• Objectives
• Introduction
• Methods
•Results & Discussion
• Future Plan
 To discuss the important causes of liver injury.
 To recall some plants from different floras are highly reputed
for their uses in folk and traditional medicine to control
hepatotoxicity.
 To Evaluate the protective effect of some medicinal plants
grown in UAE and Saudi on CCl4-induced hepatic damage in
rats.
 To Evaluate the curative effect of the plants extracts on CCl4induced hepatic damage in rats.
 To find other effective hepatoprotective and antihepatotoxic
herbal medicine from the Saudi and UAE floras
• The liver is the body's
largest and most
important metabolic
organ.
• It serves as the major
biochemical factory of
the body.
•
•
•
•
•
•
•
Major functions of the liver
Production of bile salts Synthesis of plasma proteins
Metabolic processing of nutrients
Removal of toxic or harmful substances
from the blood
Storage - Glycogen, fats, iron, copper and
vitamins
Vitamin D activation
Excretion of cholesterol and bilirubin
• Liver diseases are
considered as one of the
serious health problems.
WHO ,130–150 million people globally
have chronic hepatitis C infection.
A significant number of those who are
chronically infected will develop liver
cirrhosis or liver cancer.
848,636people die each year from liver
disease
Every year approximately 695,200
people die due to liver cancer
• A number of pharmacological and chemical agents act
as hepatotoxins and produce a variety of liver ailments.
• Drugs are an important cause of liver injury.
• More than 900 drugs, toxins, and herbs have been
reported to cause liver injury, and drugs account for 2040% of all instances of fulminant hepatic failure.
Non-steroidal anti-inflammatory drugs,
antihypertensives, antidiabetic agents,
anticonvulsants, lipid-lowering agents,
psychotropic drugs also recommended to
become hepatoxic drugs
Chitturi S and George J. Hepatotoxicity of commonly used drugs: nonsteroidal anti-inflammatory drugs, antihypertensives, antidiabetic
agents, anticonvulsants, lipid-lowering agents, psychotropic drugs. S emin Liver Dis. 2002;22(2):169-83.
• Steroids, vaccines and antiviral drugs
which have been employed as a therapy
for liver diseases, have potential adverse
effects especially when administered for
long terms.
• In absence of reliable liver-protective
drugs in modern medicine,
hepatoprotective drugs from plant
sources seem to be attractive
alternative.
Many plants of the Chinese, European,
Arabic and Indian floras are highly
reputed for their uses in folk and
traditional medicine to control
hepatotoxicity.
Milk Thistle (Silybum marianum)
• ‫ سلبين مريمي‬Milk thistle has been used for over 2,000 years
as a natural treatment for liver disorders.
•Amazing herb protects the liver cells from damaging
molecules called free radicals.
•Milk thistle is often suggested as a treatment for alcoholic
hepatitis and alcoholic cirrhosis.
•Most studies show milk thistle improves liver function and
increases survival in people with cirrhosis or chronic
hepatitis.
Agency for Healthcare Research and Quality. Milk thistle: effects on liver disease and cirrhosis and
clinical adverse effects. Summary, evidence report/technology assessment: number 21, September
2000.
Asghar Z, Masood Z. Evaluation of antioxidant properties of silymarin and its potential to inhibit peroxyl
radicals in vitro. Pak J Pharm Sci. 2008 Jul;21(3):249-54.
• Dandelion Root (Taraxacum officinale)
‫طرخشقون‬
• The bitter constituents in the root enhance
liver function by increasing production of
bile and improving gallbladder function.
Choi UK1, Lee OH, Yim JH, Cho CW, Rhee YK, Lim SI, Kim YC. Int J Mol Sci. 2010 Jan 6;11(1):67-78. doi: 10.3390/ijms11010067
.
Turmeric (Curcuma domastica) ‫كركم‬
• Turmeric's antioxidant properties protect the liver
from numerous toxic chemicals. Turmeric has been
reduced cancer-causing agents in the liver.
•MOHSEN et al . Antioxidant and Free Radical Scavenging Activities of Curcumin. Asian Journal of Chemistry; Vol. 25, No. 13 (2013), 7593-7595.
• Schisandra (Schisondra chinensis)
• The plant protects the liver from damage
by a variety of its antioxidant effects
gradually improves overall health chronic
stress is part of the life.
