Antidiabetic evaluation of vanillic acid-glyburide loaded polymeric micelles in high fat diet and streptozotocin induced diabetic rats

doi:10.21203/rs.3.rs-3138446/v1

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Antidiabetic evaluation of vanillic acid-glyburide loaded polymeric micelles in high fat diet and streptozotocin induced diabetic rats

https://doi.org/10.21203/rs.3.rs-3138446/v1

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Purpose

The present study describes oral administration of glyburide (GLY) and vanillic acid (VA) loaded polymeric micelles for the effective treatment of type 2 diabetes mellitus in high-fat diet (HFD) and a low dose of streptozotocin (STZ) rats.

Methods

The rats were divided into 12 groups (G1-G12) based on the respective treatments. All rats except G1 received high fat diet for first 15 days and then induced to STZ. Then the study was continued for another 28 days. GLY and VA loaded polymeric micelles (GV-APMs) were orally administered to the STZ induced rats. The rats of different groups received their respective treatments orally. Various parameters such as body weight, blood glucose level, biochemical parameters and histopathology of liver and pancreas. The rats of G2 received HFD-STZ alone, whereas rats of G6 received high dose of mixture of raw Gly-VA and G12 received high doses of GV-APMs, separately.

Results

The results revealed 1.43, 1.44, 1.55, 2.4, 1.66, 3.30, and 3.76-folds higher reduction in blood glucose, cholesterol (CHL), triglycerides, low-density lipoprotein (LDL), very LDL levels, CHL/high-density lipoprotein (HDL), and LDL/HDL ratio in rats receiving GV-APMs (G12) than rats receiving their physical mixture (G6). In addition to this, significant reduction in the serum inflammatory, hepatic and renal markers as well as antioxidant levels was observed in rats of G12 as compared to any other group, indicating higher efficacy of GV-APMs.

Conclusion

The study revealed that co-loading of GLY and VA in APMs showed excellent antidiabetic potential can be explored further for clinical evaluation.

Diabetes

Glyburide

Nanocombination

Polymeric micelles

Vanillic acid

Type 2 diabetes mellitus (T2DM) is a chronic metabolic and multifactorial disease characterized by hyperglycemia and dyslipidemia (Pandey 2022). Overall, 80–85% of cases of total diabetics have been diagnosed with T2DM globally. Therefore, it is considered to be the most protuberant one among all the diabetic forms worldwide. This is because of the changes in secondary lifestyle habits (Kaur et al., 2020). As per the world health organization, a healthy diet, and physical exercise on daily basis followed by maintenance of body weight (body wt.) can delay the onset of T2DM (https://www.who.int/news-room/fact-sheets/detail/diabetes, Accessed on 1 July 2022). In agreement with this, increased body mass index and central adiposity are found to be in a strong relationship with insulin resistance (IR) and are believed to be the key factors involved in the incidence of T2DM (Kaur et al., 2020). If such a condition is not controlled, it can lead to serious metabolic complications followed by hyperglycemia due to IR. To overcome such issues in extreme cases, the commencement of pharmacotherapy of anti-diabetic drugs becomes a prerequisite. These drugs provide tight control of glycosylated haemoglobin (HbA1c ≤ 7.0%) levels to prevent the risk of long-term complications and mortality in patients with T2DM.

Glyburide (Gly) is a potent drug used as a first-line treatment for the management of T2DM among patients using sulfonylureas (Wang et al. 2011). However, it has a narrow therapeutic index and is associated with hypoglycaemic risk i.e., by 80% more than other drugs upon administration at the highest dose strength 20 mg daily dose (Sola et al. 2015). It has been reported to cause weight gain of about 1.8–2.6 kg in patients with T2DM (Kaur et al., 2020). Hence, the side effects of the drug as well as secondary complications of diabetes aggravate diabetes-associated cardiovascular (CV) complications leading to atherosclerosis, dyslipidaemia, and hypertension (Kaur et al. 2020; Pandey et al. 2022). Overall, such conditions contribute to increasing the economic burden and affect the quality of life of the patients as the CV complications require angioplasty or coronary artery bypass graft surgery. In addition to this, in most cases, it has been observed that the patients having a long history of T2DM have been advised to take antihyperlipidaemic drugs such as statins to treat hyperlipidaemia as well as in many cases antihypertensives to treat hypertension (Zamorano 2011; Rosenblit 2016). Therefore, the dose-reduction of Gly becomes essential as per patients’ safety concerns in long run.

Owing to these clinically associated co-morbidities in the last two decades, a combination of Gly with other anti-diabetic drugs are extensively reported. Currently, there are various adjuvant drugs used in combination with Gly for the management of T2DM in the long run, which commonly include metformin as first-line combination therapy (Cahn and Cefalu 2016; Vijayakumar et al. 2017). Despite being effective, this risk of cardiovascular complications in diabetics is prominently higher over Gly monotherapy (Garber et al. 2003). This further urges the need for administration of other anti-hyperlipidemic drugs such as statins that are 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor as well as antihypertensive drugs to maintain a healthy life. Overall such conditions increase the complexity of antidiabetic regimens in T2DM patients (Kaur et al., 2020;Pandey et al., 2022).

Looking at these pitfalls, much attention has been given to exploring the combination of herbal drugs with marketed anti-diabetic agents (Prabhakar et al. 2014). For instance, Prabhakar and Doble, 2009, evaluated the interaction of three natural products i.e., ferulic acid, chlorogenic acid and berberine in combination with metformin and thiazolidinediones on glucose uptake by L6-myotubes. The combination of ferulic acid with the metformin or thiazolidinediones exhibited a synergistic effect and increased the uptake of glucose by 4.1 and 4.7-folds than the control respectively. Similarly, chlorogenic acid in combination with the two anti-diabetic drugs showed synergistic interaction by increasing the glucose uptake by 5.0 and 5.3-folds respectively. However, berberine in the form of combination therapy exhibited additive interaction with the two anti-diabetic drugs with maximum glucose uptake of 4.1 and 4.7-folds than the base value respectively (Prabhakar and Doble 2009). In another study, Nankar et al., 2017, used ferulic acid in combination with metformin to reduce the dose of metformin and its dose-associated side effects. The combination of ferulic acid with metformin exhibited synergistic interaction in vitro adipocyte cell lines by improving the glucose uptake as well as reducing the BGLs in diabetic rats by 2-folds than untreated diabetic rats respectively. The combination of ferulic acid caused a 3 to 5-folds reduction in metformin dose (Nankar et al. 2017).

The positive outcomes of the aforementioned studies led to the opening of new avenues for other phytoconstituents to be explored for the management of T2DM along with commercially available synthetic drugs.

Among the herbal drugs, phenolic compounds are widely being explored due to their widespread availability in various dietary and plant-based sources (Prabhakar et al. 2014). These are usually preferred in combination with anti-diabetic drugs, especially sulfonylureas such as Gly due to their high compliance, multi-faceted pharmacological actions such as anti-diabetic, antioxidant, anti-obesity, anti-hyperlipidemia, etc. (Bunyapraphatsara et al. 1996; Tongia et al. 2004; Poonam et al. 2013; Nankar et al. 2017). In addition, these are considerably free from side effects. In recent years many researchers identified and explored new phenolic compounds from natural sources for possible anti-diabetic action with minimum side effects (Nankar et al. 2017).

Vanillic acid (VA) is one of the naturally derived phenolic compounds obtained from the plants as well as other dietary sources. It is popular for its natural anti-oxidant property and has been explored well for the treatment of commonly occurring metabolic diseases including obesity and T2DM (Kaur et al., 2022b). Being a plant-derived product, it is reported to act via multiple pathways to control T2DM and its associated complications i.e., by phosphorylating ACC protein in hepatocytes, decreasing the levels of unsaturated fatty acids in liver, and by upregulating the expression of thermogenin gene and other mitochondrial specific proteins (ATP5A, NDUFB8, NRF2) in adipocytes for energy dissipation resulting in its anti-obesity action (Chang et al. 2015a; Han et al. 2018). It increases the expression of antioxidant enzymes by translocating Nrf2 into the nucleus. In addition, it increases the expression of the insulin receptor, PI3K and glucose transporter 2 (GLUT-2) protein in HFD-fed rats that making VA an effective anti-diabetic agent (Vinothiya and Ashokkumar 2017; Kaur et al. 2022).

Therefore, the combination of VA with Gly is worth exploring for the effective management of T2DM in long run. However, their confined dissolution rate assisted poor oral bioavailability due to poor water solubility of Gly and rapid elimination of VA from plasma limits their therapeutic efficacy (Cirri et al., 2016; Kaur et al., 2022b). Therefore, this arises the need for the development of oral formulation of both the drugs to overcome the aforementioned challenges for enhanced therapeutic efficacy by the virtue of effective delivery via oral route.

