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Year : 2021  |  Volume : 9  |  Issue : 2  |  Page : 95-102

Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus)

1 Department of Human Physiology, College of Medical Sciences, Ahmadu Bello University Zaria, Kaduna, Nigeria
2 Department of Human Physiology, College of Medicine, Kaduna State University, Kaduna, Nigeria
3 Department of Human Anatomy, College of Medicine, Kaduna State University, Kaduna, Nigeria

Date of Submission27-Feb-2021
Date of Decision11-May-2021
Date of Acceptance13-May-2021
Date of Web Publication10-Aug-2021

Correspondence Address:
Dr. Timothy Danboyi
Department of Human Physiology, Kaduna State University, Kaduna
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njecp.njecp_4_21

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Background: Oxidative stress is one of the major mechanisms underlying the onset and development of type-2 diabetes mellitus (T2DM). Although L-citrulline possesses antioxidant effect, little or no data exist linking such effects in diabetic setting. Objective: This study aimed to evaluate the effect of L-citrulline on biomarkers of oxidative stress in diabetic Wistar rats. Materials and Methods: Thirty male Wistar rats 10–12 weeks old and weighing 200–250 g were randomly assigned into six groups of five rats each. Group I rats were fed normal diet, while diabetes was induced in the other groups with high-fat diet (HFD) and dexamethasone intraperitoneally (1 mg/kg) for 21 days. Thereafter, Group III received metformin 100 mg/kg/day orally, and Groups IV, V, and VI received 200, 400, and 800 mg/kg/day L-citrulline, respectively, for another 21 days. Data were analyzed using SPSS and values at P < 0.05 were considered statistically significant. Results: The malondialdehyde concentrations were significantly reversed from 42.0 ± 0.42 μmol/mL in the diabetic group to 20.7 ± 0.81, 22.2 ± 0.75 and 22.1 ± 0.39 μmol/mL at 200, 400, and 800 mg/kg/day, respectively. The L-citrulline remarkably ameliorated the reduction in superoxide dismutase activity noted in the diabetic group (13.0 ± 0.44 μmol/mL) at all doses (17.8 ± 0.37, 16.0 ± 0.51, and 23.7 ± 0.78 μmol/mL at 200, 400, and 800 mg/kg, respectively). Similarly, there was a corresponding significant increase in the catalase activity, especially at 400 mg/kg (13.7 ± 0.43 ng/mL) and 800 mg/kg (14.6 ± 0.54 ng/mL) compared to the diabetic group (10.8 ± 0.41 ng/mL). The marked reduction in reduced glutathione level observed in the diabetic group (22.9 ± 0.69 mg/mL) was markedly ameliorated by L-citrulline supplementation at all doses (42.9 ± 1.08, 46.4 ± 0.53, and 45.2 ± 1.00 mg/mL at 200, 400, and 800 mg/kg, respectively). Conclusion: This study shows that L-citrulline supplementation has antioxidant effects in HFD- and dexamethasone-induced T2DM in male Wistar rats.

Keywords: Antioxidant, L-citrulline, oxidative stress, type-2 diabetes mellitus

How to cite this article:
Hassan-Danboyi E, Jimoh A, Alhassan A, Danboyi T, Mohammed KA, Dubo AB, Haruna J, Yakubu BB. Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus). Niger J Exp Clin Biosci 2021;9:95-102

How to cite this URL:
Hassan-Danboyi E, Jimoh A, Alhassan A, Danboyi T, Mohammed KA, Dubo AB, Haruna J, Yakubu BB. Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus). Niger J Exp Clin Biosci [serial online] 2021 [cited 2022 Jun 26];9:95-102. Available from: https://www.njecbonline.org/text.asp?2021/9/2/95/323670

