Hypericum perforatum L.-Mediated Green Synthesis of Silver Nanoparticles Exhibiting Antioxidant and Anticancer Activities

This contribution focuses on the green synthesis of silver nanoparticles (AgNPs) with a size < 100 nm for potential medical applications by using silver nitrate solution and Hypericum Perforatum L. (St John’s wort) aqueous extracts. Various synthesis methods were used and compared with regard to their yield and quality of obtained AgNPs. Monodisperse spherical nanoparticles were generated with a size of approximately 20 to 50 nm as elucidated by different techniques (SEM, TEM). XRD measurements showed that metallic silver was formed and the particles possess a face-centered cubic structure (fcc). SEM images and FTIR spectra revealed that the AgNPs are covered by a protective surface layer composed of organic components originating from the plant extract. Ultraviolet-visible spectroscopy, dynamic light scattering, and zeta potential were also measured for biologically synthesized AgNPs. A potential mechanism of reducing silver ions to silver metal and protecting it in the nanoscale form has been proposed based on the obtained results. Moreover, the AgNPs prepared in the present study have been shown to exhibit a high antioxidant activity for 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, and super oxide anion radical and 2,2-diphenyl-1-picrylhydrazyl. Synthesized AgNPs showed high cytotoxicity by inhibiting cell viability for Hela, Hep G2, and A549 cells.

. The data λmax (nm) of absorption peaks for different samples, Absorption max, Peak Width (FWHM), molar extinction coefficient which we obtained from the reference study [1] and the final concentration of various sizes of biosynthesized silver nanoparticles.

Isoelectric point
The point of zero charge (Isoelectric Point, IP) is the pH at which the electrical charge density on the surface is zero. Usually it is determined in relation to pH of electrolytes, and the point of zero charge value is assigned to a given colloidal particle (as in our case) or substrate. We aimed to determine the isoelectric point of the AgNPs for three reasons: First: to know at which pH value will be the electrical charges on the surfaces of silver nanoparticles in the colloidal solution zero. Second: is the stability of nanoparticles in the colloidal solution dependent on electrostatic Repulsion because nanoparticles will fall down immediately in the solution when these forces become zero. Third: if the bond between the protective layer and the surface of the nanoparticles is of the type:  Non-covalent bonds: which they are classified into various categories like hydrophobic effects, pi effects, electrostatic and van deer Waals forces.
 Chemisorptions: is a kind of adsorption which includes a chemical reaction among the adsorbate and the surface.

Thermal gravimetric analysis (TGA):
AgNPs were examined by Thermo-gravimetric analysis (TGA) (TGA/DSC 3+" from the company Mettler-Toledo) to prove the existence of biologically active secondary metabolites from H. Perforatum extract at the surfaces of silver nanoparticles. The progressive increase in temperature was adjusted between 25 °C -1000 °C at heating rate of 0.5 °C/minute in flowing N2.
The TGA diagram of the covered silver nanoparticles synthesized using Hypericum Perforatum L. extract (Figure S 3) exhibited a stable weight loss in the temperature range of 92-493 °C. The weight loss of the capped-AgNPs was a result of adsorption of bioorganic metabolites at the surface of AgNPs (protective agent) and it was almost between 40-60 % with moisture which is about 9 percent, this depends on the number of washing times with Vivaspin tubes and the conditions of centrifugation used. Figure S 3 shows the progressive degradation of capped-AgNPs in three steps with an increase in temperature. The first phase of degradation which happened between 92 and 164 °C with a weight loss of 6.93% and this weight loss may be attributed to surface adsorbed H2O molecules and some molecules of hexose ring. The second and third phase of the weight loss occurred between 170-315 and 324-492 °C consecutively, with a weight loss of 31.11 and 20.84 % respectively, this weight loss was due to combustion of the protective layer (the metabolites from St john`s extract which acts as a capping agent at the surface of AgNPs when formed) which decomposition in two steps. In summary the results of TGA analysis correspond to the results of FTIR and prove that resulted silver nanoparticles from synthesis were mixed nature with strongly coordinated metal organic framework.