With this agreement, amphiphilic polymeric micelles (APMs) formed from block copolymers was selected for formulation development due to their amphiphilic configuration, which indicated potential to entrap both the lipophilic (Gly) and amphiphilic (VA) drugs in their copolymer network.

The designing of the controlled release micellar nano-formulation i.e., APMs for the oral delivery of both the drugs (Gly and VA) using copolymer-surfactant system i.e., mPEG-b-PCL (methyl ether polyethylene glycol-block-polycaprolactone) and cetyl trimethyl ammonium bromide (CTAB) along with its in vitro characterization and pharmacokinetic data is already published in our previous work (Kaur et al., 2022a). The recent work is in continuation of our published work in terms of evaluating the therapeutic efficacy of the developed nanocombination therapy for the effective management of T2DM.

VA (99.95% purity), Gly (99.99% purity), epsilon-caprolactone (epsilon-CL), methyl ether polyethylene glycol (mPEG) (Mw ~ 2000), and stannous octoate (Sn(Oct)₂) (Tokyo chemical industry, Japan) were purchased from D.P. Traders, Mohali Punjab, India. Carboxymethyl cellulose (CMC) was purchased from Central Drug House, Mumbai, India. Triple distilled water was used throughout the study. TNF-α and IL-6 ELISA kits (Invitrogen™, US), homogenizer (RQ-127, REMI, India), pH meter (Phan, Lab India, Mumbai, India), analytical balance (AX 200; Shimadzu Analytical Pvt. Ltd., India). High-Performance Liquid Chromatography (HPLC; LC-20 AD; Shimadzu, Japan) was used for formulation characterization. EM360 automated chemistry analyser (Erba Mannheim, Mumbai, India) were used for determining serum lipid markers. The BGLs at each time interval were determined using a glucometer (Dr Morepen, BG-03) based on the glucose oxidase method. GraphPad Prism® (GraphPad Inc., La Jolla, USA) was used to carry out the statistical analysis.

In vivo antidiabetic study

3.1 Animals

Healthy SD rats of either sex (270-300g) having age ranging between 10–12 weeks were procured from Panjab University, Chandigarh, India for the pharmacodynamics study. The animals were kept in cages in the central animal house of the School of Pharmaceutical Sciences, Lovely Professional University (Phagwara, Punjab) at an ambient temperature of 25 ± 2 ℃ and relative humidity of 65% with a 12 h light/dark cycle. The experimental protocol was duly approved by the institutional animal ethics committee (Protocol no: LPU/IAEC/2021/ 78).

3.2 Obesity-induced IR and hyperglycaemia

The SD rats of either sex was randomly divided into 12 groups containing 8 rats based on dietary regimen i.e., normal pellet diet (NPD) and HFD (58%w/w fat, 25% w/w protein, and 17%w/w carbohydrate, as a percentage of total kcal) ad libitum, respectively. Group 1 received NPD while the rats allocated to the rest of the other groups received HFD for the initial period of 2 weeks. The treatment groups used in the present study are presented in Table 1. The composition and preparation of HFD were according to Srinivasan et al., 2005 as presented in Table 2 (Srinivasan et al. 2005). All the components of HFD were weighed, powdered and triturated in melted desi ghee. Afterwards, they were converted into pellets and stored at -70℃ in a deep freezer (Srinivasan et al. 2005). To each group of rats, a total of 6 pellets were given twice a day. After dietary manipulations, the rats from the HFD-fed group were injected with a single low dose of STZ (35 mg/kg; i.p.) in ice-cold 0.1 M sodium citrate buffer (pH 4.5) upon overnight fasting. The NPD-fed group received an equivalent amount of sodium citrate buffer. The HFD-fed rats with frank hyperglycaemia i.e., [blood glucose levels (BGLs) > 200 mg/dL], were considered diabetic and selected for the pharmacological screening of the test compounds.

3.3 Oral glucose tolerance test (OGTT)

The OGTT was performed on rats in all groups after overnight fasting on the 14th day (after HFD). On the 15th day, the rats were orally administered (using 16G oral feeding cannula) with 2.0g of glucose/kg body weight. The blood samples were taken by slightly pricking the rat’s tail with the help of a needle before 0 min and after 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h of glucose administration. The BGLs at each time interval were determined using a glucometer (Dr Morepen, BG-03) based on the glucose oxidase method. The obtained values of BGLs were plotted against time to develop a curve showing the changes in glucose levels with time, it was expressed as an integrated area under the curve for glucose (AUC_glucose) (Chang et al., 2015).

3.4 Treatment design and pharmacological screening

After the administration of HFD for 2 weeks followed by STZ injection on the 16th day, the respective treatments in each divided group (except group 1) were initiated from the 21st day for a consecutive period of 4 weeks (i.e., up to the 48th day) as presented in Table 1. The body weight was determined on the 0th and 15th, and on weekly basis (i.e., 20th, 27th, 33rd, 39th, and 45th day) using a digital weighing machine. The biochemical parameters such as BGLs were determined on weekly basis (i.e., 20th, 27th, 33rd, 39th, and 45th day). The serum lipid markers were evaluated on the 0th, 15th and 45th day. The inflammatory markers and histopathology were performed on the terminal day of the treatment protocol.

3.5 Biochemical parameters

For determination of biochemical parameters, blood was withdrawn from the retro-orbital plexus and collected in plain Eppendorf tubes. Blood samples were processed to separate serum and were stored in the deep freezer before biochemical analysis. The serum lipid markers such as cholesterol (CHL), High-density lipoprotein (HDL), Triglycerides (TGs), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), CHL/HDL, and LDL/HDL ratios were determined by running the samples in an automated biochemistry analyzer equipment Erba EM360 based on photometric analysis using MultiXL v2017.02 (EM360) software upon selection of the working reagent mode. The XL results were calculated automatically by the instrument. BGLs were determined using a glucometer (Dr Morepen). The serum hepatic and renal markers viz. Serum glutamic pyruvic transaminase (SGPT), Serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), Creatinine and urea were determined to evaluate the hepatic and renal functions of diabetic rats. Serum inflammatory markers (TNF-α and IL-6) were determined on a terminal day after the completion of the treatment period i.e., on the 48th day and were stored at 4°C until processing. After 30 min, the collected blood samples were centrifuged at 8000 rpm for 15 min using a cooling centrifuge. The supernatant was withdrawn in a separate Eppendorf tube and was stored at -20°C until analysis. Quantification of TNF-α and IL-6 in the blood serum was performed using an ELISA Invitrogen kit according to the manufacturer’s protocol.

Table 1

Pharmacodynamics study design
Groups	Treatment	Dose (Route of Administration)
Oral NPD treatment
I	Normal control (NC)	Ice cold 0.1 M sodium-citrate buffer (pH 4.5), p.o.
STZ (35 mg/kg) in ice cold 0.1 M sodium-citrate buffer (pH 4.5) intraperitoneal + oral HFD treatment will be given to rats of groups II to XII
II	Experimental control (EC)	HFD + STZ, i.p.
III	Placebo of APMs (P-APMs)	Placebo APMs (without drugs), p.o.
IV	Raw VA in 0.5%w/v in CMC at high dose 7.5 mg/kg [R-VA(H)]	(R-VA)-H, p.o.
V	Raw Gly in 0.5%w/v CMC at high dose 0.50 mg/kg [R-Gly (H)]	(R-Gly)-H, p.o.
VI	High dose of raw Gly (0.50 mg/kg)-VA (7.5 mg/kg) in 0.5%w/v CMC [R-Gly-VA(H)]	(R-Gly-VA)-H, p.o.
VII	VA APMs in 0.5%w/v CMC at low dose 3.7 mg/kg [VA-APMs(L)]	(VA-APMs)-L, p.o.
VIII	Gly APMs in 0.5%w/v CMC at low dose 0.25 mg/kg [Gly-APMs (L)]	(Gly-APMs)-L, p.o.
IX	VA APMs in 0.5%w/v CMC at high dose 7.5 mg/kg [VA-APMs(H)]	(VA-APMs)-H, p.o.
X	Gly APMs in 0.5%w/v CMC at high dose 0.5 mg/kg [Gly-APMs (H)]	(Gly-APMs)-H, p.o.
XI	Low dose of Gly (0.25 mg/kg)-VA (3.7 mg/kg) APMs in 0.5%w/v CMC [GV-APMs(L)]	(GV-APMs)-L+(D), p.o.
XII	High dose of Gly (0.50 mg/kg)-VA (7.5 mg/kg) APMs in 0.5%w/v CMC [GV-APMs(H)]	(GV-APMs)-H+(D), p.o.

