Antidiabetic Activity of Phytosynthesized Ag/CuO Nanocomposites Using Murraya koenigii and Zingiber officinale Extracts

In this paper, we report the phytosynthesis of Ag/CuO nanocomposites (NCs) using Murraya koenigii and Zingiber officinale green extracts containing unique metabolites. The silver and copper oxide nanoparticles, and Ag/CuO NCs were also synthesized by chemical methods for the purpose of comparison with phytosynthesized Ag/CuO NCs. All the synthesized nanomaterials were characterized by diverse spectro-analytical methods. The phytochemical analysis confirmed the presence of active phytoconstituents in the Murraya koenigii and Zingiber officinale extracts. The major biomolecules present in the plant extracts act as reducing and capping agents in the green synthesis process, as identified using FT IR spectroscopy. The maximum absorbance at ~ 290 and ~ 460 nm corresponding to CuO and Ag evidenced the formation of Ag/CuO NCs. nanomaterials ranging between 18 and 22 nm. The in vitro antidiabetic activity with α-amylase, α-glucosidase and glucose-6-phosphatase enzymes, and glucose uptake assay showed that the Ag/CuO NCs synthesized using Zingiber officinale extract displayed highest activity than the rest of the synthesized nanomaterials. ___________________________________________________________________________


Introduction
In recent years, nanotechnology has gained wide-ranging interest due to its versatile properties and potential applications in modern material science. The wide scopes of research in various scientific disciplines are due to the unique properties of nanomaterials when compared with the bulk materials. The synthesis of nanomaterials through biological routes have attracted much attention in modern nanotechnology due to their unique advantages such as simple, scalable and non-toxic process, and improved biomedical applications [1,2]. In this connection, the metal and metal oxide nanoparticles are picking up more consideration because of their surface plasmonic properties and potential technological application in biomedical fields, sensors and photocatalysis [3]. The development of nanomaterials in biological and medicinal research leads to be strong strategies to control medical issues coupled with several diseases and infections.
Nowadays, distinctive combination of nanomaterials has attracted researchers due to their stability and versatile behaviour in which the synthesis and application of metal/metal oxide nanocomposites have gained much importance as the combination of two nanomaterials provide a distinctive model for the production of nanomaterials with specified properties [4,5]. Copper oxide (CuO), a p-type semiconductor with a narrow bandgap of 1.2-3 1.9 eV and monoclinic crystal structure is well-known for diverse practical applications in heterogeneous catalysis, sensors, lithium ion electrode materials, field-emission sources, semiconductors etc., The combination of CuO with noble metal is an energizing area of research due to their enhanced biomedical, electronic, photocatalytic and other applications.
For example, in the case of CuO decorated Ag nanoparticles, the charge transfer occurred from the CuO to the Ag nanoparticles, suppressing the recombination of charge carriers, and thus exhibiting the efficient photocatalytic properties [6][7][8][9]. Additionally, the CuO nanoparticles are stable and have longer shelf-life due to which it can be utilized for many therapeutic applications [10].
Among the several green synthesis sources, the plant extracts have attracted much attention because of their advantages such as abundant availability, safe to handle, and healing as well as curing of human diseases due to the presence of various metabolites. The active biomolecules present in the plant extracts also played a crucial role in the reduction process during the synthesis of nanomaterials. Murraya koenigii from Rutaceae family, generally called curry leaves having a slight pungent taste and used as flavouring agent in food items. It is also used in Siddha medicine as tonic for stomach ache, as stimulant and carminative, hypoglycemic agent, antifungal agent, and also exhibit anticancer activity against colon cancer [11,12]. Zingiber officinale is a flowering plant belongs to Zingiberaceae family, whose rhizome, ginger root is widely used for remedial purposes over 2000 years in folk medicine [13].
The present study was designed to synthesize Ag/CuO NCs using Murraya koenigii and Zingiber officinale extracts. The preliminary qualitative and quantitative phytochemical analysis was carried out for Murraya koenigii and Zingiber officinale extracts in order to identify and quantify their phytochemical constituents. The antidiabetic potential of the synthesized Ag/CuO NCs were assessed against α-amylase, α-glucosidase and glucose-6-4 phosphatase enzymes. The gulucose uptake activity of the synthesized Ag/CuO NCs were also analysed using 3T3-L1 preadipocyte cells.

