INTRODUCTION:

Various types of health risks are generated from the heavy metal intake. Heavy metals viz. nickel, chromium, arsenic, lead, zinc and cadmium are exposed to environment from wastewater. Exposure of all these heavy metals is harmful to human health. Cadmium is a metal which exposed to environment by fertilizers, cadmium is also found in bottom sediments of natural water. Generally, Cadmium is deposited into waters or a soil, then ultimately transferred to animals and plants, and enters the human body through the food chain^1,2. When this metal gets consumed, it will aggregate inside the body all through life. Cadmium alters the integrity of blood brain barrier and reaches to CNS after exposure even in low concentration also³. Oxidative stress, transport pathways inhibition or up regulation, epigenetic changes in DNA expression and structural deformation of proteins due to cadmium are major pathway which play important role in causing Cadmium induced tissue injury⁴.

Use of antioxidants has been promoted because they prevent the occurrence of disease by virtue of its free radicals scavenger’s property⁵. Recently, the use of bioactive flavonoids, have been widely reported for the treatment of various disorders^6,7. Among such natural products Naringenin is one of the bioactive compounds (4,5,7-trihydroxyflavanone) found in citrus fruits and vegetables like tomato and cereals⁸. It is found that consumption of naringenin rich diet on regular basis is helpful in prevention of chronic disease like cardiovascular disorder, metabolic disorder, and neurological disorders.⁹ Efficient antioxidant Naringenin can readily cross the vascular endothelial barrier¹⁰viz blood brain barrier and to exert its various affects^11,12. Being a potent free radical scavenger Naringenin reduce lipid peroxidation and protect glutathione level¹³. It is well known that the cadmium causes peripheral and central toxicity by inducing free radical generation. These free radicals are dangerous to cells. They may harm various enzyme system, lipid, protein, DNA, and therefore antioxidants may be a promising approach to cure the damages caused by free radicals^14,35. Therefore, based on this fact the present study was planned to explore protective action of bioactive compound naringenin against chronic Cd‑induced neurotoxicity.

MATERIALS AND METHODS:

Animals:

Swiss albino mice weighing around 20-30g were purchased Animal science division CDRI, Lucknow and used in the present study. The animals had free access to water and a commercial standard pellet diet ad libitum. Animals were grouped as five animals per poly acrylic cage. Room temperature 25-28 ̊C, alternating light and dark cycle of 12 h each with relative humidity of 50±20% was maintained as per CPCSEA guideline. Animals was acclimatized for a week before the commencement of the experiment in order to avoid any stress due to handling. All protocols were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), Amity Institute of Pharmacy, Amity University Uttar Pradesh Lucknow, India (AUUP/AIP/M. Pharm/007/2018).

Materials:

The biochemicals like Naringenin, cadmium chloride, sodium chloride (NaCl), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA), 5,5. dithiobis (2 nitrobenzoic acid) (DTNB), acetylthiocholine iodide (AChl)., 1.1.3.3-tetraethoxypropane (TEP), sodium nitrate (NaNO₂₎were purchased from Sigma-Aldrich, USA.

Experimental Design:

Drug Administration:

Memory dysfunction was induced in mice (Group 2) by oral administration of cadmium chloride (5mg/kg body weight) for 28 days. Naringenin (20 and 40mg/kg/day in group 3 and group 4 respectively) was administered daily, one hour prior to cadmium chloride administration for 28 days¹⁵.

Experimental Protocol:

The animals were divided into followings groups:

Group 1: Control group were given distilled water (po)

Group2: Diseased control group were given Cadmium chloride (CD) 5mg/kg for 28 days (p.o.)

Group3: Mice were given with CD (5mg/kg, p.o) and Naringenin (20mg/kg) (CD+NRL) p.o for 28 days.

Group 4: Mice were given with CD (5mg/kg, p.o) and Naringenin (40mg/kg) (CD+NR) p.o for 28 days.

Group5: CD (5mg/kg, p.o) and donepezil (5mg/kg) for 28 days.

