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Green synthesis of silver nanoparticles using leaf extract of Holoptelea integrifolia and preliminary investigation of its antioxidant, anti-inflammatory, antidiabetic and antibacterial activities
Journal of Environmental Chemical Engineering 7 (2019) 103094
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Green synthesis of silver nanoparticles using leaf extract of Holoptelea integrifolia and preliminary investigation of its antioxidant, antiinflammatory, antidiabetic and antibacterial activities
T
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Vijay Kumara, ,1, Simranjeet Singhb,c,d,1, Bhavana Srivastavaa,1, Ragini Bhadouriae,1, Ravindra Singhf a
Department of Chemistry, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M.P., 474009, India Department of Biotechnology, Lovely Professional University, Punjab, 144111, India Regional Advanced Water Testing laboratory, Mohali, Punjab, 160059, India d Punjab Biotechnology Incubators, Mohali, Punjab, 160059, India e Department of Biotechnology, Kamla Raja Girls Collage, Gwalior, 474009, India f Department of Chemistry, Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi, 110058, India b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Holoptelea integrifolia Silver nanoparticles Antioxidant activity Anti-diabetic activity Anti-inflammatory activity Antimicrobial activity
In present study, silver nanoparticles (AgNPs) were synthesized from the aqueous extract of Holoptelea integrifolia (HI) leaves and characterized by UV–vis spectroscopy, Fourier transformation infrared spectroscopy (FTIR), field emission scan electron microscopy (FESEM), electron disperse X-ray (EDX) and X-ray diffraction (XRD) analysis. UV–vis study confirmed the formation of AgNPs. FTIR studies confirmed the presence of OH and NH functional groups of secondary molecules of HI capped on the AgNPs surface. FESEM studies revealed the formation of aggregates of spherical shape of size 32–38 nm. Presence of elemental silver was confirmed by EDX studies. Face centred cubic (FCC) crystal structure of biosynthesized AgNPs was governed by the XRD analysis. The biosynthesized AgNPs exhibited significant antioxidant activities (51.49 ± 3.33, 41.18 ± 2.27, and 74.59 ± 3.08% for the DPPH, metal chelating, and nitric oxide assay). Remarkable anti-diabetic (86.66 ± 5.03%), anti-inflammatory (binding constant 2.60 ± 0.05 × 10−4) and antibacterial (MIC from 75 to 150 μl) activities were noticed for biosynthesized AgNPs. This is the first report on the biosynthesis of AgNPs using leaves extract of HI. Results of present study could contribute to synthesize new and cost effective drugs from HI by using green approach.
1. Introduction Urbanization and industrialization have put the mammoth adverse effects on the environment. To conquer this, there is need of execution of sustainable processes following all fundamental principles of green chemistry, i.e. the development of simple, cost-effective and ecofriendly approaches for the production of advanced materials [1,2]. In the field of material and medical science, nanoparticles (NPs) have attained the worldwide attention. Metallic nanoparticles have shown multiple applications including antimicrobial, dental treatment, wound healing, surgery work, catalyst and in biomedical devices [3–5]. It has been found that NPs based drugs are more effective due to their unique optical and electrical properties [6,7]. Surface Plasmon Resonance mechanism is well known property of NPs which make them more
effective [4,6]. The medicinal and electrical properties of NPs depend on the shape, size and morphology of nanoparticles [8–10]. There are various traditional methods to synthesize the NPs including chemical, thermal, hydrothermal and photosynthesis methods, where hazardous and toxic chemicals like sodium borohydride and N, N-dimethyl formamide are used [11–15]. Further, these NPs have been used in various health and industry products, which could be toxic for health and environment [9,13,14,16]. Green Nanotechnology became an interesting field, where functional NPs of iron, zinc, silver and gold has been prepared without using hazardous/toxic chemicals [16–19]. Green synthesis of NPs is considered as more significant and cost-effective technique with special concern on environment and ecosystem conservation approach. Biosynthesis of NPs by using natural resources like plant extract, bacteria,
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Corresponding author. E-mail address: [email protected] (V. Kumar). 1 Equal contribution. https://doi.org/10.1016/j.jece.2019.103094 Received 5 March 2019; Received in revised form 6 April 2019; Accepted 13 April 2019 Available online 15 April 2019 2213-3437/ © 2019 Elsevier Ltd. All rights reserved.
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fungi, enzymes, algae etc. is an emerging field [16,17,20–24,44]. Natural resources are enriched with multiple secondary metabolites those could be utilized to synthesis of nanoparticles, which offers advantage like energy efficiency, cost-effectiveness and eco-friendliness chemicals [16,20–22]. Most significantly, biosynthesized NPs have good medicinal properties as compared to NPs synthesized by traditional methods. Biosynthesized NPs exhibit considerable antimicrobial, antioxidant, anti-inflammatory, anti-diabetic and anti-cancerous potential [16,17,20–24,44]. Due to these advanced properties, biosynthesized NPs are gaining attention in the field of agriculture, plant protection and food processing science [25–27]. During last decade, many researchers have explored silver nanoparticles (AgNPs) because of their applications like antimicrobial and anti-cancerous agents [22–27]. The advanced effect of AgNPs in the medical field was due to sustained release of nanoparticles from the aggregates which works as carriers for AgNPs [11,13,14,45]. AgNPs synthesized from plants have shown good biomedical activities. Various researchers have synthesized plant based AgNPs of size 5–100 nm [16,17,20–24,44]. These authors have preferred different methods and variable conditions (pH, temperature etc.) to discover the quality AgNPs [16,17,20]. AgNPs have shown various biomedical applications including antimicrobial, anti-parasitic, antioxidant, antidiabetic and anticancer [16,17,20–24,44]. Moreover, Holoptelea integrifolia (HI) is well documented plant in Ayurvedic System, having multiple applications to cure diseases like arthritis, inflammation, anaemia, skin diseases, liver disorders etc [28,29]. Researchers have observed antioxidant, antibacterial, antimutagenic, antivenom and antitumor qualities of HI [28,29]. The medicinal activities of HI depends on its chemical composition. The isolated and documented compounds of HI are 1, 4-naphthalenedione, hexacosanol, stigmasterol, β-amyrin, betulin, betulinic acid, epifriedlin, octacosanol, friedlin, holoptelin-A and holoptelin-B [28,29]. After detailed literature study, it has been found that preparation of nanoparticles by using HI have not attempted yet by any researcher. Hence the present study was designed and executed, where “green” chemistry was employed for the synthesis of AgNPs using leaves extract of Holoptelea integrifolia (HI). Nutrient and medicinal properties like antioxidant potential, total phenolic content (TPC), total flavonoids contents (TFC), anti-diabetic, protein interactions, anti-inflammatory and antimicrobial activities of biosynthesized AgNPs were evaluated.
