The effect of Argania spinosa on plasma Homocysteine ... · 2-Effect of treatment on Hcy levels, lipid profile, liver enzyme activities and antioxidants markers in mice The current

الجوهىريت الجزائريت الديوقراطيت الشعبيت Ministry of High Education and Scientific Research

N° d’ordre : 91/DS/2018

N° de série : 04/BA/2018

Thesis submitted for the degree of Doctorate in Sciences

Option: Animal physiology

Topic:

Presented by: AKLIL Badiaa

Examination board:

President: N. BAAZIZ Prof. University Frères Mentouri Constantine

Supervisor: S. ZERIZER Prof. University Frères Mentouri Constantine

Examiners: K. BOUBEKRI MC. University Frères Mentouri Constantine

S. DAHAMNA Prof. University of Sétif

S. KHENNOUF Prof. University of Sétif

C. ABDENNOUR Prof. University of Annaba

2017 / 2018

The effect of Argania spinosa on plasma

Homocysteine, Lipids, Antioxidant enzymes and

Aortas Sections in Methionine induced

Hyperhomocysteinemia in mice

University of des frères Mentouri Constantine

Faculty of life and Natural Sciences

Department of Animal Biology

جاهعت اإلخىة هنتىري قسنطينت

لىم الطبيعت و الحياة ـــليت عـــك

ىاى ــيــحــىلىجيا الـيـسن بــــق

Dedication

This thesis is dedicated to the memory of my father Omar, who would have been

happy to see me at this step.

To my mother Fatma, for her constant, unconditional love and support.

To my husband Ali, who has been a constant source of support and

encouragement during all the hard periods of research and life.

To my children: Haithem, Nouha and Assil.

To all my family, the symbol of love and giving.

And to all my friends who encourage and support me.

Acknowledgements

First, I am deeply grateful to the Almighty Allah who helped me to

complete this thesis.

I wish to express my deepest gratitude to my supervisor Prof. S. Zerizer

for introducing me to the interesting field of science and for providing me with

the opportunity to carry out this study, and for her invaluable advice, patience

and inspiring guidance throughout this work.

I am grateful to Prof. Z. Kabouche for providing me the plant material.

I would like to thank all the jury members, Prof. N. BAAZIZ

(president of the jury), Dr. K. BOUBEKRI (examiner of the doctorate thesis),

Prof. S. DAHAMNA (examiner of the doctorate thesis), Prof. S. KHENNOUF

(examiner of the doctorate thesis), and Prof. C. ABDENNOUR (examiner of the

doctorate thesis), for their interest in my work.

I wish to express my sincere thanks to Dr. Guy D’hallewin for accepting

me in his laboratory and giving me the opportunity to work with his exceptional

laboratory team; my sincere thanks also go to Pr. G. Orru to help me conduct

laboratory tests related to research.

I wish to thank Dr. G. Piacherri for her help and assistance.

I would like to thank everyone who provided any assistance even with a

single word and especially my friends.

Table of contents

LIST OF ABBREVIATIONS

LIST OF FIGURES

LIST OF TABLES

INTRODUCTION…………………………………………………………………………… 01

LITERATURE REVIEW

1- Homocysteine ………………………………………………………………...................

03

1-1 Structure and Forms of homocysteine………………………………………………. 03

1-2 Biosynthesis and Metabolism of homocysteine………………………………........... 03

1-2 Regulation of Metabolism…………………………………………………………….

05

2- Hyperhomocysteinemia …………………………………………………………………. 06

2-1 Definition…………………………………………………………………………….. 06

2-2 Causes of Hyperhomocystenemia …………………………………………………… 06

2-2-1 Genetic Defects …………………………………………………………………. 06

a- Cysthathionine beta synthase deficiency……………………………………….. 07

b- N-5, 10-Methylene tetrahydrofolate reductase deficiency……………………… 07

2-2-2 Vitamin Deficiencies …………………………………………………………….. 07

2-2-3 Other Causes of Hyperhomocysteinemia ………………………………………

08

3- Pathogenicity of Hyperhomocysteinemia ………………………………………………

08

3-1 Hyperhomocysteinemia and cardiovascular disease……………………………….. 08

3-1-1 Endothelial dysfunction………………………………………………………… 08

3-1-1-1Homocysteine-Induced Oxidative Stress Condition………………………… 10

a- Uncoupling of NO synthase ………………………………………………… 11

b- Accumulation of Asymmetric Dimethylarginine…………………………… 12

3-1-1-2Homocysteine induced protein modification and endoplasmic reticulum

stress …………………………………………………………………… …..

13

3-1-1-3 Homocysteine induced inflammatory/prothrombotic conditions…………. 13

3-1-2 Atherosclerosis……………………………………………………………………. 15

3-2 Hyperhomocysteinemia and dyslipedimia……………………………………………… 17

3-3 Hyperhomocysteinemia and hepatic disease…………………………………………… 18

3-4 Hyperhomocysteinemia and carcinogenesis …………………………………………… 18

Table of contents

4- Therapy of hyperhomocysteinemia ……………………………………………………

19

5- Argania spinosa………………………………………………………………………………….

19

5-1 Description ……………………………………………………………………………

19

5-2 Botanical classification……………………………………………………………….. 20

5-1 Therapeutic properties………………………………………………………………….

21

6- Bacterial Biofilm and cardiovascular diseases…………………………………................. 22

6-1 Definition of Biofilm………………………………………………………………… 22

6-2 Stages of Biofilm formation………………………………………………………….

22

6-3 Biofilm and cardiovascular infections ……………………………………………….. 23

6-4 Bacterial strains ……………………………………………………………………. 25

6-4-1 Streptococcus mutans…………………………………………………………………… 25

6-4-2 Streptococcus intermedius and Streptococcus anginosus …………………………. 25

6-4-1 Staphylococcus haemolyticus ………………………………………………………….. 26

6-4-3 Streptococcus uberis……………………………………………………………………..

26

MATERIALS AND METHODS

1- Biological plant…………………………………………………………………………….

27

2- Animals………………………………………………………………………………….. 27

2-1 Experimental animals…………………………………………………………….. …… 27

2-2 Experimental treatments………………………………………………………………… 28

2-3- Blood and tissue sampling………………………………………………………………

28

3- Methods…………………………………………………………………………….......... 30

3-1Chemical products……………………………………………………………………… 30

3-2 Equipments…………………………………………………………………………….. 30

3-3 Biochemical analysis………………………………………………………………….. 30

3-3-1 Plasma Hcy determination…………………………………………………………..

30

3-3-2 Lipids determination…………………………………………………………………

31

Table of contents

3-3-3 Determination of Aspartate Aminotransferase and Alanine aminotransferase

activities………………………………………………………………………………

33

3-4 Determination of oxidative stress parameters …………………………………................. 34

3-4-1 Tissue homogenate preparation………………………………………………………. 34

3-4-2 Glutathione assay……………………………………………………………………… 34

3-4-2 Catalase activity……………………………………………………………….............. 36

3-5 Histological analysis……………………………………………………………..............

37

4- Biofilm formation and quantification ……………………………………………………. 38

4-1 Strains and culture conditions……………………………………………………….. 38

4-2 Crystal Violet Biofilm formation screening assay……………………………………..

38

5- Statistical analysis…………………………………………………………………………..

40

RESULTS AND DISCUSSION

Chapter 1: Effect of treatment on body weight, Hcy levels, lipid profile, liver enzyme

activities and antioxidants markers in mice

1- Body weight …………………………………………………………………………..

41

2- Effect of treatment on lipid profile in mice……………………………………................ 41

2-1 Triglycerides……………………………………………………………………………. 41

2-2 Total cholesterol ……………………………………………………………………….. 42

2-3 HDL-c……………………..…………………………………………………………… 43

2-4 LDL-c…………… .……………………………………………………………………..

3- Effect of treatments on Homocysteine levels ……………………………………………..

44

45

4- Effect of treatment on liver enzyme activity……………………………………………… 46

4-1 Aspartate Aminotransferase………………………………………………………….. 46

4-2 Alanine aminotransferase ……………………………………………………………..

47

5- Effect of treatment on antioxidants markers ……………………………………………… 48

5-1 Reduced Gluthatione …………………………………………………………………. 48

5-2 Catalase activity……………………………………………………………………….

49

Table of contents

Chapter 2: Effect of treatment on histology of aorta heart and liver

1- Histological study of the heart…………………………………………………..............

51

2- Histological study of the aorta…………………………………………………………..

51

3- Histological study of the liver…………………………………………………...............

51

Chapter 3: Anti-biofilm formation of Argan oil “In vitro” Study

DISCUSSION……………………………………………………………………………………..

68

CONCLUSION AND PERSPECTIVES……………………………………………………….

85

REFERENCES…………………………………………………………………………………..

87

APPENDICES

PAPER

ملخص بالعربيةال

List of Abbreviations

.HRO2-: Hydroperoxyl

•NO: Nitric Oxide

•NO2: Nitrogen Dioxide

•O2-: Superoxide

•OH: Hydroxyl

•RO2: Peroxyl

5, 10-MTHF: 5, 10-Methylene Tetrahydrofolate

5, 10MTHFR: 5, 10- Methylene Tetrahydrofolate Reductase

5-MTHF: 5-Methylene Tetrahydrofolate

ADMA: Protein Asymmetric Dimethyl Arginine

AECA: Anti-Endothelial Cell Antibodies

ALT: Alanine Aminotransferase

Ang II: Angiotensin II

ANOVA: One-way Analysis Of Variance

anti-oxLDL: Anti-Oxidized LDL Antibodies

APLA: Anti-Phospholipid Antibodies

apoB: Apolipoprotein B

apoE : Apolipoprotein E

AST: Aspartate Aminotransferase

BH4: Tetrahydrobiopterin

BSA: Bovine Serum Albumin

CβS: Cystathionine β-Synthase

CoNS : Coagulase-negative staphylococci

Cth−/−: Cystathionine Deficient Mice

CVD: Cardiovascular Diseases

CYP7A1: Cholesterol 7A-Hydroxylase

DHF: Dihydrofolate

DNA: Deoxyribonucleic Acid

DTNB: Dithiobis-2-Ditrobenzoic Dcid

EDHF: Endothelium-Derived Hyperpolarizing Factor

EDTA: Tris Ethylene Di-amine Tetra Acetic acid

eNOS: Endothelial Nitric Oxide Synthase

EPS: Exopolysaccharides


ER: Endoplasmic Reticulum

ET-1: Endothelin-1

GCT: γ-cystathionase

GSH: Hepatic Reduced Glutathione

GST: Glutathione S-Transferase

H2O2: Hydrogen Peroxide

Hb: Hepatocellular Ballooning

Hcy: Homocysteine

HHcy: Hyperhomocysteinemia

HNO2: Nitrous oxide

HOCl: Hydrochlorous Acid

Hsp90: Heat Shock Protein 90

HTL: Homocysteine Thiolactone

ICAM-1: Intercellular Adhesion Molecule-1

IE: Infective Endocarditis

LCAT: Lecithin-Cholesterol Acyltransferase

LDL-c: Low Density Lipoprotein

LPS: Lipopolysaccharides

MAT I/III: Methionine Adenosyl Transferases I and III

MCP-1: Monocyte Chemoattractant Protein 1

MS: Methionine Synthase

MTHFR: Methylene Tetrahydro Folate Reductase

NAD: Nicotinamide Adenine Dinucleotide

NADH: Nicotinamide Adenine Dinucleotide

NF-κB : Nuclear Factor-kappa B

NO: Nitric Oxide

NOS: Nitric oxide Synthase

O2·−: Superoxide

ONOO−: Peroxynitrite

oxLDL: Oxidized Low Density Lipoprotein

PC: Phosphatidylcholine

PE: Phosphatidyl Ethanolamine

PEMT: Phosphatidyl Ethanolamine Methyl Transferase


PGI2: Prostaglandin I 2

PLP: Pyridoxal 5-Phosphate

PON1: Paraoxonase 1

QS: Quorum Sensing

RNS: Reactive Nitrogen Species

RONOO: Alkyl Peroxynitrates

ROS: Reactive Oxygen Species

SAG: Streptococcus Anginosus Group

SAH: S-Adenosyl –L-homocysteine

SAM: S-Adenosyl Methionine

SMCs: Smooth Muscle Cells

SPSS: Statistical Package for Social Science

SREBP-1: sterol regulatory element-binding protein

TBS: Tris-Buffered Saline

TG: Triglycerides

THF: tetrahydrofolate

TNF-α : Tumor Necrosis Factor-α

UPR: Unfolded Protein Response

VCAM-1: Vascular Cell Adhesion Molecule-1

VEC: Vascular Endothelial Cells

VLDL: Very Low Density Lipoproteins

VSMC: Vascular Smooth Muscle Cells

XDH : Xanthine Dehydrogenase

XO : Xanthine Oxidase

XOR : Xanthine Oxido Reductase

List of Figures

Figure 01. Homocysteine metabolism………………………………………………….

04

Figure 02. Potential mechanisms of homocysteine-induced endothelial dysfunction.. 09

Figure 03. Major endogenous sources of reactive oxygen species (ROS) and reactive

nitrogen species (RNS) in the cardiomyocyte…………………………………………..

10

Figure 04. Central role of endothelial NO synthase (eNOS) uncoupling in the

pathogenesis of endothelial dysfunction…………………………………………………

12

Figure 05. Regulatory circuits in inflammation and endothelial dysfunction………….

14

Figure 06. Hyperhomocysteinemia and etiopathogenesis of atherosclerosis …………..

16

Figure 07. Possible interactions between hyperhomocystenemia and hyperlipidemia in

cell pathology……………………………………………………………………………

17

Figure 08. Kernel and tegument of Argania spinosa L………………………………..

20

Figure 09. Distribution area of the Argan tree in Algeria ……………………………

21

Figure 10. Various stages of biofilm formation and development……………………

23

Figure 11. Blood and tissue sampling…………………………………………………. 29

Figure 12. Effect of L-methionine intake on mice weight during 21 days……………

41

Figure 13. Interaction of L-methionine and A. spinosa seeds on the triglycerides in

mice during 21 days of treatment……………………………………………………….

42

Figure 14. Interaction of L-methionine and A. spinosa seeds on the T-CHO in mice

during 21 days of treatment……………………………………………………………..

43

Figure15. Interaction of L-methionine and A. spinosa seeds on the HDL-c in mice

during 21 days of treatment. ……………………………………………………………

44

Figure 16. Interaction of L-methionine and A.spinosa seeds on the LDL-c in mice

during 21 days of treatment………………………………………………………………

45

Figure 17. Interaction of L-methionine and A. spinosa seeds on the homocysteine

levels in mice during 21 days of treatment…………………………………………….

46

Figure 18. Interaction of L-methionine and A. spinosa seeds on the AST in mice

during 21 days of treatment……………………………………………………………...

47

Figure 19. Interaction of L-methionine and A.spinosa seeds on the ALT in mice during

21 days of treatment…………………………………………………………………….

48

List of Figures

Figure 20. Interaction of L-methionine and A.spinosa seeds on the reduced glutathione

in mice during 21 days of treatment……………………………………………………..

49

Figure 21. Interaction of L-methionine and A.spinosa seeds on the catalase activity in

mice during 21 days of treatment……………………………………………………….

50

Figure 22. Histological sections of heart tissue in experimental groups (F, M, MP and

P)………………………………………………………………………………………..

53

Figure 23. Histological sections of the arch aorta in experimental groups (F, M, MP

and P)…………………………………………………………………………………….

55

Figure 24. Histological sections of the abdominal aorta in experimental groups (F, M,

MP and P)……………………………………………………………………….............

57

Figure 25. Histological sections of the iliac aorta in experimental groups (F, M, MP

and P)……………………………………………………………………….....................

59

Figure 26. Histological sections of liver tissue in experimental groups (F, M, MP and

P)………………………………………………………………………………………….

61

Figure 27. Inhibitory effect of Argan oil on S. intermedius biofilm formation………….

64

Figure 28. Inhibitory effect of Argan oil on S. haemolyticus biofilm formation……….

64

Figure 29. Inhibitory effect of Argan oil on S. mutans biofilm formation……………..

65

Figure 30. Inhibitory effect of Argan oil on S. anginosus biofilm formation………….

65

Figure 31. Inhibitory effect of Argan oil on S. uberis biofilm formation………………

66

Figure 32. Effect of the first dilution (100 g/mL) concentration of Argan oil on

elimination of biofilms………………………………………………………………….

67

List of Tables

Table 01: Structures and forms of Hcy and related amino acids …………………….......

03

Table 02: Composition of diet for 1 kg of food taken by the mice during 21 days

(ONAB) ………………………………………………………………………………….

27

Table 03: Treatment of mice…………………………………………………………...

28

Table 04: Concentrations and amounts of reagents needed for the dosage of catalase

activity. …………………………………………………………………………………….

36

Table 05: Reduction percent of biofilm for test bacteria treated with different

concentrations of Argan oil. ………………………………………………………………

66

Introduction

Introduction

1

Homocysteine "hypothesis of arteriosclerosis" was first proposed by McCully in 1969,

when he observed premature atherothrombosis of the peripheral, coronary, and cerebral

vasculature in children with homocystinuria, an in born error in methionine metabolism

(Rasmussen and Moller, 2000).

Homocysteine (Hcy) a type of amino acid that is naturally found in blood plasma is

not harmful at normal levels, but when its levels are too high, health problems can result. If

unhealthy levels of Hcy increase in the blood, the delicate lining of an artery (endothelium)

can be damaged. Also, Hcy can both initiate and potentiate atherosclerosis (Saleh, 2015).

Therefore, is considered as an emerging cardiovascular risk factor (Athyros et al., 2010).

Homocysteine induced injury to the arterial wall is one of the factors that can initiate

the process of atherosclerosis, leading to endothelial dysfunction and eventually to heart

attacks and strokes (Gallai et al., 2001; Papatheodorou and Weiss, 2007).

Oxidative stress induced by Hcy is reflected by a decrease in serum total anti-oxidant

capacity. The oxidative stress resulting from elevated serum Hcy can oxidize membrane lipids

and proteins and stimulate the activation of Nuclear Factor-kappa B (NFκB), and

consequently increase the expression of inflammatory factors in vivo. Hcy can be converted to

a highly reactive thiolactone which is able to react with proteins forming- NH-CO-adducts,

thus affecting body proteins and enzymes (Ramakrishnan et al., 2006).

The studies suggest that certain chronic infections increase the risk for cardiovascular

disease and that such infections may be considered novel and potentially modifiable risk

factors (Epstein et al., 1999). Specific pathogens along with their potential contribution by

direct or indirect mechanisms to atherosclerosis pathogenesis have been recently reviewed

(Rosenfeld and Campbell, 2011).

Biofilm is an aggregate of microorganisms in which cells are adhere to each other to a

surface. The adherent cells are embedded within a self-produced matrix of extracellular

polymeric substance (Gupta, 2015).

Biofilms can cause chronic infections and are associated with a number of chronic

disease states including cystic fibrosis, infectious endocarditis, and chronic wounds (Singh et

al., 2000; James et al., 2008). In this context, the development of bacterial resistance to

presently available antibiotics has necessitated the need to search for new antibacterial agents.

Introduction

2

Plants as a source of medicinal compounds have continued to play a dominant role in

the maintenance of human health since ancient times. According to the World Health

Organization plant extracts or their active constituents are used as folk medicine in traditional

therapies of 80% of the world’s population. Over 50% of all modern clinical drugs are of

natural product origin (Kirbag et al., 2009).

This study was designed to investigate the beneficial effects of Algerian plant Argania

spinosa (using the powdered seeds and oil) belongs to the family Sapotaceae. The therapeutic

benefits of A. spinosa have been claimed by previous studies which have confirmed that A.

spinosa have several biological effects including: antiproliferative (Bennani et al., 2006;

Drissi et al., 2006; Samane et al., 2006; Bennani et al., 2009), Hypolipidemic,

hypocholesterolemic ( Berrougui et al., 2003), antiatherogenic (Berrougui et al., 2004; Cherki

et al., 2005; Cherki et al., 2006), antiradical (Drissi et al., 2004; Amzal et al., 2008) and anti-

inflammatory activities (Alaoui et al., 1998).

The main objectives of this thesis are:

Induce hyperhomocysteinemia by administration of high L-methionine dose, in an in vivo

animal;

Examine the effect of L-methionine on the weight.

Examine the effect of L-methionine on some biochemical parameters such as plasma

Hcy, triglycerides (TG), Total cholesterol (T-CHO), low density lipoprotein (LDL-c),

high density lipoprotein (HDL-c), ALT, AST, reduced glutathione (GSH), and catalase

activity.

Examine the effect of L-methionine on different sections of aorta, heart and liver.

Evaluate the effect of the powdered seeds of A. spinosa seeds on hyperhocysteinemia and

other biochemical paramaters.

Evaluate the effect of the powdered seeds of A. spinosa on the structure disorders of

aorta, heart and liver induced by high L-methionine intake; and

Assess the anti-biofilm activity of Argan oil against 5 bacterial strains, which can induce

cardiovascular problems.

Literature

Review

Literature Review

3

1- Homocysteine

Homocysteine (Hcy) is a natural sulfur-containing amino acid produced in the

metabolism of the essential amino acid methionine, which is derived from dietary protein

(Narmatha et al., 2015). Normally human Hcy levels range from 4 to 12.3 μmol/l (Elhawary

et al., 2013). The levels of Hcy increase with aging, and are typically higher in men than

women (Nygard et al., 1995; Refsum et al., 2006). However, elevated plasma Hcy

concentrations have important implications for human health and disease (Jing et al., 2014).

1-1 Structure and forms of Homocysteine

Homocysteine is present in different forms (Ganguly and Alam, 2015): around 1%

circulates as free thiol, 70–80% remains disulphide-bound to plasma proteins, mainly albumin

and 20–30% combines with itself to form the dimer Hcy or with other thiols (Hankey and

Eikelboom, 1999).

Table 01: Structures and forms of Hcy and related amino acids (Miller, 2013).

1-2 Biosynthesis and metabolism of Homocysteine

The single source of Hcy in humans is dietary methionine (Miller, 2013). Methionine

is converted into S-adenosylmethionine (SAM), which then loses a methyl moiety and

becomes S-adenosyl-homocysteine (SAH), which finally hydrolyzes into Hcy and adenosine

(Tchantchou, 2006).

Homocysteine is metabolized via two pathways (Figure 01). The first one is

remethylation, where Hcy is reconverted into methionine (Tchantchou, 2006). In this

pathway, Hcy reacquires a methyl group in a reaction catalyzed by the zinc-dependent

Literature Review

4

enzyme, Methionine Synthase (MS), with methyl tetrahydrofolate serving as the methyl

donor and vitamin B12 serving as a cofactor. This reaction occurs in all mammalian cells.

Alternatively Hcy can be remethylated in a folate and vitamin B12-independent reaction using

betaine as the methyl donor and catalyzed by Betaine-Homocysteine-Methyltransferase. This

reaction occurs primarily in the liver and to a lesser extent in the kidney and possibly in the

brain (Miller, 2013).

The second pathway is transsulfuration, where Hcy is converted into cystathionine to

form cysteine by cystathionine-ß-synthase (CBS), with vitamin B6 as a co-factor

(Tchantchou, 2006; Plazar and Jurdana, 2010). Cystathionine is then cleaved to form α-

ketobutyrate and cysteine in a second PLP-dependent reaction catalyzed by cystathionase.

Further metabolism of cysteine leads to the formation of Glutathione or inorganic sulfate

(Miller, 2013).

Figure 01. Homocysteine metabolism (Škovierová et al., 2016).

Literature Review

5

1-3 Regulation of Metabolism

Perturbations in methyl group metabolism and Hcy balance have emerged over the

past few decades as having defining roles in a number of pathological conditions. Numerous

nutritional, hormonal, and genetic factors that are characterized by elevations in circulating

homocysteine concentrations are also associated with specific pathological conditions,

including cancer development, autoimmune diseases, vascular dysfunction, and

neurodegenerative disease (Schalinske and Anne Smazal, 2012).

Because Hcy has many metabolic routes for its production and utilization, a number of

key proteins involved in these processes factor heavily in the regulation of Hcy balance. When there is an excess of methionine, Hcy is metabolized via the pathway of trans-

sulfurylation, producing cystathionine and cysteine in turn. Conversely, under conditions of

methionine deficiency, Hcy is remethylated into methionine. Hcy is remethylated in the liver

via betaine-homocysteine-methyltransferase; however, in most tissues, Hcy is remethylated

into methionine by methionine synthase (MS), which uses vitamin B12 as a co-factor and 5-

Methylene Tetrahydrofolate (5-MTHF) as a substrate (Marinou et al., 2005).

An additional level on Hcy metabolism is exerted by oxidative stress, which reduces

methionine synthase activity. This may occur by oxidative inactivation of the vitamin B12

cofactor or by the oxidation of cysteine residues that are important for zinc binding. By

inhibiting methionine synthase, oxidative stress tends to divert Hcy toward cystathionine

synthesis away from methionine synthesis. This serves to increase the synthesis of

glutathione, a product of Hcy metabolism through the transsulfuration pathway and an

important intracellular antioxidant (Miller, 2013).

In addition, plasma Hcy levels are affected by menopause (Hak et al., 2000), diabetes

(Wijekoon and Brosnan, 2007), and thyroid disorders (Saleh, 2015). These observations

suggest that hormones, including estrogen, insulin, thyroxine, and thyroid stimulating

hormone, may directly or indirectly affect Hcy metabolism. The mechanisms by which these

hormones affect Hcy metabolism are poorly understood (Miller, 2013).

https://www.ncbi.nlm.nih.gov/pubmed/?term=Wijekoon%20EP%5BAuthor%5D&cauthor=true&cauthor_uid=17956306

Literature Review

6

2- Hyperhomocysteinemia

2-1 Definition

In 1969, McCully reported two patients with homocystinuria who presented with

premature atherosclerosis at the ages of 2 months and 8 years, respectively. Since then,

increasing evidence suggests that even modest elevations in plasma homocysteine called

hyperhomocystenemia( HHcy), may act as an independent risk factor for atherosclerosis in

the general population (Guthikonda and Haynes, 2006). Hyperhomocystenemia is a metabolic

systemic disorder with defects in sulphur-containing amino acid (methionine and cysteine)

metabolism leading to abnormally higher amounts of Hcy (Veeranki and Tyagi, 2013).

Several types of HHcy are classified in relation to the Hcy

concentration: moderate (16–30 μM), intermediate (31–100 μM), and severe (higher than 100

μM) (Liu et al., 2007).

Hcy occurs in human blood plasma in several forms, including the most reactive one,

homocysteine thiolactone (HTL) – a cyclic thioester, which represents less than <1 % of total

plasma Hcy. The increase in extracellular Hcy is toxic to cells and tissues and it has the

potential to initiate a broad array of vascular complications (Domagała et al., 2006;

Jakubowski, 2008).

2-2 Causes of Hyperhomocysteinemia

Elevations in Hcy concentration may be triggered by a diverse group of stimuli

internal and external to the body. Diet, genetics, medications, lifestyle, and systemic illnesses

may all, separately or in combination, result in HHcy (Refsum et al., 2004).

2-2-1 Genetic Defects

Inherited deficiencies of enzymes in the methionine-Hcy pathway produce HHcy (Guthikonda and Haynes, 2006), which are observed in individuals with homozygous genetic

defects affecting cystathionine β-synthase (CβS), N-5,10-Methylene Tetrahydrofolate

reductase (MTHFR), or any of several enzymes responsible for the conversion of vitamin

B12 to its methionine synthase-associated cofactor form (Miller, 2013).

Literature Review

7

These autosomal recessive genetic disorders, collectively termed homocystinuria

because Hcy accumulates in the urine as well as the blood, are associated with severe

premature vascular disease, including thrombosis and atherosclerosis ,mental retardation,

dislocation of the eyelens and skeletal malformations (Miller, 2013).

a- Cysthathionine beta synthase deficiency

Cysthathionine beta synthase (CβS) is responsible for the irreversible degradation of

homocysteine. Without CβS, the entire transsulfuration pathway of the methionine cycle is

shut off (Brandon, 2009).

Mutations in the gene coding for the enzyme (CβS) lead to classical homocystinuria with

severe HHcy and/or homocystinuria (Gaustadnes et al., 1998), which is an autosomal

recessive disease characterized by a severely elevated plasma Hcy and Hcy excretion in the

urine (Liselotte, 2003).

b- N-5,10-Methylene Tetrahydrofolate reductase deficiency

N-5,10-Methylene Tetrahydrofolate reductase (MTHFR) is a folate cycle enzyme that

generates the methyl donor, 5-methyltetrahydrofolate, that is used for Hcy remethylation by

MS (Lentz, 2005). The most common one that is detected worldwide and has a high

incidence in different populations, is single nucleotide polymorphisms of N-5,10-methylene

tetrahydrofolate reductase which has been associated with mild (13–24 μM) and moderate

(25–60 μM) HHcy (Curro et al., 2014). The most common enzyme defect associated with

moderately raised total Hcy is a point mutation (C-to-T substitution at nucleotide 677) in the

coding region of the gene for MTHFR, which is associated with a thermo labile MTHFR

variant that has about half-normal activity (Hankey and Eikelboom, 1999).

