Dr. Pranesh PawaskarFYR

Dept. Of Pharmacology

LTMMC, Sion, Mumbai 400022

Date : 08/08/2016

1

ANTI-OXIDATIVE EFFECTS OF HESPERETIN AGAINST LEAD

ACETATE INDUCED OXIDATIVE STRESS IN RATS.

2

ARTICLE

• AUTHOR : Jicang Wang, Huali Zhu, Zijun Yang, Zongping Liu.

• JOURNAL : Indian Journal Of Pharmacology, Year 2013, Volume 45,

Issue 4

• PLACE OF STUDY : Department Of Veterinary Medicine, College Of

Animal Science And Technology, Henan University Of Science And

Technology, No.70, Tianjin Road, 471003, Luoyang. Peoples Republic

Of China. 3

ARTICLE

• OBJECTIVES : The Current Study Was Carried Out To Evaluate

Antioxidant Activity Of Hesperetin Against Lead Acetate-induced

Oxidative Stress.

• MATERIALS AND METHODS : The Male Rats Were Treated With

Hesperetin In Combination With Lead Acetate (500 Mg/L).

• RESULTS : Hesperetin succeeded in improving lead induced deranged

levels of enzymes (SOD), (CAT) and (GPx). 4

ARTICLE

• CONCLUSION : Hesperetin Shows Antioxidant Activity And Plays A

Protective Role Against Lead-induced Oxidative Damage.

• KEYWORDS : Antioxidants, Hesperetin, Lead, Oxidative stress, Rats.

5

INTRODUCTION• Toxic Potential Of Lead.

• Food, Drinking Water, Dust.

• Oxidative Stress With Subsequent Lipid Peroxidation (LP) Induced By Production Of

Reactive Oxygen Species (ROS). The Release Of Malondialdehyde (MDA) Is An Index

Of LP.

• Bones And Blood Contain 90% And 4% Of The Total Body Lead, Respectively; The

Remaining Lead Residues Are Mainly Found In Liver And Kidneys.

• The Liver And Kidneys Play A Major Role In Elimination Of Lead; Therefore, They

Account For The Toxic Actions.

6

LEAD

DUST

FOOD

BONES

DRINKING WATER

LIVER AND KIDNEY7

• Heavy Metals Target Peroxidative Decomposition Of Membrane Lipids.

• Antioxidant Enzymes Such As Catalase (CAT), Superoxide Dismutase

(SOD) And Glutathione Peroxidase (GPx).

• Flavonoids Are The Most Abundant Antioxidants In Plants And In

Human Diet.

• They Have Attracted A Great Deal Of Attention In Recent Years For

Their Antioxidative, Antibacterial, And Hepatoprotective Activities.8

HESPERETIN• Hesperetin (5,7,3’-trihydroxy-4-methoxyl

Flavanone).

• One Of The Most Abundant Flavonoids Found

In Citrus Fruits.

• Anti-inflammatory, Anti-carcinogenic,

Antihypertensive, Antiatherogenic Effects, And

Antioxidant Properties.

• Powerful Radical Scavenger That Promotes

Cellular Antioxidant Defence-related Enzyme

Activity.

9

10

CHEMICALS

• Alanine Aminotransferase (ALT)

• Aspartate Aminotransferase (AST)

• Urea

• Uric Acid

• Creatinine

• Reduced Glutathione

• MDA

• CAT

• SOD

• GPx

• Hesperetin (Purity: >98%, High Performance Liquid Chromatography (HPLC))

11

CHEMICALS• Alanine Aminotransferase (ALT), Aspartate

Aminotransferase (AST), Urea, Uric Acid, Creatinine,

MDA, Reduced Glutathione (GSH), CAT, SOD, And GPx

Kits were purchased from Nanjing Jiancheng

Bioengineering Institute (Nanjing, P.R.China).

• Lead Acetate Was Purchased from Sigma-Aldrich (St.

