Synthesis, Characterization, and Anti-diabetic Activity of Some Novel Vanadium-Folate-Amino Acid Materials

A new six intraperitoneal injections insulin-mimetic vanadyl(IV) compounds [(VO)(FA)(AAn)] (where n = 1–6: AA1 = isoleucine, AA2 = threonine, AA3 = proline, AA4 = phenylalanine, AA5 = lysine, and AA6 = glutamine) were synthesized by the chemical reactions between folic acid (FA), VOSO4, and amino acids (AAn) with equal molar ratio 1:1:1 in neutralized media. These complexes were characterized by elemental analysis and estimation of vanadyl(IV) metal ions. The thermal stability behavior of these complexes was studied by TG-DTG-DTA analyses. The structures of these complexes were elucidated by spectroscopic methods like infrared, electron spin resonance (ESR), and solid reflectance spectroscopes. The powder X-ray diffraction (XRD) study suggested the crystalline nature of the complexes. Magnetic moments and electronic spectra revealed the square-pyramid geometrical structure of the complexes. The conductivity results refereed that all synthesized vanadyl(IV) complexes were of a non-electrolyte behavior. The infrared spectra assignments of these complexes revealed that the FAH2 and AAn chelates act as a bidentate ligation. The chelation towards vanadyl (IV) ions existed via deprotonation of one of the carboxylic groups of FAH2 drug ligand, and so amino acids act as bidentate ligands via N-amino and O-carboxylate groups. Both scanning and transmission electron microscope (SEM and TEM) techniques were used to investigate the surface morphology. The main task of this research is the aim of designing a new insulin alternative antidiabetic drug agent. The antidiabetic efficiency of these complexes was evaluated in streptozotocin-induced diabetic male albino rats. Liver and kidney functions, insulin and blood glucose levels, lipid profile, and superoxide dismutase antioxidant (SOD) are verified identifiers for the efficiency of VO(IV)/FA/AAn system compounds as antidiabetic drug agents.


Introduction
The increasing knowledge of the role of vanadium in biological systems and the potential of vanadium compounds as therapeutic agents have led to increased interest in the chemistry of coordination [1][2][3][4]. Oxovanadium(V) compounds have been utilized as a model of insulin-mimetic therapeutic agents [5,6]. Moreover, there are several medical activities regarding vanadium compounds [4][5][6][7]. Diabetes is the main causative of numerous kidney diseases as well as heart attacks that may result in patient's blindness and amputation of limbs [8,9]. The insufficiency of the pancreas' beta cells is the main cause of path physiological markers in the progression of diabetes 1 or 2 [10][11][12].
Folic acid (FAH 2 ) is commonly named as vitamin B 9 [13]; it was initially isolated from a spinach plant's leaves [14] and then used to remedy in anemia of a megaloblastic nature. Mainly, folic acid is a vitamin that is water-soluble and plays a vital role in a variety of humans' physiological functions. It played a crucial role in one-carbon metabolism for physiological DNA synthesis and cell division [15][16][17][18][19]. Nowadays, it is known that deficiency of folic acid is associated with higher plasma concentrations of homocysteine, a self-reliant risk factor for birth defects and pregnancy complications [20]. Folic acid crucially contributes to the prevention of neural tube defects in infants, not to mention its inhibition of vascular diseases and certain cancers [21].
As basic components of living organisms, amino acids take part in proteins' block building, which are chemical species indispensable to performing a large number of biological functions [22]. Amino acids and their mixed ligand complexes are found in applications of biology, industry, pharmacy, and laboratory reagents [23], with involvement in the human body various activities, e.g., transamination, pH regulation, neurotransmitter functions, cholesterol metabolism, inflammation control, decarboxylation, pain administration, and detoxification. This paper focuses on the preparation, spectroscopic, and biological characterizations of the new vanadyl(IV) folate amino acid series complexes for the utility of treatment of diabetes that was induced by streptozotocin (STZ) in male albino general rats.

Chemical and Reagents
In this study, the used chemicals and reagents were of the highest purity (Aldrich, Saint Louis, MO, USA) and without any further purifications. The folic acid pure drug, vanadyl(IV) sulfate, and amino acids (isoleucine, threonine, proline, phenylalanine, lysine, and glutamine) were bought from the Aldrich Company.