•
Cheng et al. Antioxidant and hepatoprotective effects of Schisandra chinensis pollen extract on CCl4-induced acute liver damage
in mice , Food Chem Toxicol. 2013
• Date fruit can be considered as a rich source of
antioxidant and hepatoprotective agent
•
Ebtesam et al. Phenolic Contents and Antioxidant Activity of Various Date Palm (Phoenix dactylifera L.) Fruits from Saudi Arabia Food and
Nutrition Sciences, 2011, 2, 1134-1141 doi:10.4236/fns.2011.210152 Published Online December 2011 (http://www.SciRP.org/journal/fns)
Methodology
A. Plants material and extraction
 Ten Plants belonging to different families were
collected from Western areas of Saudi Arabia
and from Dubai (UAE) during March till May
(2009-2014).
 The air-dried powdered plants (Arial parts) (500 g
each) were separately macerated in ethanol
then evaporated under reduced pressure by
using rotary evaporator .
 The residues were kept at 4°C for the biological
study.
Cucumis prophetarum L. ,
Hypoestes forsskalei
Chrozophora oblongifolia
Capparis spinosa
Cluytia myricoides
Blepharis ciliaris (L.)
Rumex nervosus
Calotropis procera
Fagonia indica
Zygophylum hamience
B. HPLC analysis of the phenolics
• The phenolic compositions were
investigated in aliquots (1 g, each) of the
hydrolysed-methanolic extracts of the
plants via RP-HPLC.
• Analyses were carried out at operating
conditions suitable for detection of either
phenolic acids or flavonoids .
 Animals. Male Wister rats, weighing
140-180 g were used.
 All animals investigations were
performed in accordance with the
guidelines of the Biochemical and
Research Ethical committee at the
King Abdalaziz University,
Jeddah, Saudi Arabia and
Research Ethical Committee ,
Dubai Pharmacy College.
Acute oral toxicity study :
• LD50s of the alcoholic extracts of
the plants under investigation
were estimated to assess their
safety (Lorke, 1983).
Lorke, D., A new approach to practical acute toxicity testing.Arch Toxicol., 54, 275-387 (1983).
Hepatoprotective effect
Group #
Type of rats
treatment
1
Control Group
liquid paraffin
2
CCl4
Without treatment
3
Positive Control
Group
standard silymarin (150 mg/kg in 1
% CMC-Na)
4-13
Treated Groups
received the plants extracts in a dose of
500 mg/kg.
The rats were received either silymarin or the different extracts by oral
gavage once daily for 7 days before induction of liver injury by CCl4.
Animals of groups 1 and 2 received 1 % CMC-Na once daily for 7 days.
 Liver injury was induced by subcutaneously (sc) injection
of CCl4 in a dose of 35 μg/100 g diluted in liquid paraffin
(0.3 ml/kg).
 Animals of all groups received CCl4 in liquid paraffin at
day 8, except the control one.
 Twenty four hours, after injecting CCl4, blood samples
were collected, plasma was separated by centrifugation
at 3500 rpm and the liver enzymes namely ALT, AST and
ALP activities were determined.
•
Sangameswaran B, Reddy TC, Jayakar B. Hepatoprotective effect of leaf extracts of Andrographis lineata nees on liver damage caused by
carbon tetrachloride in rats. Phytotherapy research 2008; 22:124-126.
Hepatotoxicity in Male Wister rats was
induced by s.c. injection of CCl4 dissolved
in liquid paraffin (30% solution), in a dose
of 1 ml/kg b.w. for 4 weeks.
Group #
Type of rats
Treatment
1
Control Group
Liquid paraffin
2
3
4-13
Negative control
Without treatment only
received 1 % CMC-Na after 29
days
Positive Control
Group
standard silymarin (150 mg/kg
in 1 % CMC-Na) after 29 days
by oral gavage
Plants Groups
received the plants extracts in
a dose of 500 mg/kg after 29
days by oral gavage
 At the end of the experiment (8 weeks), blood
samples were collected from the orbital sinus of
each rat and plasma were separated by
centrifugation at 3500 rpm. Plasma samples were
kept under -20 ºC and were used for
determination of the other parameters.
 Animals were anesthetized with diethyl ether and
sacrificed by cervical dislocation for separation of
the liver.
 Livers were dissected out, divided into two parts.
