Hypoglycaemic activity of biosynthesized copper oxide nanoparticles in alloxan‐induced diabetic Wister rats

Abstract Background Diabetes mellitus (DM) is a metabolic disorder that affects the body's ability to produce or use insulin. This study evaluated the hypoglycaemic activity of biosynthesized copper oxide nanoparticles (CuO‐NPs) in alloxan‐induced diabetic Wister rats. Methods CuO‐NPs were synthesized via the green route and characterized using different analytical tools. Diabetes was induced intraperitoneally using 90 mg/kg body weight of alloxan monohydrate in albino rats. Thirty (30) rats were randomly divided into 5 groups of 6 rats each and orally treated for 21 days. Groups I and II were treated with 300 mg/kg bwt Cereus hildmannianus extract and CuO‐NPs, respectively. Groups III and IV received 5 mg/kg bwt of Glibenclamide and 2 mL of normal saline, respectively, while Group V was left untreated as the diabetic control. Blood glucose (BG) levels and body weight changes were monitored at 3‐ and 7‐day intervals, respectively, throughout 21‐day treatment period. Lipid profiles, enzyme assays and histopathological studies of the liver were also carried out. Results Spheroidal tenorite phase of CuO‐NPs with a crystallite size of 62.57 nm, surface area (20.64 m2/g) and a UV‐maximum absorption at 214.27 nm was formed. The diabetic rats treated with 300 mg/kg bwt CuO‐NPs had the highest BG lowering ability (from 482.75 ± 27.70 to 124.50 ± 2.50 mg/dL). A significant difference (p < 0.05) in weight gain and serum enzymes was also observed in the CuO‐NPs treated group compared with other groups. The CuO‐NPs‐treated group had a significant increase (p < 0.05) in HDL‐cholesterol and a decrease in total cholesterol, triglycerides, LDL‐cholesterol and VLDL‐cholesterol compared with other groups. Conclusion The green synthesized CuO‐NPs nanoparticles significantly reduced (p < 0.05) blood glucose levels in rats and other associated indices and could serve as drug lead in the treatment of diabetes.


