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PROTECTIVE EFFECT OF AQUEOUS EXTRACT OF
ZIZYPHUSSPINA CHRISTI FRUIT AGONIST PARACETAMOL
INDUCE HEPATOAND HAEMATOTOXICITY IN ADULT MALE
RABBITS
Qayssar A. Obaid
Department of Animal’s Heath, Technical Institute, Southern Technical University
Shatra,Thi.Qar,Iraq
(Received 27 September 2016, Accepted 24 November 2016)
Keywords: Zizyphusfruit; Haematotoxicity; Paracetamol.
ABSTRACT
Zizyphusspina-christi has a common name in Iraq "Nabka",it has been used in
traditional medicine.The present study was designed to elucidate the protective effect of
aqueous extract of ziziphusspina Christi on hepato and haematotxicity induced by
paracetamol in adult male rabbits. Eighteen adult male rabbits were divided randomly into 3
groups of 6 rabbits each and were treated for four weeks as follows:control group, second
(T1) groupreceived orally (300 mg/kg B.W.) of paracetmoldailyand third (T2) Groupreceived
orally (200 mg/kg B.W.) of crude extraction of zizyphyusspinadaily with (300 mg /kg B.W.)
of paracetmoldaily. The results indicated that paracetamol administration induced hepato and
haematotoxcity manifested by significant(P<0.05) elevation in liver enzymes (AST and ALT)
and reduction in total protein and albumin concentration with significant (P<0.05)decrease in
RBC, Hb, PCV, MCH, MCHC and Neutrophiles whereas it increase in WBC, MCV,
Lymphocyte and monocytes as a compared with control group.On the other hand, animals
treatment with aqueous extract of zizyphusspinachristiin group T2 showed a significant
ameliorated in biochemical andhaematologicalparameters change induce by paracetamol.It
could be concluded that oral administration of Zizyphusextract have hepatoprotectiveand antianemic
role agonist hepato and haematotoxicity induce by paracetamol.
INTRODUCTION
Paracetamol (Acetaminophen) is a common chemotherapeutic analgesic and
antipyretic drug available as an over the counter prescription. It has only weak antiinflammatory
activity and belonging to the para-aminophenol class of the non-steroidal antiinflammatory
drugs (NSAIDs) (1). It is the active substance of phenacetin and is derived from
coal tar (2). The word ofparacetamol is derive from the chemical names for the compound:
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para-acetylaminophenol and N-acetyl-para-aminophenol (3).It is widely used for the
headache, relief of mild to moderate pain and fever, it is a major component in numerous
cold and flu remedies (4). Although, it is consider safe for human use at therapeutic doses, but
when taken acute higher dose more than 1000 mg per single dose and more than 4000 mg per
day for adults is related with significant hepatotoxicity(5). It's metabolized in the liver where
its major metabolites include inactive glucuronidation and sulfation of paracetamol, which are
excreted by the kidneys. Only a small amount of paracetamol is metabolized by the hepatic
cytochrome P450 enzyme system (its CYP2E1 and CYP1A2 isoenzymes) which is formation
toxic highly reactive metabolite N-acetyl p-benzoquinoneimine (NAPQI) (6). The NAPQI is
reactive with glutathione and formation the glutathione conjugates. About 90% from the
glutathione is exhausted following acetaminophen toxicity(7). Acetaminophen high dose
leads to the accumulation of N-acetyl-p-benzoquinoneimine, which causes oxidative stress
and glutathione depletion leads to cell injury and death (8). Excessive use of acetaminophen
causes hepatotoxicity and analgesic nephropathy in the liver and kidney (9), and induces
hepatotoxicity as an animal's models,(10). In addition to hepatotoxcity, however, it cause
activation for coagulation system and haemolyticanaemia in patient after ingestion
paracetmol(11).
Zizyphusspinachristi known as Christ's Thorn Jujube and it has a common name ‘‘Nabka”,
and locally known as sidr, it is one of the plants commonly used in Iraqi folk medicine for the
treatment of various diseases .it's a native plant that grows in warm temperature and
subtropical regions especially in Middle East. Zizyphusspina-christi belongs to the family
Rhamnaceae (12). The mature fruit is red to purplish-black and wrinkled, looking like a small
date. The fruit has a single hard seed, similar to an olive stone. It is used as both a delicious
fruit and an effective herbal remedy with therapeutic effects in various diseases (13).