nitrate solution was mixed with 100 ml of leaves extract. A colour change from light green to colloidal black indicated the formation of silver nanoparticles. After 30 min, 100 ml of reaction mixture was centrifuged for 5 min. at 5000 rpm. The solid particles as sediment were collected, washed with distilled water followed by ethanol and further dried at room temperature. The dried material was analyzed using FTIR, XRD and FESEM-EDX analysis. Formation and stability of biosynthesized AgNPs were studied using the UV–vis spectroscopic analysis. 2.3. Bio-medicinal qualities of biosynthesized AgNPs Quantitative biochemical assay like total phenolic content (TPC) and total flavonoids content (TFC), and biochemical properties like antioxidant qualities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), metal chelating (MC), and nitric oxide (NO) assays, anti-inflammatory quality (by denaturation assay), BSA proteins binding (in terms of binding constant (k)), anti-diabetic quality (using α-amylase assay) and antimicrobial properties of AgNPs were tested as per the standard protocols mentioned in our recent studies [12,30]. The detailed protocols are mentioned in Supplementary data. 2.4. Instrumentation and characterization of biosynthesized AgNPs UV-visible spectrophotometer (Shimadzu-1800; range 190–1100 cm−1) was used to characterize the AgNPs and to determine the biochemical properties. Fourier Transformation Infrared (FTIR) spectrophotometer (Shimadzu-8400; range 500–4000 cm−1) was used to characterize the AgNPs. KBr was used to prepare the disc of AgNPs. X-ray Diffraction (XRD) and Field Emission Scan Electron Microscopy with Energy Dispersive X-ray (FESEM-EDX) analysis were performed at Sophisticated Analytical Instrument Facility (SAIF) IIT Bombay. Further, the detailed conditions are mentioned in Supplementary data. 3. Results and discussions 3.1. Synthesis and characterization of biosynthesized AgNPs The size, shape and morphology of nanoparticles depend on various physicochemical conditions [30–32]. On mixing the leaves extract with AgNO3 solution, a colour change from light green to colloidal black was observed within few minutes which indicates–the formation of AgNPs (Fig. 1). In aqueous solution, AgNPs exhibit the strong surface plasmon resonance [1,7,13]. The maximum absorption of nanoparticles was about 450 nm with minimum absorption as compared to the pure extract of HI, which indicted the minimum particle size. Broadness of the absorption spectrum indicated the large distribution of particles that dominates the electronic transition [1,7,13]. According to the “Mie Theory” narrow the UV–vis spectra, larger the size of synthesized nanoparticles [1,7,13]. The AgNPs were generated from the solution of AgNO3 through the reduction of Ag+ to Ag0 by the secondary metabolites (flavonoids, terpenoids, phenols etc.) of HI. Further, excess metabolites of HI have formed the aggregates through capping mechanism [1,7,13]. Fig. 2 show the increase in absorption with passage of time due to the enhancement in the formation of AgNPs upto first 40 min. No change in absorption was noticed after 40 min indicated the reduction of all silver ions into AgNPs. Further, the stability of biosynthesized AgNPs were tested after 5 days, where small decrease in absorbance was noticed (i.e. 0.41–0.39) with minor change in wavelength (towards higher wavelength i.e. 450 nm–460 nm) (Fig. 2). Only minor decrease in the absorbance indicted the good storage ability of biosynthesized AgNPs. The slight decrease in absorbance is attributed to the change in capping ability of various molecules of HI. Initially, maximum number of molecules will adsorbed on the surface and remaining molecules will show the association and dissociation mechanisms which leads to the change in absorbance and wavelength
2. Materials and methods 2.1. Chemical and reagents Silver nitrate, gallic acid, quercetin, ascorbic acid (AA), butylhydroxytoluene (BHT), Folin Ciocalteau reagent, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ethylenediamine tetra acetic acid (EDTA), sulfanilic acid (SA), napthyldiamine dichloride (NED), bovine serum albumin (BSA), sodium carbonate, aluminium chloride salt, phosphomolybdenum reagent, ferrozine, ferrous ammonium sulphate, α-amylase and dinitro-salicylic acid were of AR grade and used without any purification. 2.2. Green synthesis of AgNPs Plant extract was prepared from the leaves of Holoptelea integrifolia (HI). Before the preparation of extract, leaves were washed with tap water followed by distilled water to remove dust particles and other impurities. Shade dried leaves were powdered by using mixture grinder. 8 gm of powder was put into 200 ml distilled water and heated at 60 °C for 15 min. cooled and the supernatant was filtered using Whatman filter paper No. 1. A light green extract as clear solution was obtained and stored at 4–10 °C. 2 mM solution of silver nitrate was prepared by dissolving 0.0225 gm of AgNO3 in 75 ml of distilled water. Then 50 ml of silver 2
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Fig. 1. Green synthesis of silver nanoparticles (AgNPs). a. Leaves of HI. 1. AgNO3 solution, 2. Aqueous extract of Holoptelea Integrifolia (HI), and 3. Colloidal solution of silver nanoparticles. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
Surface morphology, shape and approximate size of AgNPs were checked through FE-SEM analysis technique (Fig. 4). In Fig. 4, the formation of aggregates of AgNPs has been depicted (Fig. 4a & b), where silver nanoparticles were coated with secondary metabolites of HI. The aggregates of AgNPs were spherical in shape having size between the range 32– 38 nm (Fig. 4b). The main reason behind the formation of aggregates was attributed to the capping mechanism at reduced silver ion, which is surrounded with secondary metabolites of plant extract [16,17,20,21]. Sonication is the way to avoid the aggregations between nanoparticles and secondary metabolites [38]. To check the capped biomolecules with AgNPs, FTIR study was performed to confirm the functional groups and compared with FTIR spectrum of leaves extract (Fig. 5). Fig. 5 (1) has depicted peaks at 3420 ± 10 cm−1 (corresponding to eOH and eNH groups of phenols and amines), 3000 ± 10 cm−1 and 2915 ± 10 cm−1 (corresponding to asymmetric stretch of eCH), 2360 ± 10 cm−1 (corresponding to symmetric stretch of eCH) and 1654 ± 10 cm−1 (corresponding to eCH of benzene ring and eNH). The peaks at 1437, 1410, 1315, 1024, 953, 705 and 671 cm−1 corresponding to the functional basic groups of extract [22–24,44]. Broadening as well shifting of peaks was noticed in the FTIR spectrum of AgNPs (Fig. 5(2)), which indicted the reduction of AgNO3 to corresponding nanoparticles with formation of aggregates having detectable functional groups. The peaks at 3356 ± 10 cm−1 (corresponding to eOH and eNH groups of phenols and amines), 2919 ± 10 cm−1 (corresponding to asymmetric stretch of eCH), 2359 ± 10 cm−1 (corresponding to symmetric stretch of eCH) and 1651 ± 10 cm−1 (corresponding to eCH of benzene ring and eNH) were observed. The peaks at 1448, 1376, 1237, 1035, 778 and 668 cm-1 corresponding to the functional groups of extract [22–24,44].
