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76
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
HEPATOPROTECTIVE EFFECT OF MORINGA OLEIFERA LEAVES
EXTRACT AGAINST CARBON TETRACHLORIDE- INDUCED LIVER
DAMAGE IN RATS
Kadry El-bakry1
El-shahat Toson2 Mamdouh Serag
3 and Magy Aboser
2
1Zoology Department Faculty of Science Damietta University Egypt
2Chemistry Department Faculty of Science Damietta University Egypt
3Botany Department Faculty of Science Damietta University Egypt
ABSTRACT
The present study aimed to evaluate of the antioxidant and
hepatoprotective effect of Moringa oleifera leaves extract against liver
injury induced by carbon tetrachloride (CCl4) Female albino rats were
used rats were injected ip with CCl4 (05 ml kg) mixed in olive oil
(vv) twice a week for 8 weeks and treated orally with Moringa
oleifera (400 mgkg) leaves extract daily for 8 weeks and compared
with a group of rats injected ip with CCl4 (05 ml kg) mixed in olive
oil (vv) twice a week for 8 weeks as a control group Liver functions
total antioxidant capacity (TAC) blood reduced glutathione (GSH) and
malondialdehyde (MDA) in red blood cells were determined Moreover superoxide
dismutase (SOD) and catalase enzymes in liver tissue homogenate were also assayed in
addition to liver histopathology Liver enzymes and MDA were increased after injection with
CCl4 otherwise serum albumin total protein TAC and tissue SOD catalase as well as blood
glutathione level were reduced Treatment with Moringa oleifera leaves extract reduce the
level of MDA and activity of liver enzymes and increase the activity of the antioxidant
parameters Simultaneously the histological deteriorative effects of CCl4 intoxication were
also improved after treatments with this extract In conclusions Moringa oleifera leaves
extract enhanced innate antioxidant activity and ameliorate the CCl4-induced liver injury and
therefore can be used as a hepatoprotective drug in the future
KEYWORDS Moringa oleifera antioxidant hepatoprotective drugs carbon tetrachloride
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6041
Volume 5 Issue 5 76-89 Research Article ISSN 2278 ndash 4357
Corresponding Author
Kadry El-bakry
Zoology Department Faculty
of Science Damietta
University Egypt
Article Received on
01 March 2016
Revised on 21 March 2016
Accepted on 11 April 2016
DOI 1020959wjpps20165-6638
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
INTRODUCTION
Liver play a major role in the regulation of many of the physiological processes in our bodies
which include metabolic secretory and storing functions[1]
Furthermore it is involved in
detoxification of a variety of drugs and xenobiotics In this case liver becomes susceptible to
the toxicity from these agents as the metabolic products of detoxification reactions can be
destructive to the liver when in excess[2 3]
Carbon tetrachloride (CCl4) is widely used as
experimental model of liver damage The hepatotoxicity of CCl4 is due to reductive
dehalogenation products such as trichloromethyl (CCl3∙) and trichloromethyl peroxyl
(CCl3O2∙) radicals[4]
These radicals can bind to proteins and lipids or remove a hydrogen
atom from an unsaturated fatty acids thereby initiating lipid peroxidation and contributing to
liver injury[5]
Hepatocyte injury initiates the activation of Kupffer cells which secrete potent
mediators of the early inflammatory response such as reactive oxygen species (ROS)
especially superoxide anions that accounted for the formation of peroxynitrites and hydrogen
peroxides (H2O2) therefore oxidative stress can be occur[6]
The antioxidants could attenuate
this oxidative damage caused by free radicals indirectly by enhancing natural defenses of cell
andor directly by scavenging the free radicals[7]
Antioxidants such as superoxide dismutase
(SOD) can scavenges the superoxide anions whereas the glutathion reduced (GSH) is
responsible to remove H2O2 through the action of glutathione peroxidase[8]
Also H2O2 is
consumed by the action of catalase[9]
In this sense the antioxidant action plays an important
role in protecting against CCl4-induced liver injury
Recently treatment with phytochemicals was one of the best choice for overcoming hepato-
toxifications especially those which follow a free radicals mediated mechanism[10- 12]
The
polyphenol extract of these phytochemicals was found to attenuate CCl4-induced liver injury
through free radicals scavenging mechanism[13 14]
Moringa oleifera belongs to the species of Moringaceae family Its leaves are rich of macro-
and micronutrients including polyphenols phenolic acids vitamins carotenoids flavonoids
and alkaloids[15]
Therefore Moringa oleifera plant is used in nurturing both animal and
human as an excellent nutritive supplement[16]
It had been enduringly used as a traditional
medicinal source and employed for treatment of many diseases thus coined as ldquothe miracle
treerdquo Subsequently the leaves have been studied for its various therapeutic properties such
as anticancer anti-inflammatory hepato-protective and antifungal activities[17 18]
On the
other hand previous studies showed that ethanolic extract of Moringa oleifera leaves can
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
effectively protected hepatic tissue from tissue damage induced by antitubercular drugs [19]
and acetaminophen[20]
It is therefore presently a settled fact that CCl4 mediated hepato-toxicity is at least in part a
result of generation of reactive oxygen species and that Moringa oleifera leaves extract
contain active antioxidative agents The major aim of the present study is therefore to
investigate the ameliorating potentials of the leaves extract of this plant on CCl4 mediated
liver damage
MATERIALS AND METHODS
Plant materials Moringa oleifera leaves were collected from the garden of New Damietta
city authenticated by Dr Mamdouh S Serag Professor of Plant Ecology Botany
Department Faculty of Science Damietta University Leaves were air- dried at room
temperature protected from dust and sunlight and pulverized manually
Preparation of plant extract Two kilograms of plant powder were extracted by maceration
in ethanol (70 vv) for 48 h The extract was then filtered and dried under reduced pressure
in a rotary evaporator at a temperature below 50degC and stored at -20 degC until use
Experimental animals Fifty female albino rats weighing 120-150 g were obtained from the
animal station Abo Rawash Egypt and were allowed acclimatization period of 14 days in an
ambient temperature of 25 plusmn 32 degC on light dark cycle of 1212 hours according to the
criteria outlined in the bdquobdquoGuide for the Care and Use of Laboratory Animals‟‟ prepared by the
National Academy of Sciences and published by the National Institute of Health (NIH) All
rats were kept in clean polypropylene cages and administered food and water ad libitum This
was approved by the Animal House of Biochemistry Chemistry Department Faculty of
Science Damietta University Egypt
Experimental design At the end of the acclimatization period animals were randomly
divided into 5 groups (10 rats group) Rats of Group I served as the control and received
nothing but normal feed Rats of Group II were injected intraperitoneally with olive oil (05
mlkg) twice a week for 8 weeks Rats of Group III and IV were injected intraperitoneally
