INTRODUCTION:

Diabetes mellitus (DM) is one of the major health catastrophes of the present century. The global diabetes prevalence in 2019 was 9.3% (463 million people), expected to rise to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045¹. It is one of the world's oldest illnesses, recognized in historical records of civilizations². It is characterized by a persistent elevation in blood glucose level resulting from defects in insulin secretion (T1DM) and/or its action or both (T2DM).

T2DM accounts for more than 90% of the diabetic population and is multifactorial in origin. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of different organs, especially the eyes, kidneys, nerves, heart and blood vessels^3,. Several pathogenic processes are implicated in the initiation, progression and onset of diabetes and its secondary complications^4,. Numerous experimental and clinical studies established the role of chronic hyperglycemia-induced oxidative stress in the etiology of diabetes and its secondary complications. The term oxidative stress refers to the condition in which cells are exposed to excessive levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which are the terms collectively used to describe free radicals^5.. Most of the currently available drugs for the treatment of diabetes possess antioxidant properties next to their antidiabetic action⁶. Hence the search for novel drugs capable of controlling oxidative stress and hyperglycemia effectively at a low dose continues.

Zinc is one of the most abundant essential trace elements next to iron in the human body⁷. Zinc is found to be a crucial element that plays a pivotal role in numerous physiological and pathological processes including cell proliferation, differentiation, and viability including apoptosis⁸. Its catalytic functions in about 1,000 human enzymes of all six enzyme classes are used predominantly in hydrolytic reactions and at some committing steps in intermediary metabolism observed⁹. The serendipitous scrutiny by Scott and Fisher (1938)¹⁰of a 50% reduction in pancreatic zinc concentration of diabetic cadavers compared to non-diabetic cadavers illuminate interest in the interactions among zinc, the pancreas and diabetes. Zn is involved in a multitude of processes within the pancreas, including insulin secretion, storage, signaling, glucagon secretion and digestive enzyme activity¹¹. Experimental manipulations of zinc status in rodent models of experimental diabetes offer a valuable approach to investigate the mechanisms for the protective effects of zinc. Since the discovery of Insulin mimetic actions of zinc, several zinc complexes have been proposed, synthesized and their antidiabetic properties were studied both in vitro as well as in vivo^12,. However, most of the zinc complexes so far investigated for their possible antidiabetic and insulin mimic activity were poorly absorbed in their inorganic forms and required high doses, which have been associated with undesirable side effects¹³. To thwart the chronic toxicity and increase gastrointestinal tract absorption of zinc, several complexes have been formulated using ecologically derived non-nutrient plant secondary metabolites such as flavonoids as organic ligands and extensively studied for their stability, toxicity and antidiabetic properties^14,

Avicularin, a bioactive flavonol originally isolated from the leaves of Polygonum aviculare Linn., is commonly known as knotweed¹⁵. Avicularin is non-toxic and reported to possess a wide range of pharmacological properties. The pharmacokinetic studies evidenced that the oral administration of Avicularin is absorbed rapidly and the peak plasma concentration reached after approximately 30min and remained for a long time (240min). In view of the beneficial and pharmacological properties bestowed with Avicularin we have synthesized, characterized a new Zn-Avicularin complex and evaluated its toxicity and antidiabetic properties in high-fat diet fed-low dose streptozotocin-induced experimental diabetes in rats¹⁶. In the present study, an attempt has been made to evaluate the antioxidant properties new Zn-Avicularin complex.

MATERIALS AND METHODS:

Chemicals:

Zinc chloride [Zncl₂], Avicularin (Chemical Formula: C₂₀H₁₈O₁₁, Molecular Weight: 434.35) and Streptozotocin were purchased from Sigma-Aldrich, St. Louis, USA. Ultra-sensitive ELISA kit for rat insulin and C-peptide was purchased from Crystal Chem. Inc. Life Technologies, India. All other chemicals and reagents used in the present study were of analytical grade obtained from SRL chemicals, Bombay, India.

Synthesis of macrocyclic Schiff base complexes [ML]:

The Zn-Avicularin complex was synthesized as schematically represented below and characterized as previously reported by us¹⁷.

