اقليم كسدضتاى /العساق وشازة التعليم العالي والبخث العلمي جامعة الطليمانية كلية الطب
تكييم العاقة بن الركيص (اجسعة) ومكداز خاصية قهص اجروز احسة مادتي الطليبيهن ثهائي الطلطيهات والبهفوتيامن ي موذج اكطدة اهمغلوبن امطتخدث بواضطة نايرايت الصوديوم
زضالة مكدمة اى فسع اأدوية و جهة الدزاضات العليا ي كلية الطب /جامعة الطليمانية كحصء مو متطلبات نيل شهادة اماجطتر ي الصيدلة (اأدوية)
مو قبل بشسى حطو معسوف
باشساف الدكتوز ضعد عبدالسمو حطن
0202
54
Acknowledgments First and above all, I praise God, the almighty for granting me the capability to perform this work successfully. I gratefully thank my supervisor Dr. Saad A. Hussain for his valuable advice, encouragement, guidance and support from the very early stage to the final level of the work, as his positive and encouraging nature have provided a good basis for the present thesis. It is a pleasure to thank Dr. Aras A. Abdullah the dean of the College of Medicine, for his help and support. I appreciate the help of Dr. Mohammed A. Abdulla the dean of the College of Pharmacy for allowing me to work in the research lab and in the animal house of the college. I would like to thank the staff of Pharmacology Department-College of Medicine in particular Dr. Rasul M. Hasan and Dr. Zheen A. Ahmed for their kind help. I would like to thank the staff of Biochemistry Department, College of Medicine for allowing me to use the laboratory facilities of the department. I would also like to thank Dr. Munaf A. Abdulrazzaq for his help in in vitro section of the work. My special thanks to my husband for his personal support and I am grateful to him for his participation voluntarily in donating the blood samples for conducting the in vitro part of the study. Also, many thanks go to those volunteers who have given the blood for the in vitro part of the study. I am most grateful to Dr. Tavga A. Aziz for her help and providing me the gavage tube as well as demonstrating the technique of the work. Finally, I would like to show my gratitude to those who helped me in any respect during the completion of the work in particular Dr. Sana D. Jalal.
Bushra H. Marouf
I
Abstract Free radical formation in heme proteins is considered as a factor in mediating the toxicity of many drugs in oxidative stress. In addition to initiating free radical damage, heme proteins damage themselves. The xenobiotics and drug therapy-related toxicity, due to oxidative modification of hemoglobin (Hb), has been attributed in part to the uncontrolled oxidative reactions. A variety of antioxidant strategies to ameliorate potential oxidative damage in vivo have been suggested. The present study was designed to evaluate the concentration-effect relationship of the free radical scavenging properties of silibinin dihemisuccinate (SDH) and benfotiamine in nitrite-induced Hb oxidation in vitro and in vivo. Different concentrations of SDH and benfotiamine were added, before and after different time intervals of inducing Hb oxidation in erythrocytes lysate, and formation of methemoglobin (MetHb) was monitored spectrophotometrically each minute for 45 minutes; the same approach was utilized to evaluate the effect of the same concentrations of both compounds on the integrity of erythrocytes after induction of hemolysis with sodium nitrite. Moreover, the most effective dose of tested compounds was administered in rats before challenge with toxic dose of sodium nitrite, and MetHb formation was measured. The results showed that in both in vitro and in vivo models, SDH and benfotiamine successfully attenuating Hb oxidation after challenge with sodium nitrite; this protective effect was found to be not only related to the autocatalytic stage of Hb oxidation, though such effect was reported to be more prominent when both compounds administered before nitrite. In conclusion, SDH and benfotiamine can effectively, in concentrationdependent pattern, attenuate sodium nitrite-induced Hb oxidation and maintain integrity of red blood cells through free radical scavenging activity.
II
List of contents Contents
Page No.
Acknowledgments
I
Abstract
II
List of Contents
III
List of Figures
VIII
List of Tables
X
List of Abbreviations
XI
Chapter One: Introduction and literature review No.
Contents
Page No.
1
Introduction and literature review
1
1.1
Terminology and Definition of free radicals
1
1.2
Chemical Properties of Some ROS
4
1.3
Chemical Qualities and Reactivities of some ROS
6
1.3.1
Superoxide Anion Radical (O •2/HO•2)
6
1.3.2
Hydroxyl Radical (OH•)
7
1.3.3
Hydrogen Peroxide (H2O2)
8
1.4
Nitric Oxide (NO•), Peroxynitrite (ONOO –), and Other Members of the Family Sources of ROS
1.5
Free Radicals as a Cause of Oxidative Damage
11
1.5.1
Effects on Nucleic Acids
11
1.5.2
Effects on Lipids
13
1.5.3
Effects on Proteins
14
1.3.4
III
8 9
1.6
Defense Mechanisms of the Cell against Oxidative Stress
15
1.7
Methods for determination of ROS and radicals
17
1.8
Silibinin
18
1.8.1
Chemical composition
18
1.8.2
Pharmacokinetics
18
1.8.3
21
1.8.3.1
Pharmacodynamic and Medicinal properties of Silibinin Antioxidant Activity
1.8.3.2
Hepatoprotective activity
22
1.8.3.3
Effect on alcoholic liver disease
22
1.8.3.4
Anti-inflammatory activity
23
1.8.3.5
Immunomodulatory activity
23
1.8.3.6
Antiviral activity
23
1.8.3.7
Glycemic and lipidemic control
24
1.8.3.8
Anti-fibrotic activity
24
1.8.3.9
Anti-carcinogenic/anti-tumorigenesis activity
25
1.9
Benfotiamine
25
1.9.1
Chemical Composition
25
1.9.2
Pharmacokinetics
27
1.9.3
Pharmacodynamics
27
1.9.3.1
AGE-dependent pharmacological action benfotiamine AGE-independent pharmacological action benfotiamine Aim of the study
1.9.3.2 1.10
21
of
28
of
29 31
Chapter Two: Materials and Methods 2
Materials and Methods
32
2.1
Chemicals
32
IV
2.2
Instruments
32
2.3
General Consideration
32
2.4
In vitro study
35
2.4.1
Blood sample collection and preparation of lysate
35
2.4.2
Preparation of samples and analysis
35
2.4.2.2
Preparation of drug solutions
35
2.4.2.2.1
Preparation of Silibinin dihemisuccinate disodium solution Preparation of Benfotiamine solution
35
2.4.2.2.2 2.4.2.3
2.4.2.4
2.4.2.5
2.4.2.6 2.4.2.7
Evaluation of effect of different SDHS concentrations on the time course of nitriteinduced oxidation of hemoglobin Evaluation of the effect of SDHS on the time course of MetHb formation at various time intervals from nitrite addition Evaluation of effect of different benfotiamine concentrations on the time course of nitriteinduced oxidation of hemoglobin Evaluation of effect of benfotiamine on the time course of MetHb formation at various time intervals from nitrite addition Evaluation of osmotic fragility of erythrocytes
35 36
36
37
37 37 38
2.5
Evaluation of the effect of SDH on the nitriteinduced osmotic fragility of red blood cells Evaluation of benfotiamine on the nitrite-induced osmotic fragility of red blood cells In vivo study
2.5.1
Experimental animals
39
2.5.2
Treatment schedule
39
2.5.3
Measurement of methaemoglobin in blood
41
2.6
Statistical Analysis
41
2.4.2.7.1 2.4.2.7.2
38 39
Chapter Three : Results 3
Results
42
V
3.1
3.1.1
3.1.2 3.1.3
3.1.4 3.1.5 3.1.6
3.1.7 3.2 3.2.1 3.2.2 3.2.3
3.2.4 3.3 3.3.1
Time-course for oxidation of hemoglobin and MetHb formation with sodium nitrite: in vitro study Effect of different concentrations of silibinin dihemisuccinate disodium on the time-course of nitrite-induced oxidation of Hb and MetHb formation Effect of addition of silibinin dihemisuccinate disodium at different time intervals Concentration-response relationship for the radical scavenging activity of silib inin dihemisuccinate disodium in nitrite-induced Hb oxidation Effect of different concentrations of benfotiamine on the time-course of nitrite-induced oxidation of Hb and MetHb formation Effect of addition of benfotiamine at different time intervals Concentration-response relationship for the radical scavenging activity of benfotiamine in nitriteinduced Hb oxidation in vitro Comparison between radical scavenging activity of SDHS and benfotiamine in nitrite-induced Hb oxidation in vitro
42
44
47 50
52 55 58
60
Osmotic fragility test: in vitro study
62
Effect of different concentrations of SDHS on osmotic fragility of red blood cells Concentration-response relationship of SDHS on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro Effect of different concentrations of benfotiamine on osmotic fragility of red blood cells Concentration-response relationship of benfotiamine on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro In vivo study
62
Effect of silibinin dihemisuccinate on nitriteinduced MetHb formation in rats
70
VI
64 66
68 70
3.3.2
Effect of benfotiamine on nitrite-induced MetHb formation in rats
70
Chapter Four: Discussion 4 4.1
4.1.1
4.1.2 4.2
Discussion
72
Time-course for oxidation of hemoglobin and MetHb formation with sodium nitrite: in vitro study Effect of different concentrations of SDHS on the time-course of nitrite-induced oxidation of Hb and MetHb formation in vitro Effect of different concentrations of benfotiamine on the time-course of nitrite-induced oxidation of Hb and MetHb formation in vitro Osmotic fragility test: in vitro study
72
73
76 78 79
4.3
Effect of different concentrations of SDHS on osmotic fragility of red blood cells: in vitro study Effect of different concentrations of benfotiamine on osmotic fragility of red blood cells: in vitro study In vivo study
4.3.1
Protective effect of silibinin: in vivo study
83
4.3.2
Protective effect of benfotiamine: in vivo study
84
Conclusion
86
Recommendation for further work
86
References
87
4.2.1 4.2.2
Abstract in Arabic Abstract in Kurdish
VII
81 82
List of Figures Figure
Title
No. 1-1 1-2 1-3 1-4 3-1
3-2
3-3
3-4
3-5
3-6
3-7
Page No.
Interference in the balance (large arrow) between oxidant and reductant defines oxidative- or reductivestress conditions Formation of reactive species in vivo and the role of defense systems against them Chemical Structure of (1) Silybin A and (2) Silybin B Chemical structure of (A) thiamine and (B) benfotiamine Time-course for production of methemoglobin with sodium nitrite in erythrocyte lysate. Effect of different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12, 10-9, 10-6 mg/ml) on the time-course of nitrite-induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate. Effect of addition of silibinin dihemisuccinate disodium (10-6 mg/ml) at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the time course of oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate. Concentration-response relationship for the radical scavenging activity of silibinin dihemisuccinate disodium in nitrite-induced Hb oxidation in vitro. Effect of different concentrations of benfotiamine (25, 50, 100, 200 µM) on the time-course of nitrite induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate. Effect of addition of benfotiamine (200 µM) at different time intervals (10 min before, 10min after, 20min after nitrite addition) on the oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate. Concentration-response relationship for the radical scavenging activity of benfotiamine in nitrite-induced Hb oxidation in vitro.
VIII
5 12 20 26 43
45
48
51
53
56
59
3-8
3-9
3-10
3-11
3-12
Comparison between radical scavenging activity of equimolar concentrations of silibinin and benfotiamine in nitrite induced Hb oxidation in vitro. Effects of different concentrations of Silibinin dihemisuccinate disodium on the osmotic fragility of red blood cell challenged with sodium nitrite in vitro Concentration-response relationship of silibinin dihemisuccinate disodium (SDHS) on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro. Effects of different concentrations of Benfotiamine on the Osmotic fragility of red blood cell challenged with sodium nitrite in vitro Concentration-response relationship of Benfotiamine on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro.
IX
61
63
65
67
69
List of Tables Table No.
Title
Page No.
1-1
Radical and non-radical oxygen metabolites.
3
2-1
Chemicals and Their Suppliers
33
2-2
Instruments and Their Suppliers
34
Effect of different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12, 10-9, 10-6 mg/ml) on the time-course of nitrite-induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate. Effect of addition of silibinin (10-6 mg/ml) at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the time course of oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate. Effect of different concentrations of benfotiamine (25, 50, 100, 200 µM) on the time required for oxidation of hemoglobin and formation of 50% methemoglobin with sodium nitrite in erythrocyte lysate. Effect of addition of benfotiamine 200 µM at different time intervals (10 min before, 10min after, 20min after nitrite addition) on the oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate. Effects of single and multiple doses of Silibinin dihemisuccinate (100mg/kg) or Benfotiamine (70mg/kg) on nitrite-induced MetHb formation in rats.
