BIOGENIC SYNTHESIS OF SILVER NANOPARTICLES USING MANILKARA HEXANDRA (ROXB.) DUBARD STEM BARK EXTRACT AND IT’S PHYSICAL, CHEMICAL CHARACTERIZATION AND PHARMACEUTICAL EVALUATION

Objective: The present study was to synthesize nanoparticles using Manilkara hexandra stem bark extract its characterization and evaluating it by an antimicrobial and antioxidant assay. Methods: Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) was done by mixing silver nitrate (1 mmol) and aqueous stem bark extract and it was analyzed by UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Zeta potential, Field Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive Spectroscopy (EDAX), Thermogravimetry/Differential Thermal Analysis (TG/DTA Results: Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) is characterized by various techniques such as UV-visible absorption spectrum ranges from 430 nm to 440 nm indicate silver nanoparticles. The Fourier Transform Infrared Spectroscopy consists of biomolecules acts as capping agent to form silver nanoparticles. Field Emission Scanning Electron Microscopy shows particle size ranges from 15 nm to 50 nm. Energy Dispersive Spectroscopy shows the presence of Silver. X-ray Diffraction corresponds to face-centered lattice planes (111), (200), (220) and (311). Dynamic Light Scattering show the range of 68 nm and Zeta potential show the negative value of-17 nm which has high stability. Silver nanoparticles is also examined by Thermogravimetry/Differential Thermal ) and Differential scanning calorimetry (DSC). The antibacterial assay was done by a well diffusion method and also examined for antifungal assay was done by disk diffusion method and antioxidant potential Diphenyl-1-picryl hydrazyl (DPPH method)


INTRODUCTION
Indian tradition uses the medicinal plant as the predecessor for the development of the drug from the plant and is also used in modern days. It is effective because of the bio-compounds present in it has no side effects and costs less [1]. The mixture of traditional and modern ideas produces a new source of active compounds which can be used as medicine with low side effects [2]. Among various metals (Ag, Au, Pt, Pd, Cu, etc.) AgNPs having intense effect for research in recent years is due to its properties [3]. AgNPs can be synthesized by physical and chemical methods, but our aim is to develop an environmental friendly method [4]. Green synthesis is the best method its effective, novel method [5,6]. Silver nanoparticles in a biological approach involve microorganisms, plants, and viruses (or) their biomolecules [7]. Silver nanoparticles are currently applied in medicine, consumer products, even in surgical blades, catheters, and food packing [8]. The colloidal silver nanoparticles can be used as a wound dressing material, tooth cement, replacement for bones, water purifier and antimicrobial agent [9]. The botanical name of the plant is Manilkara hexandra (Roxb.) Dubard belong to Sapotaceae family, Genus-Manilkara, Species-Manilkara hexandra, in Tamil it is called as Ulakkaippaalai or Kanuppaalai. The various parts of Manilkara hexandra contain medicinal values such as astringent, colic, dyspepsia, anorexia, leprosy, anthelmintic, refrigerant, hallucination, loss of consciousness, fermentation process etc. [10].
Our aim was to develop silver nanoparticles using the stem bark of Manilkara hexandra (Roxb.) Dubard and it is novel work. In our work, we identify biocompounds first by phytochemical screening and GC-MS for aqueous stem bark extract and then we use centrifuge techniques to collect the silver nanoparticles. It has been characterized by various studies such as UV-vis absorption, FT-IR, FE-SEM, EDAX, XRD, DLS, Zeta potential, TG/DTA, DSC. The silver nanoparticles are also tested by antimicrobial assays and free radical scavenging activity (DPPH method).

Materials
All chemicals (AR grade) were purchased from Sigma-Aldrich Chemicals, India. The stem bark of Manilkara hexandra (Roxb.) Dubard was collected from Jayankondam at Ariyalur District, Tamil Nadu in India. The plant was identified in the Rapinet herbarium, in St. Joseph College, Trichy. The voucher sample was preserved (Voucher No: AAL 001) shown in fig. 1.