CCl4 (05ml CCl4kg bwt) mixed in olive oil vv twice a week for 8 weeks Simultaneously
animals of groups IV was oral administered with Moringa oleifera extract (400 mgkg bwt)
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
via a stomach tube daily for 8 weeks whereas Group V received Moringa oleifera extract
(400 mgkg bwt) via a stomach tube without CCl4 treatment daily for 8 weeks
Samples After the end of the experimental period all animals were being fasted for 12 h
then sacrificed under chloroform anesthesia Whole blood was collected in clean dry tubes
withwithout EDTA glutathione reduced (GSH) was determined in whole blood Sera and
plasma samples were then obtained by centrifugation for 15 minutes at 4000 rpm and they
were kept in Eppendorf tubes and stored at -20 ゚C until required for assay of biochemical
parameters The lower erythrocyte layer in the EDTA tubes was used for determination of
MDA while ALT AST ALP albumin total protein and total antioxidant capacity were
assayed in rats‟ sera A large lobes of liver were collected washed with normal saline and
fixed in 10 formalin for histopathological studies At the same time a certain weight of
liver tissue from each rat was washed with normal saline and then it was homogenized in ice-
cold phosphate buffer (50 mM pH 75) and the resultant homogenate (10 wv) was
centrifuged at 12000 rpm for 20 min at 4 ˚C in a cooling centrifuge then the supernatant was
collected and stored at -20 degC for subsequent biochemical assays which include SOD and
catalase
Chemicals
Alanine transaminase (ALT) and Aspartate transaminase (AST) activities in serum was
assayed according to the method of Reitman and Frankel [21]
while Alkaline Phosphatase
(ALP) serum level estimated by the principle of Tietz[22]
Serum albumin and total protein
were performed by the method of Doumas[23]
and Gornall et al[24]
respectively using kits
were obtained from BIOMED Egy-Chem Co Cairo Egypt Total antioxidant capacity
(TAC) kit was purchased from Biodiagnostic Co Cairo Egypt Other chemicals used
throughout this investigation were of the highest analytical grade available
Determination of the biomarkers of liver oxidative stress
Assay of superoxide dismutase (SOD) activity in liver was determined according to the
method described by Nishikimi et al[25]
Assay of catalase (CAT) activity in liver was
obtained by the method described by Sinha[26]
Assay of liver reduced glutathione (GSH)
concentration in blood was done by the method of Beutler et al[27]
Lipid peroxidation was
evaluated on the basis of Malondialdehyde (MDA) level MDA in RBCs was determined
using the method described by Stocks and Donnandy[28]
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Histopathological studies
Fixed liver tissue samples were dehydrated by passing in ascending series of alcohol then
cleaned with xylene and embedded in paraffin wax Sections of the tissues 5ndash6 μm thickness
were prepared by using a rotary microtone and stained with hematoxylin and eosin (HampE)
dye
Statistical analysis
The data were expressed as mean plusmn SD and statistical analysis were performed by using
ANOVA followed by post hoc test (Tukey) It is a parametric statistical analysis that
compares between-and within-groups variance to measure differences between two or more
groups The P value less than 005 was considered as statistically significant The data
analysis was done with SPSS version 20
RESULTS
Body weight Table 1 showed that intoxication of rats with CCl4 induce significant decrease
(Plt005) in the total body weight by about 2778 and increase in liver-body weight ratio by
about 164 compared to the olive oil group Also administration of Moringa oleifera alone
showed a non-significant increase in the total body weight by about 176 compared to
control group
Table 1 Effect of Moringa oleifera (MO) treatment on body weight liver weight and
liver body weight ratio of CCl4 intoxicated rats
Group body weight Increase in body
weight (gm)
Liver weight
(gm)
Liver- body weight
ratio () Initial (gm) Final (gm)
Control 1313plusmn75 1946plusmn116 634plusmn96 (326) 68plusmn04 35plusmn01
Olive oil 1332plusmn88 195 4plusmn114 623plusmn77ns
(319) 73plusmn07 37plusmn03ns
CCl4 134 4plusmn52 1794plusmn104 45plusmn12$
(2506) 78plusmn08 43plusmn03$$
CCl4-MO 1285plusmn91 2038plusmn119 753plusmn81
(37) 72plusmn09 35plusmn04
MO 1291plusmn192 2036plusmn188 745plusmn139ns
(366) 69plusmn07 34plusmn03ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant Values
between parentheses represents the percentages of the mean value of body weight increase
divided by the final weight Liver-body weight ratio calculated by the percentages of the
mean value of liver weight divided by the final weight
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
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DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
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hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
INTRODUCTION
Liver play a major role in the regulation of many of the physiological processes in our bodies
which include metabolic secretory and storing functions[1]
Furthermore it is involved in
detoxification of a variety of drugs and xenobiotics In this case liver becomes susceptible to
the toxicity from these agents as the metabolic products of detoxification reactions can be
destructive to the liver when in excess[2 3]
Carbon tetrachloride (CCl4) is widely used as
experimental model of liver damage The hepatotoxicity of CCl4 is due to reductive
dehalogenation products such as trichloromethyl (CCl3∙) and trichloromethyl peroxyl
(CCl3O2∙) radicals[4]
These radicals can bind to proteins and lipids or remove a hydrogen
atom from an unsaturated fatty acids thereby initiating lipid peroxidation and contributing to
liver injury[5]
Hepatocyte injury initiates the activation of Kupffer cells which secrete potent
mediators of the early inflammatory response such as reactive oxygen species (ROS)
especially superoxide anions that accounted for the formation of peroxynitrites and hydrogen
peroxides (H2O2) therefore oxidative stress can be occur[6]
The antioxidants could attenuate
this oxidative damage caused by free radicals indirectly by enhancing natural defenses of cell
andor directly by scavenging the free radicals[7]
Antioxidants such as superoxide dismutase
(SOD) can scavenges the superoxide anions whereas the glutathion reduced (GSH) is
responsible to remove H2O2 through the action of glutathione peroxidase[8]
Also H2O2 is
consumed by the action of catalase[9]
In this sense the antioxidant action plays an important
role in protecting against CCl4-induced liver injury
Recently treatment with phytochemicals was one of the best choice for overcoming hepato-
toxifications especially those which follow a free radicals mediated mechanism[10- 12]
The
polyphenol extract of these phytochemicals was found to attenuate CCl4-induced liver injury
through free radicals scavenging mechanism[13 14]
Moringa oleifera belongs to the species of Moringaceae family Its leaves are rich of macro-
and micronutrients including polyphenols phenolic acids vitamins carotenoids flavonoids
and alkaloids[15]
Therefore Moringa oleifera plant is used in nurturing both animal and
human as an excellent nutritive supplement[16]