Zizyphus species are commonly used in alternative medicine for the treatment of various
diseases such as digestive disorders, liver complaints, urinary troubles, weakness, loss of
appetite, obesity, diabetes, bronchitis, fever, insomnia, pharyngitis, anemia, diarrhea, and skin
infections (14).From previous studies showed the Zizyphusspina-christi have medicinal
properties as antibacterial, antidiabetes, analgesic, antifungal, anti-hyperglycemic and
antinociceptive activities (15). Chemical analysis of its fruit has shown the present
flavonoids, alkaloids, triterpenoids, saponins, lipids, proteins, free sugar and mucilage (16).
Another phytochemical studies on the different species of the genus Zizyphusrevealed its
containcyclopeptide, alkaloids, flavonoids, sterols, tannins, and triterpenoidsaponins
(17),(18). Many researcher documented the fruit extract of Z. spina Christi have
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antitumor(19), CNS depressant(20), antioxidant and antifertility activities (21).Thus, the aims
of this study were designed to determine whether the protective effect of aqueous extract of
Ziziphusspinachristifruit on the liver and hematopoietic organs agonist paracetamol which
induce hepato- and haematotoxicity in male Rabbits
MATERIAL AND METHODS
Plant:Zizyphusspina Christi fruits were purchased from Shatra market in April, 2015
andidentified and authenticated by Plant department of technical institute shatra. The fruit
were washed, then their seed were separated from fruits and removed, the samples were
shade dried at room temperature and finely powdered using mortar and pestle.
Preparation of Aqueous Fruit Extract
Zizyphusspinachristi fruits were dried and ground to powder,100 gms of the fruits powder
was dissolved in 250 ml of water for 2 hours. The suspension was then heated at 60-65ºC for
thirty min. The extract was collected separately and repeated the processes three times with
the residual powder and collected the extract in each time. The extracts were collective and
passed through fine cotton cloth. The filtrates were evaporated by rotavapor. A dark semisolid
material was obtained and stored at 0-4ºC until use (22). A known amount of the residual
extracts were dissolved in distilled water and administered orally to the rabbits via Gavage
needle.
Experimental animal
Eighteenmale local rabbits (weighing between 1,5-2kg ) were used this study. Animals were
housed in iron cages in a conditioned room (22-25°C) with light/dark cycle of 12:12 hrs per
day in animal house of the animal resources department in a technical institute in shatra.
Animals had free access to water and standard pellet diet along the experimental.
Experimental design:
The animals (male rabbits ) were divided randomly into (3 groups) 6 animals per group &
were treated daily for 30 days as follows:
A- Control group: rabbits of this group received (5 ml/kg B.W.) of tap water by oral
dosage using gavages needle.
B- T1 Group: rabbits of this group were received (300 mg/kg B.W.) of paracetamol
solution once daily (5).
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C- T2 Group: rabbits of this group were received (200 mg/kg B.W.) of crude extraction
of Zizyphusspina (21) and after 30 min. received (300 mg /kg B.W.) of paracetamol
solution daily (5).
Collection of Samples
After four weeks of treatment, the animals were anesthetized by intramuscular injection of
(ketamine 30mg/Kg B.W &Xylazine 5mg/Kg B.W). Blood samples were collected by cardiac
puncture, 5 ml of blood samples were collected from the heart andsome of the blood collected
in the EDTA tubes used to study the hematological parameters and the remaining blood
collected in test tubes without any anticoagulant stand for 30 min at 37˚C to allow coagulation
(23). Serum was separated from coagulated blood sample by centrifugation at 3000 RPM for
15 min, and kept by freezing at -20˚C until used for measuring of the biochemistry assay.
Determination of hematological parameter
Red blood cells (RBC), packed cell volume (PCV), Hemoglobin (Hb), white blood cell count
(WBC), differential WBC count were determined by standard methods. Packed cell volume
(PCV) was determined by a microhematocrit method using a microhematocrit centrifuge.
Hemoglobin concentration was determined by the Cyanomethemoglobin photometric method.