| INTRODUC TI ON
Diabetes mellitus (DM) is a chronic metabolic disorder caused by an alteration in insulin secretion, insulin action or both. 1 It is characterized by persistent hyperglycaemia and problems in carbohydrate, lipid and protein metabolism. Long-term damage is one of the outcomes of DM with diverse organ malfunction and failure. 1,2 There are an estimated 143 million persons living with diabetes globally and by 2030; this figure is expected to double. [3][4][5] There are three forms of DM. 6,7 Type 1 diabetes (insulin-dependent DM) is an autoimmune disease that occurs when the β-cells of the pancreas are damaged, resulting in the pancreas producing little or no insulin. It most commonly affects children and young adults. 6 Type 2 diabetes, commonly known as 'insulin-dependent DM', is the most common type of diabetes in adults, accounting for more than 90% of cases. Insulin resistance is a condition in which the pancreas generates enough insulin but the body is unable to use the insulin efficiently. 3,7 The third form of diabetes is gestational DM (GDM), which is a type of glucose intolerance that develops or is first noticed during the second or third trimester of pregnancy.
One of the most common metabolic disorders during pregnancy is GDM. It is caused by a lack of insulin, or a hormone produced during pregnancy. 6,7 Although a wide range of synthetic drugs have lately been used to treat T2DM, the majority of them have significant long-term side effects, including drug resistance, hepatotoxicity, abdominal pain, flatulence and diarrhoea. 3,4 As a result, a search for an alternate drug with hypoglycaemic effects on T2DM is required.
In the last few decades, advances in nanotechnology have played a key role in the medical, pharmaceutical, agricultural and textile industries. 8 Metallic nanoparticles such as silver, zinc and gold have been reported to have therapeutic potentials. 9,10 Oxides of transition metals such as ZnO, CuO, TiO 2 , Fe 3 O 4 and NiO nanoparticles have found application as advanced nanosubstances in various fields of life such as energy, environmental and biomedical field due to their high surface area and adsorption capacity. 8 Different researchers have reported that metallic oxide nanoparticles have better biological activities than metallic nanoparticles. [11][12][13][14][15] Copper is one of the most common transition metals found in metabolic pathways. Several transition metals have catalytic activity and oxygen transport potentials into the host active sites as cofactors. 3 For instance, the biocompatibility of CuO-NPs reduces the danger of toxicity, which is a key issue for other metallic nanoparticles with medical applications. Hence, biologically synthesized metallic oxide nanoparticles with a wide range of therapeutic potentials have recently gained attention from researchers. [16][17][18][19][20] CuO-NPs have also been reported to have anticancer, antimicrobial and antioxidant properties thus, suitable as a viable tool for biomedical applications. 21,22 CuO-NPs have been synthesized via different routes using physical and chemical approaches such as microwave irradiation, thermal decomposition, sol-gel, colloidal thermal synthesis, sonochemical, hydrothermal and quick precipitation. 23 However, these methods are labour and energy intensive, expensive and involve the release of hazardous chemicals into the environment. 24,25 Currently, there is a shift from the physical and chemical methods of synthesis of nanoparticles to the biological method known as biosynthesis or green synthesis. 15,26 The phytosynthesis of CuO-NPs has gained more attention recently owing to its sustainability, cost-effectiveness and simplicity. 8 Green synthesis involves the use of bacteria, fungi, algae, yeast and plant extracts or their products as reducing agents for the synthesis of metallic or metallic oxide nanoparticles, which support biocompatibility and large-scale production. 27 Several authors have reported the green synthesis of CuO-NPs using different plant extracts [27][28][29][30][31][32] including their antibacterial potentials. 33,34 In addition, Ghosh et al. 3 reported on the in vitro antidiabetic activity of CuO-NPs synthesized using Dioscorea bulbifera extract and found out that the nanoparticles significantly inhibited α-amylase and α-glucosidase, which are considered to be significant pharmacological targets for the treatment of type 2 DM. Different researchers have also investigated the in vitro antidiabetic activity of CuO-NPs [2]. This is the first comprehensive report on the in vivo hypoglycaemic activity of biosynthesized CuO-NPs in alloxan-induced Wister rats. This study evaluated the hypoglycaemic activities of biosynthesized CuO-NPs in alloxan-induced Wister rats. The green synthesis of CuO-NPs was carried out using copper (II) chloride dihydrate and an extract of Cereus hidmannianus as the precursor.
The prepared CuO-NPs were then characterized for their morphology and microstructure, elemental composition, absorption bands and surface area. The in vivo activity of the biosynthesized CuO-NPs in Wister rats was also examined. Finally, lipid profiles, enzyme assays and histopathological studies of the liver were also carried out.

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into a fine powder using mortar with a pestle and a kitchen-type blender.
Five hundred grams (500 g) of the powdered sample was extracted exhaustively with methanol under reflux at 40°C for 2 h. The extract was filtered using a muslin cloth and Whatman No 1 filter paper. The extract was left open in a fume hood for the methanol to evaporate, and a semisolid brown-like paste was finally obtained as the extract. The extract was preserved in a refrigerator at 4°C until required for use.

| Quantitative phytochemical analysis of C. hildmannianus extract
The total flavonoids, total phenols and tannins contents of the plant extract were determined using the modified method reported by Daniel et al. 1

| Green Synthesis of Copper Oxide nanoparticles
Twenty gram (20 g) of the powdered plant was weighed and put into a 500 mL beaker containing 400 mL of distilled water. The mixture was stirred using a magnetic stirrer for about 30 min at 40°C.
The brownish mixture was filtered using filter paper to obtain a to raise the pH value to 9 and stirred further for 30 min. This was accompanied by the formation of precipitates, which were left to age overnight and then filtered using a Whatman No 1 filter paper.
The precipitates were separated from the aqueous extract, first by decantation, and then washed severally with distilled water until the filtrate was clear. The washed precipitates obtained were dried in an oven at 80°C for 3 h.

| Characterization of Copper oxide Nanoparticles
The absorption band of the synthesized CuO-NPs was characterized using NOVA 4200e surface area and pore analyser instrument and X-ray photoelectron spectroscopy (PHI 5400) couple with a hemispherical sector analyser to confirm the physicochemical properties of the synthesized CuO-NPs.

| Experimental animals/ethics
Thirty (30) Wistar rats of either sex aged 8-12 weeks old weighing between 100 and 150 g were purchased from the animal farm of the University of Jos, Jos, Plateau State, Nigeria. The rats were kept in well-ventilated metal cages and maintained at room temperature of 28 ± 2°C, 45-55% of relative humidity on a 12-h light/12-h dark cycle, with access to water and pelletized commercial livestock feed (grower) ad libitum. The rats were kept for 2 weeks to acclimatize to the environmental conditions.

