1. ‫الرحي‬ ‫الرحمن‬ ‫هللا‬ ‫بسم‬
‫م‬
‫عل‬ ‫ما‬ ‫إال‬ ‫لنا‬ ‫علم‬ ‫ال‬ ‫سبحانك‬ ‫قالوا‬
‫إنك‬ ‫متنا‬
‫الحكيم‬ ‫العليم‬ ‫أنت‬
‫العظي‬ ‫هللا‬ ‫صدق‬
‫م‬
‫آية‬ ‫البقرة‬ ‫سورة‬
32

2. Biochemical studies on
the effect of Olive leaf
extracts on blood lipid
level in male albino mice
By
Amira Y. Taha
Supervisor
PRO. Maraia, F. Elmhdwi

3. Contents
• Introduction.
• Aim of the work.
• Materials and methods.
• Results.
• Conclusion.

4. Introduction

5. Introduction
• During metabolism, some oxygen molecule escape and become highly
unstable and reactive particles called free radicals.
• Free radical are essential for your survival. They perform many
functions including controlling your blood flow, fighting infection, even
killing cancer cells.
• Fortunately, the body maintains a sophisticated system of chemical
and biochemical defenses to control and neutralize free radicals.
Chemical antioxidants scavenge free radicals, that is, they stabilize
the unstable free radicals by giving them the electron they need

7. Health body
free radicals
antioxidant

9. Oxidative stress
• Oxidative stress, arising as a result of an imbalance
between free radical production and antioxidant
defenses, is associated with damage to a wide range
of molecular species including lipids, proteins, and
nucleic acids

10. Illnesses Associated With
Oxidative Stress
Free radicals
oxidative stress Skin:
melanoma
Blood vessels:
atherosclerosis
Brain:
Alzheimer
disease

11. Hyperlipidemia
• Hyperlipidemia is a term that refers to high level of
lipids (fats, cholesterol and triglycerides) circulating in
the blood. These lipids can enter the walls of arteries
and increase the risk of atherosclerosis
• Atherosclerosis is a disease process leading to
hardening and narrowing of arteries. The buildup of
fat, cholesterol, and other substances creates plaques
inside arteries, which can lead to many problems
including heart attack, stroke, and death

12. Atherosclerosis

13. Non enzymatic
enzymatic
Vitamin C
Superoxide Dismutase
Carotenoids
Catalase
Polyphenolic
Glutathione Reductase
Vitamin E
Glutathione Peroxidase
Types of antioxidants

15. Aim of the study
• the aim of this study was to determine
the antioxidant activity of the two
extracts(methanol and acetone)from
olive leaves (OLE) , also to investigate
the hypolipidemic effect of the
methanol extract on experiment animals

16. Materials and Methods

17. Material
• Plant material
The leaves of olive, were collected from Al-Jabal Al
Akhdar area in Libya during 2017. The leaves were
dried in laboratory and powder in a mixture grinder
• Animals:
eighty four healthy adult male albino mice weighing
between 20-40 g were used for this study

18. Olive leaves
Olive leaf is the leaf of the tree (olea europaea)
active compounds:
• Phenolics
- oleuropein
• Flavonids
- Elenolic acid
- luteolin

19. Historically
• The benefities of olive leaves have been used in
traditional medicine practices as folk remedies for the
treatment of variouse illnessess.
• Oleuropein a polyphenol with unique health-
improving attributes
• These extracts have been used in traditional medicine
for centuries to improve age related disease

20. Pharmacological effects of
oleuropein

21. Atorvastatin
Atorvastatin is one of the yield products which considered as a standard
hypolipidemic agent.

22. Methods
• Sample preparation (extraction
process):
The extract was obtained by macerating 50 g of the
dried leaves from olive leaves separately in
acetone(500 ml) and methanol (500 ml) for 3days.
The resultant extract was filtered, concentrated to
dryness in avacumme under reduced pressure at 40
°C, and then stored at -8 °C until use