Scheme: Schematic representation for the synthesis of a Zn-Avicularin complex

Experimental animals:

Male albino rats of Wistar strain weighing (160–180 g) were procured from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai. The rats were fed with commercial pellet rats chow (Hindustan Lever Ltd., Bangalore, India), and had free access to water ad libitum. The animal studies were designed and performed in accordance with the current ethical norms approved by the Ministry of Social Justices and Empowerment (CPCSEA), Government of India and Institutional Animal Ethical Committee (IAEC) guidelines.

High-fat diet-fed and low-dose STZ-induced Type 2 diabetes:

The High Fat Diet (HFD) to induce insulin resistance was prepared indigenously by using a normal pellet diet, raw cholesterol, a mixture of Vanaspati ghee and pure coconut oil (2:1). Briefly, the normal rat pellet diet was powdered by grinding and mixed with 2.5% cholesterol and a mixture of Vanaspati ghee and coconut oil (5%). The mixture was prepared into pellet form and orally fed to rats to induce metabolic syndrome¹⁸. The rats were divided into two dietary regimens by feeding either normal or HFD for the initial period of 2 weeks. After 2 weeks of dietary management to develop insulin resistance, the group of rats fed with HFD was intraperitoneally injected with a freshly prepared STZ (35 mg/kg b.w) dissolved in 0.1M ice-cold citrate buffer, pH 4.5. After 3 days of injection with a low dose of STZ, the experimental rats were screened for fasting blood glucose levels. The experimental rats which showed fasting blood glucose above 250 mg/dl were clinically considered as diabetic and chosen for further experimental studies¹⁹.

Experimental protocol:

The animals were divided into four groups, comprising a minimum of six animals in each group as follows:

Group 1 – Control rats.

Group 2 – HFD+STZ (i.p. 35mg/kg b.w.)
induced diabetic rats.

Group 3 – Diabetic rats treated with
Zn-Avicularin complex (5mg/kg b.w./rat/day) orally for 30 days.

Group 4 – Diabetic rats treated with
metformin (50mg/kg b.w./rat/day) in aqueous solution orally for 30 days.

At the end of the treatment period, the rats were fasted overnight, anesthetized and sacrificed by cervical decapitation. The blood was collected with and without anticoagulants for the separation of plasma and serum respectively. The liver, pancreatic, hepatic and renal tissues were selectively dissected out and washed in ice-cold saline and used for further experimental studies.

Assay of basic biochemical parameters:

The levels of fasting blood glucose, glycosylated hemoglobin, plasma protein, blood urea, uric acid and serum creatinine were determined by standardized methods. Plasma insulin and C-peptide were assayed using an ELISA kit for rats (Linco Research, Inc., USA). The presence of urine sugar was assessed using urine strips (Diastix).

In vivo antioxidant assay:

The levels of lipid peroxides²⁰, hydroperoxides²¹ and protein carbonyls (²² were determined in plasma, pancreatic and liver homogenates. The activities of enzymatic antioxidants such as Superoxide dismutase (SOD)²³, catalase (CAT)²⁴, Glutathione Peroxides (Gpx)²⁵, Glutathione S transferase (GST)²⁶ were assayed in the pancreatic and hepatic homogenates of control and experimental groups of rats. The levels of non-enzymatic antioxidants such as vitamin C²⁷, vitamin E²⁸, ceruloplasmin²⁹ and GSH³⁰ were also determined.

Statistical analysis:

All the data obtained were grouped and statistically evaluated with the aid of SPSS 16.0 software. Hypothesis testing methods included ‘One-way analysis of variance’ followed by ‘least significant difference test’ was used. A value of P < 0.05 was considered to indicate statistical significance. All results were expressed as mean ± Standard error mean (S.E.M) for six rats in each group.

RESULTS:

Table 1 portrays the levels of fasting blood glucose, hemoglobin, glycosylated hemoglobin, plasma insulin, C-peptide and the inference for the presence of urine sugar in control and experimental groups of rats. Diabetic rats showed significantly elevated levels of fasting glucose and glycosylated hemoglobin and a concomitant decrease in the levels of hemoglobin and the altered levels were significantly reverted to physiological range in Zn-Avicularin complex treated diabetic rats. Urine sugar detected in the diabetic group of rats was absent in the diabetic group of rats treated with Zn-Avicularin complex as well as metformin.

