|Year : 2020 | Volume
| Issue : 2 | Page : 100-107
Effect of L-citrulline supplementation on blood glucose level and lipid profile in high-fat diet - and dexamethasone-induced type-2 diabetes in male wistar rats
Timothy Danboyi1, Abdulwahab W Alhassan2, Abdulazeez Jimoh2, Evelyn Hassan-Danboyi2
1 Department of Human Physiology, College of Medicine, Kaduna State University, Kaduna, Nigeria
2 Department of Human Physiology, College of Medical Sciences, Ahmadu Bello University, Zaria, Nigeria
|Date of Submission||05-Jun-2020|
|Date of Decision||29-Jul-2020|
|Date of Acceptance||14-Aug-2020|
|Date of Web Publication||11-Feb-2021|
Dr. Timothy Danboyi
Department of Human Physiology, College of Medicine, Kaduna State University, Kaduna
Source of Support: None, Conflict of Interest: None
Background: Diabetes mellitus (DM) is one of the most common metabolic disorders, afflicting over 415 million people worldwide. It has been associated with several complications mainly due to hyperglycemia and dyslipidemia. L-Citrulline, a nonessential amino acid may be an efficient alternative therapy owing to its hypolipidemic and other beneficial effects which have not been extensively explored in type-2 DM (T2DM). Objective: We investigated the effect of L-citrulline supplementation on fasting blood glucose (FBG) level and lipid profile in high-fat-diet (HFD) and dexamethasone-induced T2DM in male Wistar rats. Materials and Methods: Thirty male Wistar rats, 10–12 weeks old, each weighing between 200 and 250 g were randomly assigned into six groups of five rats each. Group I was fed normal diet while diabetes was induced in the other groups with HFD and dexamethasone intraperitoneally (1 mg/kg) for 21 days. Group III which was confirmed diabetic, received metformin 100 mg/kg orally, and Groups IV, V, and VI which were also confirmed diabetic, received 200 mg/kg, 400 mg/kg, and 800 mg/kg L-citrulline, respectively, for 21 days. Serum FBG and lipid profile were obtained after humanely sacrificing the rats at the end of the treatment. Values at P < 0.05 were considered statistically significant. Results: At the end of the treatment, L-citrulline significantly reduced the FBG levels in a dose-dependent manner to 192.5 ± 3.4 mg/dL, 181.8 ± 1.2 mg/dL, and 174.8 ± 2.8 mg/dL at 200 mg/kg, 400 mg/kg, and 800 mg/kg, respectively. The total cholesterol level was significantly lowered by L-citrulline 200 mg/kg (55.2 ± 0.64 mg/dL), 400 mg/kg (57.8 ± 1.19 mg/dL), and 800 mg/kg (63.1 ± 1.50 mg/dL) compared to the diabetic control (149.8 ± 2.68 mg/dL). Similar findings were obtained for the low-density lipoprotein and triglyceride levels. There were also significant elevations in the high-density lipoprotein levels by L-citrulline at all doses compared to diabetic control (24.6 ± 1.1 mg/dL). Conclusion: L-Citrulline supplementation possesses antihyperglycemic and antidyslipidemic effects in diabetic Wistar rats.
Keywords: Antidyslipidemic, antihyperglycemic, L-citrulline, type-2 diabetes mellitus
|How to cite this article:|
Danboyi T, Alhassan AW, Jimoh A, Hassan-Danboyi E. Effect of L-citrulline supplementation on blood glucose level and lipid profile in high-fat diet - and dexamethasone-induced type-2 diabetes in male wistar rats. Niger J Exp Clin Biosci 2020;8:100-7
|How to cite this URL:|
Danboyi T, Alhassan AW, Jimoh A, Hassan-Danboyi E. Effect of L-citrulline supplementation on blood glucose level and lipid profile in high-fat diet - and dexamethasone-induced type-2 diabetes in male wistar rats. Niger J Exp Clin Biosci [serial online] 2020 [cited 2021 May 12];8:100-7. Available from: https://www.njecbonline.org/text.asp?2020/8/2/100/309165
| Introduction|| |
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia (high blood glucose level) resulting from defects in insulin secretion, insulin action, or both. According to the International Diabetes Federation, DM had an estimated global prevalence of about 8.8% in 2015 with 415 million people affected worldwide, and is predicted to rise to about 10.4% by 2040, affecting an estimated 642 million people. It has been described as the epidemic of the 21st century. In sub-Saharan Africa, about 20 million people have DM, and this is projected to reach 41.4 million by 2035, with Nigeria having the highest number (5 million) of people with DM.