46
3-1
3-2
3-3
3-4
3-5
X
49
54
57
71
List of Abbreviations AGEs
Advanced Glycation End products
CCl4
Carbon tetra chloride
CD 80
Cluster of differentiation 80
CD 86
Cluster of differentiation 86
CDK2
Cyclin-dependent protein kinase 2
CDK4
Cyclin-dependent protein kinase 4
CL
Chemiluminescence
CNS
Central nervous system
COX2
Cyclooxygenase-2
CYP3A4
Cytochrome P450 3A4
DAG
Diacylglycerol
DCs
Dendritic cells
DNA
Deoxyribonucleic acid
EDTA
Ethylene diamine tetraacetic acid
EPR
Electron pair resonance
ESR
Electron spin resonance
Fe+2
Ferrous ions
Fe+3
Ferric ions
Grx
Glutaredoxin
GSH
Glutathione
GSH
Reduced glutathione
GSSG
Oxidized glutathione
H2O2
Hydrogen peroxide
Hb
Hemoglobin
HOCl
Hypochlorous acid
Hep3B
Hepatitis B virus positive
XI
HepG2
Hepatitis B virus negative
HO•2
Hydroperoxyl
IL-10
Interleukin (IL)-10
IL-2
Interleukin (IL)-2
IL-4
Interleukin (IL)-4
LDL
Low density lipoproteins
LMWA
Low-molecular-weight antioxidants
LNCaP
Androgen sensitive human prostate adrenocarcinoma cells
MAPKs
Mitogen–activated protein kinase
MetHb
Methemoglobin
MHC
Histocompatability complex molecules
mM
Milimolar
N2O
Di-nitrogen oxide
N2O3
Di-nitrogen trioxide
NACA
N-acetylcysteine amide
NADPH
Nicotineamide adenine dinucleotide phosphate
NF-Kb
Nuclear Factor-Kappa b
NO–
Nitroxyl anion
NO+
Nitrosonium cation
NO•
Nitric oxide radical
NO2–
Nitrite ion
NOS
Nitric oxide synthase
O•2
Superoxide ion radical
1O2
Singlet oxygen
O3
Ozone
ODS
Oxygen-derived species
OH•
Hydroxyl radical
XII
ONOO–
Peroxynitrite
ONOOH
Protonated form of peroxynitrite
PBS
Phosphate Buffer Saline
PKC
Protein kinase C
RBC
Red blood cell
RNS
Reactive Nitrogen Species
RO•
Alkoxyl radicals
ROO•
Peroxyl radical
rpm
Rotation per minute
ROS
Reactive Oxygen Species
SBT
S-benzolythiamine
SDH
Silibinin dihemisuccinate
SDHS
Silibinin dihemisuccinate di-Sodium
–SH
Sulfhydryl
SOD
Super oxide dismutase
TDP
Thiamin diphosphate
TGF-
Transforming growth Factor-beta
TNF-α
Tumor necrosis Factor-alpha
Trx
Thioredoxin
-IFN µM
Interferon-gamma Micromolear
XIII
Appendix I For preparation of 1000 ml Phosphate Buffer Saline (PBS), the following chemicals were dissolved respectively in distilled water and the volume was completed to 1000 ml. The target pH was adjusted using a digital pH-meter.
NaCl
8g
KCl
0.2g
Na2HPO4 anhydrouse KH2PO4
0.9172g 0.24g
Appendix II Preparation of Phosphate buffer (PB): Solution A: 9.073 g potassium dihydrogen phosphate (KH2PO4) was dissolved in 1000ml of distilled water. Solution B: 9.473 g anhydrouse disodium hydrogen phosphate (Na2HPO4) was dissolved in 1000ml of distilled water. Then, 19.7ml of solution A was completed to 100 ml by adding solution B. final concentration of this solution will be (1/15M) i.e, 0.067M, and pH 7.4; then we dilute it to prepare 20 mM strength solution.
Appendix III Preparation of different concentrations of buffered saline solution
1
Volume of buffered Saline Solution (ml) 5
2
4.5
0.5
0.9
3
4
1
0.8
4
3.5
1.5
0.7
5
3.25
1.75
0.65
6
3
2
0.6
7
2.75
2.25
0.55
8
2.5
2.5
0.5
9
2.25
2.75
0.45
10
2
3
0.4
11
1.75
3.25
0.35
12
1.5
3.5
0.3
13
1
4
0.2
14
0.5
4.5
0.1
15
0
5
0
Tube Number
Volium of Distilled Water (ml)
Concentration of NaCl (%)
0
1
اقليم كسدضتاى /العساق وشازة التعليم العالي والبخث العلمي جامعة الطليمانية كلية الطب
تكييم العاقة بن الركيص (اجسعة) ومكداز خاصية قهص اجروز احسة مادتي الطليبيهن ثهائي الطلطيهات والبهفوتيامن ي موذج اكطدة اهمغلوبن امطتخدث بواضطة نايرايت الصوديوم
زضالة مكدمة اى جلظ كلية الطب /جامعة الطليمانية كحصء مو متطلبات نيل شهادة اماجطتر ي الصيدلة (اأدوية)
مو قبل بشسى حطو معسوف
باشساف الدكتوز ضعد عبدالسمو حطن
0202
بسم اه الرمن الرحيم
وَعََّمَمَ مَا لَمِ تَلُنِ تَعَِّمُ وَكَانَ فَضِلُ الَّهِ عََّيِمَ عَظِيماً صدق اه العظيم سورة النساء :اأية 111
To my beloved father who's life shall never be forgotten…………………………. To my mother for her love and measureless support. To my adorable husband for his love, support and encouragement, without him I would never have had the strength to perform this work…………………………………
Roya To my cute son …………… Rawsht
To my lovely daughter………
To my sisters and brothers for their never ending support.
I dedicate this thesis.
CHAPTER FOUR
DISCUSSION
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
CHAPTER THREE
RESULTS
Chapter Two
Materials and Methods
CHAPTER TWO Materials and Methods 2.1. Chemicals The chemicals used in the present study were of pure analytical grade. Chemicals with their suppliers are listed in table (2-1).
2.2. Instruments The instruments used in the present study are listed in table (2-2) with their suppliers.
2.3. General Consideration The present study was performed as three parts of experiments; first one includes an in vitro evaluation of the concentration-effect relationship for the radical scavenging activity of both SDH and benfotiamine utilizing nitrite-induced hemoglobin oxidation model; the second part includes evaluation of the protective effect of different concentrations of both SDH and benfotiamine against sodium nitrite-induced RBCs hemolysis; the third part includes evaluation of the protective effects of the maximum effective concentration of SDH and benfotiamine as determined from the first part in an in vivo model of nitrite-induced hemoglobin oxidation. All experiments were conducted at the research laboratory of the College of PharmacyUniversity of Sulaimani during the period from 1st of November 2009 to 20th of July 2010.
32
Chapter Two
Materials and Methods
Table 2-1. Chemicals and Their Manufacturers
1
2
Chemicals
Manufacturer
Di-Sodium hydrogen phosphate,
GCC: Gainland chemical company,
anhydrous powder
UK.
Potassium dihydrogen phosphate GCC: Gainland chemical company, powder
UK.
3
Sodium nitrite powder
Analar BDH, Ltd., Poole, England
4
Potassium cyanide powder
G.P.R Hopkin & Williams UK.
5
Potassium ferricyanide powder
Alpha Chemika, India
6
Triton X-100 Solution
BDH-England
7
Diethyl ether solution
Alpha Chemika, India
8 9
Silibinin dihemisuccinate disodium (Legalon® SiL) powder Madaus, GmbH, Germany for reconstitution Benfotiamine powder Polpharma, Poland
10 Sodium chloride powder
11 Potassium chloride powder
GCC: Gainland chemical company, UK. GCC: Gainland chemical company, UK.
33
Chapter Two
Materials and Methods
Table 2-2. Instruments and Their Manufacturers
Instrument name 1
Manufacturer
UV-visible spectrophotometer
Shimadzu Corporation, Kyoto, Japan
UV-1650 PC 2
Thermostated Centrifuge
United state
Jouan BR4 3
4
pH- meter
OAKTON-Eutech Instruments Pte
pH 2100 series
Ltd. Singapore
Sensitive balance
Sartarious Ag Gottiugan Germany
34
Chapter Two
Materials and Methods
2.4. In vitro study 2.4.1. Blood sample collection and preparation of lysate Blood samples (5ml) were obtained from healthy individuals by veinpuncture, and kept in Ethylene diamine tetraacetic acid (EDTA) and heparin containing tubes; which contain 1.2 mg of anhydrous salt and 1020 IU/ml of heparin respectively, the blood was utilized for separation of erythrocytes and preparation of hemolysate and red blood cell suspension for evaluation of the tested compounds in such in vitro models, based on oxidation of hemoglobin and osmotic fragility test of erythrocytes respectively.
2.4.2. Preparation of samples and analysis 2.4.2.1. Preparation of hemolysate The blood samples were centrifuged at 2500 rpm and 4 oC for 10 minutes to remove plasma and the buffy coat of white cells. The erythrocytes were washed thrice with Phosphate Buffer Saline (PBS) pH 7.4 (Appendix I) and lysed by suspending in 20 volumes of 20mM Phosphate Buffer (PB) pH 7.4 (Appendix II)
to yield the required
hemolysate concentration of 1:20, then hemolysate was centrifuged at 10000 rpm for 10 min and supernatant has been taken [184].
2.4.2.2. Preparation of drug solutions 2.4.2.2.1. Preparation of Silibinin dihemisuccinate disodium solution Different concentrations of silibinin dihemisuccinate disodium were prepared by dissolving the required quantity in deionized water to prepare stock solution of (silibinin 1mg/ml), from which serial dilutions were made to give concentrations of 10-6, 10-9, 10-12 and 10-15 mg/ml.
35
Chapter Two
Materials and Methods
2.4.2.2.2. Preparation of Benfotiamine solution Different concentrations of benfotiamine were prepared by dissolving the required quantity in phosphate buffer (pH 7.4) to prepare stock solution of benfotiamine (250µM), from which serial dilutions were made to give concentrations of 200µM, 100µM, 50µM and 25µM.
2.4.2.3. Evaluation of effect of different SDHS concentrations on the time course of nitrite-induced oxidation of hemoglobin In vitro model for oxidation of hemoglobin and formation of methemoglobin, using sodium nitrite was utilized as a model to evaluate interference of SDHS with oxidation of hemoglobin as follow [185]: To 1.5ml of freshly prepared hemolysate, 1ml of each concentration of SDHS (10-15, 10-12, 10-9 and 10-6 mg/ml) was added concomitantly with 0.1ml sodium nitrite (final concentration 6.0 mM) and the formation of MetHb was monitored spectrophotometrically at 631 nm for 50 minutes using computerized UV-visible spectrophotometer. The experiments were performed 3 times for each concentration under controlled temperature (2730oC).
2.4.2.4. Evaluation of the effect of SDHS on the time course of MetHb formation at various time intervals from nitrite addition To 1.5 ml freshly prepared hemolysate, 1.0 ml of the highly effective concentration of SDHS was added either 10 minutes before, or at 10 and 20 minutes after the addition of sodium nitrite to the hemolysate solution, and the formation of MetHb was monitored spectrophometrically at 631nm using computerized UV-visible spectrophotometer.
36
Chapter Two
Materials and Methods
2.4.2.5. Evaluation of effect of different benfotiamine concentrations on the time course of nitrite-induced oxidation of hemoglobin In vitro model for oxidation of hemoglobin with sodium nitrite was utilized for production of methemoglobin (MetHb) [185] and the antioxidant effect of different concentrations of benfotiamine was evaluated as follow: To 1.5 ml freshly prepared hemolysate, 1.0 ml of different concentrations of benfotiamine (25µM, 50µM, 100µ M and 200µM) each time were added concomitantly with 0.1 ml sodium nitrite (final concentration 6.0 mM), and the formation of MetHb was monitored spectrophotometically at 631 nm for 50 minutes using computerized UVvisible spectrophotometer.
2.4.2.6. Evaluation of effect of benfotiamine on the time course of MetHb formation at various time intervals from nitrite addition To 1.5 ml freshly prepared hemolysate, 1.0 ml of the highly effective concentration of benfotiamine was added either 10 minutes before, or at 10 and 20 minutes after the addition of sodium nitrite to the hemolysate solution, and the formation of MetHb was monitored spectrophometrically at 631 nm using computerized UV-visible spectrophotometer.
2.4.2.7. Evaluation of osmotic fragility of erythrocytes Erythrocytes suspension was prepared by mixing a volume of fresh blood with 20 volumes of Phosphate Buffered Saline (PBS, pH 7.4); the hematocrit must be adjusted to be around 2.5%. Aliquots (0.2 ml) of erythrocytes suspension (2.5% hematocrit) were added to 1.8 ml of 37
Chapter Two
Materials and Methods
buffered saline solutions of decreasing concentrations, pH 7.4, (NaCl concentration range of 9.0–1.0 g/L). The suspensions were allowed to stand for 30 minutes at room temperature, mixed again and then centrifuged for 5 minutes at 1200 rpm. The supernatants were removed and the degree of lysis was determined spectrophotometrically at 540 nm. The percentage of hemolysis was calculated from the ratios of the absorbance of the lysate [186].
2.4.2.7.1. Evaluation of the effect of SDHS on the nitriteinduced osmotic fragility of red blood cells Erythrocytes suspension was prepared by mixing a volume of fresh blood with 20 volumes of Phosphate Buffered Saline (PBS, pH 7.4); Aliquots (0.2 ml) of erythrocyte suspension (2.5% hematocrit) were added to 1.8 ml of buffered saline solutions of decreasing concentrations, pH 7.4, (NaCl concentration range of 9.0–1.0 g/L). Different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12, 10-9and 10-6 mg/ml) and 0.1 ml sodium nitrite (final concentration 6.0 mM) were incubated with the suspensions. The suspensions were allowed to stand for 30 minute at room temperature, mixed again and then centrifuged for 5 minutes at 1200 rpm. The supernatant was obtained and the level of lysis was determined spectrophotometrically at 540 nm. The percentage of hemolysis was calculated from the ratios of the absorbance.
2.4.2.6.2. Evaluation of benfotiamine on the nitrite-induced osmotic fragility of red blood cells Erythrocytes suspension was prepared by mixing a volume of fresh blood with 20 volumes of Phosphate Buffered Saline (PBS, pH 7.4); Aliquots (0.2 ml) of erythrocyte suspension (2.5% hematocrit) were added to 1.8 ml of buffered saline solutions of decreasing concentrations, pH 7.4, 38
Chapter Two
Materials and Methods
(NaCl concentration range of 9.0–1.0 g/L) (Appendix III). Different concentrations of benfotiamine (25µM, 50µ M, 100µ M and 200µM) and 0.1 ml sodium nitrite (final concentration 6.0 mM) were incubated with the suspensions. The suspensions were allowed to stand for 30 minutes at room temperature, mixed again and then centrifuged for 5 minutes at 1200 rpm. The supernatants were obtained and the level of lysis was determined spectrophotometrically at 540 nm. The percentage of hemolysis was calculated from the ratios of the absorbance.
2.5. In vivo study 2.5.1. Experimental animals Seven to eight-weeks old female Wistar rats, weighing 160-280g were purchased from the animal house of the College of Pharmacy/ Hawler Medical University. The animals were housed in the animal house of the College of Pharmacy/University of Sulaimani in well ventilated plastic cages at 24 ± 2oC and 50 ± 10 relative humidity and subjected to 12hr light/12hr dark cycle. They were acclimatized for 1 week before starting the experiments, during which they had free access to standard commercial diet purchased from (Iraqi Center for Cancer Research and Medical Genetics, Baghdad) and tap water ad libitum.
