Computational and Pharmacological Evaluation of Carveol for Antidiabetic Potential

Background Carveol is a natural drug product present in the essential oils of orange peel, dill, and caraway seeds. The seed oil of Carum Carvi has been reported to be antioxidant, anti-inflammatory, anti-hyperlipidemic, antidiabetic, and hepatoprotective. Methods The antidiabetic potential of carveol was investigated by employing in-vitro, in-vivo, and in-silico approaches. Moreover, alpha-amylase inhibitory assay and an alloxan-induced diabetes model were used for in-vitro and in-vivo analysis, respectively. Results Carveol showed its maximum energy values (≥ -7 Kcal/mol) against sodium-glucose co-transporter, aldose reductase, and sucrose-isomaltase intestinal, whereas it exhibited intermediate energy values (≥ -6 Kcal/mol) against C-alpha glucosidase, glycogen synthase kinases-3β, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and other targets according to in-silico analysis. Similarly, carveol showed lower energy values (≥ 6.4 Kcal/mol) against phosphoenolpyruvate carboxykinase and glycogen synthase kinase-3β. The in-vitro assay demonstrated that carveol inhibits alpha-amylase activity concentration-dependently. Carveol attenuated the in-vivo alloxan-induced (1055.8 µMol/Kg) blood glucose level in a dose- and time-dependent manner (days 1, 3, 6, 9, and 12), compared to the diabetic control group, and further, these results are comparable with the metformin positive control group. Carveol at 394.1 µMol/Kg improved oral glucose tolerance overload in rats compared to the hyperglycemic diabetic control group. Moreover, carveol also attenuated the glycosylated hemoglobin level along with mediating anti-hyperlipidemic and hepatoprotective effects in alloxan-induced diabetic animals. Conclusions This study reveals that carveol exhibited binding affinity against different targets involved in diabetes and has antidiabetic, anti-hyperlipidemic, and hepatoprotective actions.


INTRODUCTION
Diabetes mellitus (DM) is a leading health issue, having a highly troubling frequency in developing countries. The primary risk factors of DM include a sedentary lifestyle, poor nutritional habits, and obesity (Hu, 2011). Diabetes is an independent risk factor and important comorbidity of several human diseases and increases the risk of death by 1.5-3 times (Capes et al., 2001). The WHO ranked DM the biggest epidemic and the most common cause of functional disabilities (Mathers and Loncar, 2006;Shah et al., 2019). DM is predominantly related to increased blood glucose level associated with alteration in the breakdown of fats, proteins, and carbohydrates due to either a decrease in the production of insulin. The low insulin level in type I diabetes is due to autoimmunity in beta cells of the pancreas (Tuomi, 2005), and the resulting hyperglycemia can be managed by administering insulin injections subcutaneously. In type II diabetes, there is rather a reduction in insulin sensitivity in hepatic, cardiac, and fat cells, and this can be managed by hypoglycemic drugs (Nathan et al., 2009). The foremost risk factors for the progression of anomalous secretion of insulin or resistance to it include genetic defects, aging, viral infection, environmental factors, and a sedentary lifestyle combined with high calorie intake (Saltiel, 2001).
Type II DM has long-term health hazards, including neuropathy, nephropathy, and accelerated atherosclerosis leading to an increased risk of myocardial infarction (MI) (Hanefeld et al., 1996;Viberti, 2005). Furthermore, it makes the human body prone to dyslipidemia, high blood pressure, and obesity (Association, 2014). Moreover, dyslipidemia triggers various cardiovascular complications such as atherosclerosis, MI, hypertension, and obesity-related problems (Ashfaq et al., 2017). Stress also plays an important role in the progression of hyperlipidemia due to the generation of free radicals, which may lead to atherosclerosis and cardiovascular diseases (Arise et al., 2016).
Alloxan is a potent inducer of pancreas toxicity and is therefore used for experimental diabetes induction (Brenna et al., 2003). Alloxan induces a multiphasic blood glucose response when injected into an experimental animal, with consistent fluctuations in the plasma insulin level associated with structural damage to beta cells (Olusanya and Ifeoluwa, 2018). The pathology of DM is attenuated by insulin therapy and orally by hypoglycemic medications such as biguanides and sulfonylureas. However, the therapeutic potential of these oral synthetic antidiabetic agents has been limited by long-term microvascular and macrovascular complications (Stenman et al., 1990;Spiller and Sawyer, 2006). Carveol ( Figure 1) is a natural drug substance present in the essential oils of orange peel, dill (Anethum graveolens L.), and caraway seeds (Crowell et al., 1992). The chemical components are D-limonene, mono-terpenes carveol acetate, and trans-and cis-carveol, glyceryl esters (Singh and Lal, 2008). The seeds of caraway oil (Carum carvi L.) have been described in customary Chinese medicine as antispasmodic, carminative, astringent, anti-inflammatory, anti-cancer, and hepatoprotective (Johri, 2011;Agrahari and Singh, 2014).
This study aims to demonstrate the antidiabetic potential of carveol through molecular docking, in-vitro study, and an in-vivo animal experimentation model using alloxan-induced diabetes and to further evaluate the anti-hyperlipidemic and hepatoprotective effect of carveol.