The levels of plasma protein, blood urea, serum creatinine and uric acid were depicted in Table 2. Diabetic rats showed decreased plasma protein levels with concomitantly increased levels of renal function markers namely urea, uric acid and creatinine. The altered levels were significantly reverted to near-physiological range in Zn-Avicularin complex and metformin-treated diabetic rats.

Tables 3, 4, 5 and 6 represent the levels of lipid peroxides, hydroperoxides and protein carbonyls in plasma, pancreatic, hepatic and kidney tissues of control and experimental groups of rats respectively. The significant increase observed in the levels of lipid peroxides, hydroperoxides and protein carbonyls in plasma, pancreatic, hepatic and kidney tissues of diabetic rats were decreased to near-physiological values by the treatment of Zn-Avicularin as well as metformin to diabetic groups of rats.

Table 3: The levels of lipid peroxides, hydroperoxides and protein carbonyls in plasma of control and experimental groups of rats after 30 days of experimental period.

Groups	Lipid peroxides	Hydroperoxides	Protein carbonyls
Control	4.21 ± 0.28	11.12 ± 1.02	7.10 ± 0.36
Diabetic control	8.79 ± 0.24^a*	35.29 ± 1.12^a*	29.10 ± 1.71^a*
Diabetic + Complex	4.79 ± 0.36^b*	13.18 ± 1.12^b*	14.21 ± 0.72^b*
Diabetic + metformin	4.44 ± 0.14^b*	13.15 ± 1.11^b*	13.29 ± 0.85^b*

Units are expressed as:

nM/ml for lipid peroxides; 10^-5mM/dl for hydroperoxides; nM/mg of protein for protein carbonyls.

Table 4: Effect of Zn-Avicularin complex treatment on the levels of lipid peroxides, hydroperoxides, and protein carbonyls in the pancreatic tissues of experimental groups of rats.

Groups	Lipid peroxides	Hydro-peroxides	Protein carbonyls
Control	35.59 ± 2.06	16.16 ± 1.16	6.29 ± 0.25
Diabetic control	75.85 ± 3.02^a*	35.32 ± 1.08^a*	20.72 ± 0.84^a*
Diabetic + Complex	40.21 ± 5.53^b*	21.68 ± 1.35^b*	9.12 ± 0.51^b*
Diabetic + metformin	39.17 ± 4.29^b*	20.90 ± 1.33^b*	10.21 ± 0.62^b*

Units are expressed as:

mM/ 100g of wet tissue for lipid peroxides and hydroperoxides; nM/mg of protein for protein carbonyls. Results are expressed as mean ± S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001.

Table 5: Effect of Zn-Avicularin complex treatment on the levels of lipid peroxides, hydroperoxides, and protein carbonyls in the hepatic tissues of control and experimental groups of rats.

Groups	Lipid peroxides	Hydro-peroxides	Protein carbonyls
Control	1.89 ± 0.12	49.20 ± 3.70	4.51 ± 0.24
Diabetic control	4.69 ± 0.22^a*	126.50 ± 5.40^a*	13.72 ± 0.52^a*
Diabetic + Complex	2.30 ± 0.14^b*	72.45 ± 3.13^b*	7.24 ± 0.33^b*
Diabetic + Metformin	2.78 ± 0.20^b*	82.92 ± 2.72^b*	7.58 ± 0.33^b*

Units are expressed as: mM/ 100 g of wet tissue for lipid peroxides and hydroperoxides; nM/mg of protein for protein carbonyls.

Table 6: Effect of Zn-Avicularin complex treatment on the levels of lipid peroxides, hydroperoxides, and protein carbonyls in the renal tissue of control and experimental groups of rats.

