Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Silymarin in acute inflammation

Dose-dependent Anti-inflammatory Effect of Silymarin in

Experimental Animal Model of Acute Inflammation
Kasim M. Juma'a

*
, Saad A. Hussain

**,1
, Intesar T. Numan

**
and

Assad H. Siqar
**

*
Department of Pharmacy, Baquba General Hospital, Diyala, Iraq

**
Department of Pharmacology and Toxicology, College of Pharmacy,University of Baghdad,Baghdad,Iraq

Abstract
Silymarin, a flavolignans from seeds of „milk thistle‟ “Silybum marianum” has been widely used

from ancient times because of its excellent hepatoprotective action. It has been used clinically to treat

liver disorders including acute and chronic viral hepatitis, toxin/drug-induced hepatitis and cirrhosis

and alcoholic liver disease. The efficacy and dose-response effect of silymarin (125, 250 and 500

mg/kg) were assessed using egg albumin-induced paw edema in rats as a model of acute inflammation.

In this model, 56 rats were used and allocated into 7 subgroups each containing 8 rats. All treatments

were given intraperitonealy 30 minutes before induction of inflammation by egg albumin and then the

increase in paw edema was measured 1h, 2h and 3h after induction of inflammation by using the

vernier caliper. The results indicated that silymarin, at doses range used, significantly lowered paw

edema (P<0.05) an effect comparable to that produced by the reference drugs, acetyl salicylic acid,

meloxicam and dexamethazone. Paw edema suppressive effect of silymarin 250 and 500 mg/kg was

comparable and both of them were significantly different from that of silymarin 125 mg/kg (P<0.05).

Therefore, silymarin exert an important anti-inflammatory activity in animal model of acute

inflammation, which was significantly increased as the dose increased up to 250 mg/kg.

Key words: Silymarin, acute inflammation, dose-response

الخالصة
نمذ اسزخذو األَسبٌ ويُز انمذو يبدح انسهًُبسٍَ انًسزخهصخ يٍ ثزوس َجبد انكعىة ثسجت رأثُشهب فٍ حًبَخ انكجذ. ونمذ اسزخذيذ

سشَشَب فٍ انىلذ انحبظش نًعبندخ أيشاض انكجذ انحبدح وانًضيُخ وانُبشئخ عٍ انفُشوسبد أو انزعشض نهسًىو وانكحىل. رى فٍ هزِ

ًَىرج األنزهبة انحبد يهغى/كغى( فٍ ١٢٢، ٢١٢، ٥٢١انزأثُش انًعبد نألنزهبة ندشع يخزهفخ يٍ يبدح انسهًُبسٍَ ) انذساسخ رمُُى

خشراٌ. رى ٨يدًىعبد كم يدًىعخ ظًذ ٧أثُط لسًذ انً خشرا ١٦انًسزحذس ثىاسطخ األنجىيٍُ فٍ لذو اندشراٌ. رى اسزخذاو

ثضسق صالل انجُط فٍ دلُمخ يٍ اسزحذاس األنزهبة ۳٢ك انضسق ثبنجشَزىٌ لجم َغشعالج اندشراٌ ثدشع يخزهفخ يٍ انسهًُبسٍَ عٍ

لذو اندشراٌ، ورى لُبط يسزىي ركىٍَ انىريخ ثعذ سبعخ واحذح، سبعزٍُ وثالس سبعبد ثعذ اسزحذاس انىريخ ثبسزخذاو انفُشَُخ. أظهشد

ثبنًشكجبد انمُبسُخ انًسزخذيخ نهًمبسَخ )حبيط أسُزُم مبسَخ ثأٌ نهسهًُبسٍَ فعبنُخ يعبدح نألنزهبة رعزًذ عهً اندشعخ ي انُزبئح

بة سبنُسُهُك، دَكسبيُثبصوٌ وانًُهىكسُكبو(. ويٍ خالل انُزبئح انزٍ رى انزىصم أنُهب ًَكٍ األسزُزبج ثأٌ نهسهًُبسٍَ رأثُش يعبد نألنزه

.يهغى/كغى. ٢١٢فٍ انًُبرج انًخزجشَخ نألنزهبثبد انحبدح وانزٌ َضداد ثضَبدح اندشعخ حزً

Introduction

Inflammation is an important

physiological reaction, which occurs in

response to a wide variety of injurious agents

(bacterial infection or physical trauma)

ultimately aiming to perform the dual function

of limiting damage and promoting tissue repair

(1). It requires the participation of various cell

types expressing and reacting to diverse

mediators along a very precise sequence (2).

