Biosynthesis of silver nanoparticles using stem bark extracts of Diospyros montana and their antioxidant and antibacterial activities
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The present study reports an eco-friendly, biosynthesis of silver nanoparticles (AgNPs) using stem bark extract of Diospyros montana. Initially, the synthesis of AgNPs was confirmed by visual observation as color change. Further, the morphology of the biosynthesized nanoparticles, average size and presence of elemental silver were characterized by UV–Visible spectroscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray and dynamic light scattering spectrometer. Qualitative phytochemical screening and FTIR spectral peaks supported the role of phytochemicals in bark extract for the metal reduction, stabilization and capping of silver nanoparticles. XRD studies demonstrated that crystalline nature and their average size of nanoparticles was 28 nm as determined by Scherrer’s formula. The antioxidant ability of AgNPs and plant extract was analyzed using DPPH and Hydrogen peroxide assay. The percentage of DPPH and H2O2 activity was increased with increasing concentration of AgNPs. In vitro antibacterial effect of various concentration of AgNPs was investigated against both Gram positive (B.subtilis and S.aureus) and Gram negative (E.coli and K.aerogenes) bacterial strains. The result shows that biosynthesized AgNPs have significant antibacterial activity.
KeywordsDiospyros montana Silver nanoparticles TEM XRD Antioxidant Antibacterial activity
Nanoscience is emerging as a fast growing area with tremendous application in biomedical science and technology. Nowadays, metal nanoparticles of silver, gold and platinum have been the subject of focused research area due to their unique optical, mechanical and chemical properties that are different from those large materials . Among these metal nanoparticles, silver has become a focus of interest because they play a significant role in textile and pharmaceutical industries .
A number of approaches are available for the synthesis of silver nanoparticles (AgNPs) using electrochemical , microwave assists process , sono-chemical , radiation assist , reverse micelles process , phase transfer process  and photochemical synthesis . Most of these methods are very expensive and also uses toxic chemicals which may lead to potential environmental and health risks . In current scenario, metal nanoparticles protected by bio-organic ligands have attracted much interest due to their different applications .
In recent years, phytocompounds-facilitated synthesis of AgNPs is gaining significance due to their availability and eco-friendly . Biosynthesis of silver nanoparticles using plants such as Eucalyptus chapmaniana , Momordica charantia , Terminalia bellirica , Cochlospermum religiosum , Eucalyptus chapmaniana  and many more plants has been reported. Silver nanoparticles have numerous applications such as bio-labeling, combating of microbes, detection of cancer, drug delivery and other diseases . Antimicrobial activity of green synthesized nanoparticles allows them to use in water filtration process, textiles and food industries .
Diospyros montana also known as Bombay ebony and belong to the family of Ebanaceae. Diospyros genus is receiving increased attention as it is used in Indian traditional medicines like Ayurveda and Unani . Diospyros montana is mainly distributed in Western Ghats of India, Sri Lanka and Australia. It was reported that genus Diosyros contain various phytochemicals such as phenols, flavonoids, saponins, terpinoids and reducing sugars . Diospyros montana has significant pharmacological activities, used in the treatment of cough, ulcer, anti-hypersensitive and snake bites [21, 22]. Bark extract of D.montana contains diospyrin compound, which acts as a tumor inhibitory agent . However, to date, less attention has been paid to synthesis nanoparticles from this plant. To the best of our knowledge, this is the first report on biosynthesis of AgNPs from stem bark extract of D.montana. We have demonstrated the phytochemical screening of methanolic stem bark extract, biosynthesis, characterization of silver nanoparticles and their free radical scavenging and antibacterial activities.
Materials and methods
Chemicals and reagents
Silver nitrate (AgNO3), methanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid was purchased from Sigma Aldrich. All other reagents and chemicals used in this research were of analytical grade.
Bacterial strains Escherichia coli (MTCC 443), Klebsiella aerogenes (MTCC 98), Bacillus subtilis (MTCC 441) and Staphylococcus aureus (MTCC 3160) were used and maintained in nutrient agar (Himedia, Mumbai) slants at 4 °C.
Plant collection and authentication
The stem barks of D.montana were collected from Wayanad District (Western Ghats), Kerala, India. It was identified and authenticated (Ref. BSI/SRC/21/12/2015/Tech/1219) at Botanical Survey of India, Coimbatore, India.
Preparation of methanolic stem bark extract
The stem barks were washed thoroughly thrice with distilled water and dried for 7 days. The fine powder was obtained from stem bark using kitchen blender. About 25 g of stem bark dry powder was extracted with 250 ml of methanol solvent using soxhlet extractor. After completion of the extraction, the methanol solvents were evaporated using rotatory evaporator. Prepared extract was concentrated, dried and kept at 4 °C until further use.
Qualitative phytochemical screening
Phytochemical screening of stem bark extract of D.montana was performed for the qualitative detection of different phytocompounds such as alkaloids, flavonoids, saponins, glycosides, phenols, steroids, carbohydrates, oils and fats, proteins, amino acids, tannins, gums and reducing sugars. All phytochemical tests were performed according to the standard methods described by Al-Owaisi  and Sheel .