Table 2

Composition of HFD
Ingredients	Diet (g/kg)
Powdered NPD	365
Lard/desi ghee	310
Casein	250
Cholesterol	10
Vitamin and mineral mix	60
DL-methionine	03
Yeast powder	01
Sodium chloride	01

3.6 Euthanasia

The technique used to euthanize the experimental rats was an overdose of general anaesthesia that was performed on the terminal day of the treatment protocol i.e., on the 49th day. The experimental rats from each group were administered ketamine hydrochloride (22–24 mg/kg) via the intramuscular route. Furthermore, to ensure the death of the animal repeated intramuscular administration was employed. The liver and pancreas were isolated and preserved in 10%v/v formaldehyde until analysis.

3.7 Determination of pancreatic oxidative stress

The extent of oxidative stress in the pancreas of diabetic rats was determined by measuring the levels of antioxidant enzymes viz, catalase (CAT), glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) in the isolated pancreas of diabetic euthanized rats. After sacrificing the rats, their pancreas was quickly isolated, weighed, and washed for blood removal with phosphate buffer saline (PBS, pH = 7.4). The isolated pancreas was cut into pieces for their homogenization. For the preparation of homogenate (10%w/v), the pancreatic tissue was mixed independently with 0.1M PBS. Then the mixture was homogenized and centrifuged at 1000 rpm at 4^oC for 20 min to obtain clear supernatant (Pandey et al. 2022). The supernatant obtained was used for the assessment of CAT, GSH and TBARS respectively.

3.8 Histopathology study

Liver and pancreas of rats from each group were isolated after euthanasia and was fixed in 10%v/v formaldehyde. Further, the organs were embedded in paraffin wax and tissue sections were prepared and stained with eosin/hematoxylin dye. Photomicrographs were taken at × 400 magnification using a digital camera.

3.9 Statistical analysis

Data were presented as mean ± SD of the respective replicates. Means of different groups were compared using analysis of variance (ANOVA). P values ≤ 0.05 (95% confidence interval) were considered significant. Graph Pad Prism 7.0 version, USA was employed to carry out the statistical analysis.

4.1 OGTT

HFD administration in rats impairs hepatic glucose metabolism due to IR and reduced insulin secretion by the pancreas. This ultimately results in abnormal glucose tolerance in HFD-fed rats (Chang et al., 2015). OGTT is an important parameter used to characterize the metabolic phenotype and to determine the alteration in glucose metabolism. OGTT assesses the deposition of an orally administered glucose load over time by measuring the BGLs (Nagy and Einwallner 2018). The results of fluctuations in BGLs at different time-points post-oral glucose administration pertaining to various groups are shown in Table 3. The results indicated more than 35 mg/dL BGLs in HFD fed-rats of G2 to G12 at the end of 1.5h. This increase in BGLs at the end of 1.5h was at least + 10 mg/dL in all the treatment groups as compared to rats of G1. The objective of this study was to check the duration taken by the rats to lowering down their glucose levels to maintain glucose homeostasis. Usually, the longer duration required by the body to bring glucose levels down represents the state of IR as a measure of low glucose tolerance. In the case of G1, BGLs started lowering down after the end of 1h, indicating a higher degree of glucose tolerance in control rats in response to an oral glucose load. Further, in control rats, the secreted insulin under normal physiological functioning i.e., rats on NPD, was able to lower the BGLs. This can be understood by the decrease in BGLs by 28 mg/dL in the case of G1 rats between 1h to 4h. Whereas, in the case of rats of G2 to G12, the rise in BGLs at the end of 1h was a minimum of 24 mg/dL. Between 1h to 4h, a negligible decrease in BGLs of rats of G2, G3, G6, G7, G8, G9 and G10 was observed, whereas, rats of G4, G5, G11 and G12 showed a rise in BGLs. The overall results of treatment groups (G2-G12) indicated that their body was resistant to insulin owing to the administration of HFD. Hence, was unable to maintain glucose homeostasis at a normal level (Chang et al., 2015). The patterns for changes in BGLs as per different time intervals of different groups are shown in Fig. 1. in which all the HFD-fed rats showed significantly (p < 0.001) lower glucose tolerance than the control rats respectively.

Table 3

BGLs of different treatment groups after oral glucose load (Mean ± SD, n = 8)
Time (h)	G1 (mg/dL)	G2 (mg/dL)	G3 (mg/dL)	G4 (mg/dL)	G5 (mg/dL)	G6 (mg/dL)	G7 (mg/dL)	G8 (mg/dL)	G9 (mg/dL)	G10 (mg/dL)	G11 (mg/dL)	G12 (mg/dL)
0	116.3 ± 10.9	126.1 ± 15.2	124.3 ± 9.4	120.9 ± 14.4	122 ± 10.5	120 ± 11.8	127.8 ± 11.8	124.8 ± 10.5	123.7 ± 14.8	121.7 ± 11.3	124.6 ± 9.9	125.2 ± 5.5
1	143.5 ± 2.1	172.5 ± 3.5^ϒ	163.5 ± 2.1^β	159.1 ± 1.8 ^β	165.9 ± 3.7 ^β	163.8 ± 5.6^ϒ	160.7 ± 2.6 ^β	159.1 ± 4.1^ϒ	159.2 ± 3.3^ϒ	158.5 ± 4.1^ϒ	152.6 ± 5.6^ns	162.5 ± 5.3^ϒ
1.5	139.5 ± 2.1	173.5 ± 2.1	164.5 ± 2.1	172.8 ± 1.6	178.5 ± 1.6	180.5 ± 6.3	163.8 ± 1.2	164.2 ± 2.4	169.7 ± 3.2	161.2 ± 3.2	157.5 ± 1.5	171.3 ± 2.4
4	114.1 ± 4.1	164.6 ± 3.3^C	162.9 ± 3.5^C	162.1 ± 4.9^C	169.5 ± 4.2^C	164.9 ± 3.5^C	160.9 ± 3.5^C	170.0 ± 4.2^C	156.5 ± 2.8^C	147.5 ± 5.6^C	155.3 ± 2.9^C	171.3 ± 3.8^C
6	95 ± 7.0	124.0 ± 2.8^Z	129.4 ± 2.5^Z	136.3 ± 2.5^y	145.9 ± 3.8^y	140 ± 3.1 ^y	130.9 ± 3.2^Z	152.7 ± 5.8^y	128.7 ± 1.2^Z	126.2 ± 3.6^Z	135 ± 2.4^y	130.5 ± 3.8^Z

α, β, ϒ indicate p < 0.001, p < 0.01, p < 0.05 with respect to control at 1h; and a, b, c, indicate p < 0.001, p < 0.01, p < 0.05 with respect to control at 4h; x, y, z indicates p < 0.001, p < 0.01, p < 0.05 with respect to control at 6h. Data are presented as mean ± SD (8 rats in each group) respectively.

4.2 BGLs

In the present study, an HFD and a low dose of STZ were used to induce T2DM in rats. This model is suitable to resemble the T2DM as it replicates the natural history and metabolic characteristics of human T2DM. A high lipid-rich regimen consisting of huge caloric quantity supports obesity-triggered glucose intolerance and IR rather than frank hyperglycaemia, which displays typical characteristics of T2DM. HFD administration primarily induces hepatic IR due to alteration in hepatic glucose metabolism followed by frank hyperglycaemia upon the low dose of STZ administration in rats. Indeed, other induction models are also available such as alloxan-induced; however, are not widely preferred in terms of toxicity aspect despite being less expensive than STZ (Ighodaro et al. 2017). In addition, there are surgical models and genetically modified or engineered diabetic/obese rodents that can be used for diabetes induction. However, the major drawbacks of such models such as invasiveness, caution during grafting, high cost, and risk of early mortality due to severe diabetes make them insignificant (King 2012; Kottaisamy et al. 2021).

