باسط ,كويري

‫بسم هللا الرحمن الرحيم‬
‫قالوا سبحانك ال علم لنا إال ما علمتنا إنك‬

‫أنت العليم الحكيم‬
‫صدق هللا العظيم‬
‫سورة البقرة آية‬
‫‪32‬‬
Biochemical studies on
the effect of punica peels
extracts on blood lipid
level in male albino mice
By
Abdelbaset mohammed
Supervisor
PRO. Maraia, F. Elmhd wi
Contents
• Introduction.
• Aim of the work.
• Materials and methods.
• Results.
• Conclusion.
Introduction
What are free radicals?
• Any molecule containing one or more
unpaired electrons.
• These unpaired electrons readily form free
radical molecules which are chemically
reactive and highly unstable.
Generation of Free Radical
1. Cellular metabolism
• About 1-4% of oxygen taken up in the body is converted to
free radicals. They are constantly produced during the normal
oxidation of foodstuffs.
a) due to leaks in the electron transport chain in mitochondria.
b) Some enzymes such as xanthine oxidase and aldehyde
oxidase form superoxide anion radical or hydrogen peroxide.
c) Macrophage also produces NO from arginine by the enzyme
nitric oxide synthase. This is also an important anti-bacterial
mechanism.
Generation of Free Radical
2. Environmental effects:
a) due to drug metabolism.
b) due to damages caused by UV or X-rays
c) cigarette or alcohol.
Antioxidant
• The substance present in low concentrations relative to the
oxidizable substrate that significantly delays or reduces oxidation of
the substrate.
• They reduce the effect of dangerous oxidants by binding
together with these harmful molecules, decreasing their
destructive power.
• They can also help repair damage already sustained by cells.
• They may be considered as the scavengers of free radicals.
Antioxidants
• Prevents the transfer of electron from O2 to organic molecules

• Stabilizes free radicals
• Terminates free radical reactions
Classification of antioxidant
Ⅰ. According to their location
a) Plasma antioxidants:
– ascorbic acid (Vitamin C), bilirubin, uric
acid, transferrin, ceruloplasmin, β-
carotene;
b) Cell membrane antioxidants:
– α-tocopherol (Vitamin E)
c) Intracellular antioxidants:
– superoxide dismutase (SOD), catalase,
glutathione peroxidase (GPx)
Classification
Ⅱ. According of and
to their nature antioxidant
action
a) Enzymatic antioxidants:
– SOD, catalase, GPx, glutathione reductase
b) Non-enzymatic antioxidants:
– Nutrient antioxidants:
• β-carotene, α-tocopherol, ascorbic
acid,
– Metabolic antioxidants:
• bilirubin, uric acid, ceruloplasmin,
ferritin, transferrin, albumin,
glutathione
Health body
antioxidant free radicals
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
Oxidative Damage
Free Radicals
Proteins Lipids DNA/RNA