High-fat diet (HFD) had been used as an experimental rat model of type-2 DM (T2DM). A decrease in the glucose transporter (GLUT 4) expression in muscles and adipose tissues, associated with insulin resistance, had been implicated in the onset and progression of T2DM especially in the obese and physically inactive individuals. DM is a well-known metabolic “side effect” of glucocorticoid therapy. Exogenous glucocorticoids such as hydrocortisone and dexamethasone are potent diabetogenic agents that increase whole body insulin resistance and plasma glucose levels which may lead to the development of T2DM.,, The diabetogenic effect is mainly a result of insulin resistance in muscles and fatty tissue, increased hepatic glucose output and an impaired β-cell function., The exact mechanisms by which dexamethasone causes insulin resistance include direct injury to the β-cells, impairing their functions; direct anti-insulinic effects in liver, skeletal muscle, and adipose tissue (inhibition of insulin-induced GLUT 4 translocation to the plasma membrane); and decreasing key mediators of insulin action in peripheral tissues.
The management of DM especially the T2DM has been challenging due to the ineffectiveness of the conventional drugs to halt the progression of the disease; their high cost as well as their associated adverse effects. Some of the conventional antidiabetic drugs have severe, deleterious side effects such as severe hypoglycemia and lactic acidosis which may lead to coma and even death if not promptly and adequately treated. Hence, the quest for newer but equally potent antidiabetic drugs/supplements with less or no side effects and can that can improve, to a great extent, the quality of life of diabetic patients has been gaining universal acceptance.
L-citrulline, a naturally occurring nonessential aminoacid produced in the liver that plays a role in the production of nitric oxide (NO)., L-Citrulline however, through its role in the metabolism and regulation of NO,, prevents excessive and uncontrolled NO production. NO has a wide range of functions which are augmented by L-citrulline. These include maintenance of normal endothelial function,,, which is disrupted in the setting of DM;, and improvement of peripheral and hepatic insulin sensitivity., Therefore, L-citrulline may play a potential role in the treatment and prevention of cardiovascular diseases or events especially in the setting of T2DM.,,,
Supplementation with L-citrulline had been shown to enhance plasma citrulline concentration, and it bypasses hepatic or intestinal metabolism., It is well tolerated orally with no side effects such as gastrointestinal disturbances even at high doses.,, It is readily available and affordable especially in its natural source (Citrullus lanatus) and it has been reported to possess antioxidant activity, among several other benefits. However, to the best of our knowledge, there are very few studies that demonstrate its effects on blood glucose and lipid profile in T2DM and that is what this study aimed to investigate.
| Materials and Methods|| |
A total of 30 male Wistar rats, 8–12 weeks old and weighing 200–250 g were sourced from the animal house, Department of Human Physiology, Ahmadu Bello University (A.B.U), Zaria. They were housed in plastic cages and allowed free access to commercial grower mash feed and water.
Ethical clearance was obtained from the Ethical Committee on Research Animals' Use and Care, A.B.U, Zaria, with approval number of ABUCAUC/2020/72.
Preparation of high-fat diet
HFD was prepared by mixing normal rat chow, composed of fat (18%), proteins (54%), and carbohydrates (28%), with margarine (99.9% fats) in the ratio of 10:1, i.e., 10 g of the normal chow to 1 g of margarine.
