hepatoprotective effect of moringa oleifera leaves …

wwwwjppscom Vol 5 Issue 5 2016

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


77


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


78


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)


79


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]


80


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


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


81


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


Olive oil 516plusmn 106 ns

2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns

CCl4 365plusmn 88$ 1402plusmn142

$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$

CCl4-MO 608plusmn 93


18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns



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


82


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


83


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


84


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


85


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]


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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5 Recknagel RO Glende Jr EA Dolak JA Waller RL Mechanisms of carbon tetrachloride

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8 Dunning S Ur Rehman A Tiebosch MH Hannivoort RA Haijer FW Woudenberg J et

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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

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10 Hassan MH Edfawy M Mansour A Hamed AA Antioxidant and antiapoptotic effects of

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11 Hamadi N Mansour A Hassan MH Khalifi-Touhami F Badary O Ameliorative effects

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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

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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

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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


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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

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17 Monica PHK Sharma B Sarkar C Singh C Kinetics of drumstick leaves (Moringa

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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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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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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


77


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


78








liver damage
























79

















catalase

Chemicals























80






dye







RESULTS





control group




weight (gm)

Liver weight

(gm)

Liver- body weight


















81







CCl4 alone




intoxicated rats




97plusmn18ns

399plusmn76 ns

29plusmn02 ns

55plusmn05 ns


153plusmn19$$$

788plusmn138$$$

21plusmn07 $$

43plusmn04$$$

CCl4-MO 115plusmn23

81plusmn31

378plusmn6


MO 119plusmn45 ns

89plusmn25 ns

347plusmn37 ns

33plusmn04 ns

65plusmn05 ns







groups SOD

( inhibition)

GSH

(mMg Hb)

MDA


TAC

(mML)

Catalase

Ug tissue



2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns


$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$



18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns









82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


78








liver damage
























79

















catalase

Chemicals























80






dye







RESULTS





control group




weight (gm)

Liver weight

(gm)

Liver- body weight


















81







CCl4 alone




intoxicated rats




97plusmn18ns

399plusmn76 ns

29plusmn02 ns

55plusmn05 ns


153plusmn19$$$

788plusmn138$$$

21plusmn07 $$

43plusmn04$$$

CCl4-MO 115plusmn23

81plusmn31

378plusmn6


MO 119plusmn45 ns

89plusmn25 ns

347plusmn37 ns

33plusmn04 ns

65plusmn05 ns







groups SOD

( inhibition)

GSH

(mMg Hb)

MDA


TAC

(mML)

Catalase

Ug tissue



2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns


$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$



18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns









82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


79

















catalase

Chemicals























80






dye







RESULTS





control group




weight (gm)

Liver weight

(gm)

Liver- body weight


















81







CCl4 alone




intoxicated rats




97plusmn18ns

399plusmn76 ns

29plusmn02 ns

55plusmn05 ns


153plusmn19$$$

788plusmn138$$$

21plusmn07 $$

43plusmn04$$$

CCl4-MO 115plusmn23

81plusmn31

378plusmn6


MO 119plusmn45 ns

89plusmn25 ns

347plusmn37 ns

33plusmn04 ns

65plusmn05 ns







groups SOD

( inhibition)

GSH

(mMg Hb)

MDA


TAC

(mML)

Catalase

Ug tissue



2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns


$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$



18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns









82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


80






dye







RESULTS





control group




weight (gm)

Liver weight

(gm)

Liver- body weight


















81







CCl4 alone




intoxicated rats




97plusmn18ns

399plusmn76 ns

29plusmn02 ns

55plusmn05 ns


153plusmn19$$$

788plusmn138$$$

21plusmn07 $$

43plusmn04$$$

CCl4-MO 115plusmn23

81plusmn31

378plusmn6


MO 119plusmn45 ns

89plusmn25 ns

347plusmn37 ns

33plusmn04 ns

65plusmn05 ns







groups SOD

( inhibition)

GSH

(mMg Hb)

MDA


TAC

(mML)

Catalase

Ug tissue



2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns


$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$



18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns









82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


81







CCl4 alone




intoxicated rats




97plusmn18ns

399plusmn76 ns

29plusmn02 ns

55plusmn05 ns


153plusmn19$$$

788plusmn138$$$

21plusmn07 $$

43plusmn04$$$

CCl4-MO 115plusmn23

81plusmn31

378plusmn6


MO 119plusmn45 ns

89plusmn25 ns

347plusmn37 ns

33plusmn04 ns

65plusmn05 ns







groups SOD

( inhibition)

GSH

(mMg Hb)

MDA


TAC

(mML)

Catalase

Ug tissue



2254plusmn407ns

76plusmn09 ns

17plusmn01ns

1223plusmn241 ns


$$ 105plusmn16

$$$ 14plusmn02

$$$ 649plusmn241

$



18plusmn02

126plusmn429

MO 641plusmn127 ns

2444plusmn326 ns

71plusmn08 ns

16plusmn004 ns

1327plusmn403 ns









82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


82

























83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


83


DISCUSSION



free radicals[92]









peroxidation[30]






In the present




















and Palanivel et


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


84


al[37]








Also the







El-Meligy et al[41]


who


deteriorations












acetaminophen [43]


Diclofenac [45]

arsenic [46]

and alcohol [47]

Based









85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


85






and alcohol[47]




These phenolic
















CONCLUSION




REFERENCES



515





86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


86






1(1) 27ndash38







1992 200 248ndash254










2012 28(5) 428ndash438



Toxicol 2012 26(10) 384ndash392






58(10) 6525ndash6531



7387ndash7396


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


87








2155



400



457ndash463















1972 46 849ndash54



Lab Clin Med 1963 61(5) 882




88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


88






2008 4 325ndash346






sciences 2014 3(5)













203ndash215










89




8-13

























2015 5(2) 146ndash157


89




8-13

























2015 5(2) 146ndash157

hepatoprotective effect of moringa oleifera leaves …

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