Fig. 2. UV visible spectrum of HI (aqueous extract) and AgNPs.
[13,33,34]. The molecules extracted from the plants are mostly amorphous in nature [1]. Crystalline nature of AgNPs was confirmed through XRD analysis where 2Ѳ values were taken on X-axis and corresponding intensities were taken on Y-axis (Fig. 3). The crystalline nature of the synthesized AgNPs was checked by using XRD analysis in the range of 5 - 90° at 2Ѳ angle. Fig. 3 showed the XRD pattern of the synthesized AgNPs where four diffraction peaks at ˜38°, ˜45°, ˜64°, and ˜78° attributed to the (111), (200), (220), and (311) planes. This pattern corresponds to the FCC (Face Centre Cubic) crystal structure of biosynthesized AgNPs, which was confirmed by various researchers recently [1,34]. The XRD peaks at ˜28°, and ˜82° were attributed to the Cl in FCC structure [1,7,34]. Elemental composition of AgNPs has been analyzed by using SEM-EDX analysis. A strong peak at 3 KeV confirmed the presence of silver ions in AgNPs (Fig. 4c). The observed percentage of elemental and atomic weight of silver ions were 71.32 and 27.82% respectively. Ag was observed along with other elements like Ca, O, Cu, and Ir. Elements like O and Ca are secondary sources of elements detected in XRD which come from plant extract [1,34]. Presence of elements including Cu and Fe may attribute to contamination in raw materials. The contaminations of Cu is most likely from the TEM grid
3.2. Antioxidant potential of green synthesized AgNPs Antioxidant activity refers to the formation of non-reactive stable radicals through the inhibition of the oxidation of molecules by preventing the initiation step of the oxidative chain reaction [35–37]. The antioxidant potential of biosynthesised nanoparticles depends on the properties of various phytochemicals encrusted on their surface [21,38,44]. Here, total phenolic content (TPC) and total flavonoids content (TFC) values of the colloidal solution of synthesized AgNPs were checked and reported as gallic acid equivalent (GAE/g dry wt of leaves) and quercetin equivalent (QE/g dry wt of leaves) respectively. To calculate the TPC and TFC, standard curves of gallic acid and quercetin were plotted by taking concentration on X-axis and absorbance at Y-axis. The observed equation of line for TPC and TFC was Y = 0.009X + 0.005 with R2 = 0.999 and Y = 0.011X + 0.028 with R2 = 0.998 respectively. The TFC and TPC value of extract was check and the observed values were 7.18 ± 1.02 mg GAE/g dry wt of leaves and 1.00 ± 0.09 mg QE/g dry wt of leaves respectively which was less than silver nanoparticles of HI. The observed value of TPC and TFC for
Fig. 3. XRD pattern of aggregates of AgNPs. 3
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Fig. 4. FESEM/EDX analysis of AgNPs.
at 100 μL which was 51.49 ± 3.33% which was more than value of extract of HI i.e. 45.91 ± 2.54%. The observed percentage values (at 100 μL) for the standards AA, and BHT were 97.41 ± 4.49, and 78.93 ± 2.49% respectively. The results of present study were similar to that of study done by [28], where ethanolic extract of stem bark of HI has shown antioxidant value (65%) in DPPH assay [28,29,31]. In recent studies, for DPPH antioxidant assay, the % inhibition of various extracts of leaves of HI was 75.78% (methanolic), 89.21% (acetone), and 87.49% (ethyl acetate) [28]. Here, biosynthesized AgNPs have shown low percentage of inhibition as compared to reported data. For HI, no antioxidant study was found using MC and NO assays. In MC assay, as like DPPH assay, AgNPs have shown concentration depended activities. At higher concentration (at 100 μL), the observed values for the extract, AgNPs and EDTA (control) were 45.55 ± 1.81, 41.18 ± 2.27 and 94.24 ± 5.17% respectively. In case of NO antioxidant assay, AgNPs have shown similar trend as like that of DPPH and MC assays. The observed values for the extract, AgNPs, AA and BHT were 65.24 ± 4.11, 74.59 ± 3.08, 94.44 ± 4.88 and 91.23 ± 4.53% respectively. Here, good activity was noticed as compared to above mentioned two assays viz DPPH and MC. The lower values in case of DPPH and MC assay may be attributed to the presence of secondary metabolites in low concentrations. In case of MC assay, secondary metabolites have capping mechanism with silver metal, so they are not free, hence low MC properties are acceptable. Different bio-ingredients have different antioxidant properties with in different antioxidant assays, which help in reducing oxidative stress in cells [26,27,39–41]. Present study confirms that the antioxidant properties of HI still retained by the biosynthesized AgNPs through the capping mechanism. Colloidal solution of AgNPs exhibited average to good
Fig. 5. FTIR Spectrum of HI (1) and AgNPs (2).
the AgNPs was 11.18 ± 1.07 mg GAE/g dry wt of sample and 1.14 ± 0.15 mg QE/g dry wt of sample respectively. The observed TFC value was small, but significant TPC value was noticed, which attributed to the capped secondary metabolites of HI as confirmed by the SEM analysis. The capping of reduced silver by secondary metabolites may augment the involvement of synthesized AgNPs towards the antioxidant properties. Total antioxidant capacity of standards as well as colloidal solution of AgNPs was calculated by using Phosphomolybdenum Assay. The observed antioxidant activity for BHT (standards), extract, and AgNPs was 98.31 ± 5.17, 85.12 ± 4.44 and 90.55 ± 3.19% respectively. Clearly, AgNPs has exhibited very good total antioxidant capacity. In DPPH assay, concentration depended effect was noticed for the AgNPs (Fig. 6). AgNPs has shown highest DPPH scavenging properties 4
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Fig. 7. Binding constant study of AgNPs.