| Induction of diabetes
Diabetes mellitus was induced in the rats by a single intraperitoneal

| Experimental design
The rat groups were assigned on the basis of the treatments received by oral gavage on a daily basis for 21 days as follows: Group I: Diabetic rats treated with 300 mg/kg bwt of methanol extract of C. hildmannianus, Group II: Diabetic rats treated with 300 mg/kg bwt of CuO-NPs, Group III: Diabetic rats received 5 mg/kg bwt of Glibenclamide (standard drug), Group IV: Normal control rats received 1.0 mL/kg bwt of PBS, Group V: Diabetic control rats. The body weight of rats in each group was also determined at every 7day intervals.

| Collection and preparation of blood and tissues
On the 21st day of the experiment, overnight fasted rats were euthanized using 150 mg/kg bwt of sodium pentobarbitone anaesthesia; afterwards, blood samples were collected via cardiac puncture from each mouse into a plain sample bottle. The blood samples were kept at room temperature for 2 h to coagulate and then centrifuged at 3000x g for 10 min; then serum was separated with clean Pasteur pipette and store frozen until used for biochemical analysis. The liver of rats was excised, rinsed in normal saline and preserved in 10% formol saline (v/v) for histopathological study. 36

| Biochemical assays
The biochemical parameters of the serum samples of the treated and untreated rats at the end of 21-day treatment were assayed using appropriate kits. High-density lipoprotein (HDL), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST), Total cholesterol (TC) and triglyceride concentrations (TRIG) were determined as described by Oluba et al., 37 Additionally, serum LDL-C concentration was determined according to the formula described by Friedewald et al. 38 and reported by Oluba et al. 37 Serum VLDL-C concentration was estimated using the methods of Burnstein and Sammaille, 39 where the ratio of serum VLDL-C to triglyceride concentrations was fixed at 1:5 in fasting animals.

| Statistical and data analyses
The data collected were presented as mean ± SEM and analysed using a one-way analysis of variance (ANOVA), while treatment means were separated by the least significant difference (LSD) incorporated in the IBM statistical package for social sciences (SPSS) version 20 (IBM Corp., Armonk, N.Y., USA).

| Quantitative phytochemicals constituents of methanol stem extract of C. hildmannianus
The crude extract of C. hildmannianus leaf showed the presence of phenols, flavonoids and tannins as shown in Table 1 below. The result shows that phenol has the highest concentration (86.1 ± 0.21 mg/g) with flavonoids having the least concentration (1.61 ± 0.15 mg/g).

| Characterization of copper oxide nanoparticles
Characterization of the synthesized CuO-NPs was carried out using a UV-visible spectrophotometer, energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) pattern as shown in Figure 1A The BET plot of nitrogen adsorption-desorption isotherms of CuO-NPs is presented in Figure 2. The isotherm plot of the CuO-NPs according to IUPAC classification belongs to type IV with a narrow hysteresis which represent the mesoporous nature of CuO-NPs. 40 The nanoparticle has a large surface area of 20.640 m 2 /g, a pore diameter of 11.517 nm and a pore volume of 0.08 cc/g (Figure 2A). The surface oxidation state of the elements in CuO-NPs was examined using XPS containing Cu and O at different binding energies belonging to 1s, 3s and 3p orbitals ( Figure 2B). The XPS spectrum of Cu 2p and O 1s of CuO-NPs ( Figure 2C,D) shows that the spectrum was calibrated with a binding energy of 285.65 eV for C 1s electron.
High-resolution scanning electron microscope (HRSEM) was further used to determine the morphology of the synthesized nanoparticles, which indicated that the synthesized nanoparticles are agglomerated spherical particles of different sizes ( Figure 3A).
The high aggregation/agglomeration of the nanoparticles may be attributed to the polarity and electrostatic force of attraction between the copper salt precursor and plant extract used. 41 High-resolution transmission electron microscope (HRTEM) of the nanoparticles revealed the presence of spherical and rod-shaped nanoparticles ( Figure 3B). The HRTEM image and the selected area electron diffraction (SAED) pattern ( Figure 3C) acquired from an area containing many monodispersed nanoparticles which corroborated with the XRD data. The nanoparticles average particle size ranges between 60 nm ( Figure 3D).  with CuO-NPs in Figure 4C shows a significant decrease (p < 0.05) in plasma cholesterol, triglycerides, low-density lipoprotein (LDL) and very-low-density lipoproteins (VLDL) with an increase in high-density lipoproteins (HDL). There was a significant decrease (p < 0.05) in the serum ALT, AST and ALP of rats treated with CuO-NPs compared with the extract control and Glibenclamide groups ( Figure 4D).