Instruments
A Perkin Elmer CHN 2400 (PerkinElmer Inc., Shelton, CT, USA) was used to conduct the analysis of %C, %H and %N element content. Vanadium content was determined gravimetrically as V 2 O 5 . A Jenway 4010 conductivity meter (Jenway, Staffordshire, UK) was used for the molar conductivity measurements of the samples at 10 −3 M in dimethylsulfoxide (DMSO). A 3101 PC UV/Vis spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan) was used to scan the solid refelactance spectra for samples. A Bruker FT−IR spectrophotometer (Bruker, Billerica, MA, USA) was used to collect the IR spectra for solid samples on KBr discs within the 4000-400 cm −1 range. A Shimadzu TG/DTG-50H thermogravimetric analyzer (Shimadzu Scientific Instruments) was employed for the thermal analyses of solid samples under atmospheric nitrogen conditions. A X'Pert Philips X-ray diffractometer (Philips, Manchester, UK) was used to collect the X-ray diffraction pattern (XRD) patterns for the solid samples using CuK α1 radiation. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) micrographs were obtained using a Jeol Jem−1200 EX II electron microscope (Jeol Ltd., Akishima, Japan) operated at an acceleration voltage of 25 kV. A Gouy magnetic balance (Sherwood Scientific Ltd., Cambridge, UK) was used to measure the mass susceptibility (X g ) of the complexes at room temperature. Electron spin resonance was measured by Jeol, JES-FE2XG, ESR-spectrometer, Frequency 9.44 GHz (Jeol Ltd., Akishima, Japan).

Animal Experiments
The biological experiments were performed on male albino rats of 0.1-0.120 kg weight. These rats were received from the National Research Centre in Cairo (Egypt). These experiments were applied based on the European Community Directive (86/609/EEC) and national rules on animal care. Male rats were classified into four groups. Each group included ten rats, while Group IV included ten rats for each system.
Group I is a normal control group; Group II is a Positive control, injected intraperitoneally by a single dose of STZ (50 mg/kg body weight) [24]; Group III is a Injected i.p. by STZ, then injected each alternative day by VOSO 4 alone with a dose of 40 mg/kg body weight through 30 days and Group IV is a Injected with STZ, and then injected each alternative day by synthesized vanadyl(IV) complexes (I-VI) with a dose of 40 mg/kg body weight [25] for 30 days.

Experimental Diabetes Induction
Induction of experimental diabetes was induced in rats fasting for 18 h via a single intraperitoneally (i.p.) injection of STZ with a 50 mg/kg dose [25] freshly prepared in a cold 0.1 M citrate buffer (pH 4.5). Streptozotocin -injected rats were made to drink a 5% glucose drinking solution during the initial 24 h for survival assurance [26]. Animals were considered diabetic when their blood glucose level surpassed 220 mg/dL [27]. Afterwards, they were included in the study after 72 h of STZ injection.

Blood and Organ Collections
Blood samples of the fasting rats were collected from the medial retro-orbital venous plexus via capillary tubes (Micro Haematocrit Capillaries, (Mucaps, Fisher Scientific, Arendalsvägen, Göteborg, Sweden)) under ether anesthesia [28].

Hematological Parameters
Hemoglobin (Hb) measurements were determined using a cell counter (Sysmex, model KX21N, Sawgrass Drive Bellport, NY, USA)) in grams per deciliter (g/dL) of blood.

Insulin and Blood Glucose Levels
Insulin analysis took place via the insulin-I125 kit, according to Woodhead et al. [29], employing the use of a radioimmunoassay kit from Radio Assay System Laboratories Inc (Carson, CA, USA).

Liver and Kidney Functions
The level of LDH, ALT, uric acid, and creatinine were analyzed using the aforementioned fully auto-chemistry analyzer (Roch Integra 400 plus analyzer). The activities of G6PDH (glucose-6-phosphate dehydrogenase) were determined using commercial kits.

Determination of Blood SOD (Superoxide Dismutase)
Superoxide Dismutase was analyzed via a biodiagnostic kit. The percentage of inhibition was found to be at 560 nm as per the following equation: (1)

Histopathological Examination
Small pieces of liver and pancreas tissues were freshly collected directly following dissection, and then they were directly transferred to a 10% formalin solution for fixture [30].

Statistical Analysis
Data were analyzed using the computer program SPSS version 15.0. Duncan's multiple range test (p < 0.05) (IBM, Armonk, NY, USA) was used in accordance with Snedecor and Cochran to gauge the effect on different treated groups [31].