Experimental animal grouping
Thirty rats were randomly assigned into six groups of five rats each as follows:
- Group I (n = 5): Rats were fed normal diet for 21 days. They served as the normal control
- Group II (n = 5): Rats were fed HFD daily for 21 days plus intraperitoneal (i.p.) dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15). This served as the diabetic control
- Group III (n = 5): Rats were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral metformin (a biguanide) 100 mg/kg daily for 21 days. This served as the metformin group (i.e., Met 100 mg/kg)
- Group IV (n = 5): Rats were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 200 mg/kg body weight daily for 21 days. They served as CIT 200 mg/kg group
- Group V (n = 5): Rats were fed HFD daily for 21 days plus i.p dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 400 mg/kg body weight daily for 21 days. They served as CIT 400 mg/kg group
- Group VI (n = 5): Rats were fed HFD daily for 21 days plus i.p dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 800 mg/kg body weight daily for 21 days. They served as CIT 800 mg/kg group.
Induction of type-2 diabetes mellitus
The rats in Groups II to IV were fed with HFD for 21 days plus dexamethasone 1 mg/kg body weight intraperitoneally daily starting from day 15–21 of the experiment.
Determination of fasting blood glucose levels
Fasting blood glucose (FBG) was measured using Accu-Chek Advantage glucometer and strip (Roche Diagnostics, Germany) and expressed in mg/dL based on the glucose oxidase principle. At the end of the induction of diabetes, FBG levels were measured (after an overnight fasting for at least 8 h) on days 0, 8, 15, and 22. The blood samples were taken from the tail veins, which were slightly pierced and a drop of blood were placed on the strip of glucometer. Blood glucose levels >200 mg/dL were considered diabetic.,
Collection of blood samples
Rats were anaesthetized using 5 mg/kg body weight ketamine injection. About 4 ml of blood were drawn from each rat through cardiac puncture. Blood collected were allowed to stand and clot for 30 min, the resulting samples were centrifuged at 3000 g for 10 min. Centrifuged samples were stored in plain tubes kept in ice packs (temperature approximately between -2°C and -20°C) until assay.
Lipid profile analyses
Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels were assayed using Centronic LDL and HDL kits. Triglyceride (TG) levels were assayed using Agappe TG kit. They were assayed using enzyme-linked immunosorbent assay method.
Determination of serum total cholesterol levels
This was determined spectrophotometrically, using enzymatic colorimetric assay kits (Randox Laboratories Limited kits, United Kingdom) as follows: The serum level of total cholesterol (TC) was quantified after enzymatic hydrolysis and oxidation of the sample as described by the method of Stein (1987). Exactly 1000 μL of the reagent was added to each of the sample and standard. This was incubated for 10 min at 20°C–25°C after mixing and the absorbance of the sample (A sample) and standard (A standard) was measured against the reagent blank within 30 min at 546 nm. The value was expressed in mg/dL.
Determination of serum triglyceride levels
The serum TG level was determined after enzymatic hydrolysis of the sample with lipases as described by the method of Tietz (1990). Exactly 1000 μL of the reagent was added to each of the sample and standard. This was then incubated for 10 min at 20°C–25°C after mixing and the absorbance of the sample (A sample) and standard (A standard) was measured against the reagent blank within 30 min at 546 nm. The value was expressed in mg/dL.
Determination of serum low-density lipoprotein cholesterol levels
The serum level of (LDL-C) was measured according to the protocol described by Friedewald et al. (1972). The value was expressed in mg/dL.
Determination of high-density lipoprotein cholesterol levels
The serum level of HDL was measured by the method of Wacnic and Albers (1978). LDL and very LDL (VLDL) and chylomicron fractions in the sample was precipitated quantitatively by addition of phosphotungstic acid in the presence of magnesium ions. The mixture was allowed to stand for 10 min at room temperature and centrifuged for 10 min at 2000 g. The supernatant represented the HDL fraction. The cholesterol concentration in the HDL fraction, which remained in the supernatant, was determined. The value was expressed in mg/dL.
