Evaluation of Oxidative Stress potential of some Nsaıds against Hydrogen Peroxide in experimental animal

 

KM Diksha Singh1*,Vikram Singh2, Adity Singh1, Vipin Kesharwani1

1Department  of Pharmacology, Student of Shambhunath İnstitute of Pharmacy,

Jhalwa, Prayagraj, Uttar Pradesh - 211012, India.

2Department  of  Pharmacology, Faculty  of Shambhunath Institute of Pharmacy,

Jhalwa, Prayagraj, Uttar Pradesh - 211012, India.

*Corresponding Author E-mail: diks83282@gmail.com

 

ABSTRACT:

Background: Oxidative stress is imbalance between aggressive and defensive system. Overproduction of oxidative stress contribute in pathogenesis of many diseasesincluding Parkinsonism, Alzheimer diseases, apoptosis, hepatic fibrosis ,chronic kidney failure and liver steatosis etc . There are several OTC drugs including NSAIDs that generate oxidative stress when administered. So there is a need to explore about these drugs. Therefore this study was designed to evaluate the oxidative stress potential of Acetaminophen, acetyl salicylic acid and Celecoxib NSAIDs. Objective: The present study is design to investigate the oxidative stress of NSAIDs of acetaminophen, aspirin and Celecoxib drug with reference to the hydrogen peroxide. Material and method: The Experimental protocol was designed for estimate the level of oxidative stress in NSAIDs treated animals against hydrogen peroxides. Animal of control group received only vehicle throughout experimental protocol. Rats of AAP group, ASA group ,CX group were exposed to acetaminophen (150mg/kg; orally) acetyl salicylic acid  (300mg/kg ;orally) and Celecoxib (50mg/kg; orally) for forty two days . Rodent of HP group were challenged with Hydrogen peroxides (0.5%) with same schedule as above. At end of experimental protocols, all the animals were sacrificed and their organ were   identified and collected for oxidative stress estimation and histological examination. Result: NSAIDs administration caused increase in oxidative stress measured in terms of SOD, CAT, MDA, GSH and GPx. HP administration produced maximum oxidative stress compare to all other groups. Oxidative parameter i.e. SOD, CAT, GSH and GPx were found to be decreased as compare to control rats. However MDA were found to be increased as compare to control rats. Additionally, CX produced less oxidative stress compare to other NDAIDs. Further, histological examinations support the biochemical results. Conclusion: From the above observations it can be concluded that NSAIDs have oxidative stress potential and generate oxidative stress and damage the organs when administrated chronically. Thus, these drugs should be used judiciously.

 

KEYWORDS: Oxidative Stress; Nsaids; Superoxide Anion, Hydroxyl Radical, Hydrogen Peroxide.

 

INTRODUCTION:

Oxidative stress occurs when the production of oxidizing agents, free radicals and reactive oxygen species (ROS), exceeds the antioxidant capacity of cellular antioxidants in a biological system1. A free radical is defined as any species capable of independent existence that contains one or more unpaired electrons.  Free radicals can react with and cause damage to DNA, lipids and proteins.3 Examples of free radicals and ROS include: superoxide anion (O‾2), hydroxyl radical (OH∙), hydrogen peroxide (H2O2), nitric oxide and nitric dioxide, and peroxynitrite.2 This imbalance leads to tissue injuries and to the progression of degenerative diseases in humans, such asvarieties of neurodegenerative diseases,including Alzheimer's, Parkinson's, Hunting-ton’s, tardive dyskinesia ,epilepsy and acute diseases of the central nervous system, such as spinal cord injuries and/or brain traumatic.The acetaminophen has antipyretic and analgesic property.3,4 That is increase the pain threshold by inhibiting two isoform of cyclooxygenase, COX-1 and COX-2, which are involved in prostaglandin (PG) synthesis,PG is responsible for pain sensation. Acetaminophen is metabolized in the liver by first-order kinetics and its metabolism via the cytochrome P450 enzyme pathway and by conjugation process,Overdose of paracetamol causes hepatotoxicity, which can in severe cases lead to liver failure in experimental animals and humans . Although paracetamol directly conjugated with glucoronic acid and significant amount of acetaminophen metabolized by the cytochrome P450 system5. This leads to the formation of a reactivemetabolite, presumably N-acetyl-pbenzoquinoneimine (NAPQI) and react with glutathione (GSH).5 Thus, paracetamol metabolism causes dramatic depletion of cellular glutathione levels in the liver. If the formation of the reactive metabolite exceeds the capacity of hepatocellular glutathione, NAPQI will covalently bind to cellular proteins.6 Paracetamol challenge (300mg/kg, i.p) for 7 days caused a significant increase in the levels of bilirubin, liver enzymes, TBARS, and iron, while catalase activity and total protein level were reduced significantly in the serum and liver homogenate.7