The inflammatory response is often initiated

by the activation of resident macrophages

through pattern-recognition receptors; this

triggers the sequential release of pro-

inflammatory mediators such as eicosanoids,

cytokines, chemokines, and protease, which

drive leukocyte recruitment and activation (3).

Resolution of inflammation (anti-inflammatory

response) is an active process controlled by

endogenous mediators that suppress pro-

inflammatory gene expression and cell

trafficking, induce inflammatory cell apoptosis

and phagocytosis. An optional balance

between pro- and anti-inflammatory responses

is required to prevent the highly detrimental

effect of extensive, prolonged or unregulated

inflammation (3). Silymarin, the seed extract

of milk thistle (Silybum marianum), is an

ancient herbal remedy used to treat a range of

liver and gallbladder disorders, including

hepatitis, cirrhosis, and as a hepatoprotectant

against poisoning from wild mushroom,

alcohol, chemical, and environmental toxins

(4). Milk thistle is one of the best-studied

medicinal plants for the treatment of liver

disease (4 -7).

1
Corresponding author E-mail : saad_alzaidi@yahoo.com

Received : 13/1/2009

Accepted : 4/5/2009

mailto:saad_alzaidi@yahoo.com

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Silymarin in acute inflammation

Most of these effects have been attributed

to direct and/or indirect antioxidant capacity of

silymarin, such as being a scavenger of ROS,

phenylglyoxylic ketyl radicals and a chain

breaking antioxidant (8). The present study

was designed to evaluate the efficacy and dose

response effect of silymarin in experimental

animal model of acute inflammation.

Materials and methods
Silymarin powder used in the present was

obtained as standardized pure extract from

Luna Company (Egypt) and was dissolved in

98% dimethyl sulfoxide solution to produce

stock solution with concentration of 250

mg/ml, from which different concentrations

were obtained by dilution. Dimethyl sulfoxide

and diethyl ether solutions were obtained from

Merck Company (Germany), dexamethazone

(American regent, inc. USA), acetyl salicylic

acid (Sanofi, France) and hand caliper

(Germany). In the present study, 56 Sprague

Dawley rats of both sexes (180-220 g) were

used and allocated into 7 subgroups, each

containing 8 rats. These groups represent

control, standard and test groups. Silymarin

was tested for its ability to suppress acute

inflammation using fresh egg albumin-induced

edema in rats as a model according to the

technique established by Winter et al
(9)

. Rats

were fasted overnight, and deprived of water

during the experiment to ensure uniform

hydration and to minimize variability in

edematous response
(10)

. The rats were

separated into 7 groups (8 rats each); control

group treated with dimethyl sulfoxide 2 ml/kg;

the three standard groups treated with acetyl

salicylic acid 100 mg/kg, meloxicam 10 mg/kg

and dexamethazone 1 mg/kg respectively;

while the three test groups treated with

silymarin (125, 250 and 500 mg/kg,

respectively). All drugs were administered

intraperitonealy and 30 minutes post-treatment,

inflammation was induced by injecting 0.1 ml

of fresh egg albumin (phlogistic agent)
(11,12)

into the sub planter surface of the right hind

paw. The increase in paw edema, as a result of

inflammation, was measured using vernier

caliper before and 1 hr, 2 hr and 3 hr after

induction of inflammation. The difference in

paw thickness before and after induction of

inflammation was calculated and presented as

mean increase in paw thickness (mm). The

ability of anti-inflammatory drugs to suppress

paw inflammation was expressed as percentage

of inhibition of paw edema
(13)

. All results were

expressed as mean ± SEM. The significance of

difference between the control and treated

groups were determined using one-way

analysis of variance (ANOVA), followed by

Student's t-test. P-values < 0.05 were

considered significant.

