Comparative Evaluation of Using Intranasal Desmopressin, Parenteral Diclofenac or their Combination in the Management of Acute


Iraqi J.Pharm.Sci., Vol.16 (2)  ,2007 Effect of Silibinin on IOP

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Effect of Silibinin in Lowering the Intraocular Pressure in
Normotensive Rabbits: Interaction with Pilocarpine and

Cyclopentolate
Saad A. Hussain *,1 ,   Haider M. Mohammed* ,   Munaf H. Abdulrazzaq* ,

Ahmed T. Numan*
*Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad , Baghdad , Iraq.

Abstract
Previous data indicated the effectiveness of silibinin as intraocular pressure (IOP) - lowering agent.
The present study was performed to evaluate the interaction of silibinin with pilocarpine or
cyclopentolate in lowering IOP in normotensive rabbits. The effects of topically instilled silibinin
hemisuccinate solution (0.75%) alone or adjunctly combined with 2% pilocarpine or 1%
cyclopentolate on the IOP of normotensive rabbits were evaluated using indentation tonometry. The
results showed that 0.75% solution of silibinin was found more potent than pilocarpine (2% drops)
in lowering IOP of normotensive rabbits, while their combination results in longer duration of
action. Moreover, the elevated IOP values produced by cyclopentolate (1%drops) were decreased
by silibinin, while prior instillation of cyclopentolate did not interfere with the IOP-lowering effect
of silibinin. In conclusion Silibinin lowers IOP in normotensive rabbits more than pilocarpine, and
their combination elongates the duration of the IOP-lowering effect. This might be due to
interference with aqueous humor formation as a possible mechanism. In addition, cyclopentolate
did not significantly alter the effect of silibinin on IOP.
Key words: silibinin, IOP, pilocarpine, cyclopentolate

الخالصة
لقد ثبت في دراسة سابقة فعالیة السیلیبینین في خفض الضغط الداخلي لعین االرنب، تم تصمیم ھذه الدراسة لتقییم التداخل بین 

موضعیا في العین. تمت دراسة تأثیر االستخدام الموضعي في العین السیلیبینین والبایلوكاربین أو السایكلوبنتولیت عند استخدامھم 
% سایكلوبنتولیت على خفض الضغط الداخلي ١% قطرة بایلوكاربین أو ٢% سلیبینین لوحده أو بوضعھ قبل أو بعد ٠.٧٥لمحلول 

% سیلیبینین في خفض ضغط ٠.٧٥. أظھرت النتائج بأن تأثیر Indentation tonometryلعین االرانب الطبیعیة باستخدام تقنیة 
% بایلوكاربین وأن استخدامھما معا یؤدي الى اطالة مدى مثل ھذا التأثیر. كما ثبت بأن للسیلیبینین القدرة ٢العین الداخلي أكبر من 

ین عند استخدام %، بینما لم یرتفع ضغط الع١على تقلیل االرتفاع الحاصل في ضغط العین الداخلي الذي أحدثتھ مادة السایكلوبنتولیت 
االخیر قبل السیلیبینین. وعلیھ یمكن ان نستنتج بأن السیلیبینین افضل من البایلوكاربین في خفض ضغط العین الداخلي لعین االرانب 

یة وان استخدامھما معا یطیل مدة ھذا التأثیر، ویمكن أن یعزى ھذا التأثیر الى التداخل مع عملیة تكوین ماء العین الداخلي كمیكانیك
اضافة الى ان الساكلوبنتولیت لم یؤثر على فعالیة السیلیبینین في خفض الضغط الداخلي للعین.عمل للسیلیبینین.

