Pharmacologyonline 3: 1021-1032 (2011) Sharma and...

Pharmacologyonline 3: 1021-1032 (2011) Sharma and Kumar

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HEPATOPROTECTIVE EFFECT OF CHLOROPHYTUM

BORIVILIA�UM ROOT EXTRACT AGAI�ST ARSE�IC

I�TOXICATIO�

Sunil Kumar Sharma and Madhu Kumar*

Cell & Molecular Biology Lab, Deptt of Zoology, University of

Rajasthan, Jaipur, India

*Corresponding author: Tel: +91 9829324629. e-mail :

[email protected]

Summary

The present study was conducted to find out the hepatoprotective effect of

Chlorophytum borivilianum root extract against arsenic induced toxicity.

Three groups were made: Control(DDW), Arsenic intoxicated group (4 mg/kg

b.w) and Combination group (NaAsO2(4 mg/kg b.w)+ cb root extract(800

mg/kg b.w). Animals received their respective doses daily for 30 days orally.

Body weight, liver weight, heptopathological changes and level of ATPase

were observed. Result showed significant decrease in body and liver weight

along with disturbed hepatoarchitecture and decline in ATPase activity in

arsenic intoxicated group as compared to control. In combination group,

increased body and liver weight along with increased ATPase levels and

almost normal hepatoarchitecture were observed as compared to arsenic

treated group. Thus it can be concluded that Chlorophytum borivilianum root

extract has potential to decrease the toxic effects of arsenic.

Keywords: Cb; sodium arsenite; body weight; liver weight; ATPase .

Introduction

Arsenic, a ubiquitous metalloid which occurs naturally is a toxic pollutant. It

is ranked first in a list of 20 hazardous substances1. Its exposure occurs from

inhalation, absorption through skin, by ingestion of contaminated drinking

water and by food2. Exposure to arsenic leads its accumulation in tissues such

as skin, hair and nails, resulting in various clinical symptoms such as

hyperpigmentation and keratosis3. It affects nearly all organs. Inorganic

arsenic exposure may lead to cancer of liver, kidney, bladder, prostrate and

skin as well as to Black foot disease, heart disease, maningioma and other

adverse health effects4,5,6

.


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Arsenic causes a significant increase in the rate of formation of reactive

oxygen species (ROS) such as O2-,OH- and H2O2.

7 These are generated during

redox cycling and metabolic activation process.8 It also causes lipid

peroxidation,9 oxidation of proteins and enzymes as well as DNA and DNA

adducts.10

ATPase forms a large family of membrane proteins which

couple ATP hydrolysis to the active transport of cations or other compounds

such as phospholipids across cell membranes. 11,12

and is also considered as a

master enzyme that controls many important functions at cellular and organ

level including active tansport and electric potential across plasma membrane,

intracellular pH regulation, cell division and cell elongation. 13

Chlorophytum borivilianum (Safed musli) is a traditional rare Indian

medicinal herb.14Its roots are widely used for various therapeutic applications

in the Ayurvedic and Unani medicinal systems.15 Major phytochemical

cpmpounds reported from the roots of Chlorophytum borivilianum are

saponins, fructans, gallotanins, phenolic compounds and fructo-

oligosaccharides (FOS). 16,17,18

Saponins are steroid or triterpenoid glycosides,

common in a large number of plants and plant products that are important in

human and animal nutrition. Several biological effects have been ascribed to

saponins. Extensive research has been carried out into the membrane-

permeabilising, immunostimulant, hypocholesterolaemic and anticarcinogenic

properties of saponins and they have also been found to significantly affect

growth, feed intake and reproduction in animals. 19

In the present study, the objective is to elucidate hepatoprotective role Cb

root extract on arsenic intoxicated liver damage in mice.

Meterials and methods

Animals: Random-bred, male Swiss albino mice, (7-8 weeks) were used for

experiments. These animals were maintained in the animal house at

temperatures of 24 ± 3°C and a light of 12:12 hours of light and dark. These

animals were housed in polypropylene cages and fed standard mice feed from

Hindustan Lever Ltd. India. Tap water was provided to the animals ad libitum

and tetracycline was given to the animals against any infections.The ethical

committee of Department of Zoology, University of Rajasthan, Jaipur (India)

has approved to carry out the experiments.

Chemical:- Heavy metal Arsenic in the form of Sodium arsenite

(NaAsO2,trivalent) CAS No. 7784-465 used in the present study and was obtained

from Himedia, Mumbai, India (Batch No. 3-1621 RM-1847. The salts of Arsenic

was dissolved in double distilled water (DDW) and was administered orally. Different dose of arsenic were administered and dose was determined on basis of

LD50/30.(Fig.1)


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Figure 1. Different dose of arsenic administration and the dose selection

determination on basis of LD50/30.

