the stock solution, then added to Petri dishes (3 replicates). The Petri plates were placed in laminar flow for a night to evaporate the solvent. The next day, 20 mL of E-medium was transferred to each Petri plate. Three Petri plates were used for positive (E-medium) and negative control. To each Petri plate, ten Limna minor with 3 fronds were added and placed in growth cabinet for 7 days. The number fronds were counted on the seventh day of the activity. Growth inhibition percentage was determined using the formula of Saeed et al. (2010). % 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 = 100−

Number of fronds in the test sample Number of fronds in positive control

× 100

Furthermore, the EC50 was determined using the Biostat Software version.

Cytotoxic activity (Brine shrimp egg hatching procedure)

Brine shrimp lethality bioassay was used to study the cytotoxic potential EEE and synthesized AgNPs using the method of Atta-ur-Rhman et al. (2001). Half of the hatching tray was filled with a filtered brine solution. Perforated partition split the tray into two parts of unequal sizes. Brine shrimp eggs (25 mg) were sprinkled onto the smaller part then covered with a black paper while the larger part was kept open. The temperature of the hatching tray was kept at an optimum 24°C so that the eggs could hatch. The open part of the tray was illuminated using a lamp suspended above it.

Once hatched, the nauplii swarm moved towards the illuminated part through the perforations. A stock solution was prepared by dissolving 20 mg of EEE and AgNPs in 2 mL ethanol (70%). The concentrations 10, 100 and 1000 µg/mL were taken from the stock solution and added to each sterilized vial (3 replicates). To evaporate the solvent, the vials were kept open for a night. Five mL of brine solution was added to each vial after evaporation and 10 larvae were added to each vial after hatching through Pasteur pipette. At room temperature (25°C) the vials were placed under illumination. The positive and negative control brine solution was added to 3 vials.

Analgesic activity The hot plate method was used to determine the

analgesic potency of EEE and synthesized AgNPs. Mice weighted 15-30 g were used in this experiment. The animal response was recorded in the form of withdrawing of the paws and jumps according to Arun et al. (2014). The animals were divided into five groups of six animals each. Group 1 was treated with normal saline solution (10 mL/kg body weight, i.p) and used as negative control. Group 2 was treated with diclofenac sodium solution (10 mg/kg body weight, i.p) and used as positive control. Group 3 was treated with EEE and synthesized AgNPs (100 mg/kg of each EEE and synthesized AgNPs). Group 4 was treated with 200 mg/kg of each EEE and synthesized AgNPs. Group 5 received 300 mg/kg of each EEE and synthesized AgNPs. The mice were collected in Eddy’s hot plate at a temperature of 55 ±2ºC that was carefully regulated. If any animal had baseline latencies that were excessive (less than 5 s and greater than 30 s), they were eliminated. Cut off period of 15 s was observed to avoid paw damage. Before administration of drugs and after every 30 min and 60 min, the response times of jumping and the flicking hind and forelegs were recorded.

Statistical Analysis The data of antioxidant, phytotoxic and cytotoxic

activities were analyzed through Biostat statistical Software version 5. EC50, FI50 and LD50 were determined with 95% confidence intervals (Saeed et al. 2010). For analgesic activity, mean ± SEM of the data was determined using Microsoft excel 2013 and further analyzed by one-way ANOVA using SPSS 22 software. For multiple comparisons between the control and test treated group, Dunnet test was applied. The probability level p<0.05 was considered as significant, while p<0.001 as highly significant.

RESULTS AND DISCUSSION Characteristics of Synthesized AgNPs UV-Vis spectrophotometry The color change from greenish to dark brown

confirms the existence of AgNPs. The absorption

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spectra of a reaction mixture containing aqueous silver nitrate solution (1 mM) and EEE solution revealed the synthesis of AgNPs by the reduction of Ag ions to elemental Ag. AgNPs were synthesized using different ratio (1:1, 1:2, 1:3, 1:4, v/v) of EEE solution and silver nitrate solution. The absorption spectra of these ratios were observed using UV-Vis spectrophotometer. Four different peaks were obtained for different ratios (Fig. 1). It shows maximum absorbency at 1:1 in the range of 400-500 as shown in Fig. 1.