Except for G1, the rats of all groups were fed with HFD for 15 days and their body wt., blood sugar levels, and lipid profile was measured to assess the impact of HFD administration on the aforementioned parameters. The results indicated that all the rats from G2-G12 showed a slight rise in the BGLs. This rise in BGLs was found significant in the range of 30 mg/dL to 52.2 mg/dL upon evaluation after the 15th day (Table 4). This rise in BGLs indicated the appearance of IR in the body due to high-fat diet consumption. This HFD regimen makes our liver and other body parts fatty upon oral administration. Overall it causes gut dysbiosis and an increase in the count of gram-negative bacteria. These gram-negative bacteria cause the release of lipopolysaccharides, which make the liver fatty and make the cells resistant to insulin, thus leading to hyperglycaemia (Singh et al. 2017). However, at this stage, the rats cannot be considered diabetic and the reversal in hepatic IR at this very point is quite obvious. Therefore, to induce frank hyperglycaemia in obese rats, STZ as a diabetogenic agent is injected that causes the complete appearance of signs and symptoms of T2DM.

Hence, as a continuation of the study protocol, the obese rats of G2 to G12 were intraperitoneally administered with STZ 35 mg/kg on the 16th day. To confirm the frank hyperglycaemic state (BGLs > 200 mg/dL) in obese rats, the BGLs were monitored on the 20th day. All the obese rats of G2 to G12 on the 20th day were found diabetic by showing BGLs < 300 mg/dL with a rise in the range of + 150 mg/dL to + 253 mg/dL respectively. Such results obtained, confirmed the complete diabetic state in the obese rats of each group.

The pharmacological screening of various test formulations was started from the 21st day till the 48th day and the BGLs of all the rats were monitored on weekly basis (i.e., 27th, 34th, 41st and 48th day). A unique pattern of decrease in BGLs was observed for rats of different treatment groups based on their efficacy. The placebo-treated group (G3) and experimental control (G2) group did not show any decrease in BGLs till the 48th day of the study protocol. However, the rats of G4 to G12 showed a significant (p < 0.001) decrease in BGLs when the results of the 20th day and 48th day were compared. The decrease in BGLs between rats of G4 to G12 after the first week of treatment i.e., 27th day is shown below:

G12(-2.70-folds) > G11(-2.00-folds) > G10 (1.9-folds) > G8(-1.80-folds) > G9(-1.50-folds) > G6 (-1.30-folds) > G7(-1.20-folds) > G5(-1.15-folds) > G4(-1.11-folds)

These results (Table 4) showed that a rapid response was observed in rats of G12 receiving co-loaded GV-APMs at a higher dose followed by G11 and G10 respectively. There was a non-significant difference in the reduction of BGLs in the case of the rats of G8, G10 receiving low and high dose Gly-APMs and G11, receiving a low dose of co-loaded GV-APMs. This showed the potential of APMs in enhancing the efficacy of Gly as compared to raw Gly. A significant difference in glucose-lowering capacity (p < 0.05) of Gly-APMs (G8 and G10) was observed on the 27th day itself as compared to the rats receiving a high dose of G5. Similar observations were noted for the rats receiving VA-APMs alone at both the doses (G7 and G9) as compared to rats receiving raw VA at a high dose (G4). This indicated that developed APMs significantly enhanced the glucose-lowering potential of both the therapeutics.

Gly is a well-known anti-diabetic drug used for decades, however, its poor aqueous solubility is still a challenge for the pharmaceutical industries. VA is an antioxidant that has shown good antidiabetic potential, but its rapid elimination from the body causes its failure to maintain glucose homeostasis. Both the biopharmaceutical issues of Gly and VA have been overcome by formulating APMs as this nanoarchitecture offers enhanced solubility and controlled drug release leading to sustained and enhanced oral bioavailability. This has been proven and reported in our previous research work (Kaur et al., 2022a). Due to this fact, when both the drugs are loaded in APMs when administered alone, showed a good anti-hyperglycaemic effect. This effect got further enhanced when both Gly and VA got co-loaded in APMs, due to their action on multiple pathways in the body that cause an elevation in BGLs. Interestingly, this effect was continued for the APMs formulation till the end of the study i.e. 48th day, wherein the BGLs for rats of G11 and G12 almost reached normal (Table 4 and Fig. 2).

Table 4

Effect of different treatment groups on BGLs upon weekly basis (Mean ± SD, n = 8)
Days	G1	G2	G3	G4	G5	G6	G7	G8	G9	G10	G11	G12
0th	103.6 ± 8.0	110.2 ± 15.4	112.5 ± 7.6	107 ± 14.8	111.5 ± 7.6	118 ± 12.6	107.25 ± 10.6	111.37 ± 9.3	114.75 ± 10.9	108.125 ± 11.2	100.625 ± 13.9	101.87 ± 15.4
15th	102.3 ± 12.1	147.7 ± 5.7^β	150.1 ± 9.6 ^β	149.5 ± 11.2^ϒ	148.7 ± 15.4^ϒ	152 ± 9.1 ^ϒ	153.7 ± 14.0 ^ϒ	152.1 ± 13.9^ϒ	145.5 ± 85.0 ^ϒ	149.8 ± 13.7 ^ϒ	151.6 ± 15.9 ^ϒ	154 ± 7.8 ^ϒ
15th	102.3 ± 12.1	Rise in BGLs* by + 37.5 mg/dL	Rise in BGLs* by + 37.6 mg/dL	Rise in BGLs* by + 42.5 mg/dL	Rise in BGLs* by + 37.2 mg/dL	Rise in BGLs* by + 34.0 mg/dL	Rise in BGLs* by + 46.5 mg/dL	Rise in BGLs* by + 40.8 mg/dL	Rise in BGLs* by + 30.8 mg/dL	Rise in BGLs* by + 41.7 mg/dL	Rise in BGLs* by + 51.0 mg/dL	Rise in BGLs* by + 52.2 mg/dL
20th	101.5 ± 10.2	400.7 ± 28.6^a	389 ± 36.5 ^a	336.5 ± 70.0 ^a	334.2 ± 30.6 ^a	331 ± 85.5^a	310 ± 93.6 ^a	387.3 ± 106.3 ^a	362.8 ± 15.0 ^a	370.3 ± 96.2 ^a	344.6 ± 92.3 ^a	395.3 ± 89.4 ^a
20th	101.5 ± 10.2	Rise in BGLs** by + 253 mg/dL	Rise in BGLs** by + 238.9 mg/dL	Rise in BGLs** by + 187.0 mg/dL	Rise in BGLs** by + 185.5 mg/dL	Rise in BGLs** by + 179.0 mg/dL	Rise in BGLs** by + 156.3 mg/dL	Rise in BGLs** by + 235.2 mg/dL	Rise in BGLs** by + 217.3 mg/dL	Rise in BGLs** by + 220.5 mg/dL	Rise in BGLs** by + 193.0 mg/dL	Rise in BGLs** by + 241.3 mg/dL
27th	101.4 ± 9.8	393.3 ± 63.2^a	380.5 ± 64.9 ^a	301.1 ± 19.9^z	290.2 ± 29.3^x	251 ± 23.5 ^x	241.7 ± 37.2 ^{x, g}	210.5 ± 25.7 ^{x, ϱ}	232.1 ± 30.1 ^x,e	186.7 ± 17.8 ^{x, Ф}	167 ± 18.4 ^x,p	143.1 ± 25.9 ^x,p
27th	101.4 ± 9.8	Decline in BGLs^# by -7.4 mg/dL	Decline in BGLs^# by -8.5 mg/dL	Decline in BGLs^# by -35.4 mg/dL	Decline in BGLs^# by -44.0 mg/dL	Decline in BGLs^# by -80.0 mg/dL	Decline in BGLs^# by -68.3 mg/dL	Decline in BGLs^# by -176.8 mg/dL	Decline in BGLs^# by -130.7 mg/dL	Decline in BGLs^# by -183.6 mg/dL	Decline in BGLs^# by -177.6 mg/dL	Decline in BGLs^# by -252.2 mg/dL
48th	97.8 ± 13.2	375.2 ± 44.6^a	360.23 ± 88.0^a	258 ± 15.3^x	184.7 ± 8.0^x	157 ± 9.8^x	152.4 ± 23.4^x,e	142.1 ± 13.2^{x, ϱ}	139.6 ± 13.6^x,e	133.9 ± 24.4^{x, ϱ}	116.2 ± 14.8^x,r	109.4 ± 24.3^x,r
48th	97.8 ± 13.2	Decline in BGLs^# by -24.8 mg/dL	Decline in BGLs^# by -28.8 mg/dL	Decline in BGLs^# by -78.5 mg/dL	Decline in BGLs^# by -149.5 mg/dL	Decline in BGLs^# by -174.0 mg/dL	Decline in BGLs^# by -157.6 mg/dL	Decline in BGLs^# by -245.2 mg/dL	Decline in BGLs^# by -223.2 mg/dL	Decline in BGLs^# by -236.4 mg/dL	Decline in BGLs^# by -228.4 mg/dL	Decline in BGLs^# by -285.9 mg/dL

*Increase in BGLs in comparison to 0th day; ** Increase in BGLs in comparison to 15th day; ^# Decrease in BGLs in comparison to 20th day; α, β, ϒ, indicate p < 0.001, p < 0.01, and p < 0.05 in comparison to 0th day; a, b, c indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to normal control (G1); x, y, z indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to experimental control (G2); Ф, ¥, ϱ indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to raw Gly (G5); e, f, g indicate p < 0.001, p < 0.01, and p < 0.05 in comparison to raw VA (G4); p, q, r indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to physical mixture.

