3.1. Synthesis of ZnO Nanoparticles from Gymnema sylvestre (ZnONP-GS) and its characterization studies
Plants are instrumental in treating human diseases in almost every system of medicine worldwide . Recent advances in nano biomedicine emphasize on the importance of green synthesis of metal nanoparticles using medicinal plants. This approach is advantageous over the physical and chemical methods due to safety, cost effectiveness and absence of toxic residues . The synthesis of metal nanoparticles such as Ag, Au, Zn etc., have potential biological application especially it may inhibit cancer cell growth . In this present work, we elaborately demonstrate the biologically synthesis and characterization of ZnO NPs from the medicinal plant Gymnema sylvestre and it was evaluated in anticancer potential in MCF-7 breast cancer cells .
The biological synthesis of ZnO NPs from Gymnema sylvestre was initially conformed by the colour change of the reaction mixture from colourless to brown colour indicated the synthesis of ZnO NPs preliminarily. Then, the synthesized nanoparticles were exhibits as strongest UV absorbance peak at 300 nm (Fig. 1). Moreover, the particle size of the synthesized nanoparticles was analysed by Zeta potential and particle size analysis. The particle size and Zeta potential analysis revealed that 81.1nm average size (Fig. 2). and − 25.1 mv zetapotential value (Fig. 3) of the synthesized nanoparticles. reported the synthesis of Mg2+ doped ZnO NPs using the leaf extract of Gsylvestre. From the results it can be concluded that the synthesized nanoparticles are stable .
Moreover, active and functional biomolecules are present in the ZnO NPs synthesized from G.sylvestre which are identified and analysed from FTIR spectrum and it is shown (Fig. 4).The FTIR spectrum exhibits the peaks at 3415cm− 1 were allotted to the extending vibrations of hydroxy groups; primary and secondary amines groups were presented in the synthesized nanoparticles respectively. The presented peaks were directly equivalent to protein and enzymes molecules or polysaccharides are found in the cell biomass. The peak at 2926, 2854, 2358 and 2330cm− 1 were owed to symmetric and asymmetric stretching shaking of sp3 hybridized. The peak at 1612 and 1313cm− 1 were allocated to C = O extending vibrations of the carbonyl group in ketones, aldehydes and functional carboxylic acids. Moreover, the peak at 1163 and 1055cm− 1 were allocated to vibration of –C = C–aromatic ring stretching. In addition to this band at 995cm− 1 resembles to metal binding interact with carboxylic (M↔C ≡ O) groups, this functional group might be acts template, reducing agent and capping of nanocrystals .
The crystal nature-based structure is often crucial conformation of ZnO nanomaterials which are determined by X-Ray diffraction (Fig. 5). In this study, obtains several crystal-based aspects of ZnO nanofabricated material. The X-Ray diffraction design of ZnO nanofabricated material are wurtzite hexagonal phase which designates the well indexed XRD peaks that has corresponding to the plane’s values such as (34699), (30566), (44739), (64831) (77739) and (81869). This present result has been implying that the products comprised of pure phases. Moreover, the effective diffraction peaks were found more rigorous and narrower that implying a respectable crystalline structure of Zn nanofabricated products. The respectable of size range of ZnO nanofabricated material was from 20 nm to 100nm .
As shown in the (Fig. 6) demonstrates the surface and shape with size morphology of ZnO were characterized from the microscopical studies of SEM. This study evident that ZnO NPs were spherical and irregular in shape and were poly-dispersed. The measured average size was 50µm, Occasional agglomeration of the ZnO NPs has been observed. These all the characterization studies are scientifically evident that present nanoparticles are ZnO .
3.2. Cytotoxic effect and intracellular ROS generation in ZnO nanoparticles synthesized from Gymnema sylvestre against breast cancer MCF-7 cells
The ZnO NPs can be easily biodegraded or shall take part in nutritional cycle of the body . These nanoparticles exhibit discriminating cytotoxicity over cancer cells . ZnO NPs have ability to stimulate oxidative stress in cancer cells, which has been found to be one of the prime mechanisms of cytotoxicity. This property may be attributed to the semiconductor nature of ZnO NPs . The cytotoxic event of ZnO NPs synthesized from Gymnema sylvestre in the breast cancer MCF-7 cells were evaluated by studies of MTT assay. Here we found that increasing concentration of ZnO NPs at the ranges from 10–100 µg for 24 hrs incubation notably reduces the cell viability in MCF-7 cells. The IC 50 value of the tested ZnONP-GS exhibits 36 µg/ml. In the current work, we have chosen IC50 value of 36 µg/ml and IC25 value of 50 µg/ml of ZnONP-GS for further molecular studies . (Fig. 7).
It also induces ROS generation and leads to oxidative stress, eventually ends up in cell death when the anti-oxidative capacity of the cell is exceeded . DCFH-DA staining are highly used for detection of ZnO NP-GS treated MCF-7 cells. Untreated MCF-7 cells showed there is no significant production ROS staining. ZnO NP-GS treated MCF-7 cells showing magnificent production of ROS for 24 hours incubation by dose depended manner. This result indicated that synthesized ZnONP-GS induces oxidative stress in MCF-7 cancer cells. Our results also conform that ZnO NP produces cytotoxicity and high over production of ROS in MCF-7 cells that can be led to oxidative stress. Several in vitro studies witnessed the ZnO NPs shows selective cytotoxic activity against the cancer cells . Moreover, reported the anticancer activity of silver nanoparticles bio functionalized using aqueous extract of G.sylvestre against HT29 human colon adenoma cancer cells. Hanley also stated that ZnO NPs exhibited 28–35 times selective toxicity over cancerous cells when compared with normal cells . ZnO NPs selectively kill cancer cells by inferring selective localization and selective cytotoxicity towards them . (Fig. 8).
3.3. ZnONP-GS induces the production of ROS and apoptosis in MCF-7 cells
High over production of ROS arbitrated oxidative stress leads to the programmed cell death . Apoptosis is a crucial programmed cell death which has actively eliminated the cancer cells through whether intrinsic or extrinsic apoptotic signalling pathway . In this present work, ZnONP-GS treatment associated morphological changes of apoptotic MCF-7 cells were test by double staining of acridine orange and ethidium bromide staining. Here, we noticed ZnONP-GS treated cells showed a greater number of apoptotic cells significantly in dose depended manner. Conversely, untreated MCF-7 cells showing there is no significant apoptotic cells conformed by green fluorescence staining . (Fig. 9).
In addition, proapoptotic biomarkers such as Bax, caspases and antiapoptotic protein Bcl-2 are highly regulating apoptosis . Apoptosis has been documented as dispensation over several machineries, including amendment of the intracellular mitochondrial depended pathway to stimulate the caspase cascade activation . When the over production of ROS sprightly induces proaptotic mediators leads to apoptosis . ZnO NPs treatment arbitrated apoptotic protein appearance was studied using western blotting analysis. As (Fig. 10) confirms an extensively increased protein expression of Bcl-2 and broadly decreased expression of Bax, caspase-9 and 3 protein in untreated breast cancer cells. Suggestively, the expression of anti-apoptosis marker Bcl-2 was experientially to be less whereas the expression of proapoptotic mediators were relatively quite higher expression in ZnONPs-GS treatment in the MCF-7 cells. There are numerous documentation has been reported strongly that ZnONPs from several plant extracts induces proapoptotic markers in several cancer cell lines . This result indicates that ZnONPs-GS induces proapoptotic mediators leads to cell death .