Some other researchers also carried the UV-Visible analysis of AgNPs which were synthesized from different plants. Gnana et al. (2012) observed a change of color from yellowish to dark brown which confirmed the synthesis of AgNPs from Elettaria cardamomum. Its UV-V spectroscopy showed the absorption spectra at 460 nm. Arun et al. (2010) studied the stable AgNPs synthesized from the extract of Excoecaria agallocha. The AgNPs show maximum absorbency at the peak of

434 nm. Agrawal et al. (2014) studied the Azadirachta indica whole plant for the biological synthesis of AgNPs. Its UV-Visible spectroscopy showed the absorption spectra at the range of 400-420 nm which confirmed the formation of AgNPs. Ahmed et al. (2016) showed the formation of AgNPs by using the leaf extract of Azadirachta indica. They observed an absorption peak in the range of 436-446 nm.

EDX analysis EDX is a technique that determines the elemental

composition (Naghizadeh et al. 2017), and give information about the qualitative and quantitative status of elements which helped in the stabilization of NPs (Behravan et al. 2019). In present study, EDX was carried out to confirmed the presence of silver with no contamination. The presence of silver can be seen in the graph presented by the EDX analysis, which indicates the reduction of silver ion (Ag+) to elemental silver. As shown in Fig. 2, the spot profile of EDX of synthesized

Fig. 1. UV-Vis spectroscopy of AgNPs synthesized using different ratio (1:1, 1:2, 1:3, 1:4, v/v) of EEE solution and silver nitrate solution

Fig. 2. EDX of AgNPs synthesized from E. dracunculoides

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AgNPs showed a strong signal of the silver metal, which indicate that synthesized AgNPs contains pure silver at 3.0 KeV due to surface Plasmon resonance band. The EDX profile for the synthesized AgNPs showed strong signal silver atom. There are other elements in the graph along with silver metal which shows weaker signals such as Cl, Na, Mg, O, and C. These are biomolecules that are present in EEE bounded to the surface of synthesized AgNPs. The number of different elements present in the suspension along with silver is shown in Table 1. The EDX analysis results demonstrated that AgNPs are successfully synthesized using EEE. The EDX spectrum showed a strong signal of Ag at 3 keV which confirms the synthesis of AgNPs. Dinesh et al. (2012) synthesized AgNPs using root extract of Glycyrrhiza glabra wherein the silver absorption peak was at 3 keV due to SPR band which confirmed the synthesis of AgNPs. Akinsiku et al. (2015) studied the energy dispersive X-ray analysis of AgNPs obtained from the leaf extract of Senna occidental and Canna indica which showed the characteristic signal of the

silver element at 3 keV due to surface Plasmon resonance.

XRD analysis X-ray diffraction is an important tool, which confirms

the synthesis of AgNPs, determines its crystalline structure and calculates the crystalline particle size

(Naghizadeh et al. 2017). The crystalline nature of synthesized AgNPs from EEE was confirmed by X-Ray diffraction (XRD). XRD patterns of the synthesized AgNPs are played Bragg’s reflections representative of the fcc structure of the silver (Fig. 3). The characteristic diffraction peaks of face-centered cubic silver lattice at 38.25, 44.45, 65.00 and 77.95 in 2θ which shows corresponding planes such as (111), (200), (220), (400) indicated the crystalline shape of face-centered cubic AgNPs. The average size of synthesized AgNPs was calculated using the Scherrer’s equation by determining the full width at half maximum (FWHM) of the Bragg’s reflection, fit in card No. 4-784.