Louis, MO, USA)

• Hesperetin was purchased from Xi’an Xiaocao Botanical

Development Co., Ltd (Xi’an, P.R.China). 12

ANIMALS AND TREATMENT• Adult Male Sprague-Dawley Rats (8-weeks Old,

Weighing 180 G). Purchased from Experimental

Animal Centre Of Henan Province (Zhengzhou,

China).

• The Rats Were Maintained Under Standard

Laboratory Conditions (Temperature 24 ± 2°C,

Natural Light-dark Cycle), And Had Free Access To

Drinking Water And A Commercial Standard Pellet

Diet.

• Randomly Divided Into Four Groups, Six Rats In

Each.13

ANIMALS AND TREATMENT

All ExperimentalGroups Received Test Solutions In A Volume Of 10 Ml/Kg.

GROUP ANIMAL

Group 1 Untreated Control

Group 2 Treated Orally With Hesperetin (50mg/kg/day)

Group 3 Treated Orally With Lead Acetate (500mg Pb/L)

Group 4 Treated Orally With Hesperetin (50mg/kg/day) + Lead Acetate (500mg Pb/L)

14

ANIMALS AND TREATMENT

• The Experiments Lasted For 8 Weeks.

• At The End Of The Experimental Period, Blood Samples Were Collected

From All Animals. The Serum Obtained After Centrifugation.

• Then All Animals Were Sacrificed; The Liver And Kidney Were Removed,

Weighed And Washed Using Chilled Saline Solution.

• Tissue Minced And Homogenised with chilled saline And Then

Centrifuged Resulting Supernatant Used For Biochemical Assays.15

ASSAYS

• Spectrophotometry : AST, ALT,

Urea, Uric Acid, Creatinine.

• Analysis Of Liver And Kidney MDA

And GSH Levels Were Performed

With Commercial Kits.

• The Activities Of Liver And Kidney

SOD, CAT, And GPx Were Also

Assayed Using Commercial Kits.16

STATISTICAL ANALYSIS

• Data Were Expressed As Mean ± SE Of A

Number Of Experiments (N = 6).

• Statistical Significance Was Evaluated By

One-way Analysis Of Variance (ANOVA) Using

SPSS 15.0 Software Package

• Values Were Considered Statistically

Significant When P < 0.05. 17

18

EFFECT OF HESPERETIN ON LEAD-INDUCED SERUM BIOCHEMICAL PARAMETERS OF LIVER AND KIDNEY FUNCTION IN RATS

19

Group Parameter Control Hesperetin Lead Lead + Hesperetin

ALT (IU/L) 20.14 ± 2.10 15.17 ± 1.29 39.04 ± 3.61 21.06 ± 1.36

AST (IU/L) 61.65 ± 10.10 49.21 ± 5.49 102.55 ± 7.90 66.39 ± 8.73

Urea (mmol/L) 11.94 ± 0.61 11.74 ± 0.71 15.45 ± 0.75 12.60 ± 0.77

Uric Acid (µmol/L)

49.60 ± 6.69 52.01 ± 2.36 96.99 ± 8.97 72.09 ± 9.11

Creatinine (µmol/L)

120.48 ± 4.38 121.46 ± 6.12 202.32 ± 14.55 125.71 ± 8.80

EFFECT OF HESPERETIN ON LEAD-INDUCED LIPID PEROXIDATION AND THE ANTIOXIDANT

STATUS OF RAT LIVER

20

Group Parameter Control Hesperetin Lead Lead + Hesperetin

MDA (nmol/mgprotein)

0.85 ± 0.07 0.80 ± 0.028 1.16 ± 0.07 0.96 ± 0.05

GSH (mg/g protein) 3.23 ± 0.34 3.31 ± 0.26 2.20 ± 0.24 3.18 ± 0.37

SOD (IU/mg protein)

3.32 ± 0.22 3.37 ± 0.26 2.23 ± 0.28 3.06 ± 0.35

CAT (IU/mgprotein)