Animals
Adult Sprague-Dawley (SD) rats of either sex weighing 250-280 g and 7-11 weeks old were obtained from the local breeding facility of Riphah International University. The animals were kept in a standard animal room temperature at 18-22°C under circadian light and dark conditions with access to food and water ad libitum. All the experimental protocols were recommended and approved by the

Alpha-Amylase Inhibitory Assay
The antidiabetic potential of the test compound carveol was determined by a-amylase inhibition assay following the standard protocol with minor modification (Kim et al., 2000). The reaction mixture containing 15 ml phosphate buffer (pH 6.8), 25 ml a-amylase enzyme (0.14 U/mL), different concentrations of the sample in normal saline, and 40 ml starch solution (2 mg/mL in potassium phosphate buffer) was incubated at 50°C for 30 min in a 96-well plate followed by addition of 20 ml of 1 M HCl to stop the reaction. Afterward, 90 ml of iodine reagent (5 mM iodine, 5 mM potassium iodide) was added to each well. Negative control was prepared without the sample, whereas blank was prepared without the sample and amylase enzyme, each being replaced by equal quantities of the buffer. Acarbose (250 mM/Kg) was used as a positive control. The absorbance of the reaction plate after incubation was measured at 540 nm. The activity was expressed as percent a-amylase inhibition and calculated by the following equation: where On = Absorbance of negative control, Os = Absorbance of sample, and Ob = Absorbance of the blank well.

Blood Glucose Levels and Body Weight Measurement
SD rats were adjusted to the laboratory environments and reserved for whole night fasting (12-14 h). The animals were divided into six groups, each containing five animals (n=5). Group I and II were non-diabetic control and diabetic control groups injected with saline (10 mL/Kg) and alloxan monohydrate (1055.8 µMol/ Kg), respectively. Groups III, IV, and V were alloxan-induced diabetic rats administered with the test compound at doses of 65.7, 197, and 394.1 µMol/Kg respectively. Group VI was positive control and was injected with metformin (1207.5 µMol/Kg). The blood glucose levels were measured at days 1, 3, 6, 9, and 12, using an Accu-Chek instant glucometer. For the complete treatment period, the body weight of animals was measured at the same regular intervals. For the induction of diabetes, alloxan monohydrate was used (Dunn and McLetchie, 1943). Freshly prepared alloxan solution (1055.8 µMol/Kg) in saline was given to experimental rats via an intra-peritoneal route (Olusanya and Ifeoluwa, 2018). After 48 h, blood glucose levels of experimental rats were measured by using the tail prick methodology. Rats with blood glucose levels ≥ 200 mg/dL were demarcated as hyperglycemic (Saudek et al., 2008).

Oral Glucose Tolerance Test (OGTT)
After 18 h of fasting, rats were placed into four groups. Group I and II were non-diabetic and diabetic control and were injected with saline (10 mL/Kg) and alloxan (1055.8 µMol/Kg), respectively. Group III was a carveol-treated (394.1 µMol/Kg) group. Group IV was positive control and injected with metformin (1207.5 µMol/Kg). Each group was pre-treated, and after 30 min, the D-glucose load (3 g/Kg) was administered orally. The blood glucose level was measured at 0, 30, 60, 90, and 120 min using an Accu-Chek instant glucometer (Rubino and Marescaux, 2004).

Glycosylated Hemoglobin (HbA1C) Test
After 6 weeks of treatment, the HbA1C test was performed for all groups (Asgary et al., 2008). A cardiac puncture methodology was utilized to collect blood samples (Doeing et al., 2003). The HbA1C level was measured in a local laboratory at Islamabad, Pakistan.

Serum Biomarker Analysis
A hepatic functional test was performed for each group (n=5/ group). The cardiac puncture method was used to collect blood samples. The lipid profile in terms of TC, TGs, LDL, and HDL and the hepatic functional markers alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB) were analyzed in a local laboratory at Islamabad, Pakistan.