Data obtained were analyzed using one-way analysis of variance, followed by Tukey's post-hoc test. Results were expressed as mean ± standard error of the mean. Values at P < 0.05 were considered statistically significant using? SPSS version 23.0. (Armonk, NY: IBM Corp, 2017).
| Results|| |
Effects of L-citrulline on fasting blood glucose level
On day 0 after induction (i.e., before commencement of treatment with L-citrulline), the FBG level of the rats was >200 mg/kg in all the groups, except in the normal control (90.5 ± 1.8 mg/dL). On day 22 after induction, FBG of the diabetic control remained significantly high (195.0 ± 2.4 mg/dL) compared to the normal control (88.0 ± 2.0 mg/dL) at P < 0.05. However, L-citrulline supplementation significantly lowered the FBG levels from 210.5 ± 3.9 mg/dL, 214.5 ± 7.6 mg/dL, and 201.3 ± 1.5 mg/dL on day 0, to 192.5 ± 3.4 mg/dL, 181.8 ± 1.2 mg/dL, and 174.8 ± 2.8 mg/dL on day 22, at 200 mg/kg, 400 mg/kg, and 800 mg/kg, respectively [Table 1].
|Table 1: Effect of L-citrulline on fasting blood glucose level in high-fat-diet - and dexamethasone-induced diabetic rats|
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The trend in the FBG levels varies over the 3-week duration of treatment across all the groups with the metformin group showing the greatest change in the 1st week (27.6% decrease). The changes in the L-citrulline groups were dose dependent over the 1st and 2nd week of treatment. Elevations in the FBG levels were seen in the diabetic (3.1%) and normal (12.4%) controls in the 1st and 2nd week of treatment, respectively. Slight elevations were recorded in the L-citrulline groups at 200 mg/kg (0.2%) and 800 mg/kg (0.5%) doses in the 3rd week. However, at the end of the treatment (day 22), L-citrulline at 400 mg/kg showed greatest change (lowering) in the FBG level (15.2%), second only to metformin (40.0%), and closely followed by L-citrulline at 800 mg/kg (13.2%) compared to the initial levels on day 0 of treatment [Table 1].
Effect of L-citrulline on lipid profile
The TC level was markedly elevated in the diabetic control (149.8 ± 2.7 mg/dL) compared to the normal control (86.8 ± 1.2 mg/dL) at the end of the treatment. Similar finding was also observed in the LDL and TG levels with a marked decrease in the HDL level (24.6 ± 1.1 mg/dL).
The level of TC was significantly lowered by L-citrulline in a dose-dependent manner with the greatest effect seen at 200 mg/kg (55.2 ± 0.6 mg/dL) (at 200 mg/kg) compared to the diabetic control and metformin (92.1 ± 1.5 mg/dL); F = 512.4; P < 0.001. Similarly, the TG levels were decreased significantly by L-citrulline at 200 mg/kg (6.8 ± 0.2 mg/dL), 400 mg/kg (13.2 ± 0.6 mg/dL), and 800 mg/kg (18.2 ± 0.9 mg/dL) compared to diabetic control (141.8 ± 2.3 mg/dL) and metformin group (35.7 ± 0.9 mg/dL); F = 1582.2; P < 0.0001.
The level of LDL was reduced following L-citrulline supplementation with the greatest effect seen 400 mg/kg (26.1 ± 1.0 mg/dL) compared to the diabetic control (85.3 ± 1.7 mg/dL) and metformin group (63.3 ± 0.9 mg/kg); F = 372.3; P < 0.0001. Similarly, there was a significant alteration in the levels of HDL by L-citrulline supplementation at all doses, with the greatest effect seen at 400 mg/kg (58.8 ± 0.5 mg/kg) compared to diabetic control (24.6 ± 1.1 mg/dL) but not to the metformin group (58.4 ± 0.8 mg/dL); F = 348.3; P < 0.001 [Table 2].
|Table 2: Effects of L-citrulline on lipid profile of high-fat-diet and dexamethasone-induced diabetic rats|
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The atherogenic ratios
The low-density lipoprotein
HDL ratio was significantly reduced by L-citrulline at 200 mg/kg (1.1 ± 0.0), 400 mg/kg (0.9 ± 0.0), and 800 mg/kg (1.2 ± 0.0) compared to the diabetic control (3.5 ± 0.1). There was no significant difference in the ratios compared to the metformin group (1.1 ± 0.0) compared to the diabetic control; F = 164.2; P < 0.0001 [Figure 1].