Biosynthesis of silver nanoparticles
Silver nitrate solution (1 mM) was prepared in amber bottle and stored at dark place. The prepared stem bark extract was mixed with 1 mM AgNO3 solution in 1:9 proportions and kept at room temperature for 30 min. Purified AgNPs from the samples were obtained by centrifuging the solution at 10,000 rpm for 20 min. The precipitate was re-dispensed with 10 ml double distilled water and again centrifuged at 10,000 rpm for 10 min for removing excess biomass. The pellet of Ag nanoparticles was collected carefully and dried in desiccators for 12 h. The dried powder of AgNPs was used for further test.
Characterization of AgNPs
The formation of nanoparticles was preliminarily confirmed by visual observation as color change from watery to brown color. Further, the reduction of Ag ions in the solution mixture was confirmed by UV–Visible spectroscopy (JASCO UV–Vis NIR V-670). The spectral data of synthesized AgNPs were recorded from 200 to 600 nm at a resolution of 2 nm.
SEM and EDX
The morphology of AgNPs was determined by SEM (Hitachi S -340 N). SEM samples were prepared on a carbon coated copper grid by placing a small amount of AgNPs on the SEM grid, excess samples was cleaned by blotting paper and then samples were allowed to dry under mercury lamp. This experiment was conducted at an accelerator voltage of 20 kV. Energy dispersive X-ray analysis was also taken on the same instrument.
Transmission electron microscope (TEM) (JEOL JEM-2100) analysis was conducted by drop coating of Ag nanoparticles on a copper grid and operated at an accelerated voltage at 80 kV. The grid was dried for 2 h and imaged.
DLS particle size analysis
Average size and polydispersity index of deionised water-diluted silver nanoparticles were measured by dynamic light scattering spectroscopy (DLS) (Malvern-zeta analyser) operated with a He–Ne laser.
The structure of the AgNPs was determined using XRD technique. For X-ray diffraction studies (XRD), dried silver nanoparticles were coated on XRD grid (XPERT–PRO) and diffraction recorded in the 2θ range from 10° to 90°. The spectral data were operated at 40 kV and a current of 40 mA.
The biologically active components that are responsible for silver reduction, formation and capping were identified using Fourier Transform Infra-Red (FTIR) spectroscopy (Nicolet is 5, Thermo Scientific) by KBr pellet method. FTIR spectral reading was carried out in the range from 500 to 4000 cm−1 at a resolution of 4 cm−1.
DPPH and H2O2 free radical scavenging activity
The DPPH free radical scavenging activity of synthesized nanoparticles and plant extract were carried out spectrophotometrically (517 nm) according to the method reported by Govindappa . The Hydrogen peroxide assay, a method for measuring free radical scavenging activity, was assessed spectrophotometrically (230 nm) according to Yadav . Antioxidant activity was noted in terms of stranded control ascorbic acid antioxidant equivalents. Percentage of the activity was calculated using the following equation; Percentage of antioxidant activity = [(a − b)/a] × 100, where a was the absorbance of the control (blank) and b was the absorbance of samples.
The antibacterial activity of AgNPs were carried out by agar well diffusion method described by Gudikandula  against Gram negative bacteria such as E.coli (MTCC 443) and K.aerogenes (MTCC 98), Gram positive bacteria such as B.subtilis (MTCC 441) and S.aureus (MTCC 3160). Muller-Hinton Agar (Himedia, Mumbai) plates were prepared, sterilized and solidified. Each bacterial strain (1 × 105 CFU/mL) was swabbed uniformly on the prepared individual petri plates using sterile cotton swabs. Four wells of size approximately 6 mm are made on prepared plates using gel puncture. Different concentration of AgNPs (10, 20, 30 and 40 µL) was loaded into the wells of all plates. After incubation for 24 h at 37 °C, the plates were examined for zone of incubation in mm.
All the experiments were carried out in triplicates and the results were expressed as mean ± standard deviation. Statistical analysis was done using origin software (Origin pro evaluation, 2018).
Results and discussion
Qualitative phytochemical screening
Phytochemical screening of methanolic stem bark extract of D.montana
Methanolic stem bark extract
Oils and fates
Proteins and amino acids
Biosynthesis of AgNPs using D.Montana
Characterization of AgNPs
In this study, we have synthesized AgNPs by eco-friendly and cost effective method using D.montana stem bark extract. The phytochemicals present in the stem bark extract of plant act as a strong reducing and capping agents for the formation of AgNPs. The morphology and size of the silver nanoparticles were characterized by UV–Visible spectroscopy, SEM, TEM, EDX and DLS. XRD reports revealed that synthesized silver nanoparticles were crystalline in nature with average mean size as 28 nm and FTIR studies supported the confirmation of bioactive compounds for the Ag+ reduction. Biosynthesized AgNPs showed significant antibacterial activity against Gram negative bacteria compared to Gram positive bacteria and also exhibit remarkable free radical scavenging activity. Further studies on AgNPs will be carried out for pharmacological applications.
The authors like to thank DST-FIST for providing the laboratory facilities and we acknowledge the Department of Nanoscience and Technology, Bharathiar University, Coimbatore and Anna University for extending their nanoparticles characterization facilities established under the DST-PURSE Program.
Compliance with ethical standards
Conflict of interest
The authors have declared no conflict of interest.
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