  Introduction Top

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia (high blood glucose level) resulting from defects in insulin secretion, insulin action, or both.[1] About 422 million people had DM as of 2014.[2] DM had an estimated global prevalence of about 8.8% in 2015 with a projected rise of about 10.4% by 2040 (affecting an estimated 642 million people).[3] In sub-Saharan Africa, about 20 million people (projected to be 41.4 million by 2035) have DM, with Nigeria having the highest number (5 million) of people with DM.[4] DM is divided into two major types: type-1 (insulin-dependent and associated with absolute insulin deficiency) and type-2 (90%–95% of DM, due to insulin resistance).[1]

High-fat diets (HFDs) had been shown to increase serum glucose levels as well as insulin resistance in mice[5] and rats,[6] thereby contributing to the onset and progression of type-2 DM (T2DM). It is associated with increase in determine malondialdehyde (MDA) concentration and decrease in the activities of antioxidant enzymes and reduced glutathione (GSH) level in some tissues.[7] HFD is a model of T2DM,[6] and it is an established “side effect” of glucocorticoid therapy.[8] Exogenous glucocorticoids such as dexamethasone cause insulin resistance and hyperglycemia, which may lead to the development of T2DM.[9],[10],[11] Dexamethasone causes insulin resistance via direct impairment of beta-cell functions, decreasing key mediators of insulin action in peripheral tissues and anti-insulin effects on the liver, skeletal muscle, and adipose tissue.[10],[11],[12] In our previous study, we demonstrated that combining both HFD and dexamethasone induces diabetes in albino Wistar rats.[13]

The conventional antidiabetic drugs have been unable to halt the progression of T2DM, and coupled with their high cost as well as their associated adverse effects, the management has been a great challenge to both clinicians and patients or their relatives.[14] In addition, most of these drugs may cause serious side effects such as severe hypoglycemia, and adverse drug–drug interactions.[15] Hence, it is only logical to seek newer and (more) effective antidiabetic agents or supplements, with little or no side effects that can gain universal acceptance.

Oxidative stress is an imbalance between the production and clearance of free radicals (reactive oxygen and/or nitrogen species) with a potential damage to nucleic and cellular components.[16] Oxidative stress occurs when there is an imbalance between the body's pro-oxidant system and antioxidant defense system in favor of the former, leading to the generation of excessive amount of free radicals that attack and damage biological membranes causing lipid peroxidation, protein glycation, and nucleic acid fragmentation.[17] It leads to insulin resistance, beta-cell dysfunction, and impaired glucose tolerance and plays a significant role in the development and progression of T2DM together with its attendant complications.[16],[18],[19]

L-citrulline derives its name from Citrullus lanatus as one of the major components of watermelon.[20] L-citrulline is a nonessential amino acid mainly produced by the liver.[21] It prevents the excessive and uncontrolled production of nitric oxide (NO).[22],[23] It also augments the functions of NO,[24] which include maintenance of normal endothelial function[25],[26] and increase in peripheral and hepatic insulin sensitivity,[27],[28] which are disrupted in the setting of T2DM.[29],[30] L-citrulline may be a potential supplement in the treatment of T2DM and prevention of complications associated with it.[22],[25],[31],[32]

L-citrulline bypasses hepatic and intestinal metabolism,[26],[33] and enhances plasma L-citrulline concentration, hence a better substrate than arginine in restoring NO production.[34] It has no side effects even at high doses when taken orally.[24],[26],[35] It is affordable and readily available, especially in its natural source (C. lanatus). It possesses an antioxidant activity as it significantly decreases reactive oxygen species (ROS) production.[36] However, only very few studies exist on its effects on biomarkers of oxidative stress, especially in the setting of T2DM. This study aimed to investigate the effect of L-citrulline on biomarkers of oxidative stress in the setting of T2DM in male Wistar rats. We, therefore, hypothesized that L-citrulline has no significant effect on some biomarkers of oxidative stress in T2DM.

  Materials and Methods Top

Ethical clearance

This study was carried out according to the guidelines for the care and use of laboratory animals provided and approved by the Ahmadu Bello University (ABU) Committee on Animal Use and Care (approval number: ABUCAUC/2020/72).