3.4. Anti-diabetic qualities of green synthesized AgNPs In the era of technology, the more shocking fact is that, there is no well established medication or therapy to cure diabetes [10,23,27]. Diabetie(s) depend on the lifestyle and food habit of an individual. Plants having antidiabetic qualities can contribute as significant source for the development of secure and economical antidiabetic drugs [1–3]. Here, antidiabetic properties of the green synthesized AgNPs were tested by using the α-amylase assay. Colorimetric assay (at 540 nm) was used, where acarbose used as a standard. The antidiabetic property of green synthesized AgNPs was found increase in a dose dependent manner and 60.08 ± 3.38% of inhibition was observed by employing the 25 μL of AgNPs (Fig. 8). At same concentration (25 μL), standard (acarbose) and extract has shown 72.22 ± 4.31 and 45.88 ± 3.44% of inhibition. At highest concentration (100 μL), the observed % of inhibition of extract, AgNPs and acarbose were 71.28 ± 4.33, 86.66 ± 5.03 and 95.01 ± 5.41% respectively. Biosynthesized AgNPs exhibits very good antidiabetic activities and could be used as antidiabetic agents. In literature, various extracts of HI have shown antidiabetic applications through the inhibition of the ATP-sensitive potassium channels in pancreatic beta cells mechanism [28,29]. Here, enhanced effect of biosynthesized AgNPs was noticed (Fig. 9).
Fig. 6. Antioxidant studies of AgNPs.
antioxidant properties; it could serve as a free radical scavenger, possibly acting as a primary antioxidant having health beneficial metal like silver including various phytochemicals. Synthesized AgNPs may be a good alternate against synthetic antioxidants like butylated hydroxyl toluene, butylated hydroxyl anisole, and propyl gallate having adverse health effects [14,15,46].
3.3. Anti-inflammatory and protein binding qualities of green synthesized AgNPs
3.5. Antibacterial qualities of green synthesized AgNPs AgNPs are known for their antimicrobial properties, especially antibacterial. Higher surface to volume ratio of NPs enhanced the interactions of NPs with sulphur and phosphorus containing constituents of the bacterial cell leads to death of microbe(s) used [8–10]. In present study, biosynthesized AgNPs were checked for their antibacterial effect against two bacterial strains (Escherichia coli and Salmonella typhimurium) by using the disc diffusion method. Zone of inhibition (diameter in mm) was concentration dependent (Table 1 & Supplementary data S9). Biosynthesized AgNPs exhibited the antimicrobial effects, but
Green synthesized AgNPs were tested for their anti-inflammatory properties by employing the heat induced action on BSA protein at optimum conditions. The UV–vis spectroscopic study was performed at 660 nm. AgNPs has shown concentration dependent effects. As compared to standard (acetyl salicylic acid), AgNPs have shown 4 times lesser efficiency to reduce the heat induced effect on BSA protein. In previous studies, aqueous and ethanolic extracts of HI have shown good anti-inflammatory activities on rats (through carrageenan- induced paw edema test) as compared to standard indomethacin [28,29]. In present study, the low values efficacy of AgNPs towards inhibition of inflammation was attributed to the non-interactions of larger secondary metabolites of AgNPs with complex protein at higher temperature 51 ○C [42,43]. Protein binding ability of AgNPs was tested by using the UV–vis spectrophotometric method at 278 nm, where the complexation behaviour of the AgNPs was studied as per the protocol described by [12]. The binding ability of AgNPs with BSA was reported in terms of binding constant (k) (Fig. 7). The observed values of the binding constants (k) for the standard (acetyl salicylic acid), extract and AgNPs were 2.60 ± 0.05 × 10−4 μM-1, 3.21 ± 0.09 × 10−4 μM-1 and 2.01 ± 0.06 × 10-4 μM-1 respectively. Both, standard as well AgNPs have almost closest values which highlights the utility of biosynthesized AgNPs as a potent drug in future [29–32].
Fig. 8. Anti-diabetic study of AgNPs. 5
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Conflict of interest There is no statement on conflict of interest. Acknowledgements We are thankful to SAIF-IIT, Bombay for instrumentations. We are thankful to Director General, CCRAS for his motivation and guidance. Dr. Om Prakash, and Dr. Arjun Singh are highly acknowledge for their motivation and support. Same time RARIDD, Gwalior is highly acknowledged for laboratory and library facilities.
Fig. 9. Antimicrobial activities of the AgNPs-HI at 25 (a), 50 (b), 100 (c) and 200 μL (d).
Appendix A. Supplementary data Table 1 Antibacterial activities of AgNPs. Strain
Escherichia coli
Salmonella typhimurium
Conc (μL)
50 100 200 400 50 100 200 400
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jece.2019.103094.
Inhibition zone (mm) Extract
AgNPs
– – 4 8 – 5 9 11
– 2 4 10 2 7 11 13
Control MIC (μL)
MIC of Extract/ AgNPs (μL)
> 48
> 200/150
> 31
> 150/100
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lesser than control (Chloramphenicol). Minimum inhibitory concentration (MIC) was found by using the serial dilution method. It was found that with increase in the concentrations of AgNPs solution, the turbidity was decreased. The observed MIC values of the AgNPs against Escherichia coli and Salmonella typhimurium were approximately 150 μL and 100 μL respectively those were more significant than extract of HI leaves (Table 1). Here, we found low to mild antibacterial activities of biosynthesized AgNPs and extract (AgNPs > extract), which may be attributed to the presence of least number of secondary metabolites of HI those having antibacterial activities. The minimum or average activity of any drug against one or two strains does not mean that it is non-effective against other microorganisms [11,14,15]. Properties and effects are variable from drug to drug, which depend on the presence of chemical constituents present in drug(s) [11–15]. So, there is need to check the antibacterial activity of biosynthesized AgNPs against some other microorganisms also. Overall, if we compare the medicinal activities like antioxidant, anti-inflammatory, antidiabetic and antibacterial, as compared to extract of HI, biosynthasized AgNPs have shown good antidiabetic activities followed by antioxidant, anti-inflammatory, and antibacterial activities. In nut shell, we can say that bio-synthesized AgNPs exhibit very good antidiabetic and antioxidant activities. 4. Conclusion Green nanotechnology is considered as the eco-friendly and energy efficient technique for the production of nanoparticles without damaging the environment. Various plant materials and herbal drugs have been explored as a potential tool for the green synthesis of nanoparticles. In present study, leaves extract of HI was used for the green synthesis of spherical shape silver nanoparticles with size range 32–38 nm. The biological studies of biosynthesized nanoparticles suggests that they can serve as promising antiradical, anti-inflammatory, antimicrobial and antidiabetic agents. Moreover, further research is required to develop the nanoparticles of specific size and shape. Most importantly, there is need to check the role of most abundant chemical constituents of HI towards the synthesis of nanoparticles and their specific activities. 6