2.5.2. Treatment schedule After acclimatization of animals with the environment of the animal house, they were randomly allocated into six groups, each with six rats and treated as follows: Group I: Control group of long-term silibinin and benfotiamine exposure 1.0 ml of normal saline (0.9% sodium chloride) was given orally for 7 days, and on the seventh day, sodium nitrite (100 mg/kg) was given orally 39
Chapter Two
Materials and Methods
by gavage tube; 45 minutes later the animals were sacrificed by inhalation of high dose of anesthetic diethyl ether; intra-cardiac blood samples were collected in EDTA-containing tubes and used for measurement of MetHb level. Group II:Control group of short-term silibinin and benfotiamine exposure 1.0 ml of normal saline (0.9% sodium chloride) was given orally, 1 hour later, sodium nitrite (100 mg/kg) was given orally, after 45 minutes the animals were sacrificed by inhalation of high dose of anesthetic diethyl ether, and intra-cardiac blood samples were taken for measurement of MetHb level. Group III: Long-term silibinin exposure Silibinin dihemisuccinate (100 mg/kg) body weight was given once daily orally by gavage tube for 7 days. On the seventh day sodium nitrite (100 mg/kg) was given orally; and 45 minutes later, the animals were sacrificed by inhalation of high dose of anesthetic diethyl ether, and intracardiac blood samples were taken for measurement of MetHb level. Group IV: Short-term silibinin exposure Silibinin dihemisuccinate (100 mg/kg) body weight was given as a single oral dose, 1 hour later, sodium nitrite (100 mg/kg) was given orally; after 45 minutes the animals were sacrificed by inhalation of high dose of anesthetic diethyl ether, and intra-cardiac blood samples were taken for measurement of MetHb level. Group V: Long-term benfotiamine exposure Benfotiamine (70 mg/kg) once daily, was given orally by gavage tube for 7 days. On the seventh day sodium nitrite (100 mg/kg) was given orally, 45 minutes later the animals were sacrificed by inhalation of high dose of
40
Chapter Two
Materials and Methods
anesthetic diethyl ether, and intra-cardiac blood samples were taken for measurement of MetHb level. Group VI: Short-term benfotiamine exposure Benfotiamine (70 mg/kg) was given as a single oral dose, 1 hour later, sodium nitrite (100 mg/kg) was given orally, after 45 minutes the animals were sacrificed by inhalation of high dose of anesthetic diethyl ether, and intra-cardiac blood samples were taken for measurement of MetHb level.
2.5.3. Measurement of methaemoglobin in blood Fresh (0.2 ml) blood was lysed in a solution containing 4.0 ml of buffer (Phosphate buffer: 0.1 mol/l, pH 6.8) and 6.0 ml of non-ionic detergent solution (Triton X-100, 10ml/l), the lysate was divided into two equal volumes (A and B). The absorbance of A has measured using a spectrophotometer at 630 nm (D1). 1 drop of potassium cyanide solution (50 gm/L) has been added to A and measures the absorbance again, after mixing (D2). 1 drop of potassium ferricyanide solution (50 gm/L) has been added to B, and after 5 minutes, measure the absorbance at the same wavelength (D3). Then 1 drop of potassium cyanide solution has been added to B and after mixing a final reading (D4) has recorded. All the measurements are made against a blank containing buffer and detergent in the same proportion as present in the sample [186]. Calculation: Methaemoglobin (%) =
D1 - D2
x 100
D3 - D4
2.6. Statistical Analysis The results are expressed as mean ± standard deviation. The statistical analysis was performed using one-way ANOVA. Differences are considered significant when P< 0.05.
41
CHAPTER TWO
MATERIALS AND METHODS
CONCLUSION
Conclusion According to the results of the present study, one can conclude the following: 1- SDH and benfotiamine can effectively, in concentration-dependent pattern, attenuate sodium nitrite-induced Hb oxidation and maintain integrity of red blood cells both in in vitro and in vivo models of hemoglobin oxidation through free radical scavenging activity. 2- Both SDH and benfotiamine protect the erythrocyte plasma membrane against the damaging effects of sodium nitrite.
Recommendations for future work:
1-Evaluation of the radical scavenging activity of both SDH and benfotiamine in other models or systems that utilize other inducers of radicals generation. 2- Evaluation of the protective effects of both agents in animal models of drugs and/or toxins-induced hematological disorders including hemolytic anemia and acceleration of diabetes mellitus-induced glycated hemoglobin level.
86
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Discussion
CHAPTER FOUR Discussion 4.1. Time-course for oxidation of hemoglobin and MetHb formation with sodium nitrite: in vitro study Oxidation of hemoglobin to MetHb by nitrite has been widely studied [184]; it is one of the most employed procedures to oxidize the hemoprotein [187]. The process includes a slow initial stage followed by a rapid autocatalytic stage, which carries the reaction to completion [188]. Sodium nitrite as a pro-oxidant induce a primary extensive MetHb formation as a result of generation of several free radical species like superoxide anion, hydroxyl, peroxynitrite and nitrogen oxide radicals which are implicated in promoting the autocatalytic stage of oxidation of Hb by nitrite [189,190] this reaction takes place with a peculiar S-shaped profile, and a noticeable increase in the rate of the process after a clear lag time [187]. During the process of Hb oxidation by nitrite many reactions are involved including a chain reaction with a branching process, during which a reactive intermediate (nitrogen dioxide) is produced; this explains the observed autocatalysis, as well as the formation of nitrate ions as main reaction products [187]. The reaction of organic nitrates with Hb lead to the formation of ferric ion (Fe3+) within the hemoglobin structure; such increase in the amount of ferric ions cause an elevation of O•2 and H2O2 levels, which are delivered to the medium. Interaction between the generated O•2 and H2O2 produced the hydroxyl radicals (OH•) by the Haber Weiss reaction; Furthermore, ferrous ions together with H2O2 will generate hydroxyl radicals (OH•) through what is known as Fenton reaction [191]. In the present study, the formation of MetHb and the S-shaped curve obtained during kinetic behavior of hemoglobin oxidation may provide an evidence for the formation of various free radicals during the process of
72
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Discussion
hemoglobin oxidation, which seems compatible with previous findings reported by others [187].
4.1.1 Effect of different concentrations of SDHS on the time-course of nitrite-induced oxidation of Hb and MetHb formation in vitro Silibinin, the naturally occurring flavonolignan, is a nearly equimolar mixture of two diasteriomers; it acts mainly as an effective antioxidant [192, 193] and displays potential free radical scavenging property [194]. The radical scavenging activity of silibinin could be partly involved in cell regulatory pathways that are based on reactive oxygen species (ROS) [195]. Although the pharmacology of silibinin has been extensively studied, its molecular mechanisms of the antioxidative activity have not been systematically investigated and remain unclear [196] and few studies have been devoted to the identification of silibinin active sites [197]. In the present study, the suspected capacity of different concentrations of SDHS to scavenge various types of free radicals was evaluated, and concentration-effect relationship was reported in in vitro model of Hb oxidation and MetHb formation by nitrite in hemolysate. Erythrocytes are utilized as a traditional target for studying oxidative damage, when exposed to high oxygen tensions and in presence of high iron contents (transition metal promoting the formation of oxygen free radicals) oxidative damage occur due to both exogenous and endogenous insults [198]. In accordance with the previous findings reported by others [189,190,199] several free radical species are generated during the course of nitrite-induced oxidation of Hb. The present study has shown that SDHS can protect hemoglobin against oxidation by sodium nitrite in hemolysate, and there are many suggested theories for the mechanism through which silibinin produce such protective role. Structure-activity relationship (SAR) of flavonoids has been studied in details [200]; however, only few data are known regarding SAR of 73
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Discussion
flavonolignan, such as silibinin, and a few quantitative SAR model have also been proposed for the aim of determining the basic structural motifs of these compounds responsible for their antioxidant activity [201]. On the basis of these studies, the major determinants important for a high radical-scavenging capability of flavonoid were suggested, including the presence of catechol or pyrogallol group in ring B and the 3-OH group connected to a 2,3 double bond conjugated with the C-4 carbonyl group in the dehydrosilybin, a metabolite of silybin, this extends the possibilities for conjugation between the B and the C ring [196]. Another factor which increases the potency of flavonoid to interact with radicals is the presence of a galloyl group in the molecule [200]. The most important radical-scavenging moiety of silibinin is 20-OH, as 7-OH possesses only negligible scavenging activity and all other OH groups do not markedly participate in the overall activity [196]. Preventing the onset of the autocatalytic stage of nitrite-induced oxidation of Hb after addition of silibinin suggests that such protective effect might be due to its radical scavenging activity and not due to reduction of MetHb to Hb, because it fails to reverse the oxidized hemoglobin (i.e the formed MetHb) after 10 and 20 min of addition of nitrite. Additionally, direct interaction between nitrite and silibinin, as a reason for protection, can be ruled out because the concentration of silibinin which protect the Hb is very low. SDH proved to be a powerful scavenger of OH• through different mechanisms, including addition reaction on the aromatic rings, abstraction of phenolic hydrogen and decarboxylation reaction [202]. Furthermore, recent studies suggest that structural requirement for hydroxyl radical scavenging include the presence of the hydroxyl group in ring C. The assessment of the roles of the individual OH groups and their neighborhood in the interaction of silibinin with several radical species provide some new knowledge on the mechanism of its action [196]. Some products of the radical attack (dimers) were isolated and their structures were determined based on the presence of hydroxyl groups within the structure, 74
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Discussion
which are responsible for interaction of silybin (20-OH) and 2,3-dehydrosilybin (3-OH and 20-OH) with radicals (one electron oxidant), and thus the molecular mechanism of silybin and 2,3-dehydrosilybin radical-scavenging activity can be explain [196]. In spite of directly scavenging radicals (superoxide, hydrogen peroxide, nitric oxide and hydroxyl radicals) [203], the antioxidant effect of flavonoids may result from the interaction between flavonoids and metal ions, especially iron and copper, leading to chelate formation. It is believed that both iron chelation and free radical scavenging activities account for the antioxidative ability of flavonoids [204]. However, few studies have investigated the molecular basis for these effects, where the polyphenol structure allows both the scavenging of free radicals, with concomitant formation of fairy stable aroxyl radicals, and chelation of transition metals including iron [205]. Silibinin, in addition to its antioxidant and free radical scavenging abilities, has the ability to act as iron chelator [206]; it possesses a hydroxyl group at C5 and C3 in addition to the carbonyl group at C4 which may form chelates with divalent cation [207]; the fact that hemoglobin Fe(II) can serve as a Fenton reagent catalyzing the conversion of H2O2 to OH• and OH¯ , and as long as O•2 is available to reduce the oxidized Fe(III), the catalytic cycle can continue to generate hydroxyl radicals [208]. Although the present study did not investigate the effect of silibinin as iron chelator, but utilizing hemoglobin oxidation system as a model can not exclude this effect of silibinin. On the other hand, the study on structure-radical scavenging activity relationships of flavonoids elucidated a comprehensive model for radical scavenging reaction of flavonoids by the eases of hydrogen atom abstraction and the ease of the termination of the flavonoid aroxyl radical [209]. The data reported in the present study concerning significant inhibition of MetHb production, and the delay in time required for the formation of MetHb within the limit of concentrations used, might be closely related to the 75
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Discussion
mentioned mechanisms and structural functional groups; the protective effect of silibinin on hemolysate was found to be compatible with the reported protective effect of silymarin on erythrocyte hemolysate against benzo(a)pyren and exogenous reactive oxygen species [210]. 4.1.2. Effect of different concentrations of benfotiamine on the timecourse of nitrite-induced oxidation of Hb and MetHb formation in vitro Previously, B-group vitamins were considered to be non-antioxidant vitamins [211]. However, recent studies showed that vitamin B6 family caused suppressive effect on glucose-induced lipid peroxidation and superoxide generation in diabetic model experiments [212]. These results indicate a possibility that some of B-group vitamins possess potent antioxidant or radicalscavenging activity [211]. On the other hand, thiamin and thiamin diphosphate (TDP) caused significant inhibitory effect on protein glycation process in animal models of experimental diabetes [213], a process that involves not only the Schiff-base formation and Amadori reaction but also another oxidation process such as oxygen radical generation [214,215]. The pleotropic action of benfotiamine, a lipid soluble analogue of vitamin B1 (Thiamin) in improving the function of vascular endothelium has been partly related to inhibition of ROS formation [178]. Even though, benfotiamine and its metabolic fate products, thiamin and TDP, have been shown to prevent oxidative stress-induced pathological conditions including inflammatory disorders [156], diabetic nephropathy, retinopathy and neuropathy [216]. Additionally, benfotiamine has been shown to prevent oxidative stress-induced complications through its antioxidant properties in in vitro and in vivo models [180], as well as it inhibits the hyperglycemia-induced NF-kB pathway via PKC [162] because PKC activation has been shown to activate NADPH oxidase, which is responsible for generation ROS; the ability of benfotiamine to inhibit PKC activation may contribute to its antioxidant effects, an effect that may require relatively long 76
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Discussion
time exposure to be established [217]. Recent study showed that benfotiamine dose-dependently inhibits the expression of LPS-induced inflammatory markers which could be responsible for its protective role against oxidative stress in this model [180]. These observations are compatible with those reported in the present study concerning the concentration-dependent inhibition of MetHb formation by benfotiamine in nitrite-induced oxidation of Hb and delay the onset of autocatalytic phase of the oxidation reaction in which various free radicals endogenously generated [187], this may suggest a possibility that benfotiamine has a potent inherited antioxidant or radical-scavenging activities within its native structure. These findings are consistent with the potent radicalscavenging activities of thiamin and TDP in some in vitro experiments [211]; also there is a possibility that thiazole structure in thiamin and thiamin derivatives is responsible for the mentioned activity, which can not be excluded in ethanol-induced oxidative stress in rat alveolar cells, where a thiazole derivative showed significant radical-scavenging activity [218]. However, many studies reported controversial results which showed antioxidant and pro-oxidant activities of thiamin in some in vitro experiments [219]. Thiamin and TDP produced more specific inhibitory effect on superoxide generation than on hydroxyl radical generation because TDP possesses much higher affinity for superoxide than hydroxyl radicals [211]. In fact, the radical scavenging activity of TDP is not elucidated completely, where the metal chelating property of diphosphate may strengthen the radical scavenging activity of this compound, since hydroxyl radical generation in Fenton reaction absolutely requires metal ions such as ferrous or copper ions; the metal-chelating property of TDP may contribute to its effective-radical scavenging activity [211]. The reported data in the present study demonstrated a non-AGE dependent role of benfotiamine in reducing MetHb, and it may be important observation for initiating further investigations to explore another therapeutic potential of benfotiamine in methemoglobinemia induced by various causes; 77
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Discussion
therefore, additional clinical studies are mandatory to explore such therapeutic potential in both diabetic and non diabetic pathological conditions. 4.2. Osmotic fragility test: in vitro study The oxidation of erythrocyte membrane serves as a model for the oxidative damage of biomembranes [220]. Oxidation of erythrocyte and its ghosts membrane induced by free radicals, which are generated in the aqueous phase, attack the membrane to induce a peroxidation of the unsaturated fatty acids and phospholipids that lead to hemolysis [221]. In the present study, erythrocytes were oxidized by sodium nitrite and the protective effect of silibinin and benfotiamine on RBC hemolysis was investigated. Nitrite has a strong oxidant effect on RBC membrane and its hemoglobin [222], as it can enter erythrocyte, react with Hb and oxidize it [223]. During the course of this reaction, reactive oxygen radicals are produced and start to induce a peroxidation of the unsaturated fatty acids of the phospholipid structure of the plasma membrane. Thus it appears like an osmotic brittleness of the erythrocyte membrane as well as a disturbance of membrane transport which leads to hemolysis [221]. The osmotic fragility test was used to determine the extent of red blood cell hemolysis which may be produced by any type of osmotic stress [224]. The oxidative damage to hemoglobin molecule caused by inhaled nitrite can induce the dissociation of heme and globin chains, consequently forming polymerized globin aggregates known as Heinz bodies. These aggregates are attached to the membrane of the red blood cell, thereby altering its shape [225]. Additionally hyper-polarization of erythrocyte membranes and an increase in membrane rigidity have been observed as a result of RBC oxidation by sodium nitrite. The interaction of nitrite with cell membrane also drastically modified the erythrocyte membrane [226], and nitrite-induced methemoglobinemia in RBC lead to an inhibition of red blood cell sodium/ proton exchanger; the membrane potential changes after exposure of RBCs to nitrite which reflects the 78
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Discussion
alteration of the activity of membrane ionic exchangers. The decrease in membrane fluidity may result from the changes in the membrane structural state due to the oxidative process within the membrane provoking hemolysis [227]. Furthermore, treatment of intact RBCs with sodium nitrite causes a noticeable oxidation of oxyhemoglobin to methemoglobin through radical generation along with a decrease in glutathione level in intracellular medium and associated with membrane lipid peroxidation [221].