2-2-2 Vitamin Deficiencies

Hyperhomocystenemia can also arise from nutritional deficiencies of folate, vitamin

B6, and vitamin B12 (Curro et al., 2014) which are essential cofactors in Hcy-methionine

metabolism. Therefore, low vitamin B availability (B6, B12 and folic acid) leads to impaired

remethylation of Hcy to methionine and thus to Hcy accumulation (Mangge et al., 2014).

However, the nature of HHcy caused by vitamin B6 deficiency differs from that caused by

folate and vitamin B12 deficiencies. In vitamin B6 deficiency, fasting blood levels of Hcy are

Literature Review

8

usually not elevated or only slightly elevated. However, after a protein meal or after

consumption of an oral methionine load does plasma Hcy become abnormally elevated in

vitamin B6-deficient patients. In contrast, plasma Hcy levels tend to be elevated regardless of

prandial state in patients with folate or vitamin B12 deficiency (Miller, 2013).

2-2-3 Other Causes of Hyperhomocysteinemia

Several diseases such as renal and thyroid dysfunction, cancer, psoriasis, and diabetes

as well as various drugs, alcohol, tobacco, coffee, older age and menopause, are believed to

be associated with moderately elevated Hcy concentrations (Faeh et al., 2006).

A rise in serum creatinine also leads to a rise in fasting total homocysteine (Hankey and

Eikelboom, 1999). Other causes of HHcy include leukemia (Refsum et al., 1991), sickle cell

anemia (Houston et al., 1997), polycythemia vera, and idiopathic thrombocytosis (Gisslinger

et al., 1999).

3- Pathogenicity of Hyperhomocysteinemia

3-1 Hyperhomocysteinemia and cardiovascular disease

Cardiovascular diseases (CVD) comprise a class of diseases that involve heart and

systemic blood vessels. A large number of epidemiological studies have demonstrated that

mild HHcy is a prevalent risk factor for stroke, cardiovascular disease, and venous

thromboembolism (Den Heijer et al., 2005).

3-1-1 Endothelial dysfunction

Endothelium is composed by a single layer of endothelial cell, which lines the interior

surface of vascular lumen, between blood and vascular smooth muscle cells (VSMC) of all

kinds of blood vessels and the whole circulatory system (Lai and Kan, 2015). It plays an

important role in many physiological functions, including the control of blood cell trafficking,

vasomotor tone, vessel permeability, and hemostatic balance (Aird, 2007).

Endothelial cells produce a wide variety of substances in response to various physical

and chemical stimuli, including vasodilator substances such as nitric oxide (NO), prostacyclin

(PGI2), and endothelium-derived hyperpolarizing factor (EDHF), and vasoconstrictor

substances such as endothelin-1 (ET-1), angiotensin II (Ang II), thromboxane A2 (TXA2) or

free radicals (Aird, 2004).

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9

Endothelial dysfunction can be described as an imbalance between vasodilator and

vasoconstrictor produced by the endothelium, and it has been regarded as the core systemic

pathological status in the process of atherosclerosis and cardiovascular disease (Lai and Kan,

2015) (Figure 02).

Three mechanisms have been suggested explaining HHcy-could leads to impaired

Endothelial-dependent dilatation:

1- Oxidative stress conditions: the disruptive uncoupling of NO synthase activity,

quenching of NO, and enzymatic inhibition;

2- Endoplasmic reticulum stress with eventual endothelial cell apoptosis;

3- Chronic inflammation/prothrombotic conditions.

.

Figure 02. Potential mechanisms of homocysteine-induced endothelial dysfunction

(Lai and Kan, 2015)

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10

3-1-1-1 Homocysteine induced oxidative stress condition

Oxidative stress is defined in general as an imbalance between elevated ROS/RNS

production and removal by the endogenous antioxidant system (José, 2014). The reactive

oxygen species (ROS) include free radicals such as superoxide (•O2-), hydroxyl (•OH),

peroxyl (•RO2), hydroperoxyl (•HRO2-), as well as non radical species such as hydrogen

peroxide (H2O2).

The reactive nitrogen species (RNS) include free radicals like nitric oxide (•NO) and

nitrogen dioxide (•NO2), as well as non radicals such as peroxynitrite (ONOO-), nitrousoxide

(HNO2) and alkyl peroxynitrates (RONOO). Of these reactive molecules, •O2-, •NO and

ONOO- are the most widely studied species and play important roles in the cardiovascular

complications (Jeanette et al., 2005).

Endogenous sources of ROS can be subdivided into mitochondrial and cytosolic,

including NADPH oxidases (Nox), uncoupled NO synthases (NOSs), and xanthine oxidase

(XO). Noting that in cardiomyocytes, mitochondria are the major source of ROS, which are

generated as byproducts of electron flow through the electron transport chain (ETC),

predominantly at complexes I and III. (Figure 03).

Figure 03. Major endogenous sources of reactive oxygen species (ROS) and reactive nitro-

gen species (RNS) in the cardiomyocyte (José, 2014)

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11

a- Uncoupling of NO synthase

Nitric oxide (NO) has been shown to be a major modulator of vascular homeostasis and to

have a vasoprotective effect against atherosclerosis (Moncada et al., 1991). It inhibits platelet

aggregation, leukocyte migration, and adhesion to endothelium, and it also attenuates vascular

muscle cell proliferation and migration, which collectively promote atherosclerosis (Knowles

et al., 2000).

Nitric oxide is majorly synthesized by the endothelial isoform of NO synthase (eNOS) in

response to the vasodilation stimulus. Endothelial NO diffuses across to VSMC where it

activates cytosolic guanylylcyclase, increases cyclic GMP production, and leads to vascular

smooth muscle relaxation (Deanfield et al., 2005).

Therefore, the loss of endothelial-mediated vasodilatory ability that characterized by the

tipping of the vascular balance toward an abnormally constrictive, inflammatory and

prothrombombic state is considered to be one of the earliest manifestations of cardiovascular

damage ( lai and kan, 2015 ), and is considered to be pivotal in the initiation and progression

of atherosclerosis (Ross , 1999) .

In endothelial cells, eNOS is inactive when it is bonded with caveolin 1 (cav-1). When it

becomes active, eNOS disassociates from cav-1 and binds with calmodulin (CAM) and heat

shock protein 90 (Hsp90) and together with phosphorylation of serine sites lead to the

vasodilataion (Kietadisorn et al., 2012) (figure 04).

Tetrahydrobiopterin (BH4), an essential cofactor of eNOS, is necessary for optimal eNOS

activity (Kietadisorn et al., 2012). The reduction in BH4 availability, followed by the

uncoupling of eNOS, is the significant mark in Hcy-mediated oxidative stress (Dhillon et al.,

2003; Rochette et al., 2013). This is an exact feature of endothelial dysfunction that directly

precedes the appearance of atherosclerosis (He et al., 2010).

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12

Figure 04. Central role of endothelial NO synthase (eNOS) uncoupling in the pathogenesis

of endothelial dysfunction (Kietadisorn et al., 2012)

Homocysteine also induces NADPH oxidase activity (NOX). There are multiple

isoforms of NADPH NOX, with endothelial cell mostly exhibiting the isoform NOX2. Hcy

increased endothelial cells NADPH oxidase expression in a time- and dose-dependent manner

(Tyagi et al., 2005). The up-regulated NADPH oxidase likely represents an initiating source

of oxidative stress in endothelial cells that triggers other dormant ROS producers in HHcy (lai

and kan, 2015 ).

Homocysteine contains a highly reactive sulfhydryl (-SH) group. The sulfhydryl group

readily self-oxidizes to form disulfide linkage with other free thiols, along with the generation

of superoxide radicals as a byproduct (McDowell and Lang , 2000). In addition, the self-

oxidation of Hcy to Hcy and Hcy-thiolactone generates (ROS) and further contributes to the

vascular toxicity of homocysteinemia (Andersson, 1995).

b- Accumulation of Asymmetric Dimethylarginine

Recently, the protein asymmetric dimethylarginine (ADMA), an endogenous eNOS

inhibitor, has garnered interest as a potential biomarker for endothelial dysfunction (Zhang et

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13

al., 2012). Hcy is also known to decrease NO production by increasing the ADMA (Eren et

al., 2014). Elevated plasma ADMA is an emerging cardiovascular risk factor that is prevalent

in patients with hypercholesterolemia, hyperhomocysteinemia, diabetes mellitus, and

hypertension (Boger, 2003). In addition to inhibiting production of NO, ADMA may also

promote the uncoupling of eNOS, directly contributing to increased oxidative stress

(Vallance, 2001; Boger, 2003). Elevated plasma ADMA correlated directly with impairment

of endothelium-dependent relaxation of the carotid artery in monkeys with

hyperhomocysteinemia caused by a diet enriched with methionine and deficient in folate

(Boger, 2000). Elevated plasma ADMA was also reported to correlate with impaired

endothelial function in a rat model of HHcy (Fu et al., 2005) and in human subjects with

acute HHcy induced by oral methionine loading ( Stuhlinger et al., 2003).

3-1-1-2 Homocysteine induced protein modification and endoplasmic

reticulum stress

The Endoplasmic Reticulum (ER) plays a pivotal role in proper assisted protein

folding and post-translational modifications of proteins for appropriate function, membrane

targeting and secretion. Any process that interferes with ER function results in unfolded

protein response (UPR) and ER stress (Veeranki and Tyagi, 2013).

Elevated Hcy levels lead to ER stress and induce protein modification through an

alternative mechanism mediated by the cyclic thioester form of Hcy (homocysteine

thiolactone). Homocysteine thiolactone is formed by methionyl-tRNA synthetase as an error-

editing reaction when homocysteine becomes mis-incorporated into methionyl-tRNA in place

of methionine (Santulli and Iaccarino, 2013).

When intracellular concentrations of Hcy become elevated, Hcy can participate in

disulfide exchange reactions with ER proteins, leading to the misfolding of newly synthesized

secretory and membrane proteins such as thrombomodulin (Lentz and Sadler, 1993). The

cellular consequences of this ER stress, include dysregulation of lipid metabolism, activation

of inflammatory pathways, and impaired insulin signaling. ER stress can also lead to

apoptotic cell death (Kaufman, 2002).

3-1-1-3 Homocysteine induced inflammatory/prothrombotic conditions

Hyperhomocystenemia enhanced vascular inflammation (Durand et al., 1997).

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14

During inflammation (Figure 05), Tumor Necrosis factor-α (TNF-α) exerts its effects

on the endothelium through its receptor, TNFR. Binding of TNFR by TNF-α leads to

diminished eNOS protein expression via suppression of promoter activity and destabilization

of its mRNA. TNFR suppresses eNOS activity by preventing the degradation of its

endogenous inhibitor, ADMA. TNFR signaling also induces the transcription factor NF-κB

leading to enhanced expression of intercellular adhesion molecules: intercellular adhesion

molecule-1 (ICAM-1); vascular cell adhesion molecule-1 (VCAM-1), TNF-α and NADPH-

oxidase-1(Nox1). NF-κB induction is also mediated by oxidized low density lipoprotein

(oxLDL), ROS and binding of various autoantibodies (AECA: anti-endothelial cell

antibodies; APLA: antiphospholipid antibodies; anti-oxLDL: anti-oxidized LDL antibodies).

eNOS uncoupling, mediated in part by ROS, is associated with reduced NO production and

enhanced generation of ROS. eNOS activity is also suppressed by oxLDL (Steyers and

Miller, 2014).

Figure 05. Regulatory circuits in inflammation and endothelial dysfunction

(Steyers and Miller, 2014)

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15

HHcy also induces a prothrombotic condition (Dayal et al., 2006) including enhanced

platelet activation, enhanced coagulation (Undas et al., 2005), and attenuated fibrinolysis,

resulting from posttranslational modification of fibrinogen by homocysteinylation (Sauls et

al., 2006).

3-1-2 Atherosclerosis

The mechanism of atherosclerotic disease in HHcy is directly related to vascular

endothelial cell damage which leads to vascular endothelial dysfunction, and enhanced

oxidative stress (Malinowska et al., 2012; Yilmaz, 2012).

Atherosclerosis initiates from disrupted endothelium which allows circulating

apolipoprotein B (apoB) containing lipoproteins to penetrate and accumulate in

subendothelium where they further undergo chemical modification. Modified lipoproteins,

particularly, oxidized low-density lipoprotein (LDLs), promote the proinflammatory

phenotype of endothelial cells for increased vascular cell adhesion protein 1 (VCAM1) and

intercellular adhesion molecule 1 (ICAM1) expression and proinflammatory cytokine

production, all of which attract circulating white blood cells homing to the lesion site (Estruch

et al., 2013; Milstone et al., 2015). Following infiltration into the lesion site, monocytes,

dendritic cells and T lymphocytes uptake fat and cholesterol to become foam cells that

aggravate the inflammation cascade site (Haka et al., 2015; Cochain and Zernecke 2015)

(Figure 06).

Elevated plasma Hcy has been considered as an independent risk factor for

atherosclerotic vascular disease (Bautista et al., 2002; Cui et al., 2008).

Indeed, increased oxidative stress, alterations of lipid metabolism and induction of

thrombosis have been suggested to be pathogenic links which are present between HHcy and

atherosclerosis (Eren et al., 2014).

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16

Figure 06. Hyperhomocysteinemia and etiopathogenesis of atherosclerosis

(Sainani et al., 2008)

Homocysteine-induced histopathologic findings include thickness of the intima layer,

disruption of the elastic lamina, smooth muscle hypertrophy, platelet aggregation, and the

white thrombus formation (McCully, 1969; Tsai et al., 1994). These changes may be mediated

by several pathophysiologic mechanisms: upregulation of the monocyte chemoattractant

protein-1 and interleukin-8 expression and secretion, and subsequent leukocyte recruitment ]

(Poddar et al.,2001); binding of thiolactone with low density lipoprotein (LDL) cholesterol to

form aggregates, which are phagocytosed by macrophages in tunica intima that in turn enrich

the atherosclerotic plaques with lipid (McCully, 1996) smooth muscle cell proliferation and

increased collagen production (Majors et al ., 2002 ); attenuation of endothelial tissue

plasminogen activator binding sites; increased blood viscosity; protein C inhibition; factors

VIIa and V activation; increased fibrinopeptide A and prothrombin fragments 1 and 2;

decreased endothelial antithrombotic activity (Nappo et al., 1999); increased oxidative stress

(Mansoor et al., 1995 ) and platelet aggregation (McCully and Carvalho, 1987).

The study of (Lentz, 2005) demonstrate that HHcy was shown to accelerate the

development of atherosclerosis in susceptible models such as the apolipoprotein E (apoE)-

deficient mouse. (Hofmann et al., 2001) reported that apoE deficient mice fed a

hyperhomocysteinemic diet for 8 weeks developed atherosclerotic lesions in the aortic sinus

that were of greater size and complexity than those seen in apoE-deficient mice fed normal

chow. The vascular lesions in the hyperhomocysteinemic apoE-deficient mice contained high

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17

levels of inflammatory mediators such as the leukocyte adhesion molecule, VCAM-1

(Hofmann et al., 2001).

3-2 Hyperhomocysteinemia and dyslipedimia

Dyslipidemia, as a risk factor of CVD, is manifested by elevation or attenuation of

plasma concentration of lipoproteins. Generally, it is defined as the total cholesterol, LDL,

triglycerides, apo B or Lp (a) levels above the 90th percentile or HDL and apo A levels below

the 10th percentile of the general population (Dobsn et al., 1996).

Both Hcy and lipids are toxic in vascular cells and hepatocytes which could indicate

interactions between the two pathways. Possible mechanism might be that the intake of

saturated fatty acids can lead to increased Hcy by increasing the production of

phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via the phosphatidyl

ethanolamine methyltransferase (PEMT) pathway (Berstad et al., 2007).

Phosphatidyl ethanolamine methyltransferase consumes three SAM molecules for

transforming (PE) to (PC). The reaction produces three SAH molecules that are hydrolyzed to

Hcy via SAH-hydrolase (Figure 07). Another possible explanation could be that a diet rich in

fatty acids might contains more methionine, the precursor of Hcy (Obeid and Herrmann,

2009).

Figure 07. Possible interactions between hyperhomocysteinemia and hyperlipidemia in cell

pathology (Obeid and Herrmann, 2009)

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18

Studies have reported that oxidative stress and inhibition of NO release were induced

by Hcy, which also promoted a lower expression of paraoxonase 1 (PON1) and enhanced the

production of ROS and a lower activity of PONs, in patients with HHcy. Hcy-induced ROS

downregulates the expression of HDL-associated PON1, which accelerates the development

of atherosclerosis (Maron and Michel, 2012; Eren et al., 2013). Paraoxonase 1 is a HDL-

associated enzyme esterase which appears to contribute to the anti-oxidant and anti-

atherosclerotic capabilities of HDL (Parra et al., 2007).

3-3 Hyperhomocysteinemia and hepatic disease

The liver is central for the synthesis and metabolism of Hcy and related thiols, given

that the majority of dietary methionine is metabolized in this organ (Mato and Lu, 2005). The

Changes of Hcy metabolism were reported during liver damage associated to alterations of

lipid metabolism (Werstuck et al., 2001; Obeid and Herrmann, 2009).

Hyperhomocystenemia is also implicated in hepatic disorders, such as alcoholic liver disease

(Roblin et al., 2007), cirrhosis (Bosy- Westphal et al., 2001), steatosis and fibrosis (Adinolfi

et al., 2005; Ventura et al., 2005). This correlation is pertinent, as far as the liver is central in

Hcy metabolism (Brosnan et al., 2004).

3-4 Hyperhomocysteinemia and carcinogenesis

For the past several years, a link has been established between certain cancers and

elevated plasma Hcy. Increased plasma Hcy concentration is a risk factor for cancer and even

as a novel tumor marker (Plazar and Jurdana, 2010). Folate depletion promotes the

development of cancer, particularly colorectal cancer, whereas high doses of folic acid

enhance the growth of cancer cells. Folate, Vitamin B12, and Vitamin B6 have a number of

biologic roles that make them potentially important in cancer (Qureshi et al., 2016).

Defective metabolism of Hcy in carcinogenesis is well documented, but the

pathophysiology is not fully understood (Fassbender et al., 1999; Sun et al., 2002). Malignant

cells are characterized by high a growth rate, and the methionine requirement increases in

these cells due to increased protein synthesis and transmethylation reactions. Normal cells

meet their methionine requirement by synthesizing it from homocysteine. In contrast,

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19

methionine-dependent malignant cells in organs such as the lung, kidney, breast, colon and

bladder cannot convert Hcy to methionine, which results in Hcy accumulation (Cellarier et al.,

2003).

4- Therapy of Hyperhomocysteinemia

The primary goal of treatment is to lower blood levels of Hcy to normal. Treatment for

HHcy involves the use of vitamins, such as folic acid, Vitamin B12, and pyridoxine. Folic

acid and vitamins predominantly act under fasting condition and pyridoxine acts after meals.

Pyridoxine reduces Hcy levels by 22%. Folic acid alone reduces Hcy level by 22% and

Vitamin B12 by 11%. When both administered together, it causes a reduction of 38.5%

(Lehmann et al., 2003).

5- Argania spinosa

The herbs have been the basis for many medicinal therapies. Among these herbs: Argania

spinosa.

5-1 Description

Argania spinosa is a tropical tree that belongs to the Sapotaceae family (Chaussod et al.,

2005). This plant is endemic in southwestern Algeria and Morocco (Msanda et al., 2005).

Because of its ability to survive to arid and semi arid regions (Naggar and Mhirit, 2006), It

protects soil from desertification and erosion (Alados and El Aich, 2008).

In addition to these important ecological aspects. The Argan tree is exploited essentially

for its fruits. The endosperm seed of the fruit constitutes a good potential source of edible oil

for human consumption and is endowed with important medicinal properties (Charrouf and

Guillaume, 1999). The leaves of this tree are also used as "hanging forage" for cattle (goats

and sheep) and this forage is complemented by the energetic leftovers obtained after the oil

preparation (Charrouf and Guillaume, 1999). Indeed, Argan oil is rich in essential

polyunsaturated fatty acids. It is a source of oleic acid (47.7%) and linoleic acid (29.3)

(Rahmani, 2005) and it is rich in minor and noble compounds like tocopherols, polyphenols,

sterols, carotenoids, xanthophyls, squalen (Khallouki et al., 2005), and saponins (Guillaume

and Charrouf, 2005).

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20

5-2 Botanical classification

Figure 08. Kernel and tegument of Argania spinosa L (Ould safi, 2014)

Kingdom Plantae

Sub-division Angiospermae

Class Dicotylédonae

Sub-class Asteridae

Order Ericales

Family Sapotaceae

Genus Argania

Species Argania spinosa

Tegument

Kernel

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21

Figure 09. Distribution area of the Argan tree in Algeria (Kechairi, 2009)

5-3 Therapeutic properties

The main traditional use of Argan oil is by far for nutritional purposes. Natives either

directly eat the oil on toasts, generally for breakfast, or use it for frying.

As cosmetic, the oil is traditionally indicated to cure all kind of pimples on the skin and

more particularly juvenile acne and chicken pox pustules. It is also recommended to reduce

dry skin problems and slow down the appearance of wrinkles. It is also used in rhumatology.

For these indications, the oil is used as a skin lotion and applied on the area to be cured.

(Charrouf and Guillaume, 1999).

In addition, Many scientific studies have reported that the oil has many pharmacological

effects, such as antioxidant (El Baabli et al., 2010), antiproliferative (Bennani et al., 2007),

cardioprotective (Charrouf et al., 2007) and hypolipemiant activities (Drissi et al., 2004).

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22

6- Bacterial Biofilm and cardiovascular diseases

6-1 Definition of Biofilm

A biofilm is a structured community of bacterial cells enclosed in a self-produced

matrix that adheres to inert or living surfaces, including tissues, industrial surfaces, and

artificial devices, such as intrauterine contraceptive devices, implants and prosthetic medical

devices, catheters, dental materials, cardiac valves, and contact lenses. Biofilms form when

bacterial colonizers adhere to surfaces in aqueous environments and excrete a slimy, glue-like

substance composed of exopolysaccharides (EPS). The EPS can consist of cellulose,

alginates, poly-N-acetylglucosamine, extracellular teichoic acid, various proteins, lipids, and

extracellular RNA or DNA (Sun et al., 2013).

One of the most important characteristic of biofilms is their increased tolerance to

antimicrobial agents (Wimpenny et al., 2000). Bacteria within a biofilm are several orders of

magnitude more resistant to antibiotics, compared with planktonic bacteria (Rabin et al.,

2015)

Biofilms contain channels that allow water, nutrients and oxygen circulation (De Beer

et al., 1994). However, during biofilm formation a gradient of available substances is

established, making the outer layers becoming aerobic and metabolically active, while the

inner ones become anaerobic, nutrient deficient and slowed down growth (Werner et al.,

2004; Costerton et al., 2005; Bjarnsholt et al., 2013).

6-2 Stages of Biofilm formation

The process of biofilm formation is complex, but generally recognised as consisting of

five stages (Palmer and White, 1997) (Figure 10):

Development of a surface conditioning film;

Movement of microorganisms into close proximity with the surface;

Adhesion (reversible and irreversible adhesion of microbes to the conditioned

surface);

Growth and division of the organisms with the colonisation of the surface,

microcolony formation and biofilm formation; phenotype and genotype changes and

Biofilm cell detachment/dispersal each of these processes will be considered in turn.

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23

Figure 10. Various stages of biofilm formation and development (Gupta et al., 2016)

6-3 Biofilm and cardiovascular infections

The human body contains a large number of bacteria but their localization in healthy

individuals is normally restricted to certain body areas such as the skin, the mucosae of buccal

and nasal cavities, vagina and the gastrointestinal tract (Costello et al., 2009; Ma et al., 2012).

and endothelium (Kokare et al. 2009).

The biofilm infection strategy is one of attaching to host tissue surfaces, producing

aging of host cells in the area of infection, and providing nutrition to the biofilm constituents

through inflammatory pathways which lead to the production of plasma exudate that can be

used by the community as a nutrient source (Hall-Stoodley et al., 2004; Hall‐Stoodley and

Stoodley, 2009). Biofilms can cause chronic infections and are associated with a number of

chronic disease states including cystic fibrosis, infectious endocarditis, and chronic wounds

(Singh et al., 2000; James et al., 2008).

Infections of the cardiovascular system, including those involving prostheses and

devices, are a globally recurring problem. Vascular infections are often life-threatening,

spread easily, and costly to treat. Furthermore, infection is a common problem affecting the

success of biomedical implants, such as vascular stents (Habash and Reid, 1999). Bacteria can

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24

be introduced through surgical interventions, travel through the bloodstream and infect the

endothelial cells lining the blood vessels. Cardiovascular disease has also been linked to

microbial infection (Lowy, 1998), with attachment of bacterial pathogens to endothelium or

extracellular matrix being an initial step in the process (Beachey, 1981).

Atherosclerotic plaque contains bacteria and other microorganisms (Fabricant et al.,

1978; Ott et al., 2006). However, early efforts to determine the clinical significance of the

presence of these microorganisms in plaque proved inconclusive, mainly due to the failure of

poorly designed antibiotic trials (Rosenfeld and Campbell, 2011). Many epidemiological

studies have established positive associations between cardiovascular disease risk factors,

morbidity, mortality, and markers of infection. Specific pathogens along with their potential

contribution by direct or indirect mechanisms to atherosclerosis pathogenesis have been

reviewed (Rosenfeld and Campbell, 2011).

First, the most common source of microorganisms within atherosclerotic plaques most

closely correlate with the oral microbiome, rather than bacteria from any other niche, such as

gut, skin, or sinus (Hayashi et al., 2010; Jain and Douglas, 2014). Secondly, the arrangement

of the microorganisms within the plaques is heterogeneous (Wolcott et al., 2012), in that the

samples followed a pattern of regions of high microbial density directly adjacent to an area

which was almost void of microbial DNA. Finally, it has been shown that the regions of high

microbial density were polymicrobial. These features suggested microorganisms at the

arterial wall are in biofilm mode of growth (Dalton et al., 2011; Wolcott et al., 2012).

The general hypothesis that chronic infections can contribute to the development of

atherosclerosis has come from: direct effects of infectious agents on cellular components of

the vessel wall; increased expression of cytokines, chemokines; and cellular adhesion

molecules, resulting in local endothelial dysfunction and immune responses targeted at self-

proteins located in the vessel wall due to molecular mimicry (Epstein et al., 1999).

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25

6-4 Bacterial strains

Clinical isolated bacteria used in the study are:

6-4-1 Streptococcus mutans

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is generally

known to be a pathogen of dental caries (Hamada and Slade, 1980), and its surface protein

antigens have been investigated to clarify their role as virulence factors. It is known to be

associated with bacteremia and infective endocarditis (IE). It is also of interest that S. mutans

was shown to possess these two, indicating that the bacterium is a possible candidate for

inclusion in the group of bacterial species involved with atheromatous plaque formation

properties (Kuramitsu et al., 2001 ; Chia et al., 2004; Nakano et al., 2005).

The role of S. mutans in atherogenesis has been investigated. Although these bacteria are

capable of invading endothelial cells and stimulating the production of inflammatory markers,

in addition to being detected at a high frequency in these lesions (Nakano et al., 2006; Nakano

et al., 2009).

Several in vitro studies have shown that S. mutans has the ability to adhere to collagen

type 1 (Nomura et al., 2012), induce platelet aggregation (Matsumoto-Nakano et al., 2009),

invade human endothelial cells, and induce increased production of interleukin (IL) 1, IL-6,

monocyte chemoattractant protein 1 (MCP-1) and foamy macrophages, which are strongly

associated with the pathogenesis of atherosclerosis (Nagata et al., 2011). Studies using animal

models observed that an infection with the invasive strain of S. mutans OMZ175 accelerates

the development of atherosclerotic plaques and increases the inflammatory response in an

ApoE-null mouse when compared to the control without S. mutans infection (Kesavalu et al.,

2012). These results suggest that invasive strains of S. mutans may be related to vascular

disease in humans, possibly contributing to the progression of atherosclerotic lesions.

6-4-2 Streptococcus intermedius and Streptococcus anginosus

Streptococcus intermedius and Streptococcus anginosus are two members of the

Streptococcus anginosus group (SAG), also known as the "Streptococcus milleri" group, one

of five groups collectively known as viridans group streptococci, consists of the species S.

intermedius, S. anginosus, and S. constellatus (Whiley and Beighton, 1991) . A variety of

clinical diseases have been associated with infection with the different members of the SAG

(Claridge et al., 2001).