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Green synthesis of silver nanoparticles using leaf extract of Holoptelea integrifolia and preliminary investigation of its antioxidant, antiinflammatory, antidiabetic and antibacterial activities
T
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Vijay Kumara, ,1, Simranjeet Singhb,c,d,1, Bhavana Srivastavaa,1, Ragini Bhadouriae,1, Ravindra Singhf a
Department of Chemistry, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M.P., 474009, India Department of Biotechnology, Lovely Professional University, Punjab, 144111, India Regional Advanced Water Testing laboratory, Mohali, Punjab, 160059, India d Punjab Biotechnology Incubators, Mohali, Punjab, 160059, India e Department of Biotechnology, Kamla Raja Girls Collage, Gwalior, 474009, India f Department of Chemistry, Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi, 110058, India b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Holoptelea integrifolia Silver nanoparticles Antioxidant activity Anti-diabetic activity Anti-inflammatory activity Antimicrobial activity
In present study, silver nanoparticles (AgNPs) were synthesized from the aqueous extract of Holoptelea integrifolia (HI) leaves and characterized by UV–vis spectroscopy, Fourier transformation infrared spectroscopy (FTIR), field emission scan electron microscopy (FESEM), electron disperse X-ray (EDX) and X-ray diffraction (XRD) analysis. UV–vis study confirmed the formation of AgNPs. FTIR studies confirmed the presence of OH and NH functional groups of secondary molecules of HI capped on the AgNPs surface. FESEM studies revealed the formation of aggregates of spherical shape of size 32–38 nm. Presence of elemental silver was confirmed by EDX studies. Face centred cubic (FCC) crystal structure of biosynthesized AgNPs was governed by the XRD analysis. The biosynthesized AgNPs exhibited significant antioxidant activities (51.49 ± 3.33, 41.18 ± 2.27, and 74.59 ± 3.08% for the DPPH, metal chelating, and nitric oxide assay). Remarkable anti-diabetic (86.66 ± 5.03%), anti-inflammatory (binding constant 2.60 ± 0.05 × 10−4) and antibacterial (MIC from 75 to 150 μl) activities were noticed for biosynthesized AgNPs. This is the first report on the biosynthesis of AgNPs using leaves extract of HI. Results of present study could contribute to synthesize new and cost effective drugs from HI by using green approach.
1. Introduction Urbanization and industrialization have put the mammoth adverse effects on the environment. To conquer this, there is need of execution of sustainable processes following all fundamental principles of green chemistry, i.e. the development of simple, cost-effective and ecofriendly approaches for the production of advanced materials [1,2]. In the field of material and medical science, nanoparticles (NPs) have attained the worldwide attention. Metallic nanoparticles have shown multiple applications including antimicrobial, dental treatment, wound healing, surgery work, catalyst and in biomedical devices [3–5]. It has been found that NPs based drugs are more effective due to their unique optical and electrical properties [6,7]. Surface Plasmon Resonance mechanism is well known property of NPs which make them more
effective [4,6]. The medicinal and electrical properties of NPs depend on the shape, size and morphology of nanoparticles [8–10]. There are various traditional methods to synthesize the NPs including chemical, thermal, hydrothermal and photosynthesis methods, where hazardous and toxic chemicals like sodium borohydride and N, N-dimethyl formamide are used [11–15]. Further, these NPs have been used in various health and industry products, which could be toxic for health and environment [9,13,14,16]. Green Nanotechnology became an interesting field, where functional NPs of iron, zinc, silver and gold has been prepared without using hazardous/toxic chemicals [16–19]. Green synthesis of NPs is considered as more significant and cost-effective technique with special concern on environment and ecosystem conservation approach. Biosynthesis of NPs by using natural resources like plant extract, bacteria,
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Corresponding author. E-mail address: [email protected] (V. Kumar). 1 Equal contribution. https://doi.org/10.1016/j.jece.2019.103094 Received 5 March 2019; Received in revised form 6 April 2019; Accepted 13 April 2019 Available online 15 April 2019 2213-3437/ © 2019 Elsevier Ltd. All rights reserved.
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fungi, enzymes, algae etc. is an emerging field [16,17,20–24,44]. Natural resources are enriched with multiple secondary metabolites those could be utilized to synthesis of nanoparticles, which offers advantage like energy efficiency, cost-effectiveness and eco-friendliness chemicals [16,20–22]. Most significantly, biosynthesized NPs have good medicinal properties as compared to NPs synthesized by traditional methods. Biosynthesized NPs exhibit considerable antimicrobial, antioxidant, anti-inflammatory, anti-diabetic and anti-cancerous potential [16,17,20–24,44]. Due to these advanced properties, biosynthesized NPs are gaining attention in the field of agriculture, plant protection and food processing science [25–27]. During last decade, many researchers have explored silver nanoparticles (AgNPs) because of their applications like antimicrobial and anti-cancerous agents [22–27]. The advanced effect of AgNPs in the medical field was due to sustained release of nanoparticles from the aggregates which works as carriers for AgNPs [11,13,14,45]. AgNPs synthesized from plants have shown good biomedical activities. Various researchers have synthesized plant based AgNPs of size 5–100 nm [16,17,20–24,44]. These authors have preferred different methods and variable conditions (pH, temperature etc.) to discover the quality AgNPs [16,17,20]. AgNPs have shown various biomedical applications including antimicrobial, anti-parasitic, antioxidant, antidiabetic and anticancer [16,17,20–24,44]. Moreover, Holoptelea integrifolia (HI) is well documented plant in Ayurvedic System, having multiple applications to cure diseases like arthritis, inflammation, anaemia, skin diseases, liver disorders etc [28,29]. Researchers have observed antioxidant, antibacterial, antimutagenic, antivenom and antitumor qualities of HI [28,29]. The medicinal activities of HI depends on its chemical composition. The isolated and documented compounds of HI are 1, 4-naphthalenedione, hexacosanol, stigmasterol, β-amyrin, betulin, betulinic acid, epifriedlin, octacosanol, friedlin, holoptelin-A and holoptelin-B [28,29]. After detailed literature study, it has been found that preparation of nanoparticles by using HI have not attempted yet by any researcher. Hence the present study was designed and executed, where “green” chemistry was employed for the synthesis of AgNPs using leaves extract of Holoptelea integrifolia (HI). Nutrient and medicinal properties like antioxidant potential, total phenolic content (TPC), total flavonoids contents (TFC), anti-diabetic, protein interactions, anti-inflammatory and antimicrobial activities of biosynthesized AgNPs were evaluated.
