AIJRSTEM19-

American International Journal of Research in Science, Technology, Engineering & Mathematics Available online at http://www.iasir.net ISSN (Print): 2328-3491, ISSN (Online): 2328-3580, ISSN (CD-ROM): 2328-3629 AIJRSTEM is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by I. Association of Scientific Innovation and Research (IASIR), USA International (An Association Unifying the Sciences, Engineering, and Applied Research) SYNTHESIS AND CHARACTERIZATION OF ZINC OXIDE NANOPARTICLES USING Curcuma amada AND IT’S IN VITRO ANTIDIABETIC ACTIVITY 1* G. SUBHA1, M. KALAISELVI1* Department of Biochemistry (PG), Kongunadu Arts and Science College, Coimbatore-29, Tamil Nadu, India mksbiochem2018@gmail.com *Corresponding author: Abstract: The synthesis of nanoparticles is an expanding research area due to the potential applications in the development of novel technologies. Particularly, naturally blended nanomaterial has turned into a vital part of nanotechnology. The present work, described the synthesis of Zinc oxide nanoparticles (ZnO NPs) using rhizomes aqueous extract of C. amada its anti diabetic activities. The obtained nanoparticle was characterized by UV- visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), SEM, EDX analysis. In this study we also investigated anti diabetic activity of green synthesized ZnO NPs. The results depicted concentration of ZnO NPs was increased (100, 500 μg/ml) and also increase in anti diabetic activities α-amylase and α-glucosidase inhibitors effective. The alpha amylase inhibition assay showed that340μg/ml, 350μg/ml respectively. The α-glucosidase inhibition IC50 was 300μg/ml, 340μg/ml respectively. However, green synthesized ZnO NPs was more potent than ZnO NPs and rhizomes aqueous extract of C. amada. The results of the work therefore clearly indicate the potential of those extracts to manage hyperglycemia. Keywords: ZnO NPs, Green synthesis, FT-IR, Anti diabetic, α- amylase, α-glucosidase I. INTRODUCTION Diabetes mellitus (DM) refers to a group of common metabolic disorders that share the phenotype of hyperglycaemia with disturbance of carbohydrate, fat and protein metabolism resulting from imperfections in insulin secretion, insulin action or both. Frequent distinct types of DM are begun by a complex interaction of genetics and environmental factors. Depending on the ethology of the DM, factors contributing to hyperglycaemia include reduced insulin secretion, decreased glucose utilization and increased glucose production [1]. According to Indian Council of Medical Research India Diabetes Study (ICMR-INDIAB study) showed that India had fastest growing disease:72 million people with diabetes in 2017 figure expected to nearly double 134 million by 2025 by [2] . The application of nanotechnology to medicine is called Nano medicine. Research and technology development at the atomic, molecular and macromolecular levels in the length scale of approximately 1-100 nanometre range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices and systems that have novel properties and functions because of their small and/or intermediate size [3]. These applications take advantage of the unique properties of nanoparticles as drugs or constituents of drugs or are designed for new strategies to controlled release, drug targeting, and salvage of drugs with low bioavailability. Hopefully, the new kind of treatment may help in making the everyday lives of millions of diabetes patients more tolerable [4]. Mammalian α-amylase is a prominent enzyme in the pancreatic juice which breaks down large and insoluble starch molecules into absorbable molecules ultimately maltose [5]. α-glucosidase, on the other hand, anchored in the mucosal brush border of the small intestine catalyzes the end step of digestion of starch and disaccharides that are abundant in human diet [6]. Inhibitors of α-amylase and α-glucosidase delay the breakdown of carbohydrate in the small intestine and decrease the postprandial blood glucose excursion levels in diabetic patients. The inhibition of these two prominent enzymes has been found as a useful and effective strategy to lower the levels of postprandial hyperglycemia [7]. They serve as the major digestive enzymes and help in intestinal absorption. Alpha amylase and glucosidase inhibitors are the potential targets in the development of lead compounds for the treatment of diabetes [8]. Extensive research has been carried out to screen the bioactivity of these inhibitors because of their significant importance in health care and medicine. Plant food rich in polyphenols have been reported to cause effects AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 149 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 similar to insulin in the utilization of glucose and act as good inhibitors