3-Protective effect of polyprenols against acute liver ...Polyprenols, which have the similar structure and phar-macological effects to human body dolichols, are the lipid compounds

Journal of Pharmaceutical Medicine 2015, 3(1):17-21.

Published Online, March, 2015 (http://www.mjpub.org/journals/jpm.html)

Copyright © 2012 MJPub. ISSN: 0460-2396 JPM

M a r c & J oh n

U K P u b l is h in g

Protective effect of polyprenols against acute liver injury in-

duced by CCl4 and D-Gal

Fang-Jie Sun 1, Jun-Feng Jia

2*

1 Xinxiang Institute for New Medicine, Xinxiang, Henan, China 2 Xinjiang Nikanka Biological Co. Ltd., Yili, Xinjiang, China

Received: October 21, 2014; Received revised: January 4, 2015; Accepted: February 8, 2015

ABSTRACT

Objective: To study the protective effect of polyprenols on acute liver injury induced by CCl4 and D-Gal. Method: The

acute liver injury model induced by 0.5% CCl4 in olive oil (10ml/kg) and D-Gal (800mg/kg, i.p.) in mice and rats re-

spectively. 10, 40 and 160 mg/kg polyprenols was administered i.g., to study the protective effect for different doses of

polyprenols, the therapeutic effect of each groups was assessed using the activities of ALT and AST in serum, and ob-

served pathological changes of liver. Result: polyprenols can reduce significantly the increase of ALT and AST in se-

rum and the level of liver injury induced by CCl4 and D-Gal. Conclusion: Polyprenols has the powerful protective ef-

fect on acute liver injury induced by CCl4 and D-Gal.

Keywords: Polyprenols; CCl4; D-Gal; Liver injury

1 Introduction

Polyprenols mainly exists in ginkgo leaves, coniferous

species and spinach, mushrooms, soybeans and other

food plants, which has highest content in ginkgo leaves[1]

.

Polyprenols, which have the similar structure and phar-

macological effects to human body dolichols, are the

lipid compounds composed of a series of isoamyl alkenyl

and terminal isoamyl enol units[2,3]

. Polyprenols, which is

the carrier of the glycoprotein biosynthesis in vivo, par-

ticipate in human metabolism, improve the fluidity, re-

duce electrical stability of the cell membrane, has a cer-

tain recovery effects on cell membrane injury[4,5]

.

In recent years, the R﹠D of polyprenols com-

pounds has become a research focus on the development

of health food and medicines, which are more atten-

tion[6,7]

. Latvia and Russia and other countries were pre-

*Correspondence address: Jun-Feng Jia, Xinjiang Ni-

kanka Biological Co. Ltd., No. 1-2 Qingshui River Eco-

nomic and Technological Development Zone, Huocheng,

Yili, Xinjiang 835207, China

Tel: 09994650888; E-mail: [email protected].

pared "ROPREN" drugs, mainly used for the prevention

and treatment of diseases such as hepatitis, multiple

sclerosis caused by immune dysfunction, influenza virus

and so on, which has entered phase Ⅱ clinical trial.

Australia Solagran developed "Bioeffective R" pharma-

ceutical preparation with polyprenols as main material,

has effects on liver regeneration after partial hepatec-

tomy and liver function recovery in rat, which is in phase

Ⅱ clinical trial[8-11]

.

Toxicological safety evaluation of polyprenols

shows that its safe, non- teratogenic, carcinogenic and

mutagenicity which was done by Latvia medical scien-

cesin and Biolat according to “new drugs preclinical

toxicology test requirements” which published by the

Medical Commission of the Ministry of Health of the

former Soviet Union[12-15]

. This study mainly observed

the protective effect of polyprenols against liver injury

induced by CCl4 and D-Gal by comparing polyprenols,

Bifendate dripping pills and polyene phosphatidylcholine

capsules (Essentiale).

2 Materials and Methods

Protective effect of polyprenols against acute liver injury induced by CCl4 and D-Gal


Drugs: Polyprenols purchased from Institute of Chemi-

cal Industry of Forest Products and batch number is

051224, bifendate purchased from Beijing xiehe phar-

maceutical factory and batch number is 04060104, Es-

sentiale purchased from Beijing aventis pharmaceutical

Co., LTD and batch number is 44830A.

Reagents: D-Gal purchased from Beijing Zhognshan

Biotechnology Co., LTD and batch number is 20140521,

which is white powder and percentage of D-Gal greater

than 99.0%. The method of application is dissolve by

sterile saline solution to a solution containing 10% be-

fore use, then add NaOH to adjust pH 7.0-7.4; ALT and

AST kit which batch number is 20140723 and 20140516

respectively, were provided by Shanghai Ke-

hua-Dongling Diagnostic Products Co., LTD; Olive oil

and tween-80 were all provided by Tianjin Winterkorn

Chemical Reagent Co., Ltd.