Twenty-eight days after the start of experimental protocol, the animals were screened for behavioral test of learning and memory in Morris water maze and in Elevated plus maze.

For Elevated plus maze test, maximum effective dose was 40mg/kg of naringenin (p.o) was selected on the basis of performance of mice at both the doses in Morris Water Maze test.

On 33^rdday the animals were sacrificed, and tissues were taken for biochemical estimation and histopathology.

Behavioral Assay:

Learning and memory behavior was accesses by Morris Water Maze (MWM) test^16,17and Elevated Plus Maze (EPM)¹⁸test in various groups of mice. Behavioral test starts on 29^th day of experimental protocol after completing dosing protocol. All behavior tests have been performed in dim light by the same experimenter.

Morris Water Maze:

The Morris water maze is a large circular black pool of 120cm diameter, 50cm height, into which water was filled to a depth of 30cm at 26±2°C. Four equally spaced points viz N, E, S and W were designed around the boundary of the pool. A black colored round stand was positioned below the water surface in a constant position in middle of the NE quadrant in the pool. Nontoxic black dye was mixed with water to hide the location of this stand^36. SW quadrant was the starting point in all the trials. The mice could climb on the stand to escape the need of swimming.

In order to train mice in the Morris water maze, trials were given for 5 consecutive days. Each mouse was allowed to find the hidden stand for 60 s (cut-off time) and were permitted to stay on stand for 30 s. If a mouse was unable to find the stand, the experimenter put that mouse on stand. Daily session of three trials per day were given to each mouse to find the hidden stand. In each trial, latency time to reach the stand was recorded. Results are represented as mean latency time of all three trials. Learning is shown by a significant decrease in latency time as compared with the first trial.^16,17.

Elevated Plus Maze (EPM) Test:

This instrument consisted of 2 covered arms (16cm x 5 cm x 15cm) and 2 open arms (16cm x 5cm) extended from a central stand (5cmx 5cm) and elevated to a height of 25cm from the floor. On the first day of the trial, each mouse was placed at the end of an open arm, facing away from the central stand^34.

Transfer latency time (TL) was recorded as time taken by the mouse to shift from the open arm to one of the covered arms with all its four legs. TL was noted on the first day (29^th day of dosing) for each animal. The cutoff time was 90 sec and if the animal did not enter into one of the covered arms with in cutoff time, it was smoothly pushed into one of the two covered arms and TL was noted as 90 sec. Exploratory time was 2 minutes and then mouse was returned to its home cage. Retention trial was given after 24 h.¹⁸

Spontaneous locomotor activity:

This was assessed by actophotometer prior to trial for memory and locomotor activity test in all the groups which had been made for the study. One mouse was tested at a time. The activity meter senses movement with a grid of infrared photocells placed around then to record ambulatory activity It digitally records the horizontal movements of the animals when beam of light falling on the photo cell was cut off by the animals individual animals were reed into the activity care and left for 5 min to record the no. of movements¹⁹.

Biochemical Parameters Estimation:

Oxidative stress was determined by estimating of glutathione (GSH) level and estimation of malondialdehyde (MDA) to study lipid peroxidation, biochemical parameter for cholinergic dysfunction was estimated by measuring acetylcholinesterase (AChE) enzyme in freshly prepared brain homogenate after behavior study.

Brain Tissue Homogenate Preparation:

The mice were sacrificed under ether anesthesia. The skull was cut open and the brain was removed quickly. Thereafter brain tissue was cleaned with chilled normal saline and kept on the ice. A 10% (w/v) homogenate of brain in Sodium phosphate buffer (0.1M, pH 7.4) was prepared by using a homogenizer (Ika) and used for biochemical estimation.