Shameli et al. (2012) described NPs synthesis using green agents. They mixed silver nitrate with polyethylene glycol and green agents. X-ray diffraction showed different peaks that confirmed the face-centered cubic structure of AgNPs. Shah et al. (2014) examined the X-ray diffraction analysis of AgNPs synthesized from Terminalia tomentosa stem bark extract. Krithiga et al.

(2015) characterized AgNPs synthesized from extracts of Solanum nigrum and Clitoria ternatea. Biologically synthesized NPs showed sharpened peaks which represent the crystalline shape of NPs. Ali et al. (2015)

discussed also the X-ray diffraction analysis of AgNPs

Table 1. The percentage of elements in resulting suspension

Element Weight (%) Atomic (%) C (K) 21.75 41.56 O (K) 33.35 47.85 Na (K) 0.79 0.78 Mg (K) 0.29 0.27 Cl ( K) 0.48 0.31 Ag (L) 43.35 9.22 Total 100.0 100.0

Fig. 3. XRD of AgNPs synthesized from E. dracunculoides

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synthesized from leaf extract of Curcuma aromatic. Different diffraction pattern peaks of AgNPs indicated the cubic, face-centered phase of silver, thus it determined the crystalline size and shape of NPs. Khatami et al. (2015) observed different diffraction peaks of synthesized AgNPs at 2θ values at 38.09, 44.15, 64.67 and 77.54 which corresponds to (111), (200), (220) and (311). It showed the crystalline shape of synthesized AgNPs obtained from Sinapis arvensis seed exudates.

FTIR analysis The organic functional groups are attached to the

surface of the NPs. FTIR procedure is used to determine the surface chemistry of NPs (Chitrani et al. 2016). FTIR is a useful technique to identify functional groups between biomolecules in the extract and metallic NPs which show the chemical composition of biomolecules attached to the surface of NPs and identify the stabilization of AgNPs (Padalia et al. 2015). FTIR spectra of EEE and its silver NPs revealed the presence of various functional groups. In FTIR spectra the absorption bands are shown in Fig. 4.

FTIR analysis was carried out to identify biomolecules responsible for the reduction of silver ion and capping of synthesized AgNPs. The FTIR spectra of EEE showed characteristics bands for functional groups such as peak at 3448cm-1 showed O-H stretch H-bonded (alcohol, phenol), for (2067cm-1 ) C≡C stretch (Alkyne), for (1635cm-1) –N-H bend (amines), for (524cm-1) C-Br stretch (alkyl halides). FTIR spectra of synthesized

AgNPs showed peaks for characteristic functional group, at 3487cm-1 it showed O-H bend alcohol, and phenol, at 2052 cm-1 it showed C≡C stretch (alkyne), at the band (1635cm-1) it showed –N-H bend (amines), at (1396cm-1) it showed -C-H bending (alkane), at the band (548cm-1) it showed C-Br stretch (alkyl halides). The bands originated from the functional groups present in various plant metabolites of the plant extract. The peak (1396cm-1) present in EEE disappears in the AgNPs indicated that –C-H bending (alkane) bound to synthesized AgNPs. This result shows that the stretch which is absent in the synthesized AgNPs was responsible for the reduction of silver ion and helped in the synthesis of AgNPs.

Maheswari et al. (2012) discussed the FTIR analysis of AgNPs for the confirmation of different functional groups in the extract of Dioscorea oppositifolia. FTIR confirmed the presence of amide and polyphenols with an aromatic ring, which stabilized AgNPs. Supraja et al. (2013) observed the active biomolecules in the Cynodon dactylon leaf extract for the reduction of silver ions which resulted in the synthesis of AgNPs. Awwad et al. (2013) carried out FTIR analysis of AgNPs which synthesized from the leaf extract of carob leaves and revealed the presence of carbonyl group of amino acid which has a strong ability to bind silver, forming a layer on the surface of AgNPs. Their result suggested that the presence of proteins, which act as stabilizing and capping agent. Prusty and Parida (2014) reported FTIR of AgNPs synthesized from the leaf extract of Eichhornia

Fig. 4. FTIR Spectroscopy of AgNPs and extract of E. dracunculoides

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crassipes. Mehmood et al. (2016) carried out FTIR of AgNPs synthesized from root bark extract of Berberis lyceum and showed the stretching vibrations of the biomolecules such as phenols, alkaloids, and flavonoids in bark extract which were responsible for the capping of AgNPs and also for the reduction and stabilization of AgNPs.