 One part was kept in
liquid
nitrogen
for
determination
of • The other part was
antioxidant status by
immediately fixed in
measuring the levels of
buffered formalin 10%
tissue
contents
of
and was used for
reduced
glutathione
(GSH), malondialdehyde
histopathological
(MDA),
the
enzyme
studies.
activities of superoxide
dismutase
(SOD),
glutathione peroxidase
(GPx), glutathione –Stransferase (GST) and
glutathione
reductase
(GR).
STATISTICAL ANALYSIS
 Data are expressed as mean ± standard error (SE) of
mean. Unless otherwise indicated, statistical analyses
were performed using one-way analysis of variance
(ANOVA).
 If the overall F-value was found statistically significant
(p<0.05), further comparisons among groups were
made according post hoc Tuckey's test.
 All statistical analyses were performed using
GraphPad InStat 3 (GraphPad Software, Inc. La Jolla,
CA, USA) software.
HPLC analysis of flavonoid and phenolic
compounds of the different plants revealed that
most of the plants showed high contents from
phenolic compounds especially Quercetin,
Quercetrin, rutin and Rosmarinic acid were
detected in all plants under investigation.
LD50 :
 Oral administration of the alcoholic extracts
of different plants in doses up to about 4
g/kg body weight produced neither mortality
nor signs of morbidity or behavioral changes
in any of the treated animal groups under
examination.
 This indicates the wide safety margin of our
plants extracts.
b
b
b
b
b
b
b
b
The values are expressed as the mean ± SE of the mean. a, Significantly different from the values of the
normal rats at P<0.05. b, significantly different from the control values of CCl4 at P<0.05
a
b
b
b
b
b
b
b
Fig. 4: The effect of the plant extracts on hepatic lipid
peroxidation levels and antioxidant enzymes activities in
CCl4-induced hepatic damage in rats
a
b
b
b
b
b
b
The values are expressed as the mean ± SE of the mean of 8 rats, a, Significantly different from the values of the
normal rats at p <0.05.
b Significantly different from the control values of CCl4-induced hepatotoxic rats at p <0.05.
• Fig.1: Histopathological examination of
Liver sections from rats of the control
group rats(A), rats treated with CCl4(B),
silymarin + CCl4 (C) and extract of B.
ciliaris + CCl4 (D) (H&E X 250)
Fig.5: Histopathological examination of Liver sections from rats
of the control group rats(A), rats treated with CCl4(B), silymarin +
CCl4 (C) and extract of B. ciliaris + CCl4 (D) (H&E X 250)
 In our study, we are focusing on the liver damage which always
accompanied by cellular necrosis, increased in tissue ALP and
diminution of reduced liver glutathione.
 In addition, elevated levels of hepatic serum enzymes are indicative
of cellular leakage.
 The hepatotoxic effect of CCl4 are related to its active metabolite
trichloromethyl radical, •CCl3, this is observed by an elevation in the
serum marker enzymes namely AST, ALT, ALP. Also these effects
were coupled with a marked hepatic oxidative stress (decreased
GSH, SOD, GPx, GR and GST activities in liver tissue and
increased production of MDA activities in liver tissue).
Muriel P, Escobar Y. Kupffer cells are responsible for liver cirrhosis induced by carbon tetrachloride. Journal of Applied Toxicology 2003; 23:103108.
• It was found that phenolic and polyphenolic compounds
such as flavonoids have strong antioxidant activity
because of their molecular structure.
 Most of the plants under investigation showed
high flavonoid and phenolic contents especially
quercetirin and rosmarinic acid .
 Administration of the plants extracts illustrated
significant hepatoprotective actions against CCl4
injuring.
 The hepatoprotective activity of the plants
extracts is designated by the reduction of the
blood levels of AST, ALT and ALP
In addition, treatment with plants extracts
as well as silymarin improved the liver
function after CCl4 injures (antihepatotoxicity),
The antioxidant and hepatoprotective
activities of these plants attributed to their
phenolic contents.
In conclusion, Our plants may be added to the growing list of the
antioxidant and hepatoprotective medicinal plants.
Formulation studies involve developing a
preparation of the plants extracts which are
both stable and acceptable to the patient.
The authors are grateful to the Deanship of
Scientific Research, King Abdulaziz
University, Jeddah, Kingdom of Saudi Arabia
and Prof. Khan , Dean of Dubai Pharmacy
College, Dubai, U.A.E. for funding these
researches