Animals: Male ICR mice which weight between 25 and

28g, male SD rats, 150 to 180g, all of them were pro-

vided by Shanghai slyke experimental animal liability

Co., LTD, certification No. is SCXK(Hu)-2003-2003.

Methods:

CCl4 mouse model: Mice 70 are in 7 equal groups, i.g.

0.1% Tween-80 saline once a day for seven consecutive

days, respectively, then i.p. 0.5% CCl4 in olive oil 10

ml/kg after 1 hour, the last dose, except the mice in nor-

mal group. Drug again at 16 hours after modeling, whole

blood was collected 1 hour later from the orbit, the ac-

tivities of ALT and AST were tested using automatic

biochemical analyzer. Take the left lobe of liver into

small pieces for frozen sections, fixed with 12% formal-

dehyde, paraffin-embedded, HE and Sudan Ⅲ staining,

microscopy to observe the content and distribution of

lipid droplets in the liver cells, then score.

D-Gal rats model: Rats 60 were in 6 equal groups ac-

cording to weight, i.g. saline or medications once a day

for six consecutive days, respectively, given equivalent

normal saline in normal group, the other rats were i.p.

D-Gal (800mg/kg) on day 7, to establish the acute liver

injury model, then draw the blood from eyeball of rats

after 24h fasting and prepare serum, the activities of

ALT and AST were tested using automatic biochemical

analyzer. Take the left lobe of liver into small pieces for

frozen sections, fixed with 12% formaldehyde, paraffin-

embedded, HE staining, microscopy to observe hepato-

cyte injury level.

3 Results

3.1 The influence on the activities of serum ALT and AST in liver injury mice induced by CCl4

The activities of serum ALT and AST increase signifi-

cantly at 16h after the mice were injected with CCl4. The

activities of ALT and AST were all inhibited by Poly-

prenols, bifendate and Essentiale compared with the

model group, this result shows that polyprenols has the

remarkable protective effect on acute liver injury induced

by CCl4, the data are listed in table 1.

3.2 The influence on pathologic changes of liver tissue in liver injury mice induced by CCl4

The inflammatory cell infiltration serious in the model

group compared with the normal group, there were a

large number of hepatic steatosis around the central vein

of the liver lobule, point and piecemeal necrosis in liver

cells was everywhere obviously, swelling obviously. It

was obvious that the above symptoms eased off after

polyprenols were taken, hepatic steatosis around the cen-

tral vein of the liver lobule still existed, but the scope of

steatosis was smaller significantly, and the degree was

lighten, piecemeal necrosis existed sporadically. It shows

that polyprenols has protective effect on acute liver in-

jury induced by CCl4, the data of result are shown in

table 2.

3.3 The influence on the activities of serum ALT and AST in liver injury rats induced by D-Gal

D-Gal can lead the activities of serum ALT and AST in

liver injury rats increase significantly; The experimental

results suggest that the activities of ALT and AST de-

clined markedly in the group of polyprenols compared

with the model group, this result shows that polyprenols

has the remarkable antagonism of acute liver injury in-

duced by D-Gal, the data are listed in table 3.

3.4 The influence on pathologic changes of liver tissue in liver injury rats induced by D-Gal

The bundles of liver cells were radiate in all directions,

the structure of hepatic lobules was clear, and no in-

flammatory cell infiltration in portal area in the normal

group. Extensive hepatocyte necrosis, hepatic sinusoid

kupffer's cell proliferate and degeneration were seen in

model group, inflammatory cell infiltration was seen in

portal area, fibrous connective tissue proliferate which

had a tendency to extend to hepatic lobules. The struc-

ture of a vast majority of liver tissue which in these

groups on polyprenols, bifendate and Essentiale was

normal, we can also see the individual necrosis and aci-

dophilic degeneration of hepatocytes occasionally, the

level of inflammatory cell infiltration in portal area re-

duced significantly, the research shows that polyprenols

has the powerful protective effect on acute liver injury

induced by D-Gal, the data of result are shown in table 4.



Table 1 The influence on the activities of serum ALT and AST in liver injury mice

induced by CCl4 (x±s, n = 10)

Groups Doses

(mg/kg) ALT (U/L)

inhibition rate

(%) AST (U/L)

inhibition rate

(%)

normal group 39.9±10.1** 183.8±21.7**

model group 95.4±35.5 271.5±57.1

Polyprenols 10 47.9±18.2** 85.58 246.0±14.7 28.98

Polyprenols 40 44.7±10.1** 91.41 215.1±31.3* 64.09

Polyprenols 160 52.7±15.2* 76.94 209.4±26.2* 70.52

Bifendate 200 40.6±15.5** 98.75 203.0±31.5** 77.84

Essentiale N 360 54.5±16.6* 73.67 220.0±32.0* 58.52

The mark “*” means P < 0.05 and “**” means P < 0.01 compared with the model group.