50.66 ± 4.29 55.70 ± 2.64 37.78 ± 2.75 47.69 ± 4.11

GPx (IU/mgprotein)

2420.81 ±130.85

2797.92 ±247.55

1884.0 ±147.40

2413.61 ± 158.50

EFFECT OF HESPERETIN ON LEAD-INDUCED LIPID PEROXIDATION, AND THE

ANTIOXIDANT STATUS OF RAT KIDNEY

21

Groups Parameter Control Hesperetin Lead Lead + Hesperetin

MDA (nmol/mg protein)

0.62 ± 0.04 0.65 ± 0.05 0.97 ± 0.09 0.72 ± 0.04

GSH (mg/g protein) 2.44 ± 0.23 2.48 ± 0.22 1.70 ± 0.08 2.27 ± 0.17

SOD (IU/mg protein)

4.73 ± 0.60 4.77 ± 0.80 2.93 ± 0.45 4.70 ± 0.40

CAT (IU/mg protein)

19.80 ± 1.90 19.38 ± 1.38 10.40 ± 0.57 14.86 ± 1.44

GPx (IU/g protein) 4,304.86 ±356.71

4,323.54 ±394.60

3,159.34 ±175.79

4,081.72 ±216.93

22

DISCUSSION

Liver Is One Of The Targets For Lead Accumulation And Responds To

Toxic Insult Caused By Lead By Increasing Activities Of Transaminases.

Administration Of Hesperetin (50 Mg/Kg) Attenuated Lead-induced

Hepatotoxicity As Shown By Decreased Levels Of ALT And AST.

It Has Been Reported That Hesperetin Decreases Liver Marker Enzymes

During 7,12-dimethylbenz(a)anthracene And Cadmium induced

Hepatotoxicity Via Its Antilipoperoxidative Activity.

The Above Effects Clearly Indicate That Hesperetin May Offer Protection

By Stabilizing The Cell Membrane In Hepatic Damage Induced By Lead. 23

DISCUSSION

• The Kidney Is Vulnerable To Damage Due To Larger Perfusion And Increased Concentration Of Excreted Compounds Which Occur In Renal Tubular Cells.

• Urea : Protein Metabolism.

• Uric Acid : Purine nucleotide.

• Elevated Urea = Increased Protein Catabolism = Due To Increased Synthesis Of Arginase.

• Elevated Serum Uric Acid = Increased Endogenous Oxygen Species = Due To Uric Acid Is Scavenger Of Peroxynitrite.

• The Administration Of Hesperetin Protects Kidney Function From Lead Intoxication As Indicated By Significant Restoration Of Serum Urea, Uric Acid, And Creatinine. 24

DISCUSSION

• Lead Is Known To Produce Oxidative Damage In Liver And Kidney By

Enhancing Lipid Peroxidation.

• Glutathione (GSH) = Non Enzymatic Action = Sh Group = Reducing

Property.

• In This Study We Observed A Significant Increase In MDA Level And A

Decrease In GSH Level In Lead-treated Rat Liver And Kidney.

• Hesperetin Markedly Increased GSH Level And Decreased MDA Level In

Lead-treated Rat Liver And Kidney.25

DISCUSSION

• Lead Has High Affinity For Sh-Groups Or Metal Cofactors In

Antioxidant Enzymes And Molecules, Which Results In A Reduction

In Antioxidant Enzyme Activities, Such As SOD, CAT, And GPx. SOD,

CAT, And GPx Form The First Line Of Defence Against ROS And

Decrease In Their Activities Contribute To Oxidative Stress In The

Tissues.

• CAT Is A Major Antioxidant Enzyme Having Heme As The Prosthetic

Group. Lead Is Known To Reduce Absorption Of Iron In

Gastrointestinal Tract And To Inhibit Heme Biosynthesis.

• GPx Is A Hydroperoxide-degrading Enzyme, Which Requires

Selenium For Activity And Was Decreased In Lead poisoned Rats.26

27

CONCLUSION

• This Study Demonstrated That

Lead Acetate Was Capable Of

Causing Marked Oxidative Damage

And Inhibited Activities Of

Antioxidant Enzymes.

