Hepatoprotective Effect of Methanolic Fruit Extracts of Phoenix dactylifera (Arecaceae) on Thioacetamide Induced Liver Damage in Rats

Abstract

Background: The present study was carried out to evaluate the hepatoprotective activity of methanolic fruit extracts of Phoenix dactylifera (date palm) against thioacetamide-induced liver damage in male albino wistar rats. Methods: Twenty-five (25) male albino rats, (200-250 g) were used for the study and they were placed into five groups (A to E). Groups A, B, and C were given silymarin (50 mg/kg), low dose of the extract (300 mg/kg) and high dose of the extracts (600 mg/kg) respectively for ten (10) consecutive days through the oral route (p.o). On the eleventh (11 th) day, thioacetamide (TA) was administered at the dose of 200mg/kg subcutaneously to groups A to D. Group E was not given any form of treatment whatsoever and served as normal control. Forty-eight hours after TA administration, blood samples were collected from all the animals through the retro-orbital sinus into appropriately labeled plain bottles for assay of serum liver enzymes, albumin and total bilirubin. Also, liver was excised for histopathological assessment. Result: The fruit of Phoenix dactylifera had an oral LD 50 >6000 mg/kg in rats. Preliminary phytochemical screening revealed the presence of carbohydrates, proteins, saponins, steroids, glycosides, flavonoids, tannins, and terpenoids. There was a significant rise in the level of biochemical makers of liver damage like ALT, AST, ALP and total bilirubin and a fall in albumin in TA-treated groups when compared with the respective values for extract and silymarin treated groups (p<0.001). Also, when compared with the normal control group (Group E), with the values of 36.40±4.

Full text

American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org
Original Article
Hepatoprotective Effect of Methanolic Fruit
Extracts of Phoenix dactylifera (Arecaceae)
on Thioacetamide Induced Liver Damage in
Rats
Chukwugozie N Okwuosa*1, Theophilus K Udeani1, Joshua E Umeifekwem3, Augustine C
Onuba2, Innocent C Anioke1 and Romanus. E. Madubueze1
1
Department of Medical Laboratory Sciences, Faculty of Health Sciences & Technology, College of
Medicine, University of Nigeria, Enugu Campus
2Department of Veterinary Surgery, Faculty of Veterinary Medicine, University of Nigeria, Nsukka
3Department of Health and Physical Education, University of Nigeria, Nsukka
ABSTRACT
Background: The present study was carried out to evaluate the
hepatoprotective activity of methanolic fruit extracts of Phoenix
dactylifera (date palm) against thioacetamide-induced liver damage
in male albino wistar rats. Methods: Twenty-five (25) male albino
rats, (200-250 g) were used for the study and they were placed into
five groups (A to E). Groups A, B, and C were given silymarin (50
mg/kg), low dose of the extract (300 mg/kg) and high dose of the
extracts (600 mg/kg) respectively for ten (10) consecutive days
through the oral route (p.o). On the eleventh (11th) day, thioacetamide
(TA) was administered at the dose of 200mg/kg subcutaneously to
groups A to D. Group E was not given any form of treatment
whatsoever and served as normal control. Forty-eight hours after TA
administration, blood samples were collected from all the animals
through the retro-orbital sinus into appropriately labeled plain bottles
for assay of serum liver enzymes, albumin and total bilirubin. Also,
liver was excised for histopathological assessment. Result: The fruit
of Phoenix dactylifera had an oral LD50 >6000 mg/kg in rats.
Preliminary phytochemical screening revealed the presence of
carbohydrates, proteins, saponins, steroids, glycosides, flavonoids,
tannins, and terpenoids. There was a significant rise in the level of
biochemical makers of liver damage like ALT, AST, ALP and total
bilirubin and a fall in albumin in TA-treated groups when compared
with the respective values for extract and silymarin treated groups
(p<0.001). Also, when compared with the normal control group
(Group E), with the values of 36.40±4.28 U/L, 43.20 ± 4.31 U/L,
62.55 ± 5.8 U/L, 8.12 ± 0.52µmol/L and 49.84 ± 4.42g/L for ALT,
AST, ALP, bilirubin and albumin respectively, group B had
Address for
Correspondence
Department of
Medical Laboratory
Sciences, Faculty of
Health Sciences and
Technology, College of
Medicine, University
of Nigeria, Enugu
Campus, Nigeria
E-mail:
chukwugozie.okwuosa
@yahoo.com