Groups	Lipid peroxides	Hydro-peroxides	Protein carbonyls
Control	1.10 ± 0.05	50.41 ± 2.01	4.02 ± 0.15
Diabetic control	4.08 ± 0.19^a*	87.08 ± 3.08^a*	18.19 ± 0.66^a*
Diabetic + Complex	1.90 ± 0.12^b*	61.01 ± 3.39^b*	6.36 ± 0.33^b*
Diabetic + Metformin	1.65 ± 0.13^b*	57.15 ± 3.86^b*	5.01 ± 0.42^b*

Units are expressed as: mM/ 100 g of wet tissue for lipid peroxides and hydroperoxides; nM/mg of protein for protein carbonyls. Results are expressed as mean ± S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001.

Table 7-9 depicts the activities of enzymatic antioxidants such as SOD, catalase, Gpx and GST in pancreatic, hepatic and renal tissues of control and experimental groups of rats respectively. The activities were significantly decreased in all the tissues of diabetic groups of rats analyzed. Oral treatment of Zn-Avicularin complex at a concentration of 5mg/kg/rat/day for 30 days attenuated the altered activities of these enzymatic antioxidants to near normalcy in the diabetic groups of rats.

Table 7: Activities of Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx) and Glutathione-S-transferase (GST) in the pancreatic tissue of control and experimental groups of rats.

Groups	SOD	Catalase	GPx	GST
Control	5.42 ± 0.19	17.62± 0.21	7.11 ± 0.29	6.38 ± 0.32
Diabetic	1.35 ± 0.12^a*	5.51 ± 0.45^a*	3.04 ± 0.28^a*	1.52 ± 0.22^a*
Diabetic + Complex	3.10 ± 0.12^b*	12.22 ± 0.42^b*	5.61 ± 0.12^b*	4.57 ± 0.20^b*
Diabetic + metformin	3.54 ± 0.11^b*	13.15 ± 0.39^b*	6.05 ± 0.41^b*	4.89 ± 0.19^b*

Table 8: Activities of Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx), Glutathione-S-transferase (GST) and Glutathione reductase (GR) in the hepatic tissue of the control and experimental groups of rats.

Groups	SOD	Catalase	GPx	GST	GR
Control	11.31 ± 0.60	81.01 ± 2.54	10.77 ± 0.43	8.11 ± 0.42	27.53 ± 1.27
Diabetic	4.81 ± 0.18 ^a*	38.50 ± 2.46 ^a*	3.54 ± 0.18 ^a*	3.55 ± 0.20 ^a*	12.60 ± 0.61 ^a*
Diabetic + Complex	8.00 ± 0.19^b*	65.82 ± 2.23^b*	7.34 ± 0.26^b*	6.82 ± 0.25^b*	24.05 ± 1.39^b*
Diabetic + metformin	7.05 ± 0.24^b*	63.18 ± 3.27 ^b*	7.20 ± 0.30^b*	6.67 ± 0.23^b*	23.50 ± 1.80^b*

Activities of enzymes are expressed as: 50% of inhibition of epinephrine autoxidation/min for SOD; mM of hydrogen peroxide decomposed/min/mg of protein for catalase; mM of glutathione oxidized/min/mg of protein for GPx; U/min/mg of protein for GST; µM of DTNB-GSH conjugate formed/min/mg of protein for GR. Results are expressed as mean±S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001.

Table 9: Activities of Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx), Glutathione-S-transferase (GST) and Glutathione reductase (GR) in the renal tissue of control and experimental groups of rats.

Groups	SOD	Catalase	GPx	GST	GR
Control	17.10 ± 0.66	43.71 ± 2.54	7.71 ± 0.20	6.32 ± 0.21	34.28 ± 1.08
Diabetic	8.23 ± 0.43^a*	17.19 ± 0.91 ^a*	3.52 ± 0.15^a*	3.21 ± 0.14 ^a*	11.20 ± 0.61 ^a*
Diabetic + Complex	15.45 ± 0.42^b*	34.54 ± 1.60^b*	6.31 ± 0.22^b*	4.52 ± 0.20^b*	25.42 ± 1.14^b*
Diabetic + metformin	15.35 ± 0.43^b*	37.35 ± 2.05^b*	6.24 ± 0.17^b*	4.41 ± 0.21^b*	26.32 ± 1.42^b*

The levels of plasma non-enzymatic antioxidants such as vitamin C, vitamin E and ceruloplasmin (Table 10), and pancreatic, hepatic and renal GSH (Table 11) content are represented. HFD-STZ diabetic rats showed a significant decrease in the levels of non-enzymatic antioxidants when compared with the control group of rats. Conversely, administration of Zn-Avicularin complex as well as metformin to HFD-STZ diabetic rats significantly improved the levels to near control values.