4.2.1. Effect of different concentrations of SDHS on osmotic fragility of red blood cells: in vitro study To evaluate the possible relationship between different concentrations of silibinin (10-6, 10-9, 10-12 and 10-15 mg/ml) and free radical-mediated biological membrane damage, the effect of SDHS on the hemolysis of RBC in the presence of sodium nitrite was investigated. The present study shows that SDHS protects human erythrocytes against nitrite-induced oxidative hemolysis in concentration-dependent manner. The mechanism by which SDHS can perform this protection need to be clarified. Free radicals attack erythrocyte membrane components such as proteins and lipids, and change the structure and function of this membrane which may result in hemolysis [220]. Incubation of erythrocytes with nitrite resulted in reduced glutathione level [221], and glutathione, in fact, provides the first line of defense during oxidative insult [228].Various studies indicated that silibinin significantly increase glutathione levels, which serves as a free radical scavenger [134]. Silibinin is relatively hydrophobic and its penetration into cell is limited [229] and membrane structures are postulated to be one of the cellular targets for silibinin as it interacts with the surface of lipid bilayer [230], its effect on erythrocytes may be explained by their incorporation into this lipid bilayers of the cell membrane leading to reducing hemolysis. Silibinin on the other hand, is able to increase the activity of both superoxide dismutase and glutathione peroxidase, which 79
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Discussion
may explain the protective effect of the drug against free radicals and also stabilizing the effect on the red cell membrane [231]. The ability of flavonoids to scavenge peroxyl radicals might explain the scavenging action of silibinin to peroxyl radicals thus silibinin displays cytoprotective effect against lytic damage induced by nitrite to intact erythrocyte [232]. The possibility that silibinin exerts its cytoprotective effects at the membrane level, as a chain breaking antioxidant, should also be considered. In fact, Miki and Mino (1985) established that hemolysis happens when the erythrocyte membrane alpha tocopherol concentration has lowered to a critically low level; therefore silibinin may interact with lipoperoxyl radicals and spare alpha tocopherol molecules [233]. Moreover, iron plays a central role in generating harmful oxygen species; its redox-cycling promotes Fenton reaction [234]. When erythrocytes are incubated with an oxidizing agent, iron is released from hemoglobin [235] and the release is accompanied by MetHb formation [236]. If the erythrocytes are severely depleted of glutathione, which occur after short preincubation with oxidant, the release of iron is followed by peroxidation of membrane lipid and hemolysis. Therefore, it seems that the release of iron initiates a chain of reactions leading to lipid peroxidation and consequent hemolysis; the membrane proteins also altered resulting in the formation of senescent cell antigen, which appears to be related to the release of iron [236]. As phenolic compounds, flavonoids can scavenge free hydroxyl and peroxyl radical [237] and can react with superoxide via a one electron transfer [238]. Furthermore, as a metal chelating agent, they can extract iron ions and hinder radical reactions that set into motion by the metal redox cycling [239]. The result of the present study clearly indicated that the protection afforded by silibinin may be due, in part, to intracellular chelation of iron; this possibility must also considered that the effect of silibinin could be due to its antioxidant activity, which has been extensively documented independently of metal chelation, probably both activities are involved in the 80
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Discussion
protective effect and it is difficult to distinguish the respective roles played [240, 241].
4.2.2. Effect of different concentrations of benfotiamine on osmotic fragility of red blood cells: in vitro study Benfotiamine as well as thiamine penetrate human red cell membrane slowly, while S-benzolythiamine (SBT) penetrates RBC membrane very rapidly; thus SBT was suspected to be an actual form in the absorption of benfotiamine from intestine after dephosphorylation by alkaline phosphatase at the intestinal mucosa. In the presence of alkaline phosphatase, benfotiamine shows a high permeability through the cell membrane. The penetration of SBT into red cells proceeds through two steps, the first one includes passive diffusion of SBT through the cell membrane, and the second depends on rapid decomposition of SBT to thiamine and the resultant accumulation in the cell [157]. Whatever the metabolic fate and degradation product of benfotiamine, the reported results of the present study (concerning the effect of different concentrations of benfotiamine on osmotic fragility test) showed concentrationdependent protection of RBCs challenged with sodium nitrite; a mechanism that takes place at the cellular level and might be associated with the reduction in oxidative stress; this seems to be consistent with the reported protective effect of benfotiamine on myocardial dysfunction [242]. Additionally, a recent study has shown that at high concentration (300µM) of benfotiamine, it exerts a direct antioxidant effect independent of its transformation into thiamine and increased transketolase activity [182]. The results of the present study might be partly explained
by direct antioxidant activity of benfotiamine and
partial
dephosphorylation to S-benzoyl thiamine. Recently, a study on erythrocyte fragility in type II diabetic patients demonstrated that osmotic fragility of erythrocytes increases with the increase in the level of glycosylated hemoglobin in those diabetic patients [243], which 81
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Discussion
makes benfotiamine a proper candidate for erythrocyte protection, and might be important finding that open vista of further investigations to add another novel therapeutic potentials of benfotiamine in prevention and/or treatment of diabetic complications associated with glycosylated Hb, AGEs and generation of ROS. Moreover, lipid peroxidation involves the metal-dependent reaction associated with various oxygen radical generations [244], and the metal chelating activity of TDP inhibit generation of peroxyl radical and hydroperoxide radical [211]. Although more detailed studies for radical scavenging activity of benfotiamine are required, the reported data in the present study might give a new biochemical or pharmacological aspect of this prodrug.
4.3. In vivo study The simulation of the in vitro observation has been achieved by employing in vivo animal model to verify the validity of the protective effect of silibinin and benfotiamine in nitrite-induced oxidation of hemoglobin in experimental animals. The oxidation of oxyhemoglobin by nitrite to produce MetHb is a complex process that has been characterized by a lag phase followed by an autocatalytic phase [199]. Kohn et al (2002), in a study on pharmacokinetics of sodium nitrite-induced methemoglobinemia in rats, described the reaction of nitrite with hemoglobin by displacing the bound oxygen after binding of nitrite with oxyhemoglobin and yield MetHb, hydrogen peroxide and nitrogen dioxide in a free radical chain initiation step; nitrogen dioxide oxidizes ferrous hemoglobin to MetHb, whereas hydrogen peroxide oxidizes MetHb to a ferryl-Hb radical, and the reaction of ferryl-Hb with nitrite also produce MetHb and nitrogen dioxide; these last two reactions are the free radical chain propagation steps [245]. In the present study, acute nitrite intoxication induced an increase in MetHb up to 55%; nitrite methemoglobinemia is a potent process for free radical 82
Chapter four
Discussion
generation [245] including superoxide anion radicals. This xenobiotic is a ready source of nitric oxide which reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO¯ ) [246].
4.3.1. Protective effect of silibinin: in vivo study In the present study, an attempt has been made to validate the protective effect of silibinin against nitrite-induced hemoglobin oxidation in rats. The protective effect of silibinin was assessed by measuring the MetHb content; such protective effect was elucidated by the significant reduction in the level of MetHb, suggesting that silibinin possesses substantial protective effect and free radical scavenging mechanism against exogenous nitrite-induced oxidation of hemoglobin, which is in line with previously published findings on the protective effects of silymarin against most reactive species released by environmental toxins
[210].
The protective effect of silymarin was
demonstrated by significant reversal of the antioxidant enzymes and reduction in the level of malondialdehyde and normalizing hemolysis, methemoglobin content and protein carbonyl content [210], which can be considered as other suggested mechanisms for the protective effects of silibinin in this model. In fact, it has been reported that antioxidant property of silibinin is due to its potent ability to react with OH•, while it is not an effective scavenger of O•2 or H2O2 [247], but reaction of O•2 and H2O2 generates OH• radicals [248]. Additionally, reaction of ferrous ion in Hb with H2O2 generates OH• radical in Fenton reaction [191]. The result of the present study concerning lowering the level of MetHb during long-term exposure of animals to silibinin might be related to the scavenging of the previously mentioned radical. This finding was consistent with our current observation in the in vitro results. Furthermore, the result of this study suggests the importance of pharmacokinetic parameter in bioavailability of silibinin dihemisuccinate; since short-term silibinin exposure fails to inhibit MetHb formation compared to 83
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Discussion
control group, while long-term silibinin exposure for 7 days showed significant lowering in the level of MetHb. The poor and erratic bioavailability of silibinin might partly explain the obtained results [106]; meanwhile, parenteral administration of silibinin suspected to produce better protection against hemolysis in vivo, as seen in the case where intravenous administration of SDHS provides a highly effective hepatoprotection against damage during acute toxicity with acetaminophen or Amanita phalloid poison. Recently, nano-suspensions of silibinin with smaller particle size reveal a higher potential to increase their oral bioavailability [249], also self emulsifying drug delivery systems are a promising approach for the formulation of drug compounds with poor aqueous solubility like silibinin [250].
4.3.2. Protective effect of benfotiamine: in vivo study Oral administration of benfotiamine leads to significant increase in thiamine, thiamine monophosphate and TDP level in blood [153], the suggested mechanism of absorption and the metabolic fate of benfotiamine is dephosphorylation to S-benzolylthiamine by ecto-alkaline phosphatase present in the brush border of intestinal mucosal cells. A significant part of S -benzoyl thiamine is captured by erythrocyte [157,158] and converted to free thiamine through a slow-non enzymatic transfer of the S-benzoyl group to SH groups of glutathione [153]. Recently, a new study showed that benfotiamine reduce superoxide and hydroxyl radical levels in diabetic heart by inducing the activation of pentose phosphate pathway, which regenerates the antioxidant NADPH [251]. Significant reduction in MetHb% in both short- and long-term benfotiamine treatment approaches that followed in the present study might be explained
by radical scavenging property of benfotiamine,
because
benfotiamine exhibited antioxidant effect by reducing the oxidative stress and genomic damage caused by mitogenic model compounds, and the effect is found to be related to its direct antioxidant capacity [182]. Furthermore, we can 84
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Discussion
not exclude the other mechanisms of actions of benfotiamine and thiamine associated with the improvement in transketolase activity of erythrocytes [252,178] reducing
superoxide overproduction through directing advanced
glycation and lipoxidation end products substrate to the pentose phosphate pathway [162], decrease the level of malondialdehyde, and the increase in the levels of reduced glutathione and vitamin E levels, as reported in studies with acute ethanol and CCl4 intoxication [253,254].