of key enzymes like alpha amylase and alpha glucosidase associated with type 2 diabetes. Studies have also shown that bioactivity of polyphenols in plants is linked to their antioxidant activity and many of these plants also possess hypoglycaemic properties [9]. Traditional Indian have long used of plant and herbal extracts as an anti-diabetic agents [10]. Therefore, exploration on traditional medicinal plants has become more important and researches are challenging to find the new effective and safe therapeutic agent for the treatment of diabetes. The Indian herb C. amada commonly called as Mango ginger exhibited significant resistance to both the pathogens is an important medicinal plant belongs to the family Zingiberaceae. The major constituent found in rhizomes is curcuminoids, penolic compounds, terpinoids and essential oil. The mango ginger has the numerous biological activities such as antioxidant, antibacterial, anti-inflammatory, antiallergic, antifungal, platelet aggregation inhibition activity and analgesic activity [11]. However, the inhibitory effect of C. amada on α-amylase and α-glucosidase enzymes has not yet been reported. Hence, the present study was aimed to investigate the in vitro anti-diabetic activity of C. amada aqueous extract and ZnO NPs on α-amylase and α-glucosidase enzymes. II. MATERIALS AND METHODS 2.1 Preparation of hot water extraction The collected rhizome sample was washed, shade dried and powdered. 50 g of dried powder of C. amada rhizome was extracted in 500 ml of distilled water. The resultant rhizomes extract was concentrated to dryness and used for further studies. 2.2 Synthesis of ZnO NPs using C. amada 5 g of dried aqueous extract was dissolved in 50 ml of distilled water. From that 20 ml of extract was taken and heated at 50ºC for 10 min and 50 ml of 91 mM of zinc acetate solution (1 g of zinc acetate was dissolved in 50 ml of distilled water) was added drop wise under stirring condition. The reaction mixture became white and cream coloured precipitate of zinc hydroxide was formed. The content was left over for 30 min for complete reduction to zinc hydroxide. Then the precipitate was collected by centrifugation at 16,000 rpm for 10 min at 4ºC [12]. Green Synthesis of Zinc Oxide Nanoparticles 0.91 mM Zinc acetate dihydrate (Zn(O2CCH3)2 50ml Curcuma amada rhizomes extract Stirred for 15 minutes at 600 temperature was added drop-wise pH12 After 20 min to add sample NaOH Magnetic stirrer for 2 hrs. Filtration Drying at 80 C Gnanasangeetha and Thambavani 2013 2.3 Characterization of ZnO NPs using C. amada The bio reduction of ZnO NPs was monitored using UV–Visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, particle size analysis, Scanning electron microscope (SEM) and, EDAX analysis to determine the functional groups present, average particle size and morphology of the ZnO NPs. 2.4 In vitro anti-diabetic activity of aqueous extract and ZnO NPs from C. amada 2.4.1 Determination of α-amylase inhibitory activity 100 -500 µg/ml of different concentrations of the C. amada aqueous extract and ZnO NPs were allowed to react with 100 µl of the α-amylase enzyme and 100 µl of 2 mM phosphate buffer (pH 6.9). After 20 min of incubation, 100 µL of 1% starch solution was added. The same was performed for the control where 200 µl of the enzyme was replaced by the buffer. After incubation for 5 min, 500 µl of dinitro salicylic acid reagent was added to both control and test, and were boiled for 5 min in a water bath. The absorbance was recorded at 540 AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 150 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 nm, and the result was interpreted regarding the IC50 values (effective concentration showing 50% inhibition activity) [13]. The percentage inhibition was calculated according to the formula: Inhibition (%) = Abs 540 (control) – Abs 540 (extract) × 100 Abs 540 (control) The IC50 values were resolute from plots of percent inhibition against log inhibitor concentration and were calculated by non linear deterioration analysis from the mean inhibitory values. Acarbose was used as the reference alpha-amylase inhibitor. All the tests were performed in triplicates. 