SEM analysis SEM is a technique that describes the morphology,

size, distribution, and shape of synthesized AgNPs (Ashok 2012, Ramamurthy et al. 2013). The size and shape of AgNPs synthesized from E. dracunculoides was determined by SEM. The images obtained by SEM are shown in the Fig. 5. The shape of silver nanoparticles was spherical and its size ranged from 14-45.6nm. The average size of synthesized AgNPs was 26.8nm. Ankanna et al. (2015) analyzed SEM of the biosynthesized AgNPs from the stem bark extract of Boswelliaovali foliolata. SEM analysis indicated that the NPs ranged from size 30 to 40 nm and were highly dispersed. Rajesh et al. (2012) studied the biosynthesis

of AgNPs from the ethyl acetate extract of Ulva fasciata, wherein SEM revealed that the average size of AgNPs was 40.05 nm. Ranjitham et al. (2013) characterized AgNPs synthesized from the Cauliflower floret extract by SEM and showed that the average diameter of NPs was 40-70 nm and the shape of the particles was spherical. Medda et al. (2015) studied AgNPs synthesized from leaf extract of Aloe vera, wherein SEM analysis showed that the average size of the nanoparticles was 70 nm.

Biological Traits of EEE and Synthesized AgNPs

Antioxidant activity Table 2 shows that EEE and synthesized AgNPs

inhibited more DPPH· at higher concentrations. At 10 µg/mL, EEE showed weak inhibition of DPPH· after 30, 45 and 60 min with EC50 value 2631.78. At 100 and 1000 µg/mL, EEE showed moderate inhibition of DPPH· with EC50 value 3627.99, and 3089.43, respectively. Synthesized AgNPs showed weak inhibition at 10 µg/mL with EC50 value (214.31), while at 100 and 1000 µg/mL the inhibition of DPPH· was high with EC50 181.19, and

Fig. 5. SEM images of AgNPs synthesized from E. dracunculoides

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170.46, respectively. Ascorbic acid showed the highest inhibition of DPPH· at 10, 100, 1000 µg/mL with EC50 13859.9, 14608.3 and 7926.7, respectively. The result clearly indicated that the % inhibition of DPPH· by EEE and synthesized AgNPs was dose-dependent.

Ahmad et al. (2011) studied the radical scavenging activity of Euphorbia prostrata and concluded that by increasing the concentration of plant extract, the percentage of scavenging activity increases. Lalitha et al. (2013) carried out DPPH· and hydrogen peroxide assays for Azhadirachta indica leaf extract. The DPPH· assay of leaf extract showed the highest antioxidant activity at 250 µg/mL. Johnsona et al. (2014) carried out DPPH· assay for Sida acuta and Artemisia annua leaf extracts and synthesized AgNPs. The result showed that at lower concentrations, extracts and synthesized AgNPs exhibited excellent antiradical traits. Rajakannu et al. (2015) evaluated the antiradical activity of Garciniam angostana fruit extract and synthesized AgNPs using DPPH· assay, wherein synthesized AgNPs showed the highest antioxidant activity (Ibrar et al. 2011).