Microanalytical and Physical Data
The new six vanadyl(IV) complexes, which were synthesized in situ mixed ligands of folate and amino acid chelates, had higher melting points >260 • C with yields 80-85%. The microanalytical, physical, and chemical formulas of the synthesized complexes are listed in Table 1. The resulting data is in good agreement with the general formula of NH 4 [(VO)(FA)(AA n )] complexes. It was confirmed that SO 4 ions were not present using a 10% stock solution of BaCl 2 .2H 2 O reagent. The molar conductance data of the vanadyl(IV) folate amino acid complexes dissolved in a DMSO solvent with a concentration of 10 −3 M were found to be within the limit of 13-24 Ω −1 ·cm 2 ·mol −1 at room temperature, confirming the non-electrolytic state [32][33][34]; hence, the molar conductance values indicated the absence of SO 4 ions inside the coordination sphere. The experimental results were in agreement with the checkout of SO 4 ions using BaCl 2 ·2H 2 O reagent after the dissociation of vanadyl(IV) complexes in concentrated nitric acid. The electronic diffuse reflectance spectra of NH 4 [(VO)(FA)(AA n )] complexes have two distinguishing bands at ranges of 12,500-12,658 cm −1 and 15,625-16,000 cm −1 , which are assigned to 2 B 2 → 2 E and 2 B 2 → 2 B 1 electronic transitions [35]. The absorption band presence at ranges of 17,857-18,691 cm −1 and 21,739-24,390 cm −1 are attributed to the ligand-to-metal charge transfer (L-M CT ) band. At room temperature, the effective magnetic moment µ eff values of vanadyl(IV) folate amino acid complexes are located within 1.70-2.20 BM range; these results revealed that the geometric structure of oxovanadium(IV) complexes is square pyramid [36]. Electron Spin Resonance spectrum of the vanadyl(IV) folate amino acid complexes was measured in solid-state ( Figure 1). After calculation of g || (parallel Landé g-factor), g ⊥ (perpendicular Landé g-factor), A || (parallel hyperfine constant), and A ⊥ (perpendicular hyperfine constant) items from the ESR spectra, it was found that g || < g ⊥ < 2; these results agree with the square pyramid structure assumption [37].
Biomolecules 2020, 10, x FOR PEER REVIEW 5 of 19 room temperature, confirming the non-electrolytic state [32][33][34]; hence, the molar conductance values indicated the absence of SO4 ions inside the coordination sphere. The experimental results were in agreement with the checkout of SO4 ions using BaCl2·2H2O reagent after the dissociation of vanadyl(IV) complexes in concentrated nitric acid. The electronic diffuse reflectance spectra of NH4[(VO)(FA)(AAn)] complexes have two distinguishing bands at ranges of 12,500-12,658 cm -1 and 15,625-16,000 cm -1 , which are assigned to 2 B2→ 2 E and 2 B2→ 2 B1 electronic transitions [35]. The absorption band presence at ranges of 17,857-18,691 cm -1 and 21,739-24,390 cm -1 are attributed to the ligand-to-metal charge transfer (L-MCT) band. At room temperature, the effective magnetic moment μeff values of vanadyl(IV) folate amino acid complexes are located within 1.70-2.20 BM range; these results revealed that the geometric structure of oxovanadium(IV) complexes is square pyramid [36]. Electron Spin Resonance spectrum of the vanadyl(IV) folate amino acid complexes was measured in solid-state ( Figure 1). After calculation of g|| (parallel Landé g-factor), g ┴ (perpendicular Landé g-factor), A|| (parallel hyperfine constant), and A ┴ (perpendicular hyperfine constant) items from the ESR spectra, it was found that g||<g ┴ < 2; these results agree with the square pyramid structure assumption [37].  Table 2.
In the case of free FAH 2 , the stretching vibration band at 1694 cm −1 with very strong absorption is assigned to ν(C=O) ketonic of the carboxylic group [38]. However, this band is a little shifted to lower frequencies (1686-1688 cm −1 ) in the case of synthesized complexes' spectra due to the overlapping between C=O amid and C=O carboxylic [39][40][41][42].
The difference between the two vibration motions of carboxylate group ∆ν = ν as (COO-)-ν s (COO-)] for the vanadyl(IV) folate amino acid complexes are located within 103-74 cm −1 range; this value can be assigned to the association of bidentate-coordinated bond concerning the carboxylate group [44,45].
The δ(NH 2 ) sharp bending vibration motion regarding FAH 2 existsat 1607 cm −1 ; this band exists at the same frequencies in the case of synthesized vanadyl(IV) complexes. This can be attributed to the fact that the nitrogen of the NH 2 group is far away from the coordination process.
Regarding oxovanadium(IV) folate amino acid complexes, the ν(V=O) stretching vibration motion is present as a new band at 1114-1106 cm −1 range [46]. NH 3 to NH 2 transformation must create an upward shift in ν(NH 2 ) and free amino acids. In the current complexes, the infrared (IR) spectra show characteristic broad and split bands in the region 3187-3400 cm −1 are shown in the IR spectra, somewhat lower considering those of free ν(NH 2 ); therefore, it is fair to conclude that the nitrogen of the amino group is of a pivotal role in coordination [46]. Strong evidence is imposed by the IR spectra supporting the assumption of the coordinative role of the carboxylate group. Comparing with free amino acids, the ν as (COO − ) and ν s (COO − ) show negative shifts, confirming the carboxylate group's monodentate nature [46].
Hence, amino acids are concluded to act as a bidentate ligand in these complexes and coordinate through amino nitrogen and carboxylate oxygen. In the far IR spectra of all complexes, new bands viewed at 513-617 cm −1 and 440-460 cm −1 regions may be assigned for the ν(M-O) and ν(M-N), respectively [46].
The microanalytical and spectroscopic discussions of the vanadyl(IV) folate amino acid complexes confirm the suggested stoichiometric formulations ( Figure 3).  Table 2.
In the case of free FAH2, the stretching vibration band at 1694 cm −1 with very strong absorption is assigned to ν(C=O)ketonic of the carboxylic group [38]. However, this band is a little shifted to lower frequencies (1686-1688 cm -1 ) in the case of synthesized complexes' spectra due to the overlapping between C=Oamid and C=Ocarboxylic [39][40][41][42].
The difference between the two vibration motions of carboxylate group Δν = νas(COO-)-νs(COO-)] for the vanadyl(IV) folate amino acid complexes are located within 103-74 cm -1 range; this value can be assigned to the association of bidentate-coordinated bond concerning the carboxylate group [44,45].
The δ(NH2) sharp bending vibration motion regarding FAH2 existsat 1607 cm -1 ; this band exists at the same frequencies in the case of synthesized vanadyl(IV) complexes. This can be attributed to the fact that the nitrogen of the NH2 group is far away from the coordination process.
NH3 to NH2 transformation must create an upward shift in ν(NH2) and free amino acids. In the current complexes, the infrared (IR) spectra show characteristic broad and split bands in the region 3187-3400 cm -1 are shown in the IR spectra, somewhat lower considering those of free ν(NH2); therefore, it is fair to conclude that the nitrogen of the amino group is of a pivotal role in coordination [46]. Strong evidence is imposed by the IR spectra supporting the assumption of the coordinative role of the carboxylate group. Comparing with free amino acids, the νas(COO -) and νs(COO -) show negative shifts, confirming the carboxylate group's monodentate nature [46].
Hence, amino acids are concluded to act as a bidentate ligand in these complexes and coordinate through amino nitrogen and carboxylate oxygen. In the far IR spectra of all complexes, new bands viewed at 513-617 cm −1 and 440-460 cm −1 regions may be assigned for the ν(M-O) and ν(M-N), respectively [46].