It had been enduringly used as a traditional
medicinal source and employed for treatment of many diseases thus coined as ldquothe miracle
treerdquo Subsequently the leaves have been studied for its various therapeutic properties such
as anticancer anti-inflammatory hepato-protective and antifungal activities[17 18]
On the
other hand previous studies showed that ethanolic extract of Moringa oleifera leaves can
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
effectively protected hepatic tissue from tissue damage induced by antitubercular drugs [19]
and acetaminophen[20]
It is therefore presently a settled fact that CCl4 mediated hepato-toxicity is at least in part a
result of generation of reactive oxygen species and that Moringa oleifera leaves extract
contain active antioxidative agents The major aim of the present study is therefore to
investigate the ameliorating potentials of the leaves extract of this plant on CCl4 mediated
liver damage
MATERIALS AND METHODS
Plant materials Moringa oleifera leaves were collected from the garden of New Damietta
city authenticated by Dr Mamdouh S Serag Professor of Plant Ecology Botany
Department Faculty of Science Damietta University Leaves were air- dried at room
temperature protected from dust and sunlight and pulverized manually
Preparation of plant extract Two kilograms of plant powder were extracted by maceration
in ethanol (70 vv) for 48 h The extract was then filtered and dried under reduced pressure
in a rotary evaporator at a temperature below 50degC and stored at -20 degC until use
Experimental animals Fifty female albino rats weighing 120-150 g were obtained from the
animal station Abo Rawash Egypt and were allowed acclimatization period of 14 days in an
ambient temperature of 25 plusmn 32 degC on light dark cycle of 1212 hours according to the
criteria outlined in the bdquobdquoGuide for the Care and Use of Laboratory Animals‟‟ prepared by the
National Academy of Sciences and published by the National Institute of Health (NIH) All
rats were kept in clean polypropylene cages and administered food and water ad libitum This
was approved by the Animal House of Biochemistry Chemistry Department Faculty of
Science Damietta University Egypt
Experimental design At the end of the acclimatization period animals were randomly
divided into 5 groups (10 rats group) Rats of Group I served as the control and received
nothing but normal feed Rats of Group II were injected intraperitoneally with olive oil (05
mlkg) twice a week for 8 weeks Rats of Group III and IV were injected intraperitoneally
CCl4 (05ml CCl4kg bwt) mixed in olive oil vv twice a week for 8 weeks Simultaneously
animals of groups IV was oral administered with Moringa oleifera extract (400 mgkg bwt)
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79
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
via a stomach tube daily for 8 weeks whereas Group V received Moringa oleifera extract
(400 mgkg bwt) via a stomach tube without CCl4 treatment daily for 8 weeks
Samples After the end of the experimental period all animals were being fasted for 12 h
then sacrificed under chloroform anesthesia Whole blood was collected in clean dry tubes
withwithout EDTA glutathione reduced (GSH) was determined in whole blood Sera and
plasma samples were then obtained by centrifugation for 15 minutes at 4000 rpm and they
were kept in Eppendorf tubes and stored at -20 ゚C until required for assay of biochemical
parameters The lower erythrocyte layer in the EDTA tubes was used for determination of
MDA while ALT AST ALP albumin total protein and total antioxidant capacity were
assayed in rats‟ sera A large lobes of liver were collected washed with normal saline and
fixed in 10 formalin for histopathological studies At the same time a certain weight of
liver tissue from each rat was washed with normal saline and then it was homogenized in ice-
cold phosphate buffer (50 mM pH 75) and the resultant homogenate (10 wv) was
centrifuged at 12000 rpm for 20 min at 4 ˚C in a cooling centrifuge then the supernatant was
collected and stored at -20 degC for subsequent biochemical assays which include SOD and
catalase
Chemicals
Alanine transaminase (ALT) and Aspartate transaminase (AST) activities in serum was
assayed according to the method of Reitman and Frankel [21]
while Alkaline Phosphatase
(ALP) serum level estimated by the principle of Tietz[22]
Serum albumin and total protein
were performed by the method of Doumas[23]
and Gornall et al[24]
respectively using kits
were obtained from BIOMED Egy-Chem Co Cairo Egypt Total antioxidant capacity
(TAC) kit was purchased from Biodiagnostic Co Cairo Egypt Other chemicals used
throughout this investigation were of the highest analytical grade available
Determination of the biomarkers of liver oxidative stress
Assay of superoxide dismutase (SOD) activity in liver was determined according to the
method described by Nishikimi et al[25]
Assay of catalase (CAT) activity in liver was
obtained by the method described by Sinha[26]
Assay of liver reduced glutathione (GSH)
concentration in blood was done by the method of Beutler et al[27]
Lipid peroxidation was
evaluated on the basis of Malondialdehyde (MDA) level MDA in RBCs was determined
using the method described by Stocks and Donnandy[28]
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Histopathological studies
Fixed liver tissue samples were dehydrated by passing in ascending series of alcohol then
cleaned with xylene and embedded in paraffin wax Sections of the tissues 5ndash6 μm thickness
were prepared by using a rotary microtone and stained with hematoxylin and eosin (HampE)
dye
Statistical analysis
The data were expressed as mean plusmn SD and statistical analysis were performed by using
ANOVA followed by post hoc test (Tukey) It is a parametric statistical analysis that
compares between-and within-groups variance to measure differences between two or more
groups The P value less than 005 was considered as statistically significant The data
analysis was done with SPSS version 20
RESULTS
Body weight Table 1 showed that intoxication of rats with CCl4 induce significant decrease
(Plt005) in the total body weight by about 2778 and increase in liver-body weight ratio by
about 164 compared to the olive oil group Also administration of Moringa oleifera alone
showed a non-significant increase in the total body weight by about 176 compared to
control group
Table 1 Effect of Moringa oleifera (MO) treatment on body weight liver weight and
liver body weight ratio of CCl4 intoxicated rats
Group body weight Increase in body
weight (gm)
Liver weight
(gm)
Liver- body weight
ratio () Initial (gm) Final (gm)
Control 1313plusmn75 1946plusmn116 634plusmn96 (326) 68plusmn04 35plusmn01
Olive oil 1332plusmn88 195 4plusmn114 623plusmn77ns
(319) 73plusmn07 37plusmn03ns
CCl4 134 4plusmn52 1794plusmn104 45plusmn12$
(2506) 78plusmn08 43plusmn03$$
CCl4-MO 1285plusmn91 2038plusmn119 753plusmn81
(37) 72plusmn09 35plusmn04
MO 1291plusmn192 2036plusmn188 745plusmn139ns
(366) 69plusmn07 34plusmn03ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant Values
between parentheses represents the percentages of the mean value of body weight increase
divided by the final weight Liver-body weight ratio calculated by the percentages of the