23. Extract analysis
• Extract analysis
• GC-MS analysis.
• Measurement of the antioxidant activity of the methanol
extract by estimate:
-Total Phenolic Content.
-Total flavonoid content.
-Reducing Power assay.
-Scavenging Activity by DPPH-Radical
- Determination of lethal dose LD50

24. Induction of hyperlipidemia
• Hyperlipidemia was induced in male Swiss albino
mice by using cholesterol/cholic acid mixture (3:1)
and mixed with the synthetic diet in a dose calculated
on the basis that each mouse was received 0.5g of
this mixture/kg b.w daily for 2 weeks.

25. Ingredient Standard diet Hyperlipidemic diet
Casein 10 10
Corn oil 4 --
Saltmixture(a)
4 4
Vitaminmixture(b)
1 1
Sucrose 40.4 50
Fiber 40.4 20.8
Cholin Cl-
0.2 0.2
Cholesterol -- 3
Cholicacid -- 1
SaturatedFat -- 10

26. EXPERMENTAL
DESIGN

27. The prophylactic effect of
extracts against hyperlipidemia
• To study the protective effect of the
oils against hyperlipaemia, a total of
36 mice were used and the experiment
lasted for 2 weeks. Animals were
divided randomly into three groups
(12 mice each) as follows:

28. Group 1: mice were fed on the standard synthetic diet
(S.d) and served as negative control group (-ve).
Group 2: mice were daily attained to the hyperlipidemic
diet (H.L.D) and served as positive control group (+ve).
Group 3: mice were daily administered methanol extract a dose
of 500 mg/kg b.w oral (added to the H.L.D).

29. The curative effect of extracts on
hyperlipidaemic rats
• In this experiment, a total of 48 mice were used. 12 mice were
fed on the standard synthetic diet and served as negative
control(- ve)" Group1 "
• .The other mice were subjected to the induction of
experimental hyperlipaemia for 2 weeks as described before.
• The hyperlipidaemic mice (
48
) were divided randomly into
equal 4 sub-groups (12 mice each) as follows:
Group 2:mice were served as hyperlipidaemic animals(+ve)
Group 3:mice were daily received methanol extract at a dose of
(500 mg/kg b. w) (oral+ S.d).
Group 4:mice were daily received of Atorvastatin as a standard
hypolipidaemic agent 0.9 mg/kg b.w. (oral)

30. Blood sampling
• In the first experiment, blood samples were collected
before treatment and then after 2 weeks. In the second
experiment, blood samples were collected before and after
induction of hyperlipidemia and then again 2 weeks after
the administration of the different treatments.

31. Biochemical analysis
Determination of serum total cholesterol.
Determination of serum HDL-Cholesterol.
Determination of serum LDL-Cholesterol and VLDL-
Cholesterol.
Determination of serum triglycerides.
Determination of S. Alanine aminotransferase (ALT).
Determination of S. Aspartate aminotransferase (AST).
Determination of serum alkaline phosphatase (ALP).
Determination of serum total protein (TP).
Determination of serum Gamma-Glutamyltransferase (GGT).

32. Determination of superoxide dismutase (SOD)
Determination of Glutathione Reductase (GR).
Determination of Glutathione Peroxidase (GPx).
Determination of Catalase (CAT).
Determination of Malondialdehyde (MDE)

33. At the end of experiments, animals
in all groups were
dissected for postmortem
observation for
histopathological studies. Liver,
kidney and
heart were removed and fixed in
10% neutral formalin.

34. RESULTS

35. Antioxidant evaluation
of the two extracted
from olive leaves

36. Total phenolic content (TPC)

37. Antioxidant evaluation of the two
extracts from olive leaves
Total phenolic content (TPC)
.

38. Total flavonoids content of
qurecetin

39. Total flavonoids content of
the two extracts from olive
leaves (TFC)

40. Reducing power assay of
Vit C (RPA)

42. The DPPH radical scavenging
activity
• The results of the DPPH radical scavenging activity
of acetone and methanol extracts are shown in the
figure, these results compared with the well-known
antioxidant ascorbic acid where the percent of the
inhibition is 96.7% at 500 µg/ml of the vitamin C ;
98% at 500 µg/ml of the methanol extract and 95%
at 500µg/ml of acetone extract.