85
Concentration-Effect Relationship for the Radical Scavenging Activity of Silibinin Dihemisuccinate and Benfotiamine in Nitrite-induced Hemoglobin Oxidation
A Thesis Submitted to the council of the College of Medicine, University of Sulaimani, in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacology
By Bushra Hassan Marouf B.Sc. Pharmacy
Supervised by Prof. Dr. Saad A. Hussain September 2010
Chapter One
Introduction
Chapter one Introduction and literature review 1.1 Terminology and Definition of free radicals The related terms oxidative stress, oxidative damage, free radical, and antioxidant have become an integrated part of the scientific vocabulary and are often used in a variety of scientific discussions and issues by chemists, physicists, biologists, and researchers [1]. Free radicals, known in chemistry since the beginning of the 20th century, were initially used to describe intermediate compounds in organic and inorganic chemistry, and several chemical definitions for them were suggested; only in 1954 were these radicals suggested as important players in biological environments and responsible for deleterious processes in the cell [2]. These radicals are now considered major players in biochemical reactions, cellular response, and clinical outcome [3,4]. The toxicity of the atmospheric oxygen molecule had already been used by our ancestors for therapeutic purposes, such as treatment of sites infected with the anaerobic bacterium Clostridium by exposure to air [1]. Widespread cases of blindness in young infants born prematurely in the 1940s were associated with the high oxygen concentration in the newly invented incubators [5,6]. Chemically, every compound, including oxygen that can accept electrons is an oxidant or oxidizing agent; in contrast, a substance that donates electrons is a reducing agent [7]. In biology, a reducing agent acts via donation of electrons, usually by donation of hydrogen or removal of oxygen, while oxidation process is always accompanied by a reduction process in which there is usually a loss of oxygen, in an oxidation process there is a gain in oxygen [8]. While reducing agents and oxidants are chemical terms, in biological environments they should be termed
1
Chapter One
Introduction
antioxidant and pro-oxidant, respectively [9]. The radical group, often incorrectly called free-radical, contains compounds such as nitric oxide radical (NO•), superoxide ion radical (O•2), hydroxyl radical (OH•), peroxyl (ROO•) and alkoxyl radicals (RO •), and one form of singlet oxygen (1O 2) (Table 1.1) [10]. The occurrence of one unpaired electron results in high reactivity of these species by their affinity to donate or obtain another electron to attain stability. There is a group of non-radical compounds that contains a large variety of substances, some of which are extremely reactive although not radical by definition; among these compounds produced in high concentrations in the living cell are hypochlorous acid (HOCl), hydrogen peroxide (H2O2), organic peroxides, aldehydes, ozone (O 3), and O2 [11]. The terminologies ROS, oxygen-derived species (ODS), oxidants, reactive nitrogen species (RNS), and pro-oxidant species are often used interchangeably in the scientific literature; an antioxidant (reducing agent), therefore, can be classified as a compound capable of preventing the pro oxidation process, or biological oxidative damage [12]. Halliwell suggested a definition for antioxidant, which states that this agent, when present in low concentration, significantly prevents or delays oxidation of an oxidizable substrate [13].
2
Chapter One
Introduction
Table 1-1. Radical and nonradical oxygen metabolites [10].
3
Chapter One
Introduction
The organism must confront and control the presence of both pro-oxidants and antioxidants continuously; the balance between these is tightly regulated and extremely important for maintaining vital cellular and biochemical functions [14]. This balance often referred to as the redox potential, is specific for each organelle and biological site, and any interference of the balance in any direction might be deleterious for the cell and organism [15]. Changing the balance towards an increase in the prooxidant over the capacity of the antioxidant is defined as oxidative stress and might lead to oxidative damage; changing the balance towards an increase in the reducing power, or the antioxidant, might also cause damage and can be defined as reductive stress (Figure 1-1) [16].
1.2. Chemical Properties of Some ROS Because most radicals are short-lived species, they react quickly with other molecules; some of the oxygen-derived radicals are extremely reactive with a short half-life, for example, OH• can survive for 10–10 sec in biological systems; its reaction rate constants (m–1s –1) for biological components are extremely high (107–109 m–1s –1) and in many cases diffusion-controlled [17]. The life span of other radicals is also short but depends on the environmental medium. For example, the half-life of NO• in an air-saturated solution might be a few minutes; RO • can survive about 10–6 of a second, while the half-life of ROO• is about 17 seconds [18]. Nonradical metabolites also possess a relatively short half-life varying from parts of seconds to hours, as in the case of HOCl; obviously, the physiological environment, consisting of such factors as pH and the presence of other species, has a great influence on the half-life of ROS [19].
4
Chapter One
Introduction
Figure 1-1. Interference in the balance (large arrow) between oxidant and reductant defines oxidative- or reductive-stress conditions [16].
5
Chapter One
Introduction
Toxicity is not necessarily correlated with reactivity; in many cases a longer half-life of a species might imply a higher toxicity of the compound by allowing it adequate time to diffuse and reach a sensitive location where it can interact and cause damage a long distance from its site of production [20]. For example, the relatively long half-life of superoxide radicals permits them to move to locations where they can undergo interaction with other molecules; these radicals can be produced in the mitochondrial membrane, diffuse towards the mitochondrial genome, and reduce transition metals bound to the genome [21]. On the other hand, a highly reactive species with an extremely short life span, like OH•, is produced in locations where it can cause damage by interacting with its immediate surroundings [22]. If there is no essential biological target adjacent their production site, radicals will not cause oxidative damage. The high reactivity of radicals and their short life span illustrate the potential toxic effect and difficulties in preventing oxidative damage. To prevent the interaction between radicals and biological targets, the antioxidants should be present at the location where the radicals are being produced in order to compete with the radical for the biological substrate [9]; such information should be used as a guideline in determining appropriate antioxidant therapy.
1.3. Chemical Qualities and Reactivities of some ROS 1.3.1. Superoxide Anion Radical (O• 2/HO• 2) This species possesses different properties depending on the environment and pH due to its pKa of 4.8, superoxide can exist in the form of either O•2 or, at low pH, hydroperoxyl (HO •2) [23]. The latter can more easily
penetrate
biological
membranes
than
the
charged
form.
Hydroperoxyl radical can therefore be considered as an important species, although under physiological pH most of the superoxide is in the charged 6
Chapter One
Introduction
form. In a hydrophilic environment both the O •2 and HO•2 can act as reducing agents capable, for example of reducing ferric (Fe+3) ions to ferrous (Fe+2) ions; however, the reducing capacity of HO •2 is higher. In organic solvents the solubility of O •2 is higher, and its ability to act as a reducing agent is increased. It also acts as a powerful nucleophile, capable of attacking positively charged centers, and as an oxidizing agent that can react with compounds capable of donating H (e.g., ascorbate and tocopherol). The most important reaction of superoxide radicals is dismutation; in this reaction, superoxide radical reacts with another superoxide radical; one is oxidized to oxygen, and the other is reduced to hydrogen peroxide [24]. HO•2 /O•2– + HO•2 /O•2–
H2O2 + O2 k ≈ 106 M–1s –1
1.3.2. Hydroxyl Radical (OH• ) The reactivity of hydroxyl radicals is extremely high; in contrast to superoxide radicals that are considered relatively stable and have constant, relatively low reaction rates with biological components, hydroxyl radicals are short-lived species possessing high affinity toward other molecules [25]. OH• is a powerful oxidizing agent that can react at a high rate with most organic and inorganic molecules in the cell, including DNA, proteins, lipids, amino acids, sugars, and metals. The 3 main chemical reactions of hydroxyl radicals include hydrogen abstraction, addition, and electron transfer [26]. OH• is considered the most reactive radical in biological systems; due to its high reactivity, it interacts at the site of its production with the molecules closely surrounding it [1].
7
Chapter One
Introduction
1.3.3. Hydrogen Peroxide (H2O2) The result of the dismutation of superoxide radicals is the production of H2O2 [1]. There are some enzymes that can produce H2O2 directly or indirectly. Although H2O2 molecules are considered reactive oxygen metabolites, they are not radical by definition; they can, however, cause damage to the cell at a relatively low concentration [27]. They are freely dissolved in aqueous solution and can easily penetrate biological membranes. Their deleterious chemical effects can be divided into the categories of direct activity, originating from their oxidizing properties, and indirect activity in which they serve as a source for more deleterious species, such as OH• or HOCl. Direct activities of H2O2 include degradation of haem proteins; release of iron; inactivation of enzymes; and oxidation of DNA, lipids, SH groups, and keto acids [28].
1.3.4. Nitric Oxide (NO• ), Peroxynitrite (ONOO–), and Other Members of the Family The nitric oxide radical (NO •) is produced by the oxidation of one of the terminal guanido nitrogen atoms of L-arginine; in this reaction, catalyzed by the group of enzymes called nitric oxide synthase (NOS), Larginine is converted to nitric oxide and L-citrulline [29]. One-electron oxidation results in the production of nitrosonium cation (NO +), while oneelectron reduction leads to nitroxyl anion (NO –), which can undergo further reactions, such as interacting with NO • to yield N2O and OH• [30]. The half-life of the nitric oxide radicals depends on the square of the radical concentration; NO• can react with H2O2 and HOCl to yield a line of derivatives such as N2O3, NO2–, and NO3– [1]. One of the most important reactions under physiological conditions is that of superoxide and nitric oxide radicals resulting in peroxynitrite; this reaction helps to maintain the balance of superoxide radicals and other ROS and is also important in 8
Chapter One
Introduction
redox regulation [31]. The protonated form of peroxynitrite (ONOOH) is a powerful oxidizing agent that might cause depletion of sulfhydryl (–SH) groups and oxidation of many molecules causing damage similar to that observed when OH• is involved [32]. It can also cause DNA damage such as breaks, protein oxidation, and nitration of aromatic amino acid residues in proteins (e.g., 3-nitrosotyrosine). Under physiological conditions, ONOOH can react with other components present in high concentrations, such as H2O2 or CO2, to form an adduct that might be responsible for many of the deleterious effects seen in biological sites [33].
1.4. Sources of ROS The living cell is continuously exposed to a large variety of ROS and RNS from both exogenous and endogenous sources. Exposure of living organisms to ionizing and nonionizing irradiation constitutes a major exogenous source of ROS, such as hemolytic cleavage of H2O2 by UV radiation which yields OH• radicals [34,35]. Air pollutants such as car exhaust, cigarette smoke, and industrial contaminants encompassing many types of NO derivatives constitute major sources of ROS that attack and damage the organism either by direct interaction with skin or following inhalation into the lung [36,37]. Certain types of drugs and/or their metabolites are also considered as a major source of ROS [38,39]. There are drugs, such as belomycin [40] and doxorubicin [41], where their mechanisms of action as cytotoxic agents are mediated via production of ROS; those like nitroglycerine and other nitrates are NO• donors, and some have the ability to produce ROS indirectly. Moreover, some narcotic agents and anesthetic gases are considered major contributors for the production of ROS in the biological system [42]. A large variety of xenobiotics produce ROS as a by-product of their metabolism in vivo [43,44].
9
Chapter One
Introduction
The invasion of the biological system by pathogens, bacteria, and viruses might result in the production of many types of ROS through direct release from the invaders or as a consequence of endogenous response of the immune system, induced by phagocytes and neutrophils [45]. Food constituents can also be considered as one of the major sources for the formation of oxidant species [46,47], which includes different kinds of chemical structures such as peroxides, aldehydes, oxidized fatty acids, and transition metals [48]. Although the exposure of the organism to ROS is extremely high from exogenous sources, the exposure to endogenous sources is much more important and extensive, because it is a continuous process during the life span of every cell in the organism [45]. The mitochondria serves as the major organelle responsible for ROS production during daily activities, while enzymes comprise another endogenous source for ROS; most enzymes produce ROS as a by-product of their activity like the formation of superoxide radicals by xanthine oxidase, and there are some enzymes designed to produce ROS, such as nitric oxide synthase that yields NO• radicals [49]. White blood cells, including neutrophils [50], eosinophils, basophils, mononuclear cells (monocytes), and lymphocytes are considered as another major source for endogenously produced ROS, probably through their mechanisms of action to eradicate bacteria and other invaders [51,52]. Numerous pathologies and disease states also serve as sources for the continuous production of ROS [53, 54]. More than 200 clinical disorders have been described in the literature in which ROS were important for the initiation stage of a disease and/or produced during its course of the disease or as a consequence of tissue damage associated with it [45]. ROS may play an important role as initiators and mediators in many types of cancer [1,55,56], heart diseases, endothelial dysfunction atherosclerosis and other cardiovascular disorders [1,57,58], 10
inflammation [59], burns [60],
Chapter One
Introduction
intestinal tract diseases [61], degenerative CNS disorders [62,63,20], diabetes [64,65] and eye diseases [66]. Additionally, red blood cell has been extensively studied, both as a source of free radicals and as a target for oxidative damage. This is largely because a wide variety of drugs and xenobiotics that can undergo oxidation-reduction reactions have been reported to cause red cell destruction and hemolytic anemia. Interaction between the xenobiotic and hemoglobin is of prime importance in the process, which is usually characterized by hemoglobin oxidation to methemoglobin [67].
Among
the xenobiotics that induce methemoglobinemia is sodium nitrite; it is an inorganic salt used in the manufacture of dyes, treatment of textiles and curing of meat, which is capable of oxidizing hemoglobin and related hemoproteins; it is classified as a causative agent of methemoglobinemia [68]. Recently, an additional mechanism to generate reactive oxygen species, which involves the interaction of the ferric state of heme proteins with hydrogen peroxide, was reported during the autoxidation process in vivo [69].
1.5. Free Radicals as a Cause of Oxidative Damage Reactive oxygen species (ROS) and other free radicals are, due to their high reactivity, prone to cause damage, and are thereby also potentially toxic, mutagenic, or carcinogenic. The targets for ROS damage include all major groups of biomolecules (Figure 1-2) [70], summarized as follows:
1.5.1. Effects on Nucleic Acids Free radicals, especially reactive oxygen species (ROS), have been shown to be mutagenic [71], an effect that should be derived from chemical modification of DNA.
11
Chapter One
Introduction
Figure 1-2. Formation of reactive species in vivo and the role of defense systems against them [70]
12
Chapter One
Introduction
A number of alterations (cleavage of DNA, DNA-protein cross links, oxidation of purines) are due to reactions with ROS, especially OH•; if the DNA repair systems are not able to immediately regenerate intact DNA, a mutation will result from incorrect base pairing during replication [72]. This mechanism may partly explain the high prevalence of cancer in individuals exposed to oxidative stress [73]. The fact that apoptosis in some cases is mediated by ROS [74] may in part be due to ROS-derived damage to DNA, but is also related to increased mitochondrial permeability, released cytochrome C, increased intracellular Ca2+, and other effects [75]. The concept of ROS as an important factor in cellular and whole organism aging due to damage to mitochondrial DNA is an interesting theory [76]. This concept was recently challenged by a study based on extensive gene array analysis suggesting errors in the mitotic machinery and possibly in arachidonic acid metabolism as being important determinants for the aging process [77]. However, accumulating data do indicate that ROS contribute to aging and, conversely, that superoxide dismutase and catalase mimetics may prolong the life span of many experimental animals [78].
