Green synthesis of silver nanoparticles using the plant extract of Salvia spinosa grown in vitro and their antibacterial activity assessment
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Researchers use bionanotechnology techniques as eco-friendly and cost-effective routes to fabricate nanoparticles and nanomaterials. The present study confirms the ability of plant extract of Salvia spinosa grown under in vitro condition for the biosynthesis of silver nanoparticles (Ag NPs) for the first time. The surface plasmon resonance found at 450 nm confirmed the formation of Ag NPs. Moreover, FESEM images showed that nanoparticles had spherical morphology. Furthermore, XRD analysis confirmed the crystalline nature of the particles. FTIR analysis was carried out to identify possible biomolecules responsible in bioreduction of silver ions. Antimicrobial assay verified bactericidal activity of biosynthesized Ag NPs against Gram-positive and Gram-negative bacteria. According to the results, by growing the plants under controlled conditions, it is feasible to synthesize nanoparticles with desired properties.
KeywordsBio-nanotechnology Salvia spinosa Silver nanoparticles Bactericidal activity
Nanotechnology is the synthesis of particles with at least one dimension in the range of 1–100 nm, resulting in high surface to volume ratios. As the particle size decreases, not only does the ratio of surface area to volume increase but also the physical, chemical and biological properties of the particles differ compared to their bulk counterparts [1, 2]. Noble-metal nanoparticles exhibit incredible physicochemical, optoelectronic and biochemical characteristics. They are being used for various purposes in industrial and pharmaceutical applications [3, 4]. Despite the existence of numerous metals in nature, only a few of them such as gold, silver, palladium and platinum are synthesized extensively in nanostructured form [5, 6]. Among the above-mentioned metals, silver nanoparticles have attracted much attention due to their unique characteristics for utilizing in various applications including pharmaceutics, agriculture, water detoxification, air filtration, textile industries and as a catalyst in oxidization reactions [7, 8, 9]. Furthermore, their predominant property is their high antibacterial activity against a broad range of bacteria without any toxicity to animal cells [10, 11, 12]. The establishment of resistance to antibiotics in bacteria especially multidrug resistance compelled scientists to explore novel compounds to halt multidrug-resistant microorganisms. Bactericidal activity of Ag NPs without toxicity to human cells can make them a proper substitution for antibiotics [13, 14]. Ag NPs are being employed for eliminating microorganisms in medical devices, implants and hospital masks [15, 16]. They are also extensively being used in hospitals either supplemented with antibiotics or alone for preventing infection . Nanoparticles are traditionally synthesized broadly by physical and chemical procedures. Chemically prepared nanoparticles are not appropriate for medical usages due to hazardous chemicals binding on their surface. Furthermore, by-products produced in chemical routes are toxic for the environment. Physical routes for synthesis of NPs have some drawbacks, too. These methods require high energy and space, and are expensive [18, 19, 20]. Using biological systems such as microorganisms, plants, viruses and animal cell cultures is an alternative procedure for preparation of NPs [21, 22, 23]. In the most recent studies, researchers utilized Gelidium amansii , Enteromorpha compressa , Phanerochaete chrysosporium , Bacillus brevis  and Daucus carota  for the biosynthesis of Ag NPs and investigated their antimicrobial properties. Biosynthesis of NPs is eco-friendly, time affordable, cost effective. More importantly, the biosynthesized NPs are free of hazardous material on their surface. Also, they may be coated with bioorganic compounds that make them proper for medical applications. These give biosynthesis of NPs distinct advantages over conventional methods [29, 30]. For example, NPs are used as a new tool for targeting in cancer therapy and the most important difficulty is toxicity of NPs synthesized by previous methods. Scientists overcome this issue using biologically synthesized NPs coated with biomolecules that are more biocompatible [31, 32].
Although the exact mechanism of NPs biosynthesis by various plant extracts is ambiguous, it has been revealed that the biomolecules in plant extract such as protein, phenol and flavonoids play a significant role in the reduction of metals ions and capping the biosynthesized nanoparticles . Culturing plants in controlled environmental systems presents an approach for the production of specific bioactive molecules in plants . Since these plants are not exposed to environmental turbulence, the metabolic production of these plants is not dependent on environmental changes .
Medicinal plants have served as rich sources of pharmacologically active substances. Herbs have been used in a diverse array of purposes, including medicine, nutrition, flavoring, dying, repellents, fragrances, cosmetic, charms, smoking and industrial uses. Today, herbs are still found in 40% of prescription drugs . The genus Salvia from Lamiaceae family includes almost 900 species cultured or grown as a weed all around the world, mainly in the areas of the Central and South America, and Mediterranean and South Africa . The product of this plant is used for culinary as spices and pharmaceutical purposes. This genus offers an economical cultivate for small farmers due to its usage in folk remedies and ornamental purposes [38, 39]. The metabolite content of Salvia genus has been investigated thoroughly several times. The presence of flavonoids, caffeic acid esters such as rosmarinic acid, and phenolic diterpenes such as carnosic acid and carnosol has been detected in Salvia species extracts [34, 40].