Introduction
Two mechanisms underlay the

effectiveness of various drugs implicated in
the management of elevated intraocular
pressure (IOP), the reduction in aqueous
humor (AH) inflow and enhancement of AH
outflow (1). Reduction of aqueous inflow was
observed due to the use of ß-adrenoceptor
blockers (2), carbonic anhydrase inhibitors (3)

and others including Forskolin (4). However,
reduction of AH inflow involves either
vascular (5, 6) or ionic mechanisms (7). In a
recent study, we reported on a decrease in
IOP of normotensive rabbits after ocular
instillation of silybinin solution (8). It has been
suggested that a direct pharmacological effect
on trabecular pathway to be likely involved
in pilocarpine-induced fall in IOP (9). The
role of cAMP in AH regulation is very well
explained (10, 11). It inhibits AH inflow by

blocking ion transport across ciliary
epithelium (12). Inhibition of Na+-K+ -2Cl¯
by cAMP decreases the uptake of Cl¯  by
ciliary epithelium (13), and the increase in
cAMP level, stimulated by ß2 -adrenergic
receptor activation in the trabecular
meshwork cells, suggested to be responsible
for the increased outflow facility after
application of ß2-agonists (14). Silibinin
hemisuccinate, a powerful antioxidant
flavonoid (15), inhibits cAMP-
Phosphodiestrase enzyme, even more potent
than theophylline or papaverine (16), an effect
that can be utilized for the interference with
AH formation and IOP regulation. The present
study was designed to examine the interaction
between silibinin and pilocarpine or
cyclopentolate in lowering IOP of
normotensive rabbits, and to provide
preliminary evidence for the mechanism of
this effect.

1 Corresponding author : E-mail :  saad_alzaidi@yahoo.com
Received    :  15/7/2007
Accepted    :   30/12/2007



Iraqi J.Pharm.Sci., Vol.16 (2)  ,2007 Effect of Silibinin on IOP

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2% pilocarpine HCl

1% cyclopent olat e
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Materials and Methods
Thirty-five New Zealand white rabbits

weighing 1.5-2.5 kg were used in this study.
Animals were kept in the animal house of the
College of Pharmacy, University of Baghdad,
under standardized conditions (12 hrs light-
dark cycles at room temperature), and were fed
a standard diet (Quality Control Laboratories,
MOH, Iraq) and water ad libitum.
Drugs treatment

Silibinin hemisuccinate in pure form was a
gift from Tolbiac SRL, Argentina, and all
other compounds used during the study were
supplied by the Department of Pharmacology
and Toxicology, College of Pharmacy,
University of Baghdad. Silybinin
hemisuccinate was dissolved in arachis oil and
used freshly prepared (0.75%) solution.
Pilocarpine (2%) and cyclopentolate (1%)
were used as commercial eye drop formulas
(Alcon Pharmaceuticals LTD, Cham,
Switzerland). The animals were allocated into
7 groups (5 rabbits each) for studying the
effect of topically instilled silybinin
hemisuccinate alone, pilocarpine alone,
cyclopentolate alone, the effect of pilocarpine
or cyclopentolate instilled 30 min prior to
silybinin and the effect of prior instillation of
silybinin 30 min before pilocarpine or
cyclopentolate.
Measurement of IOP

Indentation tonometry, using Schiotz
tonometer, was utilized in this study for
measuring IOP before and after instillation of
drugs or drug vehicle (Arachis oil). Thirty
minutes before starting drug instillation, the
cornea was anesthetized with 0.5% tetracaine
HCl (Chauvin Pharmaceuticals Ltd, Surry,
England) and baseline IOP was measured
using Schiotz tonometer. After instillation of 1
drop of silibinin hemisuccinate (0.75%),
pilocarpine (2%) and cyclopentolate (1%),
measurement of IOP was performed every 30
min for 3 hr (17). After each measurement the
eyes were washed with normal saline and the
instrument was cleaned with diethyl ether. All
experiments were conducted in a masked
manner, and were performed during a fixed
time of the day (from 10:00 AM to 3:00 PM)
to exclude the effect of circadian changes in
IOP.  For assessment of the pre-instillation
effect of pilocarpine or cyclopentolate,
baseline IOP was recorded before instillation
of these drugs to both eyes, and then 1 drop of
freshly prepared silibinin hemisuccinate oily
solution was applied after 30 min to both eyes.
The IOP was measured every 30 min for 3 hr.
The same later approach was followed to
evaluate the effect of pre-instillation of
silibinin on that produced by pilocarpine and

cyclopentolate.  All results were expressed as
mean ± S.E. Comparisons with baseline were
made using Student’s paired t-test, while a
single-factor analysis of variance (ANOVA)
for repeated measurements was used to
evaluate differences between groups. P values
less than 0.05 were considered significantly
different.
Results
Effects of 0.75% silibinin, 2% pilocarpine
and 1% cyclopentolate