4.3 Body weight

The intake of HFD causes obesity in the body followed by several metabolic complications leading to cardiovascular diseases, hyperlipidaemia, gut dysbiosis, and IR (Chang et al., 2015; Singh et al., 2017; Xiong et al., 2010). Similar observations were noted in the case of rats receiving HFD treatment (G2-G12) for 15 days. In all these rats, a significant (p < 0.05) rise in body wt. was observed (Table 5). More than 50g enhancement in body wt. of all these rats was observed. In contrast to this, a significant drop in body wt. of obese rats was observed on the 27th day of the treatment as compared to the body wt. noted on the 15th day. This effect was due to the administration of STZ on the 16th day which caused frank hyperglycaemia (BGLs > 200mg/dL). Incidentally, no significant decrease (p > 0.05) in body wt. was observed on the 20th day like that on the 15th day because the rise in BGLs did not show its impact on body metabolism by the 20th day. However, continuous high BGLs over ten days (i.e., day 18 to day 28) led to the alteration in body metabolism leading to a drop in the body wt., which is a typical sign of T2DM (Chang et al., 2015). it is important to note that the treatment of test and control formulations started from the 20th day onwards. Hence, the change in body wt. noted on the 27th day was also dependent on the efficacy of the treatment. The drop in the body wt. on the 27th day in comparison to the 15th day was found in the following order as given below

G2(-1.72-folds) > G3(-1.48-folds) > G4(-1.34-folds) > G9(-1.23-folds) > G10(-1.23-folds) > G8(-1.19-folds) > G11/G12

However, no significant difference was observed in the folds-decrease in G5, G6 and G7 (-1.28-folds) and in G11 and G12 (-1.12-folds) on 27th day. When the body wt. of rats was tested on 34th, 41st and 48th days, a gradual increase in their body wt. was observed as compared to their body wt. noted on the 27th day. The improvement in body wt. of rats was based on the efficacy of treatment (Table 5 and supplementary Fig S1). The rise in body wt. of rats on 48th day as compared to 27th day was in the following order as given below

G10(+ 1.16-folds) > G9(+ 1.14-folds) > G8(+ 1.12-folds) > G11(+ 1.08-folds) > G5(+ 1.07-folds) > G4(+ 1.02-folds)

However, no significant difference was observed in folds-increase in G6, G7, and G12 (+ 1.11-folds) on 48th day. It is very pertinent and interesting to note here that the rise in body wt. of G11 and G12 rats were less than that of their body wt. on the 27th day as compared to other rats. This was because the formulation was found highly efficacious as that of other treatments. This can be easily observed from the fact that the drop in their body wt. between the 15th day and 27th day was just 35.6g (G11) and 25.0g (G12) respectively. Owing to these observations, it was noted that the rats treated with low as well as high doses of co-loaded GV-APMs were able to manage the BGLs as well as body wt. post administration of STZ and 28 days’ treatment of test formulations i.e., GV-APMs.

4.4 Lipid profile

In the present study, HFD was administered orally for 15 days to rats of G2 to G12 with the objective to develop obesity, fatty liver, and hyperlipidaemia for the induction of IR leading to T2DM. The lipid profile of all the rats was determined on the 0th day and 15th day to assess the increase in the lipid-based biochemical parameters. The test was also performed on the 48th day to check the effect of different treatments (except G2 and G3) on the decrease in elevated levels of CHL, TGs, LDL, VLDL, CHL/HDL, and LDL/HDL ratio, and an increase in the declined levels of HDL respectively. The results are shown in supplementary Table S1 and Fig. 3. On the 15th day, all the lipid-based biochemical parameters related were found elevated in all the groups except G1 and the level of HDL was found to decline, indicating the successful development of hyperlipidaemia (Pandey et al. 2022). On the 48th day, the results of lipid-based biochemical parameters were found to be more ameliorated more than that the results obtained on the 15th day, except for rats of G2 and G3 respectively.

However, the efficacy of each treatment was different based on the nature of the drugs as well as the formulation. The results were found significantly (p < 0.05) improved in the case of rats receiving nanocombination of both the drugs in APMs (G11 and G12) in a dose-dependent manner. This indicated that the developed APMs enhanced the therapeutic efficacy of both the drugs by overcoming the challenges associated with poor solubility and faster elimination.

Table 5

Effect of different treatment groups on body wt. upon weekly basis (Mean ± SD, n = 8)
Days	G1	G2	G3	G4	G5	G6	G7	G8	G9	G10	G11	G12
0th	250 ± 0	256.6 ± 0.5	250 ± 0	250 ± 0	253.3 ± 0.3	250 ± 0	249.7 ± 0.6	253 ± 0.2	251 ± 0.1	252 ± 0.2	252 ± 0.2	257 ± 0.5
15th	250 ± 0	321.8 ± 27.5^a	303.12 ± 22.5^c	316.8 ± 23.2 ^a	310 ± 28.1 ^c	317.5 ± 24.7 ^c	298.1 ± 25.3 ^c	304 ± 18.6 ^a	331 ± 42.4 ^a	319.3 ± 25.1 ^c	325.6 ± 26.2 ^a	318.7 ± 14.0 ^c
		*Increase in body wt. by + 65.2g	*Increase in body wt. by + 53.1g	*Increase in body wt. by + 66.8g	*Increase in body wt. by + 57.0g	*Increase in body wt. by + 67.5g	*Increase in body wt. by + 48.4g	*Increase in body wt.by + 51.0g	*Increase in body wt. by + 80.0g	*Increase in body wt.by + 67.3g	*Increase in body wt. by + 73.6g	*Increase in body wt. by + 61.7g
20th	253.7 ± 5.1	313.75 ± 26	290.6 ± 26.7	301 ± 23.7	297.8 ± 18.4	302.5 ± 42.7	261.8 ± 28.2	286 ± 25.3	316.2 ± 19.2	305.3 ± 19.7	303.1 ± 20.8	294.3 ± 19.5
		**Decrease in body wt. by-8.1g	**Decrease in body wt. by-12.5g	**Decrease in body wt. by -15.8g	**Decrease in body wt. by-12.2g	**Decrease in body wt. by-15.3g	**Decrease in body wt. by-36.2g	**Decrease in body wt. by -18.0g	**Decrease in body wt. by-14.8g	**Decrease in body wt. by-14.0g	**Decrease in body wt. by -22.5g	**Decrease in body wt. by -24.4g
27th	256.8 ± 5.3	186.2 ± 53.9	196.7 ± 18.8 ^n.s	235 ± 13^z	241.5 ± 10.6^x	246.2 ± 31.1^x	232.5 ± 17.5 ^z	254.3 ± 13.9 ^x	267.5 ± 13.0 ^x	259.3 ± 7.2 ^x	290 ± 18.5 ^x	283.7 ± 6.9 ^x
		**Decrease in body wt. by -135.6g	**Decrease in body wt. by -99.3g	**Decrease in body wt. by -63.8g	**Decrease in body wt. by -68.5g	**Decrease in body wt. by -71.3g	**Decrease in body wt. by -65.6g	**Decrease in body wt. by -49.7g	**Decrease in body wt. by -63.5g	**Decrease in body wt. by -60.0g	**Decrease in body wt. by -35.6g	** Decrease in body wt. by -35.0g
48th	259.9 ± 6.3	165.4 ± 47.1	173.4 ± 19.2 ^n.s	242.7 ± 11.4 ^x	258.5 ± 7.5 ^x	273.5 ± 22.6 ^x	259.5 ± 7.9 ^x	286.1 ± 4.0 ^x	305.9 ± 12.7 ^x	301.4 ± 6.1 ^x	315.9 ± 13.5 ^x	328.5 ± 16.3 ^x
		#Decrease in body wt. by -20.6g	#Decrease in body wt. by -29.6g	##Increase in body wt. by -7.7g	##Increase in body wt. by -17.0g	##Increase in body wt. by -27.3g	##Increase in body wt. by -27.0g	##Increase in body wt. by -31.8g	##Increase in body wt. by -38.4g	##Increase in body wt. by -42.1g	##Increase in body wt. by -30.9g	##Increase in body wt. by -31.8g
Increase in body wt. in comparison to 0th day; *Decrease in body wt. in comparison to 15th day; # Decrease in body wt. in comparison to 27th day; ##Increase in body wt. in comparison to 27th day; a, b, c indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to normal control (G1); x, y, z indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to experimental control (G2)