nitrate solution was mixed with 100 ml of leaves extract. A colour change from light green to colloidal black indicated the formation of silver nanoparticles. After 30 min, 100 ml of reaction mixture was centrifuged for 5 min. at 5000 rpm. The solid particles as sediment were collected, washed with distilled water followed by ethanol and further dried at room temperature. The dried material was analyzed using FTIR, XRD and FESEM-EDX analysis. Formation and stability of biosynthesized AgNPs were studied using the UV–vis spectroscopic analysis. 2.3. Bio-medicinal qualities of biosynthesized AgNPs Quantitative biochemical assay like total phenolic content (TPC) and total flavonoids content (TFC), and biochemical properties like antioxidant qualities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), metal chelating (MC), and nitric oxide (NO) assays, anti-inflammatory quality (by denaturation assay), BSA proteins binding (in terms of binding constant (k)), anti-diabetic quality (using α-amylase assay) and antimicrobial properties of AgNPs were tested as per the standard protocols mentioned in our recent studies [12,30]. The detailed protocols are mentioned in Supplementary data. 2.4. Instrumentation and characterization of biosynthesized AgNPs UV-visible spectrophotometer (Shimadzu-1800; range 190–1100 cm−1) was used to characterize the AgNPs and to determine the biochemical properties. Fourier Transformation Infrared (FTIR) spectrophotometer (Shimadzu-8400; range 500–4000 cm−1) was used to characterize the AgNPs. KBr was used to prepare the disc of AgNPs. X-ray Diffraction (XRD) and Field Emission Scan Electron Microscopy with Energy Dispersive X-ray (FESEM-EDX) analysis were performed at Sophisticated Analytical Instrument Facility (SAIF) IIT Bombay. Further, the detailed conditions are mentioned in Supplementary data. 3. Results and discussions 3.1. Synthesis and characterization of biosynthesized AgNPs The size, shape and morphology of nanoparticles depend on various physicochemical conditions [30–32]. On mixing the leaves extract with AgNO3 solution, a colour change from light green to colloidal black was observed within few minutes which indicates–the formation of AgNPs (Fig. 1). In aqueous solution, AgNPs exhibit the strong surface plasmon resonance [1,7,13]. The maximum absorption of nanoparticles was about 450 nm with minimum absorption as compared to the pure extract of HI, which indicted the minimum particle size. Broadness of the absorption spectrum indicated the large distribution of particles that dominates the electronic transition [1,7,13]. According to the “Mie Theory” narrow the UV–vis spectra, larger the size of synthesized nanoparticles [1,7,13]. The AgNPs were generated from the solution of AgNO3 through the reduction of Ag+ to Ag0 by the secondary metabolites (flavonoids, terpenoids, phenols etc.) of HI. Further, excess metabolites of HI have formed the aggregates through capping mechanism [1,7,13]. Fig. 2 show the increase in absorption with passage of time due to the enhancement in the formation of AgNPs upto first 40 min. No change in absorption was noticed after 40 min indicated the reduction of all silver ions into AgNPs. Further, the stability of biosynthesized AgNPs were tested after 5 days, where small decrease in absorbance was noticed (i.e. 0.41–0.39) with minor change in wavelength (towards higher wavelength i.e. 450 nm–460 nm) (Fig. 2). Only minor decrease in the absorbance indicted the good storage ability of biosynthesized AgNPs. The slight decrease in absorbance is attributed to the change in capping ability of various molecules of HI. Initially, maximum number of molecules will adsorbed on the surface and remaining molecules will show the association and dissociation mechanisms which leads to the change in absorbance and wavelength
2. Materials and methods 2.1. Chemical and reagents Silver nitrate, gallic acid, quercetin, ascorbic acid (AA), butylhydroxytoluene (BHT), Folin Ciocalteau reagent, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ethylenediamine tetra acetic acid (EDTA), sulfanilic acid (SA), napthyldiamine dichloride (NED), bovine serum albumin (BSA), sodium carbonate, aluminium chloride salt, phosphomolybdenum reagent, ferrozine, ferrous ammonium sulphate, α-amylase and dinitro-salicylic acid were of AR grade and used without any purification. 2.2. Green synthesis of AgNPs Plant extract was prepared from the leaves of Holoptelea integrifolia (HI). Before the preparation of extract, leaves were washed with tap water followed by distilled water to remove dust particles and other impurities. Shade dried leaves were powdered by using mixture grinder. 8 gm of powder was put into 200 ml distilled water and heated at 60 °C for 15 min. cooled and the supernatant was filtered using Whatman filter paper No. 1. A light green extract as clear solution was obtained and stored at 4–10 °C. 2 mM solution of silver nitrate was prepared by dissolving 0.0225 gm of AgNO3 in 75 ml of distilled water. Then 50 ml of silver 2
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Fig. 1. Green synthesis of silver nanoparticles (AgNPs). a. Leaves of HI. 1. AgNO3 solution, 2. Aqueous extract of Holoptelea Integrifolia (HI), and 3. Colloidal solution of silver nanoparticles. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
Surface morphology, shape and approximate size of AgNPs were checked through FE-SEM analysis technique (Fig. 4). In Fig. 4, the formation of aggregates of AgNPs has been depicted (Fig. 4a & b), where silver nanoparticles were coated with secondary metabolites of HI. The aggregates of AgNPs were spherical in shape having size between the range 32– 38 nm (Fig. 4b). The main reason behind the formation of aggregates was attributed to the capping mechanism at reduced silver ion, which is surrounded with secondary metabolites of plant extract [16,17,20,21]. Sonication is the way to avoid the aggregations between nanoparticles and secondary metabolites [38]. To check the capped biomolecules with AgNPs, FTIR study was performed to confirm the functional groups and compared with FTIR spectrum of leaves extract (Fig. 5). Fig. 5 (1) has depicted peaks at 3420 ± 10 cm−1 (corresponding to eOH and eNH groups of phenols and amines), 3000 ± 10 cm−1 and 2915 ± 10 cm−1 (corresponding to asymmetric stretch of eCH), 2360 ± 10 cm−1 (corresponding to symmetric stretch of eCH) and 1654 ± 10 cm−1 (corresponding to eCH of benzene ring and eNH). The peaks at 1437, 1410, 1315, 1024, 953, 705 and 671 cm−1 corresponding to the functional basic groups of extract [22–24,44]. Broadening as well shifting of peaks was noticed in the FTIR spectrum of AgNPs (Fig. 5(2)), which indicted the reduction of AgNO3 to corresponding nanoparticles with formation of aggregates having detectable functional groups. The peaks at 3356 ± 10 cm−1 (corresponding to eOH and eNH groups of phenols and amines), 2919 ± 10 cm−1 (corresponding to asymmetric stretch of eCH), 2359 ± 10 cm−1 (corresponding to symmetric stretch of eCH) and 1651 ± 10 cm−1 (corresponding to eCH of benzene ring and eNH) were observed. The peaks at 1448, 1376, 1237, 1035, 778 and 668 cm-1 corresponding to the functional groups of extract [22–24,44].