Thermal Analysis Study
Thermal stabilities of the NH 4 [(VO)(FA)(AA n )] complexes were investigated based on the thermogravimetric (TG), differential thermogravimetric (DTG), and differential thermal analysis (DTA) from 25-800 • C under nitrogen atmospheres. The thermal decomposition curves are displayed in Figure 4, and the assignments of the thermal decomposition results show the thermal stability of the synthesized material.

X-ray powder diffraction and Transmission Electron Microscopy Studies
The new nanostructured form of NH4[(VO)(FA)(AA1)] complex was investigated using solid X-ray powder diffraction patterns within a 4-80° range of diffraction angle (2θ) and transmission electron microscopy (TEM). The X-ray powder diffraction (XRD) patterns deduced that the vanadyl(IV) folate complex (I) has a crystalline feature. The XRD diffraction patterns of the vanadyl(IV) complex (I) ( Figure 5 and where Dis the particle size, 0.89 is the Sherrer constant, λ represents the wavelength of the radiation of the X-ray (0.154056 nm for Cu Kα), and β stands for the full-width half-maximum (FWHM) of diffraction peak measured at 2θ. The calculation of the particle size of the NH4[(VO)(FA)(AA1)] complex from the highest line diffraction peak at 6.377°. The XRD pattern of vanadyl(IV) folate complex (I) has a nanocrystalline statement with a 5 nm size. According to the TEM image ( Figure 6), the particles of NH4[(VO)(FA)(AA1)] complex exhibit irregular black stones forms, and their size is widely distributed between 50-100 nm.