The red cells (RBC) andwhite blood cells (WBC)were counted by using double improved
Neubauer counting chamber, whereas Differential white blood cell count calculate by blood
smear prepare and stained by Leishman’s stain to derive the percentage of each type of the
white blood cells.Erythrocyte indices including Mean corpuscular hemoglobin (MCH), mean
corpuscular volume (MCV) and mean corpuscular hemoglobin concentrations (MCHC) were
calculated from values of RBC, PCV and Hb as follows equations 1, 2, 3 (24):
MCH = (1)
MCV = (2)
MCHC = (3)
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Biochemical Assays:
Serum aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) activity were
enzymatically determined by using standard assay (SYRBIO chemical-kitbased on the
method of Reitman and Frankel in 1957 (25).Determination of Serum total proteins carried
out by using Biuret method, Proteins form a violet color complex in present copper ions in
alkaline solution(26).Albumin was measured in serum base on method performed by
Doumasin 1971 (27). Albumin reacts with bromocresol green to yield green color.
Statistical analysis:
Result were expressed as Mean ± standard error. Statistical analysis of data was performed on
the basis one- way analysis of variance (ANOVA) statistical SPSS software, version 21)
followed by Dunnett‟s post hoc test was performed for inter-group comparison using the
LSD.Values of p<0.05 were considered statistically significant(28).
RESULT
The result in table (1) shown that treatment of GroupT1by paracetamol daily for 30 days led
to significant (P<0.05) increase in ALT, AST concentrations as a compared with control
groups and decrease significantly (P<0.05) in total protein and albumin concentration as a
compared with control group. Results also clarified that co-administration ofaqueous extract
of Zizyphusspina-christi fruits and paracetoml significantly p<0.05decrease the change
recorded in liver enzyme concentrations and enhanced total protein and albumin concentration
near to a normal level.
Table (1) Effect of aqueous extract of Zizyphusspina-christi fruits on liver enzymes, total
protein and albumin in male rabbits treated with paracetamol.
Animal group
Parameter
Control
group
Group T1
Group T2
ALT
(U/I) 14.12 ± 0.88c 36.66 ± 1.01a 19.24 ± 0.73b
AST
(U/I) 17.76 ± 0.52c 40.50 ± 1.60a 24.96 ± 0.81b
TP
(g/dl) 6.50 ± 0.10a 3.92 ± 0.06c 5.38 ± 0.20b
Albumin
(g/dl) 3.48 ± 0.11a 2.62 ± 0.10b 3.22 ± 0.07a
-Values are expressed as mean ± SE, n = 6 each group
- Different lettersin the same raw refer to significant differences (p<0.05) vs. control.
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The obtained resultsin Table (2) revealed a significant decrease (p<0.05)the erythrocyte
values in group T1 which received paracetamol orally this observation is reflected by decline
RBC,haemoglobin concentration, haematocrit, MCH, MCHC and elevated in MCV level
when a compared with control group. On the other hand rabbit that treated with paracetamol
plus aqueous extract of ZizyphusSpina-Christi L. fruits showed improve blood component
(RBC, Hb, PCV and MCHC) as a compared with group T1 which received paracetamol.
Table (2) Effect of aqueous extract of ZizyphusSpina-Christi fruits on erythrocyte values in
male rabbits treated with paracetamol.
Animal group
Parameter
Control
group
Group T1
Group T2
RBC
(106/mm3) 5.93 ± 0.14a 4.72 ± 0.16c 5.40 ± 0.17b
Hb
(g/dl) 11.03 ± 0.27a 8.24 ± 0.27c 9.84 ± 0.19b
PCV
( %) 35.82 ± 0.18a 31.08 ± 0.25179c 33.84 ± 0.40b
MCV
(fl) 60.40 ± 1.20bc 65.84 ± 1.28a 62.66 ± 0.90ab
MCH
(Pg) 18.60 ± 0.24a 17.45 ± 0.39bc 18.22 ± 0.31ab
MCHC
(%) 30.79 ± 0.75a 26.51 ± 0.57b 29.07 ± 0.36a
- Values are expressed as mean ± SE, n = 6 each group
- Different letters in the same raw refer to significant differences (p<0.05) vs. control.
Our results about white blood cell (WBC) and differential count in table (3) indicted the
group-T1 which treated with paracetamol rabbits exhibited significant (P<0.05) increase in
white blood cell (WBC),increase in lymphocyte and monocytes and decrease neutrophils
when compared to control group.Results also clarified that co-administration ofaqueous
extract of ZizyphusSpina-Christi fruits and paracetamol significantlyp<0.05 elevation of
leucocyte count as a compared with group T1,while no significant difference of differential
count when compared with control group.
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Table (3) Effect of aqueous extract of Zizyphusspina-christifruits on leucocyte and
differential count male rabbits treated with paracetamol.