Results
The anti-inflammatory effect of silymarin

on acute inflammatory model was shown in

table 1 and figure 1. Silymarin (125, 250 and

500 mg/kg), acetyl salicylic acid, meloxicam

and dexamethazone significantly reduced egg

albumin-induced paw edema (P<0.05)

compared with control group after 1 hr, 2 hr

and 3r h from induction of inflammation. There

is significant difference between silymarin

(125 mg/kg) group and all other treatment

groups (P<0.05) along three hours of

assessment, while no significant difference

exists between silymarin doses 250 and 500

mg/kg, and with dexamethazone and

meloxicam groups at the second and third hour

of assessment. Silymarin (250 and 500 mg/kg)

produced an effect which is significantly

different from that of acetyl salicylic acid at the

second and third hour of assessment (P<0.05)

(except for silymarin 500 mg/kg group which

produces non significant effect at the second

hour).The dose-response effect of silymarin on

acute inflammation was illustrated in figure 1.

The suppressive effect of silymarin on paw

edema was significantly increased (P<0.05) as

the dose doubled from 125 to 250 mg/kg.

However, further increase in the dose up to 500

mg/kg did not show significant increase in the

anti-inflammatory activity (except after the

first hour, where silymarin 500 mg/kg

significantly differs from silymarin 250

mg/kg).

2.0 2.2 2.4 2.6 2.8 3.0

Log doses of silymarin

P
e

rc
e

n
ta

g
e

o
f

in
h

ib
it

io
n

after 1 hr.

after 2 hrs.

after 3 hrs.

Figure (1): Dose-response effect of different

doses of silymarin on egg albumin-induced
acute inflammation in rats.

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Silymarin in acute inflammation

Table 1: Effect of different doses of silymarin on egg albumin-induced acute inflammation in

rats.

% of inhibition Mean increase in paw thickness (mm)

Treatment Groups

3 h 2 h 1 h 3 h 2 h 1 h
_

1.76 ± 0.06 2.29 ± 0.05 3.06 ± 0.06 Dimethyl sulfoxide 2 ml/kg

40 41 37 1.05 ± 0.04*
a
1.35 ± 0.04*

a
1.91 ± 0.06*

a
Acetyl salicylic acid 100mg/kg

35 35 35 1.15 ± 0.05*
a
1.49 ± 0.05*

b
1.99 ± 0.05*

a
Meloxicam 10mg/kg

37 40 42 1.10 ± 0.05*
a
1.37 ± 0.06*

a
1.78 ± 0.06*

b
Dexamethazone 1mg/kg

22 23 27 1.38 ± 0.06*
b
1.76 ± 0.05*

c
2.24 ± 0.06*

c
Silymarin 125 mg/kg

29 34 33 1.25 ± 0.06*
c
1.51 ± 0.06*

b
2.05 ± 0.07*

a
Silymarin 250 mg/kg

31 37 39 1.21 ± 0.04*
c
1.45±0.04*

b
1.85 ± 0.05*

b
Silymarin 500 mg/kg

Data were expressed as mean ± SEM; number of animals = 8 in each group; *P<0.05 with respect to

control group; values with non-identical superscripts (a, b, c) among different groups are considered

significantly different (P<0.05).

Discussion
The inflammatory response is a
physiological characteristic of vascularized

tissues
(14)

. Exudation, which is a consequence

of increased vascular permeability, is

considered as a major feature of acute

inflammation
(15)

.Egg albumin-induced paw

edema in rats is an in vivo model of

inflammation used to screen agents for anti-

inflammatory effect
(16)

. The characteristic

swelling of the paw is due to edema formation.

Inhibition of increased vascular permeability

and hence the attendant edema modulate the

extent and magnitude of the inflammatory

reaction. The paw edema that induced by

injection of egg albumin is peaked after 1 h

and then progressively decreased with time.

Many chemical mediators like histamine, 5-

HT, kinins and prostanoids mediate acute

inflammation induced by phlogistic agents

including egg albumin
(17)

. In accordance with

Marsha-Lyn et al
(18)

, inflammation occurs

through three distinct phases: an initial phase

mediated by histamine and 5-HT (up to 2

hour); an intermediate phase involving the

activity of bradykinin and a third (late) phase

with prostanoid synthesis by COX
(19)

. The

anti-inflammatory activity of silymarin extract

was evaluated by egg albumin-induced paw

edema using vernier caliper method and the

results were shown in table 1 and figure 1.

Three different doses (125, 250 and 500

mg/kg) of silymarin were evaluated for the aim

of finding dose-response relationship; the

results clearly indicated the significant anti-

inflammatory activity of this flavonoid within

the dose range utilized, compared to the

standard anti-inflammatory agents used in this

respect. It efficiently suppressed early,

intermediate and late phases of acute

inflammation as illustrated in table(1) .