X-ray powder diffraction and Transmission Electron Microscopy Studies
The new nanostructured form of NH 4 [(VO)(FA)(AA 1 )] complex was investigated using solid X-ray powder diffraction patterns within a 4-80 • range of diffraction angle (2θ) and transmission electron microscopy (TEM). The X-ray powder diffraction (XRD) patterns deduced that the vanadyl(IV) folate complex (I) has a crystalline feature. The XRD diffraction patterns of the vanadyl(IV) complex (I) ( Figure 5 and where Dis the particle size, 0.89 is the Sherrer constant, λ represents the wavelength of the radiation of the X-ray (0.154056 nm for Cu Kα), and β stands for the full-width half-maximum (FWHM) of diffraction peak measured at 2θ. The

Blood Glucose and Insulin Hormone Levels
Blood glucose and insulin hormone levels in the experimental groups are illustrated as follows in Table 4. It was clear that the level of blood glucose of the diabetic groups treated with vanadyl(IV) sulfate alone and vanadyl(IV) complexes (I-VI) is significantly decreased compared with the positive control group, especially Group IV1 that was injected by the NH4[(VO)(FA)(AA1)] complex, where the blood glucose decreased from 410 ± 15 mg/dL in the positive control group to 191 ± 4 mg/dL; so that this complex is considered the most effective one.

Blood Glucose and Insulin Hormone Levels
Blood glucose and insulin hormone levels in the experimental groups are illustrated as follows in Table 4. It was clear that the level of blood glucose of the diabetic groups treated with vanadyl(IV) sulfate alone and vanadyl(IV) complexes (I-VI) is significantly decreased compared with the positive control group, especially Group IV1 that was injected by the NH4[(VO)(FA)(AA1)] complex, where the blood glucose decreased from 410 ± 15 mg/dL in the positive control group to 191 ± 4 mg/dL; so that this complex is considered the most effective one.

Blood Glucose and Insulin Hormone Levels
Blood glucose and insulin hormone levels in the experimental groups are illustrated as follows in Table 4. It was clear that the level of blood glucose of the diabetic groups treated with vanadyl(IV) sulfate alone and vanadyl(IV) complexes (I-VI) is significantly decreased compared with the positive control group, especially Group IV 1 that was injected by the NH 4 [(VO)(FA)(AA 1 )] complex, where the blood glucose decreased from 410 ± 15 mg/dL in the positive control group to 191 ± 4 mg/dL; so that this complex is considered the most effective one.
In general, these results indicated that the administration of vanadyl(IV) sulfate and vanadyl(IV) complexes had moderated activity as a hypoglycemic agent, and this effect could be linked to the mimetic effect of insulin to emulate vanadyl sulfate in different tissues [50,51].
In an in vivo study, vanadyl sulfate augments glucose transport and metabolism in skeletal muscle, adipose tissue, and liver [52]. Table 4. Effect of vanadyl(IV) complexes (I-VI) on insulin hormone, blood glucose level, serum GPT enzyme, creatinine, uric acid level, lactate dehydrogenase, glucose-6-phosphate dehydrogenase activities, hemoglobin levels, superoxide dismutase enzyme (SOD), and lipid profile in normal and diabetic rats. Vanadyl(IV) sulfate and vanadyl(IV) complexes administration to diabetic rats induced substantial changes in insulin hormone levels when compared with the untreated diabetic groups, especially Group IV 1 where the insulin increased by 56%.

Biological Test
Alternatively, the diabetic untreated group exhibited a significant marked decrease in the level of insulin compared with the normal control group alongside the other treated groups, where vanadyl salts can emulate many of insulin's metabolic actions both in vitro and in vivo and enhance glycemic control with diabetes mellitus [53].

Glutamate Pyruvate Transaminase (GPT) Enzyme Activity
Serum GPT is the major enzyme that determines liver functions, acting as an indicator of liver cell damage [54]. GPT enzyme activity in the experimental groups is illustrated in Table 4.
It was indicated that vanadyl(IV) sulfate injection alone at a dose of 40 mg/kg body weight slightly increased the activity of GPT enzyme from 112 ± 7 in the positive control group to 124 ± 11, while the injection of vanadyl(IV) complexes at the same dose slightly decreased the serum GPT activity in the other groups.
It is indicated that vanadyl(IV) complexes treatment had minimal side effects on liver cells of diabetic rats, according to GPT enzyme activity, in comparison with the positive control group, which shown a high increase in liver tissue damage; hence, this is a sign of the low toxicity effect of vanadyl(IV) complexes compared with vanadyl(IV) sulfate alone.