Table 10: Effect of Zn-Avicularin complex treatment on the levels of Vitamin E, Vitamin C, Ceruloplasmin and reduced glutathione in the plasma of control and experimental groups of rats.

Groups	Vitamin E	Vitamin C	Ceruloplasmin	GSH
Control	1.50 ± 0.02	0.80 ± 0.03	12.22 ± 0.42	38.21 ± 2.10
Diabetic	0.34 ± 0.04^a*	0.28 ± 0.04^a*	3.68 ± 0.49^a*	15.32 ± 1.03 ^a*
Diabetic + Complex	0.89 ± 0.02^b*	0.78 ± 0.03^b*	10.62 ± 0.81^b*	22.89 ± 1.60^b*
Diabetic + metformin	0.91 ± 0.03^b*	0.77 ± 0.05^b*	10.01 ± 0.67^b*	28.22 ± 1.50 ^b*

Units are expressed as: mg/dl.Results are expressed as mean±S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001.

Table 11: Effect of Zn-Avicularin complex treatment on the level of reduced glutathione in the pancreas, liver and kidney tissues of control and experimental groups of rats.

Groups	Reduced glutathione
Groups	Pancreas	Liver	Kidney
Control	23.11 ± 0.82	47.30 ± 2.13	36.25 ± 1.10
Diabetic	9.21 ± 0.32^a*	22.81 ± 0.98^a*	20.39 ± 0.72 ^a*
Diabetic + Complex	16.22 ± 0.25 ^b*	36.26 ± 2.32^b*	31.50 ± 1.35 ^b*
Diabetic + metformin	14.40 ± 0.60 ^b*	36.60 ± 1.84^b*	29.28 ± 0.78 ^b*

Units are expressed as: mg/100 g of wet tissue. Results are expressed as mean±S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001.

DISCUSSION:

One of the paradoxes of life on this planet is that the molecule that sustains aerobic life, oxygen, is not only fundamentally essential for energy metabolism and respiration, but it has been implicated in many diseases and degenerative conditions³¹. Atmospheric oxygen in its ground state has two unpaired electrons. This feature makes oxygen paramagnetic because the two unpaired electrons have parallel spins. Hence, oxygen is usually non-reactive to organic molecules, which have paired electrons with opposite spins (spin restriction). Activation of oxygen may occur by two different mechanisms; absorption of sufficient energy to reverse the spin on one of the unpaired electrons. This monovalent reduction will form the singlet state, on which the two electrons have opposite spins. The second mechanism of activation is by the stepwise monovalent reduction of oxygen to form superoxide (O2-*), hydrogen peroxide (H₂O₂), hydroxyl radical (OH-*) and finally water. However, hydroxyl radicals are the principal mechanism of oxygen activation in most biological systems.

A free radical is any atom or molecule that contains one or more unpaired electrons in its outer valence orbital. This situation is energetically highly unstable and more active. Stability is achieved by the removal of an electron from an electron pair of a surrounding molecule. Following electron transfer, the original free radical is stable. However, the donor molecule, in its turn, then has an unpaired electron, which increases its chemical reactivity. In this way, the presence of a single radical may initiate a cascade of electron transfer redox reactions. The damage that the free radicals do to cells may be quantitatively determined by measurement of levels in MDA, a product of lipid peroxidation³². In Biochemistry, the free radicals of interest are often referred to as reactive oxygen species (ROS) because the most biologically significant free radicals are oxygen-centered. But not all free radicals are ROS and not all ROS are free radicals³³

The main damage to cells results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins and DNA. Although the brain consumes 20% of oxygen in the body, it has a low content of antioxidants and high content of unsaturated fatty acids and catecholamines that are easily oxidized making it more vulnerable to oxidative damage than any other organ in the body³⁴. Cells are normally able to defend themselves against ROS damage through the aid of enzymatic antioxidants such as superoxide dismutases and catalases. Small molecules such as ascorbic acid (vitamin C) and glutathione also play important roles as cellular non-enzymatic antioxidants. The level of oxidative stress is determined by the balance between the rate at which oxidative damage is induced by free radicals and the rate at which it is efficiently repaired and removed by antioxidants³⁵.