Preparation of Chlorophytum borivilianum root extract (drug):The roots

were collected locally, air dried in shade and powdered. Powder was distilled

in Soxhlet apparatus (for 36 hours using DDW) at 400 C. The remaining

material was dried in oven at 360 C and was used as drug.

Reducing power assay: Reducing power assay done by method of Oyaizu

(1986)20.The absorbance read at 700 nm.

Figure 2. The reducing power assay of Chlorophytum borivilianum

and its comparison with ascorbic acid. Ascorbic acid served as positive

control.


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Drug Tolerance study and Selection of Dose: Mice were divided into

various groups to receive 100, 200, 400, 800 mg/kg body weight of root

extract orally for seven consecutive days. The animals were observed for 30

days. After 30 days, lipid per oxidation (LPO) and GSH content were

measured in the liver in all groups and according to the highest GSH and

minimum LPO level, the dose was decided.

Treatment of animals

The following experiment was designed to examine the effects of

Cholorophytum borivilianum on arsenic induced toxicity.

Group I (Control Group): - Animals in this group were not given plant

extract or heavy metal. Only vehicle (DDW) was given orally.

Group II (Arsenic treated group):- Arsenic 4mg/kg bodyweight was given

orally upto 30 days in the form of sodium arsenite (III).

Group III (Combination Group):- Sodium arsenite and Chlorophytum

borivilianum root extract both were given up to 30 days orally.

Autopsy Intervals: - The Animals from the above groups were autopsied at

various intervals i.e. 1, 3, 7, 15 and 30 days.

Parameters to studied: - Body weight and Liver weight changes : The animal from each group were

weighed and killed by cervical dislocation on days 1, 3, 7, 15 and 30 days

and liver was carefully excised, trimmed free of extraneous tissue, blotted dry

and weighed quickly and used for histopathological and biochemical studies (

for ATPase).

Histopathological preparations – Excised liver was fixed in Bouin's fixative

for 24 hrs. The fixed tissue was further processed by standard method and

sections were cut at 5 µ and stained with Haematoxyline and Eosine.

Total ATPase assay: - Liver homogenate was made. The total ATPase was

determined according to the method of Akagawa and Tsukada (1979).21

Statistical analysis

The statistical significance in the different parameters between control and

experimental were assessed by one way ANOVA.

Results

Body weight and Liver weight Changes :The body weight and liver weight in NaASO2 treated group were found to be significantly lower with respect to DDW (control) from day1 to 30 whereas both were recovered in combination group with respect to arsenic intoxicated group (Fig. 3, 4).


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Figure 3. Variation in body weight changes in different experimental groups. Each value

represents as mean ± SD and n = 5. Significance level was set up p< 0.05 (Almost Significant) ,

p< 0.01 (Significant) and p< 0.001(highly significant).[a=0.05,b = p< 0.01 c = p< 0.001].

Stastistical comparison were done as control Vs arsenic and arsenic Vs combination group.

Figure 4. Variation in liver weight changes in different experimental groups. Each value

represents as mean ± SD and n = 5. Significance level was set up p< 0.05 (Almost Significant) ,

p< 0.01 (Significant) and p< 0.001(highly significant).[a=0.05,b = p< 0.01 c = p< 0.001].

Stastistical comparison were done as control Vs arsenic and arsenic Vs combination group.

Histopathological alteration: Control group showed normal cellular

architecture with distinct hepatocytes (with prominent nucleus), sinusoidal

spaces and central vein. In group 2 during 30 days exposure, arsenic caused

various pathological alterations such as expanded sinusoidal spaces,

karyolysis, karyorhysis in hepatocytes, cytoplasmic vacuolization lymphocyte


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infiltration and enucleation (Fig 5, 6 & 7) in hepatocytes as compared to

control (DDW) group. In combination group, it showed recovery in the form

of maintained hepatic histoarchitecture.

Figure 5. Photomicrograph of arsenic treated group at 400X showing Cytoplasmic

Vacuolization, Expanded sinusoidal space.

Figure 6. Photomicrograph of control treated group at 400X showing central vein,

portal vein,binuceated cells and normal sinusoidal space.


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Figure 7. . Photomicrograph of combination treated group at 400X showing

hepatocytes and sinusoidal space.

