An In vitro Study on impact of Vitamin-C on Cefuroxime mediated alterations in Bio-parameters associated with free Radical linked Lipid Decomposition

 

Madhurima Ghosh1, Miltu Kumar Ghosh1, Debabrata Devbhuti2, Sandipan Dasgupta3,

Pritesh Devbhuti1*

1Department of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata - 700053, India.

2Department of Pharmacy, Jnan Chandra Ghosh Polytechnic, Kolkata - 700023, India.

3Department of Pharmaceutical Technology,

Maulana Abul Kalam Azad University of Technology, Haringhata, Nadia, India.

*Corresponding Author E-mail: ghoshmadhurima92@gmail.com, miltu.ghosh@nshm.com, ddjupt73@gmail.com, sandipan.dasgupta21@gmail.com, priteshdevbhuti@gmail.com

 

ABSTRACT:

Background: Lipid peroxidation can be interpreting as an oxidative degeneration of lipids. It happens when a hydroxyl radical removes an electron from polyunsaturated fatty acids (PUFAs), which can react with oxygen and other polyunsaturated fatty acids to produceperoxyl radicals and hydroperoxides, thus promulgating the injury. So this repeat cycle of lipid peroxidation process can be responsible of cellular damage. Drug-induced lipid peroxidation is an important phenomenon found to be involved behind its certain hazardous side effects due to the generation toxic end products of such peroxidation like malonaldehyde (MA), hydroxynonenal (HNE), etc. Antioxidants play a crucial role in modifying such processes due to their free radical scavenging capability. Objective: Keeping in mind the matter, thisin vitroinvestigation was conducted using cefuroxime, a cephalosporin antibiotic as drug of choice and vitamin C as antioxidant taking liver tissue of goat as lipid source. Methods: The liver homogenate was divided in certain experimental groups that were treated with cefuroxime and ascorbic acid for specific time periods. The level of MA and HNE in the samples was estimated and compared with control. Result: The result showed that Cefuroxime has lipid peroxidation induction capability that was counteracted by ascorbic acid. Conclusion: Thus cefuroxime-induced, peroxidation associated, toxicities may be managed well upon co-administration with the antioxidant vitamin C.

 

KEYWORDS: Lipid peroxidation, Cellular damage, Drug toxicity, Cefuroxime, Vitamin-C.

 

 


INTRODUCTION:

The aerophilous degradation of poly unsaturated fatty acids (PUFAs) known as lipid peroxidation; that consists of one carbon-carbon double bonds or multiple double bonds and peroxide radicals that initiate the chain reaction by directly reacting peroxide radicals with the multiple bonds present in fatty acid chain of the PUFA1. The essential constituents of lipoid barriers are prone to decomposition via free radicals where the mechanism happened under enzymatic or non-enzymatic control throughout the biological system2.

Lipid peroxidation (LP) is used to define membrane damage of cells and also used to identify natural antioxidants role inside human body like-vitamins, polyphenols, flavones, ginsenosides and their mechanism of action involved in various pathogenesis pathways3-4. It is an extremely damaging event that makessome broad range changes in the composition and activity of lipoid cell covering; and is an important cause of several ailments and abnormalities such as diabetes, arthritis, peptic ulceration, cataract, cancer and others1.

 

Medicinal agent associateddecomposition of lipids could be linked with various toxicities as shown by anticancer drug associated- neutropenia, thrombopenia, bone marrow depression, hematological toxicity, gastrointestinal toxicity, oral toxicity, etc. andantibiotics-induced hepatotoxicity, cardiotoxicity5, hypoglycemia, rhabdomyolisis, etc. This unwanted process may be checked by minimizing the formation of reactive oxygen species (ROS) by demolishing them inside body, providing a competitive substrate for unsaturated lipids in the cell-membrane and also stimulating the correction mechanism of mutilated cell-membrane. So, for these reasons, several natural and synthetic antioxidant compounds are used to neutralize the toxicity generated due to peroxidative process. When there islowin vivo antioxidant protection6 and hues formation of ROS that are not controlled by internal antioxidant defense system7-8, administration of antioxidants from outside sources may help prevent the oxidative injury produced by ROS.Therefore, drug-lipid interaction provides a relatively unexplored field of pharmaceutical research.