Lipid peroxidation is a free radical-induced spontaneous process involving a source of secondary free radical, which subsequently act as the second messenger or can directly react with other biomolecules such as carbohydrates, proteins, lipids and DNA and thereby causing irreversible biochemical as well as molecular lesions³⁶. Additionally, the excessive generation of cytotoxic oxidative stress markers such as lipid peroxides, hydroperoxides and protein carbonyls causes oxidative damage to proteins as well as DNA and the reduced the levels of both cellular antioxidant levels in diabetic conditions that cause organ dysfunction resulting in decreased insulin synthesis, secretion and finally β cell death. In addition to decreased levels of antioxidants in the pancreatic β cells, the supraphysiological glucose concentration is notorious to provoke oxidative stress in hepatocytes, which can cause hepatic tissue damages³⁷. In the present study, the elevated levels of lipid peroxides, hydroperoxides and protein carbonyls were significantly altered upon oral administration of Zn-Avicularin complex which demonstrates the significant free radical scavenging property of Zn-Avicularin complex in hyperglycemia mediated oxidative stress. The benefit of antioxidants is not only attributed to their radical scavenging but to their ability to interact with many basic cellular activities³⁸.

The liver is the primary organ of oxidative and detoxifying processes as well as free radical reactions and the biomarkers of oxidative stress are elevated in the hepatic tissue at an early stage in many diseases, including diabetes mellitus⁴⁷. The insulin insufficiency and hyperglycemia that result from β cell necrosis further augment liver damage through reactive free radicals mediated lipid peroxidation of the hepatocellular membrane³⁹.

Superoxide dismutase (SOD) is the pivotal antioxidant enzyme that catalyzes the dismutation of superoxide anion (O₂^-) into hydrogen peroxide and molecular oxygen⁴⁰ and in the presence of other enzymes; it is converted into oxygen and water⁴¹. Increased expression of SOD or the supplements of antioxidants including SOD mimetics, ameliorates oxidative stress, reduces ROS generation, and increases enzymatic antioxidant thereby ameliorating prevent diabetes mellitus⁴². Catalase, an antioxidative enzyme present in all living organisms, acts against oxidative stress-generated complications that occur in cancer, diabetes and cardiovascular diseases⁴³. Hydrogen peroxide, a highly reactive molecule formed in excess as a natural by-product of energy metabolism, causes significant damages to proteins, DNA, RNA and lipids⁴⁴. Catalase enzymatically processes hydrogen peroxide into oxygen and water and thus neutralizes it. Decreased catalase activity results in oxidative stress leading to β-cell dysfunction. Since β-cells are rich in mitochondria and thus this organelle might be a potential source of ROS⁴⁵. Gpx, a selenium-containing peroxidase is implicated in the detoxification of hydrogen peroxide and lipid peroxide by using GSH as a hydrogen donor and acts as a peroxynitrite reductase. Persistent hyperglycemia increases oxidative stress through diverse mechanisms; the defective antioxidant function of Gpx is a hallmark in the diabetic state. The low activity of Gpx could be directly explained by the low levels of GSH found in patients with type 2 diabetes since GSH is a substrate and cofactor of Gpx activity. Gpx is a relatively stable enzyme, but it may be inactivated under conditions of severe oxidative stress. Inactivation of this enzyme may occur through glycation governed by prevailing glucose concentration⁴⁶. Increased activity of GR may be a compensatory response to oxidative stress. Diminished glutathione peroxidase activity can be considered an adaptation of antioxidant defense against ROS. However, the altered levels of enzymatic antioxidants were significantly improved upon Zn-Avicularin complex treatment indicating the effective antioxidant as well as tissue-protective nature of the complex.