Materials and methods
Analytical grade silver nitrate was obtained from SD Fine Chemicals Limited (SDFCL), India. Copper acetate tetrahydrate, ethanol and sodium hydroxide were purchased from Fisher Scientific, India. 3T3-L1 preadipocyte cells were obtained from Microbial Type Culture Collection Center (MTCC), Chandigarh, India. Murraya koenigii and Zingiber officinale were acquired from nearby market, Royapettah, Chennai. The detailed procedure for the synthesis of silver and copper oxide nanoparticles are given in our earlier publications [14,15]. The Ag/CuO NCs were successfully synthesized using the plant extracts and chemical method by adopting the reported procedures with slight modifications [16,17].

Synthesis of Ag/CuO NC by chemical method
Copper acetate tetrahydrate (0.2 mol) and equimolar sodium hydroxide were dissolved slowly in deionized water, and stirred for 1 h at 80 ºC during which a black coloured precipitate was formed, indicating the formation of CuO nanoparticles. Then, silver nitrate (0.42 g, 0.025 mol) was added, and the mixture was stirred for 2 h at 80 ºC. The final product was collected by centrifugation at 500 rpm, washed with ethanol followed by deionized water, and dried in a hot air oven for 2 h at 120 ºC.

Synthesis of Ag/CuO NCs using Murraya koenigii and Zingiber officinale extracts
Ag/CuO NCs were synthesized by green method using Murraya koenigii and Zingiber officinale extracts. Murraya koenigii leaves were thoroughly washed with deionized water and air dried at room temperature. 10 g of air dried leaves were gauged and boiled at 100 °C with 250 mL of deionized water for 15 min. The greenish extract was separated from the 5 leaves using Whatman No.1 filter paper, and stored in refrigerator. Zingiber officinale was washed multiple times with deionized water to remove surface contaminations. The green extract was prepared by boiling 6 g of Zingiber officinale in 50 mL of deionized water and kept at normal room temperature for 24 h. The resulting solution was decanted to remove solid pieces, filtered through Whatman No. 1 filter paper and stored in refrigerator.
Copper acetate tetrahydrate (0.2 M) was dissolved in deionized water (15 mL) and silver nitrate (0.42 g, 0.025 mol) was slowly added to the solution and stirred for 10 min.
Finally, 20 mL of leaf extract (Murraya koenigii/Zingiber officinale) was added to the above mixture under constant stirring using magnetic stirrer for 10 min. After complete dissolution of the mixture, the solution was boiled at 80 ºC for 10 min. The immediate colour change after adding the leaf extract indicates the formation of Ag/CuO NCs. The final product was washed with ethanol followed by deionized water and then it was allowed to dry under room temperature to get the Ag/CuO nanocomposites.
The synthesized Ag and CuO nanoparticles are denoted as S1 and C1, respectively.
Ag/CuO NCs synthesized through chemical method is denoted as SC1, while that obtained from Murraya koenigii and Zingiber officinale extracts are denoted as SC2 and SC3, respectively.

Characterization
FT IR spectra of synthesized nanomaterials were recorded at room temperature on Perkin-Elmer spectrophotometer in the range 4000-400 cm -1 . Diffuse reflectance spectra were recorded using UV140404B spectrophotometer in the wavelength range 200-800 nm, and numerical data were plotted in the 'Origin 8' software. Photoluminescence spectra were recorded using FLUOROLOG-FL3-11fluorescence spectrometer. Powder X-ray diffraction data were collected between 10-90° (2θ) at 0.5° min −1 from a highly stabilized and automated ZESSI X-ray generator (PW 1830) operated at 40 kV and 20 mA, with Cu Kα radiation and λ 6 value is 1.5406 Å. SEM and EDX analysis were performed through JEOL 6500F instrument.
TEM measurements were performed on a JEOL model 1200EX operated at an accelerating voltage of 120 kV.