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AJPCT[2][3][2014]290-300
respective values that were significantly different from it (p<0.05)
whereas group C values were not significantly different from
group E values (p>0.05). Histopathological findings in the test
groups showed mild alteration in histoarchitecture when compared
with TA group which showed extensive vacuolation, inflammatory
cells and generalized necrosis. Conclusion: P. dactylifera has a
hepatoprotective effect in thioacetamide induced hepatic necrosis
in rats.
Keywords: Phoenix dactylifera, Hepatoprotective, Thioacetamide,
Liver enzymes, Albumin.
BACKGROUND
Hepatotoxicity from drugs and
chemicals has been incriminated as the
commonest form of iatrogenic diseases
world over1. About 5% of all hospital
admissions and 50% of all acute liver failure
has been linked to drug and chemical
induced liver injury2. Many drugs have been
responsible for liver injury and often times
led to drug withdrawal from the market3.
Chemicals that have the propensity to cause
injury to the liver are generally referred to as
hepatotoxins and they range from the
injurious, over-the-counter therapeutic drugs
like acetaminophen to more severely toxic
chemicals like thioacetamide, carbon
tetrachloride (and other halogenated
alkanes) and heavy metals. Though the exact
mechanism of hepatotoxic action (s) of some
of these agents (hepatotoxins) have not been
clearly elucidated, most of them cause liver
damage either by lipid peroxidation, protein
synthesis inhibition, enzyme poisoning,
adducts formation with DNA, or by
depriving the tissue (liver) of oxygen
leading to tissue hypoxia, or a combination
of these. Some directly cause the damage
while very many others (acetaminophen)
inflict damage through their metabolite (N-
acetyl-benzoiminoquinone). Agents that
ameliorate liver injury caused by these
chemicals or drugs are said to be hepato-
protective.
The liver is capable of detoxifying
blood by removing medications, alcohol and
other potentially harmful chemicals so that
they can be excreted through the urinary or
gastro-intestinal tracts. Exposure to
hepatotoxin may be accidental (as in the
case of therapeutic drug overdose, accidental
ingestion of Amanita muscaria, or ingestion
of aflatoxin from legume plants infested
with Aspergillus flavus), or intentional as
occurs among drug and alcohol addicts.
Liver dysfunction results in impairment of
toxin detoxification, leading ultimately to
toxin accumulation in vital organs and
consequently causing disseminated damage
to them. It has thus become particularly
imperative that a conscious effort be made
to prevent exposure to these toxic
substances, or where unavoidable, mitigate
the associated liver injury by the use of
plants or allopathic drugs known to possess
universally accepted hepatoprotective
potency. However, there is currently no
allopatic drug that is effective for the
treatment of liver diseases. On the other
hand, Medicinal plants play significant role
in the treatment/management of liver
disorders and many of them have shown
significant hepatoprotective potency4-7.
Phoenix dactylifera (date palm) has
been claimed to be used, over the years, in
Arabian and middle-eastern countries, and