 

Acetylsalicylic acid is also termed as aspirin and widely used medication for the treatment of pain and fever. This has anti inflammatory as well as antipyretic. This is also prevents stroke from blood clots and platelets aggregation used for myocardial infarction (MI). The acetylsalicylic acid (ASA) inhibits the synthesis of prostaglandin.This is non-selective for the enzymes (Flower, 2003) COX-1 and COX-2. The main metabolites of acetylsalicylic acid are salicylic acid ether or phenol glucoronide and ester or acyl glucoronide. Long term use of low dose aspirin (ASA) has been recommended to reduce the risk of heart attack but gastric bleeding and formation of gastrointestinal ulcers are the most common adverse effects. It has been shown that, ASA damages gastric mucosa by inhibiting the synthesis of protective prostaglandins and by having direct action on the mucosa.8 This results in enhancing acid back-diffusion and microvascular injury accompanied by the activation of neutrophils that produce excessive oxygen-derived free radicals which cause lipid peroxidation and tissue damage.9

 

Celecoxib inhibit both forms of cyclooxygenase (COX-1 and COX-2) which is a selective, non-competitive cyclooxygenaqse-2 (COX-2) enzyme inhibitor. Their uses minimize inflammation and pain.Celecoxib a nonselective COX inhibitor were studied in rat intestine. The study showed a significant decrease in the lipid peroxide levels as TBA reactive substances as well as the conjugated diene. H2O2, are more stable, less reactive and may act as second intracellular However; they can alter protein conformation after oxidation of cysteine and methionine residues. Moreover, in the presence of Fe2+ or Cu+, via the so-called Fenton reaction, H2O2 is converted to •OH However, although the iron-dependent formation of •OH occurs in vivo, the physiological significance of the copper-dependent formation of •OH is still debated. The amount of ROS formed is not negligible because of the high amount of O2 consumed by aerobic organisms. Indeed, approximately 2–4% of oxygen consumed in mitochondria is converted to the superoxide ion by iron-sulfur proteins. Thus, to maintain homeostasis, accumulation of excess ROS is prevented by multiple enzymatic and non-enzymatic systems that receive the generic name of ‘host antioxidant defense systems.

 

MATERIAL AND METHODS:

Reagents and chemicals:

Acetylsalicylic acid purchased from High purity laboratory chemical Mumbai. Acetaminophen and Celecoxib were procured from Sigma-Aldrich chemical   corporation India. Hydrogen peroxide solution (6%), potassium dihydrogen orthophosphate and sodium hydrogen pellets purchased from Thermo Fischer scientific India Pvt Ltd.

 

Animals and experimental protocol:

The study was carried out thirty male Wistar rats (200±20g; 8-10 week old) were procured from central animal house of Sambhunath Institute of pharmacy Allahabad and used the present study. Previously acclimatized (for 7 days) were randomly divided into equal-sized five groups. All the animals were housed in poly acrylic cage having clean paddy husk at standard condition (12:12 light/dark cycle; humidity 50±5% and temperature 26±1ş C) with food and water ad libitum throughout the experimental study. All the experimental protocol was approved by IAEA. All the experiments were performed under the guideline of CPCSEA. after acclimatization thirty male rats were randomly divided into five groups (n=6) namely control, AAP, ASA, CX and HP. Experimental protocol was designed for 42 days to estimate the level of oxidative stress in NSAIDs treated animals against hydrogen peroxides. Details of groups and their treatment were as follows:

 

Group I (Control Group): Only vehicle (distilled water, 1ml and via stomach tube) was administered to the animals of this group for 42 days

Group II (AAP Group): AAP (150mg/kg; via stomach tube) was administered to the animals of this group for 42 days