Estimation of Malondialdehyde (MDA):

Tissue homogenate was mixed with 30% trichloroacetic acid (TCA), 5N HCL and 2 % w/v 2-thaiobarbituric acid after this mixture was heated for 15 min at 90 ̊C in a water bath and then centrifuged at 12.000rpm for 10 min. Supernatant was measured spectrophotometrically at 532nm. Tetraethoxypropane was taken as standard. MDA concentration was expressed as nmol/mg protein.^20,31

Estimation of Glutathione (GSH):

Equal amount of 10% trichloroacetic acid was mixed with brain tissue homogenate and centrifuge (4°C) at 10,000rpm for 20 min. 2ml of phosphate buffer of pH 8.4, 0.5ml of DTNB and 0.4ml of double distilled water was added with 0.1ml supernatant. Quickly the absorbance was taken at 412nm. Glutathione was taken as standard. GSH concentration was expressed as µg/mg protein^21,33

Biochemical Parameters of Cholinergic Function:

Estimation of Acetylcholinesterase (AChE) Activity:

The brain homogenate was centrifuged at 4°C for 60 min. Supernatant was collected and used for acetylcholinesterase estimation.²² Enzyme activity was measured by UV visible Spectrophotometer at 412nm with an interval of 15 sec.

Protein Estimation:

Protein was measured by the method of Lowry et al., (1951).²³ Bovine Serum Albumin (BSA, 1mg/ml) was taken as standard.

Histopathological Investigations:

Mice brain tissue was dissected our and allowed to be fixed in 10% Formalin.

RESULTS:

Effect of Naringenin on Cadmium chloride Induced Memory Impairment Using MWM Test:

Spatial learning test was performed by using MWM test which is shown in Fig 1. The animals treated with Cadmium Chloride (CD) (5mg/kg), showed longer escape latency, throughout all training sessions when compared to control and treatment group (p<0.001). Cadmium chloride+ naringenin (40mg/kg.po)(CD+NR) treated mice significantly reduced mean latency time from 4^th session onwards (p<0.001). While cadmium chloride + naringenin (20mg/kg) (CD+NRL) dose did not show the significant difference in mean latency time. Further naringenin (40mg/kg), significantly reversed memory impairment induced by cadmium chloride in mice as compared to cadmium treated group and this effect is comparable to standard group (Donepezil 5mg/kg).

Figure 1: Cadmium Chloride (CD) caused constant memory impairment. CD+NRL group (naringenin dose 20mg/kg) did not show any improvement in memory throughout the all five sessions. Control group showed retention of learned task from 3^rd session onwards; CD+NR (naringenin 40 mg/kg) group showed retention of learned task from 4^th session onwards and this showed protective effect of naringenin (40mg/kg, PO) on CD induced memory impairment in MWM trial. Data values are expressed as mean latency time (s) ±S.E.M. ***Significant difference (***P<0.001) in comparison to first session. Data were analyzed by one-way ANOVA followed by Tukey's post -hoc test.

Effect of Naringenin in Elevated plus maze (EPM) Trial:

In acquisition trial (29^th day of start of experiment) naringenin treatment for 28 successive days did not significantly affect transfer latency (TL) time of mice as compare to control group and CD treated group. Naringenin treatment (40mg/kg po) significantly reduced TL time of mice on retention trial (30^th day trial) when compared to the CD treated group (negative control) (p<0.001), this showed considerable memory improvement. Naringenin (20mg/kg) did not have any significant effect. Cadmium chloride (05mg/kg. po) significantly increase TL time in mice, indicating memory loss. Naringenin treatment (40mg/kg. p.o) significantly reversed cadmium chloride induced memory impairment in mice. (Table1, Fig. 2)

Table 1: Effect of naringenin on cadmium chloride induced memory deficit on EPM:

Treatment	Dose (mg/ kg.)	TL (sec) of 29^thday	TL (see) on 30^thday
Control `	10ml/kg	78±14.69	22.75±11.17
CD Treated	5	88.75±2.16	75.75±15.93^a
CD+ NRL	20	78±16.49	64.5±5.72
CD+ NR	40	74±13.45	24.5±5.72^b
CD+DN	5	26.25 ±8.75	23±9.28^b

values are presented as mean ±SEM. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test. (n=5), comparison has been made for retention trial (30^th day) among groups, ^a p<0.001 in comparison to control group, ^b p<0001 in comparison to cadmium treated group