mean value of liver weight divided by the final weight
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81
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
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82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
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84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
effectively protected hepatic tissue from tissue damage induced by antitubercular drugs [19]
and acetaminophen[20]
It is therefore presently a settled fact that CCl4 mediated hepato-toxicity is at least in part a
result of generation of reactive oxygen species and that Moringa oleifera leaves extract
contain active antioxidative agents The major aim of the present study is therefore to
investigate the ameliorating potentials of the leaves extract of this plant on CCl4 mediated
liver damage
MATERIALS AND METHODS
Plant materials Moringa oleifera leaves were collected from the garden of New Damietta
city authenticated by Dr Mamdouh S Serag Professor of Plant Ecology Botany
Department Faculty of Science Damietta University Leaves were air- dried at room
temperature protected from dust and sunlight and pulverized manually
Preparation of plant extract Two kilograms of plant powder were extracted by maceration
in ethanol (70 vv) for 48 h The extract was then filtered and dried under reduced pressure
in a rotary evaporator at a temperature below 50degC and stored at -20 degC until use
Experimental animals Fifty female albino rats weighing 120-150 g were obtained from the
animal station Abo Rawash Egypt and were allowed acclimatization period of 14 days in an
ambient temperature of 25 plusmn 32 degC on light dark cycle of 1212 hours according to the
criteria outlined in the bdquobdquoGuide for the Care and Use of Laboratory Animals‟‟ prepared by the
National Academy of Sciences and published by the National Institute of Health (NIH) All
rats were kept in clean polypropylene cages and administered food and water ad libitum This
was approved by the Animal House of Biochemistry Chemistry Department Faculty of
Science Damietta University Egypt
Experimental design At the end of the acclimatization period animals were randomly
divided into 5 groups (10 rats group) Rats of Group I served as the control and received
nothing but normal feed Rats of Group II were injected intraperitoneally with olive oil (05
mlkg) twice a week for 8 weeks Rats of Group III and IV were injected intraperitoneally
CCl4 (05ml CCl4kg bwt) mixed in olive oil vv twice a week for 8 weeks Simultaneously
animals of groups IV was oral administered with Moringa oleifera extract (400 mgkg bwt)
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
via a stomach tube daily for 8 weeks whereas Group V received Moringa oleifera extract
(400 mgkg bwt) via a stomach tube without CCl4 treatment daily for 8 weeks
Samples After the end of the experimental period all animals were being fasted for 12 h
then sacrificed under chloroform anesthesia Whole blood was collected in clean dry tubes
withwithout EDTA glutathione reduced (GSH) was determined in whole blood Sera and
plasma samples were then obtained by centrifugation for 15 minutes at 4000 rpm and they
were kept in Eppendorf tubes and stored at -20 ゚C until required for assay of biochemical
parameters The lower erythrocyte layer in the EDTA tubes was used for determination of
MDA while ALT AST ALP albumin total protein and total antioxidant capacity were
assayed in rats‟ sera A large lobes of liver were collected washed with normal saline and
fixed in 10 formalin for histopathological studies At the same time a certain weight of
liver tissue from each rat was washed with normal saline and then it was homogenized in ice-
cold phosphate buffer (50 mM pH 75) and the resultant homogenate (10 wv) was
centrifuged at 12000 rpm for 20 min at 4 ˚C in a cooling centrifuge then the supernatant was
collected and stored at -20 degC for subsequent biochemical assays which include SOD and
catalase
Chemicals
Alanine transaminase (ALT) and Aspartate transaminase (AST) activities in serum was
assayed according to the method of Reitman and Frankel [21]
while Alkaline Phosphatase
(ALP) serum level estimated by the principle of Tietz[22]
Serum albumin and total protein
were performed by the method of Doumas[23]
and Gornall et al[24]
respectively using kits
were obtained from BIOMED Egy-Chem Co Cairo Egypt Total antioxidant capacity
(TAC) kit was purchased from Biodiagnostic Co Cairo Egypt Other chemicals used
throughout this investigation were of the highest analytical grade available
Determination of the biomarkers of liver oxidative stress
Assay of superoxide dismutase (SOD) activity in liver was determined according to the
method described by Nishikimi et al[25]
Assay of catalase (CAT) activity in liver was
obtained by the method described by Sinha[26]
Assay of liver reduced glutathione (GSH)
concentration in blood was done by the method of Beutler et al[27]
Lipid peroxidation was
evaluated on the basis of Malondialdehyde (MDA) level MDA in RBCs was determined
using the method described by Stocks and Donnandy[28]
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80
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Histopathological studies
Fixed liver tissue samples were dehydrated by passing in ascending series of alcohol then
cleaned with xylene and embedded in paraffin wax Sections of the tissues 5ndash6 μm thickness
were prepared by using a rotary microtone and stained with hematoxylin and eosin (HampE)
dye
Statistical analysis
The data were expressed as mean plusmn SD and statistical analysis were performed by using
ANOVA followed by post hoc test (Tukey) It is a parametric statistical analysis that
compares between-and within-groups variance to measure differences between two or more
groups The P value less than 005 was considered as statistically significant The data
analysis was done with SPSS version 20
RESULTS
Body weight Table 1 showed that intoxication of rats with CCl4 induce significant decrease
(Plt005) in the total body weight by about 2778 and increase in liver-body weight ratio by
about 164 compared to the olive oil group Also administration of Moringa oleifera alone
showed a non-significant increase in the total body weight by about 176 compared to
control group
Table 1 Effect of Moringa oleifera (MO) treatment on body weight liver weight and
liver body weight ratio of CCl4 intoxicated rats
Group body weight Increase in body
weight (gm)
Liver weight
(gm)
Liver- body weight
ratio () Initial (gm) Final (gm)
Control 1313plusmn75 1946plusmn116 634plusmn96 (326) 68plusmn04 35plusmn01
Olive oil 1332plusmn88 195 4plusmn114 623plusmn77ns
(319) 73plusmn07 37plusmn03ns
CCl4 134 4plusmn52 1794plusmn104 45plusmn12$
(2506) 78plusmn08 43plusmn03$$
CCl4-MO 1285plusmn91 2038plusmn119 753plusmn81
(37) 72plusmn09 35plusmn04
MO 1291plusmn192 2036plusmn188 745plusmn139ns
(366) 69plusmn07 34plusmn03ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant Values
between parentheses represents the percentages of the mean value of body weight increase
divided by the final weight Liver-body weight ratio calculated by the percentages of the
mean value of liver weight divided by the final weight
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81