Group III (ASA Group):ASA (300mg/kg; via stomach tube) was administered to the animals of this group for 42 days

Group IV (CX Group):CX (50mg/kg; via stomach tube) was administered to the animals of this group for 42 days

Group V (HP Group): Animals of thiswas exposed to H2O2 (0.5%) as drinking water for 42 days 

 

In this study we did not seen any physiological sign of toxicity in treated animal group. Different route of drugs administration (intragastrically via stomach tube or with drinking water ad libitum) required three distinct group control, standard and treatment group. Acetaminophen, acetylsalicylic acid and celecoxib solution freshly prepared daily, hydrogen peroxide is used as standard in drinking water for standard group. Water and chow utilization was estimate daily; body weight was controlled once a week. Body weights of individual animals were recorded before the experimental procedure on day (D1) - (D42). After six week exposure the animal were sacrificed and sample of blood, kidney, liver and brain were collectedThe blood samples were collected by retro-orbital sinus and heart puncture, under chloroform anesthesia using a fine capillary tube. Blood was collected into K2EDTA coated tubes for hematology estimation and tubes without anticoagulant for clinical chemistry. The blood Serum was separated by centrifugation process at 3000 rpm for 10 min at 4 ̊C. Sample of serum were frozen at -80 ˚C for further biochemical estimations. The liver, brain, and kidney was quickly removed , washed in cooled 0.9% NaCl and then homogenized in ice- cold buffer.Then, homogenates were centrifuged at 1500rpm for 15 mints at 4̊C and supernatant was collected. Their contents were examined for abnormalities and placed in 10% formal saline. The five rats that died during the course of the study were noticed after autolysis had set in. Hence, though the rats were necropsies, three organs were collected for examination.10

 

Analytical procedure:

Determination of lipid peroxidation:

Thiobarbituric acid regent, (50mM Dissolve 185mg TBA in 1ml DMSO and then make up volume up to 50 ml distilled water.)  TCA in distilled water. 100µl of sample were combined with 500µl TCA (8%w/v) and mixed thoroughly. Sample was centrifuged at 2500rpm for 5 minute to remove the protein precipitation and supernatant was collected in fresh tube. 250µl of supernatant was mixed with 1.8ml of TBA regent and the solution was heated for fifteen minutes in a boiling heating mental water bath The absorbance was analyzed at 532nm against a blank than contains all selected regent except the sample The malondialdehyde concentration was estimated using an extinction coefficient of 1.56×105 M-1 cm-1

 

Determination of catalase activity

Hydrogen Peroxide (0.065 M): Amount of 6.639ml of H2O2 (30%) was diluted to a final volume of 1 liter by Na-K-phosphate buffer. Sodium-Potassium Phosphate Buffer, pH 7.4 (60mM):  Taken a weight of 11g of disodium monohydrogen phosphate (Na2HPO4) and 2 g of potassium dihydrogen phosphate (KH2PO4) were dissolved in 1 liter of DW. The pH was adjusted to 7.4 by the addition of 1M of HCl. Ammonium Molybdate (32.4mM):Taken a weight of 40.04g of ammonium molybdate was dissolved in 1 liter of DW. All chemical compound and serum were brought to 37°C. Sample, blank, standard, and control tubes were prepared then pipette into test tubes.  Tubes were incubated for 4 min second at 37°C.

 

Determination of superoxide dismutase activity

SOD was assayed as described by The assay mixture contained 0.5ml brain, kidney, liver and blood homogenate, 1ml 50mM sodium carbonate, 0.4mi of 25μm nitroblue tetrazolium (NBT) and 0.2ml freshly prepared 0.1mM hydroxylamine hydrochloride.The changes in the absorbance were calculated at 560 nm and recorded.

 

Determination of reduced glutathione (GSH) activity:

In this method, an aliquot of 1.0ml of supernatant of the homogenatefor measurement of glutathione activity was treated with 0.5ml of Ellman’s reagent [19.8mg of 5, 5'-dithiobisnitro benzoic acid (DTNB) in 100ml of 0.1% sodium nitrate] and 3.0ml of phosphate buffer (0.2M, pH 8.0). The absorbance was calculated at 412nm. The percentage inhibition of reduced glutathione was calculated using the following equation.