Fig. 2. UV visible spectrum of HI (aqueous extract) and AgNPs.
[13,33,34]. The molecules extracted from the plants are mostly amorphous in nature [1]. Crystalline nature of AgNPs was confirmed through XRD analysis where 2Ѳ values were taken on X-axis and corresponding intensities were taken on Y-axis (Fig. 3). The crystalline nature of the synthesized AgNPs was checked by using XRD analysis in the range of 5 - 90° at 2Ѳ angle. Fig. 3 showed the XRD pattern of the synthesized AgNPs where four diffraction peaks at ˜38°, ˜45°, ˜64°, and ˜78° attributed to the (111), (200), (220), and (311) planes. This pattern corresponds to the FCC (Face Centre Cubic) crystal structure of biosynthesized AgNPs, which was confirmed by various researchers recently [1,34]. The XRD peaks at ˜28°, and ˜82° were attributed to the Cl in FCC structure [1,7,34]. Elemental composition of AgNPs has been analyzed by using SEM-EDX analysis. A strong peak at 3 KeV confirmed the presence of silver ions in AgNPs (Fig. 4c). The observed percentage of elemental and atomic weight of silver ions were 71.32 and 27.82% respectively. Ag was observed along with other elements like Ca, O, Cu, and Ir. Elements like O and Ca are secondary sources of elements detected in XRD which come from plant extract [1,34]. Presence of elements including Cu and Fe may attribute to contamination in raw materials. The contaminations of Cu is most likely from the TEM grid
3.2. Antioxidant potential of green synthesized AgNPs Antioxidant activity refers to the formation of non-reactive stable radicals through the inhibition of the oxidation of molecules by preventing the initiation step of the oxidative chain reaction [35–37]. The antioxidant potential of biosynthesised nanoparticles depends on the properties of various phytochemicals encrusted on their surface [21,38,44]. Here, total phenolic content (TPC) and total flavonoids content (TFC) values of the colloidal solution of synthesized AgNPs were checked and reported as gallic acid equivalent (GAE/g dry wt of leaves) and quercetin equivalent (QE/g dry wt of leaves) respectively. To calculate the TPC and TFC, standard curves of gallic acid and quercetin were plotted by taking concentration on X-axis and absorbance at Y-axis. The observed equation of line for TPC and TFC was Y = 0.009X + 0.005 with R2 = 0.999 and Y = 0.011X + 0.028 with R2 = 0.998 respectively. The TFC and TPC value of extract was check and the observed values were 7.18 ± 1.02 mg GAE/g dry wt of leaves and 1.00 ± 0.09 mg QE/g dry wt of leaves respectively which was less than silver nanoparticles of HI. The observed value of TPC and TFC for
Fig. 3. XRD pattern of aggregates of AgNPs. 3
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Fig. 4. FESEM/EDX analysis of AgNPs.
at 100 μL which was 51.49 ± 3.33% which was more than value of extract of HI i.e. 45.91 ± 2.54%. The observed percentage values (at 100 μL) for the standards AA, and BHT were 97.41 ± 4.49, and 78.93 ± 2.49% respectively. The results of present study were similar to that of study done by [28], where ethanolic extract of stem bark of HI has shown antioxidant value (65%) in DPPH assay [28,29,31]. In recent studies, for DPPH antioxidant assay, the % inhibition of various extracts of leaves of HI was 75.78% (methanolic), 89.21% (acetone), and 87.49% (ethyl acetate) [28]. Here, biosynthesized AgNPs have shown low percentage of inhibition as compared to reported data. For HI, no antioxidant study was found using MC and NO assays. In MC assay, as like DPPH assay, AgNPs have shown concentration depended activities. At higher concentration (at 100 μL), the observed values for the extract, AgNPs and EDTA (control) were 45.55 ± 1.81, 41.18 ± 2.27 and 94.24 ± 5.17% respectively. In case of NO antioxidant assay, AgNPs have shown similar trend as like that of DPPH and MC assays. The observed values for the extract, AgNPs, AA and BHT were 65.24 ± 4.11, 74.59 ± 3.08, 94.44 ± 4.88 and 91.23 ± 4.53% respectively. Here, good activity was noticed as compared to above mentioned two assays viz DPPH and MC. The lower values in case of DPPH and MC assay may be attributed to the presence of secondary metabolites in low concentrations. In case of MC assay, secondary metabolites have capping mechanism with silver metal, so they are not free, hence low MC properties are acceptable. Different bio-ingredients have different antioxidant properties with in different antioxidant assays, which help in reducing oxidative stress in cells [26,27,39–41]. Present study confirms that the antioxidant properties of HI still retained by the biosynthesized AgNPs through the capping mechanism. Colloidal solution of AgNPs exhibited average to good
Fig. 5. FTIR Spectrum of HI (1) and AgNPs (2).
the AgNPs was 11.18 ± 1.07 mg GAE/g dry wt of sample and 1.14 ± 0.15 mg QE/g dry wt of sample respectively. The observed TFC value was small, but significant TPC value was noticed, which attributed to the capped secondary metabolites of HI as confirmed by the SEM analysis. The capping of reduced silver by secondary metabolites may augment the involvement of synthesized AgNPs towards the antioxidant properties. Total antioxidant capacity of standards as well as colloidal solution of AgNPs was calculated by using Phosphomolybdenum Assay. The observed antioxidant activity for BHT (standards), extract, and AgNPs was 98.31 ± 5.17, 85.12 ± 4.44 and 90.55 ± 3.19% respectively. Clearly, AgNPs has exhibited very good total antioxidant capacity. In DPPH assay, concentration depended effect was noticed for the AgNPs (Fig. 6). AgNPs has shown highest DPPH scavenging properties 4
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Fig. 7. Binding constant study of AgNPs.
