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Effects of pomegranate and milk thistle extracts on chemically induced liver cancer in rats

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Saa Gamal T

on 27 November 2014

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Transcript of Effects of pomegranate and milk thistle extracts on chemically induced liver cancer in rats

Why Pomegranate ?
Liver cancer; particularly hepatocellular carcinoma (HCC) is the most common malignancy in the world.

Why Milk Thistle?
Effects of Pomegranate and Milk Thistle Extracts on Induced Liver Cancer by Diethylenitrosamine or Other Chemicals in Rats
Extraction process
The incidence of HCC is rapidly increasing in Middle East countries and this trend is expected to continue for the next 50 years because of the long latency between viral infection and development of HCC.

Consequently, there is a great demand to figure out an alternative approaches for the treatment of liver diseases

A large variety of methods are used for the treatment of liver diseases include, pharmacotherapy, surgery as well as liver transplantation, all of which have shown limited therapeutic benefits and are associated with serious complications.
The medicinal plants are considered to be the most recommended approaches for the treatment of liver disorders.
Prof./ Hekmat Mohamed Tantawy

Professor of Physiology. Zoology Department, Faculty of Science,

Suez Canal University
Dr./ Hend Maarof Tag

Lecturer of Physiology. Zoology
Department, Faculty of Science,

Suez Canal University.
Dr. Amani Kamal Ibrahim

Lecturer of Pharmacognosy
Faculty of Pharmacology,

Suez Canal University.
Pomegranate is a potent antioxidant, superior to red wine and equal to or better than green tea.

Anticarcinogenic, antioxidant and anti-inflammatory properties suggest its possible use as a therapy or adjunct for prevention and treatment of several types of cancer and cardiovascular diseases.
Pomegranate Peel
Peel is rich in:
Phenolic punicalagins
Gallic acid
Well recognized as a hepatoprotective herbal medicine.
Appears to be safe and have multiple health benefits on various liver conditions such as; liver cirrhosis, alcoholic hepatitis, alcoholic fatty liver, liver poisoning, and viral hepatitis.
Milk thistle seeds

Extraction process
Pomegranate peel
Milk thistle seed
In Vitro
Cytotoxicity Assay
Against HCC Cell line (HEP-G2)
Cell toxicity was monitored by determining the effect of both PPE and MTSE cell morphology and cell viability.
Experimental Design
Control Groups
Silymarin Control Group
Oral administration of Silymarin (4mg/Kg BW).

Three times per week for 8 weeks.
MTSE Control Group
Oral administration of milk thistle seed extract (500 mg/kg BW).

Three times per week for 8 weeks.
PPE Control Group
Oral administration of pomegranate peel extract (500 mg/kg BW).

Three times per week for 8 weeks.
Negative Control Group
Received saline only
Experimental Design
HCC Groups
Induction of HCC
PPE- HCC Group
The rats were Induced with HCC

Positive Control Group
Induced with HCC
: Intraperitoneal injection of 200 mg kg-1 diethylnitrosamine (DEN) dissolved in 0.9 normal saline.
Oral administration of pomegranate peel extract (500 mg/kg BW).

Three times per week for 8 weeks.
In Vivo
(ALT, AST, total bilirubin, LDH, total protein, Albumin)
In Vitro
Cytotoxicity assay against liver cancer cell line HEP-G2
Cytotoxicity Assay
IC50 =8.54

Biochemical Parameters
1. AlT
Biochemical Parameters
2. AST
Biochemical Parameters
3. LDH
To evaluate the anti-carcinogenic effect of pomegranate peel, milk thistle seed extracts.
To find if there is a difference between the effect of milk thistle seed extract (crude) and one of its constituents; Silymarin using the commercially available Livoprotec® drug.

- Cytotoxicity assay against liver cancer cell line
- Biochemical parameters
- Tumour marker
- Histological examinations
The parameters measured are:
Commercially available Livoprotec®
Biochemical Parameters
4. Total bilirubin
Biochemical Parameters
5. Total protein
Biochemical Parameters
6. Serum Albumin
Oxidative Stress Biomarkers
7. Lipid peroxidation content
Oxidative Stress Biomarkers
8. Glutathione
Tumour Marker
9. Alpha-feto Protein
Histological Findings
I. Control Groups
Showed normal radial liver cords of normal polygonal hepatocytes with round nuclei and eosinophilic cytoplasm, clear blood sinusoids (BS), fine arrangement of Kupffer cells.

No obvious changes were observed in livers of PPE, MTSE and Livoprotec®-treated animals.

I. Control Groups
II. HCC Group
Histological Findings
loss of normal architecture with oval or irregularly shaped neoplastic hepatocytes.
Focal necrosis associated with neutrophil infiltration.
Hydropic degenerated cells with karyolysed nuclei (KR).
Dilatation of blood sinusoids (BS).
Multiple, polymorphic and hyperchromatic nuclei.
Spindle cell/sarcomatoid hepatocytes.
II. HCC Group
Cloudy swelling and hypertrophy of some hepatocytes with multiple polymorphic and hyperchromatic nuclei
Hepatocellular carcinoma
Histological Findings
III. Plant extracts and silymarin-HCC treated groups
MTSE-HCC treated group in which hepatocytes are of normal architecture X200.
PPE-HCC group: The abnormalities are still present as neoplastically transformed cells with mild infiltration but generally less than DEN group rats indicated certain improvement.
Silymarin (Livoprotec®) -HCC treated group: mild dilatation of blood sinusoids
III. Plant extracts and silymarin-HCC treated groups
These results showed that supplementation of milk thistle, pomegranate extracts and Livoprotec® had a protective effect against chemically induced HCC in rat respectively.