2.4.2 Determination of α-glucosidase inhibitory activity 100-500 µg/ml of different concentrations of the C. amada aqueous extract and ZnO NPs were allowed to react with 100 µl of the α-glucosidase enzyme and 100 µl of 2 mM phosphate buffer (pH 6.9). After 20 min of incubation, 100 µL of 5 mM p-nitrophenyl α-D-glucopyranoside solution was added. The same was performed for the control where 200 µl of the enzyme was replaced by the buffer. After incubation for 5 min, 1000 μL Na2CO3 50 mM was added to both control and test, and were boiled for 5 min in a water bath. The absorbance was recorded at 410 nm, and the result was interpreted regarding the IC 50 values (effective concentration showing 50% inhibition activity) [14]. The percentage inhibition was calculated according to the formula. Inhibition (%) = Abs 410 (control) – Abs 410 (extract) × 100 Abs 410 (control) The IC50 values were resolute from plots of percent inhibition against log inhibitor concentration and were calculated by non linear deterioration analysis from the mean inhibitory values. Acarbose was used as the reference alpha glucosidase inhibitor. All the tests were performed in triplicates. III. RESULTS AND DISCUSSION 3.1 Synthesis of ZnO NPs from C. amada aqueous extract Using green synthesis, reduction of metal salts into metal nanoparticles is always accompanied by the colour change of reaction medium. In the present study, the colourless zinc acetate solution is changed after drop wise addition of C. amada extract at zero second. After 2 hrs, a pale white precipitate was obtained indicates the formation of ZnO NPs which was dried it in hot air oven (Figure 1). Zinc acetate soluction Zinc acetate +C.amada after (0 sec incubation) (After 2 hours) Figure 1. ZnO NPs synthesis 3.2 UV–visible spectroscopy UV–Visible absorption spectra of the ZnO particles synthesized from the mixture were shown in Figure 2. The optical properties of nanoparticle were determined using UV-Vis 2202+ Double Beam spectrophotometer. The biological synthesis of ZnO NPs is focusing on controlled the absorption peak was obtained at 290-350 nm wavelengths. It is generally recognized that UV–Vis spectra could be used to examine the size and shape controlled nanoparticles in aqueous suspension [15] the samples exhibit strong UV absorption spectra with the absorption peak ranging from 220 nm and 250 nm due to ZnONPS was stored in dried form in centrifuge tubes and was found to be stable after 4 months of room temperature storage. Arulmozhi et al. [16]revealed a green methodology for the combination of zinc oxide nanoparticles (ZnO NPs) utilizing watery concentrate of Atalantia monophylla by estimating absorbance at its qualities estimation of to be at 352 and 410 nm separately. In S. grandiflora blend of ZnO NPs shown UV-unmistakable assimilation tops at 235 nm which was accounted for by [17]. AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 151 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 Figure 2: UV-VIS spectrum analysis of ZnO NPs 3.3 FT-IR spectroscopy FT-IR spectral analysis for characteristic functional groups present in ZnONPs was shown in Figure 3. The spectrum obtained for synthesis clearly shows ZnO absorption band near 4000-500 cm1. The peak at 3454 pinnacle spoken to O-H gather extending of O-H, H-fortified single scaffold. The peaks somewhere in the range of 3340.6 and 3258.2 compare to H fortified OH stretch and N=H extend. The 1377 pinnacle results from fragrant amines and the two tops at 1040 and 1026.8 outcome from C=N stretch of aliphatic amines. The 943 and 617 pinnacles compare to alkanes and as far as anyone knows, C=H twisting in alkynes, separately. Trademark practical gatherings in charge of development of the particles in the examples were recorded. Figure 3: FTIR analysis of ZnO NPs 3.4 Scanning Electron Microscopy (SEM) Analysis The morphology of the prepared nanoparticles was examined using scanning electron microscopy (SEM). Figure 4 show the surface morphology of ZnO NPs using C. amada aqueous extract under different magnifications. SEM image showed ZnO NPs were homogenous and symmetrical in the shape. AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 152 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 Figure 4: SEM Synthesis of ZnO NPs 3.5 Energy Dispersive X-Ray Diffractive (EDX) analysis The Energy Dispersive X-ray Diffractive (EDX) study was carried out for the synthesized ZnO NPs to know about the elemental composition. EDX confirms the presence of zinc and oxygen signals of zinc oxide nanoparticle as shown in Figure 5 and this analysis showed the peaks that corresponded to the optical absorption of the produced nanoparticle. The elemental analysis of the nanoparticle yielded 42.73% of zinc and 57.27% of oxygen which proves that the produced nanoparticle is in its highest purified form. AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 153 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 Element Line OK Zn K Zn L Total Weight % Weight % Error ± 1.26 ± 7.42 --- Atom % 24.70 57.27 75.30 42.73 ----100.00 100.00 Figure 5: EDX spectrum of synthesized zinc oxide nanoparticles ZnO-NPs have been reported to zinc and oxygen was found to be 76.32 and 23.68, ZnONPs using Ocimum basilicum leaves extract 18]. 