Scanning electron microscopy (SEM):
Energy dispersive X-ray spectroscopy (EDX): Oxygen and carbon appearance clearly shows that an extracellular organic layer covers the surface of AgNPs. The occurrence of another element in the photomicrograph could be on the grating base FTO glass which is used for the analysis. Through the two images Figure S5 and the spectrum Figure S6 we note the following: in a place where there is no silver, the concentrations of all the other elements are same, especially oxygen, this proves that what we got is only the silver metal and there are no silver oxides in the samples. We note that the concentration of carbon is less where there is silver metal. This indicates that silver is surrounded by a protection layer from the organic compounds present in the extract, which means, it proves the presence of the organic layer on the surfaces of nanoparticles.

High resolution transmission electron microscopy and nano-diffraction patterns [6-11]:
SAED is a qualitative analysis method of crystal structures from a spot diffraction pattern, which is obtained from illumination of a parallel electron beam on a specimen. When entering a selector (chosen-region) slot into the image level of the objective lens, is obtained a deviation pattern from a sample area of a various 100 nm diameter. This method enables us to identify the lattice type, lattice parameters and crystallographic orientation of this selected area. To analyze patterns of SAED, we integrate the geometric relationship and Bragg's equation in the reciprocal space: Where λ is the wavelength of the electron beam, d is spacing between planes, θ is the diffraction angle, D is the distance between rings on the SAED pattern, L is camera length of TEM apparatus. For too small diffraction angle θ, tan2θ equals to 2sinθ, thus for the spots on the SAED pattern; d-spacings can be calculated by: Each ring or spot in SAED pattern corresponds to a lattice plane of a specific miller index in single crystal or a group of lattice planes of the same miller index family in the polycrystalline sample. Whole spots on the pattern could be indexed. If the electron beam is fixed and the sample rotates, several ring/spot will be activated, and another ring/spot might die far. It depends on the diffraction situations described over. The diffraction pattern can be considered as a finger print for a certain crystal. Generally, the electron beam diffracted by a single crystal produces a diffraction spots pattern, but the specimen which consist of big number of little randomly distributed grains produce a continued rings. This is because all these grains contribute to the formation of diffraction pattern. Radius of the spots is inversely proportional to the interplanar spacings dhkl of lattice planes of crystals.
The method to index the SAED pattern can be explained step by step: (1) The diameter (2R), of each ring is measured using some image processing software such as the image tool.
(2) The radius value (R) of the diameter (2R) is taken.

X-ray Diffraction (XRD):
The material consists of crystals in which the atoms are organized in a specific order. Since the wavelength of the X ray is close to the distance between atoms in these crystals, the X-ray diffraction suffers if they are coated on the material. The detector records the angles that are curving at the crystalline planes of the X-rays and the intensities of these rays. In order for the detector to collect all curvilinear rays, the detector moves around the axis of the shape on a circle where the angles and corners of the vertices are generated by a two-dimensional plan called the X-ray diagram. This diagram is characteristic for the material (fingerprint).

Determination the particles size of the silver nanoparticle from Debye -Scherrer's Equation[12]:
The broadening of the Bragg reflection peaks indicates that the crystallite domain size is in nanosizes. The broadening at half maximum intensity of the diffraction peak is related to a reduction in crystallite size, flattening and micro-strands within the diffraction domains. The average particle size D was determined by using Scherrer's formula: β is the full width at half maximum (FWHM), θ is the diffraction angle, D is the average crystallite size perpendicular to the reflecting planes and λ is the X-ray wavelength, k is constant, its value is close to one, and is related to crystalline shapes and lattice plane which corresponding to studied peak, if we assume that the shape is spherical, k will be 0.9. The FWHM for each sample were taken from the (111) Bragg's reflection and its value were estimated by fitting the peaks using origin program. Probable mechanism of biosynthesis of silver nanoparticles by plant extracts: Phenolic and hydroxyl groups of chlorogenic acid [51]