Sglt-2 Inhibitors Prevent Formation of Malondialdehyde and Scavenge Nitric Oxide Radicals in Vitro


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Canagliflozin, dapagliflozin and empagliflozin are the three phlorizin derived drugs recently approved for the treatment of type 2 diabetes mellitus. These drugs selectively inhibit SGLT-2 in kidney without affecting the intestinal SGLT-1. SGLT-2, the main reabsorption transporter of glucose in nephron, if inhibited, is likely to cause an increase in urinary glucose excretion in individuals with type 2 diabetes mellitus. The lower plasma glucose and greater urinary glucose excretion leads to a net loss of calories and weight loss and also the increased glucose in urine produce a mild osmotic diuresis that may contribute to reduction in systolic blood pressure.

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Sharpe et al., in 1998 reported oxidative stress in several human cell lines including endothelial cells under hyperglycaemic condition1. Not only O2- synthesis, hyperglycaemia also stimulate nitric oxide synthesis by increased enzymatic activity. The nitric oxide radical generated in diabetic vasculature is scavenged by O2- to form peroxynitrite [OONO-]. The reaction rate 6.7×109 ms-1 which is 3 times faster than the reaction time between O2- and SOD. Formation of peroxynitrite thus involves neutralization of potentially deleterious O2- on one hand and on the other hand, consumption of potent vasodilator nitric oxide. Peroxynitrite cause insulin secretin, cell death of human and rat islets of Langerhans, DNA damage. Thus, it acts as a double-edged sword. In electron transport chain, the electron generated during ATP production is utilized in the conversion of molecular oxygen to water by reduction at mitochondrial complex IV. If this oxygen is partially reduced, it generates highly reactive free radical O2-. This also occurs at complex I and complex III. However, in hyperglycaemic condition, this site of free radical generation is altered.

Evidences of oxidative stress have been found even in the cells lacking NADPH oxidase and mitochondria. So, there must be some other system(s) participating in ROS production. This is glucose auto-oxidation. Glucose and many metabolites of glucose are capable of reacting with cellular hydrogen peroxide in presence of Fe2+, Cu2+ and some alike transition metals to generate the most potent ROS, hydroxyl radicals ( OH)2. Non-enzymatic glycation, a process of interaction of glucose and protein yields some intermediate Amadori and Sciff base products before generation of advanced glycosylation end-products. Moreover, in hyperglycaemic condition, excess glucose is diverted to hexosamine and sorbitol pathways where ROS is generated during glucose metabolism.

In type 2 diabetes mellitus, increased oxidative modification of plasma lipoprotein leads to increased peroxide lipid and lysophosphatidylcholine4 and the peroxide lipid is accumulated in cardiovascular tissue causing macrovascular damage5. Diminished expression of nitric oxide synthase and generation of NO2, impaired expression of SOD, reduced level of antioxidant glutathione, α-tocopherol, ascorbate, hyperactivity of sorbitol pathway, enhanced protein glycosylation and AGE formation works all together in vascular endothelium to increase oxidative stress. Normal vascular endothelial functions are altered as a result and this is one of the pathogenic factor of vascular complications in several disease states including diabetes mellitus.

The current study was focused on the investigation of inhibition of production of malondialdehyde, the mediator of lipid peroxidation by the three phlorizin derived drugs viz. canagliflozin, dapagliflozin and empagliflozin and their efficacy in scavenging nitric oxide radicals in vitro. The in vitro assessment of lipid peroxidation and nitric oxide radical scavenging assay was followed by confirmatory antioxidant activity assay by DPPH radical scavenging assay and ferric ion reduction by FRAP.

Materials and Methods

Materials- All the chemicals used were analytical grade. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Himedia laboratories, Mumbai, India. L-ascorbic acid, methanol, sulphanilamide, N-(1-naphthyl)-ethyl-enediamine dihydrochloride, orthophosphoric acid, sodium, ethanol, hydrochloric acid, potassium ferrocyanide, ferric chloride, sodium dodecyl sulphate, ammonium molybdate, disodium hydrogen phosphate dihydrate, sulphuric acid, ferrous sulphate heptahydrate, glacial acetic acid, trichloroacetic acid, thiobarbituric acid and butan-1-ol were purchased from Merck, Mumbai, India.


DPPH Radical Scavenging Assay

The DPPH scavenging assay was performed for all the three drugs at concentration 50-800μg/ml against reference compound ascorbic acid by previously described method6 with some modifications. The drugs were solubilized in suitable solvents with vortexing for five minutes or until complete solubilization. 50μL of the drug solutions of each concentration were taken in a test tube followed by addition of methanol up to 3 ml. 150μL of 0.13% w/v solution of DPPH in methanol was then added in each test tubes and sufficient time was provided for the antioxidant action to take place. The absorbance of the solution was measured with UV-visible spectrophotometer (Schimadzu UV-1800) at 517 nm. The decrease in absorbance upon increasing drug concentration implicates more scavenging of free radicals. The % inhibition was calculated from the formula- % inhibition = (Abs of control-Abs of test)/(Abs of Control)×100