3.6 In vitro anti-diabetic activity 3.6.1 α-amylase inhibitory activity Alpha-amylases are digestive enzymes which hydrolyze glycosidic bonds in starch to glucose, maltose. With the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into many other fields, such as clinical, medicinal and analytical chemistry [19]. The in vitro α-amylase inhibitory activity of aqueous extract and ZnO NPs from C. amada was tested. The result of α-amylase inhibitory activity of C. amada aqueous extract and ZnO NPs was shown in Figure 6. The percentage inhibition of aqueous extract, ZnO NPs and standard acarbose was found to 71 %, 74 % and 88.2% at a maximum concentration 500 μg/mL respectively with its IC50 value of was found to be 340μg/ml and 350μg/ml mL compared to reference acarbose 290μg/ml respectively. Hence from the above results, it is clearly indicates that ZnO NPs showed maximum inhibition when compared to that of aqueous extract of C. amada which shows ZnO NPs have a better antidiabetic activity. Figure 6: α-amylase inhibitory activity AIJRSTEM 19-230; © 2019, AIJRSTEM All Rights Reserved Page 154 Kalaiselvi et al.., American International Journal of Research in Science, Technology, Engineering & Mathematics,26(1), March-May 2019, pp. 149-156 Previous studies concerning other plants were screened for α-amylase activity and showed inhibitory activity. They been reported that α-amylase inhibitory activity of M. lucida aqueous extract showed IC50 value of 2.30mg/mL were reported by [7]. Laoufi et al reported that O. angustissima have more potent inhibitor of αamylase showed at 77% at a concentration of 3.3 mg/mL respectively. Our studies was also coincidence with the previous report of α-amylase inhibitory activity of G. officinalis, P. vulgaris and T. indica obtained 35, 45-75, 90% inhibition of α-amylase at concentration of 200 mg/mL [21]. 3.6.2 α-glucosidase inhibitory activity The result of α-glucosidase inhibitors activity of C. amada aqueous extract and ZnO NPs was shown in Figure 7. The percentage inhibition of aqueous extract, ZnO NPs and standard acarbose was found to 77 %, 72 % and 94% at a maximum concentration 500 μg/mL respectively with its IC50 value of was found to be 300μg/ml and 340μg/ml mL compared to reference acarbose 270μg/ml respectively. Hence from the above results, it is clearly indicates that ZnO NPs showed maximum inhibition when compared to that of aqueous extract of C. amada which shows ZnO NPs have a better antidiabetic activity. Figure 7: α- Glucosidase inhibitory activity Previous studies concerning other plants were screened for α-glucosidase activity and showed inhibitory activity. Nair et al., reported that methanolic extracts of A. heterophyllus, A. altilis, P. betel and C. zeylanicum showed IC50 value of 129.85, 76.90, 140.01 and 96.56 μg/ml respectively. Kazeem et al. reported that P. nitida have more potentials inhibitor of acetone extract of were IC50 value of 3.00 mg/ml respectively. They been reported that α- glucosidase inhibitory activity of T. fragosa extracts showed 55% inhibition of the activity were reported by [22]. From the above results, it can be concluded that C. amada water extracts and ZnONPs can be excellent choice of drug with α-glucosidase inhibitory activity and can thus reduce the rate of digestion and absorption of postprandial hyperglycemia. IV. CONCLUSION The synthesis of ZnO NPs was characterized using UV–Vis, FTIR, SEM, EDX investigation. Anti-diabetic studies results proved that the synthesized ZnO NPs and C. amada extract can be showed the maximum alpha amylase and alpha glucosidase inhibitory activity. Biosynthesized ZnO NPs prepared from C. amada extracts are expected to have significant applications in pharmaceutical and biomedical fields such as drug delivery more research is required for developing a potential and valuable anti diabetic therapy using alpha amylase and alpha glucosidase inhibitors of plant origin. In conclusion, the synthesis of ZnONPs using C. amada aqueous extract were biosynthesis through green approach, pollution-free and eco-friendly approach for anti-diabetic activity. V. CONFLICT OF INTEREST We, the authors declare that they have no conflict of interests. VI. ACKNOWLEDGEMENTS We acknowledge our sincere thanks to our management of Kongunadu arts and Science College, Coimbatore for providing all the facilities to carry out this work. VII. REFERENCES 1. 2. Arun Chaudhury , Chitharanjan Duvoor , Vijaya Sena Reddy Dendi , Shashank Kraleti , 2017; Clinical Review of Antidiabetic Drugs: Implications for Type 2 Diabetes Mellitus Management; Frontiers in Endocrinology ; 10.3389/fendo.2017.00006 Ranjit Mohan Anjana, Mohan Deepa, et al For The ICMR–INDIAB Collaborative Study Group 2017; Prevalence of diabetes and prediabetes in 15 states of India:results from the ICMR–INDIAB population-based cross-sectional study; Articles. 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