A similar observation was noted for APMs containing VA and Gly during the pharmacokinetic study, wherein both the drugs showed enhanced oral bioavailability as that of their raw form as well as their administrations alone respectively. Interestingly, on the 48th day, the efficacy of raw VA and VA-APMs was found to better in lowering the levels of CHL, TGs, LDL, VLDL, CHL/HDL, and LDL/HDL ratio as well as elevating the levels of HDL as compared to rats receiving treatment of raw Gly alone and in the form of APMs respectively. This could be attributed to the anti-obesity and anti-hyperlipidaemic activity of VA that works by stimulating AMPKα- and thermogenesis in obese rats, increasing hepatic phosphorylated acetyl Co-A carboxylase protein expression, and reducing the levels of circulating leptin hormone secreted by adipose tissues (Chang et al., 2015;Kaur et al., 2022a). Previous studies on VA also indicated about its anti-hyperlipidemic potential by reducing serum leptin hormone having direct co-relation with lipid utilization by the body (Chang et al., 2015).

Whereas, the rats receiving Gly showed a comparatively lesser effect in lowering lipid-based biochemical parameters than that of VA. This could be because Gly is an anti-hyperglycemic drug and have no direct action on attenuation of lipid/CHL levels. The slight attenuation observed in the rats receiving Gly could be due to better maintenance of BGLs that would have led to restriction in dyslipidaemia. The combination therapy of VA and Gly either in their raw form or in the form of APMs showed significantly better attenuation of elevated lipid levels due to lipid-lowering properties of VA and reduction of BGLs by both the drugs (i.e. Gly and VA) respectively.

4.5 Hepatic and renal markers

Continuous administration of HFD causes hyperlipidaemia leading to fatty liver followed by alteration in liver metabolic enzymes. Certain studies reported the co-relation of hepatic IR with elevated SGPT, SGOT and ALP levels owing to HFD in obese/overweight populations without frank hyperglycaemia (Liu et al. 2021). Therefore, obesity is a main contributing factor to hepatic IR that amplifies the detrimental effects of IR on liver functions (Liu et al. 2021). In addition to this, hyperglycaemia being a major characteristic of T2DM is reported to cause serious complications in diabetic patients if not controlled. The most prevalent one is the alterations in renal markers such as creatinine and urea due to the reduced functioning of the kidney's filtering system in diabetics. In agreement with this, similar observations were observed in our study, after the complete induction of T2DM followed by HFD administration and a low dose of STZ in the rats of each group. However, in the treatment groups reduction in the hepatic and renal markers was found based on the therapeutic efficacy of different treatments.

Similar to the results of lipid profile, on the terminal day (48th day) of the study, the levels of SGPT, SGOT, ALP, creatinine and urea were found high in case of G2 and G3 rats and normal in case of rats G4 to G12, as compared to rats of G2. However, a significant decrease (p < 0.05) in enzymes level was observed in the case of rats receiving treatment of APMs of single drugs (VA-APMs and Gly-APMs) and their nanocombination (GV-APMs) i.e., from G7 to G12. The values of SGPT, SGOT and ALP for all the twelve groups and their fold decrease with respect to G2 are given in Table 6. In these cases, also the potential of VA in lowering the enzyme levels was found high as compared to Gly owing to the antioxidant, anti-obesity and anti-hyperglycaemic potential of VA as it can via multiple targets. However, Gly has shown its indirect action by maintaining glucose homeostasis in rats. The nanocombination therapy was found highly effective owing to the multiple health benefits of VA and Gly. Therefore, the decrease in the hepatic and renal markers by the different treatments was found in the following order as given below.

G1 > G12 > G11 > G9 > G6 > G7 > G10 > G4 > G8 > G5

However, no significant difference was observed in decrease in hepatic and renal markers in G2 and G3 on the 48th day.

4.6 Serum inflammatory markers

An inflammatory condition in diabetic rats is found to be in a positive co-relation with HFD consumption that is considered to be a contributing factor in the development of IR in rats (Chang et al., 2015). In addition, the low dose of STZ used in this model is also a chief factor that causes oxidative stress-induced activation of inflammatory signalling in diabetic rats (Samarghandian et al. 2017). With an agreement to this, the serum inflammatory markers viz. TNF-α and IL-6 were found to be elevated in the rats of the experimental control group and placebo group i.e., G2 and G3. However, the administration of different treatment groups based on their efficacy and dose reduced the level of inflammatory markers in the following order as given below.

G1 > G12 > G11 > G10 > G9 > G8 > G7 > G6 > G5 > G4

However, no significant difference was observed in decrease in inflammatory markers in G2 and G3. Similar to the results of hepatic and renal markers on the terminal day of the study, the levels of serum inflammatory markers were found normal in the case of G1. Whereas in treatment groups i.e., from G4 to G12, a significant difference (p < 0.05) was observed in the levels of inflammatory markers in comparison to G2 respectively.

Table 6: Effect of different treatments on hepatic/renal markers on 48^th day (Mean±SD, n=8)

Hepatic/renal markers	G1	G2	G3	G4	G5	G6	G7	G8	G9	G10	G11	G12
SGPT	46.2 ± 1.04	97.4 ± 1.18	92.2 ± 0.93^ns	66.5 ± 0.11^x	78.3 ± 0.57 ^x	58.1 ± 1.67 ^x	62.4 ± 3.21 ^{x, f}	69.8 ± 2.88 ^{x, ϱ}	56.7 ± 3.06 ^{x, g}	63.1 ± 2.13 ^{x, Ф}	47.2 ± 1.46 ^{x, e}	40.5 ± 0.48 ^{x, e}
SGPT	NA	NA	NA	##Decreased by -1.46-folds	##Decreased by -1.24-folds	##Decreased by -1.67-folds	##Decreased by -1.56-folds	##Decreased by -1.39-folds	##Decreased by -1.71-folds	##Decreased by -1.54-folds	##Decreased by -2.06-folds	##Decreased by -2.40-folds
SGOT	28.3 ± 2.55	69.8 ± 0.27	71.6 ± 1.20 ^ns	47.0 ± 1.37 ^x	61.0 ± 0.23 ^z	40.0 ± 1.22 ^x	40.3 ± 2.02 ^{x, g}	51.1 ± 0.87 ^{x, ϱ}	37.1 ± 3.44 ^{x, e}	46.2 ± 1.85 ^{x, Ф}	36.6 ± 1.93 ^{x, r}	30.1 ± 4.22 ^{x, r}
SGOT	NA	NA	NA	##Decreased by -1.48-folds	##Decreased by -1.14-folds	##Decreased by -1.74-folds	##Decreased by -1.73-folds	##Decreased by -1.36-folds	##Decreased by -1.88-folds	##Decreased by -1.51-folds	##Decreased by -1.90-folds	##Decreased by -2.31-folds
ALP	42.8 ± 1.71	97.9 ± 1.36	94.6 ± 0.78 ^ns	61.6 ± 2.76 ^x	72.8 ± 3.17 ^x	55.7 ± 0.06 ^x	58.2 ± 1.51 ^{x, g}	66.5 ± 0.06 ^{x, ϱ}	50.1 ± 2.66 ^{x, f}	60.0 ± 1.33 ^{x, ϱ}	45.6 ± 2.11 ^{x, r}	39.8 ± 3.66 ^{x, r}
ALP	NA	NA	NA	##Decreased by -1.58-folds	##Decreased by -1.34-folds	##Decreased by -1.75-folds	##Decreased by -1.68-folds	##Decreased by -1.47-folds	##Decreased by -1.95-folds	##Decreased by -1.63-folds	##Decreased by -2.14-folds	##Decreased by -2.45-folds
Creatinine	0.36 ± 0.34	0.78 ± 1.87	0.73 ± 0.51 ^ns	0.58 ± 0.23 ^z	0.67 ± 1.88 ^y	0.49 ± 0.82 ^x	0.52 ± 0.03 ^y	0.59 ± 0.51 ^{z, ϱ}	0.45 ± 2.20 ^z	0.56 ± 2.07 ^{z, ϱ}	0.41 ± 1.12 ^{z, r}	0.31 ± 0.02 ^{z, q}
Creatinine	NA	NA	NA	##Decreased by -1.34-folds	##Decreased by -1.21-folds	##Decreased by -1.59-folds	##Decreased by -1.50-folds	##Decreased by -1.30-folds	##Decreased by -1.73-folds	##Decreased by -1.39-folds	##Decreased by -1.90-folds	##Decreased by -2.51-folds
Urea	24.62 ± 2.02	62.40 ± 0.67	65.80 ± 0.32 ^ns	47.52 ± 1.71 ^z	53.30 ± 1.02 ^z	40.86 ± 0.71 ^x	42.64 ± 1.31 ^{x, g}	46.78 ± 0.83 ^z,¥	36.52 ± 2.16 ^{x, g}	41.36 ± 3.65 ^{x, Ф}	32.27 ± 1.28 ^{x, r}	26.17 ± 0.19 ^{x, r}
Urea	NA	NA	NA	##Decreased by -1.31-folds	##Decreased by -1.17-folds	##Decreased by -1.52-folds	##Decreased by -1.46-folds	##Decreased by -1.33-folds	##Decreased by -1.70-folds	##Decreased by -1.50-folds	##Decreased by -1.93-folds	##Decreased by -2.38-folds

