Nowadays, interest of nanobiotechnology is the development of environmentally benign technology for the synthesis of metal/metal oxide nanoparticles with miraculous and boundless applications in the agriculture, cosmetics, defense, environmental safety, food, health and pharmaceutical [1-9]. Metallic nanoparticles exhibit enormous chemical, optical, physical, and thermal properties due to large surface area, spatial confinement and reduced imperfections. The reduction of material size and shape has pronounced effects on the physical properties that may be significantly different from the corresponding bulk material . Among all metals nanoparticles, AgNPs has gained significant boundless interest because of their wide range of application in catalysis , sensors [12-14], photonics , and photocatalysis . Moreover, AgNPs have great applications in antibacterial , antifungal , antiviral , ant angiogenesis , anti-inflammatory , etc. and which can be incorporated into cryogenic superconducting material, composite fibres, cosmetic products, electronic components and food industry . AgNPs are competent biological properties, which are almost used in antiseptic sprays, fabrics, topical creams, wound dressing and successfully used in the cancer diagnosis and treatment [21-22].
Hitherto, several chemical and physical methods were employed for the synthesis of AgNPs in the past such as including microwave assisted , electrochemical , chemical reduction method , radiation assisted , thermal decomposition, chemical and photochemical reactions . However, these methods have some disadvantages like the use of toxic chemicals, need of special instruments, long reaction time, and requirement of external additives during the reaction. But green synthesis protocol is economically affordable, environmentally benign, no need of high temperature or pressure and importantly toxic chemicals. Therein, ecofriendly methods for metal nanoparticles synthesis using the microorganism , fungi , enzyme , plant extracts , etc. are given much more attention. Among the several biosynthetic approaches, the use of plant extracts has received much remarkable as they are safe to handle, easily available and have a broad variability of phytochemicals. Moreover, synthesis of functionalized AgNPs using photochemical transformations in test tube play an indispensable role; because the functional groups of various phytochemicals enhance the reduction of silver ions to elemental silver. The phyto constituents such as tannins, carbohydrates, flavonoids, saponins, coumarins, proteins, amino acids, and terpenoids present in the plant extracts play an important role in the synthesis of nanoparticles. The scrutiny of the literature revealed some notable plant parts extract used for facile synthesis of AgNPs. For example Leucaena leucocephala L. , Ziziphora tenuior , Azadirechta indic , Pistacia atlantica , Nervalia zeylanica , Ipomoea digitata , Caesalpinia pulcherrima , Hedichium spicatum , Gymnema sylvestre , Pomegranate peel , Buddleja globosa , Caralluma fimbriata , Peganum harmala  and unripe fruit of Annona reticulata  has been already reported.
Herein, we investigate the efficient, cost effective, and safe and ecofriendly green synthesis of AgNPs using leaves extracts of Rosa chinensis L. and their antibacterial activity against some human pathogens has been evaluated. The results would help to utilize as synthesized AgNPs effectively in future biomedical concerns.
MATERIALS AND METHODS
Preparation of Rosa chinensis L. extract
The fresh leaves (Fig. 1) of Rosa chinensis L. were collected from the campus of G. M. V. College of Science. The collected leaves were washed thoroughly with distilled water and incised into small pieces. About 5 g of finely cut leaves of Rosa chinensis L. were weighed and transferred into a washed 250 ml beaker containing 100 ml distilled water, mixed well and boiled for 15 minutes at 80-100 ºC. Mixture was cooled at room temperature and then filter through ordinary filter paper. The extract obtained was filtered through Whatmann number 41 filter paper and the filtrate was collected in 250 ml Erlenmeyer flask. The collected filtrate is used for further synthesis of AgNPs.
Bio-fabrication of AgNPs
The aqueous solution of 0.01 M silver nitrate (AgNO3) was prepared by using double distilled water and used for the synthesis of AgNPs. The leaves extract of Rosa chinensis L. Was mixed to 0.01M of AgNO3 solution in 1:9 ratios in a conical flask and color of medium changed to brown within 1 min.The solution turned to brown color, indicating the formation of AgNPs. Then solution was incubated for 24 h at room temperature. The resultant solutions were centrifuged at 8000 rpm for 5 min (200C) and the mixture was collected after discarding the supernatant. The collected AgNPs were allowed to dry in a watch glass. A fine shiny black colored material was obtained and this was carefully collected for characterization purposes.