43. The DPPH radical scavenging
activity of the two extracts
from olive leaves

44. the chemical composition of the acetone
extracted from olive leaves.
As can be seen from GC.MS compounds
representing about 99.78%. The major
components are as follows: α–pinene
(34.70), Oleuropein (24.70), 2,6-
Dimethyloctane(10.57) and
a-2-Methoxy-3-isopropylpyrazine (6.01)

45. Conc. (%)
R.I.
RT
Constituent
1.51
868
63.40
(z)-3-hexanol
1.26
875
67.36
(E)-2-Hexenol
24.70
885
68.90
Oleuropein
34.70
910
70.24
α–pinene
2.46
958
81.40
β– pinene
0.61
1012
63.346
(Z)-3-Hexenyl acetate
6.01
1045
66.857
2-Methoxy-3-isopropylpyrazine
1.46
1159
69.309
(E,Z)-2,6-Nonadienal
0.74
1163
70.479
(E,Z)-2,6-Nonadienol
0.45
1171
78.775
1,4-Dimethoxybenzene
1.05
1198
97.798
Decanal
10.57
1210
47.336
2,6-Dimethyloctane
0.50
1233
47.695
2,4-Dimethyldodecane
0.43
1281
48.941
2,6,11-Trimethyldodecane
1.45
1400
53.080
α-ionone
0.84
1465
54.436
α-humulene
0.78
1471
55.782
1-Dodecanol
0.89
1499
55.861
β -Ionone
0.65
1760
58.396
Hexadecanoicacid
0.81
1860
59.550
Oleic acid
3.58
2210
60.770
Docosane
4.38
2340
60.892
Tetracosane

46. Biological
experimental

47. Effect of methanol extract in
prophylactic experiment

48. Biological
experimental
Determination of lethal dose LD50

49. Effect of methanol extract in
prophylactic experiment

50. Effect of methanol extract
from olive leaves on HDL-
Cholesterol

51. Effect of methanol extract from
olive leaves on
(T.CHOL/HDL.CHOL)

52. Effect of methanol extract
from olive leaves on VLDL-
Cholesterol

53. Effect of methanol extract
from olive leaves on LDL-
Cholesterol

54. Effect of methanol extract
from olive leaves on
S.Triglycerides

55. Effect of methanol extract
from olive leaves on S.ALT

56. Effect of methanol extract
from olive leaves on S.AST

57. Effect of methanol extract
from olive leaves on S.GGT

58. Effect of methanol extract
from olive leaves on S.ALP

59. Effect of methanol extract
from olive leaves on S.TP

60. Effect of methanol extract
from olive leaves on plasma
SOD

61. Effect of methanol extract
from olive leaves onplasma
MDA

62. Effect of methanol extract
from olive leaves on plasma
GR

63. Effect of methanol extract
from olive leaves on
plasmaGPX

64. Effect of methanol extract
from olive leaves on plasma
CAT

65. Effect of methanol extract
from olive leaves on blood
urea

66. Histopathology

67. The liver tissue of control
mice , showing normal
histology
.
The liver tissue of positive control
mice,showing micro vesicular fatty
change distributed throughout the
liver lobules accompanying with
focal necrosis

68. The liver tissue of mice
treated the methanol
extract of olive leaves
(500mg/kg b. w ),
showing normal histological
change with reduction in fat
deposits in liver tissues

69. The heart tissue of control
mice for 2 weeks , showing
normal architecture.e
.
The heart tissue of The
positive control mice for 2
weeks , showing mild
groups of fatty cell