Table 2 The influence on pathologic changes of liver tissue in liver injury mice induced by CCl4

The score of content on lipid droplets in the liver cells Groups

Doses (mg/kg)

The number of mice (piece)

0 1 2 3

Value P*

normal group 10 10 0 0 0 <0.01

model group 10 0 4 5 1

Polyprenols 10 10 4 3 3 0

Polyprenols 40 10 4 6 0 0 <0.01 Polyprenols 160 10 7 3 0 0 <0.01

Bifendate 200 10 7 2 1 0 <0.01

Essentiale 360 10 7 3 0 0 <0.01

The number “0” represents that no lipid droplets has been found in the liver cells; “1” represents that lipid droplets

can be found in individual liver cell; “2” represents that lipid droplets were flakily or focally but all small; “3” repre-

sents that lipid droplets was widespread in liver cell. The mark “*” means compared with the model group.

Table 3 The influence on the activities of serum ALT and AST in liver injury rats

induced by D-Gal (x±s, n = 10)

Groups Doses

(mg/kg) ALT (U/L) AST (U/L)

normal group 202±35** 55±9**

model group 4454±1947 1222±598

Polyprenols 10 1876±517** 1098±510

Polyprenols 40 1632±513** 897±587*

Polyprenols 160 961±371** 705±295*

Bifendate 200 1482±467** 667±228**

Essentiale 360 1569±938** 732±246*

Table 4 The influence on pathologic changes of liver tissue in liver injury rats induced by D-Gal

inflammatory cell infiltration necrosis acidophilic degeneration Groups

Doses

(mg/kg) - + ++ +++ - + ++ +++ - + ++ +++

normal group 10 0 0 0** 10 0 0 0** 10 0 0 0**

model group 0 1 4 5 0 2 5 3 1 2 5 2 Polyprenols 10 5 3 2 0 6 2 2 0 6 2 2 0

Polyprenols 40 6 4 2 0** 6 3 1 0** 6 2 2 0**

Polyprenols 160 7 3 0 0** 7 2 1 0** 7 3 0 0** Bifendate 200 5 4 1 0** 7 3 0 0** 7 3 0 0**

Essentiale 360 5 3 2 0** 6 3 1 0** 6 2 2 0**

4 Discussion

CCl4 is classic liver toxicity substances, the mice acute

liver injury model induced by CCl4 is the classic model

of experimental liver injury and good repeatability,

which can accurately reflect the function of liver cells,

metabolic and morphological changes[16]

. Lipid peroxi-

dation is the main mechanism of liver injury induced by

CCl4[17,18]

. CCl4 is metabolized by the liver cell micro-



somal cytochrome P450 oxidase system into the body,

generates trichloromethane radicals (•CCl3), methylene

chloride radicals (•CCl2) and peroxide trichloromethane

radicals (•OOCCl3) after activated, attacks the liver cell

membrane or activates phosphorylase peroxide on the

membrane, result in the cell membrane and organelles

membrane injury[19]

. Successful modeling if the activities

of serum ALT and AST increasing, fibrous hyperplasia

on liver tissue and inflammatory cell infiltration can be

seen in portal area after modeling by ip. CCl4 in rats[20]

.

D-Gal can cause diffuse hepatic necrosis and in-

flammation, induce lipid peroxidation by generate free

radicals, eventually causes liver injury, which is liver cell

phosphate uridine disruptors, histopathological changes

of toxic liver injury model caused by D-Gal is similar to

viral hepatitis[21,22]

. Liver injury model caused by D-Gal

is considered the classic animal model to study liver in-

jury mechanisms and treatment drug development. The

main mechanism of liver injury have five ways: first,

inhibited the biosynthesis synthesis of nucleic acid, pro-

tein, fat and other substances and limit the generate of

organelles and enzymes by uridine diphosphate (UDP),

competitive capture UTP produce uridine diphosphate

galactose (UDP-gal) to exhaust UDP[23,24]

; second, affect

the integrity of liver cell membrane by specific binding

with it; third, increase the Ca2+

concentration; fourth,

exhaust glutathione; finally, cause apoptosis by release

tumor necrosis factor TNF-d[25]

.

Serum transaminases are sensitive indicators of liv-

er injury, ALT and AST are most commonly used, ALT

and AST are soluble enzyme present in the cytoplasm of

liver cells, the level of serum ALT can reflect the sensi-

tivity of liver cell degeneration and the extent of necrosis,

when the liver is damaged, the permeability of cell

membrane increase, ALT and AST in intracytoplasmic

released into the blood, then elevate serum ALT and

AST[22-25]

.

This study showed that CCl4 and D-Gal can signifi-

cantly increase the activities of serum ALT and AST in

mouse, given different doses polyprenols can block sig-

nificantly, and significantly reduced the degree of liver

injury, the result suggest that polyprenols play a signifi-

cant role in protective effect on acute liver injury induced

by CCl4 and D-Gal, the study provides pharmacody-

namic basis for clinical application.

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Documents

3-Protective effect of polyprenols against acute liver ...Polyprenols, which have the similar structure and phar-macological effects to human body dolichols, are the lipid compounds