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26

Streptococcus intermedius has a tendency to cause abscess formation commonly found in

the liver and brain, but is rarely the etiologic agent in infective endocarditis (Whiley et al.,

1992; Rashid et al., 2007). Previous studies on infective endocarditis caused by SAG have

relied on phenotypic methods for identification. (Sussman et al., 1986) studied 36 patients

with viridans streptococcal endocarditis, and identification at the species level was determined

by using biochemical tests. Four of the cases were found to be S. intermedius.

Recently, (Cooper and Gotoff, 2016) reported a case of purulent pericarditis with associated

subdiaphragmatic and hepatic collections due to S. anginosus.

6-4-3 Staphylococcus haemolyticus

Staphylococcus haemolyticus plays an important role in hospital-acquired opportunistic

infections related to implanted medical devices (Mack et al., 1996; Mehta et al., 1997; Mack

et al., 2006). Furthermore, S. haemolyticus has the highest level of antimicrobial resistance

among all CoNS (Coagulase-negative staphylococci) (Froggatt et al., 1989; Chiew et al.,

2007).

The study of Ott et al., (2006) reported that S .haemolyticus among bacterial agents could

have secondarily colonized atheromatous lesions and could act as an additional factor

accelerating disease progression.

6-4-4 Streptococcus uberis

Streptococcus uberis is an environmental Gram-positive bacterium belonging to the

Streptococcaceae family. It is responsible for a high percentage of mastitis in dairy cattle and

it is rarely associated with human infections (Zadoks, 2007). Only a few case reports have

described human infections with this microorganism, which is widely difficult to identify.

In vitro experiments demonstrated that S. uberis can readily develop penicillin

resistance, and microbial analysis of bacterial population in treated milk showed that S. uberis

can grow even in cold storage (Haenni et al., 2010; Rasolofo et al., 2010). It has been

hypothesized that the stability of this pathogen under various environmental conditions and

the expression of virulence factors (Haenni et al., 2010), may expand the pathogenicity of

this bacterium from cattle to humans as described for Streptococcus agalactiae, a pathogen

that is associated with both animal and human diseases ( Zadoks et al., 2011).

Recently, Gulen and his collabators (2013) identified S. uberis from urine samples of

seven of 148 patients by phenotypic methods.

Materials and

Methods

Materials and methods

27

1- Biological plant

In this study, Argania spinosa seeds were collected from Tindouf which is located in

South west of Algeria, on December 2011 (L.O.S.T As. 12. 11).

The powdered seeds of A. spinosa seeds were obtained by cutting the fruits into pieces

to obtain seeds, and then the seeds were subjected to size reduction to a coarse powder using a

mechanical grinder.

2- Animals

2-1- Experimental animals

The experiments were performed on 28 adult male abino Mus Musculus mice (2.5- 3

month old), weighing (30– 35g), given from central pharmacy, Algeria.

The animals were separated and housed 7 per plastic cages covered with a stainless

wire netting, a layer of sawdust is placed at the bottom of each cage, and it is regularly renewed

every two days. They were maintained under standard laboratory conditions of humidity,

temperature and light.

Animals were fed with normal commercial pellet diet (LA RATION, Bouzeriaa, Algeria) and

water ad libitum. The animals were acclimatized to laboratory conditions for one week prior

to experiment.

Table 02: Composition of diet for 1 kg of food taken by the mice during 21 days (ONAB):

Composition Amount in g / kg Percentage %

Corn 620 62

Soja 260 26

Phosphate 16 1,6

Limestone 9 0,9

Cellulose 10 1

Minerals 10 1

Vitamins 10 1


28

2-2- Experimental treatments

Animals were divided into four groups of similar mean body weights and fed for 21

days with control and experimental diet:

The control group (F) was fed with white bread (0.50 mg/mice);

The second group (M) was fed with L-methionine (500 mg/kg/day);

The third group (MP) was fed with L-methionine (500 mg/kg/day) and

treated with A. spinosa powdered seeds (150mg/kg/day), While;

The positive control group (P) was treated with the powdered seeds of A.

spinosa (150 mg/kg/day) only.

The methionine and plant extract were given in white bread (0.50mg/mice), and animals

were allowed free access to food and water.

During all the treatment period (21 days), body weight of the mice was measured daily

at the same time.

Each dose of methionine or plant is incorporated into a flour ball (0.50 mg) and then

administered to the mouse orally.

Table 03: Treatment of mice (n=7, for 21 days).

2-3- Blood and tissue sampling

At the end of the experiments, blood samples were collected after fasting, from the retro

orbital vein into EDTA tubes by using glass capillaries. They were centrifuged immediately,

and plasma was frozen under -20°C until assay time.

Experimental

group

Substance

administered

Daily dose

F

Flour 0,5 mg/mice

M

Flour +L-Methionine 500 mg/kg/mice

MP

Flour+ L-methinine +

plante

500 mg/kg+150mg/kg/mice

P

Flour +Plante 150 mg/kg/mice


29

After the blood samples collection, the animals were sacrificed. Then, organs used for

histological analysis (Aorta, Heart and Liver) were quickly removed, rinsed with saline solution

(0.9%), and fixed in formalin 10%. The techniques used in this research study are summarized

in (figure 11).

Figure 11. Blood and tissue sampling.

28 adult male Abino Mus

Musculus mice

The positive

control group (P) d’engraissement

(15 semaines)

Blood sample

Histological investigations of aorta, heart and liver

The third group

(MP)

iment

d’engraissement

(15 semaines)

8 lapins nourris

à l’aliment

d’engraissement

The second group

(M) d’engraissement

(15 semaines)

The control group

(F)

d’engraissement

(15 semaines)

Tissue sample

Blood sample Blood sample Blood sample

Tissue sample Tissue sample Tissue sample

Dosage of biochemical

parameters: Hcy, TG,

CHO, HDL-c , LDL-c,

ALT, AST

FSH, LH, Testostérone,

Oestradiol

Determination of

oxidative stress

parameters : GSH and

catalase activity



CHO, HDL-c , LDL-c,

ALT, AST


Oestradiol

Determination of

oxidative stress

parameters: GSH and

catalase activity



CHO, HDL-c , LDL-c,

ALT, AST


Oestradiol

Determination of

oxidative stress


catalase activity



CHO, HDL-c , LDL-c,

ALT, AST


Oestradiol

Determination of

oxidative stress


catalase activity


30

3- Methods

3-1Chemical products

Chemical products used in our study are:

L-methionine, chloroform, NaCL 0.9%, formalin 10%, dithiobis-2-nitrobenzoic acid (DTNB),

sulfo-salicylic acid (0.01M), Bovine Serum Albumin (BSA), orthophosphoric acid (85%), Tris

Ethylene Di-amine Tetra Acetic acid (EDTA, 0.02M), different concentrations of ethanol

(50%, 70%, 95% and 96%), HCl, NaOH, NaCl, butanol, xylene, paraffin and glycerin.

3-2 Equipments

Precision weighing balances (readability 0.01g) to determine the weight of the mice,

Precision Weighing Balances (readability 0.0001g) to determine the quantity of methionine,

Heating magnetic stirrer, pH meter, Centrifuge, Spectrophotometer , Oven, Microtome and

Photo microscope connected to computer.

3-3 Biochemical analysis

Plasma Hcy and lipids status determination were performed in the medical laboratory of

IBN SINA, Constantine.

3-3-1 Plasma Hcy determination

Homocysteine levels were measured by competitive solid phase chemiluminescance

immunoassay (IMMULITE).

Homocysteine involved a preliminary manual sample pretreatment step. Hcy in the

plasma sample is released from its binding proteins and converted to SAH by an off-line 30

minute incubation at 37°C in the presence of SAH hydrolase and Dithiothreitol. The treatment

sample and alkaline phosphate –labeled-anti-SAH antibody are simultaneously introduced into

a test unit containing an SAH coated polystyrene bead. During 30 minutes of incubation, the

converted SAH from the sample completes with the immobilized SAH for binding the alkaline

phosphatase labeled-anti SAH antibody conjugated. Unbound enzyme conjugated is removed

by centrifugal wash. The substrate is added and the procedure continues as described for the

typical immunoassays.


31

3-3-2 Lipids determination

Total cholesterol, HDL-c, LDL-c and triglycerides concentrations were assessed using

colorimetric automatic procedures (Auto-analyzer type Integra 400).

Clinical significant of total cholesterol

Cholesterol is an unsaturated alcohol of the steroid family of compounds and found in blood,

bile, and brain tissue. It is synthesized in many types of tissues, but particularly in the liver and

intestinal wall. It serves as a precursor to bile acids, adrenal and gonadal steroid hormones and

vitamin D (Cox and Garcia-Palmieri, 1990).

Epidemiological studies have shown a positive relationship between total cholesterol

concentrations and mortality from coronary heart disease (Obeid and Herrmenn, 2009).

The series of reactions involved in the assay system are as follows:

Cholesterol oxidase

Cholesterol esters + H2O Cholesterol + fatty acids

Cholesterol Oxidase

Cholesterol + O2 Cholest-4-ene-3-one + H2O2

Peroxidase

2 H2O2 + amino-4-antipyrine + phenol Quinoneimine dye + 4 H2O

The intensity of the color produced is directly proportional to cholesterol concentration. It is

determined by measuring the increase in absorbance at 500 – 550 nm.

The concentration of cholesterol was calculated by using the following formulae:

Absorbance of Sample

Cholesterol Concentration= (Cholesterol standard)*200 mg/dl

(mmol/L) Absorbance of Standard

Clinical significant of triglyceride

Triglyceride (TG) is water insoluble lipids, synthesized in the intestinal mucosa by the

esterification of glycerol and free fatty acids. They represent a concentration source of

metabolic energy.


32

Triglyceride are transported in the blood as core constituents of all lipoproteins, but the

greatest concentration of these molecule is carried in the TG-rich chylomicrom and very low

density lipoproteins (VLDL) (Rifai et al., 2001).

The triglycerides are determined after enzymatic hydrolysis with lipases. The indicator is a

quinoneimine formed from hydrogen peroxide, 4-aminophenazone and 4-chlorophenol under

the catalytic influence of peroxidase (Young, 2001).

Lipoprotein Lipase (LP)

Triglycerides (TG) Glycerol+ fatty acids

Glycerol Kinase (GK)

Glycerol +ATP Glycerol-3-phosphate (G3P) + ADP

Glycerol Phosphate Oxidase (GPO)

G3P + O2 Dihydroxyacetone phosphate (DAP) + 2 H2O2

Peroxidase (POD)

2 H2O2+ 4-AAP + 4-Chlorophenol Quinoneimine dye + 4 H2O

The concentration of triglycerides was calculated by using the following formulae:


TG concentration = * 200 (Standard concentration)

Absorbance of Standard

= mg/ml *0.0114 mmol/L.

Clinical significant of HDL-c

High density lipoprotein cholesterol (HDL-c) also known as "good" cholesterol, molecules

consisting of cholesterol and protein that carry cholesterol from cells back to the liver (Obeid

and Herrmenn, 2009).

HDL-c was determined with enzymatic procedure after lipoproteins were precipitate by

phosphotungstate in the presence of magnesium ions. After centrifugation, the HDL cholesterol


33

in the supernatant is determined by the same technique as the total enzymatic cholesterol, and

the calculation as shown below:


HDL-c Concentration = (Standard concentration)*200 mg/dL

Absorbance of Standard

Clinical significant of low density lipoprotein cholesterol

Low density lipoprotein cholesterol (LDL-c) particle carry cholesterol from the cell back to

the tissue. LDL-c is known as bad cholesterol because high levels are thought to increase the

risk of heart disease.

LDL-c concentration was obtained by direct calculation according to Friedwald formula:

LDL = total cholesterol - HDL - triglycerides / 5

When the level of TG is greater than 3.4 g / l (3.75 mmol / L), LDL cholesterol cannot be

calculated by this formula, it should be assayed by a direct enzymatic method.

3-3-3 Determination of Aspartate Aminotransferase and Alanine

aminotransferase activities

Aspartate Aminotransferase (AST) and Alanine aminotransferase (ALT) values were

assessed using colorimetric automatic procedures (Auto-analyzer type Integra 400).

Aspartate Aminotransferase

Aspartate Aminotransferase is a cellular enzyme present in many tissues such as heart,

skeletal muscles, kidney, brain, liver, pancreas or erythrocytes. It exists in two isoforms,

cytoplasmic and mitochondrial. The determination of AST activity in serum is used mainly to

assess the liver damage.


34

Principle:

Aspartate Aminotransferase catalyzes the transfer of an amino group from glutamic acid

to oxaloacetic acid with the formation of α-ketoglutarate and L-aspartate. In the Alera assay,

the reaction mixture contains an excess of malate dehydrogenase. In the presence of reduced

nicotinamide adenine dinucleotide (NADH), the malate dehydrogenase converts oxaloacetic

acid to malic acid with the oxidation of NADH to nicotinamide adenine dinucleotide (NAD).

NADH absorbs strongly at 340 nm, whereas NAD does not. Therefore the rate of conversion of

NADH to NAD can be determined by monitoring the decrease in absorbance bichromatically at

340-647 nm.

Alanine aminotransferase

Alanine aminotransferase is a cytoplasmic enzyme. It is primarily localized in

hepatocytes. It is released into the blood during the cell damage. The determination of ALT

activity in serum is used mainly to assess the liver damage.

Principle:

Alanine aminotransferase in serum converts the L-alanine and α-keto-glutarate in the

reaction to L-glutamate and pyruvate. The pyruvate that is formed reacts with reduced

nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase to form

lactic acid and oxidized nicotinamide adenine dinucleotide (NAD). The rate of conversion of

the reduced cofactor to the cofactor can be determined by monitoring the decrease in

absorbance bichromatically at 378 nm- 505 nm.

3-4 Determination of oxidative stress parameters

3-4-1 Tissue homogenate preparation

0,5g of the liver was homogenized in 2ml of TBS (Tris 50 mM, NaCl 150 mM, pH 7.4).

The homogenates were centrifuged at 9000 g for15 min at 4˚C. And the supernatant was used

for determination of reduced glutathione, the catalase activities and protein concentrations.

3-4-2 Glutathione assay

Glutathione (GSH) is a water-soluble tripeptide (γ-glutamyl-cysteinylglycine) produced

naturally by the liver. Due to the thiol function of cysteine, glutathione is an important

compound in maintaining the redox balance of the cell. It maintains in the proper redox state


35

OD * 1*1.525

GSH (nmol/mg of protein) =

13100*0.8*0.5.mg protein

the thiol groups of soluble and structural proteins, and participates in the detoxification of

hydroxyperoxides. In addition to detoxification, GSH plays a role in other cellular reactions,

including, the glyoxalase system, reduction of ribonucleotides to deoxyribonucleotides,

regulation of protein and gene expression via thiol disulfide exchange reactions (Townsend et

al., 2003).

The concentrations of the GSH are proportioned by the method of Weckbecker and Cory as

shown below, The spectrophotometric reader assay method for GSH involves oxidation of

GSH by the sulfhydryl reagent 5,5′-dithio-bis2-nitrobenzoic acid (DTNB) to form the yellow

derivative 5′-thio-2-nitrobenzoic acid (TNB),which is measurable at 412 nm.

Liver homogenate sample (0.8ml) was deproteinized with (0.2ml) of 5-sulfosalicylic acid

solution (0.25%) and was allowed stand on ice for 10 min. Following centrifugation at 1000

tours/mn) during 5minutes to remove the precipitated protein. (0.5 ml) of supernatant was

mixed with 1 ml Tris/EDTA buffer (pH 9.6) and (0.025 ml) of DTNB-reagent (0.01M 5,5'-

dithiobis-2-nitrobenzoic acid) and left at room temperature for 5 min. Then the absorption was

measured at 412 nm using a spectrophotometer by comparing to the blank reaction.

Glutathione concentration was obtained by direct calculation of the following formulae:

OD: optical density

1: total volume of solutions in the deproteinisation (0.8ml homogenate+ 0.2ml 5-

sulfosalicylic acid).

1.525: total volume of the solutions used in the assay of GSH (0.5ml supernatant+ 1 ml

Tris/EDTA+ 0.025 ml DTNB).

13100: absorbance coefficient at Groupment—SH to 412nm.

0.8: volume of homogenat sample.

0.5: volume of supernatant.


36

Protein determination

Protein concentration was measured by the method of Bradford (1976), using bovine serum

albumin as standard. The procedure is based on the formation of a blue complex between the

comaissie bruillant blue G-250 dye, and proteins in solution. The amount of absorption is

proportional to the protein present.

Liver homogenate sample 0.1ml was mixed with 5ml Bradford reagent and was allowed

stand for 5min. Then the absorbance was measured at 595 nm using a spectrophotometer by

comparing to the blank reaction.

The protein concentration of a test sample is determined by comparison to that of a standard

series of bovine serum albumin to reproducibly exhibit a linear absorbance profile in this assay

(Figure 01 annex).

3-4-3 Catalase activity

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. It

catalyzes the decomposition of hydrogen peroxide (H2O2) to water and oxygen. Catalase is a

tetramer of four polypeptide, It contains four porphyrin heme (iron) groups that allow the

enzyme to react with the hydrogen peroxide. It was estimated in the liver homogenate in a UV

spectrophotometer as described by Aebi (1984). The specific activity of catalase has been

expressed as mmol of H2O2 consumed/min/ mg protein. The difference in absorbance at 240

nm per unit time is a measure of catalase activity.

The reaction is believed to occur in two stages:

Catalase-(Fe III) + H2O2 [Catalase-H 2O-(Fe V)] + H2O

[Catalase-H 2O-(Fe V)]+ H 2O2 Catalase-(Fe III) + H2O + O2

2 H2O2 2 H2O + O2

Table 04: the concentrations and amounts of reagents needed for the dosage of catalase

activity.

Sq: supernatant quantity

Reagents Sample (μl) blank (μl)

Phosphate buffer (100Mm, PH7.5) 790 800

H2O2 (500Mm) 200 200

Sq (1 to 1.5 mg prt/ml) 10 0


37

The activity of catalase was estimated by the decrease of absorbance at 240 nm for 1 min

(15 and 60 seconds ) as a consequence of H2O2 consumption.

Catalase activity was obtained by direct calculation of the following formulae:

Catalase activity (mmol H2O2/min/ mg prot) =

ΔDO

ε ×L × χ × Fd

ε : extinction coefficient (= 0.043 mM-1.cm -1).

L : The length of the cuvette used (1 cm).

χ : protein quantity mg/ml.

Fd : 0.02 (dilution factor of the H2O2 in the buffer).

λ : 240 nm.

3-5 Histological analysis

After the blood samples collection, the animals were sacrificed, and samples for light

microscopic investigations were obtained from aorta, heart and liver. For histological

investigations the aorta was divided into 4 sections (arch, thoracic, abdominal, and iliac).

The samples were rinsed of all adherent tissues with saline solution (0.9%).

Then, they are kept in small containers filled with diluted formol 10%. Liver, heart and the

different parts of the aorta were included in Bouin solution for 5 min (until colored), and the

deshydration was performed through a series of ethanol solution (50 %, 70 %, 96 %), each step

was placed for approximately 30 min (3×30 min=1h 30min).

The tissues were then kept in small containers filled with butanol for 3 days. After that they

were cleared in Xylene for 10 min with two exchanges.

For the next step, the organs were immersed in paraffin at 60 °C for 1h and 30 min at

three exchanges. The sectioning was performed with a microtome (Leica RM 2135, laboratory

EL-YASSEMINE, ANNABA).

Paraffin slices, 5μm thick were stained following the heamatoxylin eosin staining

protocol (Appendix).


38

4- Biofilm formation and quantification

4-1 Strains and culture conditions

The following strains were used in this study:

Staphylococcus haemoliticus clinical isolate NC1 ;

Streptococcus intermedius DSMZ 20573 (German Collections of

Microorganisms and Cell Cultures);

Streptococcus anginosus, clinical isolate NC10 ;

Streptococcus mutans CIP103220 (Collection Institut Pasteur) and

Streptococcus uberis human clinical isolate NC6.

These strains were identified by 16S rRNA gene sequencing, using the Sanger capillary

sequencing procedure with ABI 310 apparatus (Applied Byosystem). The bacteria prior the

use were stored at – 80°C in a tube contained the proper medium broth (Microbiol, UTA,

Cagliari, Italy) with 20% glycerol.

4-2 Crystal Violet Biofilm formation screening assay

Preparation of inoculum:

The bacteria (Streptococcus spp.) were grown in anaerobic Schaedler agar (Microbiol,

UTA, Cagliari, Italy) at 37°C for 24 hours with a CO2 concentration of 5% and in Muller-

Hinton agar (Microbiol, UTA, Cagliari, Italy) for aerobic bacteria (S. haemolyticus).

After the incubation, 5 ml of SH Broth was inoculated with the following bacterial

species until a final concentration of 106cell/ml

for each bacterial species using a

spectrophotometer at 620 nm (DMS100s, Varian, NH, USA).

Protocol for quantification of biofilm on microplate

For an in vitro biofilm evaluation we used the protocol described by (Merritt et al.,

2005).

Briefly, each antibacterial combination was suspended in a 96 well microplate, where

100 L of SH Broth and 100 L of the Argan oil were added in the first well. From the first

well, 12 serial dilutions were done (from 100 to 0,04 g/ml). Next, 100 L of microbial


39

suspension was added into each well. However, positive control wells contained SH Broth and

microbial suspension, and negative control wells contained SH Broth and Argan oil only.

The experiment was performed in duplicate.

Then, The plate was agitated on microplate shaker and incubated for 37°C for 48h

aerobically (S. haemolyticus), or in 5% CO2 (Streptococcus spp.) to permit biofilm formation.

After this, the plate samples were gently washed three times with Phosphate-buffered

saline (PBSGibco®) to eliminate planktonic cells and the adhering cells in the biofilm were

stained with 100 µl of 0.1 % v/v of crystal violet solution (Microbial, Uta , Cagliari) for 10

minutes at 25°C. After three washes with PBS solution, as described previously, 200 µl of 30%

v/v acetic acid were added in every well to solubilize the dye from the bacterial biomass. The

biofilm amount was measured with a plate reader spectrophotometer (SLT-Spectra II, SLT

Instruments, Germany) at 620 nm.

In these conditions, the minimum biofilm inhibition concentration (MBIC) is defined as

the lowest concentration of an antimicrobial required to inhibit the formation of novel biofilms,

and it was determined by observing a reduction of 90% in color intensity at 620 nm ABS, as

compared to positive controls in the microplate wells.

The absorbance of biofilm formation was calculated using the ratio between the values

of wells with and without Argan oil.

The percentage of biofilm inhibition was calculated by the formula:

C= mean OD of positive control,

B = mean OD of negative control,

T= mean OD of test wells.


40

5- Statistical analysis

Values obtained were expressed as mean ± SEM and subjected to statistical analysis

using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. The

programme used for the analysis was SPSS (version 20). P < 0.05 values were considered as

significant different.

The comparison is considered, according to the probability (P) as:

No significant if P >0.05.

Significant (*) if P <0.05.

Highly significant (**) if P <0.01.

Very highly significant (***) if P <0.001.

Results and

Discussion

Chapter 1

Effect of L-methionine and A. spinosa

powdered seeds on body weight, Hcy levels,

lipid profile, liver enzyme activities and

antioxidants markers in mice

Results and Discussion

41

1- Body weight

In the present study, we examined the effects of of L-methionine on the weight of mice.

No significant difference was found in body weight among the four groups (F), (M),

(MP) and (P) during the experimental period, indicating that (500mg/ Kg) of L-Methionine

or/and (150 mg/ Kg) of the powdered seeds of A. spinosa supplementations do not affected

significantly mice growth P> 0.05 (Figure 13).

Figure 12. Effect of L-methionine intake on mice weight during 21 days.

F: Control group received flour at 0.5mg/mice; M: group received methionine at dose 500mg/kg; MP: group

received methionine at dose 500mg/kg + Argania spinosa powdered seeds at dose 150mg/kg; P: group received

Argania spinosa powdered seeds at dose 150mg/kg.

2- Effect of treatments on lipid profile in mice

2-1 Triglycerides

As shown in the (Figure 13), the concentrations of the TG were in: the (F) group (0.61

±0.05 g/L), (M) group (0.81 ± 0.06 g/L), (MP) group (0.57 ± 0.05 g/L) and (P) group (0.55±

0.04

15,00

20,00

25,00

30,00

35,00

40,00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

wei

gh

t (g

)

F

M

PM

P

Days


42

g/L) .Our data indicated that there is a high significant difference between groups (P =

0.002).

The Tukey test revealed significant difference between the groups (F and M) P= 0.029,

and a high significant difference between the groups (M and MP) (P=0.006) and the groups

(M and P) (P= 0.003).

Figure 13. Interaction of L-methionine and A. spinosa seeds on the triglycerides in mice

during 21 days of treatment.

Values are shown as mean ±SEM (n = 5); *p<0.05 and **p<0.01 .

F: Control group received flour at 0.5mg/mice; M: group received L-methionine at dose 500mg/kg; MP: group

received L-methionine at dose 500mg/kg + Argania spinosa powdered seeds at dose 150mg/kg; P: group

received Argania spinosa powdered seeds at dose 150mg/kg.

2-2 Total cholesterol

The concentrations of the T-CHO were in: the (F) group (1.09 ±0.05 g/L), (M) group

(1.29 ± 0.08 g/L), (MP) group (1.04 ± 0.04 g/L) and (P) group (0.95± 0.11 g/L). Our results

shown that there is a significant difference among the four groups (P = 0.017).

The Tukey test revealed significant difference between the groups (M and P) P= 0.012.

(Figure 14)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

F M MP P

Tri

gly

ceri

des

(g

/L)

* ** **


43

Figure 14. Interaction of L-methionine and A. spinosa seeds on the T-CHO in mice during 21

days of treatment.

Values are shown as mean ±SEM (n = 7); *p<0.05.




2-3 HDL-c

The present data showed that there is a significant difference in the concentrations of the

HDL-c among the four groups: (F) group (0.88 ±0.02 g/L), (M) group (0.70± 0.07 g/L), (MP)

group (0.84 ± 0.03 g/L) and (P) group (0.72± 0.03 g/L) (P = 0.017).

The Tukey test revealed significant difference between the groups (F and M) (P= 0.033).

(Figure 15)

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

F M MP P

Tota

l Ch

ole

ste

rol (

g/L)

*


44

Figure15. Interaction of L-methionine and A. spinosa seeds on the HDL-c in mice during 21

days of treatment.



received L- methionine at dose 500mg/kg + Argania spinosa powdered seeds at dose 150 mg/kg; P: group


2-4 LDL-c

The Figure 16 demonstrated that the concentrations of the LDL-c were in: the (F)

group (0.09 ±0.02 g/L), (M) group (0.42 ± 0.12 g/L), (MP) group (0.08 ± 0.02 g/L), (P) group

(0.06± 0.6 g/L). Our results showed that there is a high significant difference among the four

groups (P = 0.002).

The Tukey test revealed a high significant difference between the (F and M) P= 0.008,

the groups (M and MP) P=0.005 and the groups (M and P) P= 0.003.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

F M MP P

HD

L-c

(g/L

)

*


45

Figure 16. Interaction of L-methionine and A. spinosa seeds on the LDL-c in mice during 21

days of treatment.

Values are shown as mean ±SEM (n = 7); *p<0.05 and **p<0.01.




3- Effect of treatments on Homocysteine levels

After 21 days of experiments, results showed that there is a very significant difference

(p=0.007) between 4 groups : plasma t-Hcy of the (M) group achieved higher levels, and the

average was 10.04±0.83 μmol/l, significantly higher than that of the control group F

(6,84±0.51 μmol/l) and control positive group (P) (7.1±0.88 μmol/l). However, in the

combined treatments of A. spinosa crude extract with L-methionine, the Hcy concentration

was (7.26±0.46 μmol/l), showed a significant decline in plasma t-Hcy, demonstrating that

A.spinosa crude extract appears to be effective in preventing the increase of t-Hcy levels.

The Tukey test revealed a high significant difference between the groups (F and M) (P=

0.01), the groups (M and P) (P= 0.01) and a significant difference between the groups (M and

MP) (P=0.028). (Figure 17)

0

0,1

0,2

0,3

0,4

0,5

0,6

F M MP P

LD

L-c

(g

/L)

** ** **


46

Figure 17. Interaction of L-methionine and A. spinosa seeds on the Homocysteine levels in

mice during 21 days of treatment.





4- Effect of treatment on liver enzymes activities

4-1 Aspartate Aminotransferase

The concentrations of the AST were in: the (F) group (129 ±65 UI/L), (M) group

(386.14 ± 53.13 UI/L), (MP) group (308.37 ± 55.5 UI/L) and (P) group (197.18± 35.72 UI/L).

Our data shown that there is a very high significant difference among the four groups (P =

0.001).