Phytochemical studies
The aqueous Murraya koenigii and Zingiber officinale were subjected to qualitative and quantitative analysis for the identification of their active phytoconstituents [18][19][20]. The detailed procedures are reported in supplementary material (S1 and S2).

α-Amylase inhibitory activity
α-Amylase (0.05 g of α-amylase in 100 mL of ice-cold distilled water) was premixed with the synthesized nanomaterials at different concentrations (100-1.52 g/mL) and sonicated at room temperature for 30 min. Starch as a substrate was added as a 0.5% starch solution in phosphate buffer to start the reaction. The assay mixture was incubated at 37 o C for 15 min and terminated by adding 2 mL of DNS reagent (1% 3,5-dinitrosalicyclic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The reaction combination was then heated for 15 min at 100 o C in a boiling water. The α-amylase inhibitiory activity was calculated as percentage inhibition using the following formula; A 540 control -A 540 sample I α-Amylase (%) = × 100 A 540 control where A540 control = absorbance without nanomaterials, and A540 sample = absorbance with nanomaterials.

α-Glucosidase inhibitory activity
α-Glucosidase (0.05 g of α-glucosidase in 100 mL of ice-cold distilled water) was pre-mixed with the synthesized nanomaterials at different concentration (100-1.52 g/mL) and sonicated at room temperature for 30 min. In order to start the reaction, p-nitrophenyl--D-glucopyranoside (3 mM) as a substrate in potassium phosphate buffer was added to the mixture. The reaction was incubated at 37 o C for 20 min and terminated by the addition of Na2CO3 (2 mL, 0.1 M). A tube containing α-glucosidase without nanomaterials served as the control with 100% enzyme activity. α-Glucosidase inhibitory activity was determined by measuring the release of p-nitrophenyl--D-glucopyranoside as percentage inhibition using the following formula; where A405 control = absorbance without nanomaterials, and A405 sample = absorbance with nanomaterials.

Glucose-6-phosphatase inhibition activity
The glucose-6-phosphatase activity was carried out using glucose-6-phosphatase from a rabbit liver (Sigma, G5758), and was determined by measuring the release of glucose-6phosphate at 660 nm. Inhibition rates were calculated by using the following formula; IC50 values were determined from dose-response curve of percentage inhibition versus nanomaterials concentration and compared with the IC50 value of the synthetic inhibitor of glucose-6-phosphatase (metformin), under similar conditions. 8

Glucose uptake determination
The 3T3-L1 preadipocyte cells were cultured and plated in 24-well plate, and incubated for 24 h in DMEM growth medium containing glucose (5 mM). Initially (1 st day), the growth medium was replaced by DMEM supplemented with 10% fetal calf serum (FCS), 10 μg/mL insulin, dexamethasone (DEX; 10 -8 M) and 3-isobutyl-1-methylxanthine (IBMX; 0.1 mM). After 70 h (4 th day), this medium was replaced with growth medium. After 48 h incubation (6 th day), the cells were analyzed by removing 10 µL of the media and placing in the 96 well plates to which GOD-POD reagent (200 μL) was added and incubated for 15 min at 37 ºC. The amount of glucose content (mg/dL) in each well was determined by using the following formula; Finally, the glucose uptake over control was calculated as the difference between initial and final glucose content in the incubated medium.

Results and discussion
Ag/CuO nanocomposites (NCs) were synthesized by green route using aqueous extracts of Murraya koenigii and Zingiber officinale. The use of these extracts leads to the reduction of metal ion to zero valent metal nanoparticles by avoiding the usage of common reducing agents such as sodium borohydride. In order to carry out comparison study, we have also synthesized the same nanocomposite by chemical methodology.  (Table S1). Gums and mucilages, and terpenoids are missing in both the extracts.
Rest of the compounds including alkaloids and phytosterols are available only in Zingiber officinale extract. In this regard, we have also quantitatively determined the essential phytoconstituents present in both the extracts ( Table S2). The biomolecules such as falvonoids and phenolic compounds may have the potential to bind metal ions and cause reduction to metal nanoparticles. The results support the role of flavonoids and phenolic compounds in the bioreduction.