Animal Group
Parameter
Control group Group T1
Group T2
WBC 103/mm3 9.68 ± 0.23c 16.04 ± 0..37a 10.98 ± 0.34b
Lymphocytes 71.96 ± 0.72b 79.66 ± 0.73a 73.74± 0.96b
Neutrophils 22.40 ± 0.72a 12.90 ± 0.68b 20.80 ±0.68a
Monocytes 4.14 ± 0.20b 5.08 ± 0.29a 4.36 ±0.19b
- Values are expressed as mean ± SE, n = 6 each group
- Different letters in the same raw refer to significant differences (p<0.05) vs. control.
DISCUSSION
Paracetamol is commonly used as an analgesic and antipyretic drug at therapeutic doses.
However, the overdose of paracetamol cause hepatatoxicity and oxidative stress, the obtain
result indicated chronic paracetamol consumption induces severe liver injury and liver
necrosisas monitored by the elevation liver enzyme (AST and ALT) and reduction in total
protein and albumin concentration in group T1 which received paracetamol may be
paracetamol overdose caused formation reactive oxygen speciesand induce oxidative stress
which led to hepatotoxcicity, also elevation may be attributed to the release of these enzymes
from the cytoplasm into the blood circulation after rupture of the plasma membrane and
cellular damage. Serum AST and ALT are biomarkers in the diagnosis of hepatic damage
because they are released into the circulation after cellular damage (29). This result in
agreement withRadosavljevićin 2010 who reported overdose of paracetamol produce
oxidative stress caused severe liver injury with liver cell necrosis(30). Liver enzymes
elevation may be a reflection of radical-mediated lipid peroxidation of liver cell membrane,
this result similar with Reid in 2005 who observed paracetamol metabolic in liverby
cytochrome p450 which produce a toxic metabolite, N-acetyl-p-benzoquinone imine(NAPQI)
which caused depletes liver glutathione thereby inducing oxidative stress and binds to hepatic
cell and mitochondrial proteins leading to hepatocellular necrosis and increased lipid
peroxidation in hepatic tissues(31). Also NAPQI were binds to cysteine residues on proteins
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to form acetaminophen adducts which excellent correlate of acetaminophen toxicity (32).It
can be propose that this may be this binding led to protein miss folding –unfolding causing its
reduction. therefore, free radicals produced after paracetamol treatment may mediated
oxidation, proteolysis and degradation of albumin leading to its reduction (33). Also
prolonged paracetamol consumption caused destruction of the hepatic cells and hepatic
dysfunction led to impairs protein synthesis and decrease in the serum total protein, albumin
and globulin concentration (34).Our result agree with previous study that animal exposure to
paracetamol caused elevation in activity of ALT and AST and depression in total protein.
(35).
The current study revealed that oral administration of aqueous extract of Zizyphusspinachristifruits
in group T2have ameliorated effect agonist hepatotoxicity which induce by
paracetamol, our result shown reduction liver enzymes AST and ALT in addition elevation
total protein and albumin in group T2 when compared with group T1 which expose to
paracetamol may be due to phytochemical compound in extract of Zizyphusspinachristiwhich
act as antioxidant substance serve a inhibition free radicals-induced lipid
peroxidation and suppression paracetamol toxicity. phytochemical analysis of
Zizyphusspina-christi revealed present cyclopeptide alkaloids, flavonoids, sterols, tannins,
and triterpenoidsaponins (18), therefor, this compound act antioxidant activity for scavengers
free radical ( 14 ), This result agree with Yossef in 2011 who reportedZizyphusspina
reduction the serum levels of liver enzymes and restored normal levels of endogenous
antioxidants and it have protective effect and antioxidant activity agonist oxidative stress and
hepatic toxicity(12).
Oral administration of paracetamol induced anemia indicated from result obtain shown
decrease in the erythrocytic count, haemoglobin content and haematocrit value, MCH and
MCHC in groupT1 which treated with paracetamol as a compared with control group. The
reduction in erythrocyte value may be due to increase free radicals, reactive oxygen species,
and peroxide radicals after paracetamol administration which lead to hemolysis anemia(36),
also paracetamol caused hepatotixcity and impairs protein synthesis and decrease in the
serum total protein, albumin and globulin concentration, therefore, insufficiency of protein
synthesis that mainly induces decrease of essential amino acids and shortage of energy source
of protein synthesis incorporated in hemoglobin production and anemia (36). Our result in
accordance with previous study that paracetamol caused destruction RBC, and induce
thrombocytopenia and haemolyticanaemia(37).