(-SH) (R-OO.) (-OH.)
Harmful effects of free radicals
B. Diseases
1. Cardiovascular diseases (CHD): ox-LDL, formed by
the action of free radicals, promote CHD and
atherosclerosis (AS).
2. Cancers: damage DNA and cause mutation and
cytotoxicity, play a key role in carcinogenesis.
3. Inflammatory diseases: damage on the extracellular
components such as collagen and hyaluronic acid, promote
glomerulonephritis and ulcerative colitis.
4. Respiratory diseases: destroy endothelium and cause
lung edema. Cigarette smoke contains free radicals and
promotes the production of more free radicals.
Harmful effects of free radicals
B. Free Radical and diseases
5. Diabetes mellitus: Destruction of islets results in
pathogenesis.
6. Cataract
7. Male infertility: reduce sperm motility and
viability.
8. Aging process
9. Others: such as Parkingson’s disease, Alzheimer’s
disease, multiple sclerosis, liver cirrhosis, muscular
dystrophy.
Illnesses Associated With
Oxidative Stress
Free radicals
:Brain oxidative stress :Skin
Alzheimer melanoma
disease
:Blood vessels
atherosclerosis
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
Aim of the study
• the aim of this study was to determine
the antioxidant activity of the two
extracts(aqueous and acetone)from
punica peels(pp) , also to investigate
the hypolipidemic effect of the
aqueous extract on experiment
animals
Materials and Methods
Material
• Plant material
The peels of punica, were collected from benghazi
area in Libya during 2017. The peels 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
Taxonomy
• Kingdom: Plantae
• Phylum: Tracheophyta
• Class: Magnoliopsida
• Order: Myrtales
• Family: Punicaceae
• Genus: Punica
• Species: granata
http://forum.canucks.com/lofiversion/index.php/t184247.html
Modern Uses
• Powerful Antioxidant
• Has strong degree of free radical scavenging,
absorbs oxygen radicals, lowers LDL levels in blood
• Shows in vitro anticancer properties
www.pomwonderful.com/juice.html
Atorvastatin
Atorvastatin is one of the yield products which considered as a standard
hypolipidemic agent.
Methods
• Sample preparation (extraction
process):
The extract was obtained by macerating 50 g of the
dried peels from punica granatum separately in
aqueoues (500 ml) and acetone (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
Extract analysis
• Extract analysis
• HPLC-MS analysis.
• Measurement of the antioxidant activity of aqueous extract
by estimate:
-Total Phenolic Content.
-Total flavonoid content.
-Reducing Power assay.
-Scavenging Activity by DPPH-Radical
- Determination of lethal dose LD50
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.
Ingredient Standard diet Hyperlipidemic diet
Casein 10 10
Corn oil 4 --
Salt mixture(a) 4 4
Vitamin mixture(b) 1 1
Sucrose 40.4 50
Fiber 40.4 20.8
Cholin Cl- 0.2 0.2
Cholesterol -- 3
Cholic acid -- 1
Saturated Fat -- 10
The prophylactic effect of
extracts against hyperlipidemia
• To study the protective effect of the
aqueous extract 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:
Group 1: mice were fed on the standard synthetic diet
.and served as negative control group (-ve) )S.d(
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 aqueous extract a

dose
.of 300 mg/kg b.w oral (added to the H.L.D)
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 (36) were divided randomly
into equal 3 sub-groups (12 mice each) as follows:
Group 2:mice were served as hyperlipidaemic animals(+ve)
Group 3:mice were daily received aqueous extract at a dose of
(300 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)
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.
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).
Determination of superoxide dismutase (SOD)
.Determination of Glutathione Reductase (GR)
.Determination of Glutathione Peroxidase (GPx)
.Determination of Catalase (CAT)
Determination of Malondialdehyde (MDA)

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.
RESULTS
Antioxidant evaluation
of the two extracted
from punica peels
Total phenolic content (TPC)
Antioxidant evaluation of the two
extracts from punica peels
Total phenolic content (TPC)
.
Total flavonoids content of
qurecetin
Total flavonoids content of the
two extracts from punica peels
(TFC)
Reducing power assay of
Vit C (RPA)
The DPPH radical scavenging
activity
• The results of the DPPH radical scavenging
activity of acetone and aqueous extracts are shown
in the figure, these results compared with the well-
known antioxidant ascorbic acid where the percent
of the inhibition is 92.3%at 500 µg/ml of the
vitamin C ; 98.7%at 500 µg/ml of the aqueous
extract and 93.2% at 500µg/ml of acetone extract.
The DPPH radical scavenging
activity of the two extracts from
punica peels
the chemical composition of the acetone extracted
from punica peels. As can be seen from HPLC.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)
Constituent RT R.I. Conc. (%)
(z)-3-hexanol 63.40 868 1.51
(E)-2-Hexenol 67.36 875 1.26

Oleuropein 68.90 885 24.70
α–pinene 70.24 910 34.70

β– pinene 81.40 958 2.46
(Z)-3-Hexenyl acetate 63.346 1012 0.61

2-Methoxy-3-isopropylpyrazine 66.857 1045 6.01
(E,Z)-2,6-Nonadienal 69.309 1159 1.46
(E,Z)-2,6-Nonadienol 70.479 1163 0.74
1,4-Dimethoxybenzene 78.775 1171 0.45