Ferric Ion Reduction by FRAP

The method of Prieto et al7 was followed with some modifications. The principle behind this test is the ability of antioxidant to reduce Fe3+ to Fe2+ by electron donation. To the test tubes containing 100μL drug solution with concentration ranging from 50-800μL. Three ml deionized water were added along with 90μl of 95% ethanol. In the next step, addition of each of 150μL of 1M hydrochloric acid and 1% w/v potassium ferrocyanide was followed by addition of 50μL of 1% sodium dodecyl sulphate. Finally, 20μL of 0.2% ferric chloride was added which imparts Prussian blue colour. For proper distribution of colour, all test tubes were vortexed for 15 minutes. The absorbance was measured using UV-visible spectrophotometer (Schimadzu UV 1800) at 700 nm. The antioxidant activity of the drugs was evaluated taking ascorbic acid as standard.

Assessment of Inhibition of Lipid Peroxidation

Thiobarbituric Acid Reactive Substances (TBARS) method as described by Roberto et al8 was adopted with slight modification for the assessment of extent of inhibition of lipid peroxidation using egg yolk homogenate as lipid rich media. 500μL of 10% egg yolk homogenate was added to tubes containing 100μL of drug solution of concentration ranging from 50-800μg/ml. The volume of the tubes was made 1 ml with deionized water. 50μL of 0.07M ferrous sulphate was added to all the tubes and incubated for 20 minutes to induce lipid oxidation. Then 1.5 ml of each of 20% acetic acid and 0.8% w/v thiobarbituric acid prepared in 1.1% sodium dodecyl sulphate were added to all tubes followed by addition of 50μL of 20% w/v trichloroacetic acid. Tubes were then vortexed and heated for 1 hour at 95° C. After cooling to room temperature, 5 ml of n-butanol was added to each test tubes and centrifuged at 5000 rpm for 5 minutes. The organic upper layer was collected and absorbance was measured of each sample at 532 nm. The antioxidant capacity of the drugs was calculated as % inhibition taking ascorbic acid as standard.

Determination of Nitric Oxide (NO) Scavenging Assay

The method of Bajpai et al.,9 was followed for the determination of nitric oxide radical scavenging potential. 5mM Sodium nitroprusside (SNP) in phosphate buffer saline pH 7.4 was used to generate NO which intermingles with oxygen to generate nitrite ions. Scavenging of the nitrite ions was assessed with Griess reagent (1% w/v sulphanilamide, 5% w/v phosphoric acid, 0.1% w/v N-(1-naphthyl)-ethyl-enediamine dihydrochloride). 1ml of sodium nitroprusside was added to each drug concentration ranging from 50-800μg/ml followed by incubation at 25 ̊C at light for 60 minutes and addition of equal volume of Griess reagent and incubation at same temperature at dark condition for 30 minutes. The diazotization of nitrite ions with sulphanilamide and subsequent coupling reaction with N-(1-naphthyl)-ethyl-enediamine dihydrochloride generates a pink chromophore, absorbance of which was measured at 546 nm. The % inhibition was calculated using the same formula as used in DPPH radical scavenging assay process.


Determination of DPPH Radical Scavenging Assay

A dose dependent DPPH radical scavenging activity was shown by ascorbic acid and all the gliflozin analogues. At 800 µg/ml concentration, extent of DPPH radicals scavenging by ascorbic acid, canagliflozin, dapagliflozin, empagliflozin (table 1), was 97.87%, 87.18±0.080%, 94.47±0.010% and 60.83±0.030%, respectively. The IC50 value shown by these drugs were 117.4 ± 0.02, 103.6 ± 0.06, 130.1 ± 0.06 and 294.1 ± 0.04 respectively for ascorbic acid, canagliflozin, dapagliflozin and empagliflozin (table 5).

Ferric Ion Reduction by FRAP

The dose dependent increase in radical scavenging activity of ascorbic acid measured by FRAP process. All the drugs followed similar dose dependent radical scavenging activity. At 800 µg/ml, the % radical scavenging activity of ascorbic acid, canagliflozin, dapagliflozin and empagliflozin (table 2) was found to be 98.47%, 74.18±0.080, 90.22±0.105 and 94.70±0.085 respectively. The IC50 data shown by these drugs and ascorbic acid were 271.2 ± 0.06, 350.6 ±0.09, 281.1 ±0.07, 264.1 ±0.16 (table 5).

Nitric Oxide Scavenging Method

A dose dependent nitric oxide radical scavenging activity was established taking ascorbic acid as antioxidant. All the gliflozin also followed more or less similar pattern. At 800 µg/ml, the extent of NO radical scavenging by ascorbic acid, canagliflozin, dapagliflozin and empagliflozin (table 3) was 87.16%, 66.77±0.140%, 68.23±0.080% and 72.80±0.115% respectively. The IC50 data of ascorbic acid, canagliflozin, dapagliflozin and empagliflozin (table 5) were 93.64 ± 0.115, 331.5 ± 0.16, 210.9 ± 0.19 and 92.71 ± 0.06.