Figure 2: Transfer latency time (Day 1) on acquisition trial and (Day 2) on retention trial, n=5 in each group, values are expressed as mean ± SEM. Data were analyzed by one- way ANOVA followed by Tukey’s post hoc test. [*** (p<0.001) CD treated (5mg/kg, p.o) when compared with control group. ###(p<0.001) when compared with CD Treated group]

Effect of Naringenin on Cadmium Chloride Induced Toxicity in Acetylcholinesterase (AChE) Activity:

Acetylcholinesterase activity (µmole/min/mg protein) was increased significantly (p< 0.001) in cadmium chloride treated (5mg/kg, p.o) group when compared with control group which was reversed by naringenin 40 mg/kg (p< 0.001) (Figure 3)

Figure: 3 Effect of Naringenin treatment (20 and 40mg/kg, p.o.) on Acetylcholinesterase activity. N=5, the results are presented as mean ± SD. All tests were performed by using One-way ANOVA followed by Tukey's post hoc test. *** (p<0.001) when compared with control group, ### (p<0.001) when compared with CD treated group.

Effect of Naringenin on Oxidative Stress in Cadmium Chloride Treated Mice:

Reduced Glutathione (GSH) Level:

Cadmium chloride treated (5mg/kg p.o) animal showed marked (p< 0.01) depletion of GSH level when compared to control group. This depletion was significantly prevented by Naringenin (40mg/kg.2.0) (p <0.001) and Donepezil (5mg/kg. p.o) group (p<0.001). (Figure 4)

Malondialdehyde (MDA) Level:

Cadmium chloride treatment (5mg/kg. p.o) group showed significant increase in MDA level (p<0.001) in comparison to control group. This rise in MDA level was attenuated in the brain of naringenin treated (40mg.p.) mice and Donepezil treated (5mg/kg, po) mice. MDA level in naringenin treated group was significantly (p<0.001) lower than CD treated mice. (Figure 5)

Figure: 4 Effect of Naringenin treatment (40 mg/kg, po) on GSH level, n=5, the results are expressed as mean± SEM. All tests were performed by using One-way ANOVA followed by Tukey's post hoc test. [** (p<0.01) when compared with control group. ### (p<0.001) when compared with CD treated group].

Figure: 5 Effect of Naringenin treatment (20 and 40mg/kg, p.o) on MDA level. n=5, the results are expressed as mean ± S.E.M All tests were performed by using One-way ANOVA followed by Tukey’s post -hoc test. [***(p<0.001) significance difference as compared with control group. ##(p<0.01) significance difference as compared with CD treated group].

Histopathological Study:

In control group microscopic examination of cortex revealed normal cell bodies and cortical tissues (Fig.6a). In cerebellum normal granular and molecular layers as well as purkinje cells were visible (Fig 6b). In CD treated group microscopic examination of mid brain revealed presence of degenerative changes in neurons (Fig 6c). In hippocampus degenerative changes in detatus gyrus and increase in neurophil content were visible (Fig 6d). In cerebellum degeneration of purkinje cells as well as degeneration in granular layer was found (Fig 6e). In CD+ NR group (40mg/kg) microscopic examination revealed the presence of proportionally normal neurons along with the degenerated neuronal cell bodies in cerebral cortex (Fig.6 f). In mid brain gliosis was reported to protect the neurons (fig 6g). In donepezil treated group microscopic examination of selection of cortex revealed presence of majority of neuronal cell bodies were normal with few degenerating neurons (fig 6h). In mid brain gliosis to protect neurons (fig 6i).