0

2

4

6

8

10

12

F M MP P

Hcy

(µ

mo

lL)

** * **


47

The Tukey test revealed a very high significant difference between the groups (F and

M) (P= 0.001), and a significant difference between the groups (F and MP) (P=0.02) and a

high significant difference between the groups (M and P) (P= 0.01). (Figure 18)

Figure 18. Interaction of L-methionine and A. spinosa seeds on the AST in mice during 21

days of treatment.

Values are shown as mean ±SEM (n = 7); *p<0.05, **p<0.01 and ***p<0.001.




4-2 Alanine aminotransferase

The concentrations of the ALT showed that there is a difference among the four groups

but not significantly (P> 0.05): in the (F) group (38.89 ±4.01 UI/L), (M) group (49.04.14 ±

5.31 UI/L), (MP) group (45.14 ± 15.14 UI/L) and (P) group (35.29± 10.34 UI/L).

ALT levels were increased in (M) group and decreased in (MP) group but not significantly

compared to the other groups (Figure 19).

0

50

100

150

200

250

300

350

400

450

500

F M MP P

AS

T (

UI/

L)

*** ** *


48

Figure 19. Interaction of L-methionine and A.spinosa seeds on the ALT in mice during 21

days of treatment.

Values are shown as mean ±SEM (n = 7).




5- Effect of treatment on antioxidants markers

5-1 Reduced Gluthatione

The concentrations of the reduced glutathione (GSH) were in: the (F) group (8.03±0.55

nmol/mg protein), (M) group (4.48±0.55 nmol/mg protein), (MP) group (6.1±0.5 nmol/mg

protein) and (P) group (7.36±1.28 nmol/mg protein). Our data showed that there is a high

significant difference among the four groups (P = 0.01).

The Tukey test revealed a high significant difference between the groups (F and M) (P=

0.01) and a significant difference between the groups (M and P) (P=0.048) (Figure 20).

0

10

20

30

40

50

60

F M MP P

AL

T (

UI/

L)


49

Figure 20. Interaction of L-methionine and A.spinosa seeds on the reduced glutathione

in mice during 21 days of treatment.





5-2 Catalase activity

After 3 weeks of treatment, our results showed that there is a significant difference

(p=0.02) among the four 4 groups: Hepatic catalase activity was in: the (F) group (61.37

±6.39 mmol/mg protein), (M) group (45.82±5.83 mmol/mg protein), (MP) group (62.26±3.32

mmol/mg protein) and (P) group (52.16±3.19 mmol/mg protein).

The Tukey test revealed a significant difference between the groups (F and M) (P=

0.03) and the groups (M and MP) (P= 0.02) (Figure 21).

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

9,00

10,00

F M MP P

GSH

(n

mo

l/m

g p

rote

in)

**

*


50

Figure 21. Interaction of L-methionine and A.spinosa seeds on the catalase activity in mice

during 21 days of treatment.





0

10

20

30

40

50

60

70

80

F M MP P

Ca

tala

se a

ctiv

ity

(mm

ol/

mg

pro

tein

)

* *

Chapter 2

Effect of L-methionine and A. spinosa

powdered seeds on histology of aorta, heart

and liver


51

The administration of L-methionine (500mg/Kg/animal) during 3 weeks appears to alter

the aorta, heart and liver structures compared to the control group.

1- Histological study of the heart

Changes observed by light microscopy were more apparent in the sections of (M)

group than in (F) group-stained, paraffin-embedded sections. These changes consisted of

presence of lysis, and architectural changes of cardiomyocytes (Figure22: (B) and (C). There

were no apparent differences in severity of lesions among samples obtained from group (MP)

and (P) (Figure22: (D) and (E)).

2- Histological study of the aorta

In the group (M), the aortic intima showed degeneration and desquamation of

endothelial cells with fenestration, it is observed in the media lysis, formation of foam cells

laden with small lipid droplets and oval nuclei, disappearance of elastics fibers especially in

the arch aorta. (Figure 23: (B) and (C); Figure 24: (B); Figure 25: (B), (C), (D) and (E)).

However, in the control group (F), the aortic sections have intact endothelium and

spindle shaped mediocytes nuclei. Also we have observed intact aorta in the group (MP)

treated with L-methionine and A. spinosa (Figure 23: (D); Figure 24: (C); Figure 25: (F)) and

in the group (P) treated with A. spinosa only (Figure 23: (E); Figure 24: (D); Figure 25: (G)).

Noting that all these microscopic changes were observed in the histological sections of

(arch, abdominal and iliac) aorta. For the thoracic aorta sections in experimental groups (F,

M, MP and P), any change was observed.

3- Histological study of the liver

The microscopic analysis of the liver of the control group (F), showed the

parenchymal cells which are hepatocytes. These polygonal cells are joined to one another

anastomosing plates, with borders that face either the sinusoid or adjacent hepatocytes (Figure

26: (A)).

For the group (M), our data showed various pathological alterations in liver of mice

induced by the oral L-methionine administration. These alterations were marked by


52

destruction of membrane cells, hepatocellular ballooning, hypertrophy of some hepatocyte

nuclei, cytoplasmic vacuolization, and macrovesicular hepatic steatosis with a large lipid

vacuole filling the hepatocyte cytoplasm and around the blood vessels. (Figure 26: (B), (C),

(D), (E) and (F)).

The liver of (P) group showed a normal structure (Figure 26: (H). Thus, in

combination group (MP), were A. spinosa powdered seeds were administered with 500

mg/Kg of L-methionine showed reparative changes (Figure 26: (G)).


35

Figure 22. Histological sections of heart tissue in experimental groups (F, M, MP and P)

X 100 X400

X 100 X 100 X 100


54

(A). Histological sections of the cardiac muscle. Control’s Hematoxylin eosin staining (x100).

The heart histology of the group (F) was intact, with the presence of cardiac muscle fibers and

their nuclei.

(B) and (C). Histological sections of the cardiac muscle. 21 days of oral L-methionine

application (500 mg/kg/day). Hematoxylin eosin staining (Bx400, Cx100).

We observed in group (M), lysis and architectural changes of cardiomyocytes.

(D). Histological section of the cardiac muscle. 21 days of oral L-methionine (500 mg/kg/day)

+ A. spinosa powdered seeds (150mg/kg/day) application. Hematoxylin eosin staining (x100).

In the histological sections of the cardiac muscle of group (PM), no lysis was observed.

(E). Histological section of the cardiac muscle. Positive Control’s Hematoxylin eosin

staining (x100).

No change was observed.

ACC. Architectural Changes of Cardiomyocytes, CMF. Cardiac Muscle Fibers, MCN. Muscle Cell Nuclei,

L. Lysis.


55

Figure 23. Histological sections of the arch aorta in experimental groups (F, M, MP and P)

X 100

X 100

X 400 X 100 X 100


56

(A). Histological sections of the arch aorta. Control’s Hematoxylin eosin staining (x100).

The endothelium of the arch aorta of the group (F) was intact.

(B) and (C). Histological sections of the arch aorta. 21 days of oral methionine application

(500 mg/kg/day). Hematoxylin eosin staining (B x 100, C x400).

We observed in group (M), in the media the presence of lipids droplets, and the disappearance

of elastics fibers. We noted also appearance of the oval nuclei.

(D). Histological section of the arch aorta. 21 days of oral methionine (500 mg/kg/day) +

A.spinosa powdered seeds (150mg/kg/day) application. Hematoxylin eosin staining (x100).

In the histological sections of the arch aorta of group (PM), no change was observed.

(E). Histological section of the arch aorta. Positive Control’s Hematoxylin eosin staining

(x100).


FC. Foam Cells, FN. Fibroblast Nuclei, IEND. Intact Endothelium, LD. Lipid Droplets, Lu. Lumen, ON.

Oval Nuclei, SN. Spindle Nuclei.


57

Figure 24. Histological sections of the abdominal aorta in experimental groups (F, M, MP and P)

X 100 X 100 X 100 X 100


58

(A). Histological sections of the abdominal aorta. Control’s Hematoxylin eosin staining

(x100).

This photograph showed the intact endothelium of the abdominal aorta of the group (F).

(B). Histological section of the abdominal aorta. 21 days of oral methionine application (500

mg/kg/day). Hematoxylin eosin staining (x100).

The aortic intima of group (M), showed endolysis.

(C). Histological section of the abdominal aorta. 21 days of oral methionine (500

mg/kg/day) + A. spinosa powdered seeds (150mg/kg/day) application. Hematoxylin eosin

staining (x100).

In the histological section of the abdominal aorta of group (PM), no lysis was observed.

(D). Histological section of the abdominal aorta. Positive Control’s Hematoxylin eosin

staining (x100).

In the positive control group (P), the aortic sections have intact endothelium and spindle

shaped mediocytes nuclei.

END. Endolysis, FN. Fibroblast Nuclei, IEND. Intact Endothelium , LU. Lumen, SN. Spindle Nuclei.


Figure 25. Histological sections of the iliac aorta in experimental groups (F, M, MP and P)

59

X 100 X 100 X 400

X 400 X 400 X 100 X 100


60

(A). Histological sections of the iliac aorta. Control’s Hematoxylin eosin staining (x100).

The histology of the iliac aorta of the group (F) was intact.

(B), (C), (D) and (E). Histological section of the iliac aorta. 21 days of oral L-methionine

application (500 mg/kg/day). Hematoxylin eosin staining (B x 100; D, E, Cx400).

Changes observed by light microscopy in the group (M), were degeneration and desquamation

of endothelial cells, we also observed in the media lysis, fenestration, formation of foam cells,

lipids droplets and oval nuclei.

(F). Histological section of the iliac aorta. 21 days of oral L-methionine (500 mg/kg/day) +

A. spinosa powdered seeds (150mg/kg/day) application. Hematoxylin eosin staining (x100).

In the histological section of the iliac aorta of group (PM), no change was observed.

(G). Histological section of the iliac aorta. Positive Control’s Hematoxylin eosin staining

(x100).


D. Desquamation, END. Endolysis, F. Fenestration, FC. Foam Cells, FN. Fibroblast Nuclei, IEND. Intact

Endothelium , LU. Lumen, ON. Oval Nuclei, SN. Spindle Nuclei.


61

Figure 26. Histological sections of liver tissue in experimental groups (F, M, MP and P)

X 100 X 100 X 400 X 400

X 400 X 400 X 100 X 100


62

(A). Histological section of the liver. Control’s Hematoxylin eosin staining (x100).

The liver of control group showed a normal structure.

(B), (C), (D) (E), and (F). Histological sections of the liver. 21 days of oral L-methionine

application (500 mg/kg/day). Hematoxylin eosin staining ((B) x(100); (C), (D) (E), and (F)

x400).

We observed in the liver of the group (M), destruction of membrane cells, hepatocellular

ballooning, hypertrophy of some hepatocyte nuclei, cytoplasmic vacuolization, and

macrovesicular hepatic steatosis with a large lipid vacuole filling the hepatocyte cytoplasm

and around the blood vessels.

(G). Histological section of the liver. 21 days of oral L-methionine (500 mg/kg/day) +

A.spinosa powdered seeds (150mg/kg/day) application, Hematoxylin eosin staining (x100).

The histological section of the liver of group (PM), showed reparative changes.

(H). Histological section of the liver. Positive Control’s Hematoxylin eosin staining (x100).

Any change was observed in this histological section.

CV: Centro Lobular Vein, Ds. Destruction of membrane cells, H: Hepatocyte, S: Sinusoid. Hb. Hepatocellular

Ballooning, Hy. Hypertrophy of Hepatocyte Nuclei, ICF. Inflammatory Cell Foci, S. Sinusoid, V. Vacuole.

Chapter 3

Anti-biofilm formation of Argan oil

“In vitro” Study


63

Results of the biofilm assay are shown in (Figures 27, 28, 29,30, 31).

These results showed that there was a trend of increasing inhibition of S. intermedius,

St haemolyticus, S. mutans, S. anginosus and S. uberis biofilm formation as the Argan oil was

getting more concentrated.

As known that the minimum biofilm inhibition concentration (MBIC) is defined as

the lowest concentration of an antimicrobial required to inhibit the formation of novel

biofilms, and it was determined by observing a reduction of 90% in color intensity at 620 nm,

as compared to positive controls in the microplate wells.

Argan oil had a MBIC value of 100 g/mL when tested against S. intermedius, St

haemolyticus, S. mutans, and S. anginosus. Of all the 12 concentartions that were investigated

for their effects on growth, the first concentration (first dilution) was the most effective in the

inhibition of growth of these mentioned bacteria (Table 05, Figure 32), and produced a

percent reduction of 93.81 %, 100 %, 100 %, 100 %, 78.78 % for. S. intermedius, St

haemolyticus, S. mutans, S. anginosus and S. uberis respectively. However, the lesser

concentrations did not significantly inhibit biofilm formation.

The acceptable concentration of Argan oil (100 µg/mL) indicated the highest

antibacterial effect on biofilms of S. intermedius, St haemolyticus, S. mutans, S. anginosus S.

but not S. uberis (78.78 % lower than 90%).

Our study leads to the conclusion that Argan oil is able to efficiently reduce biofilms

formation of (Streptococcus mutans, Streptococcus anginosus, Streptococcus intermedius,

and staphylococcus haemolyticus) strains.


64

Figure 27. Inhibitory effect of Argan oil on S. intermedius biofilm formation.

Figure 28. Inhibitory effect of Argan oil on S. haemolyticus biofilm formation.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

100 50 25 12,5 6,25 3,12 1,56 0,78 0,39 0,19 0,09 0,048

Bio

film

fo

rma

tio

n a

t 6

20

nm

Concentrations of dilutions (µg/mL)

S intermedius +

argan oil

S intermedius

0,0

0,5

1,0

1,5

2,0

2,5

3,0

100 50 25 12,5 6,25 3,12 1,56 0,78 0,39 0,19 0,09 0,048

Bio

film

fo

rma

tio

n a

t 6

20

nm

concentrations of dilutions (µg/mL)

S. haemolyticus +

argan oil

S.haemolyticus


65

Figure 29. Inhibitory effect of Argan oil on S. mutans biofilm formation.

Figure 30. Inhibitory effect of Argan oil on S. anginosus biofilm formation.

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

100 50 25 12,5 6,25 3,12 1,56 0,78 0,39 0,19 0,09 0,048

Bio

film

fo

rmat

ion

at

62

0 n

m


S.mutans + argan

oil

S. mutans

0,0

0,5

1,0

1,5

2,0

2,5

100 50 25 12,5 6,25 3,12 1,56 0,78 0,39 0,19 0,09

Bio

film

fo

rma

tio

n a

t 6

20

nm


S. anginosus+ argan oil

S.anginosus


66

Figure 31. Inhibitory effect of Argan oil on S. uberis biofilm formation.

Table 05: Reduction percent of biofilm for test bacteria treated with different concentrations

of Argan oil.

Bacteria

Concentrations

S intermedius

S. haemolyticus

S. mutans

S. anginosus

S. uberis

100

g/mL

93.81 % 100 % 100 % 100 % 78.78 %

50 g/mL 18.05 % 22.64 % 57.57 % 63.36 % 55.11 %

25 g/mL / 44.71 % 6.19 % / 1.44 %

12.5 g/mL / / / / 70.57 %

6.25 g/mL 42.76 % / / / 27.18 %

3.12 g/mL / / / / 2.09 %

1.56 g/mL 24.03 % / / / 21.29 %

0.78 g/mL / / 11.32 % 10.85 % 17.37 %

0.39 g/mL / / / 25.40 % 10.70 %

0.19 g/mL 11.3 % / / 5.32 % /

0.09 g/mL / / / 9.00 % /

0.048 g/mL / / 4.82 % / 11.41 %

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

100 50 25 12,5 6,25 3,12 1,56 0,78 0,39 0,19 0,09 0,048

Bio

film

fo

rma

tio

n a

t 6

20

nm


S. uberis +

argan oil

S. uberis


67

Figure 32. Effect of the first dilution (100 g/mL) concentration of Argan oil on

biofilm inhibition.

0%

20%

40%

60%

80%

100%

93,81% 100% 100% 100%

78,78% R

edu

ctio

n p

erce

nt%


68

DISSCUSION

This research investigated the relationship between dietary intake of L-methionine and

total plasma Hcy concentration (t-Hcy). This (t-Hcy) is a biomarker of the homocysteine-

methionine cycle and dysfunction in this cycle can lead to several cardiovascular diseases. In

other hand, we evaluate the protective and the preventive effect of the powdered seeds of A.

spinosa against HHcy, hyperlipidemia, oxidant status and damages in the aorta, heart and

liver induced by high methionine intake in mice. Also, this study investigated the antibacterial

effect of Argan oil on biofilm formation.

1- Body weight

The first experiment evaluates the effect of L-methionine on the weight. The results

showed that there is no significant difference in weight of mice among the four groups, which

means that the body weight of mice was not affected by L-methionine intake.

Our results are in agreement with (Lamda, 2000) who indicated a stable weight despite

the applied treatment (L-methionine and cholesterol). Another work of (Zarrouk et al., 2010)

who reported that a similar evolution of weight between animals subjected to methionine and

the control group during the first month of the experiment (administration of 70 mg/kg during

6 months in an athero-sensitive model (Psammomys obesus)). However, the study of (Zerizer,

2006) reported an increased in the weight of rats treated with 200 mg/kg of L-methionine for

21 days. Also, the study of (Boudebouz, 2013; Sakhri, 2014) showed an increase in the weight

of mice treated with 400 mg/kg of L-methionine for 3 weeks. However, the work of

(Benmebarek, 2013) indicated a significant decrease in the weight of mice treated with

200mg/kg of L-methionine for the same period. Given the conflicting results, we cannot

conclude that there is a relationship between HHcy and the weight of mice.

2- Effect of treatment on Hcy levels, lipid profile, liver enzyme activities

and antioxidants markers in mice

The current study has shown that high L-methionine diet in mice clearly caused a

significant increase in plasma t-Hcy levels, T-CHO, TG , AST activities, and significant

decrease in HDL-c, reduced glutathione, and catalase activity in liver.


69

Hyperhomocysteinemia results from deficiency of one or more vitamin cofactors,

genetic conditions as reduced activity of enzymes involved in Hcy metabolism, such as CβS

deficiency, deficient activity of methionine adenosyl transferases I and III (MAT I/III), or

non-genetic conditions included liver disease, or high dietary methionine intake (Mudd, 2011;

Hoffman, 2011).

Methionine feeding has often been used to elevate serum and tissue Hcy levels to

study the pathogenesis of HHcys-related metabolic disturbances (Yalçınkaya et al., 2009).

In the present study, only the higher L-methionine dose (500mg/kg) for 21 days of the

experiment, was sufficient to induce HHcy in mice notable by a highly significant increase of

plasma Hcy compared to the normal group; suggesting a detrimental effect of excess dietary

methionine, which may have a practical significance in humans consuming large amounts of

foods high in methionine such as animal proteins (Bhandari et al., 2011).

Acquired results are similar to several studies which have shown that methionine

enriched-diet induced a significant increase of plasma t-Hcy (Mudd, 2011; Viggianoa et al.,

2012; Yamada et al., 2012; Kirac et al., 2013).

This potential effect of high L-methionine diet could possibly be due to up-regulation

in the enzymes that metabolize Hcy, for example cystathione β-synthase. It is clear that tissue

concentration of Hcy is maintained at low levels by regulating production and efficient

removal of this thiol (Stipanuk, 2004).

When methionine is in excess, Hcy is directed to the transulfuration pathway. In this

way, Hcy is converted to cystathionine and then cysteine by the pyridoxal 5-phosphate, the

physiological active coenzyme form of vitamin (B6) dependent enzyme. Cysteine is the rate-

limiting component in the synthesis of glutathione.

Studies have shown that high Hcy concentration could cause oxidative damage to

cells, and cysteine may act as a pro-oxidant agent causing the overproduction of free radicals

and hydrogen peroxide, and may further lead to gene mutation and subsequent development

of cancer (Hogg, 1999; Stipanuk, 2004) and cardiovascular disease.


70

Actually, Hcy can be harmful to cells because it evokes oxidative stress through the

production of ROS, binds to nitric oxide (NO) and produces homocysteinylated proteins, or

led to the accumulation of its precursor, S-adenosyl homocysteine, a potent inhibitor of

biological transmethylations (Kamal and Thanaa, 2009).

In this context, the study of (Heydrick et al., 2004) indicated that high Hcy

concentrations increased formation of ROS and lipid oxidation in endothelial cells but not

vascular smooth muscle cells. Indeed, Hcy induced expression of iNOS and decreased eNOS

expression, which led to a decreased NO bioavailability (Kamal and Thanaa, 2009).

Also, other studies have demonstrated the involvement of Hcy actions linked to

oxidative stress (Streck et al., 2003; Matté et al., 2009). It was previously shown that acute

Hcy administration decreased TRAP,Na+, K+-ATPase and catalase (CAT) activities by 20%,

60% and15%, respectively in hippocampus of rats (Wyse et al., 2002). In addition, it was

recently shown that chronic Hcy administration increased DNA damage and disrupted

antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood plasma

(Matté et al., 2009).

Meanwhile, we found that the diet supplemented with the powdered seeds of A.

spinosa was effective in prevention against HHcy in mice exposed to a L-methionine enriched

diet, indicating that this medicinal plant has the potential to reduce t-Hcy levels in vivo. Some

evidences showed that lowering Hcy by nutrition interventions might offer preventive or

therapeutic benefits against cardio- and cerebrovascular diseases, although controversy still

exists (Spence, 2007; Abraham and Cho, 2010). Several dietary factors, including folate,

vitamins B6 and B12, and betaine have been demonstrated to be effective in decreasing

plasma Hcy level (Lin et al., 2006; Spence, 2007).

Our results are in agreement with (Hamelet et al., 2007; Yalcınkaya et al., 2009 ;

Meng et al., 2013) who reported that quercetin, catechin and taurine supplementation are

effectives in attenuating the increase of the serum Hcy level as induced by a Methionine

enriched diet in rats and mice respectively.

The protective effect of Argan oil is probably due to its high contents of antioxidants,

particularly polyphenols, tocopherols and sterols, which are known as powerful antioxidants

(Masella et al., 2001).


71

As found in the present investigation, mice fed with high Methionine enriched diet

have shown an increase in serum T-CHO, LDL-C, and TG concentrations; however, HDL-C

values were lower.

An association between hyperlipidemia and HHCY has been suggested. Results

obtained are in agreement with the previous experimental studies of (Lee et al., 2002; Obeid

and Herrmenn, 2009; Tselmin et al., 2013; Boudebouz, 2013, Sakhri, 2014). However, it’s

differed from studies of (Zerizer, 2006 and Zerizer et al., 2008) who observed no relationship

between HHcy and lipid status.

Both Hcy and lipids are toxic in vascular cells and hepatocytes, which could indicate

interactions between the two pathways (Obeid and Herrmann, 2009).

Low HDL-c in combination with raised triglyceride levels is considered an

atherogenic lipid profile (Obeid and Herrmann, 2009). In addition, LDL-c elevation in

hyperhomocystenemic rats may be attributed to the reduction in the number of LDL receptor

or reduced LDL binding to its receptor in rats. Changes in LDL- receptor contribute to the

elevation in serum cholesterol levels induced by methionine (Bhandari et al., 2011).

Also, the low methyl group availability decreases the synthesis of

phosphatidylcholine and the secretion of VLDL, causing triglyceride accumulation in the liver

(Li and Vance, 2008; Bravo et al., 2011). It has been reported that severe HHcy, caused by

CβS deficiency, leads to disturbances in the regulation of lipid metabolism and fat

accumulation in the liver (Gaull Get al., 1974; Namekata et al., 2004).

An inverse association between Hcy and lipoproteins, especially HDL, has been well

described in humans (Moat et al., 1999; Poloni et al., 2012) and various animal models of

HHcy (Poloni et al., 2015). It has been reported that HHcy alters intracellular lipid

metabolism (Werstuck et al., 2001) and may be associated with hepatic fat accumulation

(Carvalho et al., 2013). It seems that hypomethylation associated with HHcy is responsible for

lipid accumulation in tissues. HHcy and hypercholesterolemia are linked to the development

of atherothrombotic diseases (Obeid and Herrmann, 2009). However, many studies have

indicated that cholesterol homeostasis is a major mechanism for suppressing cardiovascular

disease (Koyama et al., 2015).


72

HDL is involved in several biological processes that counteract inflammation and

oxidative stress, by beneficially influencing, such as pancreatic beta-cell function, endothelial

vasoreactivity, endothelial apoptosis, restorative processes and monocyte activation as well as

adhesion molecules expression, thus being highly vasculoprotective (Chapman et al., 2011).

The effect of Hcy on HDL is probably related to inhibiting enzymes or molecules

participating in HDL-particle assembly (Obeid and Herrmann, 2009). Therefore, increased

risk for atherosclerosis and dysfunctional HDL particles in HHcy subjects might be related to

low activity of Paraoxonase-1 PON1 (Holven et al., 2008). PON1 is a serum HDL-associated

phosphotriesterase secreted mainly by the liver and shows a Hcy thiolactonase activity

(Domagala et al., 2006) thus protecting from atherosclerosis (Bhattacharyya et al., 2008).

In addition, HDL-c are antiatherogenic lipoproteins implicated in the protection of

LDL against oxidation (Mackness et al., 1993). Low HDL-c attributed to its central function

in the reverse of CHO transport, a process whereby excess cell CHO is taken up and

processed by HDL particles for further delivery to the liver for metabolism (Martinez et al.,

2004). Also the study of (Tselmin et al., 2013) suggests that the decrease in HDL is due to an

inhibition synthesis of Apo AI, the major apolipoprotein of HDL by Hcy.

On the other hand, A. spinosa powdered seeds administration caused a significant

reduction of TG, t-CHO and LDL-c showing the beneficial effect of this plant in the treatment

of the hyperlipidemia. This protection related to the decrease level of Hcy and therefore the

suppression of their cytotoxic effects on different organs. These corrections related to the

antioxidants and anti-inflammatory components of A. spinosa seeds.

Recent experimental study has shown that Argan oil blunted the increases of T-Cho,

LDL-C and TG concentrations in a high-fat diet (Sour et al., 2015).

Virgin Argan oil induces a lowering of the triglycerides level in men (Derouiche et al.,

2005) and of plasma LDL-c in healthy subjects (Drissi et al., 2004; Cherki et al., 2005).

Similar studies have demonstrated that the phenolic extract of Argan oil inhibits

human LDL-c oxidation and increases the cholesterol efflux from human T-helper precursor-1

macrophages (Berrougui et al., 2006). (Drissi et al., 2004) demonstrated that regular

consumption of virgin Argan oil induces a lowering of LDL-c and has antioxidant properties.


73

The decrease in cholesterol concentration could be due to a low intestinal absorption of

cholesterol because of the activity of saponins in Argan oil (Berrougui et al., 2003).

The impact of Argan oil consumption on oxidative stress plasma markers and HDL

paraoxonase 1 (PON1) activity has been evaluated. After three weeks of daily Argan oil

consumption (25 mL/day), plasma PON1 activity, antioxidant vitamins, and LDL

susceptibility to oxidation were measured. A significant increase in PON1 activity and

benficial effects on plasma lipid peroxide, conjugated dienes, and vitamin E concentration

were observed (Cherki et al., 2005).

Epidemiological studies have shown that consumption of food and beverages rich in

phenols can reduce the risk of heart disease by slowing the progression of atherosclerosis

principally by protecting LDL from oxidation (Duffy and Vita, 2003).

Argan oil contains a high amount of Vitamin E but also a non-negligible proportion of

phenolic compounds. The study of (Berrougui et al., 2003; Berrougui et al., 2004) have

previously demonstrated that chronic ingestion of crude Argan oil not only reduces plasma

cholesterol and LDL levels in rats fed with hypercholesterolemic diet , but also improve

endothelial function and prevents high blood pressure.

These effects are principally related to the richness of this oil in oleic and linoleic

acids and tocopherol.

The antioxidant activity of polyphenolics is principally defined by the presence of

orthodihydroxy substituents, which stabilize radicals and chelate metals. The antioxidant

effect of phenolic acids and their esters depends on the number of hydroxyl groups in the

molecule. This antioxidant compound protects against LDL-oxidation (Chen et al., 2004).

Concerning the anti-oxidative parameters, results shown that the content of GSH and

catalase activity in liver tissue was significantly decreased in response to the oral methionine

administration. Our findings are supported by the study of (Sakhri, 2014) who reported a

decrease in GSH levels of mice treated with 400 mg/kg of L-methionine for 21 days. Recently

(Meng et al, 2013) also found that the content of GSH in serum or liver tissue was decreased

in response to quercetin administration in rats fed a Methionine enriched diet.

It was reported that lower level of glutathione, the major intracellular antioxidant, is

accompanied by decreased level of S-adenosylmethionine (SAM), methyl donor in reactions


74

catalyzed methyltransferase and the main metabolic regulator of Hcy synthesis (Li and Vance,

2008; Bravo et al., 2011).