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
wwwwjppscom Vol 5 Issue 5 2016
82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
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83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
via a stomach tube daily for 8 weeks whereas Group V received Moringa oleifera extract
(400 mgkg bwt) via a stomach tube without CCl4 treatment daily for 8 weeks
Samples After the end of the experimental period all animals were being fasted for 12 h
then sacrificed under chloroform anesthesia Whole blood was collected in clean dry tubes
withwithout EDTA glutathione reduced (GSH) was determined in whole blood Sera and
plasma samples were then obtained by centrifugation for 15 minutes at 4000 rpm and they
were kept in Eppendorf tubes and stored at -20 ゚C until required for assay of biochemical
parameters The lower erythrocyte layer in the EDTA tubes was used for determination of
MDA while ALT AST ALP albumin total protein and total antioxidant capacity were
assayed in rats‟ sera A large lobes of liver were collected washed with normal saline and
fixed in 10 formalin for histopathological studies At the same time a certain weight of
liver tissue from each rat was washed with normal saline and then it was homogenized in ice-
cold phosphate buffer (50 mM pH 75) and the resultant homogenate (10 wv) was
centrifuged at 12000 rpm for 20 min at 4 ˚C in a cooling centrifuge then the supernatant was
collected and stored at -20 degC for subsequent biochemical assays which include SOD and
catalase
Chemicals
Alanine transaminase (ALT) and Aspartate transaminase (AST) activities in serum was
assayed according to the method of Reitman and Frankel [21]
while Alkaline Phosphatase
(ALP) serum level estimated by the principle of Tietz[22]
Serum albumin and total protein
were performed by the method of Doumas[23]
and Gornall et al[24]
respectively using kits
were obtained from BIOMED Egy-Chem Co Cairo Egypt Total antioxidant capacity
(TAC) kit was purchased from Biodiagnostic Co Cairo Egypt Other chemicals used
throughout this investigation were of the highest analytical grade available
Determination of the biomarkers of liver oxidative stress
Assay of superoxide dismutase (SOD) activity in liver was determined according to the
method described by Nishikimi et al[25]
Assay of catalase (CAT) activity in liver was
obtained by the method described by Sinha[26]
Assay of liver reduced glutathione (GSH)
concentration in blood was done by the method of Beutler et al[27]
Lipid peroxidation was
evaluated on the basis of Malondialdehyde (MDA) level MDA in RBCs was determined
using the method described by Stocks and Donnandy[28]
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80
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Histopathological studies
Fixed liver tissue samples were dehydrated by passing in ascending series of alcohol then
cleaned with xylene and embedded in paraffin wax Sections of the tissues 5ndash6 μm thickness
were prepared by using a rotary microtone and stained with hematoxylin and eosin (HampE)
dye
Statistical analysis
The data were expressed as mean plusmn SD and statistical analysis were performed by using
ANOVA followed by post hoc test (Tukey) It is a parametric statistical analysis that
compares between-and within-groups variance to measure differences between two or more
groups The P value less than 005 was considered as statistically significant The data
analysis was done with SPSS version 20
RESULTS
Body weight Table 1 showed that intoxication of rats with CCl4 induce significant decrease
(Plt005) in the total body weight by about 2778 and increase in liver-body weight ratio by
about 164 compared to the olive oil group Also administration of Moringa oleifera alone
showed a non-significant increase in the total body weight by about 176 compared to
control group
Table 1 Effect of Moringa oleifera (MO) treatment on body weight liver weight and
liver body weight ratio of CCl4 intoxicated rats
Group body weight Increase in body
weight (gm)
Liver weight
(gm)
Liver- body weight
ratio () Initial (gm) Final (gm)
Control 1313plusmn75 1946plusmn116 634plusmn96 (326) 68plusmn04 35plusmn01
Olive oil 1332plusmn88 195 4plusmn114 623plusmn77ns
(319) 73plusmn07 37plusmn03ns
CCl4 134 4plusmn52 1794plusmn104 45plusmn12$
(2506) 78plusmn08 43plusmn03$$
CCl4-MO 1285plusmn91 2038plusmn119 753plusmn81
(37) 72plusmn09 35plusmn04
MO 1291plusmn192 2036plusmn188 745plusmn139ns
(366) 69plusmn07 34plusmn03ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant Values
between parentheses represents the percentages of the mean value of body weight increase
divided by the final weight Liver-body weight ratio calculated by the percentages of the
mean value of liver weight divided by the final weight
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81
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
wwwwjppscom Vol 5 Issue 5 2016
82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
wwwwjppscom Vol 5 Issue 5 2016
83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
80
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Histopathological studies
Fixed liver tissue samples were dehydrated by passing in ascending series of alcohol then
cleaned with xylene and embedded in paraffin wax Sections of the tissues 5ndash6 μm thickness
were prepared by using a rotary microtone and stained with hematoxylin and eosin (HampE)
dye
Statistical analysis
The data were expressed as mean plusmn SD and statistical analysis were performed by using
ANOVA followed by post hoc test (Tukey) It is a parametric statistical analysis that
compares between-and within-groups variance to measure differences between two or more
groups The P value less than 005 was considered as statistically significant The data
analysis was done with SPSS version 20
RESULTS
Body weight Table 1 showed that intoxication of rats with CCl4 induce significant decrease
(Plt005) in the total body weight by about 2778 and increase in liver-body weight ratio by
about 164 compared to the olive oil group Also administration of Moringa oleifera alone
showed a non-significant increase in the total body weight by about 176 compared to
control group
Table 1 Effect of Moringa oleifera (MO) treatment on body weight liver weight and
liver body weight ratio of CCl4 intoxicated rats
Group body weight Increase in body
weight (gm)
Liver weight
(gm)
Liver- body weight
ratio () Initial (gm) Final (gm)
Control 1313plusmn75 1946plusmn116 634plusmn96 (326) 68plusmn04 35plusmn01
Olive oil 1332plusmn88 195 4plusmn114 623plusmn77ns
(319) 73plusmn07 37plusmn03ns
CCl4 134 4plusmn52 1794plusmn104 45plusmn12$
(2506) 78plusmn08 43plusmn03$$
CCl4-MO 1285plusmn91 2038plusmn119 753plusmn81
(37) 72plusmn09 35plusmn04
MO 1291plusmn192 2036plusmn188 745plusmn139ns
(366) 69plusmn07 34plusmn03ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant Values
between parentheses represents the percentages of the mean value of body weight increase
divided by the final weight Liver-body weight ratio calculated by the percentages of the
mean value of liver weight divided by the final weight
wwwwjppscom Vol 5 Issue 5 2016
81
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
wwwwjppscom Vol 5 Issue 5 2016
82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