DISCUSSION:

Cadmium is widely used in various industries however it is toxic heavy metal; Cadmium has long retention time in tissues and by inducing oxidative stress it contributes to serious pathological conditions. Various mechanisms have been suggested to explain brain injury induced by Cadmium, which includes increased lipid peroxidation and its effects on membrane components, cadmium also deplete glutathione level thereby inducing oxidative stress²⁴. Therefore, mitigation of cadmium toxicity can be achieved through the complementing antioxidant therapy. In this perspective the present study also confirmed that the administration of naringenin (40mg/kg) significantly protect neurodegeneration against the toxic effects exerted by cadmium. Cadmium represents a dangerous environmental and industrial pollutant ³⁰. We have studied various parameters such as GSH level, MDA level, AchE level in cadmium induced memory impairments and further explored the effect of naringenin on cadmium chloride induced oxidative stress, and cholinergic dysfunction for the memory impairment. We have evaluated behavioral parameters by using MWM and Elevated EPM.

Cadmium chloride (5mg/kg. po) administration for 28 days, showed persistent memory deficit in MWM and EPM as well as oxidative stress which may be a cause of memory impairment. Necrosis in brain part was also found in histopathology of brain tissue. Cadmium chloride treated group showed decreases level of GHS, increased level of MDA which are hallmark sign of oxidative stress and leads to impairment of memory and presence of neurotoxicity in mice brain. In MWM trial, cadmium chloride treated mice were not able to reach to the stand with in the latency period. In EPM transfer latency of cadmium chloride treated mice was greater in comparison with control group, which indicate memory impairment.

Treatment of Naringenin (40mg/kg p.o) in cadmium chloride treated mice improved spatial and condition avoidance memory as evidenced by improved performance in MWM and EPM test. Earlier studies in different memory impairment models had been done but effect of bioactive flavonoid naringenin neurotoxicity induced by cadmium chloride have not been reported. In Morris water maze lest administration of cadmium chloride in combination with naringenin (CD+NR) (40mg/kg po) showed improvement in learning behavior^29,32.In EPM trial in CD+ NR group decrease in transfer latency in retention trial showed the memory improvement in mice brain in comparison with CD treated mice.

Further Acetyl cholinesterase (AChE) is a key enzyme in detecting the neurotoxic effect of certain heavy metals. Number of studies has reported that the free radicals production could be associated with the decreased activity of brain AChE. In this study we have observed significant increase in AChE activity in cadmium treated group as compared to control group. However, treatments with naringenin (40mg/kg, (CD+NR) decrease the acetyl cholinesterase level as compared to CD Treated group. Hence, it was confirmed that treatment with naringenin showed protective activity against cadmium induced neurotoxicity in brain. Pretreatment with naringenin (40mg/kg) may have toned up the antioxidant defense in the brain of mice and conferred protection against oxidative stress.

CONCLUSION:

Human are highly exposed to cadmium through occupational, food contaminant, smoking and environment. Various studies reported the importance of bioactive food compounds in reversing the damage caused by these heavy metals. Flavonoids, which are also freely available as dietary supplements, represent the most plant polyphenols in human diet ^25. Polyphenols are free radical scavengers and also help in metal sequestration²⁶. Naringenin (40mg/kg) treatment in diseased mice improved spatial memory and condition avoidance memory evidenced by improved performance in MWM and EPM test. Naringenin protected cadmium induced neurodegeneration by its antioxidant effect and inhibition of ACE activity ^27. The effect of naringenin in neurotoxicity induced by cadmium chloride exposure has not been reported yet. In the present study naringenin showed its potential against memory deficit induced by cadmium chloride. Hence it be concluded that Naringenin protected cadmium induced neurodegeneration attributed to its antioxidant properties and inhibition of ACE activity ²⁸. This activity of Naringenin can be further explored in neurodegenerative conditions for detecting brain damage by reducing oxidative stress and by enhancing AChE activity.

REFERENCE:

1. K. Hans Wedepohl, “The composition of the continental crust,” Geochimica et Cosmochimica Acta. vol. 59, no. 7, pp. 1217–1232, 1995.

2. D. R. Abernethy, A. J. DeStefano, T. L. Cecil, K. Zaidi, and R. L. Williams, “Metal impurities in food and drugs. Pharmaceutical Research. 2010; 27(5): 750-755.

3. Shukla A, Shukn GS and Srimal RC (19% Cadmium Induced alterations in Blood-brain barrier permeability and its possible correlation with decreased microvessel antioxidant potential in mat. Man and Experimental Toxicology. 2007; 13(1): 400-405.