70. The heart tissue of group treated with the treated the
methanol extract of olive leaves (500mg/kg b. w ) for 2
weeks, showing vacuolar changes in myocardial
muscles and improves compared to the positive group

71. The kidney tissues of control
group showing normal renal
structure with regulatednuclear
arrangement of uriniferous
tubules
The kidney tissue of positive control
mice degenerative changes in
epithelial lining of convoluted tubules

72. The kidney tissue of mice treated the
methanol extract of olive leaves (500mg/kg b. w
), showing significantly improved renal
morphology compared to the positive group

73. Effects of methanol extract
from olive leaves in curative
experiment

74. The curative effect of methanol
extract from olive leaves on
S.T.cholesterol

75. The curative effect of methanol
extract from olive leaves on
S.T.HDL

76. The curative effect of methanol
extract from olive leaves on
T.CHOL/HDL.CHOL

77. The curative effect of methanol
extract from olive leaves on
S.LDL.Chol

78. The curative effect of methanol
extract from olive leaves on
S.VLDL-Chol

79. The curative effect of methanol
extract from olive leaves on
S.Triglycerides

80. The curative effect of methanol
extract from olive leaves on blood
urea

81. The curative effect of methanol
extract from olive leaves on S.ALT

82. The curative effect of methanol
extract from olive leaves on S.AST

83. The curative effect of methanol
extract from olive leaves on
S.GGT

84. The curative effect of methanol
extract from olive leaves on S.ALP

85. The curative effect of methanol
extract from olive leaves on
S.T.protein

86. The curative effect of methanol
extract from olive leaves on
plasma SOD

87. The curative effect of methanol
extract from olive leaves on
plasma MDA

88. The curative effect of
methanol extract from olive
leaves on plasma GR

89. The curative effect of methanol
extract from olive leaves on
plasma GPX

90. The curative effect of methanol
extract from olive leaves on
plasma CAT

91. Hepatic tissue suffering hyperlipidemia
showing fatty liver tissue with disrupted
celvacuolated cytoplasm and necrosis
with disrupted hepatic strands (arrows)
Hepatic tissues of control group
showing hepatic strands of cells
(arrow).

92. Hepatic tissues pretreated
methanol extracted
from olive leaves shows be
restoring the normal
appearance of
hepatic strands (arrow).
Hepatic tissues pretreated
Atorvastatin (0.9mg/kg b.
w)shows no necrotic
hepatic tissues with normal
hepatic strands
(arrows).

93. • Heart tissues of control group
showing normal structure
of cardiac muscles consist of
muscle fibers

94. The heart tissue of methanol
extract of olive leaves (500 mg/kg
.b.w.) for 2 weeks, showing
vacuolar degenerative changes
in myocardial muscles
Representing the heart
of positive control mice for
2 weeks and showing
mild groups of fatty cells
(arrow).
The heart of Atorvastatin treated
(0.9mg/kg.bw) for2 weeks, showing
dilated and congested Blood
vessels (arrow) .

95. Renal tissues of control group
showing normal renal structure
The kidney of positive control mice
revealing focal areas of vacuolar
degenerative changes in epithelial
lining of convoluted tubules (arrow).
aphotomicrograph of a kidney
section of methanol extract of olive
leaves treated (500mg/kg. b.w.),
showing few aggregates of
inflammatory cells (arrow) in
perivascular channels and hyaline
casts (arrow head) in the Lumen of
some Lubules

96. The kidney of atorvastatin treated (0.9mg/kg.b.w.) for 2
weeks, showing few calcium depositis (arrow) in some
number of collecting tubules

97. Conclusion
• The results obtained revealed that the methanol
extracted from olive leaves has an antioxidant activity,
in addition to prophylactic and curative effect
against hyperlipidemia compared with the reference
standard atorovastatin˝.
• In general, to use these extracted as safe prophylactic
and curative agents, more studies should be carried
out to know all the active / inactive components and
their mechanism of actions