 

Estimation of glutathione peroxidase (GPx) activity:

The activity of glutathione peroxidase (GPx) was measured by the method gunzler and flohe-clariborne (1985) method. Take 100µL of tissue supernatant of liver kidney, brain and blood was put on 10µL of glutathione reductase and 290µL of buffer solution (50mM K2HPO4), (50mM KH2PO4) ph7.0, 3.5mM reduced glutathione, 1mM sodium azide and 0.12mM NADPH and 100µLH202 of 0.8mM their absorbance was read at 340 nm at 0 and 60 s .

 

Histopathology examination:

Histological estimation was go through on the selected organs of representative rat of each group. The tissues were processed for routine paraffin embedding and sections were stain with Haematoxylin and Eosin stain.

 

Statistical analysis:

All values were expressed as mean± standard error of mean (SEM). All the statistical analysis of data was done using one way analysis of variance (ANOVA) followed by student – Newman –Keuls post hoc test to monitor significance among group p<0.05 was considered as significant.

 

RESULT:

Effect of ASA, AAP and CX treatment on catalase activity in discrete organs of rat:

Effect of ASA, AAP and CX treatment on catalase activity in discrete organ [(brain; A) (liver; B) (kidney; C)] of rat are depicted in figure (1). Statistical analysis revealed that there was significant difference in catalase activity in brain [F (4, 25) =26.34, P < 0.05], liver [F (4, 25) = 46.55, P <0.005] and kidney [F (4, 25) = 25.91, P < 0.05] between the group. Post hoc study illustrated that HP significantly attenuated the catalase activity in above organs of rats as compare to control rat. Further, ASA, AAP and CX also significantly abolished the catalase activity in all above organs as compare to control rat decrement in the activity was found to be less as compare to HP rat. However, CX showed less decrement in catalase activity as compare to ASA and AAP.

 

Effect of ASA, AAP and CX treatment on SOD activity in discrete organs of rat:

Effect of ASA, AAP and CX treatment on SOD activity in discrete organ [(brain; A) (liver; B) (kidney; C)] of rat are depicted in figure (4). Statistical analysis revealed that there was significant difference in SOD activity in brain [F (4, 25) =24.88, P < 0.05], liver [F (4, 25) = 22.85, P <0.005] and kidney [F (4, 25) = 18.62, P < 0.05] between the group. Post hoc study illustrated that HP significantly attenuated the SOD activity in above organs of rats as compare to control rat. Further, ASA, AAP and CX also significantly abolished the SOD activity in all above organs as compare to control rat decrement in the activity was found to be less as compare to HP rat. However, CX showed less decrement in SOD activity as compare to ASA and AAP.

 

Figure 1:- Effect of treatment on oxidative stress marker in terms of catalase activity in brain (A) liver (B) and kidney (C). All values are Mean ± standard Error of Mean (SEM, n=6). aP < 0.005 compared to control, bP < 0.005 compared to ASA, Cp < 0.005 compared to AAP and dP < 0.005 compared to CX. (One way ANOVA followed by student- Newman – kelus post –hoc test).

 

Figure 2:- Effect of treatment on oxidative stress marker in terms of SOD activity in brain (A) liver (B) and kidney (C). All values are Mean ± standard Error of Mean (SEM, n=6). aP < 0.005 compared to control, bP < 0.005 compared to ASA, Cp < 0.005 compared to AAP and dP < 0.005 compared to CX. (One way ANOVA followed by student- Newman – kelus post –hoc test)

 

Effect of ASA, AAP and CX treatment on MDA activity in discrete organs of rat:

Effect of ASA, AAP and CX treatment on MDA activity in discrete organ [(brain; A) (liver; B) (kidney; C)] of rat are depicted in figure (3) . Statistical analysis revealed that there was significant difference in MDA activity in brain [F (4, 25) =14.74, P < 0.05], liver [F (4, 25) = 17.41, P <0.005] and kidney [F ( 4, 25 ) = 15.25, P < 0.05] between the group . Post hoc study illustrated that HP significantly increased the MDA activity in above organs of rats as compare to control rat. Further, ASA, AAP and CX also significantly increased the MDA activity in all above organs as compare to control rat but increment in the activity was found to be less as compare to HP rat. However, CX showed less increment in MDA activity as compare to ASA and AAP.