Materials used were 1 ml, 2 ml, and 5 ml disposable syringes; distilled water, Accu-Chek advantage glucometer and strips (Roche Diagnostics, Germany); electronic weighing balance, surgical blades, cotton wool, methylated spirit, ketamine, plain tubes, ice packs, masking tape, and marker. Others include commercial grower feeds (Chikun Feeds Company, Nigeria), distilled water, dissecting board, latex gloves, bench centrifuge, and feeding bowls. Dexamethasone injection and L-citrulline oral supplement (MedChemXpress, USA) were of analytical grade.

Experimental animals and housing conditions

A total of 30 male albino Wistar rats (Rattus norvegicus) 8–12 weeks old and weighing 200–250 g were used due to their genetic, biological, and behavioral similarities with humans and are the most commonly used strains in metabolic and other research. They were sourced from the animal house, Department of Human Physiology, ABU, Zaria. They were housed in plastic cages with commercial bedding materials which were changed every other day and allowed free access to commercial grower mash feed and water under standard laboratory conditions (temperature- and humidity controlled with 12-h light-dark cycle and well ventilated). There were 5–10 rats per cage depending on the grouping. They were all drug-and test-naïve and certified healthy.

Preparation of high-fat diet

This study was part of a multistage research using the same experimental design. HFD was prepared by mixing normal feed (crude fats: 18%, crude proteins: 47%, carbohydrates: 28%, crude fiber: 5%, methionine: 0.5%, lysine: 1.1%, and phosphorus: 0.4%) with margarine (99.9% fats) in the ratio of 10:1, i.e., 10 g of normal feed to 1 g margarine[37] with some modifications.

Drug formulation, dosage, and route of administration

The L-citrulline was constituted with distilled water at 100 mg citrulline/ml distilled water. It was administered orally because of its water solubility, ready absorbability, and by-pass of the first-pass effect following oral ingestion. The dexamethasone was constituted with distilled water at 2 mg/ml. It was administered intraperitoneally (i.p.) at a dose of 1 mg/kg. The rats received the L-citrulline and dexamethasone in the mornings after their breakfast at a well-ventilated area within the laboratory, where they were kept for about 1–2 h after the administration before returning them to their cubicles. The remaining formulated drugs after administration were returned to the refrigerator maintained at 2°C–5°C.

Induction of type-2 diabetes mellitus

The rats were grouped into six groups (I–VI), but those in Groups II–IV were fed with HFD for 21 days in addition to dexamethasone 1 mg/kg body weight i.p. daily starting from day 15 to 21 of the experiment according to a previously described method[38] with some modifications.

Determination of fasting blood glucose levels

Fasting blood glucose (FBG) levels were measured (after overnight fasting for 8 h) using the glucometer and strips and expressed in mg/dL prior to induction of diabetes. It is according to the glucose oxidase principle.[39] The measurement of the FBG levels took place on day 0 (after induction of diabetes) and day 22. A drop of blood obtained from slightly piercing the tail veins was placed on the glucometer strip. Blood glucose levels >200 mg/dL were considered diabetic.[40],[41]

Experimental animal grouping

The study involved 30 male Wistar rats randomly assigned into six groups of five rats each.

  • Group I (n = 5): received normal diet for 21 days. This group serves as normal control
  • Group II (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight[38] daily for 7 days (starting from day 15). This group serves as diabetic group
  • Group III (n = 5): 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[42]
  • Group IV (n = 5): 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[43] daily for 21 days
  • Group V (n = 5): 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[43]
  • Group VI (n = 5): 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.[43]

Collection of blood samples

The rats were anesthetized using 75 mg/kg ketamine and 50 mg/kg diazepam i.p. Two milliliter of blood was drawn from each rat via cardiac puncture using a 5 mL syringe. Blood collected was allowed to stand and clot for 30 min and 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 ready for analysis.

Assay for biomarkers of oxidative stress

The centrifuged blood samples were used to determine MDA concentrations; activities of superoxide dismutase (SOD) and catalase (CAT); and GSH levels.