##Decrease in hepatic and renal markers with respect to G2; x, y, z indicates p < 0.001, p < 0.01, p < 0.05 in comparison to G2; Ф, ¥, ϱ indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to raw Gly (G5); e, f, g indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to raw VA (G4); p, q, r indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to G6.

However, a significant decrease in the cytokines levels (TNF-α and IL-6) was observed in the case of rats receiving single drug-loaded APMs (VA-APMs and Gly-APMs) and their nanocombination therapy (GV-APMs) i.e., from G7 to G12, respectively. The results obtained for each treatment group in reducing the serum TNF-α and IL-6 levels along with their fold reduction in comparison to G2 are presented in Table 7. The results obtained indicated a higher potential of VA in lowering serum cytokines levels than that of Gly which could be due to its natural antioxidant and anti-inflammatory activity i.e., by modulating nrf2/NF-κB pathway (Kaur et al., 2022b). In the case of Gly, the reduction in serum cytokine levels with respect to G2 has been achieved due to its anti-hyperglycemic activity. Therefore, the effect of nanocombination therapy at a high dose i.e., G12 (GV-APMs) was found highly effective among all the different treatment groups owing to the multiple health benefits of VA used in combination with Gly. In addition, the combination of both the drugs in nanoform provided higher absorption, higher mean residence time in the systemic circulations, and controlled-release properties leading to higher oral bioavailability of both the drugs (Kaur et al., 2022a). Overall, the administration of nanocombination therapy provided higher therapeutic efficacy than their individual APMs and raw drugs respectively. Hence, it indicated improved additive pharmacological interaction than the physical mix used.

Table 7

Effect of different treatments on serum inflammatory markers on 48th day (Mean ± SD, n = 8)
Groups	Mean ± SD (TNF-α)	Fold-decreased^## (TNF-α)	Mean ± SD (IL-6)	Fold-decreased^## (IL-6)
G1	6.87 ± 0.32	NA	8.74 ± 0.87	NA
G2	33.56 ± 1.21	NA	44.61 ± 2.65	NA
G3	32.43 ± 0.42^ns	NA	46.34 ± 0.38 ^ns	NA
G4	17.41 ± 1.53 ^x	1.92-folds	26.54 ± 0.20 ^x	1.68-folds
G5	25.30 ± 0.03 ^x	1.32-folds	32.67 ± 0.54 ^x	1.36-folds
G6	14.41 ± 1.12 ^x	2.25-folds	21.46 ± 0.86 ^x	2.15-folds
G7	15.32 ± 0.10 ^{x, f}	1.13-folds	20.23 ± 1.48 ^{x, g}	1.31-folds
G8	19.85 ± 3.24 ^{x, ϱ}	1.27-folds	26.31 ± 2.05 ^{x, ϱ}	1.24-folds
G9	14.67 ± 0.45 ^{x, f}	0.98-folds	15.87 ± 1.07 ^{x, g}	1.35-folds
G10	16.13 ± 1.85 ^{x, Ф}	0.94-folds	19.64 ± 2.04 ^{x, ϱ}	1.03-folds
G11	11.45 ± 1.65 ^{x, r}	1.73-folds	12.07 ± 2.16 ^{x, r}	2.17-folds
G12	7.20 ± 2.92 ^{x, p}	2.03-folds	9.17 ± 3.17 ^{x, r}	1.73-folds

^##Decrease in TNF-α and IL-6 level with respect to G2; x, y, z indicates p < 0.001, p < 0.01, p < 0.05 in comparison to G2; Ф, ¥, ϱ indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to raw Gly (G5); e, f, g indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to raw VA (G4); p, q, r indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to physical mixture (G6).

4.7 Pancreatic oxidative stress

STZ being a cytotoxic agent causes degeneration of pancreatic β-cells leading to decreased insulin production causing frank hyperglycaemia followed by abnormal glucose homeostasis. Pancreatic β-cells are highly vulnerable to oxidative stress due to their lower antioxidant capacity and higher metabolic activity (Wang et al. 2018). Due to their vulnerability, STZ injection promotes the generation of reactive oxygen species in pancreatic cells along with mitochondrial dysfunction that contributes to its cytotoxic effect. Hence, results in impairment of redox metabolism (Nahdi et al. 2017). In this context, in the present study, reduction in the antioxidant enzymes i.e., CAT, GSH and elevated TBARS levels were observed in the experimental control group and placebo i.e., G2 and G3. Whereas, it was found normal in the case of the normal control group i.e., G1.

On the terminal day of the present study, a significant difference (p < 0.05) in CAT, GSH and TBARS levels was observed in all the treatment groups i.e., from G4 to G12. The results obtained in each treatment group are presented in Table 8. The results indicated a higher antioxidant potential of the nanocombination therapy with respect to single drugs loaded in APMs, their raw form and their physical mixture, respectively. This increment in their antioxidant efficacy was attributed to the use of VA in combination with Gly in nanoform that overcome the challenge of rapid elimination of VA from plasma. As a result, provided enough time for the drug for interacting with the biological targets leading to enhanced therapeutic efficacy than their raw forms respectively. The improvement in the CAT, GSH and TBARS levels by the different treatment groups based on their efficacy and dose is shown in the following order as given below.

G1 > G12 > G11 > G9 > G10 > G7 > G6 > G8 > G4 > G5

However, no significant difference was observed in the improvement in antioxidant levels in G2 and G3 on 48th day.

Table 8

Effect of different treatment groups on pancreatic oxidative markers on 48th day (Mean ± SD, n = 8)
Groups	Mean ± SD (CAT)	Fold-increased** (CAT)	Mean ± SD (GSH)	Fold-increased** (GSH)	Mean ± SD (TBARS)	Fold-decreased## (TBARS)
G1	97 ± 0.14	NA	80 ± 1.02	NA	1.97 ± 0.16	NA
G2	23 ± 1.23	NA	16 ± 2.44	NA	7.78 ± 1.16	NA
G3	26 ± 2.04	NA	20 ± 1.51	NA	7.4 ± 0.40	NA
G4	51 ± 2.76^z	2.21-folds	47 ± 1.10 ^z	2.93-folds	4.01 ± 0.27 ^z	1.94-folds
G5	45 ± 1.88^z	1.95-folds	35 ± 1.62 ^z	2.18-folds	5.68 ± 1.56 ^z	1.36-folds
G6	60 ± 1.62^z	2.60-folds	54 ± 2.38 ^x	3.37-folds	3.24 ± 0.87 ^z	2.40-folds
G7	63 ± 2.34^{z, g}	2.73-folds	58 ± 2.03 ^{x, f}	3.62-folds	3.05 ± 1.24 ^{z, g}	2.55-folds
G8	55 ± 2.13^{z, ϱ}	2.39-folds	43 ± 1.18 ^{z, ¥}	2.68-folds	4.72 ± 1.04 ^{z, ϱ}	1.64-folds
G9	71 ± 1.45^{x, f}	3.08-folds	65 ± 2.92 ^{x, g}	4.06-folds	2.76 ± 0.41 ^{z, g}	2.81-folds
G10	64 ± 2.92^{z, Ф}	2.78-folds	55 ± 1.43 ^{x, ϱ}	3.43-folds	3.18 ± 2.38 ^{z, ϱ}	2.44-folds
G11	89 ± 1.57^{x, r}	3.86-folds	68 ± 1.08 ^{x, q}	4.25-folds	1.78 ± 2.21 ^{z, q}	4.37-folds
G12	95 ± 1.20^{x, r}	4.13-folds	76 ± 0.05 ^{x, r}	4.75-folds	1.11 ± 1.94 ^{z, r}	7.00-folds

**Increase in CAT/GSH level with respect to G2; ##Decrease in level of TBARS with respect to G2; x, y, z indicates p < 0.001, p < 0.01, p < 0.05 in comparison to G2; Ф, ¥, ϱ indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to raw Gly (G5); e, f, g indicates p < 0.001, p < 0.01, and p < 0.05 in comparison to raw VA (G4); p, q, r indicates p < 0.001, p < 0.01 and p < 0.05 in comparison to physical mixture (G6).