Total ATPase Assay: The total ATPase activity in NaASO2 treated group

was found to be significantly lower with respect to DDW (control), whereas

combination group showed significant elevation in total ATPase with respect

to their control (arsenic treated group) during 30 days experimental period

(Fig.8).

Figure 8.Variation in ATPase activity in different experimental groups. Each value represents as

mean ± SD and n = 5. Significance level was set up p< 0.05 (Almost Significant) , p< 0.01

(Significant) and p< 0.001(highly significant).[a=0.05,b = p< 0.01 c = p< 0.001]. Stastistical

comparison were done as control Vs arsenic and arsenic Vs combination group.


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Discussion

Reduction in body weight is used as an indicator for the deterioration of

general health status. It has been reported that arsenic could induce

toxicological effects and biochemical dysfunctions representing serious health

hazards.22

The findings from the present study indicate that arsenic exposure

caused decrease in the body and liver weight which are in support of the

findings by Yousef et al. (2008) and El-Demerdash et al. (2009)23

who

reported that high arsenic exposure have significantly induced disturbances

the total body weight and liver weight. Oral administration of arsenic is

associated with severe gastrointestinal and liver side effects24

. Arsenic

increases permeability of intestinal lining and causes the gut leaky. This

increased permeability of intestinal lining may be responsible for improper

absorption of nutrients and loss of appetite and weakness25

. and reduction in

body weight26. Reduction in liver weight is due to damaged hepatic

histoarchitecture. Arteel et al.(2008) 27 suggested that arsenic exposure caused

a change in the balance between cell death and proliferation coupled with loss

of body weight. Arsenic produces ROS during its cycles between different

oxidation states,28

which appears to be involved in the mechanism of various

types of cell injury 29,7,9

. Liver cells have particularly high probability of being

subjected to ROS induced toxicity because hepatocytes produces large amount

of ROS during the dexoification of xenobiotics and toxic substances30

.

Arsenic induced reactive oxygen species and subsequent depletion of

antioxidant cell defenses can result in disruption of the pro-

oxidant/antioxidant balance in mammalian tissues31,32

. Consequently, ROS

directly react with cell biomolecules, causing damages to lipids, proteins and

DNA, and hence leading to cell death33,34

.

Present study revealed decreased ATPases activity after arsenic intoxication

at all autopsy intervals. Arsenic induced reactive oxygen species impairs cell

membrane stability7,8

and damages mitochondrial membrane severely. It is

well established that mitochondria are the major site of utilization of oxygen

and many of the mitochondrial enzymes contain essential sulfhydryl groups.

In addition, since the inner and outer mitochondrial membranes contain

unsaturated lipids, mitochondria are more susceptible to arsenic attack as well

as by the free radicals produced by it than other organelles35

.The damaged

membrane cannot develop a proton motive force that is preliminary

requirement of cellular energy production36

. Arsenic disrupts mitocondrial

membrane potential and increases ROS generation37

that causes depletion of

ATP.38

It uncouples oxidative phosphorylation, thus inhibiting energy-linked

reduction of NAD+, mitochondrial respiration, and ATP synthesis

39. Thus

ATPase activity is significantly reduced after arsenic intoxication.

In combination group modulation by Cb root extract was observed in terms

of increased body and liver weight, less damage in hepatocytes and increased

ATPase level. Cb root extract contains Saponins, Gallotannins and Fructans.

Saponins40,Gallotanins41 and Fructans18have antioxidant activity. Saponins


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inhibit intracellular ROS formation, reduces the level of the lipid peroxidation

(MDA) and maintains cellular antioxidant enzymes activities 42. Saponins

have hypocholesteric properties 19. Cholesterol enrichment was shown to have

an inhibitory effect on many membrane ATPases, as it may directly interact

with the boundary lipids of ATPase and alter the intermolecular hydrogen

bonds of the protein.43,44 Saponin (Ginsenosides) interacts with membrane

cholesterol and displace it from surrounding lipids environment of ATPases.

Removal of cholesterol will lead to an increase in membrane fluidity which

facilitates conformational changes of ATPases during their transport cycle that

controls the enzyme activity of biological membrane and has important role in

ion transport.45

Saponins maintains Na+- K

+-ATPase activity against ROS

induced reaction.46

Saponins reduces ROS formation due to their antioxidant

property. Since cell membrane is protected, GIT is not leaky,So body weight

is increased. Hepatohistoarchitecture injuries induced by ROS, are reduced

due to antioxidant property of saponins.The body and liver weight are also

recovered. Thus it can be concluded that cb root extract can ameliorates

arsenic induced toxicity in terms of increased body and liver weight, lessens

the hepatotoxicity and maintains ATPase level.

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