Determination of malondialdehyde concentrations

The level of thiobarbituric-acid reactive substance (TBARS), MDA concentrations, as an index of lipid peroxidation was determined. The principle is based on the reaction of MDA with thiobarbituric acid (TBA), forming an MDA-TBA or TBARS adduct that will absorb strongly at 532 nm. It was based on the colorimetric method described by Satoh.[44] Exactly 150 μl of serum was treated with 2 ml of TBA-tricarboxylic acid-hydrochloric acid reagent (1:1:1 ratio) and placed in a water bath at 90°C for 60 min. The mixture was cooled and centrifuged at 3000 rpm for 5 min and the absorbance of the pink supernatant (TBA-MDA complex) was then measured at 535 nm. The MDA formed was then calculated using the molar extinction coefficient of 1.56 × 10−5 cm−1 M−1.

Determination of superoxide dismutase activity

Activities of SOD in the rat serum were determined according to the method described by Misra and Fridovich.[45] This is based on the principle of superoxide inhibition of auto-oxidation of adrenaline at pH 10.2. Serum of 0.1 ml was diluted in 0.9 ml of distilled water to make 1:10 dilution of microsome. An aliquant mixture of 0.2 ml of the diluted microsome was added to 2.5 ml of 0.05 M carbonate buffer. The reaction was started with the addition of 0.3 ml of 0.3 mM adrenaline. The reference mixture contained 2.5 ml of 0.05 M carbonate buffer, 0.3 ml of 0.3 mM adrenaline, and 0.2 ml of distilled water. The absorbance was measured over 30 s up to 150 s at 480 nm.

Determination of catalase activity

CAT activity was measured according to the method described by Aebi.[46] Exactly 100 μl of serum was added to a test tube containing 2.80 ml of 50 mM potassium phosphate buffer (pH 7.0). The reaction was initiated by adding 1 ml of freshly prepared 30 mM H2O2 and the decomposition rate of hydrogen peroxide (H2O2) was measured at 240 nm for 5 min on a spectrophotometer. A molar extinction coefficient (E) of 0.041 mM−1 mm−1 was used to calculate the CAT activity. One unit is the amount of CAT that decomposes 1 μmol of H2O2 per minute at pH 7.0.

Determination of reduced glutathione level

GSH concentration measurement was done according to the method described by Rukkumani et al.[47] The procedure was based on the reaction of 5, 5-dithiobis nitro benzoic acid and GSH. To 150 μl of serum or tissue homogenate (in phosphate-saline buffer pH 7.4), 1.5 ml of 10% TCA was added and centrifuged at 1500 g for 5 min. One milliliter of the supernatant was treated with 0.5 ml of Ellman's reagent and 3 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. The quantity of GSH was obtained from the graph of the GSH standard curve.

Statistical analysis

Data obtained were analyzed using International Business Machine (IBM) Statistical Package for Social Sciences (SPSS) Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp., United States of America, 2015), using one-way analysis of variance, followed by Tukey's post hoc test. The format of result presentation was mean ± standard error of the mean. Any value at P < 0.05 is statistically significant.

  Results Top

Serum malondialdehyde concentrations

There was a significant increase in the MDA concentrations observed in the diabetic group (42.0 ± 0.42 μmol/mL) compared to the normal control (30.7 ± 0.78 μmol/mL). Marked decreases were recorded in the citrulline 200 mg/kg (20.7 ± 0.81 μmol/mL), citrulline 400 mg/kg (22.2 ± 0.75 μmol/mL) and citrulline 800 mg/kg (22.1 ± 0.39 μmol/mL), compared to the diabetic group as well as metformin group (25.6 ± 0.56 μmol/mL). F =160.2; P < 0.0001 [Figure 1].
Figure 1: Effect of L-citrulline on serum malondialdehyde concentrations of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, and c denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, and metformin group, respectively

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Level of serum superoxide dismutase activity