In normotensive rabbits, instillation of
0.75% silibinin decreases IOP significantly for
2.5 hr, and remains significantly different with
respect to baseline value at all measured time
points (Figure 1). The maximal decrease in
IOP was achieved after 1 hr of instillation
(38.56 %) compared to baseline (P<0.05).
Pilocarpine eye drops (2%) significantly
reduces IOP after 30 min of instillation
(21.5%, P<0.05), decreased gradually to non-
significant value (0.46%, P>0.05) at the end of
3 hr compared to baseline (Figure 1).
Instillation of 1 drop cyclopentolate (1%)
significantly elevates IOP, reaching maximum
value after 0.5 hr (34.76 %); then gradually
decreases with time (4.24 %) after 2.5 hr of
instillation.

Figure 1: Effect of 0.75% silibinin
hemisuccinate, 2% pilocarpine HCl and
1% cyclopentolate HCl on IOP in
normotensive rabbits; results are presented
as mean of percent changes ± SEM; n=10
eyes for each group; * P < 0.05 with respect
to baseline; † P < 0.05 with respect to 0.75%
silibinin alone.



Iraqi J.Pharm.Sci., Vol.16 (2)  ,2007 Effect of Silibinin on IOP

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Effects of pre-treatment with 2% pilocarpine
and 1% cyclopentolate

The IOP-lowering effect of pilocarpine
was enhanced when silibinin instilled 30 min
latter, 26.87% compared to 19.0% produced
by pilocarpine alone (P<0.05). The reduction
in IOP observed in this procedure remains
significantly high (40.88%, 49.5%, 43.34%,
and 26.87%) compared to that produced by
pilocarpine alone (9.23%,4.16%,1.81% and
0.46%) for the time intervals 1.0, 1.5, 2.0, and
2.5 hr respectively (P<0.05) (Figure 2).
Moreover, this level reduction in IOP was
significantly different compared to that
produced by silibinin alone along the entire
period beyond the addition of silibinin. When
cyclopentolate instilled 30 min prior to
silibinin, the latter still have the ability to
reverse the elevation of IOP produced by
cyclopentolate (from 34.76% to 17.3%) after
30 min of instillation (P<0.05); these changes
are not significantly different at the time
intervals 1.0, 1.5, and 2.0 hr after instillation
of silibinin, P>0.05 (Figure 3).

Figure 2: Effect of pre- and post treatment
with 0.75% silibinin hemisuccinate on IOP
lowering effect of 2% pilocarpine in
normotensive rabbits; results are presented
as mean of percent changes ± SEM; n=10
eyes for each group; * P < 0.05 with respect
to baseline; † P < 0.05 with respect to 2%
pilocarpine alone.

Figure 3: Effect of pre- and post treatment
with 0.75% silibinin hemisuccinate on IOP
rising effect of 1% cyclopentolate in
normotensive rabbits; results are presented
as mean of percent changes ± SEM.  n=10
eyes for each group; * P < 0.05 with respect
to baseline; † P < 0.05 with respect to 1%
cyclopentolate alone; Pre-treatment with
silibinin hemisuccinate did not significantly
differ from silibinin hemisuccinate alone P
> 0.05.

Effects of pre-treatment with silibinin
Pre-treatment with silibinin, 30 min

before instillation of pilocarpine resulted in
additive effect on the IOP lowering activity of
pilocarpine (37.5%), which was significantly
different, compared to the effect of pilocarpine
alone (21.5%). This reduction in IOP remains
significantly different with respect to that
produced by pilocarpine after 1.0, 1.5 and 2.0
hr. However, the effect of pre-treatment with
silibinin did not significantly differ compared
to that produced by silybinin alone during all
time intervals (P>0.05) (Figure 1). Even when
cyclopentolate instilled 30 minutes latter, pre-
treatment with silibinin results in highly
significant reduction in IOP (31.47% P<0.05),
and remains significantly high 1.0, 1.5, and 2.0
hr after instillation of cyclopentolate (-20.08%,
-8.92%, -4.12% respectively) compared with
the rise in IOP (28.36%, 18.29%, 10.18% and
10.18%) produced by cyclopentolate alone
(P<0.05). Cyclopentolate appeared to slightly
reverse the action of silibinin when instilled 30
min latter; however, such effect did not
significantly differ from that produced by
silibinin alone (P>0.05).