• Treatment With Hesperetin Could

Minimize These Hazards, And May

Be Useful And Reasonable In The

Treatment Of Lead Toxicity.28

CRITICISM

29

TITLE

• Title : Incomplete.

• Original Title : Antioxidative Effects Of

Hesperetin Against Lead Acetate-induced

Oxidative Stress In Rats.

• Proposed Title : Effect Of Anti-oxidant

Hesperetin Against Oxidative Stress Of Lead In

Sprague Dawley Rat.

30

ABSTRACT• Abstract : Incomplete.

• Research Objective - Clear.

• Keywords - Adequate.

• Result And Conclusion Of Study - Proper.

• Species And Strain Of Animal - Not Mentioned.

• Method Of Experiment - Not Mentioned. 31

BACKGROUND• It Does Not Include Sufficient Scientific

Background (Including Relevant References To

Previous Work).

• However Motivation Of Study Was Clear.

• Still Background Lacks Experimental Approach And

Context.

• Does Not Explain Why The Animal Species And

Model Being Used Can Address The Scientific

Objectives.

• Primary Objective Of Study Is Clearly Described.

• Study Is Relevant To Human Biology. 32

ETHICS

• No Permission From

Animal Ethical

Committee Is

Taken.

33

STUDY DESIGN

• The Number Of Experimental And Control Groups Is Given.

• Any Steps Taken To Minimise The Effects Of Subjective Bias When Allocating

Animals To Treatment (E.G. Blinding of Procedure) And When Assessing Results

Is Not Mentioned.

34

EXPERIMENTAL PROCEDURE

• Dose Of Drug, Site And Route Of Administration

Is Mentioned But Reason Of Selecting Oral Route

Is Not Mentioned.

• Anaesthesia Or Analgesia Is Not Mentioned.

• Method Of Euthanasia Is Not Mentioned.

• Time Of Administration Of Drug Is Not

Mentioned.

• Also The Method Of Blood Sample Collection Is

Also Not Mentioned.35

EXPERIMENTAL ANIMALS

• Animal Species, Strain, Weight, Age And Sex Is

Mentioned.

• Source Of Animal Is Mentioned.

• Husbandry Conditions Like Temperature, Food, Water Is

Mentioned.

• Genetic Modification Status (E.G. Knockout Or

Transgenic), Genotype, Health/Immune Status, Drug Or

Test Naïve, Previous Procedures, Etc. Is Not Known.

• Type Of Cage, Bedding Materials Is Not Mentioned. 36

EXPERIMENTAL ANIMALS

• Assessments And Interventions That Were Carried

Out Prior To, During, Or After The Experiment Is Not

Mentioned.

• No. Of Animal In Each Group Is Mentioned However

Total No. Of Animals Used Is Not Mentioned.

• No Details Of Sample Size Calculation Is Provided.

• Animals Were Divided Into Groups Randomly.

• Experimental Outcomes Were Clearly Defined.37

STATISTICAL METHODS

• Details Of Statistical Method Is Given.

• N Value Is Mentioned

• P Value Is Mentioned

38

STATISTICS

• Number Of Animals Analysed In Each Group

Have Been Given In Absolute Numbers.

• Report The Results For Each Analysis Carried

Out, With A Measure Of Precision Is

Mentioned.

• Baseline Data : Relevant Characteristics And

Health Stats For Each Group Is Not Given.

• Values Of Standard Error And Confidence

Interval Are Lacking.39

ADVERSE DRUG EVENT

• Adverse Drug Event Is Not Mentioned.

• No Modification Or Protocol To Reduce Adverse Drug Event Is Done.

40

CONFLICT OF INTEREST

• Not Declared.

41

FUNDING

• Source Of Funding Or

Grants Are Not Mentioned.

42

REFERENCES STYLE

• Vancouver Style

Performed For

References.

43

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