4. Kumar R. Agarwal KA and Seth KP: Oxidative stress mediated neurotoxicity of Cadmium toxicology Letters;1996; 89(1): 63-69.

5. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG: Selenium: Biochemical role as a component of glutathione peroxidase. Science. 973; 179(1): 588-590.

6. Bhosale UA, Yegnanarayan R, Pophale PD; Study of central nervous system depressant and behavioral activity of an ethanol extract of Achyranthes aspera (Agadha) in different animal models. International Journal of Applied and Basic Medical Research. 2011; 1(2); 104-108.

7. D. Dhingra, M. Parle, and S. K. Kulkarni. Memory enhancing activity of Glycyrrhiza glabra in mice. Journal of Ethnopharmacology. 2014; 91(1): 361–365.

8. Ankita Tripathi, Himani Awasthi, Dan Bahadur Rokaya, Dipti Srivastava and Vivek Srivastava; Antimicrobial and wound healing potential of dietary flavonoid naringenin. Natural Product Journal. 2018;61-68.

9. A.M. Alam, N. Subhan, M.M. Rahman, S.J. Uddin, H.M. Reza, S.D. Sarker, Effect of citrus flavonoids, naringin and naringenin, on metabolic syndrome and their mechanisms of action. Adv. Nutr. 2018; 5(1), 404–417.

10. Y. Chtourou, H. Fetoui, R. Gdoura, Protective effects of naringenin on iron-over load induced cerebral cortex neurotoxicity correlated with oxidative stress, Biol. Trace Elem. Res. 2014; 158(1), 376–383.

11. K. Gopinath, G. Sudhandiran, Naringin modulates oxidative stress and inflammation in 3nitropropionic acid-induced neurodegeneration through the activation of Nrf2 signaling pathway. Neuroscience. 2014; 227(1),134–143.

12. Youdim KA, Dobbie MS, Kuhnle G, Proteggente AR, Abbott NJ, Rice-Evans C: Interaction between flavonoids and the blood–brain barrier: in vitro studies. J Neurochem. 2003; 85(1): 180–192.

13. Monica Cavia-Saiz, Mari Busto, Maria Concepción Pilar-Izquierdo, Natividad, Ortega, Manuel Perez-Mateos, Pilar Muñiz; Antioxidant Properties, Radical Scavenging Activity and Biomolecule Protection Capacity of Flavonoid Naringenin and Its Glycoside Naringin: A Comparative Study. J Sci Food Agric. 2010; 90(7): 1238-1244.

14. Naskar S, Islam A, Mazumder UK, Saha P, Haldar PK and Gupta M. In vitro and in vivo antioxidant potential of hydromethanolic extract of Phoenix dactylifera fruits. J Sci Res. 2010; 2(1): 144‑157.

15. R.S. Adnik P.S. Gavarkar and S. K. Mohite, UPSR, Evaluation of antioxidant effect of Citrullus vulgaris against cadmium-induced neurotoxicity in mice brain. 2015; 6(10).

16. Tota S, Awasthi H, Kamat PK, Nath C, Hanif K; Protective effect of quercetin against intracerebral streptozotocin induced reduction in cerebral blood flow and impairment of memory in mice. Behavioural Brain Research. 2013; 209(1): 73-79.

17. R. Morris, “Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods. 1978: 11(1) 47–60.

18. D. Dhingra, M. Parle, and S. K. Kulkarni, “Memory enhancing activity of Glycyrrhiza glabra in mice. Journal of Ethnopharmacology. 91: 361–365, 2004.

19. Bhosale UA, Yegnanarayan R, Pophale PD; Study of central nervous system depressant and behavioral activity of an ethanol extract of Achyranthes aspera (Agadha) in different animal models. International Journal of Applied and Basic Medical Research. 1(2); 104-108,2011.

20. Colado M.I., Green A.R.: A study of the mechanism of MDMA (‘Ecstasy')-induced neurotoxicity of 5-HT neurones using chlormethiazole, dizocilpine and other protective compounds. Br. J. Pharmacology. 1994; 111(1):131–136.