| DISCUSS ION
The synthesis of CuO-NPs was successfully carried out via the green methods using an extract of C. hildmannianus. The synthesis of metallic oxide nanoparticles requires three major components, which are reducing agents, stabilizing agents and solvent medium. [42][43][44] Although different metallic nanoparticles have been synthesized using biological materials such as plants, bacteria, fungi and algae, the actual mechanism of synthesis remains unknown with novel methods evolving each day hence, presenting opportunities for further study. 45  With further stirring and oven drying at 80°C for 3 h, copper (I) oxide was converted to CuO-NPs via a series of reaction steps (3)(4)(5). 47 CuO-NPs were synthesized as spherical-shaped nanoparticles after the reduction of CuO material by the action of plant phytochemicals. 48 Further confirmation of the biosynthesized CuO-NPs using energy-dispersive X-ray spectroscopy (EDX) shows that the nanoparticles consist majorly of Cu and O with a trace of C with an atomic weight of 55.72%, 34.57% and 9.70%, respectively ( Figure 1B). The presence of carbon maybe attributed to the phytochemical constituents of the extract such as phenols, flavonoids and tannins, which are present in the extract. 49 The crystal formation of the synthesized CuO nanoparticles was determined using X-ray diffraction (XRD) analysis ( Figure 1C). The result revealed the spheroi-  (1) High-resolution scanning electron microscopy image, (B) High-resolution transmission electron microscopy image, (C) selected area electron diffraction patterns and (D) particle size distribution of green synthesized CuO-NPs using stem extract of C. hildmannianus.
hysteresis, which represents the mesoporous nature of CuO-NPs. 40 The nanoparticle has a large surface area of 20.640 m 2 /g, a pore diameter of 11.517 nm and a pore volume of 0.08 cc/g. The result of this study is at variance with the report of Manasa et al., 40 and Navada et al., 50 who reported a surface area of 17.10 m 2 /g. The variation in the result may be because of different reaction conditions used in the synthesis of the nanoparticles. Small particle size and high surface area play a key role in determining the biological activity of nanoparticles causing an improvement in surface reactivity. 51,52 The surface oxidation state of the elements in CuO-NPs was examined using XPS as shown in Figure 2B. The nanoparticle-containing Cu and O at different binding energies belonging to 1s, 3s and 3p orbitals. The binding energy of 934.10 and 943.56 eV corresponds to Cu 2p 3/2 and Cu 2p 1/2 , respectively, which is similar to the reported data of Saif et al. 53 The splitting between these two states is about 9.46 eV, which confirmed the existence of Cu in +2 oxidation state.
The binding energy at 532.69 eV corresponds to O 1s of CuO-NPs. Similar binding energy (529.8 eV) was also reported by Tamuly et al. 54 and Saif et al. 53 The XPS analysis strongly confirmed the absence of Blood glucose is the key biomarker used for diagnosis and prognosis of DM. According to Figure 4A, it was noticed that treatment with 300 mg/kg bwt of CuO-NPs decreased blood glucose levels significantly (p < 0.05) when compared to 300 mg/kg bwt of C. hildmannianus and the diabetic control ( Figure 4A). This agrees with a previous report by Martín Giménez et al. 55 that CuO-NPs are good therapeutic agents for the management of type 2 DM. One of the most promising tools in type 2 diabetes management is the use of natural health products. Several trace metals such as chromium, selenium, vanadium, molybdenum and magnesium have been reported to be a good therapeutic agent for lowering blood glucose 33 because of their insulin-mimetic actions, they have hypoglycaemic activity. [56][57][58][59] The ability of alloxan to induce weight loss in untreated rats' mimics what is commonly observed in clinical diabetes. Alloxan induces diabetes by destroying the β-cells of the islets of Langerhans in the pancreas prompt a decreased degree of endogenous insulin accordingly influencing glucose uptake by tissue. 60 Glibenclamide, an example of sulfonylurea drugs, fortifies β-cells to deliver insulin along these lines bringing down the blood glucose level. 61  which might be due to the breakdown of muscle fat and proteins. 