Decanal 97.798 1198 1.05
2,6-Dimethyloctane 47.336 1210 10.57

2,4-Dimethyldodecane 47.695 1233 0.50
2,6,11-Trimethyldodecane 48.941 1281 0.43

α-ionone 53.080 1400 1.45
α-humulene 54.436 1465 0.84

1-Dodecanol 55.782 1471 0.78
β -Ionone 55.861 1499 0.89

Hexadecanoicacid 58.396 1760 0.65
Oleic acid 59.550 1860 0.81

Docosane 60.770 2210 3.58
Tetracosane 60.892 2340 4.38

Biological
experimental
Effect of aqueous extract in
prophylactic experiment
Biological
experimental
Determination of lethal dose LD50
Effect of aqueous extract from
punica peels on S.T

90
cholesterol zero time
80 14 days
S. T. Choleterol (mg/dl)
70
60
50
40
30
20
10
0
Control Positive aqueous
extract(300mg/kg b. w)
The prophylactic effect of aqueous
extract on S. T. Cholesterol.
punica peels on HDL-
Cholesterol
35 zero time
14 days
S. T. Cholesterol (mg/dl)
30
25
20
15
10
0
Control Positive control Aqueous extract
(300mg/kg b.w)
The prophylactic effect of aqueous extract on S. T. HDL Cholesterol

Effect of aqueousextract from
punica peels on
(T.CHOL/HDL.CHOL)
zero time
4.5 14 days
4
(T.CHOL / HDL. CHOL)
3.5
3
2.5
2
1.5
1
0.5
0
Control Positive aques extract( 300 mg/kg
b.w)
The prophylactic effect of aques extract of punica peels on ( T.
CHOL / HDL. CHOL.)
punica peels on VLDL-
Cholesterol
zero time
14 days
20
18
S. VLDL Cholesterol (mg/dl)
16
14
12
10
8
6
4
2
0
Control Positive aqueous extract
(300mg/kg b.w)
The prophylactic effect of fixed oil on S.VLDL.Cholesterol.
punica peels on LDL-
Cholesterol
zero time
50 14 days
45
S. T. LDL Cholesterol (mg/dl)
40
35
30
25
20
15
10
5
0
Control Positive Aqueous extract
(300mg/kg b.w)
The prophylactic effect of fixed oil on S. T. LDLCholesterol
punica peels on Triglycerides
100
zero time
S. Triglycerides( mg/dl)
90 14 days
80
70
60
50
40
30
20
10
0
Control Positive aques extract
(300mg/kg b. w)
The prophylactic effect of aques extract of punica peelson S. T.

Triglycerides.
punica peels on S.ALT
zero time
60
14 days
50
S. ALT (U/ml)
40
30
20
10
0
Control Positive aques extractl
(300mg/kg b. w)
The prophylactic effect of aques extract of punica peels on S. ALT

punica peels on S.AST
zero time
100
14 days
90
80
70
S. AsT (U/ml)
60
50
40
30
20
10
0
(300mg/kg b. w)
The prophylactic effect of aqueous extract of punica peels on S. AsT
punica peels on S.GGT
zero time
30
14 days
25
20
S. GGT (U/L)
15
10
0
(300mg/kg b. w)
The prophylactic effect of aqueous extract of punica peels on S. GGT
punica peels on S.ALP
zero time
80
14 days
70
60
S. ALP (IU/L)
50
40
30
20
10
0
(300mg/kg b. w)
The prophylactic effect of aqueous extract on S. ALP