21. Ellman GL. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics.1959; 82 (1), 70-77.

22. Ellman GL, Courtney KD, Andres V, Jr., Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 1961; 7(1), 88-95.

23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951; 193 (1), 265-275.

24. Sandeep K Agnihotri , Usha Agrawal , Ilora Ghosh. Brain Most Susceptible to Cadmium Induced Oxidative Stress in Mice. J Trace Elem Med Biol. 2015 Apr; 30; 184-93.2015.

25. M. Ashraful Alam, Nusrat Subhan, M. Mahbubur Rahman, Shaikh J. Uddin, Hasan M. Reza, Satyajit D. Sarker. Effect of Citrus Flavonoids, Naringin and Naringenin, on Metabolic Syndrome and Their Mechanisms of Action, American Society for Nutrition. Adv. Nutr. 2014; 5(1): 404–417.

26. Pandey K B, Syed Ibrahim Rizvi Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longev. 2009; 2(5): 270–278.

27. V. Uma Rani, M. Sudhakar, A. Ramesh. Protective effect of Pueraria tuberosa Linn. in arsenic induced nephrotoxicity in rats. Asian J. Pharm. Res. 2017; 7(1): 15-20.

28. Dibyajyoti Saha, Ankit Tamrakar. Xenobiotics, Oxidative Stress, Free Radicals Vs. Antioxidants: Dance of Death to Heaven’s Life. Asian J. Res. Pharm. Sci. 1(2): April-June; Page 2010; 36-38.

29. Ghanshyam B. Jadhav, Ravindranath B. Saudagar. Free radical Scavenging and Antioxidant Activity of Punica granatum Linn. Asian J. Res. Pharm. Sci. 2014; 4(2): 51-54.

30. Vadapalli Umarani, Muvvala Sudhakar, Alluri Ramesh. Protective Potential effect of Gloriosa superba Linn. against lead Nitrate Induced Oxidative stress in Rats. Asian J. Res. Pharm. Sci. 2019; 9(3): 186-192.

31. Samir Derouiche, Khaoula Zeghib, Safa Gherbi, Yahia Khelef. Protective effects of Aristolochia longa and Aquilaria malaccensis against lead-induced oxidative stress in rat cerebrum. Asian J. Res. Pharm. Sci. 2019; 9(1): 57-63.

32. M. Padmaja, B. Praveen, B. Raghunath Reddy, P. Venu Madhav. Therapeutic Efficiency of Leucas aspera (Lamiaceae) on Lead Acetate Induced Nephrotoxicity and Oxidative Stress in Male Wistar Rat. Asian J. Research Chem. 2011; 4(10): Oct., Page 1515-1519.

33. Ena Gupta, Abubakar Mohammed, Shalini Purwar, Syed Ibrahim Rizvi, Shanthy Sundaram. Diminution of oxidative stress in alloxan-induced diabetic rats by Stevia rebaudiana. Res. J. Pharmacognosy and Phytochem. 2017; 9(3): 158-166.

34. Vadivelan R, Umasankar P, Dipanjan Mandal, Shanish A, Dhanabal SP, Elango K. Oxidative Stress Induced Diabetic Nephropathy. Research J. Pharmacology and Pharmacodynamics. 2010; 2(5): 321-323.

35. AV Jaydeokar, DD Bandawane, SS Nipate, PD. Chaudhari. Natural Antioxidants: A Review on Therapeutic Applications. Research J. Pharmacology and Pharmacodynamics. 2011; 4(1): 55-614.

36. Nirjala Laxmi Madhikarmi, Kora Rudraiah Siddalinga Murthy. Study of oxidative stress and antioxidants status in iron deficient anemic patients. Research J. Science and Tech. 2012; 4(4): 162-167.

Received on 21.07.2020 Modified on 18.08.2020

Research J. Pharm. and Tech. 2021; 14(8):4053-4059.

DOI: 10.52711/0974-360X.2021.00702