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northern parts of Nigeria to treat liver
disease-related clinical symptoms such as
jaundice. It has also been claimed that the
fruits of the very plant protect the liver form
chemical insult and alcohols8. Therefore, the
aim of this study is to verify these claims
using experimental animal model of liver
injury.
MATERIALS AND METHODS
Animals
Twenty-five (25) apparently healthy
male albino Wistar rats weighing 200-250 g
were used for the experiment. They were
obtained from and housed in the Animal
House of the College of Medicine, University
of Nigeria Teaching Hospital, Enugu. They
were weighed and grouped into five
according to their weights (X ± 20 g) and
allowed two weeks to acclimatize. They were
maintained in a room under Standard
environmental condition and controlled
temperatures (30 ± 20C). The animal house
was regularly cleaned and disinfected with
Isol to reduce the risk of infection and the
animals were fed ad libitum with feed and
water. All the animals used for this study
were treated according to international
guidelines for experiments involving the use
of animals9 and the guidelines from the
institutional animal research ethical
committee (UNTH/CSA. 457/VOL. 8).
Collection and Preparation of P. dactylifera
The fruits of P dactylifera were
obtained from Kano, a state in Northern
Nigeria, in the month of April, 2013. The
seeds were authenticated by a taxonomist in
the Department of Plant Science and
Biotechnology, University of Nigeria, Nsukka
(UNH/75c). The seeds of the fruits were
carefully removed and the fleshy part dried at
room temperature prior to extraction.
Extraction of plant material
After drying the flesh of P dactylifera
at room temperature, they were crushed into
powder (500 g) and then macerated with 1.5
litres of analytical grade of methanol for 48
hours. This was filtered through muslin and
then through Whatman No 1 filters. The
filtrate was concentrated by evaporating in an
oven (Gallenkamp, UK) at 60°C. The yield of
the syrupy methanol extract was 10.6 %
(w/w). The appropriate dose for the
experiment was reconstituted from the
concentrate (20 g) using physiological saline
as the solvent or diluent (200 mg/ml). This
was labeled the methanol extract of Phoenix
dactylifera (MEPD).
Acute toxicity test (LD50)
This was performed on rats and the
Lorke procedure of LD50 determination was
used10.
Preliminary Phytochemical screening
This was done according to the
procedure of Trease and Evans11.
Experimental design and conduct
Twenty-five (25) apparently healthy
male albino Wistar rats were used for the
study. They were placed into five (5) groups
labeled A to E according to their body
weights. Group A was the positive control
group and received silymarin (50 mg/kg p.o),
groups B and C were the test groups and
received 300 and 600 mg/kg MEPD, p.o,
respectively while group D served as the
thioacetamide (TA) control and received only
thioacetamide 200 mg/kg s.c. Group E served
as the normal control and received no
treatment whatsoever. Drug and extracts were
administered to groups A, B, and C
respectively for ten (10) consecutive days
through the oral route (p.o). On the eleventh
(11th) day, TA (hepatotoxin) was
administered at the dose of 200 mg/kg
subcutaneously to groups A to D; group E

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was not given any form of treatment
whatsoever and it served as a normal control.
Forty-eight (48) hours after TA
administration, all the animals (groups A to
E) were bled through the retro orbital sinus
into appropriately labeled plain bottles. Also,
liver tissues were harvested from the rats after
euthanasia under ether anaesthesia. The liver
tissues excised were washed in cold normal
saline, fixed in 10% formal saline in universal
containers and used for histopathological
studies. The blood samples were allowed to
clot and sera separated promptly from cells
after centrifugation at 3000 r.p.m for 10 mins.
The sera were stored deep frozen until
analyzed. They were analyzed for liver
marker enzymes (Aspartate transaminase,
Alanine transaminase and alkaline
phosphatase), total bilirubin and albumin.
Biochemical methods
The serum samples obtained were
assayed for Alanine transaminase (ALT) and
Aspartate transaminase (AST) using Reitman
and Frankel method12, Alkaline phosphatase
using King and king method13 and Total
Bilirubin using Powell’s method14. The dye
binding method (bromocresol green) of
Bartholomew and Delaney15 was used to
assay albumin.
Histopathology
The formalin-fixed tissues were
processed and embedded in paraffin wax.
Thin sections (3-5µm) were made and stained
with hamatoxylin and eosin staining
technique for light microscopy. Photo-
micrographs of the Liver were taken for
permanent record.
Statistical analysis
Statistical analysis was done with
SPSS software package version 15. The
results of biochemical assay were expressed
as mean ± SEM. The level of significance
was tested using one way analysis of variance
(ANOVA) followed by Tukey’s post-hoc
multiple comparisons. p<0.05 was considered
statistically significant.
RESULTS
Result of acute toxicity study showed
that the fruit of Phoenix dactylifera had an
oral LD50 >6000 mg/kg in rats. Preliminary
phytochemical screening revealed the
presence of Carbohydrates, proteins,
saponins, steroids, glycosides, flavonoids,
tannins, and terpenoids.
The result presented in table 1 showed
statistically significant differences between
the mean values of all the parameters
measured in all the groups (p<0.001). The
extract treated groups had significantly lower
mean values for the biomarkers of liver injury
when compared with the thioacetamide
control (p<0.001). The values of ALT, AST,
ALP and Bilirubin were 61.20±2.14 U/L,
72.40±2.99 U/L, 81.42±2.2 U/L and
10.8±0.48 µmol/L respectively for group B,
and 41.0±2.96 U/L, 52.80±4.66 U/L,
71.16±1.98 U/L, and 8.68±0.54 µmol/L
respectively for group C and these were
significantly lower than their corresponding
values in the thioacetamide control (p<0.001).
On the other hand, albumin values of
36.87±3.82 g/L for group B and 48.86±2.4g/L
for group C were significantly higher when
compared with that of group D (thioacetamide
control) which was 20.83±3.38g/L. Results
also showed that there was no significant
difference (p>0.05) between mean values of
parameters measured in groups A and C when
compared with their counterparts in the
control group (group E). However, when
those of group B were compared with the
same control group, a significant difference in
the mean values of these analytes was
observed (p<0.05).
The histological picture corroborated
the hepatoprotective activity of P. dactylifera
extracts observed from the biochemical