 

Cefuroxime is a semi synthetic β-lactam antibiotic; more specific it is a second generation cephalosporin.It is used to prevent several types of bacterial infections9 caused by bacteria including pneumonia, lower respiratory tract infections, meningitis, gonorrhea, skin, blood, bone joint and urinary tract infections10. But cefuroxime is associated with a number of serious adverse reactions and has ability to produce ocular toxicity11, hepatotoxicity12, and neurotoxicity13. Such reactions may manifest long after therapy had been completed and in severe cases may result in life-long disabilities and some uncommon adverse effects like- respiratory, cardiovascular, musculoskeletal disorders have also been reported with the use of cefuroxime14. Vitamin C, is a natural antioxidant. It has free radical scavenging property. It is an important nutritional factor involved in the recreation of tissue and the enzyme-linked formationof some neurotransmitters and also needed for the proper functionalityof immune system15-16. So, the use of free radical scavengers as adjunct of noxious drugs may be an effective tool for eliminating toxicities of drugs17. Many naturally occurring products are also there that could control peroxidative degradation of lipids and corresponding damaging effects to cells and tissues18-21.

 

Considering ourcontinuous involvement in searching the interaction ability of drugs withlipid, we tend to mention in this study thein-vitroexamination of cefuroxime-induced lipid peroxidation and its suppression with the water-soluble vitamin, ascorbic acid.

 

MATERIAL AND METHODS:

In the present study, drug-induced lipid peroxidation and its suppression with antioxidant has been focused in an attempt to correlate toxicity of drugs to its peroxidation induction capacity. The antioxidant co-administration with the drug in vivo, might reduce toxicity and increase the therapeutic index of the drug. The study was performed using goat (Capracapra) liver homogenate22 as lipid source for in-vitro study because it is very much similar with human liver. Here we observed some common parameters of peroxidative degradation of lipids like- malonaldehyde (MA) andhydroxynonenal (HNE).

 

Preparationof goat liver homogenate and grouping for experimental study:

Goat liver homogenate was prepared by using phosphate buffer (PH7.4) at a concentration of 1g/ml and fractionated to three investigational batches: untreated (UT), cefuroxime-treated (CT), cefuroxime with vitamin C-treated (CVCT). Drug and vitamin C, at definite concentration, were added to the respective groups and then these were incubated at room temperature up to 5 hours. The samples were withdrawn after 3hours and 5 hours of incubationand transferred to marked centrifuge tubes, and treated with same quantity of TCA solution. Upon mixing properly and centrifuging at a speed of 3000rpm for 20 minutes, the supernatant was filtered and therefore used for the estimation of MA and HNE content.

 

1.     Estimation of malonaldehyde (MA) by thiobarbituric acid(TBA) method:

As malonaldehyde serves as an indicator of lipid peroxidation, various methods have been proposed for its estimation. To estimate lipid peroxidation, the procedure that had been followed in the study involved treatment of supernatant filtrate with equal volume of thiobarbituric acid (TBA) solution in graduated glass tubes with stopper. The mixture was heated using a water bath for a period of 30 minutes and then allowed to cool to surrounding temperature and therefore the absorbance of the solution was measured at a wave length of 530nm against a blank containing equal volume of TBA solution and water. The content of MA was estimated from the standard curve, that indicates the extent of lipid peroxidation23.

 

2.     Estimation of hydroxynonenal (HNE):

HNE could be a major organic compound generated throughout peroxidation of unsaturated fatty acids. HNE in biological fluids may be used as an indicator of peroxidation of lipids23. 2ml of the supernatant filtrate was treated with dinitrophenyl hydrazine solution and after1 hour it was extracted with hexane and the extract was evaporated at low temperature. The remnant was solubilized in 2ml of methyl alcohol and thenabsorbance was measured at 350 nm against methyl alcohol blank24.