Glutathione is a key buffer of intercellular oxidative reduction reaction, and its

dependent antioxidant enzymes include glutathione S-transferase (GST) and glutathione

peroxidase (GPx), which play a fundamental role in cellular defense against reactive free

radical and other oxidant species (Stipanuk et al., 2006). Organic peroxides can also be

reduced by GPx and GSH S-transferase. Catalase can also reduce hydrogen peroxide but it is

present only in peroxisome. This makes GSH particularly important in the mitochondria in

defending against both physiologically and pathologically generated oxidative stress

(Fernández-Checa et al., 1997; Garcia-Ruiz and Fernández-Checa, 2006). There is

accumulating data that reduced GSH levels occur in many human diseases and they contribute

to worsening of the condition (Ballatori et al., 2009). While, oxidative injury plays a dominant

role in GSH depletion in many of these disorders, some are causally related to reduced

expression of GSH synthetic enzymes (Lu, 2009).

More, hyperaccumulation of methionine sulfoxide in the liver may induce more

serious (oxidative) hepatotoxicity in Cth−/− mice, whose levels of several antioxidative

cysteine metabolites, including GSH, and taurine/hypotaurine, were all downregulated

(Yamada et al., 2012). Hyperhomocysteinemia leads to increased oxidative stress via the

generation of ROS which weaken intracellular antioxidation defense systems or elicit

intracellular redox controlled inflammation responses (Welch et al., 1998).

In this study, we also verified the effect of Hcy on catalase, since this enzyme has the

higher activity among the hepatic antioxidant enzymes (Polavarapu et al., 1998; Kasdallah-

Grissa et al., 2007).

A significant inhibition of hepatic catalase activity was observed in

hyperhomocysteinemic mice. In agreement with our data, other studies suggest a negative

correlation between plasma Hcy levels and catalase activity in liver of rats, pointing a

significant reduction of hepatic antioxidant defenses (Woo et al., 2006; Chanson et al., 2007;

Matté et al., 2009).

In addition, it has been shown that Hcy can directly act on catalase and inhibit the

breakdown of H2O2 by conversion of the enzyme into the inactive form (Milton, 2008). Loss


75

of catalase activity is associated with increased susceptibility to oxidative stress (Góth et al.,

2004; Ho et al., 2004). The mechanism of Hcy inhibition of catalase is shared with a number

of inhibitors including 3-amino-1:2:4:- triazole (Margoliash and Novogrodsky , 1958; Putnam

et al., 2000) and amyloid-ß ( Milton, 1999).

Indeed, we demonstrated that A. spinosa powdered seeds when given in combination

with high L-methionine diet increased not significantly liver GSH level and significantly

catalase activity, indicating its beneficial effect in prevention against oxidative stress in vivo.

In accordance with our results, (Necib et al., 2013) demonstrated that Argan oil treatment

augments the GSH against mercuric chloride induced oxidative stress in experimental rats.

The elevated level of GSH protects cellular proteins against oxidation through glutathione

redox cycle and directly detoxifies reactive species (Ketterer, 1998). Also, our results showed

an increase of catalase activity in (MP) group. These results are in agreement with those of

(Benajiba et al., 2002) who showed that the activities of cytosolic CAT were significantly

higher in Wistar rats treated with Argan oil in comparison with untreated rats.

Argan oil is rich en polyphenols. These compounds have been found to modulate

expression and activity of catalase and eNOS in several tissues, increases catalase activity in

guinea pig cardiac tissue (Floreani et al., 2003), or rat liver (Kasdallah-Grissa et al., 2007),

while red wine polyphenols extract increases phosphorylation of eNOS in porcine coronary

arteries (Madeira et al., 2009) and in aorta and carotid artery of Zucker Fatty rat, a model of

obesity, with an increase of NO bioavailability (Agouni et al., 2009).

For liver enzyme activities, data have shown a significant increase of AST in (M)

group but not ALT values. We believe that the absence of significant differences in ALT

levels in our study could be due to the short period of treatment with Hcy (21 days only).

Liver injury could be identified by serum markers, such as (ALT) and (AST), which

are increased as a result of hepatic necrosis (Ozer et al., 2008). However, increased activity of

AST indicated cardiac disorders (Killip and Payne, 1960).

(Yalcınkaya et al., 2009) have shown that a high methionine diet supplemented for 6

months caused HHcy, and increased serum ALT and AST levels in rats. The increment of the


76

activities of AST and ALT in serum may be mainly due to the leakage of these enzymes from

the liver cytosol into the blood stream (Navarro et al., 1993).

In this context, Hcy has been related to hepatotoxic conditions in numerous reports,

which have showed a positive correlation between HHcy and plasma aminotransferases

activities in clinical and experimental studies (Huang et al., 2001 ; Frelut et al., 2006; Woo et

al., 2006) associated with ROS production and hepatic lipid peroxidation (Huang et al., 2001 ;

Woo et al., 2006).

Previously, we demonstrated that the administration of A.spinosa powdered seeds

increased tissue antioxidant capacity and significantly protected the liver against liver injury

in mice. Simultaneously, the serum activity of AST was significantly decreased, indicating

that a toxic action was displayed with excessive intake of A. spinosa extract crude.

3- Effect of treatment on histology of aorta heart and liver

Histological analysis showed that HHcy induced by the high methionine intake

prompted an angiotoxic activity on the aorta (arch, abdominal and iliac), cardiac and liver

tissue damages. This was observed through the loss and degeneration of endothelium,

formation of foam cells in the different sections of the aorta, alteration of the cardiac muscle,

and liver macrovesicular steatosis.

In our experimental situation, is due to elevated Hcy levels, which perturbs lipid

profile, and decreases tissue level of reduced GSH, and catalase activity, the well known bio-

markers of oxidative stress.

The obtained data in the current investigation were in agreement with the studies of

(Boudebouz, 2013; Sakhri 2014). They reported that the high dose of methionine perturbed

the structures of aorta, heart and liver.

It is belived that HHcy leads to endothelial cell damage, reduction in the flexibility of

vessels, and alters the process of homeostasis (Baszczuk and Kopczynski, 2014).

Homocysteine-induced injury to the arterial wall is one of the factors that can initiate

the process of atherosclerosis, leading to endothelial dysfunction and eventually to heart

attacks and strokes (Gallai et al., 2001; Papatheodorou and Weiss, 2007). Evidence from


77

animal models of HHcy suggest that endothelial dysfunction is largely due to oxidative stress

and decreased bioavailability of NO (Pacher, 2007), NO may protect against the onset of

vascular diseases (Cooke and Dzau, 1997).

Homocysteine promoted oxidative stress through production of reactive oxygen

species (ROS). ROS disrupts endothelial cell integrity, which in turn, can cause endothelial

cell damage predispose affected vessels to the subsequent development of atherosclerosis

(Kanani et al., 1999).

Recently, the study of (Chen et al., 2015) demonstrated that HHcy promotes

atherosclerosis progress. This may be associated with decreased levels of endothelial or aortic

protein S-nitrosylation which plays an important role in the regulation of cardiovascular

functions in nitric oxide (NO) Pathway.

Another possible mechanisms by which Hcy must be contributing to atherogenesis

and thrombosis include increased smooth muscle cell proliferation, cytotoxicity, increased

oxidative stress, stimulation of low density lipoprotein oxidation, induction of endothelial

dysfunction, enhanced coagulability and platelet activation (Willoughby et al., 2002; Luo et

al., 2006).

In addition, in the present study an increase in atherogenic index was found,

consequently of the increase of cholesterol and decrease of HDL-c. This atherogenic index

indicates the disposition of foam cell or fatty infiltration or lipids in heart, coronaries, aorta

liver and kidneys. The higher the atherogenic index, the higher is the risk of the above organs

for damage (Mehta et al., 2003).

Studies in several animal species, including rabbits, baboons, and rats, have

demonstrated desquamation of endothelial cells, fragmentation of the internal elastic lamina,

disruption of elastic fibers, and focal areas of smooth muscle hyperplasia (Rolland et al.,

1995). (Zulli and Hare, 2009) has been reported that the combination of high methionine and

cholesterol increased the alterations of the arterial wall structures and the thickness of the

aortic wall in animal models.

However, in methionine-treated animals, it was shown an aortic angiotoxic action

following to an increase of plasma Hcy levels. (Raghuveer et al., 2001) have reported that,


78

acute elevations in plasma Hcy after methionine loading causes vessel endothelial dysfunction

and this could be reversed by administration of vitamin E in humans.

It has been documented that Hcy can interact with different plasma and cellular

proteins and by forming mixed disulfide conjugates, alters the physicochemical properties of

the affected proteins. This has been also proposed as a potential mechanism for Hcy induced

cellular dysfunction (Barbato et al., 2007).

Accumulating evidence indicates that oxidative stress was the major mechanism of

vascular injury caused by Hcy. The active free sulfhydryl contained in Hcy is prone to self-

oxidation under catalysis of the copper found in blood, forming homocysteine–homocysteine

mixed bisulfide. In this course, many series of oxygen reactions with associated toxic effects

could be produced, including the formation of hydrogen peroxide (H2O2), superoxide anion,

and the hydroxyl radical. This induced membrane lipid peroxidation, directly resulted in

chemical injury of vascular endothelial cells (VEC) and impaired endothelial function

(Nygard et al.,1997). Then, by carboxyl methylation, Hcy decreased activity of membranes.

Accordingly, Hcy inhibited the cell cycle of VEC, decreased the regeneration capacity of the

endothelium and induced endothelial dysfunction (Lee and Wang, 1999). Therefore, the

supplementation with antioxidants like the crude extract of A.spinosa to reduce some of the

prooxidant activity of Hcy is beneficial to alleviate endothelial injury

In addition to early cardiovascular events, patients with homocysteinuria develop

hepatic steatosis or ‘‘fatty liver” which is characterized by enlarged, multinucleated

hepatocytes containing microvesicular lipid droplets (Mudd et al., 1995), which can progress

into hepatocellular carcinoma (Dara and Kaplowitz, 2011). It has been reported that Hcy

elicits hepatic damage in experimental models by three mechanisms: oxidative stress,

endoplasmic reticulum stress, and activation of proinflammatory factors (Ji and Kaplowitz ,

2004; Robert et al., 2005). (Woo and Siow, 2008) showed that HHcy, induced by high-

methionine diet for 4 weeks, elevated the expression and the protein synthesis of monocyte

chemoattractant protein-1 (MCP-1) in plasma and in liver tissue homogenate, due to

hepatocyte production, suggesting that Hcy may contribute to chronic inflammation in this

organ studied hyperhomocysteinemic CβS-deficient mice and demonstrated foci of

perilobular mononuclear inflammatory infiltrate around the vessels.


79

Also, (Ji and Kaplowitz, 2004; Matté et al., 2009) showed that HHcy increased

mediators of inflammation, such as nuclear factor kappa B NFκB, interleukin (IL)-1b, IL-6

and IL-8 in liver.

We also observed the disruption of the elastic lamina and disorganization of elastic

fibers. It has been reported that the disappearance of the aortic elastic lamina might be due to

production of elastase, a serine protease, by increased number of SMCs induced by Hcy

(Jourdheuil-Rahmani et al., 1997; Zulli et al., 1998).

In addition, A.spinosa powdered seeds treatment was found to decrease highly and

significantly serum AST activities and to ameliorate histopathologic changes in the liver,

heart due to the high-fed methionine diet, and prevents the endothelial alteration as shown by

the morphological data. The study of (kumar et al., 2010) suggested that an increase intake of

antioxidants appeared to be protective in cardiovascular diseases. Also, epidemiological

studies have shown that consumption of food and beverages rich in phenols can reduce the

risk of heart disease by slowing the progression of atherosclerosis principally by protecting

LDL from oxidation (Duffy and Vita, 2003).

The work of Zerizer & Naimi (2004), and Benmebarek et al., (2013) proved that , the

administration of L-methionine (200 mg/kg) to rats and mice respectively during 21 days,

could damage the aorta and heart tissue and the treatment of these animals with vitamins B9,

B12 , B6 and Stachys mialhesi extract respectively corrected these alterations.

Another work of (Zerizer et al., 2008) established that higher level of Hcy could

stimulate the angiogenesis on the aorta of rats, and the treatment with the extracts of

medicinal plants Stachys mialhesi and Chrysanthemum Macrocarpum could inhibit the

angiogenesis.

4- Anti- biofilm effect of Argan oil

Bacterial infectious diseases represent an important cause of morbidity and mortality

worldwide. Therefore, the development of new antimicrobial agents for the treatment of

bacterial infections is of increasing interest.

Normally, bacterial infectious diseases could be assumed as being a “biofilm type of

infection” (Draelos, 2010), and recent research has shown that generally these bacteria


80

structured in a sessile form are more resistant to various antimicrobial treatments (Olsen,

2015).

One of the most important characteristic of biofilms is their increased tolerance to

antimicrobial agents (Wimpenny et al., 2000). The presence of exopolysaccharides, which

accounts for a majority of the dry mass of the biofilm, is known to act as a mechanical barrier,

thereby diminishing the effectiveness of various antibiotics (Stewart, 1996). Moreover, the

negatively charged polymers present on the surface of the biofilm matrix are known to

interact with positively charged antibiotics (such as aminoglycoside), thereby

limiting/slowing the penetration of these drugs (Nichols et al., 1988).

Additionally, altered environmental conditions may trigger differential oxygen

concentration, such as the surface of the biofilms may show higher oxygen levels, whereas a

scarcity of oxygen may be observed toward the core of the biofilms, thereby making the core

less accessible to the antibiotics (De Beer et al., 1994; Yang et al., 2008).

Biofilms exhibit regions of oxygen limitation and nutrient deficiency that, in contrast

to planktonic cultures, are non-uniformly distributed (Xu et al., 1998). Bacteria located

within starvation zones may be metabolically dormant and thus it has been suggested that

growth-dependent changes in cellular processes may contribute to reduced sensitivity to many

classes of antimicrobial agents (Borriello et al., 2004 ; Field et al., 2005).

Many bacteria are known to regulate their cooperative activities and physiological

processes through a mechanism called quorum sensing (QS), in which bacterial cells

communicate with each other by releasing, sensing and responding to small diffusible signal

molecules. A quorum sensing mechanism, including symbiosis, formation of spore or fruiting

bodies, bacteriocin production, genetic competence, programmed cell death, virulence and

biofilm formation (Li and Tian, 2012).

Many naturally occurring compounds found in plants, herbs, and spices have been

shown to possess antimicrobial functions and serve as a source of antimicrobial agents against

pathogens (Deans and Ritchie 1987; Kumar et al., 2006).


81

Thus, the antibacterial agents derived from the natural source (plants) may serve as an

effective alternative, due to the presence of secondary metabolites, which are known to enjoy

selectional advantages against the resistance organisms (Butler and Buss, 2006).

In this study, the anti-biofilm formation of the Algerian Argan oil was assessed against

5 species of bacteria, that belong to Gram positive (Streptococcus mutans, Streptococcus

anginosus, Streptococcus intermedius, Streptococcus uberis and Staphylococcus

haemolyticus) strains.

The results obtained with the broth microdilution test showed for the first time, that

there was a trend of increasing inhibition of S. intermedius, St haemolyticus, S. mutans and S.

anginosus biofilm formation as the Argan oil was getting more concentrated.

The first one (100 µg/ml) of Argan oil was effective to remove S. uberis biofilm but

not to kill the cells. However, the highest effect of this oil was on S. intermedius, St

haemolyticus, S. mutans, S. anginosus biofilms with reduction percent greater than 90%.

The inhibitory activity produced by the Argan oil could be due to

the presence of individual phytochemicals, which are active against this strains of bacteria or

could be as a result of the synergistic effect of two or more phytochemicals that are

contained in the oil (Da Silva et al., 2016). The Argan oil was found to display antibiofilm

properties, probably by the richness of natural phenols such as: caffeic acid, vanillic acid,

tyrosol, epicatechin …. (Charrouf and Guillaume, 2007).

Argan oil has been confirmed to contain phytochemicals such as steroids,

tannins, flavonoids and saponins (Charrouf and Guillaume, 2007). And these phytochemicals

have been demonstrated to associate with bacterial proteins and inhibit microbial adhesion,

enzymes, cell envelop and transport proteins (Samy et al., 2010; Upadhyay et al., 2014). It

is, therefore, likely that the Argan oil inhibited biofilm formation through some of these

mechanisms. Bacterial adhesion is important during biofilm formation (Rabin et al., 2015)

and agents that disrupt bacterial adhesion to surfaces have the potential to act as anti-biofilm

agents.

In this context, the study of (Stojkovic et al., 2013) demonstrated a good antioxidant

and antimicrobial activity of caffeic acid. Also, a number of polyphenols (hydroxycinnamic


82

acid, rutin, epicatechin) have been found to block quorum sensing in Chromobacterium

violaceum (Nazzaro et al., 2013).

The microbial biofilm inhibition can be managed either by preventing the attachment

of the organism to the surface or by breaking the structure of the biofilm

if they formed (Gupta et al., 2016).

Another explanation that Argan oil might have contained antibacterial phytochemicals

that stimulate a stress response in the biofilm. Several studies have demonstrated that when

microbes are stressed, such as under antibiotic treatment, genes associated with biofilm

formation are stimulated and the bacteria convert to the biofilm phenotype (Ackart et al.,

2013).

Recently, Lotfi et al., (2015) revealed that Argan oil (extracted from the kernels of the

Argan tree of Bechar, Algeria ) has shown a positive effect on resistant planktonic bacteria

studied in particular staphylococcus aurus and staphylococcus white.

According to our finding, spices such as garlic (Bjarnsholt et al., 2005), ginger,

cinnamon (Niu et al., 2006) , clove (Khan et al., 2009), cumin (Packiavathy et al., 2012) and

turmeric (Packiavathy et al., 2014) have been found to display QS-mediated biofilm

inhibitory properties.

The findings suggest that curcumin derived from C. longa significantly inhibits the

formation of biofilm by reducing exopolysaccharide as well as alginate production, probably

by interfering with the signal molecules of the QS system. Studies have also shown that

curcumin enhances the susceptibility of the organism to conventional antibiotic (Packiavathy

et al., 2014).

Zingerone, one of the main chemical constituents of ginger (Zingiber officinale),

displays the ability to inhibit as well as eradicate biofilms formed by P. aeruginosa. In a study

conducted with ginger extract, pretreatment with the extract effectively reduced the

production of EPS (Kim and Park, 2013). Zingerone also increased the susceptibility of the

organism to conventional antibiotics, when used in combination with the standard antibiotics

such as ciprofloxacin (Kumar et al., 2013).


83

Garlic (Allium sativum) was found to block QS mediated biofilm formation and

virulence development by inhibition of LuxR-type receptor (Prigent-Combaret et al., 1999)

Essential oils are aromatic oily liquids from plant materials and are well known for

their antibacterial properties (Burt, 2004). A variety of researches have shown that the

biofilm could be removed effectively by essential oils for example essential oils of Piper

bredemeyeri, Piper brachypodom, Piper bogotence, Gaultheria procumbens L., Achillea

millefolium (yarrow), Syzygium aromaticum (clove), Coriandrum sativum (coriander),

Cinnamomum verum (cinnamon), thyme and Origanum vulgare (oregano) showed inhibitory

effects on biofilm formation at sublethal concentrations (Khan et al., 2009; Musthafa et

al.,2010; Olivero et al., 2011; Jadhav et al., 2013)

Traditional Chinese medicinal herbs have also been screened for antibiofilm properties

against microbes like C. violaceum and P. aeruginosa (Koh and Tham, 2011).

Also, Alcoholic extracts of some medicinal plants of Egypt were investigated for anti-

QS activity against C. violaceum (Zaki et al., 2013). Findings also reveal information about

the effectiveness of some plants (leaves of Adhatoda vasica Nees, Bauhinia purpurea L.,

Lantana camara L., Myoporum laetum G. Forst., the fruits of Piper longum L., and aerial

parts of Taraxacum officinale) against the QS system (Zaki et al., 2013).

The microorganisms used in this study are generally known to be a pathogen of dental

caries. In this context, infections, including those caused by oral bacteria, are more likely

involved in CVD progression than previously thought. Many studies have established a

plausible link between oral bacteria and atherosclerosis, which is extremely complex and it’s

highly likely that more than one mechanism is involved (Leishman et al., 2010).

Endocarditis is the interaction between the surfaces of the

endothelium and bacteria. Though the early association is weak, but with the advent of any

wound, the microbes turn opportunistic and form a strong biofilm-aided association which can

damage heart valves (Kokare et al., 2009). The organisms can primarily enter into the blood

stream through oropharynx, genitourinary tract and gastrointestinal tract. Generally, the

adherence of microbes to the intact endothelium is very poor, but in case of a wounded or

damaged epithelium, a condition namely nonbacterial thrombotic endocarditis develops


84

(Kokare et al., 2009). In this condition, the red blood cells, platelets and fibrin accumulate at

the site of injury. Endothelial cells secrete fibronectin which have the ability to bind to

collagen, fibrin, human cell as well as bacteria. Microbes like Staphylococcus and

Streptococcus sp. have fibronectin receptors which can form biofilms on the site of injury as

well as damage the tissue of the valves (Kokare et al., 2009). Therefore, an invasion of

endothelial cells by oral bacteria may lead to changes in the proinflammatory and

proatherogenic properties of endothelial cells as well as programmed cell death, all of which

are indicative of endothelial dysfunction (Leishman et al., 2010).

Through the results obtained, Argan oil can be used in the manufacturing of the herbal

toothpaste, which prevents cardiovascular disease indirectly.

Conclusion

and

Perspectives

Conclusion and perspectives

85

The purpose of this study was to induce hyperhomocysteinemia by administration of

high dose of L-methionine (500mg/kg) during 21 days in an in vivo animal; therefore evaluate

the protective and preventive effect of the powdered seeds of A.spinosa against the metabolic

and structural disorders induced in L-methionine treated mice. On the other hand, investigated

the antibiofilm effect of Argan oil againt 5 species of bacteria. These bacteria can lodge on

heart valves and cause infection of the endocardium.

The current study has shown that high L-methionine diet clearly caused some metabolic

disorders manifested by:

Hyperhomocysteinemia.

Hyperlipedemia with an increase in the total lipid rate: the total cholesterol and

triglycerides.

Dyslipoproteinemia with a decrease in HDL-c and an increase LDL-c.

An increase in the atherogenic index, consequently of the increase of cholesterol and

decrease of HDL-c.

Anti-oxidative disorder by the depletion of the reduced glutathione and the catalase

activity.

Increase of liver enzyme especially AST.

In addition, histology analyses showed cellular damages of aorta, heart and liver

characterized by:

Loss and degeneration of endothelium.

Formation of foam cells in the different sections of the aorta.

Formation of lipids droplets in aorta and liver.

Disappearance of elastics fibers especially in the arch aorta sections.

Alteration of the cardiac muscle.

Destruction of membrane cells, hepatocellular ballooning, hypertrophy of

some hepatocyte nuclei, cytoplasmic vacuolization, and macrovesicular

hepatic steatosis.

Conclusion and perspectives

86

Meanwhile, A diet enriched with (150mg/kg/day) of Algerian Argania spinosa

seeds was effective in preventing the increase of these metabolic disorders, endothelial

alteration, and the heart and liver damages induced by methionine enriched diet in mice.

Also, Considering the results obtained from the in vitro study, we can conclude that

the Algerian Argan oil was able to efficiently reduce biofilms formation of (Streptococcus

mutans, Streptococcus anginosus, Streptococcus intermedius and staphylococcus

haemolyticus) strains, when oil was getting more concentrated.

This preventive protection of Argania spinosa (seeds and oil ) might be related to its

content of many antioxidants and anti inflammatory compounds such as tocopherols (vitamin

E), phenols (caffeic acid, oleuropein, vanillic acid, tyrosol, catechol, resorcinol,

epicatechin and catechin), carotenes, squalene, and fatty acids, (80% unsaturated fatty acids).

Based on the findings of this study our future work and perspectives can evaluate

many topics:

Purification and determination of the bioactive molecules presented in the

powdered seeds of Argania spinosa.

Determination of antioxidant enzyme superoxide dismutase and glutathione-s-

Transferase.

Study the gene expression of antioxidant enzymes.

Determination of pro-inflammatory cytokines in mice administered with high

dose of L-Methionine and treated with Argania spinosa.

Isolate the specific antibacterial principles in Argan oil.

Determine the activity of the plant extract on other types of bacteria as

staphylococcus aurus, in addition to the synergistic activity of this medicinal

plant with antibiotics.

https://en.wikipedia.org/wiki/Tocopherol

https://en.wikipedia.org/wiki/Caffeic_acid

https://en.wikipedia.org/wiki/Oleuropein

https://en.wikipedia.org/wiki/Vanillic_acid

https://en.wikipedia.org/wiki/Tyrosol

https://en.wikipedia.org/wiki/Catechol

https://en.wikipedia.org/wiki/Resorcinol

https://en.wikipedia.org/wiki/Epicatechin

https://en.wikipedia.org/wiki/Catechin

https://en.wikipedia.org/wiki/Carotene

https://en.wikipedia.org/wiki/Squalene

https://en.wikipedia.org/wiki/Fatty_acid

https://en.wikipedia.org/wiki/Saturated_fat

https://en.wikipedia.org/wiki/Fatty_acids

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الملخص بالعربية

الملخص

1

المقدمة

عندما الحظ "الشرايينتصلب " قترح فرضية إول من أهو McCully كان 9191في عام

ين يعانون من تراكم ذطفال ال أللدى ا طراف ألشرايين الدماغ و ا تجلط شرايين القلب التاجية و

.ستقالب الميثيونينإمتعلق ب جيني أنتيجة خط ،ينيبيلة الهوموسيست يسمى و ماأ ين في البوليالهوموسيست

ا زاد تركيزه عن ذإلكن ،الخالياو في بالزما الدميوجد ميني طبيعيأن هو حمض يالهوموسيستي

تدمير الجدار الداخلي المبطن إلىدي ؤمما ي ،ن يسبب مشاكل صحيةأمعدله الطبيعي في الدم يمكن

. وعية الدمويةأليعتبر عامل خطر بالنسبة للقلب و ا لك ذل ،صابة بتصلب الشرايينإللى درجة اإللشرايين

إلىالتي تودي حد العوامل أين هو يرتفاع مستويات الهوموسيستإن خدش بطانة الشرايين بسبب إ

ختالل في وظيفة بطانة إلي إدي بدوره ؤمما ي ، atherosclerosis بـو ما يعرف أين بداية تصلب الشراي

.النوبات القلبية و السكتتات الدماغية إليا ما يتسبب في نهاية المطاف ذو ه الشرايين

رتفاع إناتج عن يعتبر عامل مهم في تصلب الشرايين كسدي أجهاد التإلن اإف، لكذلى إضافة إلبا

غشية و ألكسدة دهون و بروتينات األى إدي ؤمما ي ،كسدةألنخفاض مضادات اإ، ينعكس بن يالهوموسيستي

. ة داخل العضويةبيلتهاإلنتاج العوامل اإزيادة

يعتبر عنصر شديد الذيلى ثيوالكتون و إن يتحول أن ييخرى، يمكن للهوموسيستأمن جهة

بروتيناتو التي توثر بدورها على NH-COضافية إمما يسمح بتشكيل معقدات بروتينات الالتفاعل مع

.نزيمات الجسمإو

صابة بأمراض إللتهابات المزمنة تزيد من خطر اإلن بعض اأخرى تشير الدراسات أمن جهة

.تصلب الشرايين فيو غير مباشرة أليات مباشرة آفبعض الجراثيم تساهم ب ،وعية الدمويةالقلب و األ

هي عبارة عن مستعمرات من الكائنات الحية الدقيقة بحيث تكون غشية الحيويةو األأ البيوفيلم

و ما يطلق عليه عديد أفراز نسيج خارجي إبو تتسم ، ملتصقة ببعضها البعض و مرتكزة على سطح

. extracellular polymeric substanceالسكاريديد الخارجي

لتهابات الشغاف المعدية، و إالكيسي، التليف مراض المزمنة مثل ألترتبط البيوفيلمات عادة مع عديد ا

جل تطوير مقاومة بكتيرية بالمضادات الحيوية أا الصدد وجب البحث من ذفي ه ،لخإ....الجروح المزمنة

.مواد الطبيعيةالمستخلصة من ال

الملخص

2

هاما في اكمصدر للمركبات الطبية، و التي لعبت دور النباتاتعتبرت أالعصور القديمة ذمن

ستخدام المكونات الفعالة في الطب إن إف نظمة الصحة العالمية مووفقا ل ،نسانإلالمحافظة على صحة ا

دوية ألمن ا % 08كثر من أن أكما ،من سكان العالم %08 لى عالج ما يقاربإدت أالتقليدي الشعبي

.صل نباتي طبيعيأالحديثة من

Arganiaثير الفعال و المفيد لنبتة طبية جزائرية ألى التعرف على التإه الدراسة ذتهدف ه

spinosa لى عائلةإو التي تنتمي Sapotaceae، ور شجرة ذستخدام المستخلص الخام لبإب سواء

ه النبتة ذن لهأثبتت أستنادا للدراسات السابقة و التي إا ذو ه ،ورذه البذو الزيت المستخلص من هأ رغانألا

لتهابات، مضادة لتكاثر الخاليا إلمخفضة للشحوم و الكوليسترول، مضادة ل: فوائد عالجية على غرار

.لخإ.....السرطانية

: ه الدراسة في ذهداف هأا المنطلق تتمحور ذو من ه

جرعات عالية من باستخداملك ذين في الدم و يالتحفيز على تكوين حالة فرط الهوموسيست

.الميثيونين لدى الفئران

ه الجرعات على وزن الفئرانذتقييم تأثير ه.