However, the effect of silymarin on early and

intermediate phases was better than that on the

late phase as shown in figure (1). The anti-

inflammatory effect of silymarin, especially at

the doses 250 and 500 mg/kg was comparable

to that of standard drugs and there was no

significant difference between them.

Additionally, silymarin shows a dose-

dependent effect up to 250 mg/kg, and further

increase of the dose was not associated with

further increase in activity. The in vivo anti-

inflammatory activity of silymarin was tested

in different experimental models of

inflammation, and the results suggested that an

important anti-inflammatory action was

achieved by inhibition of neutrophils migration

into the inflamed site which lead to the release

of ROS, RNS and proteolytic enzymes

resulting in microvascular endothelial injury,

increase endothelial barrier permeability and

edema
(20)

. Silymarin at doses range 25, 50 and

100 mg/kg, when administered orally,

significantly reduced papaya latex-induced

paw inflammation. However, it was not

effective against carrageenan-induced

inflammation; it also reduced experimentally-

induced ear edema in mice (36.84%) but the

reduction was statistically not significant
(21)

Silymarin has many biological activities as

antioxidant, anti-inflammatory, cytoprotective

and anticancer effects, it scavenges free

radicals, increases cellular GSH content and

induces superoxide dismutase (SOD) activity

and causes a significant reduction in lipid

peroxidation and consequently protecting and

stabilizing cell membranes
(22, 23)

. The cell

membrane stabilizing effect of silymarin on

Iraqi J Pharm Sci , Vol.18 (Suppl.), 2009 Silymarin in acute inflammation

mast cell
(20)

may explain its effect on the

initial phase of acute inflammation model

mediated by histamine and 5-HT. It may

suppress mast cell degranulation and inhibits

the release of these mediators that initiate early

phase of paw inflammation. Silymarin blocked

TNF-α-induced activation of NF-κB which

regulates the expression of various genes

involved in inflammation, cytoprotection and

carcinogenesis in a dose and time dependent

manner
(24)

, and it is completely blocked the

mRNA expression of IL-1β and COX-2 in

lipopolysaccharide (LPS) stimulated RAW

264.7 cells
(25)

. Additionally, the strong

inhibition of leukotriene B4 (LTB4) formation

by silybinin, which is the major component

extracted from milk thistle and considered to

be most biologically active in terms of its

antioxidant and anti-inflammatory properties
(26)

, was confirmed in experiments with

phagocytic cells isolated from human liver,

with a significant inhibition of 5-LOX

achieved in vivo
(27)

. Inhibition of PG synthesis

and COX expression by silymarin may explain

its effect on the third phase of inflammatory

reaction. It is now clear that silymarin

suppresses early, intermediate and late phase

of acute inflammatory model through

interference with the initiation and propagation

phases of acute inflammation. However, there

is limited data about the role of silymarin in

the resolution phase of inflammation, which is

recently considered as an important target for

drugs used in inflammatory diseases.

Leukocytes migration contributes to initiation

and propagation of acute inflammation which

was significantly inhibited by silymarin.

Propagation phase also mediated by a diverse

number of inflammatory mediators whose

release, activity and genetic expression was

significantly inhibited by silymarin
(25)

. In

comparison with silymarin, NSAIDs inhibit

propagation phase of inflammation through

inhibition of PG synthesis, but they have

negative effect on the resolution phase of

inflammation (except acetyl salicylic acid)

through inhibition of 15d-PGJ2 synthesis,

which is important pre-resolving anti-

inflammatory mediator
(28)

. On the other hand,

steroids are considered as a gold standard of

anti-inflammatory drugs where they inhibit all

phases of acute inflammation. They inhibit

initiation and propagation phases through

suppression of leukocytes migration and

inflammatory mediators genetic expression

while enhance resolution phase of

inflammation through augmentation of

macrophages capacity for phagocytosis of

apoptotic cells
(29)

. In conclusion, silymarin in

a dose dependent pattern was effective in

decreasing acute inflammatory reactions in

experimental animal models. The anti-

inflammatory activity of silymarin increased

up to 250 mg/kg and further increase of the

dose will not result in further increase in

activity.

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