 

Figure 3:- Effect of treatment on oxidative stress marker in terms of MDA activity in brain (A) liver (B) and kidney (C). All values are Mean ± standard Error of Mean (SEM, n=6). aP < 0.005 compared to control, bP < 0.005 compared to ASA, Cp < 0.005 compared to AAP and dP < 0.005 compared to CX. (One way ANOVA followed by student- Newman – kelus post –hoc test).

 

Effect of ASA, AAP and CX treatment on GSH activity in discrete organs of rat:

Effect of ASA, AAP and CX treatment on GSH activity in discrete organ [(brain; A) (liver; B) (kidney; C)] of rat are depicted in figure (4). Statistical analysis revealed that there was significant difference in GSH activity in brain [F (4, 25) =18.62, P < 0.05] , liver [F ( 4,25) = 22.85, P <0.005] and kidney [F ( 4, 25 ) = 16.21, P < 0.05] between the group . Post hoc study illustrated that HP significantly attenuated the GSH activity in above organs of rats as compare to control rat. Further, ASA, AAP and CX also significantly abolished the GSH activity in all above organs as compare to control rat decrement in the activity was found to be less as compare to HP rat. However, CX showed less decrement in GSH activity as compare to ASA and AAP.

 

Figure 4:- Effect of treatment on oxidative stress marker in terms of GSH activity in brain (A) liver (B) and kidney (C). All values are Mean ± standard Error of Mean (SEM, n=6). aP < 0.005 compared to control, bP < 0.005 compared to ASA, Cp < 0.005 compared to AAP and dP < 0.005 compared to CX. (One way ANOVA followed by student- Newman – kelus post –hoc test).

 

Histological studies on liver, brain and kidney

Figure (5), and (6) illustrated the effect of treatment on rat brain (A), kidney (B) and liver (C). Histological examination suggested that hydrogen peroxide caused maximum damage to the all above organs compare to all other organs. Further, ASA and AAP damaged the all selected organs more as compare to control animal but less as compare to HP group rats. Additionally, CX also damaged the organ but less than all other treatment group except control.

 

 

Figure 5: histological representative of brain (control, ASA, AAP, CX and HP)

 

Figure 6 :- histological representative of kidney (control, ASA, AAP, CX and HP)

 

DISCUSSION:

The present study was designed to demonstrate the oxidative stress potential of NSAIDs against hydrogen peroxide. ASA, AAP and CX were selected as treatment drugs and administered successively for 42 days to evaluate the oxidative stress potential. This study reports for first time about comparative study of ASA, AAP and CX. All these drugs generate oxidative stress when administered. Further, ASA and AAP produced more oxidative stress as compare to CX. The oxidative stresses were evaluated in terms of, SOD, MDA, GSH, GPx, and catalase. Body weight of each animal was recorded every week. At last animals were sacrificed and brain, kidney and liver were collected and applied for histological examination.

 

Free radical and reactive oxygen species are toxic to biological system.The toxicity is concern in particular to hydroxyl radical, which in turn, may react with the molecule component of the cell and produce second radical that interact with other molecule which is continue to the radical chain reaction.11 Oxidative stress associated with the formation of ROS, play an important role in the pathogenesis of various deleterious processes and diseases in human, such as brain related diseases12, amyotrophic lateral diseases.13 and Huntington diseases,14 renal fibrogenosis (Masyuk et al., 2008) and liver fibrosis, Apoptosis, liver aging  etc. ROS are particularly active in the brain and neuronal tissue as the excitatory amino acid and neurotransmitter, whose metabolism is a factory of ROS which are unique to the brain serve as source of oxidative stress.15

 

In recent year mitochondria have been gradually recognized as the main source of oxidative stress after an overdose of acetaminophen due to excessively formation of NAPQI which was reduce the amount of GSH and attach on the cellular protein According to researcher AAP excess used caused direct molecular changes in mitochondrial electron transport chain (ETC) which was damage to chain of electron caused free radical formation in renal and liver function. According to Hayrettin ozturk Celecoxib responsible for liver injury due to oxidative stress, and other researcher reported that the chronic administration of Celecoxib may have damaging effect in kidney and this damage by oxidants. The evidences of non selective NSAIDs show that they cause more liver toxicity and brain toxicity due to known COX1/COX2 relationship which is responsible for oxidative stress.