Assessment of Inhibition of Lipid Peroxidation

The dose dependent increase in antioxidant power of ascorbic acid was established in the assessment of inhibition of lipid peroxidation. The potential to inhibit the lipid peroxidation by ascorbic acid, canagliflozin, dapagliflozin and empagliflozin (table 4), at concentration 800 µg/ml was 98.31%, 85.46±0.035%, 81.39±0.065% and 70.80±0.075% respectively. The IC50 values shown by ascorbic acid, canagliflozin, dapagliflozin and empagliflozin were 141.8±0.09, 207.5 ± 0.10, 243 ± 0.23, 264.6 ± 1.07 (table 5).


2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable nitrogen centred free radical widely used in the screening of antioxidant assay. Every antioxidant has some reducing property that can cause reduction of DPPH. After accepting one electron, hydrazyl group is converted to hydrazine and the deep violet colour of DPPH radical changes to light yellow, absorbance of which is measured at 517 nm. The decrease in absorbance indicates scavenging of DPPH free radicals by the antioxidant. All the gliflozin contains 4 electron-rich hydroxyl group and at least one oxane ring. Due to this electron availability, gliflozins should have good antioxidant activity which is shown in table 5. The IC50 value of canagliflozin is lower than ascorbic acid which indicates canagliflozin as better antioxidant than the standard. Iron exists in dual oxidation state (Fe2+ and Fe3+) and is able to accept of donate electrons through redox system and is very essential in certain cellular functions.

A slight disturbance in the balanced redox system leads to reaction between iron and hydrogen peroxide and superoxide radical and through Haber-Weiss reaction, convert them in hydrogen peroxide radical which is responsible for protein damage, membranes and DNA injuries10. Hydroxyl radicals also decompose lipid hydroperoxides into peroxyl and alkoxyl radicals and initiate chain reaction of lipid peroxidation11. From the study it was evidenced that canagliflozin have more ferric ion reducing capacity than the standard and is followed by empagliflozin. This proved significant radical scavenging and antioxidant potential of the three drugs.

Iron catalyse the generation of hydroxyl radical or ferry-perferryl complex in the lipid media and accelerates the decomposition of lipid hydroperoxides into peroxyl and alkoxyl radicals12. The extremely reactive hydroxyl radicals react with polyunsaturated lipids of cell membrane and yields several aldehydes like malondialdehyde (MDA). This MDA further form adducts with amino group of proteins, biomolecules and DNA and is thus mutagenic as well as carcinogenic. In the reaction system, MDA forms a pink chromophore with thiobarbituric acid and addition of the drugs have prevented the formation of MDA, hence indicating their efficacy, although not as potent as standard, to inhibit lipid peroxidation.

The source of nitric oxide in human body is L-arginine and nitric oxide synthase13. Apart from some normal physiological action, nitric oxide is associated with inflammatory conditions and several carcinomas12. In the structure of nitric oxide radical, the oxygen atom has a single unpaired electron. Nitric oxide is a very less reactive radical but it is often converted to nitrite and peroxynitrite radicals in physiological system which are highly reactive and deleterious. The secondary process of generation of nitric oxide free radical is reaction between peroxy radical and NO14. Constant production of nitric oxide causes septic shock and tissue toxicity and chronic over expression of nitric oxide radicals lead to juvenile diabetes, arthritis, ulcerative colitis and multiple sclerosis15. Nitric oxide extracts labile hydrogen by attaching itself with the double bonds of the carbon skeleton and thus initiates lipid peroxidation and generation of other free radicals9. The oxygen atom forms peroxide compound which in turns generate more free radicals. Peroxidation in presence of nitric oxide generates a metabolite peroxynitrite (ONOO-) which is extremely reactive free radical. Peroxynitrite directly induce cytotoxic reactions like SH- group oxidation, lipid peroxidation, tyrosine nitration and DNA damage16. In the reaction system, nitric oxide generated from sodium nitroprusside reacts with oxygen to form nitrite ions. The drugs and ascorbic acid directly compete with the oxygen to prevent the formation of nitrite ions17. The study indicated empagliflozin as the best scavenger of nitric oxide radicals. Canagliflozin and dapagliflozin also showed some activity in scavenging nitric oxide radicals, although not close to ascorbic acid.

The current study reveals that the empagliflozin is more efficacious in scavenging nitric oxide radicals than ascorbic acid and canagliflozin, although not equipotent with ascorbic acid, is the best amongst the three drugs in inhibiting formation of malondialdehyde. Dapagliflozin also have significant antioxidant activity as indicated by DPPH radical scavenging assay and ferric ion reduction by FRAP. So, these drugs, especially canagliflozin and empagliflozin, when are in use clinically, they may elevate the quality of health of the patient by decreasing the diabetes induced oxidative stress and related complications. There may be a variation of these results in vivo as the therapeutic antidiabetic dose and plasma protein binding of these drugs vary significantly, leading to a variation in plasma drug concentration for effective antioxidant activity. Therefore, these drugs should be tested for the maximum plasma drug concentration at therapeutic dose and the activity of these drugs in that plasma drug concentration in vivo.

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