Creatinine and Uric Acid Levels
As per the results in Table 4, diabetic rats treated with vanadyl(IV) sulfate alone and vanadyl(IV) complexes had diminished creatinine level when compared with diabetic rats, especially Group IV 3 that was injected with the NH 4 [(VO)(FA)(AA 3 )]·H 2 O complex, which had serum creatinine decreased from 1 ± 0.2 mg/dL in the positive control group to 0.6 ± 0.2 mg/dL, and thus indicating that VO(IV) complexes had no side effect on kidneys tissue in the animal model system and greatly improved kidney functions, and stating VO(IV) complexes had an ameliorating effect on the functions of kidneys [55,56].
Uric acid increased value (hyperuricemia) observed in diabetic rats coincides with Edwards' [57] findings that uric acid surged in diabetic mice; this may be caused by the breakage of uric acid, in case of diabetics, into substances called purines. According to our study, VO(IV) complexes decreased this value in treated diabetic rats, especially in Group IV 4 , where the uric acid decreased by 35%, and this decline in uric acid can be explained by the impaired oxidative phosphorylation processes that inhibit protein synthesis [58].

Lactate Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Activities
LDH is often used as an indicator of tissue breakdown due to its abundance in red blood cells and its functionality as an indicator of hemolysis [59]. LDH enzyme activity in the experimental groups is illustrated in Table 4. In general, serum LDH activity in STZ diabetic rats exponentially surged compared with the normal control group. VO(IV) complexes administration to STZ diabetic rats caused a significant decline in LDH activities compared with the diabetic positive control groups, especially Group IV 5 , that afforded a significant decrease in LDH by 24%. The increase of LDH activity in diabetic rats caused by LDH leakage into the blood owing to the toxicity of STZ in the liver; these results agree with who stated the same response in alloxan diabetic mice [60].
G6PD is the main source of the major intracellular reductant, NADPH, which is a necessity for many enzymes, including enzymes of the antioxidant pathway [61]. G6PD level decreased in all STZ diabetic rats as compared with the normal control (Table 4); such observations were reported beforehand with the exact parameters [62,63]. Diabetic rats' treatment with vanadyl complexes increased the G6PD activity as compared with the positive diabetic groups, especially Group IV 6 , that showed a significant increase in G6PD activity. Table 4 illustrates the extent of hemoglobin (Hb) levels in the experimental groups. The results indicate that hemoglobin levels diminished compared with the normal control group. The reduction of hemoglobin and anemia that occur in diabetic rats mainly results from the increased RBC membrane proteins nonenzymatic glycosylation, which correlates with hyperglycemia [64]. VO(IV) complexes administration fended the increase in the level of Hb content at the end of the study compared with the untreated diabetic group. Simultaneously, the diabetic group treated with VO(IV) complexes incited a substantial surge in Hb level with reference to the normal control group, especially Group IV 1 that afforded a significant increase in Hb by 8%, and this is an indicator of VO(IV) complexes' low toxicity effect on the living systems of experimental animals.

Superoxide Dismutase Enzyme (SOD)
The superoxide dismutase (SOD) is a crucial enzyme of the antioxidant system that increase their activities in the case of positive effects of an antioxidant agent in a living system. SOD scavenges the superoxide radical by transforming it into H 2 O 2 and molecular oxygen. The SOD activity is minor in diabetes mellitus [65]; SOD diminished activity could a result of its degradation or inhibition due to increased production of free radicals. In Table 4, our results indicate the significant decrease of SOD in diabetic rats as compared with the normal control group, unlike the slight decrease in SOD in VO(IV) complexes-treated diabetic group. The treatment with VO(IV) complexes motivated the activity of SOD and may assist in controlling diabetic rats' free radicals. Table 4 illustrates total cholesterol levels (TC), triglycerides (TG), HDL-c, and LDL-c. The results exhibit that total cholesterol levels (TC), triglycerides (TG), and LDL-c substantially surge in diabetic STZ rats as compared with the normal control group, while inhibition of the levels of serum HDL-c in diabetic rats was noticed, agreeing with Bolkent et al.'s [66,67] reports beforehand. The aberrant serum lipids' high levels in diabetic animals are mainly resulting from the enhanced mobilization of free fatty acids from the peripheral deposits, as insulin hinders the hormone-sensitive lipase [68]. Extra fatty acids in diabetic mice's serum are transformed into phospholipids and cholesterol in the liver. It is an insulin-dependent tissue that plays crucially affects glucose and lipid homeostasis and severely affected in the case of diabetes [69]. Diabetes induces a decrease in the utilization of glucose and a surge in the production of glucose in insulin-dependent tissues, e.g., liver [70]. Hypercholesterolemia's degree is directly proportional to the severity of diabetes. This study showed that VO(IV) complexes administration significantly improved the parameters of lipid metabolism where total cholesterol (TC), triglycerides (TG), and LDL-c are substantially diminished, while HDL-c levels are significantly increased in the serum of diabetic rats. The underlying mechanism of vanadyl sulfate exertion of cholesterol's lowering effect seems to be an inhibition in cholesterol absorption from the intestine through binding with the intestine's bile acids and increasing bile acids excretion [71]. However, Sharma et al. [72] reported the decreasing effect of vanadyl sulfate on the cholesterol biosynthesis, especially the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, a key enzyme of cholesterol biosynthesis, and/or the reducing effect that NADPH requires for fatty acids and cholesterol biosynthesis. Additionally, vanadyl may enhance hypercholesterolemia by altering lipoprotein metabolism: enhancing uptake of LDL by increasing LDL receptors [73], and/or surging the activity of lecithin cholesterol acyl transferase [74], which play a role in blood lipid regulation.