|Figure 1: Effect of L-citrulline on the LDL: HDL ratio in HFD- and dexamethasone-induced diabetic Wistar rats. CIT: Citrulline, Met: Metformin, HDL: High-density lipoprotein, LDL: Low-density lipoprotein. Superscriptsa, b ande denote statistically significant difference at P < 0.05 compared to normal control, diabetic control and CIT 400 mg/kg groups respectively|
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Similarly, the total cholesterol
HDL ratio was significantly higher in the diabetic group (6.1 ± 0.5) compared to the normal control (1.6 ± 0.0). However, L-citrulline, especially at 400 mg/kg markedly reduced this ratio (2.0 ± 0.2) compared to the diabetic control. The ratio in the metformin group (1.6 ± 0.1) was much smaller compared to those of the L-citrulline groups; F = 54.4; P < 0.0001 [Figure 2].
|Figure 2: Effect of L-citrulline on the TC: HDL ratio in HFD- and dexamethasone-induced diabetic Wistar rats. CIT: Citrulline, Met: Metformin, TC: Total cholesterol, HDL: High-density lipoprotein, LDL: Low-density lipoprotein. Superscriptsa, b, andc denote statistically significant difference at P < 0.05 compared to normal control, diabetic control and Met 100 mg/kg groups, respectively|
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The atherogenic indices
The atherogenic index (calculated as [TC-HDL]/HDL) in the diabetic control was significantly higher (5.1 ± 0.1) compared to the normal control (0.6 ± 0.0). This was markedly reduced by L-citrulline supplementation especially at 400 mg (1.0 ± 0.0) compared to the diabetic control and again, the index in the metformin group was much lower (0.6 ± 0.2) compared to all the L-citrulline groups; F = 54.4; P < 0.0001 [Figure 3].
|Figure 3: Effect of L-citrulline on the atherogenic indices in HFD- and dexamethasone-induced diabetic Wistar rats. CIT: Citrulline, Met: Metformin, TC: Total cholesterol, HDL: High-density lipoprotein, LDL: Low-density lipoprotein. Superscriptsa, b andc denote statistically significant difference at P < 0.05 compared to normal control, diabetic control and Met 100 mg/kg groups respectively. It is calculated as (TC-HDL)/HDL|
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| Discussion|| |
At the end of the treatment (day 22), L-citrulline at all doses employed in this study, and in a dose-dependent manner, significantly lowered the FBG level compared to the diabetic control. Although not measured in this study, L-citrulline could produce such effect due to its ability to increase hepatic and peripheral sensitivity to insulin., However, metformin, which inhibits hepatic glucose release and increases peripheral insulin sensitivity, showed a greater effect (suggesting greater efficacy) over the 3-week duration of treatment, compared to the L-citrulline. It also showed the greatest change (lowering) in the FBG level within the 1st week and at the end of the treatment.
There was a dose-dependent change (lowering) in the FBG level by L-citrulline within the 1st week of treatment but at the end of the treatment, the greatest change was noted at the 400 mg/kg. This shows that the optimal dose for antidiabetic effect of L-citrulline is 400 mg/kg. Before the commencement of the L-citrulline supplementation (day 0), the FBG level was lowest in the CIT 800 mg/kg group hence, not surprising that the level was also the lowest after the treatment (i.e., day 22), even though the reduction in the FBG level was dose dependent.
Both metformin and L-citrulline at all doses employed in this study showed a classical of their effect on the FBG level over the 3-week duration of treatment, with the L-citrulline at 200 mg/kg and 800 mg/kg showing a mild but unexpected elevation in the FBG level in the 3rd week of treatment. This might have been the reason why the 400 mg/kg was optimal in lowering the FBG level. The anti-hyperglycemic effect of L-citrulline has also been well documented.