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parameters. Group C which received a high
dose of the extract appeared almost the same
as group E (normal control) even after TA
treatment. Group D (TA control) sustained
very severe hepatic injuries as evident from
high infiltration of inflammatory cells,
disseminated necrosis and vacuolations.
Group B that received a lower dose of MEPD
showed less severe hepatic injuries when
compared with those of group D. The
necrosis observed in group B was zonal rather
than disseminated as for group D. Group A
which is the positive control wass nearly the
same as the normal control group but had
moderate vacuolation and inflammatory cell
infiltration. However, there was no evidence
of necrosis. These histopathological findings
are summarized in table 2 and the
photomicrographs of these tissues are
presented in figures 1-5.
DISCUSSION
Phoenix dactylifera fruits (palm dates)
are consumed as staple food in the middle-
east, and Northern parts of Nigeria usually
because of their medicinal values. Middle
easterners believe that consumption of the
fruits especially in the morning on an empty
stomach protects the liver from the action of
toxic materials that the subject may have been
exposed to16. This claim/belief was
investigated, in the present study, against
thioacetamide induced liver damage in rats.
Though the exact mechanism of
hepatoprotective activity of the P. dactylifera
was not evaluated in this study, β-sitosterol, a
constituent of the fruit may be partly
responsible17. Also, flavonoid is suggested to
contribute to its hepatoprotective activity
through the inhibition of cytochrome P450
aromatase18. P. dactylifera has a high
antioxidant activity, being equipped with at
least five vitamins-Vitamin C, Vitamins B1
and B2, Niacin and Vitamin A19 and the
enzyme peroxidase20. The significant
antioxidant capacities possessed by this fruit
have been surmised to be possibly responsible
for the hepatoprotective effects21.
Thioacetamide–induced liver damage
probably depends on the formation of S-
Oxide which in turn causes membrane
peroxidation and consequent liver damage.
The results of biochemical assay revealed that
P. dactylifera fruits possess significant
hepatoprotective activity as evident from the
significant decrease in the mean liver marker
enzyme levels of the test groups when
compared with TA group. Bilirubin was
equally elevated and albumin decreased
significantly in the thioacetamide group than
in extract and silymarin treated groups,
further indicating the hepatoprotective
potency of the P. dactylifera fruit extract. The
ability of the extract to antagonize the rise in
serum alkaline phosphatase and bilirubin
induced by thioacetamide is indicative of
membrane stabilizing action. Flavonoids have
been reported to exert membrane stabilizing
action22. It is, therefore, likely that the
flavonoids present in P dactylifera extract
could be responsible for the membrane
stabilizing property. The significant reduction
in mean serum albumin level of the
thioacetamide group is as a result of
thioacetamide-induced cellular toxicity which
affected the synthetic capacity of the liver.
Interestingly, extract and silymarin treatment
preserved the synthetic capacity of the liver as
evident from the non-significant differences
in their mean albumin levels when compared
with the normal control. The
histopathological findings support the
hepatoprotective effect of P. dactylifera as
evident from biochemical parameters. Mild
changes in liver histo-architecture of the test
when compared with TA control group
(group D) correlates well with the
biochemical findings. The thioacetamide
control group showed loss of details with
increased vacuolation, inflammatory cells and
disseminated or generalized necrosis. Group