FT IR analysis
The FT IR spectra revealed the bioreduction role of major phytoconstituents present in the extracts and the effective incorporation of green exracts on the synthesized nanomaterials (Fig. 1)

UV-Vis DRS spectral studies
The UV-Vis DRS spectroscopy is used to understand the formation of Ag/CuO NCs (Fig. 2).

Powder XRD analysis
The XRD analysis was performed to ascertain the crystalline nature and phase purity of the synthesized nanomaterials (Fig. 4)

SEM and EDX analysis
The SEM images of CuO and Ag/CuO NCs revealed the spherical shapes of the synthesized nanomaterials [31,36].

TEM analysis
The TEM images of the synthesized Ag/CuO NCs exhibit the uniform dispersion by contrast and colour differences with an average size between 18 and 22 nm (Fig. 7). The size of the nanomaterials calculated from XRD analysis is closely matched with TEM results. The AgNPs appeared to be a black in colour and CuO was light due to high mass thickness of Ag

Antidiabetic activity
Diabetes is a tricky disease that refers to a metabolic disorder distinguished by higher range of blood glucose level, and classified into Type 1, Type 2 and gestational type diabetes.
The Type 2 diabetes received considerable attention as it influences a large proportion of populates worldwide by increasing the blood glucose level to insulin resistance in adipose tissue, muscle and liver [38]. The inhibition of carbohydrate enzymes prevent the increase of glucose level in blood. Sulfonylureas, biguanides, genistein, astilbin and hesperidin are generally recommended antidiabetic drugs for the purpose of controlling the blood glucose level [39,40]. The carbohydrate digesting enzymes such as α-amylase and α-glucosidase avoid the sudden raise of glucose level in blood. Acarbose, miglitol and voglibose are commonly utilized as standards in α-amylase and α-glucosidase inhibiton activity [41].

α-Amylase and α-glucosidase inhibion activity
The in vitro α-amylase and α-glucosidase inhibition activity of the synthesized nanomaterials were carried out with respect to the standard drug acarbose ( Based on the obtained results, the phytosynthesized Ag/CuO NCs could be suggested for further antidiabetic studies in order to explore the medicinal applications. Table 2 Inhibitory effect of nanomaterials on α-amylase and α-glucosidase activity.
Data are expressed as mean ± SEM; *Average of three independent determinations.

Glucose-6-phosphatase inhibitory activity
Glucose-6-phosphatase is an enzyme made up of proteins, phosphate group and glucose. Glucose exported from cell via glucose transporter membrane proteins. This catalysis is used in gluconeogenesis and plays a key role in the homeostatic regulation of blood glucose level [23]. Generally, glucose-6-phosphatase targets insulin action by inhibiting hyperglycaemia state and it prevent the production of glucose. In type II diabetes, liver becomes resistant to insulin and overexpression of glucose-6-phosphatase leads to uncontrolled gluconeogenesis [42,43]. The inhibition activity of the synthesized nanomaterials was carried out against glucose-6-phosphatase with respect to the standard drug, metformin under identical conditions ( Table 3 and Fig. 9). The obtained results revealed that Ag and CuO NPs, and the nanocomposite synthesized by chemical method showed weak activity when compared to the nanocomposites synthesized by green methods.
The nanocomposite synthesized using Zingiber officinale extract (SC3) showed higher activity among the nanomaterials with respect to their IC50 values because of the rich amount of phytochemicals present in the green extract, which leads to reduce the glucose formation.  Fig. 9. Bar diagram showing the inhibition of glucose-6-phosphatase enzyme by nanomaterials.

Glucose uptake assay
Glucose uptake assay is a simple, non-radioactive assay, which helps in measuring the uptake of glucose inside the cells. The Type 2 diabetes is associated with lack of insulinstimulated glucose uptake, while high glucose uptake is a sign of high glucolytic rate associated with cancer. Glucose uptake assay was carried out against 3T3-L1 adipocyte cells at a concentration of 100 µg/mL in the presence of insulin and metaformin, and the synthesized nanomaterials, both separately and in combination to each other ( Table 4 and  Glucose uptake %

Compliance with Ethical Standards
Conflict of interest The authors declare that they have no competing interest.