Phytotoxic activity The phytotoxic activity was evaluated for EEE and

synthesized AgNPs against Lemna minor. The result showed the % inhibition of fronds using different doses of EEE and synthesized AgNPs (Table 3). At the dose 10, 100 and 1000 µg/mL, the EEE showed 15.46%, 22.68% and 40.20% inhibition of frond, respectively with FI50 value of 1419.9 µg/mL. Synthesized AgNPs showed the % inhibition of fronds 21.6%, 46% and 69.0%, respectively at the doses 10, 100 and 1000 µg/mL with FI50 value 483.94 µg/mL. The result obtained from the experiment showed that the inhibition of fronds was dose-dependent.

Gubbins et al. (2011) investigated the toxic effect of AgNPs against Lemna minor. Their study revealed that at low concentration (5 μg/L), AgNPs inhibited the plant growth after small exposure (~20 nm) and high exposure (~100 nm). With longer exposure time, their effect becomes more potent. Ullah et al. (2012) observed the phytotoxic activity of Calendula arvensis against Lemna minor and grains pests. Their result showed that at 10 µg/mL and 100 µg/mL, C. arvensis had low toxicity while at 1000 µg/mL it showed moderate toxicity. Oukarroum et al. (2013) studied the effect of AgNPs on Lemna gibba plant and found that AgNPs were a potential source of toxicity to the plant. Ibrar et al. (2015) investigated Callicarpa macrophylla bark and leaves extracts for its phytotoxic cytotoxic and acute toxicity. The phytotoxic activity of the plant leaf extract showed FI50 value of 464.55 against Lemna minor, while bark extract has no effect. Therefore, the C. macrophylla leaf extract was proved to be an advantageous natural herbicidal agent.

Cytotoxic activity Compounds isolated from natural sources are a huge

source of anti-cancer drugs and make up nearly 50% of all drugs used in clinical trials against cancer (Newman and Gragg 20007). The cytotoxic assay against brine shrimp is a reliable, cost-effective and effective process for analyzing test plant samples for anti-cancer traits (Ibrar et al. 2011). In our study, cytotoxic activity of EEE and synthesized AgNPs was carried out. The result in Table 4 showed that the % mortality using different doses of EEE and synthesized AgNPs. EEE showed 30% mortality at 10 µg/mL, at 100 µg/mL it was 43.3% and at 1000 µg/mL it showed 100% mortality with LD50 value 256.78 µg/mL. Similarly, synthesized AgNPs showed 36.6% mortality at 10 µg/mL, 60% at100 µg/mL and 100% mortality 1000 µg/mL with LD50 value 98.40

Table 2. Antiradical activity of EEE and synthesized AgNPs Concentration

(µg/mL) % DPPH· inhibition by EEE % DPPH· inhibition by synthesized AgNPs % DPPH· inhibition by ascorbate

30 min 45 min 60 min 30 min 45 min 60 min 30 min 45 min 60 min 10 23.12 24.36 22.24 24.85 27.41 28.38 71.93 71.84 72.55 100 28.59 33.89 34.06 67.16 67.25 66.90 73.08 72.02 75.19

1000 34.42 34.06 34.24 72.46 71.31 71.40 73.96 73.34 76.52 EC50 (µg/mL) 2631.78 3627.99 3089.43 214.31 181.19 170.46 13859.9 14608.3 7926.7

Table 3. Phytotoxic activity of EEE and synthesized AgNPs Dose (μg/mL) No. of fronds in test No. of fronds in control (+) Inhibition (%) FI50

EEE 10 82

97

15.46 1419.9 100 75 22.68

1000 58 40.20

AgNPs 10 76 21.64

483.94 100 54 46 1000 30 69.07

Table 4. Cytotoxic activity of EEE and synthesized AgNPs Dose (μg/mL) Total no. of larvae No. of survival larvae No. of death of larvae % mortality LD50 (mg/mL)

Control 30 30 0 0 - -

EEE 10 30 21 09 30.0

256.78 100 30 17 13 43.33 1000 30 0 30 100

Synthesized AgNPs 10 30 19 11 36.66

98.40 100 30 12 18 60 1000 30 0 30 100

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µg/mL. EEE and synthesized AgNPs showed significant cytotoxic activity by increasing the doses.