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B showed minor changes like mild
vacuolation, inflammatory cells and zonal
necrosis whereas group C had histological
features that were indistinguishable from the
normal control and also to some extent
showed better protection than the reference
drug silymarin. This study is in agreement
with those of several workers who
demonstrated that Phoenix dactylifera is
hepatoprotective in carbon tetrachloride and
dimethoate induced liver injury respectively23-
27.
CONCLUSION
From the experimental result, it can
be concluded that P. dactylifera fruits possess
hepatoprotective effect in thioacetamide-
induced liver injury in rats.
ACKNOWLEDGEMENT
We wish to express our gratitude to
the Technical and Scientific staff of the
Department of Medical Laboratory Sciences,
University of Nigeria, Enugu Campus for
their assistance with slide preparation and
taking of photomicrographs. Many thanks to
Dr Ukekwe, pathologist, Morbid Anatomy
Department, University of Nigeria Teaching
Hospital, Ituku-Ozalla, for the histopathology
report. Finally, we thank the entire staff of the
Animal House of the College of Medicine,
University of Nigeria for their co-operation
during the period of the study.
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Table 1. Effect of the extracts of P dactylifera on biochemical markers of liver damage in rats
Groups ALT (iu/L) AST (iu/L) ALP (iu/L) Bilirubin (µmol/L)
Albumin (g/L)
A (50mg/kg
silymarin, 39.6 ± 2.06a 52.6±3.26 a 67.2±2.4 a 8.52±0.48 a 48.24±2.5 a
B (300mg/kg
MEPD 61.2±2.14 ac 72.4±2.99 ac 81.42±2.2bc 10.80±0.48 ac 36.87±3.82 ac
C (600mg/kg
MEPD 41.0 ± 2.96 a 52.8±4.66 a 71.16+1.98 a 8.68±0.54 a 48.86±2.48 a
D (200mg/kg of
TA only) 107.2± 4.58d 106.2±5.94 d 139.71±2.5 d 17.44±0.46 d 20.83±3.38 d
E (Normal
control) 36.4 ± 4.28 43.2±4.31 62.55±5.8 8.12±0.52 49.84±4.42
F–Ratio 309.93 133.17 365.14 243.3 51.65
P-value < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
(MEPD = Methanol extract of P. dactylifera, S.C = subcutaneous route, TA = thioacetamide)
a = p < 0.001 with respect to the thioacetamide control. b = p < 0.01 with respect to the
thioacetamide control. c = p < 0.05 with respect to the normal control; d = p < 0.001 with
respect to the normal control. n = 5.
Table 2. Histopathological findings
Groups Vacuolation Necrosis Inflammatory cells
A (50mg/kg silymarin) + TA + - +
B (300mg/kg MEPD) + TA
++ ++z ++
C (600mg/kg MEPD) + TA
- - -
D (200mg/kg of TA S.C only) +++ +++D
+++
E (Normal control) - - -
+ = mild, ++ = moderate, +++ = severe, D = Disseminated necrosis, Z = zonal necrosis.

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Figure 1. (H & E x 200) photomicrograph of the liver of rats in group a showing
moderate vacuolation and inflammatory cells at the periportal areas. PV =
Portal hepatic vein, h = hepatocyte, ic = Inflammatory cells
Figure 2. (H & E x 200) Liver photomicrograph of animals in group B showing
zonal necrosis, moderate vacuolation, and the presence of inflammatory cells.
CV = central vein, V= vacuolation, Z = zonal necrosis, IC = inflammatory cells

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Figure 3. Photomicrograph of the liver of rats in group C showing normal hepatic
chords. PV = portal hepatic vein, h = hepatocyte
Figure 4. (H & E x 200) Photomicrograph of the liver of rodents in group D
showiing a preponderance of inflammatory cells, vacuolations, and
disseminated necrosis. CV = Central vein, IC = inflammatory cells, D=
Disseminated necrosis, V = Vacuolation

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Figure 5. (H & E: x 200) Photomicrograph of the liver of the normal control group
showing normal hepatic chords