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From result Zizyphusspina-christi have protective effect agonist haematotoxicity induce by
parcetamol in group T2 which received Zizyphusspina-christi and paracetamol when
compared with group T1 which treated with paracetamol may be due Zizyphusspinachristicontain
phytochemical compound include peptide,cyclopeptide, alkaloids, balsams,
carbohydrates, saponins, steroids, terpenes. polyphenol, flavonoids, sterols, tannins, betulinic
acid, triterpenoidal and glycosides (38),(39).These compound are well known haemopoietic
factors that have direct effect on the production of blood and antioxidant substance sever on
inhibition free radical, prevent hemolytic anemia and improve blood compenent
(40),(41).This result agree withPitchaiahin 2015who reported Zizyphus fruit have antianaemic
activity by enhanced the red blood cell count and hemoglobin concentration(42).
In the current work, chronic administration of paracetamol inducedsignificant increase in
total WBC, lymphocytes and monocytes may be due to oxidative stress which induce by
paracetamol, (43), this oxidative stress caused elevation WBC count(44), Our result agree
with Samuel in 2015 who reported paracetamol consumption reduced RBC count and PCV,
and elevated WBC count (45). Co-adminstration of ZizyphusSpina-Christi L fruit and
parcetamol shown decrease WBC as a compared with group T1 and restore differential count
near to control group. It has been suggested Zizyphusspina-christi fruit have antioxidant
compound act to inhibition oxidative stress and have antiflammatery activity led to return
WBC count and differential count near to normal value(46).
It may conclude that paracetamol induce hepato and haematotoxicity and Zizyphus extract
have hepatoprotective, antioxidant and antianemicactivity agonist hepato-and haematotoxicity
induce by paracetamol.
ضد البراستیمول zizyphusspinachristi الدور الوقائی للمستخلص المائی لفاکھة نبات السدر
المستحدث التسمم الکبدی والدموی فی ذکور الارانب البالغة
قیصر عبدالحسین عبید
قسم الصحة الحیوانیة، المعهد التقنی الجامعة التقنیة الجنوبیة ، الشطرة، ذی قار،الع ا رق
الخلاصة
یمتلک اسم شائع فی العراق "النبق " والذی استخدم فی الطب الشعبی. صممت ھذه Zizyphusspina-christi نبات السدر
الدراسة لتوضیح الدور الوقائی للمستخلص المائی لفاکھة نبات السدر ضد التسمم الکبدی والدموی الذی یحدثھالبراستیول
فی ذکور الارانب البالغة . استخدم ثمانیة عشر من ذکور الارانب البالغة قسمت عشوائیا الى ثلاثة مجامیع ست ارانب لکل
تتناول فمویا 300 ملغرام T مجموعة وعولجت لمدة اربع اسابیع وکالاتی :الاولى مجموعة السیطرة ، الثانیة مجموعة 1
تتناول فمویا 200 ملغرام/ کیلو غرام من وزن T /کیلو غرام من وزن الجسم من البراستیمول یومیا والثالثة مجموعة 2
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الجسم من المستخلص المائی لفاکھة نبات السدر یومیا مع 300 ملغرام / کیلو غرام من وزن الجسم من البراستیمول یومیا.
فی (P< اکدت النتائج ان اعطاء البراستیمول استحدث التسمم الکبدی والدموی الذی تصف بالارتفاع المعنوی ( 0.05
فی عدد (P< وانخفاض فی ترکیز البروتین الکلی والالبومین مع نقصان معنوی ( 0.05 (ALT وAST ) انزیمات الکبد
کریات الدم الحمر ،خضاب الدم ، معدل حجم کرات الدم المرصوص ، متوسط ھیموغلوبین الکریة ومتوسط ترکیز
ھیموغلوبین الکریة والعدلات بینما ارتفاع فی متوسط حجم الکریة ،عدد کریات الدم البیض ،الخلایا اللمفاویة وخلایا وحیدة
فی Ziziphusspina Christi النواة . من الناحیة الاخرى ، الحیوانات التی عولجت بالمستخلص المائی لثمار نبات السدر
لوحظ تحسن معنوی فی تغیرات القیم الکیموحیویة والدمویة التیاستحدثة بواسطة البراستیول . نستنتج من ذلک T مجموعة 2
ان اعطاء مستخلص نبات السدر یمتلک دور وقایة الکبد ومضاد فقر الدم ضدالتسمم الکبدی والدموی الذی استحدث بواسطة
البراستیمول.
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