punica peels on S.TP
zero time
7
14 days
6
5
S. TBIL ( g/dl)
0
(300mg/kg b. w)
The prophylactic effect of aqueous extract on S. TBIL
punica peels on plasma SOD
zero time
14 days
9
8
plama SOD. (U/mol)
7
6
5
4
3
2
1
0
(300mg/kg b. w)
The Prophylactic effect of aqueous extract on plasma SOD
punica peels on plasma MDA
zero time
14 days
30
plama MDA. (nmol/ml)
25
20
15
10
0
(300mg/kg b. w)
The Prophylactic effect of aqueous extract of punica peels on plasma
MDA.
punica peels on plasma GR
zero time
14 days
20
18
16
plasma GR. (U/L)
14
12
10
8
6
4
2
0
(300mg/kg b. w)
The Prophylactic effect of aqueous of punica peels on plasma GR.
punica peels on plasm GPX
zero time
14 days
35
30
plasma GPX. (mu/ml)
25
20
15
10
0
(300mg/kg b. w))
GPX.
punica peels on plasma CAT
zero time
14 days
40
35
plasma CAT. (U/L)
30
25
20
15
10
5
0
(300mg/kg b. w)
CAT.
punica peels on blood urea
zero time
40
14 days
35
30
S. TBIL ( g/dl)
25
20
15
10
0
(300mg/kg b. w)
The prophylactic effect of aqueous extract on S. TBIL
Histopathology
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
The liver tissue of mice treated
the aqueous extract of punica peels
(300mg/kg b. w ), showing normal
histological change with reduction in fat
deposits in liver tissues
.
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
The heart tissue of group treated with the treated the aqueous extract of
punica peels (300mg/kg b. w ) for 2 weeks, showing vacuolar
changes in myocardial muscles and improves compared to the positive
group
The kidney tissues of control group showing
normal renal structure with regulated nuclear
arrangement of uriniferous tubules
The kidney tissue of positive control mice

degenerative changes in epithelial
lining of convoluted tubules
The kidney tissue of mice treated the aqueous extract of punica peels
(300mg/kg b. w ), showing significantly improved renal morphology compared
to the positive group
Effects of aqueous extract from
Ponica peels in curative
experiment
The curative effect of aqueous
extract from punica peels on S.T.
cholesterol
160
Before
After
140
120
S.T.Cholesterol (mg/dl)
100
80
60
40
20
0
Control Positive aqueous extract (300 Atorvastatin
mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract aS.T. Cholesterol.
extract from punica peels on
S.T.HDL
40 before
after
35
S. HDL-Cholesterol (mg/dl)
30
25
20
15
10
0
Control Positive aqueous extract Atorvastatin
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S.HDL-Cholesterol.
T.CHOL/HDL.CHOL
9
Before
8 After
7
T. CHOL/ HDL-CHOL
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on T. CHOL/ HDL-CHOL.

S.LDL.Chol
120
Before
After
100
S. LDL-Chol ( mg/dl)
80
60
40
20
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. LDL-Chol.

S.VLDL-Chol
Before
30
After
25
S. VLDL-Chol ( mg/dl)
20
15
10
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. VLDL-Chol.

S.Triglycerides
140
Before
After
120
S. Triglycerides ( mg/dl)
100
80
60
40
20
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. Triglycerides.

extract from punica peels on blood
urea
S.ALT
70
Before
After
60
50
S. ALT ( U/ ml)
40
30
20
10
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. ALT.

S.AST
140
Before
After
120
100
S. AST ( U/ ml)
80
60
40
20
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqeuous extracton S. AST

S.GGT
30
Before
After
25
20
S. GGT ( U/L)
15
10
0
Control Positive aqueous extractl Atorvastatin
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. GGT

S.ALP
Before
70
After
60
50
S. ALP ( IU/L)
40
30
20
10
0
Control Positive aqueous extract( Atorvastatin
300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. ALP.

S.T.protein
6.4
Before
After
6.2
S. T. Protein (g/dl)
5.8
5.6
5.4
5.2
5
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on S. T. Protein.