3.4. Anti-diabetic qualities of green synthesized AgNPs In the era of technology, the more shocking fact is that, there is no well established medication or therapy to cure diabetes [10,23,27]. Diabetie(s) depend on the lifestyle and food habit of an individual. Plants having antidiabetic qualities can contribute as significant source for the development of secure and economical antidiabetic drugs [1–3]. Here, antidiabetic properties of the green synthesized AgNPs were tested by using the α-amylase assay. Colorimetric assay (at 540 nm) was used, where acarbose used as a standard. The antidiabetic property of green synthesized AgNPs was found increase in a dose dependent manner and 60.08 ± 3.38% of inhibition was observed by employing the 25 μL of AgNPs (Fig. 8). At same concentration (25 μL), standard (acarbose) and extract has shown 72.22 ± 4.31 and 45.88 ± 3.44% of inhibition. At highest concentration (100 μL), the observed % of inhibition of extract, AgNPs and acarbose were 71.28 ± 4.33, 86.66 ± 5.03 and 95.01 ± 5.41% respectively. Biosynthesized AgNPs exhibits very good antidiabetic activities and could be used as antidiabetic agents. In literature, various extracts of HI have shown antidiabetic applications through the inhibition of the ATP-sensitive potassium channels in pancreatic beta cells mechanism [28,29]. Here, enhanced effect of biosynthesized AgNPs was noticed (Fig. 9).
Fig. 6. Antioxidant studies of AgNPs.
antioxidant properties; it could serve as a free radical scavenger, possibly acting as a primary antioxidant having health beneficial metal like silver including various phytochemicals. Synthesized AgNPs may be a good alternate against synthetic antioxidants like butylated hydroxyl toluene, butylated hydroxyl anisole, and propyl gallate having adverse health effects [14,15,46].
3.3. Anti-inflammatory and protein binding qualities of green synthesized AgNPs
3.5. Antibacterial qualities of green synthesized AgNPs AgNPs are known for their antimicrobial properties, especially antibacterial. Higher surface to volume ratio of NPs enhanced the interactions of NPs with sulphur and phosphorus containing constituents of the bacterial cell leads to death of microbe(s) used [8–10]. In present study, biosynthesized AgNPs were checked for their antibacterial effect against two bacterial strains (Escherichia coli and Salmonella typhimurium) by using the disc diffusion method. Zone of inhibition (diameter in mm) was concentration dependent (Table 1 & Supplementary data S9). Biosynthesized AgNPs exhibited the antimicrobial effects, but
Green synthesized AgNPs were tested for their anti-inflammatory properties by employing the heat induced action on BSA protein at optimum conditions. The UV–vis spectroscopic study was performed at 660 nm. AgNPs has shown concentration dependent effects. As compared to standard (acetyl salicylic acid), AgNPs have shown 4 times lesser efficiency to reduce the heat induced effect on BSA protein. In previous studies, aqueous and ethanolic extracts of HI have shown good anti-inflammatory activities on rats (through carrageenan- induced paw edema test) as compared to standard indomethacin [28,29]. In present study, the low values efficacy of AgNPs towards inhibition of inflammation was attributed to the non-interactions of larger secondary metabolites of AgNPs with complex protein at higher temperature 51 ○C [42,43]. Protein binding ability of AgNPs was tested by using the UV–vis spectrophotometric method at 278 nm, where the complexation behaviour of the AgNPs was studied as per the protocol described by [12]. The binding ability of AgNPs with BSA was reported in terms of binding constant (k) (Fig. 7). The observed values of the binding constants (k) for the standard (acetyl salicylic acid), extract and AgNPs were 2.60 ± 0.05 × 10−4 μM-1, 3.21 ± 0.09 × 10−4 μM-1 and 2.01 ± 0.06 × 10-4 μM-1 respectively. Both, standard as well AgNPs have almost closest values which highlights the utility of biosynthesized AgNPs as a potent drug in future [29–32].
Fig. 8. Anti-diabetic study of AgNPs. 5
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Conflict of interest There is no statement on conflict of interest. Acknowledgements We are thankful to SAIF-IIT, Bombay for instrumentations. We are thankful to Director General, CCRAS for his motivation and guidance. Dr. Om Prakash, and Dr. Arjun Singh are highly acknowledge for their motivation and support. Same time RARIDD, Gwalior is highly acknowledged for laboratory and library facilities.
Fig. 9. Antimicrobial activities of the AgNPs-HI at 25 (a), 50 (b), 100 (c) and 200 μL (d).
Appendix A. Supplementary data Table 1 Antibacterial activities of AgNPs. Strain
Escherichia coli
Salmonella typhimurium
Conc (μL)
50 100 200 400 50 100 200 400
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jece.2019.103094.
Inhibition zone (mm) Extract
AgNPs
– – 4 8 – 5 9 11
– 2 4 10 2 7 11 13
Control MIC (μL)
MIC of Extract/ AgNPs (μL)
> 48
> 200/150
> 31
> 150/100
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lesser than control (Chloramphenicol). Minimum inhibitory concentration (MIC) was found by using the serial dilution method. It was found that with increase in the concentrations of AgNPs solution, the turbidity was decreased. The observed MIC values of the AgNPs against Escherichia coli and Salmonella typhimurium were approximately 150 μL and 100 μL respectively those were more significant than extract of HI leaves (Table 1). Here, we found low to mild antibacterial activities of biosynthesized AgNPs and extract (AgNPs > extract), which may be attributed to the presence of least number of secondary metabolites of HI those having antibacterial activities. The minimum or average activity of any drug against one or two strains does not mean that it is non-effective against other microorganisms [11,14,15]. Properties and effects are variable from drug to drug, which depend on the presence of chemical constituents present in drug(s) [11–15]. So, there is need to check the antibacterial activity of biosynthesized AgNPs against some other microorganisms also. Overall, if we compare the medicinal activities like antioxidant, anti-inflammatory, antidiabetic and antibacterial, as compared to extract of HI, biosynthasized AgNPs have shown good antidiabetic activities followed by antioxidant, anti-inflammatory, and antibacterial activities. In nut shell, we can say that bio-synthesized AgNPs exhibit very good antidiabetic and antioxidant activities. 4. Conclusion Green nanotechnology is considered as the eco-friendly and energy efficient technique for the production of nanoparticles without damaging the environment. Various plant materials and herbal drugs have been explored as a potential tool for the green synthesis of nanoparticles. In present study, leaves extract of HI was used for the green synthesis of spherical shape silver nanoparticles with size range 32–38 nm. The biological studies of biosynthesized nanoparticles suggests that they can serve as promising antiradical, anti-inflammatory, antimicrobial and antidiabetic agents. Moreover, further research is required to develop the nanoparticles of specific size and shape. Most importantly, there is need to check the role of most abundant chemical constituents of HI towards the synthesis of nanoparticles and their specific activities. 6
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