As a result; the present study suggested that PPE and MTSE are useful therapeutic agents in hepatocarcinogenesis.


MTSE-HCC: -31.69%
HCC: 551.5%
PPE-HCC: -46.48%
Silymarin-HCC: -35.73%
: IC50 =10.95
IC50 =49.23
Silymarin-HCC: -30.64%
HCC: 362.48 %
PPE-HCC: -40.9%
MTSE-HCC: -29.42%
HCC: 900%
Silymarin-HCC: -14%
MTSE-HCC: -26%
PPE-HCC: -44%
HCC: -36.6%
PPE-HCC: 30.13%
Silymarin-HCC: 16.74%
4 * (n=6)

HCC: -38.95%
PPE-HCC: 27.93%
Silymarin-HCC: 1.72%
MTSE-HCC: 13.1%
MTSE-HCC: -48.68%
Silymarin-HCC: -25.89%
PPE-HCC: -24.69%
HCC: 254.03%
Silymarin-HCC: 144.71%
HCC: -77.63%
PPE-HCC: 148.24%
MTSE-HCC: 210.59%
Rats were left for 2 weeks.
injected intraperitoneally for 2 weeks (3 times per week) with 1 ml/kg BW dissolved in olive oil of a 50 percent solution to initiate hepatic carcinogenesis.
administration of AAF for 2 weeks with 3 mg/kg BW (3 times per week) as a promoter for hepatocellular carcinoma.
The rats were Induced with HCC
Oral administration of milk thistle seed extract (500 mg/kg BW).
Three times per week for 8 weeks.
Silymarin- HCC Group
The rats were Induced with HCC
Oral administration of milk thistle seed extract (4 mg/kg BW).
Three times per week for 8 weeks.
Tumour marker AFP
Oxidative stress parameters
MTSE-HCC: -62.09%
PPE-HCC: -68%
Silymarin-HCC: -54.83%
HCC: 489.58%
Focal necrosis associated with lymphatic inlfiltration
Hydropic degenerated cells with karyolysed nuclei (KR).
Hepatocellular hyper trophy (Hepatonodular adenoma).
Sarcomatoid or spindle cell hepatocyte
Dilated blood sinusoid
Neutrofil infiltrate
hepatocyte necrosis
diploid nuclei
In Egypt, 4.7% of chronic liver disease patients develope HCC.
In many researches, pomegranate was found to inhibit prostate and breast cancer growth.
it has anticarcinogenic effects on cervical, breast and prostate cancer.
Cytotoxic activity of both extracts may be attributed to their powerful antioxidant content

Milk thistle: Rich in flavonoids

Pomegranate: Rich in punicalagins such as elagic acid elagitannins which also inhibits carcinogenesis and have anticancer activity

ALT and AST are located in the cytoplasm and the rise in their activities may be attributed to damaged structural integrity of hepatocytes due to lipid peroxidation of the cell membrane.

Administration of PPE, MTSE and silymarin conditioned the hepatic cells and prevented any further damage to the liver parenchyma decreasing the leakage of these enzymes into the circulation.

Enhancement of liver function tests may occur via several mechanisms:

Inhibition of lipid peroxidation.
Protection of glutathione from being depleted.

Protein Synthesis
Increased protein synthesis promotes hepatic tissue regeneration.

LDH activity

It is a marker of xenobiotic induced injury, the rise in its activity indicates the loss of membrane integrity due to carcinogen administration.

Total Bilirubin
The rise in its serum concentration is due to:

MDA indicates liver lipid peroxidation with reduction of hepatic glutathione.
Increased generation of ROS which play a major role in tumour promotion through interaction with critical molecules such as membrane lipids, DNA and DNA repair systems.
It also causes the production of MDA "toxic.
The disturbance in the antioxidant status leads to oxidative stress and carcinogenesis.
The reduction of MDA levels with the rise of GSH could be explained by the presence of strong antioxidant such as polyphenols: such as quercetin and catechin

They have antioxidant activity:
by scavenging ROS like which can be very damaging to lipid, protein and DNA.
The phenolic core can act as a buffer and capture electron s from ROS making them less reactive OR the can chelate metal like ions involved in free radicals formation.

they interfere with the cellular detoxification systems such as super oxide dismutase SOD, catalase or glutathione peroxidases.
They also can inhibit enzymes generating ROS as xanthine oxidase.
Thank you
Examiner committee
My supervisors
Zoology Department
Thank You
Thank You
Essential oil (mucilage)
Gamma linoleic acid
The decreased uptake of unconjugated bilirubin and decreased glucuronidation.

The elevated conc. after administration of the 3 drugs may be attributed to:

Enhanced detoxification

By the enhancement of glucuronidation.
AFP is the major glycoprotein in foetal life, it begins to distinguish after year 1, it is also secreted by endodermal sinus tumours such as HCC.

It reflects larger and altered liver cell regeneration.
PPE mild infilteration
MTSE appears as normal
Silymarin: mild dilatatin of BS
Full transcript