4.8 Histopathology study

HFD administration primarily induces excessive insulin discharge from the pancreatic β-cell, which is then reduced by the fractional destruction of the functioning β-cells and frank hyperglycemia upon low dose of STZ administration in non-genetic SD rats. Thus, T2DM and obesity are claimed to co-exist in strong relationship (Samuel 2011). With this agreement higher levels of plasma inflammatory markers, circulating fatty acids and TGs in the obese people results in liver steatosis followed by IR.

The excess intake of HFD increases BGLs that initially causes hyperinsulinemia in response to compensate glucose levels upon regular ingestion of HFD, whereas higher levels of circulating fatty acids triggers hepatic lipogenesis that impairs insulin signal transduction pathway for glucose uptake upon activation of protein kinase Cε (PKCε). This eventually results in the development of hepatic IR (Samuel 2011). In addition, circulating fatty acids activate protein kinase C, which subsequently affect the expression of inhibitor of kappa β kinase, c-Jun N-terminal kinases, and p38 mitogen-activated protein kinases, stimulating the release of inflammatory factors such as TNF-α and IL-6, and causing internal inflammatory responses (Qatanani and Lazar 2007).

After the termination of the study, the pancreas and liver of rats in each group were isolated and were fixed in 10%v/v formaldehyde to identify the effect of each treatment given to the diabetic rats based on their efficacy. The changes in the cellular architecture of isolated pancreas and liver of diabetic rats in each group were determined by sequentially embedding them in paraffin blocks according to standard procedure. These samples were sectioned at 6 µm thickness, and then stained for functional pancreatic/liver cells with hematoxylin and eosin.

The consumption of HFD in rats induced obesity, metabolic alterations followed by changes in the level of lipid markers i.e., CHL, TGs, HDL, LDL, VLDL, CHL/HDL and LDL/HDL ratio as discussed above in biochemical study leading to steatosis in liver tissues. This condition leads to the development of IR in liver and the other peripheral cells. With this agreement, the histopathological images obtained for EC and placebo confirmed the occurrence of steatosis i.e., formation of multiple lipid droplets with cellular infiltration and cytoplasmic vacuolation. Whereas, in case of rats that received VA showed higher preservation of liver cells with minimal lipid droplets than that of rats that received Gly. This could be because of the direct action of VA on liver lipogenesis in obese-diabetic rats either by inhibiting release of leptin or by phosphorylating Acetyl CoA carboxylase, which regulate adiposity (Chang et al., 2015).

However, Gly showed slight effect on liver steatosis that could be because of its indirect anti-hyperglycaemic action. Overall, this could be the possible reason for the improved efficacy of both drugs in the form of combination therapy for the effective management of T2DM. In case of rats that received GV-APMs showed combined effect of both the drugs as a measure negligible lipid deposition than that of their physical mix. Whereas, the rats of group VA-APMs and Gly-APMs showed good reversal from liver steatosis with slight appearance of lipid droplets, which was comparatively higher than that of their raw form. In comparison to the lower dose, higher dose of the single drug loaded APMs showed good reversal of steatosis while complete restoration of liver cells was observed in rats that received GV-APMs.

The histology of islet cells of rats in normal control were normal. In diabetic rats without treatments i.e., EC and placebo showed breakdown of micro-anatomical characteristics including pancreatic β-cells degranulation, decreased cellular density, atrophy of islet cells, vacuolar and necrotic changes. These changes were found moderate in case of raw Gly, raw VA, physical mix, and slight in case of single drug loaded APMs and GV-APMs.

The histopathological images of rats of EC and placebo group showed damaged islet of Langerhans due to cytotoxic effect of STZ. Since, Gly is known to improve the insulin secretion by modulating ATP-sensitive potassium ion channels on islet cells, it showed improvement in cellular architecture of pancreatic cells after 28 days of treatment. Further, the effect of VA was found good in improving the micro-anatomical features of islet cells but its efficacy was lower than that of Gly. This could be because of the direct action of Gly onto the pancreatic islet cells or could be based on their bioavailability in the body. The loading of both the drugs in APMs either single loaded or co-loaded showed good recovery owing to their enhanced oral bioavailability.

The images of histopathology of different groups for liver is shown in Fig. 4 and pancreas in Fig. 5. The inference from these images is summarized in supplementary Table S2.

The study indicated the successful development of nanocombination therapy using APMs co-loaded with Gly and VA. The developed GV-APMs have shown significant improvement in levels of blood glucose, LDL, VLDL, TG, CHL, SGOT, SGPT, ALP, urea, creatinine, IL-6 and TNF-α in HFD- low dose STZ induced rat model. Even the diabetic rats receiving GV-APMs at low as well as high doses (G11 and G12) have shown good recovery in the tissue architecture of liver and pancreas as compared to any other treatment groups receiving monotherapy of VA and Gly in their raw form or, in the form of APMs, indicating the superiority of GV-APMs. This indicated that both the drugs were found more effective upon delivery via a nanocarrier system. It is important to note that the combination of both drugs was found to be more effective than their individual doses. As enhanced pharmacodynamics efficacy of any drug is one of the alternative ways to find out bioavailability of drugs; Hence, the study entailed that improved in vivo pharmacodynamics performance of GV-APMs in managing T2DM is a result of improved oral bioavailability of Gly and VA. Thus, the current findings are in concordance with our previous reports wherein, it was reported that the GV-APMs showed higher and sustained release profile, improved glucose uptake in HepG2 cells and enhanced oral bioavailability. The developed herbal-synthetic drug combination therapy in nano form has been found successful in treating diabetes owing to multiple mechanistic pathways of VA and glucose-lowering potential of Gly. The positive outcomes of pre-clinical studies indicated their further exploration for clinical trials so that the research should reach in the form of product for the effective management of T2DM.

Acknowledgement All the authors are thankful to their respective institutions for providing facilities and support to complete this research work.

Ethics Approval and Consent to Participate: The experimental protocol was duly approved by the institutional animal ethics committee (Protocol no: LPU/IAEC/2021/ 78) and performed as per CPCSEA guidelines

Consent for Publication: Yes

Availability of Data and Materials: Not Applicable

Competing Interests: None

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' Contributions:

S.N.	Authors	CRediT role(s)
1	Jaskiran Kaur	Methodology, Data Curation and Writing - original draft
2	Monica Gulati	Supervision
3	Narendra Kumar Pandey	Methodology
4	Gopal Lal Khatik	Methodology
5	Swathi Sudhakar	Methodology
6	Faisal Alotaibi	Methodology
7	Deepak Kumar	Methodology
8	Monu Kumar Shukla	Methodology
9	Parveen Kumar	Methodology
10	Paras Famta	Methodology
11	Poonam Arora	Methodology
12	Kunle Okaiyeto	Methodology
13	Oluwafemi Omoniyi Oguntibeju	Review & editing
14	Gaurav Gupta	Review & editing
15	Keshav Raj Paudel	Review & editing
16	Kamal Dua	Supervision
17	Sachin Kumar Singh	Conceptualization, Validation, Supervision and Writing - review & editing

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No competing interests reported.

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Antidiabetic evaluation of vanillic acid-glyburide loaded polymeric micelles in high fat diet and streptozotocin induced diabetic rats

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

In vivo antidiabetic study

3.1 Animals

3.2 Obesity-induced IR and hyperglycaemia

3.3 Oral glucose tolerance test (OGTT)

3.4 Treatment design and pharmacological screening

3.5 Biochemical parameters

3.6 Euthanasia

3.7 Determination of pancreatic oxidative stress

3.8 Histopathology study

3.9 Statistical analysis

Results and discussion

4.1 OGTT

4.2 BGLs

4.3 Body weight

4.4 Lipid profile

4.5 Hepatic and renal markers

4.6 Serum inflammatory markers

4.7 Pancreatic oxidative stress

4.8 Histopathology study

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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