1.5.2. Effects on Lipids Lipid peroxidation is probably the most explored area of research when it comes to ROS [79]. Polyunsaturated fatty acids are, because of their multiple double bonds, excellent targets for free radical attacks; such oxidation is also essential for the generation of atherosclerotic plaques [80]. The mechanism for plaque formation involves oxidation of low density lipoproteins (LDL), uptake of those particles by phagocytes in the subendothelial space via their scavenger receptor, and finally, accumulation of these phagocytic cells in the sub-endothelial space, where they stimulate formation of atherosclerotic plaques [81]. Cardiovascular disease with plaque formation constitutes a large part of the total burden of disease, at 13
Chapter One
Introduction
least in western countries; therefore, prevention or decrease of lipid peroxidation is of significant medical importance [82].
1.5.3. Effects on Proteins Proteins, also major constituents of membranes, can serve as possible targets for attack by ROS, which have been shown to react with several amino acid residues in vitro, generating anything from modified and less active enzymes to denatured, nonfunctioning proteins [83]. Among the most susceptible amino acids are sulfur- (or selenium)-containing residues. General antioxidant systems such as thioredoxin (Trx), glutaredoxin (Grx) or glutathione (GSH), or specific systems, such as methionine sulfoxide reductases to which Trx serves as electron donor [84], all uphold the protection of proteins from such modification. Among the various ROS, the OH•, RO•, and nitrogen-reactive radicals predominantly cause protein damage. Hydrogen peroxide itself and superoxide radicals in physiological concentrations exert weak effects on proteins; those containing SH groups, however, can undergo oxidation following interaction with H2O2 [85]. Proteins can undergo direct and indirect damage following interaction with ROS, including peroxidation, damage to specific amino-acid residues, changes in their tertiary structure, degradation, and fragmentation [86]. The consequences of protein damage as a response mechanism to stress are loss of enzymatic activity, altered cellular functions such as energy production, interference with the creation of membrane potentials, and changes in the type and level of cellular proteins [87]. Protein oxidation products are usually aldehydes, keto compounds, and carbonyls; one of the major adducts that can easily be detected and serve therefore as a marker for protein oxidative damage is 3nitrotyrosine, this adduct is produced following the interaction between ONOO and other nitrogen reactive radicals with the amino acid tyrosine [1]. Following OH• attack, a series of compounds can be formed, including 14
Chapter One
Introduction
hydroxyproline, glutamyl semialdehyde, and others. Following protein oxidation, modified proteins are susceptible to many changes in their function; these include chemical fragmentation, inactivation, and increased proteolytic degradation [88].
1.6. Defense Mechanisms of the Cell against Oxidative Stress Continuous exposure to various types of oxidative stress from numerous sources has led the cell and the entire organism to develop defense mechanisms for protection against reactive metabolites. These mechanisms encompass both indirect and direct activities. Indirect approaches may involve control of the endogenous production of ROS [89], one of the most important methods for the organism to cope with oxidative damage, consists of enzymes and small molecules that can efficiently repair the radical-induced
damage at many sites
on
macromolecules and tissue components. Physical defense of biological sites, such as biological membranes, is also an important mechanism allowing the cell to cope with oxidative stress [45]. Moreover, endogenous compounds such as albumin, tocopherols and many others provided enhanced stability to cellular membranes, and some times through steric interference, they can prevent ROS from approaching the target [45]. Among the various defense mechanisms, the one involving antioxidants is extremely important due to its direct removal of pro-oxidants, oxidants and potentially toxic radicals; in the biological system, there are many compounds that can act as antioxidants and ensure maximum protection for biological sites [45]. The uniqueness of this system is its direct interaction with ROS of various kinds and its provision of protection for biological targets. The system contains two major groups; antioxidant enzymes and low-molecular-weight antioxidants
(LMWA).
The enzyme-containing
group is composed of direct-acting proteins, such as SOD [1]; there are 15
Chapter One
Introduction
many isoforms of proteins in this family, which differ in their structure and the associated cofactors. It is capable of enhancing the spontaneous dismutation of superoxide radicals to H2O2 [90]. The end product of the dismutation reaction (H2O2) can be removed by the activity of the enzyme catalase and some members of the peroxidase family, including glutathione peroxidase [91]. The low-molecular-weight antioxidant (LMWA) group contains numerous compounds capable of preventing oxidative damage by direct and indirect interaction with the generated ROS [89]. The indirect mechanism involves the chelation of transition metals (iron, copper) and prevents them from participating in the metal mediated chain reactions, which are responsible for continuous ROS production [92]. The direct acting molecules donate electrons to the oxygen radical so that they can scavenge the radical and prevent it from attacking the biological target. Scavengers possess many advantages over the group of enzymatic antioxidants. Because scavengers are small molecules, they can penetrate cellular membranes and be localized in close proximity to the biological target [45]. They possess a wide spectrum of activities toward a large variety of ROS, and the scavenging mechanism can proceed only if the concentration of the scavenger is sufficiently high to compete with the biological target on the deleterious species [89]. Direct radical scavengers are characterized by their common mechanism of activity; they react directly with the radical and neutralize it by donating an electron(s) to these reactive species [45]. Among LMWA is glutathione (GSH); a lowmolecular-mass, thiol-containing tripeptide, glutamic acid-cysteine-glycine (GSH) in its reduced form and (GSSG) in its oxidized form [1]. GSH is present in humans, animals, plants, and aerobic bacteria at high concentrations reaching the millimolar range; it acts as a cofactor for the enzyme peroxidase, thus serving as an indirect antioxidant donating the electrons necessary for the decomposition of H2O2 [45]. 16
Chapter One
Introduction
Melatonin, another LMWA, a hormone synthesized by the pineal gland, possesses a powerful antioxidant capacity in vitro, and may scavenge a variety of ROS, probably through donation of the hydrogen atom by the (NH) group [93]. Uric acid is a cellular waste product, originating from the oxidation of hypoxanthine and xanthine by xanthine oxidase and dehydrogenase. It provides efficient antioxidant activity for the organism, and its formation was found to be up-regulated during the state of oxidative stress [94]. Large numbers of antioxidant molecules exist in green vegetables, fruits, and many other natural sources; they include small molecules such as ascorbic acid, a water-soluble antioxidant that can be synthesized by plants and some animals; ascorbate can act as an efficient antioxidant and scavenge a variety of ROS including hydroxyl, peroxyl, thyil, and oxosulphuric radicals [95]. Ascorbate is also a powerful scavenger of HOCl and peroxynitrous acid and can inhibit the peroxidation process [96]. Additionally, vitamin E, lipophilic LMWA derived from dietary sources in the family of tocopherol antioxidants, this compound acts as a chainbreaking antioxidant, and can scavenge many types of ROS generated within the lipoidal structures to inhibit the lipid peroxidation process in biological membranes [97]. There are many drug molecules that have inherited radical scavenging activity, and such property may constitute part of the mechanisms through which they produce their pharmacological response; these include carvedilol [98], N-acetylcysteine amide (NACA) [99], pramipexol [100] and Silibinin dihemisuccinate among others [101].
1.7. Methods for determination of ROS and radicals Many approaches allow evaluation and demonstration of the participation of
ROS
in
biochemical events.
The
methods
of
chemiluminescence (CL) and electron (spin, pair) resonance (ESR,EPR) 17
Chapter One
Introduction
are direct methods for radical investigation [102,103], and spin trapping method also is another technique in which it is suitable to detect a highly reactive radicals such as OH˙; the principle is based on the reaction of the radical with a trap molecule to produce a stable radical product that can be evaluated; other trapping procedures are hydroxylation of salicylic acid [104], the deoxyribose assay [25], the cytochrome C reduction assay for detection of superoxide radicals [105].
1.8. Silibinin 1.8.1. Chemical composition Silibinin or (silybin), a flavonolignan extracted from milk thistle, is a semipurified, commercially available fraction of silymarin; it was once thought to be a single compound and is often treated so in the literature. In fact silibinin is a roughly 1:1 mixture of diasterioisomeric compounds, silibinin A and silibinin B (Figure 1-3) [106]. It is considered as a major constituent of silymarin (total extract of Silybum marianum seeds) with the greatest degree of biological activity, which accounts for 90% of the total extract components in most preparations [106,107].
1.8.2. Pharmacokinetics Flavonolignans bioavailability [106].
are
notorious
for
their
poor
and
erratic
To maximize oral bioavailability, silibinin was
formulated with phosphatidylcholine as (Silipide).
Pharmacokinetic
studies with this complex (Silipide) have shown an increase in the oral bioavailability of silibinin in healthy subjects, probably by an enhancing role of the drug complex on the passage of the parent drug across the gastrointestinal tract [108,109]. Low- and high-dose pharmacokinetic studies provided the best evidence that silibinin can be administered to human at doses producing anticancer-relevant concentration with minimum 18
Chapter One
Introduction
or no side effects [110,111], although asymptomatic hyperbilirubinemia may appear due to the inhibition of glucuronyl-transferase activity in the liver [111].
Drug
interaction
between
silibinin
and
conventional
pharmaceuticals appears to be quite low [112], particularly at doses less than 1 to 5g/day; however, several reports have appeared suggesting that silibinin can inhibit some isoforms of cytochrome P450 family, including CYP34A and CYP2C9, which may predispose to a pharmacokinetically relevant drug-drug interactions [113,114,115,116]. In cancer chemotherapy, these findings are important because several conventional chemotherapeutic agents are also metabolized by CYP3A4, raising some concerns regarding other silibinin-drugs interactions in oncology or other settings [106].
19
Chapter One
Introduction
(1) Silybin A
(2) Silybin B
Figure 1-3. Chemical Structure of (1) Silybin A and (2) Silybin B [106]
20
Chapter One
Introduction
1.8.3. Pharmacodynamic and Medicinal properties of Silibinin 1.8.3.1 Antioxidant Activity Silibinin works as an antioxidant, scavenging free radicals and inhibiting lipid peroxidation; it provides protection against genomic injury, increases hepatocyte protein synthesis, decreases the activity of tumor promoters, stabilizes mast cells, chelates iron and slow down calcium metabolism [117]. It influences the enzyme system associated with glutathione metabolism and superoxide dismutase activity [118]. A significant increase in the amount of reduced glutathione (GSH) content was found in the liver, intestine and stomach after treatment with silibinin intravenously [119]. It protects the brain from oxidative damage for its ability to prevent lipid peroxidation and replenishing the GSH level in the CNS [120]. Silibinin displays powerful cytoprotective properties [121] and it may protect blood constituents from oxidative damage induced by many events, including xenobiotics and endogenous toxins [122]. The antioxidant properties were evaluated by studying the ability of this drug to react with relevant biological oxidant, such as superoxide anion radical (O•2), hydrogen peroxide (H2O2), hydroxyl radical (OH•) and hypochlorous acid (HOCl) [101]. Both Silibinin, as a base, and silibinin dihemisuccinate (SDH) proved to be a strong scavengers of hypochlorous acid (HOCl) but not of superoxide anion radical (O•2) [121], which is produced by human granulocytes, and no reaction with H2O2 was detected; however, SDH reacts rapidly with hydroxyl radical (OH•) and appears to be a weak iron ion chelator. The studies on rat liver microsome lipid peroxidation induced by FeIII/ascorbate showed that SDH has an inhibitory effect, which is dependent on its concentration and the magnitude of lipid peroxidation [101].
21
Chapter One
Introduction
1.8.3.2. Hepatoprotective activity Silibinin significantly inhibits concanavalin A-induced liver disease [123]; it also provides hepatoprotection against poisoning by phalloidin [124], hallothan [125], and ischemic injury [126]. SDH interacts with many hepatotoxic xenobiotics and drug molecules; it protects liver against glutathione depletion and lipid peroxidation induced by acetaminophen and prevents or attenuates hepatotoxicity [127]. Silibinin has a strong affinity for the cytochrome P450 enzymes, and there is a possibility that modulation of their activities by Silibinin may play a role in this respect, through interference with the metabolic activation of many chemicals invading the biological system [128]. It protects the exocrine pancreas from cyclosporine-induced toxicity, inhibits lipid peroxidation in hepatic microsomes, mitochondria of rats and is also able to reduce the activity of various
monooxygenases
with
consequent
interference
with
the
biotransformation of many xenobiotics [127]. In this respect, the effect of silibinin on cyclosporine biotransformation in the liver is thought to be via modulation of the catalytic activity of cytochrome P450 [129]. It also can lessen or protect against the toxic effects of cisplatin and vincristine [130,131].
1.8.3.3. Effect in alcoholic liver disease Ethanol metabolism is directly involved in the production of reactive oxygen species and reactive nitrogen species; these form an environment favorable to oxidative stress [132]. Silibinin was found to be effective in alcoholic cirrhosis [133] and was able to protect rats from ethanol-induced oxidative stress in the liver tissue [134].
22
Chapter One
Introduction
1.8.3.4. Anti-inflammatory activity Silibinin showed anti-inflammatory effects [135], which may be attributed to inhibition of neutrophil migration [136] and kuppfer cell inhibition [137]. It has been found to interfere with the formation of leukotrines and prostaglandin formation from polyunsaturated fatty acids in the liver, via its inhibition of the enzyme lipoxygenase; these leukotrines are very well characterized as potential mediators of harmful inflammatory cascades that leads to tissue damage [138].
1.8.3.5. Immunomodulatory activity Silibinin significantly suppress the expression of CD 80, CD 86, MHC (Histocompatability complex molecules) class I and class II in the murine bone marrow-derived dendritic cells (DCs); it is reported to inhibit the lipopolysaccharide-induced activation of MAPKs (Mitogen–activated protein kinase) and nuclear translocation of the NF-kB p56 subunit [139]. Additionally,
silibinin
inhibits
interferon-gamma ( -IFN),
intra-hepatic
expression
of
TNF-α,
interleukin IL-4,
IL-2 and
iNOS; and
augmenting synthesis of IL-10 [123]; such types of reported activities strongly suggesting silibinin as a good candidate to be evaluated for its clinical use in many disorders related to impaired or dysregulated immune function.