 

 


 

Table 1: Changes (%) in peroxidation parameter (MA)

Parameter

Mean ( SE) change (%) at time gap

3 Hours

5 Hours

CT

CVCT

ANOVA

CT

CVCT

ANOVA

MA

27.93

( 4.1)

6.72

( 2.76)

F1=58.38 (df 1,4)

F2= 6.05 (df 4,4)

Pooled variance=19.26

LSD = 6.39

Ranked means = (CT) (CVCT)

62.88

( 7.91)

21.95

( 6.17)

F1=20.15 (df 1,4)

F2= 1.40 (df 4,4)

Pooled variance=207.88

LSD = 21.02

Ranked means = (CT) (CVCT)

Mean alteration (%) in comparison to untreated sample of that particulartime period are shown. Replicability estimated considering t test and the resultsare convincing at p value < 0.05. F1 and F2 reflects variance ratio between different samples (CT, CVCT) and that between liver homogenate groups, respectively. LSD stands forcritical difference. CT and CVCT indicate cefuroxime-treated and cefuroxime and vitamin C-treated samples respectively. SE is standard error (df= 4); * indicates that two averages not present insidethe sameparenthesisaredifferent more significantlyatp<0.05

 

Table 2: Changes (%) in peroxidation parameter (HNE)

Parameter

Mean ( SE) change (%) at time gap

3 Hours

5 Hours

CT

CVCT

ANOVA

CT

CVCT

ANOVA

HNE

46.64

( 5.19)

7.33

( 2.72)

F1=105.94 (df 1,4)

F2= 3.72 (df 4,4)

Pooled variance=36.44

LSD = 8.80

Ranked means* = (CT) (CVCT)

58.96

( 4.56)

16.90

( 3.27)

F1=106.22 (df 1,4)

F2= 2.78 (df 4,4)

Pooled variance=41.64

LSD = 9.41

Ranked means* = (CT) (CVCT)

Mean alteration (%) in comparison to untreated sample of that particular time period are shown. Replicability estimated considering t test and the resultsare convincing at p value < 0.05. F1 and F2 reflects variance ratio between different samples (CT, CVCT) and that between liver homogenate groups, respectively. LSD stands forcritical difference. CT and CVCT indicate cefuroxime-treated and cefuroxime and vitamin C-treated samples respectively. SE is standard error (df= 4); * indicates that two averages not present inside the sameparenthesisaredifferent more significantlyatp<0.05

 


RESULT AND DISCUSSION:

Table 1represents the percent change in MA content in cefuroxime treated (CT) as well as both cefuroxime and ascorbic acid treated (CVCT) samples with respect to control at different time intervals. Table 1 reveals that the MA content of liver homogenate has been increased with respect to control group upon treatment with cefuroxime. On the other hand, when the homogenate was treated with both cefuroxime and ascorbic acid, the cefuroxime-induced MA content enhancement was suppressed by the antioxidant. In many studies it was found that antioxidants could suppress MA level25. The table says that the drug cefuroxime possesses significant capability to induce peroxidation of lipids that actually caused MA content enhancement which was further suppressed by the water soluble antioxidant vitamin ascorbic acid. Table 2 discloses the fact that cefuroxime-induced peroxidation of lipids is responsible behind the enhancement of HNE level26 in homogenate samples with respect to untreated group and this capability of cefuroxime was minimized by ascorbic acid.

 

CONCLUSION:

The drug cefuroxime belongs to cephalosporin class and generally known as a β-lactam antibiotic is widely used as an antimicrobial agent in different bacterial infections. This compound also possesses several types of adverse drug reactions or toxicities. From the in vitro experimental study it was found that the drug cefuroxime has significant interactive effects on lipid component of liver homogenate that leads to its oxidative decomposition. This might be one of the reasons behind the toxic effects shown by cefuroxime. Since peroxidation is a free radical mediated process, the free radical scavengers like theantioxidant vitamin ascorbic acid might play enough role in minimizing toxicities likely to be induced by the drug cefuroxime. Thus, to achieve maximum benefit from cefuroxime therapy, an important way would beantioxidant co-administration.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this study.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the authorities of NSHM Knowledge Campus, Kolkata for providing necessary facilities to carry out the work successfully.

 

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Received on 29.06.2023 Modified on 21.10.2023

Accepted on 27.12.2023 RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(4):1795-1798.

DOI: 10.52711/0974-360X.2024.00285