Histopathology of Pancreas
Pancreas cells from the normal control group were all normally proportional and structured in terms of pancreatic tissue and size, as shown by the normal-sized islet of Langerhans surrounded by normal pancreatic acini. The islets consist of glucagon-secreting alpha cells and insulin-secreting beta cells, as shown in Figure 7A. On the other hand, pancreatic tissues in STZ diabetic control rats showed dilated congested vascular spaces surrounded by inflammatory cells and pancreatic acini's aggregations. The islets are mainly inhabited by a uniform material of eosinophilic nature and few atrophic cells with reduced size. Eosinophilic materials also surround the blood vessel, as shown in Figure 7B,C. Pancreatic tissues in diabetic rats treated with only VOSO 4 showed mild improvement of the size of the islet of Langerhans, with an expanded congested vascular space surrounded by a few inflammatory cells aggregates, as shown in Figure 7D,E. Pancreatic tissues in diabetic rats treated with folic acid/vanadyl/isoleucine system complex NH 4 [(VO)(FA)(AA 1 )] showed a good response with a return of islet of Langerhans to its normal size and absence of inflammatory cells and no eosinophilic deposits were seen, as shown in Figure 7F.
In the present study, the histopathology of the pancreas of the normal control rats yielded no notable changes in its histology across the 30-day study. On the other hand, administrating STZ incited severe pancreatic injury, decreasing the number of the islet cell and the diameter of the pancreatic islets where the islets shrunk in the diabetic rats compared with the normal ones. The islets destruction absolutely diminishes insulin, characteristic of diabetes mellitus. The administration of VOSO 4 showed a mild islet expansion and substantially decreased pancreatic injuries within 30 days of treatment and recovered pancreatic tissue damage. The treatment of the diabetic rats with vanadyl complexes, especially NH 4 [(VO)(FA)(AA 1 )] complex, return to the normal pancreas histological structure with rich vascular supply and this may be due to the role of the prepared complexes in recovering the damage of pancreatic tissue that is caused by STZ-induced diabetes. In summary, this study assessed the STZ effect on β-cells and focused on vanadyl complexes potential in the prevention or treatment of diabetes. response with a return of islet of Langerhans to its normal size and absence of inflammatory cells and no eosinophilic deposits were seen, as shown in Figure 7F.
In the present study, the histopathology of the pancreas of the normal control rats yielded no notable changes in its histology across the 30-day study. On the other hand, administrating STZ incited severe pancreatic injury, decreasing the number of the islet cell and the diameter of the pancreatic islets where the islets shrunk in the diabetic rats compared with the normal ones. The islets destruction absolutely diminishes insulin, characteristic of diabetes mellitus. The administration of VOSO4 showed a mild islet expansion and substantially decreased pancreatic injuries within 30 days of treatment and recovered pancreatic tissue damage. The treatment of the diabetic rats with vanadyl complexes, especially NH4[(VO)(FA)(AA1)] complex, return to the normal pancreas histological structure with rich vascular supply and this may be due to the role of the prepared complexes in recovering the damage of pancreatic tissue that is caused by STZ-induced diabetes. In summary, this study assessed the STZ effect on β-cells and focused on vanadyl complexes potential in the prevention or treatment of diabetes. 3.2.9. Histopathology of Liver From a microscopic perspective, liver samples from the normal control group showed normal structure consisting of the central vein surrounded by cords and rows of optimally healthy hepatocytes with a central nucleus and blood sinusoids, as shown in Figure 8A. Alternatively, liver