Dyslipidemia, characterized by high levels of TC, TGs, and LDL cholesterol and low HDL level, has been a consistent finding in the setting of T2DM as revealed by several studies.,,,,, From our findings, there were significant derangements in the lipid profile of the diabetic control compared to the normal control, suggesting abnormalities in lipid metabolism typically seen in T2DM which might be due to abnormality in glucose metabolism (hyperglycemia) recorded in this study and supported by other studies.,,,, These changes, especially the elevated LDL level are often pro-atherogenic. Patients with T2DM are at a significantly higher risk of developing a major cardiovascular event compared to nondiabetic individuals and this risk can be significantly reduced with lipid-lowering drugs.,,
L-Citrulline, in the present study, had shown a remarkable improvement in the dyslipidemic changes noted in the diabetic control, suggesting a potent antidyslipidemic effect of L-citrulline. Although not measured in this study, L-citrulline might have down-regulated the production of VLDL, a major contributor in the dyslipidemia seen in T2DM, or improved the sensitivity of the adipose tissue and liver to insulin,, thereby decreasing the synthesis of free fatty acids (FFAs) which are used by the liver to increase secretion of VLDL and TGs. L-citrulline can also decrease HDL catabolism through its inhibition of hepatic lipase, thereby increasing the HDL level. In addition, L-citrulline might have enhanced the reverse cholesterol transport by the HDL (i.e., the cholesterol efflux capacity of the HDL), which is impaired in T2DM, leading to high cholesterol level, and increase the risk of atherosclerosis.
Although the effects of L-citrulline on lipid profile especially the TC and TGs, have been reported to be inconsistent, our findings showed a significant lowering of the TC and TG levels by L-citrulline, in a dose-dependent manner. However, this was surprisingly in an “inversely” dose-dependent manner and the mechanism behind this is not clear. Moreover, L-citrulline at 400 mg/kg markedly reduced the LDL and elevated the HDL levels compared to the other doses. This suggests that the optimal dose for the antidyslipidemic effect of L-citrulline may be between 200 and 400 mg/kg.
The LDL: HDL and TC: HDL ratios as well as the atherogenic index (calculated as [TC-HDL]/HDL) showed a similar pattern (reduction) across the metformin and L-citrulline groups. In the diabetic group, both ratios and index were significantly higher compared to the normal control. This is in line with the findings of Ali et al., who found markedly increased in these ratios among diabetic atherosclerotic patients but were significantly reduced in T2DM patients on atorvastatin in addition to metformin. Again, L-Citrulline supplementation at 400 mg/kg, was able to optimally and significantly reduced these ratios and index in our study, suggesting the antidyslipidemic effectiveness of L-citrulline at 400 mg/kg.
As expected, metformin also markedly reduced the TC, LDL, and TGs levels and increased the HDL level from the present study. This is in line with the findings of other studies,,,, noting that antidiabetic therapies including metformin, are also capable of slowing down the progression of atherosclerosis due to dyslipidemia, which contributes greatly to increased risk of cardiovascular disease and events in T2DM.,,,,,
Dyslipidemia, among other factors such as high HbAIC level, duration of disease, obesity, age and sex, contributes greatly to the development of cardiovascular disease and events in T2DM,,,,, and even with good glycemic control, sometimes the lipid derangements persist., Hence, an antidyslipidemic therapy in addition to the antidiabetic medication is highly recommended in T2DM. It has been recommended that adult diabetic patients do check their lipid profile at the time of diagnosis, and monitor it at least once every 5 years thereafter and at least once yearly when on lipid lowering medications.
| Conclusion|| |
The present study revealed that L-citrulline supplementation, especially at 400 mg/kg possesses a potent antidiabetic and antidyslipidemic effects in the diabetic Wistar rats. This shows a great potential in reversing the micro- as well as macro-vascular complications associated with T2DM. Where the conventional antidiabetic drugs fail, L-citrulline may be a good substitute.
Limitation of the study
The levels of NO or its metabolite, nitrite, and the eNOS expression were not assessed in this study. The mechanisms by which L-citrulline lowers the blood sugar and corrects dyslipidemia need to be explored further and other parameters such as insulin level or expression, HOMA-IR, HbA1C, VLDL, and FFAs which were not measured in this study may be considered in future studies. The interaction between L-citrulline and drugs such as antidiabetic, antidyslipidemic, antihypertensive, and other drugs can be explored too.
Financial support and sponsorship
This research work was carried out through personal funding.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]