The extract of various plants grown in farms is used permanently for the biosynthesis of nanoparticles, but whether the plants grown under in vitro condition have this ability or not is still under scrutiny. The main purposes of this work are considering the potential of the plant extract of S. spinosa grown in vitro in Murashige and Skoog (MS) medium for the biosynthesis of Ag NPs and investigation of their antibacterial activities against both Gram-positive and Gram-negative species of bacteria.
Materials and methods
Plant growth under in vitro condition
Seeds of S. spinosa were purchased from Qazvin farmers. They were surface sterilized by ethanol 70% for 1 min. After rinsing with sterile distilled water, sterilization was continued by soaking the seeds in sodium hypochlorite 2.5% for 5 min. This process is followed by three times rising with sterile distilled water for 10 min. Mineral salts, 3% sucrose and 4.5% agar were combined for Murashige and Skoog medium preparation and finally pH was adjusted at 5.8. After autoclaving, seeds were explanted in Petri dishes for germination. After germination, seedlings of the same age were subcultured into jars containing the same medium. These cultures were placed in the growth room at 25 ± 2 °C, in 16-/8-h period of day/night. After 2 months, plants were harvested and dried in oven at 37 °C for 48 h.
Preparation of plant extract
All chemicals were provided from Merck or Aldrich. The dried plants were powdered with a mortar and pestle. 10 ml of distilled water was poured to 0.2 g of plant powder. This combination was boiled for 5 min and then was cooled. The cooled solution was filtered with Whatman No. 1 filter paper.
Biosynthesis of Silver nanoparticles
Aqueous solution of silver nitrate (1 mM) was prepared and mixed with fresh plant extract of S. spinosa at a ratio of 9:1. This solution was placed on a shaker with constant rotation in the room temperature at 27 ± 2°C for 6 h. All stages of the experiment were implemented in three replicates.
Characterization of the silver nanoparticles
Conventionally, characterization of particles is implemented by UV–Vis spectroscopy. An UV–Vis spectrophotometer (UVD 3200) was employed for verifying the biosynthesized Ag NPs. An Equinox 3000 diffractometer was utilized for XRD studies of Ag NPs. Furthermore, biosynthesized Ag NPs were observed by field-emission scanning electron microscopy (FESEM, HITACHI, S-4160). Particle size distribution of biosynthesized Ag NPs was obtained using Dynamic Light Scattering Malvern-Zetasizer (Nno-z 590). This study was undertaken to know the functional groups existed in plant extract responsible in silver ion reduction, so the pellets of plant extract and biosynthesized Ag NPs with potassium bromide (1:100) were used for FTIR spectrum analysis of plant extract and biosynthesized Ag NPs by a Bruker Tensor 27 spectrophotometer.
Antibacterial activity of biosynthesized Ag NPs
The antibacterial activity of the biosynthesized Ag NPs against Gram-positive and -negative bacteria species was done by disk diffusion method. Experimented bacteria were Bacillus subtilis (accession number: M59 KP406766), Bacillus vallismortis (accession number: M92 KP406765) and Escherichia coli (PTCC: 1276). Bacterial strains were spread on the Petri dishes which contained autoclaved Luria–Bertani (LB) medium containing agar. Then, the disks (6 mm diameter) soaked in distilled water as a control, plant extract and biosynthesized Ag NPs were separately placed on Petri dishes containing LB media. Petri dishes were incubated at 37 °C. Inhibition zone of each disk was measured by ruler after 18 h.
Results and discussion
Color change of solution
X-ray diffraction analysis
FESEM and DLS analysis
Fourier infrared spectroscopy analysis
Antibacterial activity of the Ag NPs
Biosynthesis of Ag NPs using plant extract of S. spinosa grown in vitro under controlled condition was carried out for the first time in this study. This study confirmed S. spinosa’s (cultured under controlled condition) capability for the biosynthesis of Ag NPs. The characteristics of the biosynthesized Ag NPs were measured by different equipments. Moreover, bactericidal activity assessment of the biosynthesized Ag NPs showed their inhibitory function against both Gram-positive and Gram-negative bacteria. In this study, possible functional groups and effective compounds responsible in reduction of silver ions were assigned. The speculated mechanism in this work elucidates the involvement of carboxylic acid functional group presented in carnosic acid and flavonoids in the bioreduction process and the stabilization of biosynthesized Ag NPs.
Authors are thankful for Imam Khomeini International University authorities for supporting this work.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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