1.8.3.6. Antiviral activity Silibinin strongly inhibited the growth of both HepG2 (hepatitis B virus negative; p53 intact) and Hep3B (hepatitis B virus positive; p53 mutated) cells with a relatively stronger cytotoxicity in Hep3B cells, which was associated with induction of apoptosis; it also caused G1 arrest in HepG2 and both G1 and G2-M arrest in Hep3B cells. Furthermore,
23
Chapter One
Introduction
silibinin strongly inhibited CDK2 and CDK4 activity in the HCC cells [140].
1.8.3.7. Glycemic and lipidemic control Silibinin shows a favorable impact on glycemic and lipidemic control in type 2 diabetics with liver cirrhosis [141]. A commercial pharmaceutical preparation of silibinin is being successfully used in human medicine to improve glycemic control in type 2 diabetics, as it induced a decrease in both fasting insulin and exogenous insulin requirements during mealtime [142]. Furthermore, recent evidence shows that silibinin induced a shortterm inhibition of gluconeogenisis in hepatocytes perifused with a variety of gluconeogenic sources, an event which can be correlated with the decrease in glucose-6-phosphate hydrolysis [143]. Silibinin also has inhibitory properties on LDL oxidation in vitro, and might represent a novel tool in the prevention and therapy of atherosclerosis [144]. A thermal burn which is associated with extensive oxidation of polyunsaturated fatty acids can be antagonized by silibinin [145].
1.8.3.8. Anti-fibrotic activity Hepatic stellate cells and the derived myofibroblasts play a central pathogenic role in liver fibrogenesis. Silibinin reduced the transformation of these cells towards myofibroblasts, and down-regulated the gene expression of extracellular matrix components and the profibrogenic transforming growth beta (TGF- ) [146]. Alterations of TGF- 1 and c-myc expression in the liver may be involved in the hepatoprotective effects afforded by silibinin [147]. Inhibition of hepatic stellate cell proliferation and transformation might be one of the important aspects of the potential antifibrotic properties of silibinin [146]. 24
Chapter One
Introduction
1.8.3.9. Anti-carcinogenic/anti-tumorigenesis activity Treatment with silibinin results in a highly significant inhibition of both cell growth and DNA synthesis in a time-dependent manner, with large loss of cell viability only in case of cervical carcinoma cells [148]. Silibinin also significantly induces growth inhibition, a moderate cell cycle arrest and a strong apoptotic death in both small cell and non-small cell human lung carcinoma cells. It inhibits cell growth via G1 arrest, leading to differentiation of androgen-dependent human prostate carcinoma LNCaP cells [149]. Silibinin caused hypophosphorylation of Rb-related proteins, so it may, in part, be responsible for its cancer preventive and anticarcinogenic efficacy in different cancer models including prostate cancer cells; this effect was mainly attributable to a large decrease in the amount of Rb phosphorylated at specific serine sites [150]. Silibinin was found to suppress the growth and induce the apoptosis of ECV304 cells, at least partly, by inhibiting angiogenesis via modulation on NF-қB, Bcl-2 family and caspases [151].
1.9. Benfotiamine 1.9.1. Chemical Composition Benfotiamine, a unique pro-drug derivative of thiamine, is the most potent of allithaimines, a group of lipid-soluble forms of thiamine [152]. Chemically
known
as
S-benzoylthiamine-O-monophosphate,
an
amphiphilic S-acylthiamine derivative; it has different mechanisms of action and a different pharmacological profile than lipid-soluble thiamine disulfide derivatives [153], which are found in trace amounts in roasted garlic and other herbs of the genus Allium.
25
Chapter One
Introduction
Figure 1-4. Chemical structure of (A) thiamine and (B) benfotiamine containe an open thiazole ring that helps benfotiamine readily enter the cell through the plasma membrane, increasing its bioavailability. Once in the cytoplasm the ring closes and gives it a structure similar to that of thiamine [152]
26
Chapter One
Introduction
Compared to thiamine, benfotiamine has a unique open thiazole ring structure (Figure 1-4) which allows it to pass directly through cell membranes [152].
1.9.2. Pharmacokinetics The chemical structure of benfotiamine has an open thiazole ring enables it to enter directly through the cell membranes resulting in enhanced bioavailability [154,155,156]. Open thiazole ring closes once the compound is taken up by the cells, producing the biologically active thiamine.
Benfotiamine
after
its
oral
administration
is
first
dephosphorylated to S-benzoylthiamine by ecto-alkaline phosphatase present in brush borders of intestinal mucosal cells. The lipophilic Sbenzoylthiamine is absorbed and then diffuses by passive diffusion through the membranes of intestinal and endothelial cells and subsequently appears in circulation. A significant part of S-benzoylthiamine is captured by erythrocytes and is converted to free thiamine through a slow nonenzymatic transfer of the S-benzoyl group to SH groups of glutathione [157]. In the liver the remainder can be enzymatically hydrolyzed to thiamine and benzoic acid by thioesterases present in hepatocyte [158]. Oral administration of benfotiamine results in the availability of at least five times greater plasma concentration of thiamine than an equivalent dose of thiamine [159]. It strongly increases thiamine levels in blood and liver while has no significant effect in the brain, which explains the beneficial effects of the drug, that concern with peripheral tissues but not the central nervous system [153].
1.9.3. Pharmacodynamics The Advanced Glycation End products (AGEs) are heterogeneous class of compounds that are formed by non-enzymatic reaction between 27
Chapter One
Introduction
reducing sugars and amino acids on proteins, lipids and nucleic acid. AGEs have been implicated in the induction and progression of various diseases [160]. Benfotiamine as an inhibitor of the formation of AGEs exerts its beneficial effects through a diverse mechanism, based on AGE-dependent and AGE-independent actions [161].
1.9.3.1. AGE-dependent pharmacological action of benfotiamine In diabetes, benfotiamine blocks three major biochemical pathways implicated in the pathogenesis of chronic hyperglycemia-induced vascular damage, i.e. hexosamine pathway, AGE formation pathway and diacylglycerol (DAG)-protein kinase C (PKC) pathway, which are activated by the high availability of the glycolytic metabolites such as glyceraldehyde-3-phosphate
and
fructose-6-phospahete
[162].
Benfotiamine prevents the progression of diabetic complications by increasing tissue levels of thiamine diphosphate and subsequently enhancing transketolase activity, that directs the elevated levels of hexose and trios phosphate to the pentose phosphate pathway leading to a reduction in tissue AGEs in experimental diabetic neuropathy [163,164]. The most significant effect in reducing the neuropathic pain was noted in patients receiving high-dose benfotiamnie [165]. Administration of benfotiamine in diabetic patients having thiamine deficiency markedly ameliorated neuropathic symptoms by neutralizing the damaging effects of hyperglycemia on neuronal vascular cells [166]. It has been recently suggested to be considered as a first choice nutritional supplement in preventing the progression of diabetic neuropathy based on its efficacy and safety data [167]. Diabetic nephropathy is a major cause of end-stage renal failure [168], and benfotiamine in high dose prevented the development of 28
Chapter One
Introduction
diabetic nephropathy by increasing transketolase expression in renal glomeruli, triggering the conversion of triosphosphate to ribos-5phosphate, and inhibiting the incidence of microalbuminuria, which is associated with decreased activation of PKC and reduced formation of protein glycation and oxidative stress [169]. Diabetic retinopathy is a major cause of blindness [170], and administration of high-dose benfotiamine in diabetic rats prevented the development
of
retinopathy
by
halting
AGEs
formation
[171].
Benfotiamine decreased the retinal capillary changes in streptozotocininduced diabetic rats by increasing the activity of transketolase in the retina. Additionally, it blocks the three major pathways of hyperglycemic damage to prevent the progression of diabetic retinopathy [162]. Moreover, benfotiamine prevents human pericyte apoptosis, which reveals its additional role in preventing diabetic complication [172]. It has been recently
shown
that
benfotiamine
prevents
experimental
diabetic
retinopathy by increasing extracellular matrix turnover [173].
1.9.3.2. AGE-independent pharmacological action of benfotiamine It has been reported that benfotiamine reduced diabetic-induced increase in oxidized glutathione (GSSG) levels and oxidative stress independent of AGE-inhibitory mechanism [174]. In addition, treatment with benfotiamine antagonizes the impaired cardiomyocyte contractile function in the streptozotocin-induced diabetic mouse by altering glucose metabolism and protein kinase C activation independent of its AGEinhibitory mechanism [175]. Recently growing body of evidence demonstrated the novel non-AGE-dependent role of benfotiamine in reducing oxidative stress and improving the function of vascular endothelium [176]. The vascular protective potential of benfotiamine has 29
Chapter One
Introduction
been confirmed, in which benfotiamine was noted to reduce oxidative stress and activate endothelial nitric oxide synthase to enhance the generation and bioavailability of nitric oxide and subsequently improve the integrity of vascular endothelium [177]. Benfotiamine has been shown to prevent macro/microvascular endothelial dysfunction and oxidative stress following a meal rich in AGEs in individuals with type 2 diabetes [178]. It also accelerates the healing of ischemic diabetic limbs in mice through protein kinase B/Akt-mediated potentiation of angiogenesis and subsequent inhibition of apoptosis [179]. Benfotiamine has been noted to prevent endotoxin-induced uveitis in rats by suppressing oxidative stress-induced NF-kB dependent inflammatory signaling [156]. Additionally, protective role of benfotiamine in lipopolysaccharide-induced cytotoxic signals in murine macrophage by blocking the LPS-induced increased expression of cytokines and chemokines and the inflammatory markers, such as iNOS and COX2, activation of PKC and NF-kB, and expression of apoptotic proteins and decreases the mitochondrial membrane potential, suggest a possible therapeutic application of benfotiamine supplementation for the prevention of inflammatory complications [180]. Through its suggested property as an antioxidant, benfotiamine has been shown to prevent high glucose-induced increase in DNA fragmentation and caspase-3- activity and consequently endothelial cell damage and apoptosis in endothelial cells and pericytes [181]. Furthermore, benfotiamine has the ability to reduce the oxidative stress and genomic damage caused by the mutagenic model compounds in renal cells, indicating its direct antioxidant capacity [182]. Benfotiamine also has antinociceptive, anti-hyperalgesic and antiallodynic activity in rats, suggesting the possible documented clinical use of this drug [183].
30
Chapter One
Introduction
1.10. Aim of the study This study was designed to: 1. Evaluate the possible concentration-effect relationship for radical scavenging property of silibinin dihemisuccinate and benfotiamine in an in vitro model of methemoglobin production by sodium nitrite. 2. To verify the validity of nitrite-induced hemoglobin oxidation model through in vivo study for evaluation of the suspected protective
activity
of
both
silibinin
dihemisuccinate
and
benfotiamine against chemicals-induced hemoglobin oxidation. 3. To investigate the concentration-effect relationship of both silibinin dihemisuccinate and benfotiamine for their membrane stabilizing activity in vitro model of erythrocyte hemolysis.
31
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CCl4-induced
REFERENCES
Chapter Three
Results
CHAPTER THREE Results 3.1. Time-course for oxidation of hemoglobin and MetHb formation with sodium nitrite: in vitro study Addition of sodium nitrite to the erythrocytes lysate resulted in rapid oxidation of hemoglobin to methemoglobin. The oxidation was slow at the initial stage and became rapid later (Figure 3-1). The process shows a characteristic pattern of lag phase followed by a rapid autocatalytic phase. MetHb levels are followed and evaluated by measurement of light absorbance at 631 nm. It has been reported that maximum level of MetHb was produced 53-55 min after initiation of Hb oxidation with sodium nitrite (Figure 3-1).
42
Chapter Three
Results
1.4 1.2
Absorbance
1 0.8 0.6 0.4 0.2 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 Time (min)
Figure 3-1. Time-course for production of methemoglobin with sodium nitrite in erythrocyte lysate
43
Chapter Three
Results
3.1.1. Effect of different concentrations of silibinin dihemisuccinate disodium on the time-course of nitrite-induced oxidation of Hb and MetHb formation In the presence of different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12 , 10-9 and 10-6 mg/ml) the time-course of oxidation of hemoglobin shows a concentration-dependent slowing in the rate of increase in light absorbance, which seems to be due to interference with the process o f oxidation and decrease in the levels of methemoglobin formation. Linear relationship
was
reported
between
silibinin
dihemisuccinate
disodium
concentrations and percent inhibition of methemoglobin formation (64.3%, 72%, 79.5% and 82.4% respectively, Figure 3-2, Table 3-1), and show a delay in the oxidation process in a concentration-dependent manner. The time required to convert 50% of the available hemoglobin to methemoglobin was 25 min in the absence of silibinin dihemisuccinate disodium (control), whereas it was delayed to 70, 89.4, 122 and 150 min in the presence of 10-15, 10-12 , 10-9 and 10-6 mg/ml silibinin dihemisuccinate disodium respectively (Table 3-1).
44
Chapter Three
Results
1.4
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 22 23 24 25 26 27 28 29 30 3132 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Time (min) Control
Silibinin 10-6 mg/ml
Silibinin 10-12 mg/ml
Silibinin 10-15 mg/ml
Silibinin 10-9 mg/ml
Figure 3-2. Effect of different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12, 10-9, 10-6 mg/ml) on the time-course of nitrite-induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate
45
Chapter Three
Results
Table 3-1. Effect of different concentrations of silibinin dihemisuccinate disodium (10-15, 10-12, 10-9, 10-6 mg/ml) on the time-course of nitrite-induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate Silibinin Concentrations (mg/ml) Control Silibinin 10-15 Silibinin 10-12 Silibinin 10-9 Silibinin 10-6
% formation of MetHb 100 35.7 28 20.5 17.6
Values represent mean of 3 experiments.