Histopathology of Liver
From a microscopic perspective, liver samples from the normal control group showed normal structure consisting of the central vein surrounded by cords and rows of optimally healthy hepatocytes with a central nucleus and blood sinusoids, as shown in Figure 8A. Alternatively, liver tissues in the diabetic control rats group exhibited a substantial area of hepatic necrosis infiltrated with inflammatory cells with a markedly expanded congested central vein laden with red blood cells and surrounded by aggregates of inflammatory cells with rows and cords of swelled and degenerated hepatocytes with a severe fatty change, as shown in Figure 8B. Liver tissues in diabetic rats treated with only VOSO 4 showed a mild improvement of hepatocytes with a moderately dilated congested central vein surrounded by rows and cords of hepatocytes showing a moderate degree of fatty change with normal parenchymal histology, as shown in Figure 8C. Liver tissues in diabetic rats treated with folic acid/vanadyl/isoleucine system complex NH 4 [(VO)(FA)(AA 1 )] showed good improvement of the liver tissues and returned to the normal state to the normal size of the central vein surrounded by rows and cords of normal hepatocytes and absence of inflammatory cells, as shown in Figure 8D.
In the current study, the results showed that treatment of the diabetic rats with vanadyl complexes, especially the NH 4 [(VO)(FA)(AA 1 )] complex, return the normal liver histological structure and this may be due to the role of the prepared complexes in diminishing the oxidative stress on hepatic cells and diminishing hepatocellular damage and suppression of gluconeogenesis; consequently, this may ameliorate liver damage caused by STZ-induced diabetes, which agrees with Subash et al.'s results as they studied the same effect of cinnamaldehyde on liver tissues [75].
Biomolecules 2020, 10, x FOR PEER REVIEW  15 of 19 with inflammatory cells with a markedly expanded congested central vein laden with red blood cells and surrounded by aggregates of inflammatory cells with rows and cords of swelled and degenerated hepatocytes with a severe fatty change, as shown in Figure 8B. Liver tissues in diabetic rats treated with only VOSO4 showed a mild improvement of hepatocytes with a moderately dilated congested central vein surrounded by rows and cords of hepatocytes showing a moderate degree of fatty change with normal parenchymal histology, as shown in Figure 8C. Liver tissues in diabetic rats treated with folic acid/vanadyl/isoleucine system complex NH4[(VO)(FA)(AA1)] showed good improvement of the liver tissues and returned to the normal state to the normal size of the central vein surrounded by rows and cords of normal hepatocytes and absence of inflammatory cells, as shown in Figure 8D.
In the current study, the results showed that treatment of the diabetic rats with vanadyl complexes, especially the NH4[(VO)(FA)(AA1)] complex, return the normal liver histological structure and this may be due to the role of the prepared complexes in diminishing the oxidative stress on hepatic cells and diminishing hepatocellular damage and suppression of gluconeogenesis; consequently, this may ameliorate liver damage caused by STZ-induced diabetes, which agrees with Subash et al.'s results as they studied the same effect of cinnamaldehyde on liver tissues [75].

Conclusions
In conclusion, the study demonstrates the synthesis of a new six intraperitoneal injections insulin-mimetic vanadyl(IV) compounds [(VO)(FA)(AAn)]. These compounds were synthesized by the chemical reactions between folic acid (FA), VOSO4, and amino acids (isoleucine, threonine,

Conclusions
In conclusion, the study demonstrates the synthesis of a new six intraperitoneal injections insulin-mimetic vanadyl(IV) compounds [(VO)(FA)(AA n )]. These compounds were synthesized by the chemical reactions between folic acid (FA), VOSO 4 , and amino acids (isoleucine, threonine, proline, phenylalanine, lysine, and glutamine) with equal molar ratio 1:1:1 in neutralized media. The characteristics of these compounds were discussed based on spectroscopic techniques such as infrared, electron spin resonance (ESR), and solid reflectance spectroscopes. An elemental analysis (XRD) study suggested the crystalline nature of the complexes; the transmission electron microscope (TEM) technique was used to investigate the surface morphology. These six newly synthesized complexes were tested in rats using a 30-day STZ-induced diabetic model. In this in vivo study, the insulin and blood glucose levels, the lipid profiles, and the histology of the pancreas and liver of the animals are qualified factors to identify the efficiency of these complexes as an alternative antidiabetic drug model.