plasma SOD
8 Before
After
7
6
Plasma SOD.(u/mol)
0
(300 mg/b.w) (0.9mg/kg b. w)
The curative effect of aqueous extract on plasma SOD.
plasma MDA
50 Before
45 After
40
Plasma MDA.(nmol/ml)
35
30
25
20
15
10
5
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on plasma MDA.
extract from punica peels on GR
30 Before
After
25
Plasma GR.(U/L)
20
15
10
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous extract on plasma GR.
plasma GPX
40 Before
35 After
30
Plasma GPX.(mu/ml)
25
20
15
10
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueoes extract on plasma GPX.
plasma CAT
60
Before After
50
Plasma CAT.(U/L)
40
30
20
10
0
(300mg/kg b. w) (0.9mg/kg b. w)
The curative effect of aqueous on plasma CAT.
Hepatic tissues of control group
showing hepatic strands of cells
Hepatic tissue suffering hyperlipidemia

showing fatty liver tissue with disrupted
celvacuolated cytoplasm and necrosis
with disrupted hepatic strands (arrows )
Hepatic tissues pretreated aqueous
extracted from punica
showing aggregation of
lymphocytes in portal areas.
Effect of atorvastatin treated

(0.9mg/kg .b. w.) for 2 weeks, on
lipid accumulation in livers mice
showing the disappearance of fat
cells compared to the positive
• Heart tissues of control group showing normal
structure of cardiac muscles consist of
muscle fibers
Representing the heart of positive
control mice for 2 weeks and
showing mild groups of fatty cells
The heart tissue of aqueous extract of

punica peels (300 mg/kg .b.w.) for
2 weeks, showing vacuolar
degenerative changes in myocardial
muscles
The heart of Atorvastatin treated

(0.9mg/kg.bw) for2 weeks, showing
dilated and congested Blood
vessels (arrow) .
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 aqueous

extract of punica peels treated (300mg/kg. b.w.),
showing few aggregates of inflammatory cells
(arrow).
The kidney of atorvastatin treated (0.9mg/kg.b.w.) for 2
weeks, showing few calcium depositis (arrow) in
some number of collecting tubules
Conclusion
• The results obtained revealed that the aqueous
extracted from punica peels 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

باسط ,كويري

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‫بسم هللا الرحمن الرحيم‬

‫قالوا سبحانك ال علم لنا إال ما علمتنا إنك‬

• Prevents the transfer of electron from O2 to organic molecules

Proteins Lipids DNA/RNA

• Hyperlipidemia was induced in male Swiss albino

Group 2: mice were daily attained to the hyperlipidemic

Group 3: mice were daily administered aqueous extract a

• In the first experiment, blood samples were

.Determination of Glutathione Reductase (GR)

.Determination of Glutathione Peroxidase (GPx)

.Determination of Catalase (CAT)

Determination of Malondialdehyde (MDA)

(E)-2-Hexenol 67.36 875 1.26

α–pinene 70.24 910 34.70

(Z)-3-Hexenyl acetate 63.346 1012 0.61

(E,Z)-2,6-Nonadienal 69.309 1159 1.46

(E,Z)-2,6-Nonadienol 70.479 1163 0.74

1,4-Dimethoxybenzene 78.775 1171 0.45

2,6-Dimethyloctane 47.336 1210 10.57

2,6,11-Trimethyldodecane 48.941 1281 0.43

α-humulene 54.436 1465 0.84

β -Ionone 55.861 1499 0.89

Oleic acid 59.550 1860 0.81

Tetracosane 60.892 2340 4.38

punica peels on S.T

The prophylactic effect of aqueous extract on S. T. HDL Cholesterol

The prophylactic effect of aques extract of punica peelson S. T.

The prophylactic effect of aques extract of punica peels on S. ALT

The prophylactic effect of aqueous extract on S. ALP

The liver tissue of positive control

The heart tissue of control mice for 2

The heart tissue of The positive

The kidney tissue of positive control mice

The curative effect of aqueous extract on T. CHOL/ HDL-CHOL.

The curative effect of aqueous extract on S. LDL-Chol.

The curative effect of aqueous extract on S. VLDL-Chol.

The curative effect of aqueous extract on S. Triglycerides.

The curative effect of aqueous extract on S. ALT.

The curative effect of aqeuous extracton S. AST

The curative effect of aqueous extract on S. GGT

The curative effect of aqueous extract on S. ALP.

The curative effect of aqueous extract on S. T. Protein.

Hepatic tissue suffering hyperlipidemia

Effect of atorvastatin treated

The heart tissue of aqueous extract of

The heart of Atorvastatin treated

The kidney of positive control mice

aphotomicrograph of a kidney section of aqueous

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