46
% Inhibition of MetHb 0 64.3 72 79.5 82.4
Time to form 50% MetHb (t½)(min) 25 70 89.4 121.9 150
Chapter Three
Results
3.1.2. Effect of addition of silibinin dihemisuccinate disodium at different time intervals Addition of the highly effective concentration of SDHS (10-6 mg/ml) to the hemolysate mixture, at different time intervals (10 min before nitrite, 10 min after and 20 min after nitrite addition i.e during autocatalytic phase) produced remarkable decrease in methemoglobin related absorbance of light, and resulted in significant inhibition of MetHb formation (83%, 77.4% and 75.3% respectively, Figure 3-3, Table 3-2). The time required to convert 50% of the available hemoglobin to methemoglobin was 25min in the absence of SDHS (control), whereas it was delayed to 146, 110 and 101 min when SDHS (10-6 mg/ml) was added 10 min before nitrite, 10 min after and 20 min after nitrite addition respectively (Figure 3-3, Table 3-2).
47
Chapter Three
Results
1.4
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0 1 2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Time (min) Control
10 min before nitrite
After 10 min
After 20 min
Figure 3-3. Effect of addition of silibinin dihemisuccinate disodium (10-6 mg/ml) at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the time course of oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate
48
Chapter Three
Results
Table 3-2. Effect of addition of silibinin (10-6 mg/ml) at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the time course of oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate % formation of MetHb
% inhibition of MetHb
Time to form 50% MetHb (t½)(min)
100
0
25
17
83
146
Addition after 10min
22.6
77.4
110
Addition after 20min
24.7
75.3
101
Time-course of addition of 10-6 mg/ml silibinin Control Incubation before 10 min
Values represent mean of 3 experiments.
49
Chapter Three
Results
3.1.3. Concentration-response relationship for the radical scavenging activity of silibinin dihemisuccinate disodium in nitrite-induced Hb oxidation Figure 3-4 shows concentration-response relationship for the radical scavenging activity of SDHS in nitrite-induced hemoglobin oxidation in vitro. Silibinin dihemisuccinate disodium remarkably inhibits MetHb formation; the degree of inhibition increased with increasing the concentration of SDHS, reaching its maximum level at the highest concentration used. However, best linearity for such relationship was reported below the minimum effective concentration (10-15 mg/ml), which indicates the highly reactivity at low concentrations (Figure 3-4).
50
Chapter Three
Results
% inhibition of MetHb formation
90 80 70 60 50 40 30 20 10 0 0
-15
-12
-9
-6
log conc.of silibinin
Figure 3-4. Concentration-response relationship for the radical scavenging activity of silibinin dihemisuccinate disodium in nitrite-induced Hb oxidation in vitro
51
Chapter Three
Results
3.1.4. Effect of different concentrations of benfotiamine on the time-course of nitrite-induced oxidation of Hb and MetHb formation In the presence of different concentrations of benfotiamine (25, 50, 100 and 200 µM) the time-course of oxidation of hemoglobin shows a slow increase in light absorbance related to reduced rate of Hb oxidation and inhibition of methemoglobin formation. The linear relationship was reported between benfotiamine concentrations and inhibition of MetHb formation (62%, 66%, 71%, and 75% respectively, Figure 3-5, Table 3-3), indicating a delay in the oxidation process in a concentration-dependent manner. The time required to convert 50% of the available hemoglobin in the erythrocyte lysate to methemoglobin was 25min in the absence of benfotiamine (control), whereas it was delayed to 66, 76, 88 and 100 min in the presence of 25, 50, 100 and 200 µM of benfotiamine respectively (Table 3-3).
52
Chapter Three
Results
1.4
1.2
Absorbance
1
0 .8
0 .6
0 .4
0 .2
0 1 2
3
4
5
6 7
8
9 10 11 12 13 14 15 16 17 18 19 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0
Time (min) Control
Benfotiamine 25µM
Benfotiamine 50µM
Benfotiamine 100µM
Benfotiamine 200µM
Figure 3-5. Effect of different concentrations of benfotiamine (25, 50, 100, 200 µM) on the time-course of nitrite induced oxidation of hemoglobin and methemoglobin formation in erythrocyte lysate
53
Chapter Three
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Table 3-3. Effect of different concentrations of benfotiamine (25, 50, 100, 200 µM) on the time required for oxidation of hemoglobin and formation of 50% methemoglobin with sodium nitrite in erythrocyte lysate Benfotiamine Concentrations
% formation of MetHb
%inhibition of MetHb
Time to form 50% MetHb (t½)(min)
Control
100
0
25
Benfotiamine 25µM
38
62
66
Benfotiamine 50µM
34
66
76
Benfotiamine 100µM
29
71
88
Benfotiamine 200µM
25
75
100
Values represent mean of 3 experiments.
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3.1.5. Effect of addition of benfotiamine at different time intervals Addition of the highly effective concentration of benfotiamine (200 µ M) to the hemolysate mixture at different time intervals (10 min before nitrite, 10 min after and 20 min after nitrite addition i.e during autocatalytic phase) decreases absorbance of light attributed to methemoglobin formation, which is an index for protection of Hb oxidation due to the addition of sodium nitrite to the lysate (79.4%, 78% and 75% respectively, Figure 3-6, Table 3-4). The time required to convert 50% of the available hemoglobin to methemoglobin was 25 min in the absence of benfotiamine (control), whereas it was delayed to 122, 114 and 101 min when benfotiamine (200 µM) was added 10 min before nitrite, 10 min after and 20 min after nitrite addition respectively as shown in table 3-4.
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1.4
1.2
Absorbance
1
0 .8
0 .6
0 .4
0 .2
0 1 2
3
4
5
6 7
8
9 10 11 12 13 14 15 16 17 18 19 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0
Time (min) Control
10 min before nitrite
After 10 min
After 20 min
Figure 3-6. Effect of addition of benfotiamine (200 µM) at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate
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Table 3-4. Effect of addition of benfotiamine 200 µM at different time intervals (10 min before, 10 min after, 20 min after nitrite addition) on the oxidation of hemoglobin and formation of methemoglobin in erythrocyte lysate Time-course of addition of 200µM benfotiamine Control Incubation before 10 min Addition after 10 min Addition after 20 min
% formation of MetHb
% inhibition of MetHb
Time to form 50% MetHb(t½)(min)
100
0
25
20.6
79.4
121.5
22
78
114
24.9
75.1
101
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3.1.6. Concentration-response relationship for the radical scavenging activity of benfotiamine in nitrite-induced Hb oxidation in vitro Figure 3-7 shows concentration-response relationship for the radical scavenging activity of benfotiamine in nitrite-induced hemoglobin oxidation in vitro. Benfotiamine remarkably inhibits MetHb formation; the degree of inhibition increased with increasing the concentration of benfotiamine, reaching its maximum level at the highest concentration used. However, best linearity for such relationship was reported below the minimum effective concentration ( 25 µM), which indicates the relatively high reactivity at low concentrations (Figure
3-7).
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% inhibition of MetHb formation
80 70 60 50 40 30 20 10 0 0
1.39
1.69
2
2.3
Log conc.of Benfotiamine
Figure 3.7. Concentration-response relationship for the radical scavenging activity of benfotiamine in nitrite-induced Hb oxidation in vitro
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3.1.7. Comparison between radical scavenging activity of SDHS and benfotiamine in nitrite-induced Hb oxidation in vitro Comparison between the effects of equimolar concentrations of silibinin dihemisuccinate disodium and benfotiamine in nitrite-induced Hb oxidation in vitro was demonstrated in figure 3-8. Very low molar concentration of SDHS (1.6 x 10-15 µM) showed 50% inhibition of methemoglobin formation, whereas the same equimolar concentration of benfotiamine did not show any effect in this respect, which indicates that, at molar ratio bases, silibinin dihemisuccinate disodium was extremely highly effective compared to benfotiamine.
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60%
MetHb
% inhibition of MetHb
50% 40% 30% 20% 10% 0% Silibinin (1.6 X 10 -15 µM)
Benfotiamine (1.6 X 10 -15 µM)
Figure 3-8. Comparison between radical scavenging activity of equimolar concentrations of silibinin and benfotiamine in nitrite induced Hb oxidation in vitro
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3.2. Osmotic fragility test: in vitro study 3.2.1. Effect of different concentrations of SDHS on osmotic fragility of red blood cells The resistance of erythrocytes to lysis by diluted buffer saline solutions of decreasing concentrations (NaCl range of 9.0-1.0 g/l) was assayed. In figure 3-9 the present study, 50% RBC hemolysis occurred with 0.49% NaCl buffer saline solution when RBCs were treated with sodium nitrite alone; whereas a 0.47% , 0.45%, 0.43% and 0.36% NaCl buffer saline solution were needed to cause 50% lysis of RBCs when solutions of different concentrations of SDHS (10-15, 10-12, 10-9,
10-6 mg/ml SDHS respectively, Figure 3-9) were added to the
incubation mixture in addition to sodium nitrite. The figure shows the difference in susceptibility of human RBCs to osmotic lysis when subjected to increasing hypotonicity with and without addition of SDHS, which shift the curve toward that of control RBCs which are not challenged by sodium nitrite.
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120
%
Hemolysis %
100 80 60 40 20 0 0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
NaCl conc. g/l Control Silibinin (1x10-9) +Nitrite
Nitrite only Silibinin (1x10-12) +Nitrite
Silibinin (1x10-6) +Nitrite Silibinin (1x10-15) +Nitrite
Figure 3-9. Effects of different concentrations of Silibinin dihemisuccinate disodium on the osmotic fragility of red blood cell challenged with sodium nitrite in vitro
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3.2.2 Concentration-response relationship of SDHS on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro Figure 3-10 shows relationship between logarithms of concentration of SDHS and the concentration of NaCl solution required to produce 50% hemolysis of RBCs. The percent of NaCl resulted in 50% lysis of RBCs was markedly decreased with increasing the concentration of SDHS. The relationship showed best linearity and a plateau shape curve there after, indicating no remarkable increase in the membrane stabilizing activity of SDHS with increasing the concentration in the incubation mixture.
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0.4 RBC
% NaCl solution produce 50% hemolysis of RBC
0.5 0.45 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
-15
-12
-9
-6
Log Concentration of SDHS
Figure 3-10. Concentration-response relationship of silibinin dihemisuccinate disodium (SDHS) on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro
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3.2.3. Effect of different concentrations of benfotiamine on osmotic fragility of red blood cells The resistance of erythrocytes to lysis by diluted buffer saline solutions of decreasing concentrations (NaCl range of 9.0-1.0 g/l) was assayed. In figure 311 the present study, 50% RBC hemolysis occurred with 0.49% NaCl buffer saline solution when RBCs were treated with sodium nitrite alone; whereas a 0.485 %, 0.475%, 0.465% and 0.425% buffered saline solutions were needed to cause 50% lysis of RBCs when solutions of different concentrations of benfotiamine (25, 50, 100 and 200 µM respectively, Figure 3-11) were added to the incubation mixture in addition to sodium nitrite. The figure shows the difference in susceptibility of human RBCs to osmotic lysis when subjected to increasing hypotonicity with and without addition of benfotiamin, which slightly shifts the curve toward that of control RBCs which are not challenged by sodium nitrite.
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12 0
10 0
Hemolysis %
80
60
40
20
0 0.5
1
1. 5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
NaCl g/l
Control
Nitrite only
Benfotiamine 25 µM + Nitrite
Benfotiamine 50 µM + Nitrite
Benfotiamine 100 µM + Nitrite
Benfotiamine 200 µM + Nitrite
Figure 3-11. Effects of different concentrations of Benfotiamine on the Osmotic fragility of red blood cell challenged with sodium nitrite in vitro
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3.2.4. Concentration-response relationship of benfotiamine on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro Figure 3-12 shows the relationship between logarithms of concentration of benfotiamine and the concentration of NaCl solution required to produce 50% hemolysis of RBCs. The percent of NaCl resulted in 50% lysis of RBCs was decreased with increasing the concentration of benfotiamine. The relationship showed best linearity and a plateau shape curve there after, indicating no remarkable increase in the membrane stabilizing activity of benfotiamine with increasing its concentration in the incubation mixture.
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% NaCl solution produce 50% hemolysis of RBC
0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0
1.397
1.689
2
2.3
Log concentration Benfotiamine
Figure 3-12. Concentration-response relationship of Benfotiamine on the sensitivity of erythrocytes to hemolysis, when challenged with sodium nitrite in vitro
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3.3. In vivo study In order to develop an in vivo model that could simulate the in vitro observations for radical scavenging activity of both SDH and benfotiamine, nitrite-induced oxidation of hemoglobin was utilized for production of methemoglobin in rats and percent of methemoglobin formed were measured in all animal groups.
3.3.1. Effect of silibinin dihemisuccinate on nitrite-induced MetHb formation in rats The results obtained in this section of the study showed that MetHb% formed was significantly decreased (44.6%) in animals treated with silibinin dihemisuccinate (100mg/kg) once daily for 7 days (long term silibinin exposure) before induction of methemoglobinemia with orally administered sodium nitrite (100mg/kg), compared with saline treated only animals (P<0.05, table 3-5); whereas non-significant differences were observed with silibinin dihemisuccinate (100mg/kg) treatment for one hour (short term silibinin exposure) compared to control group as shown in table 3-5.
3.3.2. Effect of benfotiamine on nitrite-induced MetHb formation in rats Pre-treatment of animals with benfotiamine (70mg/kg), both in long-term approach (single daily dose for 7 days before administration of sodium nitrite) and short-term-approach (single dose 1 hour before administration of sodium nitrite) decreases the level of MetHb% (40.1% and 35.6% respectively) compared to saline treated only group (P<0.05, table 3-5).
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Table 3-5. Effects of single and multiple doses of Silibinin dihemisuccinate (100 mg/kg) or Benfotiamine (70 mg/kg) on nitrite-induced MetHb formation in rats % MetHb formation Type of treatment Saline treated only Pre-treatment for 7 days Treatment for one hour
n
Silibinin (100mg/kg)
Benfotiamine (70mg/kg)
6
55.6 ± 6.0
55.6 ± 6.0
6
30.8 ± 13.6 * a
33.3 ± 11.1* a
6
55.9 ± 9.3 b
35.8 ± 16.4 * a
Each value represents mean ± SD; n= number of animals; *significantly different compared to saline treated group (P<0.05); values with non-identical superscripts (a,b) were considered significantly different within the same group (P<0.05)
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