Mechanism of AgNPs formation
The bioactive compounds present in the extract react with AgNO3 to form AgNPs [11]. The biosynthesis mechanism of nanoparticles is explained below.
Ag + NO3 - The stem bark of Manilkara hexandra was washed in tap water and again washed with distilled water, then allowed to air-dry under the shade for two weeks. After it was crushed into a fine powder using pestle mortar 10g of Manilkara hexandra stem bark powder was soaked in 100 ml of double-distilled water for 30 min. This mixture is then heated at 60 °C for 20 min, after which it is cooled to room temperature. Finally, the filtrate is stored in the refrigerator for further investigations. Chemical compounds were analyzed by using phytochemical screening [13]. Six ml of stem bark was added to the 120 ml of the 10 +Stem bark extract → Ag °NPs+by-products The bioactive compounds present in stem bark extract act as reducing agent. The reduction process is mainly due to flavonoids which act as a strong reducing agent. The biological compounds act as capping, reducing agent and is a major cause for the stability of compounds [12].

Synthesis of silver nanoparticles
-3 M silver nitrate (AgNO3 ) solution. When the reaction started, we observe the change in color from yellow to dark brown. These silver nanoparticles were separated using a highspeed centrifuge (Remi RM-12C) at 12,000 rpm for 15 min. After separation, nanoparticles is purified with alcohol and dried in a hot air oven at 200 °C for 2 h. The product is then powdered and stored in a properly labelled container for further studies. The characterization instruments and its uses are listed in table 1.

Antibacterial assay
The antibacterial activity of the stem bark extract, Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs), silver nitrate (AgNO3) solution, and standard were determined by the well diffusion method against various bacteria, such as Klebsiella pneumonia, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumonia on Muller Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI) [14]. The testing sample (75 µl/ml) is loaded with 6 mm of an antibiotic disc and compared with Amoxicillin as standard.

Antifungal assay
The antifungal activity of stem bark extract, Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs), silver nitrate (AgNO3 2,2-diphenyl-1-picrylhydrazyl (DPPH) is used for examining free radical scavenging activity. DPPH radicals (0.2 mmol) are prepared in methanol solution. Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) concentration varied from 20-100µg/ml with water mixed with one ml of prepared 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution in a test tube. It is shaken vigorously and kept in a dark room for 30 min after absorbance is measured. Similarly, ascorbic acid is used as a standard to compare the silver nanoparticles. After measuring the IC ) solution, and standard were determined by disk diffusion method against two fungal stains such as Candida albicans and Aspergillus niger on Sabouraud's dextrose agar [15]. The testing sample (10 µl/ml) is loaded in 6 mm of an antifungal disc and is compared with Fluconazole as standard. 50 The scavenging ability is calculated using formula.

Free radical scavenging activity on DPPH method (in vitro)
value is calculated [16].

Where I (%) is inhibition percentage
A-Absorbance of control reaction B-Sample absorbance of test compound.

Synthesis and characterization of silver nanoparticles
The primary identification of silver nanoparticle is the change of color from yellow to dark brown and is shown in fig. 3.
The Manilkara hexandra stem bark extract and MHSB-AgNPs were first determined by UV-visible spectrum wavelength ranging from 300-1100 nm. Where double distilled water was used as reference [17]. The UV-visible absorption spectrum of bio-synthesized Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) is analyzed at various intervals of time. It has an optical absorption range from 430-440 nm and for the stem bark extract, it is 311 nm shown in fig. 4a and fig. 4b.  The surface morphology, shapes, size distribution in nano (10 corresponding to-OH is stretching alcohol, O꞊C꞊O stretching, NH is bending amine, C-H bending alkane, C-H bending alkane methyl group, C-Br stretching halo compounds, respectively. Fourier Transform Infrared spectrum shows the presence of bio-molecules and metal nanoparticles present in the synthesized nanoparticles [18]. -9 ) and micro (10 -6 ) scales were identified by using Field Emission scanning Electron Microscope (FE-SEM) [19]. Particles are spherical and size ranges from 15-50 nm as shown in fig. 6a. Energy Dispersive Spectroscopy (EDAX) shows the presence of Silver (Ag) which confirms the formation of Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) as shown in fig. 6b. There are also other peaks such as Carbon (C), Chlorine (Cl), Oxygen (O), Potassium (K) they are due to biocompounds from the plant. The Silicon (Si) is due to glass wafer used for coating the nanoparticles.