Statistical Analysis
Data are presented as mean ± standard error of the mean (SEM). The significance of results was evaluated by analysis of variance (ANOVA), followed by a multiple comparison test. p < 0.05 was considered to be statistically significant. Statistical assessment, preparation of graphs, and evaluation were performed by using Graph Pad Prism 6.

a-Amylase Inhibition
Carveol and acarbose caused a-amylase enzyme inhibition at different concentrations; the results are shown in Table 2.

DISCUSSION
Virtual screening, or the in-silico approach, is a procedure through which ligands are docked with respective target proteins by using a fast and cost-effective technique, and it Values expressed as percentage inhibition mean ± SEM. One-way ANOVA was used for statistical analysis, ***p < 0.001 compares the percentage inhibition of carveol group vs. the acarbose group.
FIGURE 3 | Bar graph representing blood glucose level on different treatment days of the saline-treated group (non-diabetic control), alloxan-treated group (diabetic control), carveol-treated groups at different doses (65.7, 197, 394.1 µMol/Kg), and metformin-treated group against alloxan-induced hyperglycemia in rats. Data presented as mean ± SEM. Statistical analysis used one-way ANOVA, followed by post-hoc Tukey's test. ### p < 0.001 vs. saline group, **p < 0.01 and ***p < 0.001 comparison of the blood glucose levels of carveoland metformin-treated groups vs. diabetic control group. Values expressed as mean ± SEM. One-way ANOVA was used for statistical analysis, followed by Bonferroni's multiple comparisons test. ## p < 0.01, ### p < 0.001 vs. saline group, **p < 0.01, ***p < 0.001 comparison of the blood glucose levels of carveol-and metformin-treated groups vs. diabetic control group.
FIGURE 4 | Bar graph representing blood glucose levels at different time intervals (0-120) after administration of oral glucose load (non-diabetic control), alloxan-treated (diabetic control), carveol-treated, and metformintreated groups. Values expressed as mean ± SEM. One-way ANOVA was used for statistical analysis, followed by post-hoc Tukey's test. ### p < 0.001 vs. saline group and ***p < 0.001 comparison of the blood glucose levels of carveol-and metformin-treated groups vs. diabetic control group.
requires computer-assisted programs and software (Prakhov et al., 2010). We demonstrated a comparative study by comparing the results for ligand-protein complexes to those for the standard drugs, obtained from the RCSB and PubChem database. Auto-Dock vina, patch dock, gromacs, and gold suite provide docking of ligands with a possibility of a dozen torsional degrees of freedom. A lower binding value (kcal/mol) reveals reduced energy of desolvation, which depicts stability of the ligand-protein complex (Pecsi et al., 2010). Hydrogen bonding, pi-pi bonding, and other hydrophobic interactions provide valuable strength for structurally complex stabilization. Other hydrophobic interactions play a vital role in increasing the affinity of the ligand towards the protein receptor (Dallakyan and Olson, 2015). E-value, H bonding, and hydrophobic interactions play a key role in the assessment of the binding affinity of ligand and protein complexes. We demonstrated here that carveol manifested the best binding score with the lowest E value against SGLT. Based on the E value against different protein targets, the order of ligand affinity was found to be SGLT > AR > SIMI > PI3K > 11b-HSD1 > FBP1 > C-AG > PEPCK > GSK-3b > AA > PPAR-g. The enzyme alpha-amylase and alpha-glucosidase are responsible for the breakdown of carbohydrates and for hydrolyzing starch into glucose before absorption (Chelladurai and Chinnachamy, 2018). Reduction in postprandial hyperglycemic level is due to alpha-amylase inhibition, which delays the small intestine carbohydrate digestion and diminishes the postprandial blood glucose level (Kwon et al., 2007). One of the approaches for treating DM is to inhibit the carbohydrate digesting enzymes such as a-amylase in the GIT and thereby reduce postprandial glucose (Raman et al., 2012). Previous studies demonstrated that flavonoids, tannins, and terpenoids could effectively inhibit alpha-amylase and alpha-glucosidase (Khan et al., 2014). Similarly, we demonstrated that carveol can inhibit a-amylase concentration-dependently, which may be due to its mono-terpenoid nature. Moreover, a-amylase inhibition was performed at various concentrations and compared with the standard drug acarbose at the same concentrations.
In the present study, the antidiabetic effect of carveol against alloxan-induced diabetes in rats was investigated. Administration of alloxan leads to inhibition of insulin secretion, resulting in persistent hyperglycemia or diabetes (Yang et al., 2010). Carveol reversed the blood glucose level in a dose-dependent and time-dependent fashion when compared to the alloxan-treated diabetic group. Furthermore, the results for carveol were comparable to those for the