There was a marked decrease in SOD activity in the diabetic group (13.0 ± 0.44 μmol/mL) compared to the normal control (18.4 ± 0.54 μmol/mL). The activity in the citrulline 400 mg/kg group (16.0 ± 0.51 μmol/mL) was significantly increased compared to the diabetic group and decreased compared to metformin (19.5 ± 0.89 μmol/mL) and citrulline 800 mg/kg groups (23.7 ± 0.78 μmol/mL) (F = 33.7; P < 0.0001) [Figure 2].
Figure 2: Effect of L-citrulline on level of serum superoxide dismutase activity of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, c, d, and e denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, metformin, CIT 200 mg/kg, and CIT 400 mg/kg groups, respectively

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Level of serum catalase activity

The CAT activity was significantly increased in the citrulline 400 mg/kg (13.7 ± 0.43 ng/mL) and citrulline 800 mg/kg (14.6 ± 0.54 ng/mL) groups, compared to the diabetic (10.8 ± 0.41 ng/mL) and metformin (11.0 ± 0.69 ng/mL) groups (F = 14.6; P < 0.0001) [Figure 3].
Figure 3: Effect of L-citrulline on level of serum CAT activity of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, c, and d denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, metformin, and CIT 200 mg/kg groups, respectively

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Serum-reduced glutathione level

There was a significant reduction in the level of GSH seen in the diabetic group (22.9 ± 0.69 mg/mL) compared to the normal control (28.3 ± 1.17 mg/mL). There was a significant increase seen in the metformin (39.7 ± 0.69 mg/mL), citrulline 200 mg/kg (42.9 ± 1.08 mg/mL), citrulline 400 mg (46.4 ± 0.53 mg/mL), and citrulline 800 mg (45.2 ± 1.0 mg/mL) groups (F = 118.2; P < 0.0001) [Figure 4].
Figure 4: Effect of L-citrulline on serum reduced glutathione level of high-fat diet- and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, and c denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, and metformin group respectively

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  Discussion Top

All doses of L-citrulline employed in this study were able to lower the FBG levels on day 22 compared to the diabetic control, with the greatest effect observed at 400 mg/kg. This hypoglycemic effect of L-citrulline is probably due to its ability to improve peripheral and hepatic insulin sensitivity.[27],[28] However, in a study among overweight and obese individuals, a daily intake of watermelon (the main natural source of L-citrulline) over a period of 4 weeks did not produce any significant change in the blood glucose level[20] compared to the baseline. Metformin, one of the most commonly used hypoglycemic medications,[16] known to ameliorate hepatic and peripheral insulin resistance,[48] and improved endothelial dysfunction in type-2 diabetes,[49] had shown a superior effect compared to L-citrulline in the present study.

There was a marked increase in the MDA concentration, a biomarker for lipid peroxidation in the diabetic control group compared to the normal control in our study. A study reported a marked increase in MDA level among diabetic patients compared to the nondiabetic control.[50] Nevertheless, L-citrulline was able to ameliorate this in a dose-dependent pattern, suggesting a protective effect. In a study by Liu et al.,[51] L-citrulline was able to protect against glycerol-induced oxidative damage characterized by significant dose-dependent decreases in MDA concentrations in rats. Similarly, Lum et al.[20] demonstrated a significant decrease in the MDA concentration after 4 weeks of watermelon consumption compared to the baseline in overweight and obese individuals.

In the present study, there were decreases in SOD activity and GSH level in the diabetic group compared to the normal control, which is probably due to excessive generation of free radicals leading to oxidative/nitrosative stress (though not measured in this study). As the SOD was consumed in the diabetic group, less amount of hydrogen peroxide (H2O2) is expected to be produced, which may explain the slight increase in CAT activity in the diabetic group. The significant decrease in GSH level is probably due to its depletion while scavenging free radicals that were likely generated. Yarube and Gwarzo[50] had also demonstrated such decreased antioxidant capacity among diabetic patients compared to their nondiabetic counterparts.