Ali et al. (2009) studied the cytotoxic activity of Euphorbia wallichii extracts and showed that at 10 µg/mL, cytotoxicity was 25-70%, while at 1000 µg/mL toxicity was 50-100%. Biswas et al. (2014) studied the cytotoxic activity of the ethanol extract of Mentha arvensis and reported that the lethality of an extract with a LC50 value of 40 µg/mL against brine shrimp and LC90 value of 160 µg/mL. Parvez et al. (2015) examined the cytotoxic traits of Euphorbia granulate Forssk against brine shrimp. At 0.1 and 1.0 mg/mL of extract, no lethality was recorded. At 2.0 mg/mL, it showed 2.5% lethality, while at 25 mg/mL; it showed significant toxicity (97.5%). Asha et al. (2015) studied the cytotoxic activity of AgNPs synthesized from Sargassum polycystum against brine shrimp and DLA Cell lines. They demonstrated that synthesized AgNPs had high lethality against brine shrimp and DLA Cell lines.

Analgesic activity The analgesic activity of the EEE and synthesized

AgNPs were studied (Table 5). EEE at various doses through i.p route (100, 200 and 300 mg/kg b.wt.) showed an increase in MLT value in different tested groups at different times. After 30 min of drug administration, the MLT value of positive control (diclofenac sodium) was 15.02 (100%). EEE at 100 mg/kg b.wt., showed MLT 13.06 (86.95%) compared to positive control MLT 15.02 (100%). At 200 mg/kg, it showed MLT 18.09 (120.43%), and at 300 mg/kg it showed MLT 19.45 (129.49%). After 60 min of drug administration, EEE showed MLT 13.89 (98.09%), at 200 mg/kg it showed 14.07 (99.36%), while at 300 mg/kg it showed 18.94 (133.75%), compared to positive control. On the other hand, after 30 min of AgNPs administration, it showed MLT 14.20 (94.54%) at 100 mg/kg compared to diclofenac sodium MLT 15.02

(100%). At 200 mg/kg, it showed 16.49 (109.78%), and at 300 mg/kg showed 16.62 (110.65%). After 60 min of AgNPs administration, it showed MLT 14.47 (102.18%) at 100 mg/kg, at 200 mg/kg it showed 17.89 (126.34%), while at 300 mg/kg it showed 19.22 (135.73%). This result showed that both EEE and synthesized AgNPs are analgesic and proved to be excellent analgesic. The effect of EEE and synthesized AgNPs was dose-dependent as shown in Table 5.

Jami et al. (2014) evaluated Terminalia chebula ethanol extract for analgesic traits and showed that at 250 mg/kg and 500 mg/kg b.wt., plant extract extended the reaction time in hot plate method. Das et al. (2015) performed the analgesic activity of leaf, flower, and stem of Tabernaemontana corymbosa extracts and reported that all extracts showed excellent analgesic traits at high doses. Rajalakshimi et al. (2015) conducted analgesic activity of Seenthil churanam using eddy’s hot plate method and acetic acid writhing test. At high dose (400 mg/kg), Seenthil churanam showed greater analgesic activity than at a low dose (200 mg/kg).

CONCLUSION EEE has a great ability for the reduction of the silver

nitrate solution which resulted in the formation of AgNPs. In the present study, synthesized AgNPs was confirmed by EDX profile, UV-Vis spectroscopy, XRD and FTIR. EEE and synthesized AgNPs comparatively showed good biological activities including antioxidant, phytotoxic, cytotoxic and analgesic traits, while synthesized AgNPs were most effective in all the respective doses. This can be beneficial for pharmacologists to discover safe and cost-effective drugs instead of synthetic drugs for the treatment of haphazard ailments.

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Table 5. Analgesic activity of EEE and synthesized AgNPs Mean Latency time in seconds (mean ± SEM)

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