 

SOD superoxide dismutase is the first detoxification enzyme. It is act like first line defense against free radicals. Superoxide anion constantly generated by endogenously and exogenously and produces hydroxyl radical and indirectly from hydrogen peroxide (Tarrunet al., 2003). Further, researcher reported that SOD deficiency causes neurodegenerative disease, myocardial disease and (Strange et al., 2003) death, and deficiency of SOD isoenzyme SOD1 cause amyotrophic lateral sclerosis’s (Roberts  et al., 2007). Recently (Dayal et al.2017) says that SOD deficiency cause cerebral vascular hypertrophy.  In the current study we estimated the SOD level in the different organ such as liver, brain, kidney and blood exposed to NSAIDs against hydrogen peroxide. We found that NSAIDs administration decreased the in SOD level in selected rodent tissue compare to control group. 

 

Malondialdehyde is one of the final products of polyunsaturated fatty acid peroxidation in the cells. It increases due to oxidant species attack lipids containing products and those lipids can oxidize by enzyme cyclooxygenase, cytochrome P450 and lipoxygenase in response to cell death.16 In the current study we evaluated the MDA level in the different organ such as liver, brain, kidney and blood exposed to NSAIDs against hydrogen peroxide. NSAIDs treatment caused increase in MDA level in selected tissue of rats compare to control group.

 

Glutathione reductase is an enzyme which catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). GSH serve as an antioxidant, reacting with free radical and organic peroxides. GSH play a central role in cell death .In the current study we estimated the GSH level in the different organ such as liver, brain, kidney and blood exposed to NSAIDs against hydrogen peroxide. We found that NSAIDs administration caused decrease in GSH level in all the tissue of rodents compare to control group.

 

Glutathione peroxidase is similar to catalase enzyme which is breakdown the hydrogen peroxide to water and lipid peroxides.18 These GPx is important role in the blocking lipid peroxidation and prevent for oxidative damage.19 The deficiency of GPx can cause neurotoxic damage cancer and cardiovascular disease.20 In the current study we evaluated the GPx in the different organ such as liver, brain, kidney and blood exposed to NSAIDs against hydrogen peroxide. Result indicated that NSAIDs administration caused decrease in GPx level in selected tissue of rats compare to control group.

 

Catalase is a tetrameric protein which has four subunits. Each subunit has contained single ferri protoporphyrin . The ability of catalase reduces the concentration of hydrogen peroxide which is important for physiological process. The reduction of this catalase enzyme can cause various disease and abnormalities in the cell.21 Previous study reported that reduction of catalase can cause risk of cancer and mental disorder.22 In the present study we evaluated the CAT level in the different organ such as liver brain kidney and blood exposed to NSAIDs against to hydrogen peroxide. We found that NDAIDs administration caused significant decrease in CAT level in the all above tissue of animals compare to control group. Tissues of all the selected organs were implied for histological examinations that confirm the biochemical results.23 Histological examination suggested that hydrogen peroxide caused maximum damage to the all above organs compare to all other organs. Further, ASA and AAP damaged the all selected organs more as compare to control animal but less as compare to HP group rats. Additionally, CX also damaged the organ but less than other NSAIDs treated groups.

 

CONCLUSION:

The obtained results indicated the NSAIDs have oxidative stress potential and generate oxidative. NSAIDs administration caused increase in oxidative stress measured in terms of SOD, CAT, MDA, GSH and GPx. HP administration produced maximum oxidative stress compare to all other groups. Oxidative parameter i.e. SOD, CAT, GSH and GPx were found to be decreased as compare to control rats. However MDA were found to be increased as compare to control rats. Additionally, CX produced less oxidative stress compare to other NDAIDs. Further, histological examinations support the biochemical results. From the above observations it can be concluded that NSAIDs have oxidative stress potential and generate oxidative stress and damage the organs when administrated chronically. These drugs are OTC but should be administered with precaution and should be used judiciously

 

ACKNOWLEDGEMENT:  

I am thankful to the management of sambhunath institute of pharmacy, Praygraj for providing best lab facilities necessary for completion of my research.

 

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Received on 01.09.2020            Modified on 15.12.2020

Accepted on 04.01.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6194-6200.

DOI: 10.52711/0974-360X.2021.01072