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83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
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El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
81
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
Liver function tests Table 2 showed that intoxication of rats with CCl4 induce significant
elevations (Plt0001) in serum ALT AST and ALP activities accompanied with a significant
reduction in serum albumin and total proteins levels compared with those of the olive oil
group On the other hand rats received CCl4 plus Moringa oleifera extract showed significant
improvements in all of these liver function tests compared with those of animals received
CCl4 alone
Table 2 Effect of Moringa oleifera (MO) treatment on the mean activities of alanine
transaminase (ALT) aspartate transaminase (AST) and alkaline phosphatase (ALP)
enzymes as well as the mean levels of serum albumin and total protein of CCl4
intoxicated rats
Group ALT (UL) AST (UL) ALP (UL) Albumin (gdl) T protein (gdl)
Control 11plusmn30 99plusmn16 402plusmn28 32plusmn02 64plusmn04
Olive oil 108plusmn40 ns
97plusmn18ns
399plusmn76 ns
29plusmn02 ns
55plusmn05 ns
CCl4 228plusmn19 $$$
153plusmn19$$$
788plusmn138$$$
21plusmn07 $$
43plusmn04$$$
CCl4-MO 115plusmn23
81plusmn31
378plusmn6
28plusmn03 52plusmn02
MO 119plusmn45 ns
89plusmn25 ns
347plusmn37 ns
33plusmn04 ns
65plusmn05 ns
Data were expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$)
very significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive
oil group versus those of the CCl4 group ( and ) and (ns) not significant
Table 3 Effect of Moringa oleifera(MO) on the mean activities of superoxide dismutase
(SOD) and catalase enzymes as well as the mean levels of reduced glutathione (GSH)
malondialdehyde (MDA) and total antioxidant capacity (TAC) of CCl4 intoxicated rats
groups SOD
( inhibition)
GSH
(mMg Hb)
MDA
(microMml packed cell)
TAC
(mML)
Catalase
Ug tissue
Control 59plusmn73 2539plusmn585 73plusmn1 16plusmn01 1264plusmn237
Olive oil 516plusmn 106 ns
2254plusmn407ns
76plusmn09 ns
17plusmn01ns
1223plusmn241 ns
CCl4 365plusmn 88$ 1402plusmn142
$$ 105plusmn16
$$$ 14plusmn02
$$$ 649plusmn241
$
CCl4-MO 608plusmn 93
2172plusmn261 81plusmn08
18plusmn02
126plusmn429
MO 641plusmn127 ns
2444plusmn326 ns
71plusmn08 ns
16plusmn004 ns
1327plusmn403 ns
Data are expressed as mean plusmn SD (n = 10 in each group) ($)significant Plt 005 ($$) very
significant Plt 001 and ($$$) extremely significant Plt 0001 versus those of the olive oil
group versus those of the CCl4 group ( and ) and (ns) not significant
Antioxidant biomarkers Table 3 showed that intoxication of rats with CCl4 induce
significant (Plt0001) increase in MDA level a significant (Plt005) decrease in TAC
concentration and activities of both hepatic SOD and CAT as well as GSH content compared
with those of olive oil group On contrast the administration of Moringa oleifera extract
wwwwjppscom Vol 5 Issue 5 2016
82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
wwwwjppscom Vol 5 Issue 5 2016
83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
82
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
resulted in significant improvement (Plt005) in all of these parameters compared with those
of rats administered CCl4 alone
Histopathological analysis As reflected from Fig 1 the liver of control rat showed normal
liver structure including central vein (CV) lined by endothelial cells (en) hepatic strands
(hs) hepatic cells (hp) kupffer cells (K) and normal sinusoids (S) (Fig 1A) Also olive oil
rat group showed normal cellular articulation with distinct hepatic cells sinusoidal spaces
(S) billary duct (B) portal vein (PV) and hepatic artery (ha) (Fig 1B) Furthermore CCl4
administration showed histopathological changes including hepatic architecture
disorganization with severe fatty degeneration of hepatocytes and inflammatory cells
infiltration (f) as well as necrotic cells number (n) elevation and most of the blood sinusoids
appeared narrow or even obliterated (Fig 1C) On the other hand using Moringa oleifera
extract along with CCl4 showing improvement in liver histology most areas appear to have
recovered hepatocytes well preserved and no area of necrosis (Fig 1D) Administration of
Moringa oleifera extract alone showed normal hepatocellular architecture and there were no
significant pathological changes compared to the control group (Fig 1E)
Figure 1 Photomicrograph of liver sections (stained with haematoxylin and eosin times
200) from (a) control rat showing normal liver structure (B) olive oil rat group
showing normal cellular articulation of the portal area (C) CCl4 intoxicated rat
showing vascular degeneration (D) CCl4-MO group showing improvement in liver
histology (E) MO group showing normal hepatocellular architecture CV Central
vein en Endothelial cells hs Hepatic strands hp Hepatic cells K Kupffer cells S
Sinusoids B Billary duct PV Portal vein ha Hepatic artery
wwwwjppscom Vol 5 Issue 5 2016
83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
83
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
DISCUSSION
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the
experimental studies to investigate the liver injury that associated with oxidative stress and
free radicals[92]
This is because the metabolism of CCl4 begins with the formation of
trichloromethyl free radical (CCl3) which was formed by the action of the mixed function of
the cytochrome P450 oxygenase system This free radical reacts very rapidly with oxygen to
yield a highly reactive trichloromethyl peroxy radical (CCl3OO)
[4] Both radicals are capable
of binding to proteins or lipids thus initiating tissue lipids peroxidation inflammation
hepato-toxification and MDA accumulation[5]
The latter is one of the final products of lipid
peroxidation[30]
The results of the present study showed that CCl4 induced a significant reductions in the total
body weights of the intoxicated animals Such reduction may be due to the hepatotoxic
effects of CCl4 administration which may contribute to the reduction of serum albumin and
total protein concentration which is actually the case in this study (Table 2)
Hepatomegaly is one of the most common complications of liver fibrosis[31]
In the present
study Moringa oleifera leaves extract could improve the hepatomegaly induced by chronic
CCl4 administration in rats This may lead one to conclude that this plants extract can retard
the progression of liver fibrosis Concerning the results of the present study are in agreement
with those obtained by Ezejindu et al who found that the final body weights of their
experiment on normal rats fed with low and high doses of Moringa oleifera leaves extract
were significantly increased compared to that of the control group[32]
Based on the previous
data one can conclude that the extract of Moringa oleifera leaves in this instance functions
primarily as dietary supplement which can enhance body weight growth This is due to this
part of the plant is considered as a high delivery source of protein β-carotene vitamins A B
C E riboflavin nicotinic acid folic acid and pyridoxine amino acids minerals and various
phenolic compounds[20 33 34]
The results of the present study revealed that CCl4 administration induces oxidative stress