Group
HbA1c level (%) Non-Diabetic Control (Saline, 10 mL/Kg) 5.0 ± 0.05 Diabetic Control (Alloxan 1055.8 µMol/Kg) 7.1 ± 0.11 ### Alloxan (1055.8 µMol/Kg) + Carveol (65.7 µMol/Kg) 5.6 ± 0.14*** Alloxan (1055.8 µMol/Kg) + Carveol (197 µMol/Kg) 5.2 ± 0.11*** Alloxan (1055.8 µMol/Kg) + Carveol (394.1µMol/Kg) 4.9 ± 0.06*** Alloxan (1055.8 µMol/Kg) + Metformin (1207.5 µMol/Kg) 5.1 ± 0.10*** Values expressed as mean ± SEM. One-way ANOVA was used for statistical analysis, followed by post-hoc Dunnett test. ### p < 0.001 vs. saline group, and ***p < 0.001 comparison of the HbA1C levels of carveol-and metformin-treated groups vs. diabetic control group. Values expressed as mean ± SEM. One-way ANOVA was used for statistical analysis, followed by post-hoc Dunnett test. ## p < 0.01, ### p < 0.001 vs. saline group, and *p < 0.05, **p < 0.01, and ***p < 0.001 comparison of the blood glucose levels of carveol-and metformin-treated groups vs. diabetic control group. standard metformin group, which was used as a positive control. Metformin lowers the blood glucose level by numerous pathways, including decreasing biogenesis in hepatic tissue, minimizing glucose absorption by the intestine, and improving its peripheral utilization through enhancing insulin sensitivity (Klip and Leiter, 1990). We demonstrated that carveol reduced the body weights of test animals at regular daily intervals. As obesity is directly related to diabetes, a drug with the dual benefits of glycemic and weight control is of interest in diabetes, so this is an attractive outcome of carveol usage. In the glucose-loaded hyperglycemia model, which aims to assess oral glucose tolerance, carveol exhibited considerably better tolerance of glucose overload at regular intervals than did the metformin group. Carveol produced a dose-dependent effect of reducing the HbA1C level and was found to be effective as a long-term antidiabetic agent. Diabetes is also associated with a variable lipid profile (Virdi et al., 2003), which can be further linked to obesity and renal impairment (Al-Shamaony et al., 1994). The marked hyperlipidemia that characterizes the diabetic state may be a consequence of the abnormal function of lipolytic hormones on the fat depots (Ayeleso et al., 2012). Lowering of serum lipid levels through dietary or drug therapy seems to be associated with a decrease in the risk of vascular disease in diabetes (Ayeleso et al., 2012). The results of the present investigation show that carveol produced a significant decrease in TC, TG, and LDL and a significant increase in HDL as compared to diabetic control. This improvement in the lipid profile status of alloxan-treated rats revealed the anti-hyperlipidemic properties of carveol. It has previously been reported that about 30% of blood cholesterol is circulated in the form of HDL-cholesterol (HDL-C). HDL-C removes cholesterol atheroma from arteries and transports it to the liver for excretion or re-utilization, which is a beneficial approach in cardiovascular diseases (Owolabi et al., 2010). Therefore, the increase in the serum HDL-C level by carveol in hyperglycemic rats indicates that carveol can augment HDL-C effects. Furthermore, our study demonstrated that various hepatic enzymes, such as AST, ALT, and ALP, were significantly increased in the alloxan-induced diabetic control group. However, carveol significantly decreased the level of these enzymes compared to diabetic control. Furthermore, increased gluconeogenesis and ketogenesis might be due to elevated activity of transaminases (Gandhi et al., 2011) associated with hepatocyte damage in experimental animals (Abolfathi et al., 2012). The ability of carveol to attenuate the serum level of ALT, AST, and ALP suggests its hepato-cellular protective effect.

CONCLUSIONS
The present study demonstrated that carveol exhibited maximum binding affinity against SGLT, intermediate binding affinity against FBP1, and lower energy values against PEPCK and GSK-3b. Moreover, our in-vitro and in-vivo study suggests that carveol could be a promising therapeutic agent in the management of diabetes. However, extensive exploration is still required to delineate the underlying protective mechanisms of carveol.

DATA AVAILABILITY STATEMENT
All datasets generated and analyzed for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by Ethical Committee, Riphah Institute of Pharmaceutical Sciences (Ref. No.: REC/RIPS/2019/03).

AUTHOR CONTRIBUTIONS
MA carried out the computational studies, in-vitro and in-vivo experimentation, evaluation of results, and documentation. A-uK, and FS supervised the research project and drafted the initial and final version of the manuscript. LK help in data analysis, revision and with drug Carveol. All authors contributed to the article and approved the submitted version.