Characterization of the synthesized AgNPs
The powder of green synthesized AgNPs was used for the Fourier transform Infra-red (FT-IR) (JASCO 4100) analysis. Find the exact morphological structures and size of the AgNPs using transmission electron microscopic (TEM) with selected area electron diffraction(SAED)analysis is done by using a Philips CM 200 operated at accelerating voltages of 20 and 200 kV. X-ray diffraction (XRD) pattern of AgNPs was obtained using Bruker D8-Advanced Diffractometer (λ=1.54 Å) from which average crystallite size of AgNPs was calculated. The surface morphology, purity and chemical composition study of synthesized AgNPs were carried out by field emission scanning electron microscopy (FESEM) and Energy Dispersive Spectroscopy (EDS) (JEOL JSM-6701).
Fresh aqueous leave extract of Rosa chinensis L. were used for phytochemical screening. Phytochemical screenings were carried out by standard method .
Antibacterial activity of synthesized AgNPs
The Minimum inhibition concentration (MIC) of biogenically fabricated AgNPs and leaves extract of Rosa chinensis L. were carried out by broth micro-dilution protocol . DMSO was used as diluents to get desired concentration of drugs to test upon standard pathogenic bacterial strains. Serial dilutions were prepared in primary and secondary screening. The control tube containing no antibiotic was immediately subculture by spreading evenly over a plate of medium suitable for the growth of the test bacterial pathogens and incubated overnight at 37 ºC. The MIC of the control bacterial strain was measured to check the accuracy of the drug concentrations. The lowest concentration inhibiting growth of the bacterial pathogen was recorded as the MIC. The amount of growth from the control tube before incubation was compared. Subcultures might evince same number of colonies indicating bacteriostatic, a reduced number of colonies indicating a slow bactericidal activity and no growth if the whole inoculums has been killed. The test must include a second set of the same dilutions inoculated with a bacterial pathogen. A synthesized AgNPs and leaves extract was diluted obtaining 2000 µg/ml concentration, as a stock solution. In primary screening, 500, 250 and 125 µg/ml concentrations of the synthesized AgNPs and leaves extract were taken. The synthesized AgNPs and leaves extract found in this primary screening were further tested in a second set of dilution against all microorganisms. The silver nanoparticles found active in primary screening were similarly diluted to obtain 100, 50, 25, 12.5, 6.250, 3.125 and 1.5625 µg/ml concentrations. The highest dilution showing at least 99 % inhibition is taken as MIC .
RESULTS AND DISCUSSIONS
Structural & crystallographic analysis
X-ray diffraction analysis (2θ range is 30º-80º) clearly illustrates crystalline nature of the synthesized AgNPs in (Fig. 2.)The prominent peaks at 2θ=38.10º, 44.40º, 64.50º and 77.40º corresponding to the (111), (200), (220) and (311) Bragg’s reflections of the Face Cubic Centered crystal structure (JCPDS card no. 04-0783) of AgNPs, respectively. The average crystallite sizes (D) of AgNPs were calculated by using Debye-Scherrer’s equation.
D = Kλ /β COS θ (1)
Where, D is the crystal size of synthesized AgNPs (nm), θ is Bragg angle (degrees), λ is the wavelength of the X-ray source used (1.54060 Å), β is the angular width at the half maximum of the diffraction peak (radians) and K is the constant of Debye-Scherrer’s equation which is generally, for the spherically grown nanoparticles 0.94. The average crystallite size of the synthesized AgNPs is found 42±5 nm. Thus, XRD pattern clearly illustrated that the AgNPs formed in this study are pure face-centered cubic crystalline in nature and was good agreement with TEM and FESEM results.