لبيوكيميائية مثل قياس ه الجرعات العالية من الميثيونين على بعض التحاليل اذتقييم تأثير ه

، الكوليسترول، البروتينات الدهنية(Triglycerides) ، الدهون الثالثية (Hcy) ن الهوموسيستيي

نزيمات الكبدية إل، ا(HDL-c)، البروتينات الدهنية عالية الكثافة (LDL-c)منخفضة الكثافة

(ALT, AST)مثل كسدةأللى مضادات اإضافة إل، با(Gluthatione ) نشاط الكاتاالز و

Catalase activity.

ورطي و ألالشريان ا ،لك على المقاطع النسيجية لكل من القلبذه الجرعات كذختبار تأثير هإ

.الكبد

ين و باقي التحاليل البيوكيميائيةيرغان على الهوموسيستألور شجرة اذتقييم المستخلص الخام لب.

القلب، الشريان األورطي ) رغان على المقاطع النسيجيةألور شجرة اذتقييم المستخلص الخام لب

(.و الكبد

نواع من البكتيريا و التي قد تسبب مشاكل أ 0ان ضد رغألتقييم المفعول المضاد للبيوفيلم لزيت ا

.وعية الدموية ألللقلب و ا

الطرق و الوسائل المستعملة

:المستخلصات النباتية

الملخص

3

( نرغاألستخدام نبتة طبية جزائرية معروفة في الطب الشعبي و هي اإه الدراسة تم ذخالل ه

(Argania spinosa رغان و الزيت المستخلص ألور شجرة اذالمستخلص الخام لبستعمل كل من أو لقد

.ورذه البذمن ه

:الحيوانات

تم الحصول عليها Mus musculusه الدراسة هي فئران تجارب من نوع ذالحيوانات المستعملة في ه

عطيت فترة للتأقلم مع ظروف أن أبعد ما التجارب فأجريت على الحيوانات أ ،من معهد الصيدلة بقسنطينة

.بل كل تجربةق ألمخبريالعمل

:التجارب

مجموعات بحيث تحتوي كل 4لى إنقضاء فترة التكيف، تم تقسيم الفئران إبعد : ولىألالتجربة ا

:و عوملت كما هو موضح في الجدول التالي ،حيوانات 7مجموعة على

(ميو 19لكل مجموعة لمدة 7: عدد الحيوانات ) .المعاملة التجريبية للفئران : 10الجدول

المجموعة التجريبية

المادة المستخدمة

الجرعة اليومية

(F)المجموعة

مسحوق الدقيق اللين رأف/ مغ 0.5

(M) المجموعة

+مسحوق الدقيق اللين

الميثيونين

+ فأر /مغ 8.0

كغ/مغ088

(MP) المجموعة

+اللين مسحوق الدقيق

+ الميثيونين

(م خ ب ش أ)

+فأر /مغ8.0

(+ميثيونين)كغ /مغ088

كغ /مغ908

(م خ ب ش ا )

(P ) المجموعة

+مسحوق الدقيق اللين

(م خ ب ش أ)

+فأر فا/مغ 8.0

كغ/مغ908

(م خ ب ش ا)

الملخص

4

OD * 1*1.525

GSH (nmol/mg of protein) =

13100*0.8*0.5.mg protein

.ور شجرة األرغانذالمستخلص الخام لب(: أم خ ب ش )

ن ييهوموسيستالخد عينات من الدم لقياس كل من أتم ،(سابيع أ 3) التجريبيةالمدة نقضاء إبعد

(Hcy) الدهون الثالثية ،(Triglycerides) الكوليسترول، البروتينات الدهنية منخفضة الكثافة ،

(LDL-c) البروتينات الدهنية عالية الكثافة ،(HDL-c)نزيمات الكبدية إل، ا(ALT, AST)، كما تم

لى تجميد إروطي و الكبد للدراسة النسيجية باإلضافة ألتخدير الفئران ألخذ عينات من القلب و الشريان ا

Catalase ))نشاط الكاتاالز و ( Gluthatione)كسدة مثل ألجزء من الكبد لمعايرة مضادات ا

activity في المسحوق الخلوي الكبدي.

:التحاليل البيوكيميائية -0

بروتينات الدهنية منخفضة ن ، الدهون الثالثية، الكوليسترول، الييقياس كل من هوموسيستتم

بن إ" نزيمات الكبدية في مخبر التحاليل البيولوجية إل، البروتينات الدهنية عالية الكثافة، و االكثافة

.بقسنطينة" سينا

:كسدة ألمعايرة نشاط مضادات ا -2

قياس الجلوثاثيون المختزل:

لقياس كمية الجلوثاثيون المختزل في الكبد، اتبعت طريقة (Weckbeker و األخرون

. في ذلك( 9100،

بعد إنقضاء فترة التجربة، تم تخدير الحيوانات و تشريحها لنزع الكبد بصفة فورية، في

حالته الرطبة، بعد ذلك تم إستخدام مسحوق الكبد المتجانس لمعايرة كمية الجلوثاثيون

:حسب المعادلة التالية DTNBختزل طيفيا بإستعمال كاشف التلوينالم

:حيث

OD : الكثافة الضوئية.

لى خط إجراء مقارنة إعن طريق ( Bradford) ،9179د تركيز البروتينات بواسطة طريقة تم تحدي -

.BSAالمعايرة

الملخص

5

تاالزقياس نشاط الك:

(Aebi ،9104)نزيم الكتاالز وفق طريقة إمسحوق الكبد المتجانس كذلك لمعايرة نشاط أستخدم

الطريقة ،كسجينأجزيئة ماء و لى إ H2O2 نزيم له القدرة على تحليل بروكسيد الهيدروجينإلا اذفه،

: المتبعة موضحة في الجدول التالي

:12الجدول

µL))العينات µL))الشاهد

الكواشف

088 718 , µM محلول الفوسفات

pH=7.5)988 (

188 188

H2O2

- 98

السائل الطافي لمسحوق الكبد

المتجانس

:كما هو موضح في العالقة التاليةنزيم إلبعدها تم تقدير فعالية ا ،تم قياس نشاط الكتالز طيفيا

:حيث

OD : الكثافة الضوئية

الملخص

6

ε :متصاصية الموليةمعامل اإل (0.043 mM-1.cm -1) .

L :طول كوفيت المستعملة (1 cm). .

X :البروتين كمية (mg/ml).

Fd : التخفيف عامل.

:تحضير القطاعات النسيجية

روطي و جزء من الكبد ووضعها في قارورات ألخد كل من القلب و الشريان اأبعد تشريح الفئران تم

.بغرض تثبيتها % 98بـ صغيرة تحتوي على الفورمول المخفف

مدة % ) 19، %78، %08) يثانول بتراكيز متزايدة إلحواض كحول اأستخدام إلك تم نزع الماء بذبعد

(.مرات 3تعاد كل مرحلة ) د 38كل مرحلة حوالي

د في كل 98لمدة (xylène)بعدها وضعت العينات في البوتانول، ليتم غمرها الحقا مرتين في زيلين

.مرة

مع تغيير البرافين كل و نصف ساعات 4ما في المرحلة الموالية، طمرت العينات في شمع البرافين لمدة أ

.(Hematoxylin eosin) ها خير قطعت العينات بالمقطاع المجهري ليتم تلوينألو في ا ،ساعة و نصف

:التجربة الثانية

: البيوفيلم تشكل رغان علىألتأثير زيت ا

:السالالت المستخدمة

:وهي نواع من السالالت البكتيرية أ 0ستعمال إه الدراسة تم ذفي ه

Staphylococcus haemoliticus

Streptococcus intermedius

Streptococcus anginosus

Streptococcus mutans

Streptococcus uberis

(.إيطاليا) تم الحصول على هذه السالالت من مخبر الميكروبيولوجيا بـكالياري

الملخص

7

و SH agar, Muller-Hinton agar : زرعت كل بكتيريا على حدة في وسط زرع المناسب -

بالنسبة للبكتيريا ساعة 14ثاني أكسيد الكربون لمدة %0و 37°حضنت في درجة حرارة

( Staphylococcus haemoliticus)اما البكتيريا الهوائية . (Streptococcus) الالهوائية

.فحضنت في وسط هوائي

أي وسط زرع سائل يحتوي على بكتيريا، و تم inoculum)) إينوكولوم بعد ذلك تم تحضير -

98الحصول على تركيز 9

و ذلك بإستخدام قياس مل لكل نوع بكتيري، /خلية بكتيرية

(.نانومتر 918) اإلمتصاصية على الطول الموجي

حيث وضعت : كما يلي well microplates-96 ))فيما بعد قمنا بعمل التجربة التالية على -

100 L 100من وسط الزرع و L ليتم بعدها اجراء .من زيت االرغان في الحفرة االولى

من L 988بعدها تمت اضافة ، (g/ml 988الى 8.84من )سلسلة من التخفيفات

. الى كل الحفر االنوكولوم

رت حففي حين احتو ،على وسط الزرع مع االنوكولوم( positive control) احتوت حفر -

( negative control ) على وسط الزرع مع زيت االرغان فقط.

كسيد الكربون لمدة أثاني % 0و 37° درجة حرارة في well microplates-96))ن يحضتتم -

.و في وسط هوائي بالنسبة البكتيريا الهوائية بالنسبة للبكتيريا الالهوائية ساعة 40

.PBSلك ذزالة إليتم فيما بعد PBSثالث مرات بمحلول و غسلت الحفر زيل محتوىألك ذبعد -

،د 98لمدة % 8.9تركيزها ) Crystal violet ( بصبغةالخطوة الموالية تمثلت في تلوين الحفر -

.زالة الصبغةإ بعدها تمت

ستخالص الصبغة الملتصقة بالحفر إنتاج المادة المخاطية نوعيا، تم إولتقدير قدرة البكتيريا على -

.لكل حفرة % 38و تركيز ذمن حامض الخليك (µl)188بإضافة

.نانومتر 918الطول الموجي متصاصية علىإلو من ثم قياس ا -

:تم استعمال العالقة التالية ، و لحساب نسبة تثبيط البيوفيلم -

الملخص

8

:حيث

C :امتصاصية positive control

B :امتصاصية negative control

T :امتصاصية test wells

النتائج المتحصل عليها

و أ( كغ/مغ088)جرعة الميثيونين بأخذسواء يتأثرن وزن الحيوانات لم أولية ألبينت النتائج ا

خالل (F ,M, MP, P)ربعة ألبين المجموعات ا ،(كغ/مغ908) رغانألور شجرة اذالمستخلص الخام لب

. (93)الفترة التجريبية كما هو موضح في الشكل

المنخفض ن كل من الكولسترول و الكولسترول أظهرت النتائج أبالنسبة للتحاليل البيوكيميائية فقد

البروتينات الدهنية عالية الكثافة ما أرتفعت بقيمة معتبرة ، إقد (TG)و الدهون الثالثية (LDL-c)الكثافة

(HDL-c) لك لدى المجموعة الثانية ذنخفضت بقيمة معتبرة كإفقد(M) مقارنة بالمجموعة الشاهدة(F).

(TG)و الدهون الثالثية (LDL-c)خرى، عرفت مستويات كل من الكولسترول أمن جهة

جرعات عالية من الميثيونين عطيتأ، و التى (MP)نخفاضا بقيمة معتبرة لدى المجموعة الثالثة إ

،ا المستخلصذمما يثبت فعالية ه (كغ/مغ908) رغانألور شجرة اذو المستخلص الخام لب( كغ/مغ088)

(.97، 99، 90، 94)شكال ألا

(M)رتفاعا معتبرا لدى المجموعة الثانية إعرفت (AST)نزيمات الكبد إن ألى إتشير النتائج

ن أفي حين ،ي تغير ملحوظألم يشهد ALT) (نزيمإ، بينما (F , MP, P)خرى ألا مقارنة بالمجموعات

ن المستخلص أمما يدل على ، بقيمة معتبرة (AST) نخفضت لديها مستوياتإ (MP)المجموعة الثالثة

(.91،20)الشكلين ،نزيمات الكبدإرغان له دور في تعديل ألور شجرة اذالخام لب

رتفعت بقيمة إن في الدم قد ين مستويات الهوموسيستيألى إلك ذتبين النتائج المحصل عليها ك

ا ما قورنت مع إذ( كغ/مغ088)عطيت جرعات عالية من الميثيونين أمعتبرة لدى المجموعة التي

بينما المجموعة التى تلقت معالجة مضاعفة بالميثيونين و . (F , MP, P)خرى ألالمجموعات ا

ا ذين، و هيمعتبرا في مستويات الهوموسيست نخفاضا إشهدت فقد رغانألور شجرة اذالمستخلص الخام لب

(. 90)الشكل ،ا العاملذرتفاع هإا المستخلص في الوقاية والحد من ذما يثبت نجاعة ه

الملخص

9

ن كمية الجلوثاتيون المختزل في أكسدة، فقد كشفت النتائج المتحصل عليها ألبالنسبة لمضادات ا

إذا، (M)نخفاظا ملحوظا بقيمة معتبرة لدى المجموعة الثانية إنزيم الكتالز قد عرفا إلك نشاط ذالكبد و ك

رتفاعا إفقد سجلت (MP)ن المجموعة أفي حين ،(F MP, P) األخرىما قورنت بالمجموعات

رتفعت لكن ليس بشكل إما كمية الجلوثاثيون لدى نفس المجموعة فقد أ، معتبرا بالنسبة إلنزيم الكتالز

(.11و 19)الشكلين ،(يوم 19)ن المدة التجريبية كانت غير كافية ألملحوظ، ربما

، األخرىما قورنت مع المجموعات إذا ن تحليل المقاطع النسيجية لفئران المجموعة الثانيةإ

نتكون خاليا رغوية في الطبقة الوسطى للشريا كذلكروطي ، ألتقشر البطانة الداخلية للشريان ا يظهر لنا

عضلة القلب تخريبا في بعض المناطق باإلضافة شهدت بينما ،مع تمركز بعض الدهون في حويصالت

خر تغيرات آل، عرف نسيج الكبد هو ا أخرىمن جهة .خالياه الذهتغيير في البنية المورفولوجية ل إلى

نوية، ظهور حويصالت أللبعض الخاليا الكبدية، تضخم بعض اشية البالزمية غألتمثلت في تخريب ا

(. 17، 19، 10، 14، 13)شكال األ .قاطع النسيجيةالمكما هو موضح في ،ليبيدية

تم تغذيتها يروطي و القلب و الكبد لدى المجموعة التألبينما المقاطع النسيجية لكل من الشريان ا

. رغان و الميثيونين معا فقد ظهرت بشكل سليمألور شجرة اذبالمستخلص الخام لب

بالنسبة لألنواع رغان فعاليته في القضاء على تشكل البيوفيلمألزيت اثبت أخر، آفي سياق

: البكتيرية التالية

Staphylococcus haemoliticus

Streptococcus intermedius

Streptococcus anginosus

Streptococcus mutans

و 39، 38، 11، 10)شكال ألا ا بوضوح فيذيتجلى لنا ه ،رغانألتوافقا مع زيادة تركيز زيت اا ذو ه

31.)

الملخص

10

:المناقشة

:خذ جرعات عالية من الميثيونين على وزن الفئرانأتأثير -0

خذ جرعات عالية من الميثيونين لم يوثر على وزن أن تأثير أ بينت النتائج المتحصل عليها

بحاث أ كذلك (.Lamda, ،1888)هذه النتائج تتوافق مع ،األربع المجموعات بين الفئران

(Zarrouk 1898، خرون آو ) على عكس(Zerizer 1889، خرون آو ) رتفاع محسوس إذكرت

، 2013)بحاث أكذلك . يوما 19كغ من الميثيونين لمدة /مغ188 بـالفئران التي عولجت في وزن

Boudebouz; Sakhri، 2014 ) كغ من /مغ 488 ـالفئران التي عولجت بن وزن أشارت أفقد

(Benmebarek بحاثأو على عكس . رتفاعا محسوساإخر عرف هو اآل سابيعأ 3الميثيونين لمدة

3كغ من الميثيونين لمدة /مغ 188 ـالفئران التي عولجت بنخفاض في وزن إين الحظت أ (1893،

.سابيعأ

.الفئران ووزن HHcy بين عالقة هناك نأ نستنتج نأ يمكننا ال ، المتضاربة النتائج لىإ وبالنظر

نزيمات إين، الدهون و يخذ جرعات عالية من الميثيونين على مستويات الهوموسيستأتأثير -1

:كسدة ألنشاط مضادات ا و الكبد

رتفاع مستويات إلى إدى أو جرعات عالية من الميثيونين قد ذ لغذائيان النظام أه الدراسة ذهظهرت أ

كسدة و البروتينات الدهنية ألنزيمات الكبد، بينما عرفت مضادات اإين، الدهون، يكل من الهوموسيست

ضرار في بنية كل من الشريان ألحاق إلى إدى أنخفاضا محسوسا مما إ (HDL-c)عالية الكثافة

.روطي و القلب و الكبد ألا

.كرذالتغيرات السالفة ال رغان فعاليته في تعديل ألور شجرة اذثبت المستخلص الخام لبأخرى أمن جهة

لى إدت أيوم 19للفئران لمدة ( كغ/مغ088)جرعات عالية من الميثيونين ذخأن أظهرت أنتائجنا

. ين في الدم يتكوين حالة فرط الهوموسيست

تنظيم تغير في إلىيرجع أن يمكن العالي من الميثيونين الغذائي النظام من المحتمل التأثير نإ

على أن الحفاظ الواضح ومن. سينثاز β سيستاثيون المثال سبيل على موسيستين،واله ستقالبإ إنزيمات

)ثيولال هذا من فعالة وإزالة نتاجإ لى تنظيمإراجع منخفضة من الهوموسيستين في األنسجة مستويات

Stipanuk ، 1884.)

الملخص

11

ين في الدم يرتفاع الهوموسيستإلى إدى أالغني بالميثيونين الغذائين النظام أبينت عديد الدراسات

(Mudd ،1899 ؛ Viggianoa ؛1891خرون، آو Yamada ؛1891خرون، آو Kirac خرون، آو

1893.)

مسار إلى الهوموسيستيين توجيه يتم ، الميثيونين يكون هناك فائض في عندما

transulfuration .الهوموسيستيين إلىتحويل يتم هذا السياق، في cystathionine ثم ومن cysteine

.الجلوتاثيون تركيب يدخل في عنصر هو pyridoxal 5-phosphate .cysteine قبل من الى

األكسدة ضرار نتيجة أ يسبب أن يمكنالهوموسيستيين تركيز رتفاعإ أن الدراسات أظهرت وقد

وبيروكسيد الحرة الجذور في اإلفراط في سببي مما مؤكسد عامل بمثابة يكون قد والسيستين ،الخلوية

؛ Hogg ، 9111) اتسرطانالتطورو الجينية اتطفرال من مزيد إلى يؤدي أن ويمكن الهيدروجين،

Stipanuk ، 1884 )الدموية واألوعية القلب وأمراض.

لى تشكيل إدى أين يرتفاع مستويات الهوموسيستإن أ( 1884خرون، آو Stanley)بينت دراسة

رتفاع إلك ذلى إ باإلضافة ،وعية الدمويةألكسدة دهون خاليا بطانة اأ، و (ROS)الحرة الجذور

NOنخفاض إ إلى دىأمما eNOSنتاج إو تثبيط iNOSلى تحفيز تكوين إدى أالهوموسيستين في الدم

(Kamal و Thanaa ،1881.)

كسدي أجهاد التإلن و ايمستويات الهوموسيستي رتفاعإثبتت العالقة الموجودة بين أخرى أدراسات

(Streck ؛1883خرون، آو Matté 2009خرون، آو.)

التيين لدى الفئران يالهوموسيست مستوياتنخفاضا كبيرا في إن هناك أظهرت نتائجنا أكما

لى الدور الفعال إمشيرة . المجموعات السابقة عمقارنة مرغان ذور شجرة األبالمستخلص الخام لب تغذت

خرون، أو Hamelet)ه النتائج تتوافق مع ذه. ينيه النبتة الطبية في خفض مستويات الهوموسيستذله

ن كل من أكرت ذ التيو ( 1893خرون، أو Meng ؛1881خرون، أو Yalcınkaya ؛1887

(quercetin )و (catechin) و( taurine )ين في الدم يساهمت في خفض مستويات الهوموسيست

.بالترتيب

رغان يكمل في محتوياته العالية من مضادات ذور شجرة األلمستخلص الخام لبلالوقائي التأثيرن إ

(. 1889خرون، أو Masella)كسدة القوية مثل البوليفينول، و توكوفيرول و ستيروالت ألا

تلقت جرعات عالية من يمستويات الشحوم في الدم لدى المجموعة الترتفاع إفيما يخص

، Herrmenn و Obeid ؛1881، خرونأو Lee)الميثيونين، فنتائجنا تتوافق مع دراسات كل من

الملخص

12

على عكس دراسة (. Sakhri ،1894 ؛Boudebouze ،1893 ؛1893خرون، آو Tselmin ؛1881

(Zerizer ،1889؛ Zerizer 1880خرون، آو ) ين يرتفاع الهوموسيستإلم تجد أي عالقة بين التيو

.رتفاع مستويات الشحومإو

وعية الدموية و خاليا الكبد بطانة األة بالنسبة لخاليا ين و الشحوم سامييعتبر كل من الهوموسيست

(Obeid و Herrmenn ،1881 .) ن ويوجود عالقة عكسية بين الهوموسيستي أثبتتدراسات كثيرة

و Poloni ؛9111خرون، آو Moat)نسان سواء عند اإل HDL-c))البروتينات الدهنية عالية الكثافة

.(1890خرون، آو Poloni)و عند الحيوان أ( 1891خرون، آ

وإفراز phosphatidylcholine نتاجإ من يقلل الميثيلية المجموعة نخفاض في إ أيضا،

VLDL ، الكبد في الثالثية الدهون تراكم في يتسبب مما قد (Li and Vance ، 1880؛ Bravo

يؤدي ، CβS deficiency نقص عن الناجمة فرط الهوموسيستيين في الدم، أنكما (. 1899 وآخرون،

؛9174 ،وآخرون Gaull) الكبد في الدهون وتراكم للدهون الغذائي التمثيل تنظيم في ضطراباتإ إلى

Namekata ،1884 وآخرون.)

جيد بشكل ، HDL وخاصة الدهنية، بين الهوموسيستيين والبروتينات عكسية عالقة وصف تم وقد

وآخرون، Poloni) و عند الحيوانات( 1891 وآخرون، Poloni ؛9111 وآخرون، Moat) عند البشر

وآخرون، Werstuck و) الخاليا داخل الدهون ستقالبإ يغير الهوموسيستيين كما أن(. 1890

أن يبدو و (.1893 وآخرون، Carvalho) الكبدية الدهون تراكم مع ترافقي أن ويمكن ،(1889

hypomethylation األنسجة في الدهون تراكم عن المسؤول هو مع فرط في مستويات الهوموسيستيين .

atherothrombotic (Obeid أمراض بتطور في الدم الكوليستيرول وفرط الهوموسيستيين ويرتبط

and Herrmann ، 1881 .)،توازن أن إلى الدراسات من العديد أشارت فقد وعلى عكس ذلك

(.1890 وآخرون، Koyama) الدموية واألوعية القلب أمراض لقمع رئيسية آلية هو الكوليسترول

تتصدى التي البيولوجية العمليات من العديد في HDL)) البروتينات الدهنية عالية الكثافة تشارك

الدموية بطانة األوعية ،(بيتا)البنكرياس خاليا وظيفة على التأثير خالل من التأكسدي، واإلجهاد لتهابلإل

جزيئات عن التعبير عن فضال األحادية، الخاليا وتنشيط الدموية، المبرمج للبطانة األوعية الخاليا موت ،

(.1899 وآخرون، Chapman)لتصاق اإل

الملخص

13

المشاركة الجزيئات أو اإلنزيمات بتثبيط مرتبطا HDL)) تأثير الهوموسيستيين على يكون وربما

الشرايين تصلب خطر زيادة فإن ولذلك،HDL (Obeid and Herrmann ، 1881 .) تجمع في

Paraoxonase-1 PON1 (Holven نشاط نخفاضإب صلة ذات تكون قد المختلة HDL وجزيئات

thiolactonase (Domagala نشاط ويظهر الكبد من أساسا يفرز PON1(. 1880 وآخرون،

(.1880 وآخرون، Bhattacharyya) الشرايين تصلب من حماية وبالتالي( 1889 وآخرون،

رتفاع مستويات الدهون إقتران مع باإل HDL-c))البروتينات الدهنية عالية الكثافة نخفاض إن إ

لها دور HDL-c))لك جزيئات ذلى إ باإلضافة. بتصلب الشرايين لإلصابةالثالثية يعتبر عامل خطر

(.9113خرون، آو Macknesset) (LDL-c)كسدة الكولسترول الضارأوقائي ضد

التي الجرذانلدى LDL))جزيئات رتفاع إ ، (1898خرون، آو Bhandari)حسب دراسة

في عدد نخفاضإلى إين راجع ربما يميني الهوموسيستآلتعاني من مستويات عالية من الحمض ا

LDL))مستقبالت في تغييرات أو. مع مستقبالته LDL)) نخفاض ترابط إو أ LDL))مستقبالت

.د جرعات عالية من الميثيونينأخرتفاع مستويات الكوليسترول بسبب إدي الى ؤت

رغان في تعديل ألور شجرة اذالمستخلص الخام لبظهرت نتائجنا فعالية أخرى، أمن جهة

الذينخفاض الهوموسيستين لدى نفس المجموعة و إلى إا راجع ذمستويات الدهون السالفة الذكر، و ه

.كسدةأله النبتة بمضادات اذه غنى لى إبدوره راجع

نخفاضا إتالز فقد عرفا انزيم الكإلى كمية الجلوثاثيون المختزل في الكبد و نشاط إبالنسبة

فيما يخص الجلوثاثيون المختزل فنتائجنا . معتبرا لدى الحيوانات التي تلقت جرعات عالية من الميثيونين

لميثيونينعولجت با التيالفئران نفس النتائج لدى تحصلت علىو التي ( Sakhri ،1894) تتوافق مع

.(كغ/مغ488) و لكن بجرعة

نخفاض إن أ، (1899و آخرون، Bravo ؛ Vance ،1880و Li)ظهرت دراسات كل من أ

. ينييضي في تصنيع الهوموسيستأليعتبر المنظم ا الذي (SAM)نخفاض مستوى إالجلوثاثيون يتوافق مع

كسدة القوية التي تلعب دورا هاما في حماية ألمضادات احد أالمختزل هو الجلوثاثيون

(. 1889، آخرون و free radical reactive( )Stipanuk )نسجة من الجذور الحرة ألا

نزيماتاإل وتشمل الخاليا، بين األكسدة تفاعل ساسية للحد منوامل األعحد الأ هو الجلوتاثيون

glutathione peroxidaseو glutathione S-transferase (GST) لها التابعة لألكسدة المضادة

الملخص

14

(GPx)، المؤكسدة واألنواع الحرةللجذور الفعل رد ضد الخلوي الدفاع في أساسيا دورا تلعب والتي

-GSH S و GPx بواسطة العضوية البيروكسيدات تخفيض أيضا يمكن(. Stipanuk ، 1889) األخرى

transferase. في فقط موجود ولكنه الهيدروجين بيروكسيد يقلل أن أيضا يمكن كاتاالزال إنزيم

التأكسدي اإلجهاد من كل ضد الدفاع في خاص بشكل مهما الجلوثاثيون ليجع وهذا. بيروكسيسوم

-Fernándezو Garcia-Ruiz ؛9117 وآخرون، Fernández-Checa) الفسيولوجي والمرضي

Checa ، 1889 .)كثير حدثي الجلوثاثيون مستويات اضخفنإ نأشارت أ التي البحوثالعديد من هناك

بسبب مهيمنا دورا تلعب التأكسدية اإلصابات أن حين في(. 1881 وآخرون، Ballatori) األمراض من

نزيماتإ نتاج إ نخفاضإب سببيا يرتبط وبعضها ضطرابات،اإل هذه من العديد في الجلوثاثيون نخفاضإ

(.Lu ، 1881)نتاج الجلوثاثيون إ

الكبدي لدى التأكسد خطورة من المزيد يسبب قد الكبد في الميثيونين سولفوكسيد تراكم فرط نإ

،جلوثاثيون ذلك في بما السيستين،مثل األكسدة مضادات مستوياتها كانت والتي ، −/−Cth الفئران

taurine/hypotaurine,، غيرمعدلة (Yamada ،1891 وآخرون .)وسيستين فيهومال فرط ويؤدي

لألكسدة المضادة الدفاع أنظمة يضعف الذي ROSنتاج إ طريق عن لتأكسديا اإلجهاد زيادة إلى الدم

(.9110 وآخرون، Welch) أو الخاليا داخل

و Woo) تالز، فنتائجنا تتجه في نفس السياق معانزيم الكإنخفاض نشاط إفيما يخص ما أ

ه الدراسات وجدت ذه(. 1881و آخرون، Matté ؛ 1887و آخرون، Chanson ؛ 1889آخرون،

.نزيم الكتالز في الكبدإنخفاض إين و يرتفاع مستويات الهوموسيستإعالقة عكسية بين

ين بسبب عديد من المثبطات على ينزيم الكتالز من طرف الهوموسيستإنخفاض نشاط إيعود

و Putnam ؛Novogrodsky ،9100و amino-1:2:4: triazole (Margoliash-3غرار

amyloid-ß (Milton ،1999 .)، و (1888خرون،أ

رغان قد ساهم في رفع و ور شجرة األذالمستخلص الخام لب ن أ ه الدراسةذنتائج هظهرت أ

رتفعت لكن ليس بقيمة معتبرة، إخرى ألبينما مستويات الجلوثاثيون هي ا ،تالزانزيم الكإ تحسين مستويات

. فترة التجريبية لراجع لقصر ا ربما اذو ه

نزيم إ نتاجإلى تعديل إدت أه المركبات ذه. الذكرسلفنا أرغان غنية بالبوليفينول كما ألن نبتة اإ

و (1883خرون، آو Floreani )نسيج عضلة القلب نسجة على غرار ألفي عديد ا eNOSتالز و االك

. (1887خرون، آو Kasdallah-Grissa )نسيج الكبد

الملخص

15

رتفاع معتبر في إ، فقد تحصلنا على (AST، ALT)فيما يخص النتائج المتعلقة بإنزيمات الكبد

و التي تتوافق مع دراسة . رتفاعا بقيمة غير معتبرةإشهدت ( ALT)، بينما مستويات (AST)مستويات

(Yalcınkaya 1881خرون، آو )رتفاع إلى إدت أاشهر 9بالميثيونين لمدة الجرذان تغذيةن أثبت أى ذال

(.ALT)، و(AST)ين، و يمستويات كل من الهوموسيست

يدل على تخرب ( AST، ALT) رتفاع هده األنزيمين إن فإ، (1880و آخرون، Ozer)حسب

، Payneو Killip)فهو يعكس تغيرات في نسيج العضلة القلبية (AST)رتفاع نشاط إما أ الكبد، نسيج

9198) .