Vitamin C, a hydrophilic antioxidant sequesters the singlet oxygen radicals, stabilizes the hydroxyl radical and regenerates reduced vitamin E back to its active state. Vitamin E, a lipophilic antioxidant, transfers its phenolic hydrogen to a peroxyl free radical of peroxidized polyunsaturated fatty acids, thereby quenching the radical chain reaction and averting the peroxidation of membrane lipids⁴⁷. Ceruloplasmin is a powerful non-enzymatic antioxidant that inhibits lipid peroxidation by binding with copper. The observed decline in plasma ceruloplasmin in diabetic rats may be due to elevated lipid peroxidation which was normalized upon treatment with Zn-Avicularin complex. Reduced glutathione (GSH), a tripeptide, γ- Lglutamyl -Lcysteinylglycine, is present in all mammalian tissues at 1–10mm concentrations as the most abundant nonprotein thiol that defends against oxidative stress. GSH is known to maintain SH groups of proteins in a reduced state, participate in amino acid transport, detoxify foreign radicals, act as a coenzyme in several enzymatic reactions and also prevent tissue damage⁴⁸. It is an effective antioxidant present in almost all living cells and is also considered as a biomarker of redox imbalance at the cellular level⁴⁹. Decreased GSH level is a contributory factor in the oxidative DNA damage in type 2 diabetes mellitus⁵⁰. Similarly, the lowered GSH levels observed in diabetic rats were increased upon oral administration of the Zn-Avicularin complex further amplified the antioxidant potential of the complex. The altered levels of biochemical indices were reverted to the physiological range after oral treatment with the complex and the data obtained on the status of oxidative stress markers and the levels of both enzymatic and non-enzymatic antioxidants provide evidence the antidiabetic and antioxidant properties of the Zn-Avicularin complex.

In the present study, the altered levels of important biochemical parameters were reverted to near-normal levels after oral treatment with the Zn-Avicularin complex. Additionally, the elevated levels of oxidative stress markers were significantly decreased upon oral administration of Zn-Avicularin complex which demonstrates the antioxidant potential of Zn-Avicularin complex under chronic hyperglycemia-induced oxidative stress environment. The Zn-Avicularin complex treatment improved the antioxidant status in pancreatic, hepatic and renal tissues of diabetic rats. Thus, it can be concluded that the Zn-Avicularin complex recuperates antioxidant status and protects the pancreatic, hepatic and renal tissues from hyperglycemia mediated oxidative stress suggesting that the antioxidant properties of the complex may be partially due to its antidiabetic potential.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 21.06.2021 Modified on 28.12.2021

Research J. Pharm. and Tech 2023; 16(1):145-152.

DOI: 10.52711/0974-360X.2023.00027

Groups	Fasting Blood Glucose (mg/dl)	HbA1c (% Hb)	Hemoglobin (%)	C. Peptide (μU/ml)	Plasma Insulin (µU/ml)	Urine sugar
Control	92.21 ± 5.75	6.01 ± 0.57	14.51 ± 0.68	262.81 ± 1.54	15.37 ± 1.75	Nil
Diabetic	277.25 ± 16.73^a*	10.20 ± 1.06^a*	9.15 ± 0.55^a*	125.61 ± 0.96^a	9.15± 0.44^a*	+++
Diabetic + complex	118.11 ± 10.13^b*	6.42 ± 0.34^b*	12.25 ± 0.49^b*	210.24 ± 1.61^b*	12.80 ± 1.05^b*	Nil
Diabetic + Metformin	107.05 ± 7.07^b*	6.12 ± 0.56^b*	12.28 ± 0.50^b*	227.03 ± 1.45^b*	14.19 ± 1.28^b*	Nil

Groups	Total protein (g/dl)	Blood urea (mg/dl)	Serum uric acid (mg/dl)	Serum creatinine (mg/dl)
Control	8.85 ± 0.56	22.30 ± 2.20	2.51 ± 0.53	0.54 ± 0.07
Diabetic	6.30± 0.39^a*	44.74 ± 5.16^a*	5.35 ± 0.67^a*	1.21 ± 0.31^a*
Diabetic + Complex	7.22± 0.30^b*	28.32 ± 1.82^b*	3.09 ± 0.30^b*	0.67 ± 0.07^b*
Diabetic + Metformin	7.74± 0.58^b*	30.60 ± 2.93^b*	2.71 ± 0.22^b*	0.62 ± 0.08^b*