The supplementation with L-citrulline was able to augment the endogenous antioxidant enzymes (SOD and CAT) activities and level of GSH at all doses from this study. This is in line with the findings of Oseni et al.[52] that demonstrated a significant increase in GSH level and SOD activity with a mild increase in CAT activity after a week of administration of watermelon crude extract containing a large amount of L-citrulline. It may be due to the sparing of the endogenous antioxidant enzymes by the L-citrulline, especially at 800 mg/kg. As the activity of SOD increases (probably to scavenge the excessive superoxide ion generated), a large amount of H2O2 is produced which might have triggered the increase in the CAT activity (to scavenge the H2O2). This is necessary because, in the presence of reduced metals such as ferrous iron (Fe2+), H2O2 can be converted by the Fenton reaction into the highly reactive hydroxyl radical (OH), one of the most harmful of all ROS.[53] To further support the antioxidant efficacy of L-citrulline, Liu et al.[51] also found a significant increase in SOD activity and GSH level in rats when supplemented with L-citrulline.

Endogenous GSH can augment the effect of oral L-citrulline on NO synthesis, protecting against the oxidative reactions of NO by increasing the activity of NO synthase in rodents and humans.[43] GSH plays a significant role in intracellular defense against oxidative stress.[54] In the present study, there was a marked reduction in the GSH level in the diabetic group. This is in line with some studies,[55],[56] which reported reduced synthesis and level in patients with uncontrolled diabetes. However, with L-citrulline supplementation at all doses, there was a significant increase in the GSH level. This is probably due to sparing of the GSH by L-citrulline in the rats. In addition, L-citrulline might enhance the synthesis of cysteine and glycine, which are amino acid precursors for GSH synthesis.[55]

L-citrulline supplementation, from our finding, had shown a superior effect compared to metformin in ameliorating the oxidative stress noted in the diabetic group. Although not measured in this study, L-citrulline could exert such effects via its ability to significantly decrease ROS formation[36] and at the same time, regulate the production and metabolism of NO,[22] preventing its excessive and uncontrolled production.[23]

Some studies showed an improvement in antioxidant status and vascular function by watermelon consumption, preventing oxidative stress.[57],[58] It causes such effects via the production of NO[58] that has a vasodilator effect and prevents oxidative stress by scavenging hydroxyl radicals.[59] These results show that L-citrulline has a great potential in the treatment of cardiovascular diseases and diabetic complications with endothelial dysfunction as the underlying mechanism.[33],[36]

  Conclusion Top

The present study revealed that L-citrulline possesses antioxidant effects in diabetic rats in an almost dose-dependent manner. This shows a great potential in reversing the vascular complications as well as halting the progression of T2DM. Where the conventional antidiabetic drugs fail, L-citrulline may be a good alternative.

Limitations of the study

The levels of NO, endothelial NO synthase expression, ROS such as superoxide, and reactive nitrogen species such as peroxynitrite were not assessed in this study. NO may have oxidant effects when combined with superoxide (in the presence of oxidative stress), forming a reactive NO and other reactive nitrogen species. In type-2 diabetes, in which there is an enhanced production of superoxide, the superoxide-NO interaction may reduce flow-mediated dilation of the coronary arterioles which may contribute to the development of coronary artery disease. The question is could L-citrulline alter such interaction? Moreover, could diabetic angiopathy (the main cause of morbidity and mortality in DM), which is caused by endothelial dysfunction[30],[60],[61] be due to such interaction? Tissues such as the liver, kidneys, brain, and heart and adipose tissues that are involved in glucose metabolism are also potential areas to explore in further studies, especially the effect of L-citrulline on the tissue markers for oxidative stress.

Furthermore, the mechanism(s) by which L-citrulline ameliorate the biomarkers of oxidative stress in the diabetic setting need is not explored. Other parameters such as serum insulin level or expression, HOMA-IR, and HbA1C not measured in this study are the next targets in future studies.


The authors would like to thank all staff of the Department of Physiology, College of Medical Sciences, Ahmadu Bello University, Zaria, who contributed to the success of this work.

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], [Figure 4]


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