which was manifested by the significant increase in MDA levels in blood as a consequence of
the significant reduction in TAC GSH content and both SOD and CAT activities The
mechanism may involve the participation of the latters in the scavenging ability of the cells
and thus the reductions in their mean values mediated the overproduction of MDA These
results were coincide with Gangarapu et al[35]
Najappaiah and Hugar [36]
and Palanivel et
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
84
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
al[37]
in that CCl4 administration resulted in a significant elevation in MDA and reduction of
GSH content and activity of antioxidant enzymes SOD and CAT in hepatic tissue
In the present study the serum activities of ALT AST and ALP were increased after CCl4
intoxication This result may be based on liver cell plasma membrane alteration via oxidative
stress mediation a process which can participate in cytosolic enzymes including ALT AST
and ALP liberation with a simultaneous serum level elevations Therefore the measurement
of the activities of these serum enzymes can help in liver functions investigation[38]
Also the
reduction in serum total protein and albumin concentrations after CCl4 intoxication may be
due to considerable liver damage through induction of lipids peroxidation cellular membrane
inflammation andor degradation and synthetic function inactivation The latter may be due to
trichloromethyl free radical-cell membrane conjugation[39]
A somewhat similar results were
obtained by Bahashwan et al[40]
El-Meligy et al[41]
and Gangarapu et al[35]
who
demonstrated the same hepatic enzymes elevations and serum total protein and albumin
deteriorations
The efficacy of any hepatoprotevtive drug depends on its capacity of either reducing the
harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4
andor other hepatotoxicants[42]
The present study revealed that Moringa oleifera
administration causes significant serum levels of ALT AST ALP albumin and total protein
amelioration after CCl4-intoxication The reduction in the activities of ALT AST and ALP as
a result of Moringa oleifera leaves extract point us towards an early improvement in the
cellular membrane integrity of the hepatic cell which is a clear manifestation of anti-
hepatotoxic effect of its administration The data of the present study were in consistence
with previous studies who found that the extract of Moringa oleifera leaves causes significant
reduction the elevated levels of ALT AST and ALP in animals model intoxicated by
acetaminophen [43]
cadmium chloride [44]
Diclofenac [45]
arsenic [46]
and alcohol [47]
Based
on the data of the previous and the present study one can suggest that the protective
mechanisms of Moringa oleifera leaves extract may follow an antioxidant mediated
mechanism This was obvious from the results of antioxidant enzymes including enzymatic
(SOD and catalase) and non enzymatic (GSH) as well as total antioxidant capacity which was
found to be elevated after treatment of CCl4 intoxicated animals with this extract At the same
time the simultaneous reduction of MDA level after the latter enzymatic elevations confirm
the antioxidant mediated mechanism incorporation Moreover previous studies have shown
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
85
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
that Moringa oleifera leaves extract increased the liver SOD and CAT activities as well as
GSH content and significantly reduced elevated MDA in rats intoxicated by
acetaminophen[20 43]
cadmium chloride [44]
and alcohol[47]
The antioxidant activity of Moringa oleifera is mainly due to their content of phenolic
compounds as the principal polyphenol compounds in Moringa oleifera leaves are
kaempferol rhamnetin quercitin chlorogenic acid rutin apigenin[48 49]
These phenolic
compounds are multifactorial defenders against oxidative stress This is because they can act
as reducing agents via singlet oxygen scavengers and hydrogen atom donators with
subsequent stabilization of the produced free radicals forming stable compounds that do not
initiate or propagate oxidation In addition the involvement of the anti-inflammatory and
analgesic activities of such phenolic compounds in both plants in the protective mechanisms
cannot be neglected Further phenolic compounds including flavonoids can protect the cells
against emptying reduced glutathione via activating the activity of glutathione reductase as
well as increasing the activities of other antioxidant enzymes which are ultimately helpful in
hepatoprotection[50]
Histopathological results of the present study not only confirm the hepatotoxic effect of CCl4
on hepatic cells but also the hepatic recovery after treatment of intoxicated rats with Moringa
oleifera extract This is due to the improvement in hepatic architecture disorganization
severe hepatic fatty degeneration together with inflammatory cells infiltration after Moringa
oleifera extract administration A somewhat similar results were obtained by Taha et al after
Diclofenac intoxication[45]
CONCLUSION
The present study concluded that Moringa oleifera leaves can not only protect against liver
intoxication but also participate in body weight amelioration and when the antioxidant
mechanisms be one of our target investigation
REFERENCES
1 Kumar CH Ramesh A Suresh Kumar JN Mohammed Ishaq B A review on
hepatoprotective activity of medicinal plants Int J Pharmaceut Sci Res 2011 23 501ndash
515
2 Muriel P Some experimental models of liver damage In Sahu SC editor
Hepatotoxicity From genomics to in vitro and in vivo models West Sussex England
Wiley 2007 119ndash137
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
86
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
3 Muriel P Cytokines in liver diseases In Sahu SC editor Hepatotoxicity From genomics
to in vitro and in vivo models West Sussex England Wiley 2007 pp 371ndash389
4 Brattin WJ Glende Jr EA Recknagel RO Pathological mechanisms in carbon
tetrachloride hepatotoxicity Journal of Free Radicals in Biology and Medicine 1985
1(1) 27ndash38
5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride
toxicity Pharmacology and Therapeutics 1989 43(1) 139ndash154
6 Agarwal R Hennings L Rafferty TM Letzig LG McCullough S James LP et al
Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide
synthase knockout mice J Pharmacol Exp Ther 2012 340(1) 134ndash142
7 Krinsky NI Mechanism of action of biological antioxidants Proc Soc Exp Biol Med
1992 200 248ndash254
8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et
al Glutathione and antioxidant enzymes serve complementary roles in protecting
activated hepatic stellate cells against hydrogen peroxide-induced cell death Biochim
Biophys Acta 2013 1832(12) 2027ndash34
9 Salvi M Battaglia V Brunati AM LaRocca N Tibaldi E Pietrangeli P Marcocci L
Mondovi B Rossi CA Toninello A Catalase takes part in rat liver mitochondria
oxidative stress defense J Biol Chem 2007 282 24407ndash24415
10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of
capsaicin against carbon tetrachloride-induced hepatotoxicity in rats Toxicol Ind Health
2012 28(5) 428ndash438
11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects
of resveratrol on liver injury in streptozotocin-induced diabetic rats J Biochem Mol
Toxicol 2012 26(10) 384ndash392
12 Soylu AR Aydogdu N Basaran UN Altaner S Tarcin O Gedik N et al Antioxidants
vitamin E and C attenuate hepatic fibrosis in biliary-obstructed rats World J
Gastroenterol 2006 12 6835ndash6841
13 Yang JY Li Y Wang F Wu C Hepatoprotective effects of apple polyphenols on CCl4-
induced acute liver damage in mice Journal of Agricultural and Food Chemistry 2010
58(10) 6525ndash6531
14 Yeh YH Hsieh YL Lee YT Effects of yampeel extract against carbon tetrachloride-
induced hepatotoxicity in rats Journal of Agricultural and Food Chemistry 2013 61(30)
7387ndash7396
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
87
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
15 Leone A Spada A Battezzati A Schiraldi A Aristil J Bertoli S Cultivation Genetic
Ethnopharmacology Phytochemistry and Pharmacology of Moringa oleifera Leaves An
Overview Int J Mol Sci 2015 16 12791ndash12835
16 Siddhuraju P Becker K Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa
oleifera Lam) leaves Journal of Agricultural and FoodChemistry 2003 51(8) 2144ndash
2155
17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa
oleifera) during convective drying African Journal of Plant Science 2010 4 (10) 391ndash
400
18 Patel RK Manish MP Nilesh RK Kirit RV Patel RK In vitro hepatoprotective activity
of Moringa oleifera Lam Leave on isolated Rat hepatocytes Int J Ph Sci 2010 2(1)
457ndash463
19 Pari L Kumar NA Hepatoprotcetive activity of Moringa oleifera on antitubercular drug-
induced liver damage in rats J Med Food 2002 5 171ndash177
20 Fakurazi S Sharifudin SA Arulselvan P Moringa oleifera hydroethanolic extracts
effecively alleviate acetaminophen-induced hepatotoxicity in experimental rats through
their antioxidant nature Molecules 2012 17 8334ndash8350
21 Reitman S Frankel S Colorimetric methods for aspartate and alanine monotransferases
Am J Clin Path 1957 28 55ndash60
22 Tietz NW Fundamentals of Clinical Chemistry WB Saunders Co Philadelphia 1976
23 Doumas BT Watson WA Biggs HG Albumin standards and the measurement of serum
albumin with bromocresol green Clinica Chimica Acta 1971 31 87ndash96
24 Gornall AC Bardawill CJ David MM Determination of serum proteins by means of
biuret reaction J Biol Chem 1949 177 751ndash766
25 Nishikimi M Roa NA Yogi K The occurrence of superoxide anion in the reaction of
reduced phenazine methosulfate and molecular oxygen Biochem Biophy Res Commun
1972 46 849ndash54
26 Sinha AK Colorimetric assay of catalase Anal biochem 1972 47 389ndash394
27 Beutler F Duron O Kelly MB Improved method of estimation of blood glutathione J
Lab Clin Med 1963 61(5) 882
28 Stocks J Donnandy T The autoxidation of human red cell lipids induced by hydrogen
peroxide Br J Haematol 1971 20 95ndash111
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
88
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
29 Dahiru D Mamman DN Wakawa HY Ziziphus mauritiana fruit extract inhibits CCl4-
induced hepatotoxicity in male rats Pak J Nutr 2010 9 990ndash993
30 Mohamed AM Mahmoud SS Farag ARA Influence of stiva seeds against liver fibrosis
and consequences complications in murine schistosomiasis Int J Biotechnol Biochem
2008 4 325ndash346
31 Gill MA Kircbain WR Alcoholic liver disease In Dipiro JT Talbert RL Yee GC
Matzke GR Wells BG Poser LM (eds) Pharmacotherapy a pathophysiologic approach
Stamford Appleton and Lange 1997 pp 785ndash800
32 Ezejindu DN Udemezue OO Chinweife KC Hepatoprtective effects of Moringa oleifera
extract on liver of wistar rats International journal of research in medical and health
sciences 2014 3(5)
33 Anwar F Latif S Ashraf M Gilani AH Moringa oleifera A food plant with multiple
medicinal uses Phytother Res 2007 21 17ndash25
34 Khalafalla MM Abdellatef E Dafalla HM Nassrallah AA Aboul-Enein KM Lightfoot
DA El-Deeb FE El-Shemy HA Active principle from Moringa oleifera Lam leaves
effective against two leukemias and a hepatocarcinoma African Journal of
Biotechnology 2010 9(49) 8467ndash8471
35 Gangarapu V Gujjala S Korivi S et al Combined effect of curcumin and vitamin E
against CCl4 induced liver injury in rats Am J Life Sci 2013 1(3)117ndash124
36 Nanjappaiah HM Hugar S Prophylactic and curative effects of Moringa oleifera Lam
pods in CCl4 damaged rat liver Indian J Nat Prod Resour 2012 3(4) 541ndash546
37 Palanivel MG Rajkapoor B Kumar RS et al Hepatoprotective and antioxidant effect of
Pisonia aculeate L against CCl4-induced hepatic damage in rats Sci Pharm 2008 76
203ndash215
38 Jaeschke H Williams CD McGill MR Xie Y Ramachandran A Models of drug-induced
liver injury for evaluation of phytotherapeutics and other natural products Food Chem
Toxicol 2013 55 279ndash89
39 Lee KJ Woo E Choi CY Shin DW Lee DG You HJ Jeong HG Protective effect of
acteoside on carbon tetrachloride-induced hepatotoxicity Life Sci 2004 74 1051ndash1064
40 Bahashwan S Hassan MH Aly H Ghobara MM El-Beshbishy HA Busati I Crocin
mitigates carbon tetrachloride-induced liver toxicity in rats Journal of Taibah University
Medical Sciences 2015 10(2) 140ndash149
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
wwwwjppscom Vol 5 Issue 5 2016
89
El-bakry et al World Journal of Pharmacy and Pharmaceutical Sciences
41 El-Meligy RM Zain ME Ahmed FA Protective role of Cynanchum acutum L extracts
on carbon tetrachloride-induced hepatotoxicity in rat Int J Chem Appl Biol Sci 2014 1
8-13
42 Senthilkumar MKT Rajkapoor B Kavimani S Protective effect of Enicostemma littorale
against CCl4-induced hepatic damage in rats Pharmaceuti Biol 2005 43 485ndash487
43 Karthivashan G Arulselvan P Tan S Fakurazi S The molecular mechanism underlying
the hepatoprotective potential of Moringa oleifera leaves extract against acetaminophen
induced hepatotoxicity in mice Journal of Functional Foods 2015 17 115ndash126
44 Toppo R Roy BK Gora RH Baxla SL Kumar P Hepatoprotective activity of Moringa
oleifera against cadmium toxicity in rats Veterinary World 2015 8(4) 537ndash540
45 Taha NR Rabah SO Shaker SA Mograby MM Effect of Moringa oleifera Leaves on
Diclofenac Sodium Induced Hepatic Injury in Albino Rats Ultrastructural and
Immunohistochemical Studies J Cytol Histol 2015 6 315
46 Sheikh A Yeasmin F Agarwal S Rahman M Islam K Hossain E et al Protective effects
of Moringa oleifera Lam leaves against arsenic-induced toxicity in mice Asian Pac J
Trop Biomed 2014 4(1) 353ndash358
47 Saalu LC Ogunlade B Ajayi GO Oyewopo AO Akunna GG Ogunmodede OS The
hepato-protective potentials of Moringa oleifera leaf extract on alcohol-induced hepato-
toxicity in wistar rat Am J Biotechnol Mol Sci 2012 2(1) 6ndash14
48 Ezuruike UF Prieto JM The use of plants in the traditional management of diabetes in
Nigeria Pharmacological and toxicological considerations Journal of
Ethnopharmacology 2014 155(2) 857ndash924
49 Karthivashan G Tangestani FM Arulselvan P Abas F Fakurazi S Identification of
bioactive candidate compounds responsible for oxidative challenge from hydroethanolic
extract of Moringa oleifera leaves Journal of Food Science 2013 78(9) 1368ndash1375
50 Samani MA Farkhad NK Azimi N Fasihi A Ahandani EA Kopaei MR Medicinal
plants with hepatoprotective activity in Iranian folk medicine Asian Pac J Trop Biomed
2015 5(2) 146ndash157
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