خرى في تعديل مستوياتأرغان مرة ألور شجرة اذالمستخلص الخام لبظهرت نتائجنا فعالية أ

(. AST) نزيمإ

تأثير اخذ جرعات عالية من الميثيونين على المقاطع النسيجية للقلب و الكبد و الشريان -3

.روطي األ

لية من الميثيونين ، فيتجلى المقاطع النسيجية للمجموعة التي تلقت جرعات عا ما فيما يخص تحليلأ

ل من البنية النسيجية حداث بعض تغيرات لكإن في يرتفاع مستويات الهوموسيستيإبوضوح تأثير لنا

نخفاض مستويات الجلوثاثيون المختزل إعلى الدهون و تأثيرهبسبب . روطي و القلب و الكبدللشريان األ

.كسديجهاد التأو التي تعكس حالة اإل نزيم الكتالز إو نشاط

اللتان ( Sakhri ،1894 ؛Boudebouze ،1893 ( تتوافق مع دراستي كل من ه النتائج فهيذه

.السالفة الذكرنسجة نفس األ بنية لى التأثير علىإدت أجرعات عالية من الميثيونين ن أذكرتا

األوعية، مرونة من والحد البطانية، الخاليا تلف إلى يؤدي ينيرتفاع مستويات الهوموسيستإ أن يعتقد

نهاية و التي تنتهي في (.Kopczynski ،1894 و Baszczuk)توازنها الداخلي عملية كما يغير من

و Papatheodorou ؛1889و آخرون، Gallai) الدماغية والسكتات القلبية النوبات إلى المطاف

Weiss ،1887.)

و التي بدورها تساهم في ( ROS)نتاج الجذور الحرة إلى إن يالهوموسيستي ارتفاع ديؤكذلك ي

و Kanani) وعية الدموية مما يعزز تطور عملية تصلب الشرايينألتالف خاليا بطانة اإتخريب و

(. 9111آخرون،

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16

ين تساهم في يرتفاع مستويات الهوموسيستإن أ مؤخرا، ( 1890و آخرون، Chen) دراسة أظهرت

في البطانة S-nitrosylation بروتين مستويات نخفاضإ مع ا يتوافق ذو ه الشرايين تصلب تقدم

واألوعية القلب وظائف تنظيم في هاما دورا تلعب وعية الدموية ، و التيألو اأروطي ألالداخلية للشريان ا

. الدموية

والجلطات هي الشرايين تصلب فيين يرتفاع الهوموسيستإخرى التي يساهم فيها ألليات اآلو من بين ا

البروتينات الدهنية كسدة أالتأكسدي، اإلجهاد الخاليا، زيادة العضلية، سمية الخاليا تحفيز تكاثر :

Willoughby) ، و المساهمة في عملية التخثر و تنشيط الصفائح الدموية LDL) ) منخفضة الكثافة

(.1889و آخرون، Luo ؛ 1881و آخرون،

atherogenic ـو ما يعرف بأوجدنا سابقا في هذه الدراسة زيادة في مؤشر تصلب الشرايين كما

index الكوليسترول في الدم وانخفاض نتيجة لزيادةHDL . حسب(Mehta ،1883و آخرون) ،

عضلة القلب، ىلإو تسرب الدهون أ( foam cells) المؤشر وجود الخاليا الرغوية اذيعكس ه

. بها ضرارأالشريان األورطي، الكبد و الكلى ، مما يلحق وعية التاجية، ألا

في عدة نماذج ي، نسيج الكبدالتالف إين يسبب يالهوموسيست فرطن ألك وجد ذلى إباإلضافة

اإلندوبالزمية، الشبكة التأكسدي، إجهاد اإلجهاد زيادة :ليات، و التي تتمثل في أتجريبية عن طريق ثالث

و من بين هده (.1880 وآخرون، Robert ؛Kaplowitz ،1884و Ji) لتهابيةو تنشيط العوامل اإل

و IL-8 (Ji و 1b ، IL-6-(IL)نترلوكينات ، اإلNFkBالعامل النووي : لتهابيةالعوامل اإل

Kaplowitz ،1884؛ Matte ،1881 وآخرون) .

روطي والكبد، ألنسجة القلب، الشريان اأالمقاطع النسيجية لكل من نأه الدراسة ذظهرت هأو قد

بشكل سليم ظهرت رغان، ور شجرة األذعولجت بالميثيونين و المستخلص الخام لب فئران التيلل بالنسبة

.ه النبتةذكسدة التي تحتويها هألا راجع لمضادات اذو ه

الجرذان أن( 1893 وآخرون، Benmebarek) و( Naimi ، 1884 و Zerizer) عمل أثبت

الحقت بأضرار يوما 19 من الميثيونين خالل كغ/ مغ 188 ب التوالي على عولجت التي والفئران

إلى أدى B6 و B9، B12 الحيوانات بالفيتامينات هذه عالج أنكما والقلب األورطي الشريان باألنسجة

. الدموية واألوعية القلبب الملحقة األضرار هذه تصحيح

الملخص

17

البيوفيلم تشكيل تأثير زيت االرغان على -4

أنحاء جميع في والوفيات األمراضفي عديد من هاما سببا البكتيرية المعدية األمراض تمثل

.البكتيرية االلتهاباتلعالج هذه للميكروبات مضادة جديدة عوامل تطوير وجب ولذلك. العالم

، Draelos" )الحيوية العدوى أنواع من نوع" هي البكتيرية المعدية األمراض أن يفترض عادة،

العالجات لمختلف مقاومة أكثر عام بشكل األغشية الحيوية هذه أن األخيرة األبحاث أظهرت وقد ،(1898

(.Olsen ، 1890) للميكروبات المضادة

للميكروبات المضادة لعواملل مقاومتها زيادة هيو البيوفيلم أ الحيوية األغشية سمات أهم من واحدة

(Wimpenny ،1888 وآخرون .)وجود أن المعروف ومن EPS ، من الجافة الكتلة أغلبية يمثل الذي

(. 9119 ستيوارت،) المختلفة الحيوية المضادات فعالية من يقلل مما ميكانيكي، حاجز بمثابة بيوفيلم،ال

مع تفاعلت بيوفيلمال سطح على الموجودة الشحنة سالبةال البوليمرات أن المعروف من ذلك، على وعالوة

Nichols) األدوية هذه تغلغل من يحد مما ،(أمينوغليكوزيد مثل) الموجبة ذات الشحنة الحيوية المضادات

(.9100 وآخرون،

األغشية سطحعلى مثال ، األكسجين تركيزك المتغيرة البيئية الظروف تأثير ذلك، إلى باإلضافة

األغشية جوهر بينما تنخفض هذه النسبة في ،عالية األوكسجين مستويات تكون (وفيلميالب) الحيوية

Yang ؛9114 وآخرون، De Beer) الحيوية مضاداتال وصولل عرضة أقل النواة يجعل مما الحيوية،

(.1880 وآخرون،

تسمى آلية خالل من والفسيولوجية التعاونية أنشطتها تنظيم البكتيريا من العديد أن المعروف ومن

quorum sensing (QS)، اإلفراج طريق عن البعض بعضها مع تتواصل البكتيرية الخاليا حيث

تشمل تشكيل ، quorum sensing آلية. لإلنتشار قابلة صغيرة إشارة لجزيئات واإلستجابة واإلستشعار

و Li) البيوفيلم وتكوين المبرمج، الخاليا وموت الوراثية، والكفاءة البكتريوسين، وإنتاج ، الجراثيم

Tian ، 1891.)

تمتلك والتوابل واألعشاب النباتات في الموجودة الطبيعية المركبات من العديد أن تبين وقد

) األمراض مسببات ضد الميكروبات مضادات لعوامل كمصدر وتعمل الميكروبات مضادات وظائف

Deans and Ritchie ،91107؛ Kumar ،1889 وآخرون.)

الملخص

18

تكون أن يمكن( النباتات) الطبيعي المصدر من المستمدة للبكتيريا المضادة العوامل فإن وهكذا،

ضد نتقائيةإ بمزايا تتمتع أنها المعروف من التي الثانوية، األيضات وجود بسبب وذلك فعاال، بديال

(.Buss ، 1889 و Butler) المقاومة الكائنات

0 ضد الجزائري األرغان لزيت للبيوفيلم المضاد المفعول تقييم لىإهدفت دراستنا ،اقيسال هذا في

: و هي وعية الدمويةألنها تساهم في تشكيل مشاكل للقلب و اأيعتقد بكتيريا أنواع

Streptococcus mutans ، Streptococcus anginosus،Streptococcus intermedius

Streptococcus uberis و Staphylococcus haemolyticus .

و . الذكربكتيرية السالفة أنواع 4رغان كان فعاال في تثبيط ألزيت ا أن بيوفيلمال فحص نتائج أظهرت

حمض كافييك : مثل الطبيعي الفينول من مادة الزيت هذا ثراء ربما بسبب .ا تزامنا مع زيادة تركيزهذه

caffeic acid حمض الفانيليك، vanillic acid تيروسول ، tyrosol و ايبيكاتشين ، epicatechin

(charrouf وGuillaume ،1887.)

زيت من( مل/ ميكروغرام 988) تركيز أول كان اكيز المذكورة ،رالت بينن من أكما بينت النتائج

الزيت تأثيرن أغير . الخاليا لقتل ليس ولكن Streptococcus uberis بيوفيلملتقليص فعال األرغان

Streptococcus mutans ، Streptococcus anginosus على كل من بيوفيلم أعلى كان

،Streptococcus intermedius Staphylococcus haemolyticus كثر من أ بـ نخفاضإ مع

.لتثبيطا نسبةبالنسبة ل 18٪

ضد تنشط و التي النباتية، الكيميائية المواد وجود إلى األرغان لزيت المثبط النشاط يرجع أن ويمكن

الكيميائية المواد من أكثر أو ثنينإل التعاوني للتأثير نتيجة يكون أن يمكن أو البكتيريا من السالالت هذه

المضادات يعتبر من األرغانزيت أن يعني وهذا(. 1899 وآخرون، Da Silva) لهذا الزيت النباتية

الفانيليك، حمض الكافيك، حمض: مثل الطبيعية الفينوالت من الزيت هذا ثراء خالل من ربما الحيوية،

يحتوي انغاألر زيت أن ثبت وقد(. Guillaume ، 1887و Charrouf.... ) إبيكاتشين تيروسول،

و Charrouf ) والصابونين الفالفونويدات العفص، ،ستيروييدال مثل نباتية كيميائية مواد على

Guillaume ، 1887 .)وتمنع البكتيرية بالبروتينات ترتبط النباتية الكيميائية المواد هذه أن ثبت وقد

؛1898 وآخرون، Samy) النقل وبروتينات الخلوي، والمغلف واإلنزيمات، الميكروبات، لتصاقإ

Upadhyay ،1894 وآخرون .)

الملخص

19

. اآلليات هذه من بعض خالل من بيوفيلم تشكيل تثبيطقام ب اناألرغ زيت أن المرجح من وبالتالي،

لتصاقإ تعطل التي والعوامل( 1890 وآخرون، Rabin) بيوفيلم تشكيل أثناء مهم البكتيريا لتصاقإ

.للبيوفيلم مضادة كعوامل العمل على القدرة لديها األسطح إلى البكتيرية

نشاطا ظهرأالكافيك قد ن حمضأ ( 1893 وآخرون، Stojkovic) دراسة أظهرت السياق، هذا في

هيدروكسيناميك، حمض) البوليفينول من عدد على العثور تم أيضا،. للميكروبات ومضاد لألكسدة مضادا

(.1893 وآخرون، Nazzaro) Chromobacterium violaceum لمنع( إبيكاتشين روتين،

كسر طريق عن أو السطح على الحي الكائن تعلق منع طريق عن يتم نأيمكن البيوفيلم تثبيط ن إ

(.1899 وآخرون، Gupta)تشكل إذا البيوفيلم هيكل

النباتية الكيميائية المواد على حتواءهإليرجع قد االرغان في تثبيط البيوفيلم زيت لدور آخر تفسير

تمي عندما أنه الدراسات من العديد أظهرت وقد. البيوفيلم في اإلجهاد ستجابةإ تحفز التي للبكتيريا المضادة

بالبيوفيلم و المرتبطة تحفيزالجينات يتم الحيوية، بالمضادات العالجك الميكروبات، على تطبيق اجهاد

(.1893 وآخرون، Ackart) يوفيلمبلل الظاهري النمط اختفاءبالتالي

ؤ بشار أرغان شجرة ورذب من المستخرج األرغان زيت أن( 1890 آخرون، و Lotfi) خرا، كشفم

ة البكتيريا على إيجابيا في القضاء أثرا أظهر قد ،الجزائر على مقاومةال (البالنكتوني ة)العائمة والحر

العنقودية والمكورات ( Staphylococcus aurus)الذهبية العنقودية المكورات الخصوص وجه

.(Staphylococcus white)البيضاء

Bjarnsholt) الثوم مثل البيوفيلم ثبتت فعاليتها في تثبيط أبهارات ل بحاث أ نتائجنا تتوافق مع عدة

، (1881خرون، أو Khan)القرنفل ، (1889خرون، أو Niu)والقرفة والزنجبيل،، (1880خرون، أو

. ( 1894خرون، أو Packiavathy)والكركم ، ( 1891خرون، أو Packiavathy)الكمون

بخصائصها عرفت النباتية المواد عطرية مستخلصة من زيتية سوائل هي عبارة األساسية ن الزيوتإ

: ثبتت فعاليتها في تثبيط تشكل البيوفيلمأهده الزيوت التي من بين (. Burt ، 1884) للجراثيم المضادة

، Piper bredemeyeri ، Piper brachypodom ، Piper bogotenceلـ الزيت االساسي

Gaultheria procumbensوش دكحزنبل، القرنفل الزعتر و المر، ال(Khan ؛1881خرون، أو

Musthafa ؛ 1898خرون، أو Olivero ؛ 1899خرون، أو Jadhav 1893خرون، ألو.)

الملخص

20

وفي. األسنان لألمراض مسببة بأنها عموما الدراسة هذه في المستخدمة الدقيقة الحية الكائنات تعرف

في للتقدم عرضة أكثر تكون الفموية، البكتيريا تسببها التي تلك ذلك في بما لتهابات،إلا فإن السياق، هذا

البكتيريا بين صلة وجود الدراسات من العديد أثبتت فقد. سابقا يعتقد كان مما الوعائية القلبية األمراض

واحدة آلية من أكثر هناك أن جدا المرجح ومن للغاية، معقد أمر وهو الشرايين، وتصلب الفموية

(Leishman ،1898 وآخرون.)

تتحول جرح، أي ظهور مع. والبكتيريا البطانة الشريان أسطح بين تفاعل هو الشغاف التهابن إ

وآخرون، Kokare) القلب صمامات تتلف أن يمكن التي قوية و بيوفيلمات لىإ نتهازيةإلا الجراثيم

التناسلي البولي الجهاز البلعوم، خالل من الدم مجرى في في تدخل أن لهذه الجراثيم يمكن(. 1881

فإنهالتصاقها إ حالة في ولكن ،ضعيفة الشريان بطانة في جراثيمال تصاقلإ عموما،. الهضمي والجهاز

الحالة، هذه في(. 1881 وآخرون، NBTE( )Kokare) الخثاري الشغاف لتهابإ تلف أوجرح تسبب

فبرونيكتين تفرز البطانية خاليا. اإلصابة موقع في والفيبرين الدموية والصفائح الحمراء الدم خاليا تتراكم

.البكتيريا وكذلك البشرية الخلية الفيبرين، الكوالجين، ربط على القدرة لديها التي

يمكن التي فبرونيكتين مستقبالت لديها والعقدية العنقودية المكورات مثل الميكروباتخرى أمن جهة

وآخرون، Kokare) الصمامات أنسجة تلف وكذلك اإلصابة موقع على الحيوية األغشية تشكل أن

في تغيرات إلى تؤدي قد الفم طريق عن البكتيريا من طرف البطانية الخاليا غزو ولذلك،(. 1881

إلى تؤدي كلها وهي المبرمج،الخاليا موت وكذلك البطانية الخاليا proatherogenicوها مثل خصائص

(.1898 وآخرون، Leishman) البطانية الخاليا في وظيفة خلل

معجون تصنيع في تستخدم أن يمكن رغاناأل ن زيتإف عليها، الحصول تم التي النتائج خالل من

.الدموية بطريقة غير مباشرة واألوعية القلب أمراض يمنع والذي العشبية، األسنان

الخاتمة

ية الفئران تغذ طريق عن الدم في ينالهوموسستي فرطه الدراسة تكوين حالة ذكان هدف ه

يوما، و تقييم التأثير 19 بـخالل مدة تجريبية تراوحت ( كغ/مغ088) بجرعات عالية من الميثيونين

يضية و ألضد التغيرات ا (كغ/مغ908) رغانألور شجرة اام لبذللمستخلص الخالوقائي و العالجي

واع بكتيرية نأ 0ختبار المفعول ضد تشكل البيوفيلم ضد إخرى، أمن ناحية . البنيوية التي سببها الميثيونين

.وعية الدمويةألمقاومة و التي قد تتسبب في مشاكل للقلب و ا

األيضية اإلضطرابات بعض واضح بشكل سبب رتفاع جرعات الميثيونينإ أن الدراسة هذه أظهرت قد

:على غرار

الملخص

21

؛لدى الفئران الدم في نييستيالهوموس فرطتكوين حالة

؛رتفاع مستويات الكوليسترول، الدهون الثالثية إ

يبوبروتينات لختالل في الإ(Dyslipoproteinemia ) نخفاض مستوى إمعHDL-c رتفاع إو

؛LDL-c مستوى

مستوى الدم في الكوليسترول مستوى رتفاعإ بسبب الشرايين، تصلب مؤشر في زيادةHDL-c؛

؛ختالل في مستويات مضادات االكسدة إ

الكبد خاصة نزيماتإ في زيادة ALT.

والكبد والقلب األورطي للشريان بنيوية ضرارأ النسيجية تحليل المقاطع أظهر ذلك، إلى باإلضافة

:بما يلي تميزت

؛األورطي البطانة الداخلية للشريان وتخرب فقدان

؛األورطي الشريان من مختلفة أقسام في رغوية خاليا تشكيل

؛والكبد األبهر الشريان في كل من ظهور حويصالت ليبيدية

؛األورطي للشريان المطاطية األلياف ختفاءإ

؛ القلب لبعض خاليا تغيرات في البنية العامة

الفجوات تشكلنوية، و ألغشية البالزمية لبعض الخاليا الكبدية، تضخم بعض األتخريب ا

. السيتوبالزمية

رغان ألور شجرة اذبالمستخلص الخام لب غني غذائي نظام تباعإ ن أتفيد دراستنا

و البنيوية السالفة األيضية، ضطراباتإلثبت فعاليتة في تحسين اأقد (كغ/مغ908) الجزائرية

. لدى الفئران لميثيونينل عن الجرعات العالية الناجمة الذكر،

المفعول الحيوي و المثبط لتشكل فيما يخص عليها الحصول تم التي النتائج إلى بالنظر و

سالالت 4خرى ضد أثبت نجاعته مرة أقد الجزائري األرغان زيت أن نستنتج أن يمكننا البيوفيلم

. تركيزها توافقا مع زيادة ذبكتيرية مقاومة، و ه

لما تحتويه ربما يعود ( المستخلص الخام للبذور والزيت)رغان ألالوقائي لنبتة ا التأثيرهذا

tocopherolsالتوكوفيرول لتهابات مثل إلعلى العديد من مضادات األكسدة والمركبات المضادة ل

حمض فانيليك ،oleuropein، الوغوبين caffeic acidحمض كافييك ) ، الفينول (Eفيتامين )

https://en.wikipedia.org/wiki/Tocopherol

https://en.wikipedia.org/wiki/Caffeic_acid

الملخص

22

vanillic acid ، تيروسول tyrosolو كاتشين ، catechin) الكاروتينات ،carotenes ،

(.٪ األحماض الدهنية غير المشبعة08)، واألحماض الدهنية، squaleneالسكوالين

:لى القيام بما يليإنطمح في المستقبل الدراسة هذه نتائج على وبناء

؛النباتي مستخلصال في الواردة بيولوجيا النشطة الجزيئات وتحديد ستخالصإ

نشاط مضادات أكسدة أخرى مثل قييمت superoxide dimustaseو glutathione

transférase.

ظهار إو ،النباتي المستخلص يحتويها التي لألكسدة المضادة لإلنزيمات الجيني التعبير دراسة

؛ .الدموية واألوعية القلب عالجات فيدورها

؛اإللتهابية السيتوكينات الجيني ضد التعبير إبراز دور النبتة في تنظيم

؛األرغان لزيت للجراثيم المضادة المركبات عزل

الذهبية ، العنقودية المكورات مثل خرىأبكتيرية نواعأرغان ضد ألختبار فعالية زيت اإ

. الحيوية المضادات بعض معا الزيت ذله التعاوني النشاط إلى باإلضافة

https://en.wikipedia.org/wiki/Vanillic_acid

https://en.wikipedia.org/wiki/Catechin

https://en.wikipedia.org/wiki/Carotene

https://en.wikipedia.org/wiki/Squalene

الملخص

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:االشكال

.يوما 19تأثيرالميثيونين على وزن الفئران خالل فترة : (02)الشكل

تأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات الدهون الثالثية : ( 03)الشكل

.يوما من التجربة 19خالل

تأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات الكوليسترول : (04)الشكل


البروتينات الدهنية المستخلص الخام لبذور شجرة األرغان على مستويات تأثيرالميثيونين و: (05)الشكل

.يوما من التجربة 19خالل منخفضة الكثافة

البروتينات الدهنية تأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات : (06)الشكل

.يوما من التجربة 19خالل عالية الكثافة

تأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات الهوموسيستيين : (07)الشكل


AST نزيم الكبد إتأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات : (08)الشكل


ALT نزيم الكبد إالمستخلص الخام لبذور شجرة األرغان على مستويات تأثيرالميثيونين و : (01)الشكل


تأثيرالميثيونين و المستخلص الخام لبذور شجرة األرغان على مستويات الجلوثاثيون : (21)الشكل

.يوما من التجربة 19المختزل خالل

نزيم الكتاالز إلبذور شجرة األرغان على مستويات تأثيرالميثيونين و المستخلص الخام : (20)الشكل


(P) و (MP) ، (M) ، (F)المقاطع النسيجية للقلب للمجموعات : (22)الشكل

(P) .و (MP) ، (M) ، (F)المقاطع النسيجية للشريان األورطي للمجموعات : (23)الشكل

(P). و (MP) ، (M) ، (F)المقاطع النسيجية األورطي للمجموعات : (24)الشكل

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(P). و (MP) ، (M) ، (F)المقاطع النسيجية األورطي للمجموعات : (25)الشكل

(P). و (MP) ، (M) ، (F)المقاطع النسيجية للكبد للمجموعات : (26)الشكل

S. intermedius.بيوفيلمالرغان على تشكيل ألالتأثيرالمثبط لزيت ا: (27)الشكل

St. haemolyticus بيوفيلم العلى تشكيل األرغانالتأثيرالمثبط لزيت : (28)الشكل

S. mutansبيوفيلم العلى تشكيل األرغانالتأثيرالمثبط لزيت : (21)الشكل

S. anginosus بيوفيلم العلى تشكيل األرغانالتأثيرالمثبط لزيت : (31)الشكل

.S. uberisبيوفيلم العلى تشكيل األرغانالتأثيرالمثبط لزيت : (30)الشكل

.على تثبيط البيوفيلم األرغانتأثير تركيز زيت : (32)الشكل

Paper

THE PROTECTIVE EFFECTS OF ARGANIA SPINOSA SEEDS AGAINST HYPER-HOMOCYSTENEMIA INDUCED BY HIGH METHIONINE DIET IN MICE

Original Article

BADIAA AKLIL1,2, SAKINA ZERIZER*1,2, ZAHIA KABOUCHE1 1.2

Received: 10 Mar 2017 Revised and Accepted: 16 Oct 2017

Université Des Frères Mentouri-Constantine, Département de Biologie Animale, Laboratoire d’Obtention de Substances Thérapeutiques (L. O. S. T), 25000 Constantine, Algeria

Email: [email protected]

ABSTRACT

Objective: Hyperhomocysteinemia (HHCY), oxidative stress and decreased antioxidant capacities lead to several clinical manifestations and particularly, cardiovascular and liver diseases. Our aim in this study was to investigate the protective effects of Argania spinosa powdered seeds against high methionine diet-induced HHCY, oxidative stress and damages in the aorta, and heart of mice.

Methods: Adult male Mus Musculus was systematically divided into four groups of similar mean body weights and fed for 21 d with control and experimental diets. The control group (F) was fed with white bread (0.50 mg/mice), group (M) was fed with L-methionine (500 mg/kg/day), group (MP) was fed with L-methionine (500 mg/kg/day) plus A. spinosa powdered seeds 150 mg/kg), and the group (P) was treated with A. spinosa powdered seeds (150 mg/kg/day). The experimental diets were given in white bread (0.50 mg/mice). After 3 weeks of treatments, homocysteine (HCY) concentrations, hepatic antioxidant status and histological sections of aorta and heart were determined.

Results: Consumption of high methionine diet led to an increase in plasma HCY, reduced the concentrations of GSH, and the enzyme catalase. These were associated with the loss and degeneration of endothelium, fenestration and formation of foam cells of the aorta, also the alteration of the cardiac muscle. However, administration of A. spinosa powdered seeds in combination with methionine decreased the concentration of HCY from (10.04±0.83 μmol/l) to (7.26±0.46 μmol/l), increased catalase activity from (45.82±5.83 m mol/mg protein) to (62.26±3.32 m mol/mg protein), and ameliorated histological changes.

Conclusion: A. spinosa powdered seeds were effectives in decreasing plasma HCY level as induced by methionine-enriched diet in mice, and improved the antioxidants defence.