Fig. 6: a.) Field Emission Scanning Electron Microscopy (FE-SEM) image of MHSB-AgNPs and b.) Energy Dispersive Spectroscopy (EDAX) spectrum of MHSB-AgNPs
X-ray diffraction analysis (XRD) of Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) shows the different peaks at (111), (200), (220), (311) and is face-centered cubic (FCC). Standard data file for silver (JCPDS No. 04-0783). Bragg reflection is based on the crystal of silver nanoparticles (AgNPs). These phases were indexed to a spherical shape. The Scherrer equation was used to calculate the particle size and found to be in the range of 38 nm as in fig. 7.

Fig. 7: X-ray powder diffraction (XRD) of MHSB-AgNPs
Dynamic light scattering (DLS) analysis shows the average size to be at 68 nm. Dynamic light scattering (DLS) shows a minor peak at 3.7 nm, medium peak at 16.5 nm and a major peak at 121 nm as in fig. 8a. The size of the particles is high in Dynamic light scattering (DLS) compared to Field Emission scanning Electron Microscope (FE-SEM). The Dynamic light scattering (DLS) is measured based on the hydrodynamic diameter of the particles and it provide average particle size whereas Field Emission scanning Electron Microscope (FE-SEM) is examined by dry particles [20]. The high stability of zeta potential (-17 nm) is confirmed from fig. 8b. In zeta potential, the magnitude is predictive for the stability of the nanoparticles [21].

Fig. 8: a.) The hydrodynamic size of MHSB-AgNPs and b.) Zeta potential of MHSB-AgNPs
The thermal stability of Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) was analyzed using Thermogravimetry/Differential Thermal Analysis (TG/DTA). In TG curve, we observe weight loss from 4.4340 mg to 2.806 mg from room temperature to 820C and the residue is 63.29% after 820˚C it is constant there is no weight loss. The weight was lost at three stages from room temperature to 130C, 130˚C to 450˚C and 450˚C to 820˚C. The primary weight loss is due to the moisture content present in the nanoparticles. The major weight loss is at 130C to 450˚C it is due to decomposition of materials and is shown in fig. 9. The Differential Thermal Analysis (DTA) reveals two exothermic peak at 120˚C and 300˚C. The peak at 300˚C shows the melting point of silver nanoparticles. The release of energy at 300C is depicted in DTA curve shown in fig. The crystallinity and thermal decomposition can be examined for silver nanoparticles in DTA [22].

Fig. 9: Thermogravimetry/Differential Thermal Analysis (TG/DTA ) of MHSB-AgNPs
We observe three exothermic peaks at 35˚C , 192.3˚C, 315˚C and four endothermic peak at 57.1C, 135˚C , 290˚C and 312˚C. The nanoparticles melt in an exothermic peak at 315C. This stage is called as melting stage and the energy is been released by the material at this stage. The nanoparticles are stable until 315C.
There are also small endothermic and exothermic peaks present in the Differential Scanning Calorimetry (DSC) curve. There are also peaks present in glass transition stage and crystalline stage. These peaks are due to change properties of nanoparticles shown in fig. 10.

Antimicrobial assay
The bio-synthesized Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs), stem bark extract, silver nitrate (AgNO 3 ) solution, and standards were subjected to bacterial assays. Five different bacteria were analyzed they are Klebsiella pneumonia, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumonia among these Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) have high Zone of Inhibition (ZOI) for Klebsiella pneumoniae and Escherichia coli at 11.5 mm as in fig. 11. There is no ZOI for the silver nitrate (AgNO 3 ) solution and a stem bark extract. This explains that the stem bark extract is non-toxic and adaptable to nature as shown in table 3.