60 In Figure 4B, it was noticed that the body weight of diabetic rats treated with the CuO-NPs increased significantly (p < 0.05) when compared to groups treated with extract and Glibenclamide. This suggests that the nanoparticles were able to reverse the weight loss because of alloxan induction in the animals. The improvement in the body weight seen in hyperglycaemic rats treated with CuO-NPs might be because of improved metabolic activities making the body more capable of maintaining blood glucose haemostasis. 60 Diabetes is also associated with an alteration in lipid profiles and other biochemical parameters of patients. In Figure 4C, there was a significant decrease in plasma cholesterol, triglycerides, LDL, and VLDL with an increase in HDL levels for diabetic rats treated with nanoparticles compared with the diabetic control and extract group. On the contrary, a significant increase (p < 0.05) in serum lipids level was observed in the alloxan-induced rats treated with CuO-NPs when compared to the normal control. This may be a result of a disturbance in the regulation of the activity of the hormonesensitive enzyme lipase by insulin due to its deficiency or absence caused by the destruction of the β-cells of the islet of Langerhans by alloxan. 62 The decrease in the various lipid profile parameters of the diabetic rats may be due to impaired fatty acids synthesis, enhanced catabolism of VLDL, activation of Lecithin: Cholesterol Acyltransferase (LCAT), tissue lipases, inhibition of acetyl-CoA carboxylase and production of triglycerides precursors such acetyl-CoA and glycerol phosphate by CuO-NPs. 63 Also, a significant decrease (p < 0.05) in the serum ALT, AST and ALP of rats treated with CuO-NPs compared with the extract control and Glibenclamide groups is observed in Figure 4D. Serum AST, ALT and ALP are enzyme biomarkers used in monitoring liver structural integrity and help in the clinical diagnosis of liver toxicity. 64 Increased serum liver enzymes are an indication of hepatocellular destruction typical to diabetic complications. 65 The result, therefore, indicates that the nanoparticles may not be responsible for any injury in the liver of the rats as observed in the histopathological study of the liver of rats treated with the extract and CuO-NPs ( Figure 5). The report of this study is contrary to that of Abdelazeim et al. 62 who observed an elevation in serum enzymes as a result of an injury in the liver of rats treated with CuO-NPs. Mohammadyari et al. 66 also reported that CuO-NPs increase the ratio of ALT/AST in the serum, which is an indication of cellular leakage. Finally, elevated level of stress has also been reported to be responsible for triggering several ailments, particularly hepatic inflammation. 35 Therefore, the observed alteration in the serum enzymes of the experimental animals may be due to the stress they undergo because of alloxan induction and the process of treatment for 21 days.
In conclusion, the antidiabetic potentials of CuO-NPs were successfully evaluated in this study. The nanoparticles significantly lowered (p < 0.05) blood glucose levels, plasma cholesterol, triglycerides, LDL and VLDL and an increase in HDL levels of alloxan-induced diabetic rats after 21 days of treatment. Antidiabetic activities of the CuO-NPs were linked to its crystalline nature and enhanced F I G U R E 5 Histoarchitecture of liver sections of diabetic rats treated with 300 mg/kg bwt of C. hildmannianus extract (A), 300 mg/kg bwt of CuO nanoparticles (B), 5 mg/kg bwt of Glibenclamide (C), diabetic control (D) and normal saline (E), respectively, showing hepatic tissue with preserved architecture composed of cords of normal hepatocytes, normal portal tracts and central vein. There are no features of acute or chronic damage in the organs of the experimental rats. Keys: HC, Hepatic cells; PT, Portal tract.
surface area for improved absorption and distribution. Rats treated with CuO-NPs show no alteration of liver architecture or injury. Therefore, it is recommended that further study on the toxicity and safety of this nanoparticle in animal models should be carried out so as to validate the report of this study and justify its usage and safe dose.

ACK N O WLE D G E M ENTS
We thank the support of the Technologists and Technical staffs of the Department of Biochemistry, Federal University of Technology, Minna, for their assistance and granting us access to the Laboratory facilities.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.