Keywords: Homocysteine, Argania spinosa, Antioxidant enzymes, Methionine, Oxidative stress, Cardiovascular diseases

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2017v9i12.18275

INTRODUCTION

Both cardiovascular disease and liver injury are major public health issues [1]. It has been reported that elevated plasma HCY concentration is an important risk factor for vascular diseases [2-3], and defects in methyl group metabolism, often resulting in HHCY, are among the key molecular events postulated to play a role in liver injury [1]. Plasma HCY levels can be determined by genetic, biological, nutritional, hormonal and lifestyle factors [4].

HHCY caused by excessive methionine intake is a classical attribute cardiovascular [5] and hepatic diseases [6-7]. Due to the presence of the highly reactive sulfhydryl group, HCY can undergo auto-oxidation to generate oxygen radicals [8].

Studies have shown that high HCY concentration could cause oxidative damage to cells [9-10]. HHCY induces endothelial dysfunction [11], which played an important role in the early stages of the atherogenic process by decreasing the availability of NO, stimulating the activation of nuclear factor kappa B and consequently increasing the expression of ICAM-1 [12]. Indeed, the oxidative stress resulting from elevated serum Hcy can oxidize membrane lipids and proteins and stimulate the activation of NF-B, and consequently increase the expression of inflammatory factors in vivo [13].

Hyperhomocysteinemia leads to increased oxidative stress via the generation of reactive oxygen species which weaken intracellular antioxidation defence systems [14]. ROS (including superoxide and hydrogen peroxide) are produced by endothelial cells and the adjacent smooth muscle cells, adventitial fibroblasts and inflammatory cells. ROS can affect the NO pathway [15]. The need for protection against ROS and other reactive molecules has led to

the specious theory that a high intake of exogenous antioxidants protects the body from oxidative stress, which is also widely known as the antioxidant hypothesis [16-17].

A high intake of fruit and vegetables rich in natural antioxidants, such as vitamins C and E, polyphenols, carotenoids, terpenoids, and phytomicronutrients, show an inverse association with the risk of cancer and the development of cardiovascular diseases.

The argan tree (Argania spinosa (L.) Skeels is a tropical plant, which belongs to the Sapotaceae family and is endemic in southwestern Morocco [19], and Algerian region of Tindouf [20]. It is exploited essentially for its fruits. The endosperm seed of fruit constitutes a good potential source of edible oil for human consumption and endowed with important medicinal properties [19].

Considering its rich composition in antioxidant compounds and unsaturated fat, A. spinosa can be used as a nutritional intervention in the CVD diseases prevention [21].

This study was designed to investigate the beneficial effects of powdered seeds of A. spinosa against hyper-homocystenemia, antioxidant status and damages in the heart and aorta induced by high methionine intake in mice.

MATERIALS AND METHODS

Plant material

Seeds of Argania spinosa were collected from a region near Tindouf (Southwest of Algeria). The fruits were cut into pieces to obtain seeds, which were subjected to size reduction to a coarse powder using a mechanical grinder. The powder was then used for treatment preparations.

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 9, Issue 12, 2017

http://creativecommons.org/licenses/by/4.0/�

Zerizer et al.

Int J Pharm Pharm Sci, Vol 9, Issue 12, 64-69

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Chemicals and reagents

L-Methionine purity 98% was obtained from across organics (Belgium). Total HCY levels were measured using the Immulite HCY kit (Siemens, Finland), on Immulite 2000 system. Other chemicals used were of Sigma chemical company.

Animals

Experiments were performed on 28 adult male Albino Mus Musculus mice weighing (30–35g), given from central pharmacy, Algeria. Animals were housed 7 per cage, and maintained under standard laboratory conditions of humidity, temperature (25 °C) and light (12 h day: 12 h night). After the adaptive period, they were divided into four groups of similar mean body weights and fed for 21 d with control and experimental diets. The control group (F) was fed with white bread (0.50 mg/mice), the second group (M) was fed with L-methionine (500 mg/kg/day), the third group (MP) was fed with L-methionine (500 mg/kg/day) in combination with powdered seeds of A. spinosa (150 mg/kg/day), while the group (P) was treated with powdered seeds of A. spinosa (150 mg/kg/day). The experimental diets were given in white bread (0.50 mg/mice) and allowed free access to food and water. After 3 w* of feeding, blood samples were collected after fasting, from the retro orbital plexus into EDTA tubes by using glass capillaries. They were centrifuged immediately, and plasma was frozen under-20 °C until assay time. The experiments were conducted in strict compliance according to ethical principles and provided by the committee for the purpose of control and supervision of Experiments on the animal (CPCSEA).

Determination of homocysteine level

The levels of total homocysteine (t-HCY) were assayed by competitive solid phase chemiluminescence immunoassay.

Tissue homogenate preparation

0.5 g of the liver was homogenized in 2 ml of TBS (Tris 50 mmol, NaCl 150 mmol, pH 7.4). The homogenates were centrifuged at 9000 g for 15 min at 4˚C, and the resultant supernatant was used for determination of: reduced glutathione, the catalase activities and protein concentrations.

Protein quantification

Protein was measured by the method of Bradford (1976) [22], using bovine serum albumin as the standard.

Determination of reduced glutathione (GSH)

The glutathione reduced content in the liver was measured spectrophotometrically by using 5, 5′ -dithiobis-(2 nitrobenzoic acids) (DTNB) as a coloring reagent, following the method described by Weeckbeker and cory (1988) [23].

Determination of catalase (CAT)

Tissue CAT activity was determined according to Aebi’s method (1974) [24]. The principle of the assay based on the determination H2O2 decomposition rate at 240 nm.

Histological sections

After the blood samples collection, the animals were sacrificed and organs designed for morphological analysis (heart, and aortas) were quickly removed, rinsed with saline solution (0.9%), and fixed in formalin 10%. The processed tissues were embedded in paraffin, sectioned at 5 μm thickness, and stained following the haematoxylin eosin staining method.

Statistical analysis

The values obtained were expressed as mean±SEM and subjected to statistical analysis using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test (SPSS version 20). P<0.05 values were considered as significantly difference.

RESULTS

Effects of treatments on HCY

At the third week, plasma HCY of the (M) group achieved higher levels, and the average was 10.04±0.83 μmol/l, significantly higher

than that of the control group (F) (6, 84±0.51 μmol/l) and control positive group (P) (7.1±0.88 μmol/l). However, the combined treatments of A. spinosa powdered seeds with methionine (7.26±0.46 μmol/l) showed a significant decline in serum t-HCY, demonstrating that A. spinosa appears to be effective in preventing the increase of t-HCY levels (fig. 1).

Fig. 1: The interaction of L-methionine and A. spinosa seeds on the plasma homocysteine in mice during 21 d of treatment,

Values are the means±SEM (n); *p<0.05 and **p<0.01

Effects of treatments on hepatic anti-oxidative stress parameter

The present data showed that there is a highly significant depletion in reduced glutathione level (4.48±0.55 n mol/mg protein) (P<0.01) and a significant decrease in catalase activity (45.82±5.83 m mol/mg protein) (P<0.05), for the group (M) in comparison with control group (F), where the reduced GSH level was (8.03±0.55 n mol/mg protein) and the concentration of catalase was (61.37±6.39 m mol/mg protein). However the concentration of reduced GSH increased significantly in the group (P) (7.36±1.28 n mol/mg protein) (P<0.01), and not significantly with the group (MP) (6.1±0.5 n mol/mg protein) (P>0.05). On the other hand, the concentration of catalase increased significantly in the group (MP) (62.26±3.32 m mol/mg protein) (P<0.05), but not significantly in the group (P) (52.16±3.19 m mol/mg protein) (P>0.05) (fig. 2).

Histological impact of A. spinosa powdered seeds on heart and aortas tissues

Our data shows various pathological alterations in the heart and aorta of mice induced by the oral methionine administration.

In the second group (M) which had been fed with 500 mg/kg of methionine, Microscopic observation of heart was characterized by the presence of lysis, and architectural changes of cardiomyocytes as shown in (fig. 3-B and 3-C). In the other groups: (F), (MP) and (P), we have not observed any alteration in contrast to methionine intake group (fig. 3-A, 3-D and 3-E).

In the group (M), the aortic intima showed degeneration and desquamation of endothelial cells with fenestration, we also observed in the media lysis, formation of foam cells laden with small lipid droplets and oval nuclei as illustrated in (fig. 4-B and 4-C), (fig. 5-B) and (fig. 6-B, 6-C, 6-D and 6-E).

However, in the control group (F), the aortic sections have intact endothelium and spindle-shaped meiocytes nuclei as shown in (fig. 4-A, 5-A and 6-A). Also, we have observed intact aorta in the group (MP) treated with L-methionine and A. spinosa (fig. 4-D, 5-C and 6-F), and in the group (P) treated with A. spinosa only (fig. 4-E, 5-D and 6-G).

DISCUSSION

Hyper-homocystenemia can arise from nutritional deficiencies of folate, vitamin B6, and vitamin B12 [25]. Several diseases such as renal and thyroid dysfunction cancer, psoriasis, and diabetes as well as various drugs, alcohol, tobacco, coffee, older age and menopause, are believed to be associated with moderately elevated HCY concentrations [26]. There has been an indication towards a

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significant correlation between HHCY and cardiovascular disease and its complications such as heart attacks and strokes [27]. It is believed that hyperhomocysteinemia leads to endothelial cell damage, reduction in the flexibility of vessels, and alters the process of homeostasis [27]. HHCY may lead to an enhancement of

the adverse effects of risk factors like hypertension, smoking, lipid and lipoprotein metabolism, as well as the promotion of the development of inflammation. Physical activity, moderate alcohol consumption, good folate and vitamin B12 status are associated with lower HCY levels.

Fig. 2: The interaction of L-methionine and A. spinosa seeds on the reduced glutathione levels, and catalase activity in mice during 21 d of treatment, values are the means±SEM (n); *p<0.05, and **p<0.01

Fig. 3: Histological sections of the heart of (A) control, (B), (C) treated with Methionine, (D), treated with Methionine and A. spinosa powdered seeds and (E) treated with A. spinosa powdered seeds for 3 w*. Hematoxylin-Eosin Staining (A, C, D and E X100, and B X 400).

ACC. Architectural Changes of Cardiomyocytes, CMF. Cardiac Muscle Fibers, MCN. Muscle Cell Nuclei, L. Lysis

Fig. 4: Histological sections of the arch aorta of (A) control, (B), (C) treated with Methionine, (D), treated with Methionine and A. spinosa powdered seeds and (E) treated with A. spinosa powdered seeds for 3 w*. Hematoxylin-Eosin Staining (A, B, D and E X100, and C X 400).

FC. Foam Cells, FN. Fibroblast Nuclei, IEND. Intact Endothelium, LD. Lipid Droplets, Lu. lumen, ON. Oval Nuclei, SN. Spindle Nuclei

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Fig. 5: Histological sections of the abdominal aorta of (A) control, (B) treated with Methionine, (C) treated with Methionine and A. spinosa powdered seeds and (D) treated with A. spinosa powdered seeds for 3 w*. Hematoxylin-Eosin Staining (A, B, C and D X100). END.

Endolysis, FN. Fibroblast Nuclei, IEND. Intact Endothelium, LU. Lumen, SN. Spindle Nuclei

Fig. 6: Histological sections of the iliac aorta of (A) control, (B), (C), (D) and (E) treated with Methionine, (F), treated with Methionine and A. spinosa powdered seeds and (G) treated with A. spinosa powdered seeds for 3 w*. Hematoxylin-Eosin Staining (A, B, D and E X100, and C X 400). D. Desquamation, END. Endolysis, F. Fenestration, FC. Foam Cells, FN. Fibroblast Nuclei, IEND. Intact Endothelium, LU. Lumen, ON.

Oval Nuclei, SN. Spindle Nuclei

The current study has shown that high Met-diet in mice during 21 d clearly caused a significant increase in HCY level and decreased the reduced glutathione, and catalase concentrations in liver, meanwhile our results confirm that high level of HCY might be an effect of aorta and heart damages.

Several studies have shown that methionine enriched-diet induced a significant increase of plasma t-HCY [28-31].

Further, we found that the content of GSH and catalase activities in liver tissue was significantly decreased in response to the oral methionine administration.

hyperaccumulation of methionine sulfoxide in the liver may induce more serious oxidative hepatotoxicity in Cth−/− mice, whose levels of several antioxidative cysteine metabolites, including GSH, and taurine/hypotaurine, were all downregulated [30]. HHCY leads to increased oxidative stress via the generation of reactive oxygen species (ROS) which weaken intracellular antioxidation defence systems or elicit intracellular redox-controlled inflammation responses [32].

Glutathione is a key buffer of intercellular oxidative reduction reaction, and its dependent antioxidant enzymes include glutathione S-transferase (GST) and glutathione peroxidase (GPx), which play a fundamental role in cellular defense against reactive free radical and other oxidant species [33]. In addition, it has been shown that Hcy can directly act on catalase and inhibit the breakdown of H2O2 by

conversion of the enzyme into the inactive form [34]. Loss of catalase activity is associated with increased susceptibility to oxidative stress [35-36]. The mechanism of HCY inhibition of catalase is shared with a number of inhibitors including 3-amino-1:2:4:-triazole [37-38] and amyloid-ß [39].

Histological analysis showed that HHCY induced by the high methionine intake prompted an angiotoxic activity on the aorta and cardiac tissue damages. This was observed through the loss and degeneration of endothelium, formation of foam cells in the different sections of the aorta, alteration of the cardiac muscle.

In our experimental situation, is due to elevated HCY levels, which decreases the reduced GSH, and catalase activities, the well-known biomarkers of oxidative stress. Our results are in agreement with [40, 31], who reported that HCY-induced injury to the arterial wall is one of the factors that can initiate the process of atherosclerosis, leading to endothelial dysfunction and eventually to heart attacks and strokes [41-42]. Another work of [43] showed that elevated plasma HCY increase cholesterol synthesis, exerts an angiotoxic action direct to the aorta, by the loss of endothelium and degeneration partly with the dissolution of media cells.

Evidence from animal models of HHCY suggests that endothelial dysfunction is largely due to oxidative stress and decreased bioavailability of NO [44], NO may protect against the onset of vascular diseases [45].

Zerizer et al.


68

HCY promoted oxidative stress through the production of reactive oxygen species (ROS). ROS disrupts endothelial cell integrity, which in turn, can cause endothelial cell damage predispose affected vessels to the subsequent development of atherosclerosis [46].

Studies in several animal species, including rabbits, baboons, and rats, have demonstrated desquamation of endothelial cells, fragmentation of the internal elastic lamina, disruption of elastic fibers, and focal areas of smooth muscle hyperplasia [47-48] has been reported that the combination of high methionine and cholesterol increased the alterations of the arterial wall structures and the thickness of the aortic wall in animal models.

In methionine-treated animals, it was shown an aortic angiotoxic action with alterations not observed in the arterial vascular system of other organs. [49] have reported that acute elevations in plasma HCY after methionine loading causes vessel endothelial dysfunction and this could be reversed by administration of vitamin E in humans.

It has been documented that HCY can interact with different plasma and cellular proteins and by forming mixed disulfide conjugates, alters the physicochemical properties of the affected proteins. This has been also proposed as a potential mechanism for Hcy induced cellular dysfunction [50].

On the other hand, we found that the diet supplemented with the extract crude of A. spinosa was effective in prevention against HHCY in mice exposed to a Met-enriched diet by lowering the concentrations of HCY and increasing the concentrations of glutathione reduced and catalase, indicating that this medicinal plant has the potential to reduce t-HCY levels in vivo.

In addition, other investigators [51-53] reported that catechin, taurine and quercetin supplementation are effective in attenuating the increase of serum HCY level as induced by a Met-enriched diet in rats and mice respectively. The protective effect of argan oil is probably due to its high contents of powerful antioxidants, particularly polyphenols,tocopherols and sterols, which are known as powerful antioxidants [54]. Indeed we demonstrated that powdered seeds of A. spinosa when given in combination with high methionine diet increased significantly the catalase activity, but not significantly the GSH level, indicating its benefic effect in prevention against oxidative stress in vivo. In accordance with our results, [20] demonstrated that argan oil treatment increased the GSH against mercuric chloride induced oxidative stress in experimental rats. The elevated level of GSH protects cellular proteins against oxidation through glutathione redox cycle and directly detoxifies reactive species [55]. Our results showed an increase of catalase activities in group (MP) which administered with L-methionine (500 mg/kg) and treated with A. spinosa (150 mg/kg), these results are in agreement with those of [56], who showed that the activities of cytosolic CAT were significantly higher in Wistar rats treated with argan oil in comparison with untreated rats.

The work of [57, 40] proved that the rats and mice respectively administered with 200 mg/kg during 21 d could damage the aorta and heart tissue and the treatment of these animals with vitamins B9, B12 and B6 and Stachys mialhesi extract corrected these alterations. Another work of [58] established that the high level of Hcy could stimulated the angiogenesis on the arota of rats, and the treatment with the extracts of medicinal plants Stachys mialhesi and Chrysanthemum Macrocarpum could inhibited the angiogenesis.

Antioxidants are emerging as prophylactic and therapeutic agents [59]. Furthermore, another study of [60] suggested that an increased intake of antioxidants appeared to be protective in cardiovascular diseases. Epidemiological studies have shown that consumption of food and beverages rich in phenols can reduce the risk of heart disease by slowing the progression of atherosclerosis principally by protecting LDL from oxidation [61].

CONCLUSION

The current study has shown that the powdered seeds of A. spinosa were effective in attenuating the increase of HCY level, improved the antioxidants defence and prevented the endothelial, cardiac alterations, as induced by a Met-enriched diet in mice. It may be

interesting in the development of new drugs for cardiovascular diseases induced by hyperhomocysteinemia.

ACKNOWLEDGEMENT

The authors are grateful to the MESRS (Ministery of Scientific Research, Algeria).

AUTHORS CONTRIBUTION

BADIAA Aklil carried out the experimental part of the work, performed data analysis and drafted and revised the manuscript. The design of the work and correction of the manuscript was done by the corresponding author Mrs ZERIZER Sakina and ZAHIA Kabouche carried out the phytochemical part. All authors read and approved the final manuscript.

CONFLICT OF INTERESTS

Declared none

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Paciaroni%20M%5BAuthor%5D&cauthor=true&cauthor_uid=11239192�

http://www.ncbi.nlm.nih.gov/pubmed/?term=Cardaioli%20G%5BAuthor%5D&cauthor=true&cauthor_uid=11239192�

http://www.ncbi.nlm.nih.gov/pubmed/?term=Arning%20E%5BAuthor%5D&cauthor=true&cauthor_uid=11239192�

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bottiglieri%20T%5BAuthor%5D&cauthor=true&cauthor_uid=11239192�

http://www.ncbi.nlm.nih.gov/pubmed/?term=Parnetti%20L%5BAuthor%5D&cauthor=true&cauthor_uid=11239192�

Appendices

1- Treatment dose calculation

A. spinosa crude extract given dose (150 mg/kg)

0.15 g 1000g

X g Mouse weight g

A. spinosa crude extract given dose

Methionine given dose (500 mg/kg)

0.50 g 1000g

X g Mouse weight g

Methionine given dose

2- Hematoxylin eiosin staining:

Dip slides in alcohol for 5 minutes;

Rinse with water;

Stain slides in hematoxylin for 4 minutes;

After rinsing, stain with eosin for 10 minutes;

Rinse with water;

Dip slides in alcohol for 1 minute;

After rinsing and drying, the editing is done using xylene

3- PBS preparation

Nacl : 8g

Kcl : 0,2g

NaH2PO4 : 1,15g

KH2PO4 : 0,2g

Mgcl2 6H 2O: 0,1g

Cacl 2 2H 2O : 0,137g

QS: 1 L of water

Table 1: 21 days of average weight of mice.

Groups

D1

D2

D3

D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20 D21

(F) :

Average

weight

(g)

32,10 30,18 28,22 27,82 27,82 27,40 26,98 27,20 27,37 23,88 27,00 26,83 26,85 26,88 26,92 26,92 27,22 26,72 31,70 32,34 32,98

(M) :

Average

weight

(g)

35,01 34,46 33,91 33,67 33,27 33,47 33,88 33,12 33,46 33,81 34,33 33 34,1 34,1 34,39 31,41 28,44 33,59 33,89 34,79 32,04

(MP) :

Average

weight

(g)

31,84 31,74 32,32 32,08 31,7 31,77 32,05 32,33 31,76 32,24 32,49 29,3 28,17 27,03 32,16 31,56 30,41 32,06 32,8 30,45 28,1

(P) :

Average

weight

(g)

30,58 29,97 30,32 30,66 30,74 30 30,81 30,93 31,17 30,86 30,6 31,1 30,2 30,86 30,69 30,51 30,21 29,91 30,33 30,77 31,19

Abstract

Hyperhomocysteinemia (HHcy), oxidative stress and decreased antioxidant capacities

lead to several clinical manifestations and particularly, cardiovascular and liver diseases.

In this study, we evaluated the protective and the preventive effect of the crude extract

of Argania spinosa against HHcy, hyperlipidemia, oxidant status and damages in the aorta,

heart and liver induced by high L-methionine intake in mice.

After 3 weeks of treatments, Hcy concentrations, lipid parameters, liver enzyme

activities, hepatic antioxidant status and histological sections of aorta, liver and heart were

determined. Our results showed that consumption of high L-methionine diet (500mg/Kg) led

to an increase in plasma Hcy, CHO, LDL-c, TG, AST, corresponding with decrease of HDL-

c, reduced GSH, and catalase activity. These were associated with the loss and degeneration

of endothelium, fenestration and formation of foam cells in the media of the aorta, also the

alteration of the cardiac muscle and liver tissue. However, the administration of the crude

extract of A. spinosa (150mg/Kg) in combination with L-methionine ameliorated all these

changes.

Moreover, in the present thesis, we conducted a study in vitro to evaluate the

antibacterial effect of Argan oil on biofilm formation.

The results showed that there was a trend of increasing inhibition of 4 species of

bacteria (Streptococcus mutans, Streptococcus anginosus, Streptococcus intermedius, and

staphylococcus haemolyticus), that belong to gram positive strain as the Argan oil was getting

more concentrated.

Keywords: Homocysteine, Argania spinosa , Antioxidant enzymes, Methionine, Oxidative

stress, Cardiovascular diseases, biofilm.

Résumé

L'hyperhomocystéinémie (HHcy), le stress oxydatif et la diminution des capacités

antioxydantes entraînent plusieurs manifestations cliniques et en particulier des maladies

cardiovasculaires et hépatiques.

Dans cette étude, nous avons évalué l'effet protecteur et préventif de l'extrait brut d’A.

spinosa contre l'HHcy, l'hyperlipidémie, le statut oxydant et des lésions de l'aorte, du cœur

et du foie induits par une forte prise de méthionine chez la souris.

Après 3 semaines de traitement, les concentrations de Hcy, les paramètres lipidiques,

les activités des enzymes hépatiques, le statut antioxydant hépatique et les sections

histologiques de l'aorte, du foie et du cœur ont été déterminés. Nos résultats ont montré que la

consommation d'une diète riche en méthionine (500 mg / kg) peut entraîner une

augmentation des taux de Hcy, CHO, LDL-c, TG, AST, avec une diminution des

concentrations des HDL-c, GSH réduit et des catalases, associés à la perte et à la

dégénérescence de l'endothélium, à la fenestration et à la formation de cellules spumeuses

dans l'aorte, ainsi qu'à l'altération du muscle cardiaque et du tissu hépatique. Cependant,

l'administration de l'extrait brut d’A. spinosa (150 mg / kg) en combinaison avec de la

méthionine a amélioré tous ces changements.

De plus, dans le présent travail, nous avons mené à une étude in vitro pour évaluer

l'effet antibactérien de l'huile d'Argan sur la formation de biofilm.

Les résultats ont montré qu'il y avait une inhibition croissante de 4 espèces de

bactéries (Streptococcus mutans, Streptococcus anginosus, Streptococcus intermedius, and

staphylococcus haemolyticus), appartenant à la souche Gram positif, lorsque l'huile d'Argan

devenait plus concentrée.

Mots-clés: Homocystéine, Argania spinosa, Antioxydant, Méthionine, Stress

oxydant, Maladies cardiovasculaires, Biofilm.

ملخص

وانخفاض مضادات األكسدة تؤدي إلى ظهور سديكاإلجهاد التأ ،فرط الهوموسيستيين في الدم

.األمراض خاصة أمراض القلب واألوعية الدموية والكبد من العديد

A.spinosa رغان ور نبتة األذفي هذه الدراسة، قمنا بتقييم التأثير الوقائي للمستخلص الخام لب

األكسدة باإلضافة إلى مضاداتالهوموسيستيين، ارتفاع مستوى الدهون في الدم وضد ارتفاع مستوي

القلب والكبد الناجمة عن ارتفاع تناول جرعات عالية من ،األضرار النسيجية في الشريان األورطي

.الميثيونين لدى الفئران

،إنزيمات الكبد ،ليبيداتتحاليل ال، الهوموسيستيين بعد ثالثة أسابيع من العالج، تم قياس مستويات

. الكبد والقلب،الشريان األورطي تحضير القطاعات النسيجية لكل من كما تم األكسدةنشاط مضادات

الهوموسيستيين أدى إلى ارتفاع( كغ/مغ/ 055)ميثيونين بالنتائج أن استهالك نظام غذائي غني الأظهرت

، (AST) و (TG) الدهون الثالثية ، (LDL-c)المنخفض الكثافةالكولسترول و الكوليسترول، في الدم

الجلوثاثيون، و نشاط انزيم ، (HDL-c)العالي الكثافةالكولسترول انخفاض في مستوىمع تسجيل

تزامنت هذه النتائج مع فقدان وتدهور البطانة ، نوفذة وتشكيل خاليا رغوية في الشريان . الكاتاالز

كل من اخذ أنفي حين بينت النتائج . وأيضا تم تسجيل تغيير في عضلة القلب وأنسجة الكبد األورطي،

.تحسين كل هذه التغييرات إلى أدىمع الميثيونين معا ( كغ/مغ005)األرغان ور ذالمستخلص الخام لب

من جهة أخرى ، أجرينا دراسة أخرى من اجل تقييم تأثير مضاد للجراثيم لزيت األرغان ضد

.شكيل البيوفيلمت

Streptococcus)أنواع من البكتيريا 4وقد أظهرت النتائج أن زيت األرغان أدى إلى تثبيط

mutans, Streptococcus anginosus, Streptococcus intermedius, and

(Staphylococcus haemolyticus,إيجابية و ذلك كلما كان أكثر غرام كتيرياي، التي تنتمي إلى ب

.تركيزا

:الكلمات المفتاحية

، سديكاإلجهاد التأ ، اإلنزيمات المضادة لألكسدة، ميثيونين،Argania spinosaالهوموسيستيين،

.أمراض القلب واألوعية الدموية، البيوفيلم

Academic year: 2017-2018 Family Name: AKLIL

First Name: BADIAA

Title: The effect of Argania spinosa on plasma Homocysteine, Lipids,

Antioxidant enzymes and Aortas sections in methionine induced

Hyperhomocysteinemia in mice

Thesis submitted for the degree of DOCTORAT IN SCIENCES

Hyperhomocysteinemia (HHcy), oxidative stress and decreased antioxidant capacities

lead to several clinical manifestations and particularly, cardiovascular and liver diseases.

In this study, we evaluated the protective and the preventive effect of the crude

extract of Argania spinosa against HHcy ,hyperlipidemia, oxidant status and damages in

the aorta, heart and liver induced by high L-methionine intake in mice.

After 3 weeks of treatments, Hcy concentrations, lipid parameters, liver enzyme

activities, hepatic antioxidant status and histological sections of aorta, liver and heart were

determined. Our results showed that consumption of high L-methionine diet (500mg/Kg)

led to an increase in plasma Hcy, CHO, LDL-c, TG, AST, corresponding with decrease of

HDL-c, reduced GSH, and catalase activity. These were associated with the loss and

degeneration of endothelium, fenestration and formation of foam cells in the media of the

aorta, also the alteration of the cardiac muscle and liver tissue. However, the administration

of the crude extract of A. spinosa (150mg/Kg) in combination with L-methionine

ameliorated all these changes.

Moreover, in the present thesis, we conducted a study in vitro to evaluate the

antibacterial effect of Argan oil on biofilm formation.

The results showed that there was a trend of increasing inhibition of 4 species of

bacteria (Streptococcus mutans, Streptococcus anginosus, Streptococcus intermedius, and

staphylococcus haemolyticus), that belong to gram positive strain as the Argan oil was

getting more concentrated.

Keywords: Homocysteine, Argania spinosa, Antioxidant enzymes, Methionine, Oxidative

stress, Cardiovascular diseases, biofilm.

Documents

The effect of Argania spinosa on plasma Homocysteine ... · 2-Effect of treatment on Hcy levels, lipid profile, liver enzyme activities and antioxidants markers in mice The current