Antioxidant assay (in vitro DPPH method)
The free radical scavenging activity in vitro is examined for various concentrations of Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) from 20 µg/ml to 100µg/ml. They are alternatively compared with ascorbic acid, which is a standard for antioxidant assay. Here Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) show a good antioxidant property but low compared with the ascorbic acid standard. But, Manilkara hexandra stem bark silver nanoparticles (MHSB-AgNPs) is non-toxic and has no side effect when compared with ascorbic acid. The IC50 value is calculated as in fig. 13. and table 5.

DISCUSSION
It is stated that active bio-compounds present in dried materials are more concentrated than that of fresh materials [23,24]. Here silver nanoparticles were synthesized by using Manilkara hexandra stem bark extract belong to the family of sapotaceae. The change of color from yellow to dark brown was observed. Which is similar to the results of previous studies Adhatoda vasica leaf extract [25]. In Aegle maremelos leaf extract silver nanoparticle shows the spectral studies close to 450 nm this confirms the formation of silver nanoparticles and this absorption depends on the dielectric medium, particle size and chemical surrounding [26] which corresponds to the formation of AgNPs (430-440 nm) from Manilkara hexandra stem bark. The DLS report of MHSB-AgNPs (68 nm) correlate with the Lantana camara flower extract for silver nanoparticles was 62.8 nm [27]. The long term stability of AgNPs can be examined by zeta potential [28,29]. In general, the stability of zeta potential which is negatively charged surfaces to prevent the aggregation of nanoparticles and also control the size and shapes of silver nanoparticles [30]. The Caralluma umbellate silver nanoparticles the particle size is examined by SEM which is in the range 50 nm to 85 nm [31]. This corresponds to the size of MHSB-AgNPs this shows the silver nanoparticles is synthesized in a proper method. Antimicrobial report of silver nanoparticle was not clear. Due to the three different mechanisms of action, first silver ions attached to the bacterial cell membrane and cause plasmolysis (bacterial cell wall is separated from the cytoplasm of bacteria), this inhibit synthesis of bacterial cell membrane [32]. Secondly, silver nanoparticles combined with S (sulphur) and P (phosphorus) contains compounds which present inside and outside of the bacterial cell, this affect respiratory chain reaction, cell division and finally, it leads to cell death [33]. Finally, Ag ions were released from silver nanoparticles and this penetrate into the cell wall, this cause the condensation of DNA damage and also affect protein synthesis [34]. Overall, results show the involvements of all these three phenomena of synthesized particle cause the killing of pathogens. Antioxidant agents are that which restricts the deleterious effect of oxidant reactions. This restriction involves preventing the radical formation or scavenging free radicals and thereby can enhance the immune defense and lower the possibility of diseases occurrence [35]. In our present study, we use the DPPH method of free radical scavenging activity. The percentage of inhibition increases with increases in the concentration of silver nanoparticles. This shows that silver nanoparticles may donate electrons to DPPH and a lipophilic radical accept that electron thereby conversion of color from purple to yellow is detected [36].

CONCLUSION
The present work focused on the synthesis of silver nanoparticles using the stem bark of Manilkara hexandra. The silver nanoparticles is then characterized by various techniques such as UV-vis, FT-IR, FE-SEM, EDAX, XRD, DLS, Zeta potential, TG/DTA, DSC. The GC-MS analysis, project 20 bio-compounds for aqueous stem bark extract and are also evaluated by phytochemical screening. These biocompounds play important role to synthesize the nanoparticles after it is validated by various microbial pathogens. They are indulged in antioxidant assay in vitro and nanoparticles show good report for microbial pathogen and antioxidant assay. The particle size is 15-50 nm. Here we eventually say that this method is simple, eco-friendly, and is a natural method. This can be implied in the drug production.

AUTHORS CONTRIBUTIONS
All the author have contributed equally

CONFLICTS OF INTERESTS
Declared none