The acquaintance about surface morphology and crystal size of the synthesized AgNPs has been analyzed by FE-SEM microphotographs. Figs. 3 (a, b, c, d) shows FE-SEM images at different magnifications. It seems that surface is spongy and it can be observed that the average crystal grain size of the quasi-spherical morphology AgNPs was mainly 35-50 nm except for slight agglomeration. This result exceeds the literature result which quasi-spherical shape of AgNPs was prepared by green synthesis method .
To get better understanding of the morphology of AgNPs is shown in TEM images [Figs. 4 (a,b,c,d)]. It indicates the presence of AgNPs with size 25-60 nm which form bead type of aggregation throughout the region, on the contrary the image shows distinct nanoparticles of nearly spherical structure which are correlated well with the XRD results. SAED pattern also shows the spot type pattern which is indicative of the presence of single crystallite particles. No evidence was found for more than one pattern, suggesting the single phase crystalline nature of the material.
The composition of green synthesized AgNPs has been analyzed by investigating the energy-dispersive X-ray spectroscopy (EDS) and exhibits strong peak at 3eV (Fig. 5) confirms the formation of AgNPs. This quantitative data confirms the purity, elemental composition and formation of AgNPs nanoparticles.
FTIR spectroscopic studies were carried out to find out the possible chemical changes present in the extract. The spectra were recorded before and after addition of AgNO3 solution. The broad peaks of the leaves extract and nanoparticles shown in (Fig.6 a) The peak at 3284 cm-1 belongs to O-H stretching of phenolic compounds and the bands at 2916 and 2849 cm-1 region arising from C-H stretching of aromatic compound were observed. The bands at 1733cm-1areobserved as amide, ester, and acids arise due to carbonyl group stretching vibration. The stretching vibration of -C-C- in aromatic ring causes an absorption peak at 1610 cm-1. The band at 1027 cm-1 corresponds to C-OH stretching in leaves extract. Simultaneously, in AgNPs (Fig.6 b) the band at 3041 cm-1 shows the O-H stretching in phenols and alcohols. The band at 1701cm-1areobserved as amide, ester, and acids arise due to carbonyl group stretching vibration and 1592 cm-1 represents the −C-C- in aromatic ring. The strong peak at 1019 cm-1 denotes the C-O stretching of phenolic compounds. Thus, mostly phenolic and flavonoid compounds are involved in the biosynthesis of nanoparticles.
Phytochemical screening studies
Table 1 describes the qualitative pharmo-cognostic evaluation of aqueous leaf extract of Rosa chinensis L. highlighted the presence of tannins, saponins, coumarins, flavonoids, cardial glycosides, steroids, phenols, carbohydrates, amino acids, etc.which can play a role in reduction and stabilization in the biosynthesis of nanoparticles.
Antibacterial activity of AgNPs
The results of antibacterial activity of the synthesized AgNPs are presented in Table 2. Moderate to good antibacterial activity is observed against some bacteria. Synthesized AgNPs exhibited potent and good antibacterial activity against S. aureus, S. pyogenus and moderate activity against other bacteria with ampicillin were used as the reference drug.
Bio-fabrication of stable AgNPs (using Rosa chinensis L. Leaves) was reported in this work. AgNPs were successfully biosynthesized by this facile, rapid, cost effective, environmentally benign, and efficient method, which excludes external reagents. In this process, the plant extract is ascribed to the relative levels of phenols, flavonoids, tannins and emodins which act as reducing as well as capping agents AgNPs. In addition, the biosynthesized AgNPs are found satisfactory antibacterial agents and thus it can be used as potential candidates for various bioengineering applications and will play a vital role in nanobiotechnology.
The author S. V. Bangale are thankful to SAIF IIT Bombay, CIF Savitribai Phule Pune University and Microcare Laboratory, Gujrat for providing the technical, instrumental and biological activities supports. Authors are also thankful to N. G. Vedak and N. S. Yadav for providing necessary facilities and encouragement during research work.
CONFLICT OF INTEREST STATEMENT
The authors declare that there is no conflict of interests regarding the publication of this article.