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Pre-treatment with 0.75% silybinin hemisuccinate
post-treatment with 0.75% silybinin hemisuccinate

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P re-t reat ment  wit h 0.75% silybinin hemisuccinat e

0.75% silybinin hemisuccinat e alone

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Iraqi J.Pharm.Sci., Vol.16 (2)  ,2007 Effect of Silibinin on IOP

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Discussion
It has been reported previously in our

laboratory that corneal instillation of silibinin
lowers IOP in normotensive rabbits in a dose
dependent manner. It also delays IOP recovery
rate after I.V infusion of 20% sodium chloride
solution (8). Although inhibition of cAMP-
phosphodiesterase is proposed as a suspected
mechanism for the action of silybinin on IOP
(18), interference with the cholinergic influence
in this respect was evaluated. In the present
study, the effect of silibinin on IOP was higher
than that produced by pilocarpine, and their
combination results in an additive effect.
Muscarinic agonists, including pilocarpine,
lower IOP through enhancing AH outflow due
to contraction of the iris sphincter (19).
According to the reported mechanisms of
action of silibinin, targeting AH formation and
interference with ion transport can be
suggested as possible mechanisms (20).
Consequently, the mechanisms through which
pilocarpine and silibinin produce their effects
can be utilized for explaining the additive
effect reported when both of them are used at
the same time. To confirm the idea that
silibinin lowers IOP through a mechanism not
related to the cholinergic system, the
interaction of silibinin with anticholinergic
agent like cyclopentolate was evaluated.
Although there is no practical evidence on
elevation of IOP in rabbits due to instillation
of cyclopentolate, the results reported in this
study demonstrated such effect, which can be
attributed to the abnormal sensitivity of the
locally bred strain of rabbits to the effect of
cyclopentolate. In the present work, the rise in
IOP produced by instillation of cyclopentolate
was effectively reversed by silibinin;
meanwhile, the IOP lowering effect of
silibinin was not affected by postinstillation of
cyclopentolate. Based on these data, one can
postulate that silibinin interferes with IOP
regulation through reduction of AH inflow.
Taken together with the data obtained in
previous study (8) about the effect of silibinin
on IOP recovery rate and its contralateral
effect, one can suggest the interference with
AH inflow as a mechanism involved in
pilocarpine-silibinin and cyclopentolate-
silibinin interactions. The IOP-lowering effect
of cholinomimetics is mediated via the
activation of the inositol triphosphate (IP3)
pathway that linked to M3-receptors in the
ciliary and iris-sphincter muscles (21). Silibinin,
on the other hand, strongly inhibits cAMP
phosphodiesterase with consequent elevation
of cAMP levels (16). The later mediates many
biological effects including the inhibition of
ion transport by Na+-K+- 2Cl– co-transporter

across ciliary epithelium and trabecular
meshwork (13, 22), which is similar to that
produced by activation of the IP3 pathway
initiated by pilocarpine. Regulation of cell
volume is very important phenomenon that
involved during exposure to hypo- or
hypertonic environment, and Na+ - K+- 2Cl –

co-transporter is the system responsible for
such regulation (23, 7, 24). Infusion of hypertonic
sodium chloride solution resulted in shrinkage
of ciliary epithelium, and to retain the original
volume, ion transporters should be activated to
transport Na+ and Cl– across ciliary epithelium
(25). Inhibition of this co-transporter by cAMP
delayed osmotic recovery rate, an effect
reported after silibinin administration. In
conclusion, corneal instillation of silibinin
lowers IOP in normotensive rabbits, probably
through a mechanism not related to the
interference with cholinergic influence on IOP.

Acknowledgment
The authors gratefully thank College of
Pharmacy, University of Baghdad for
supporting the project and Tolbiac SRL,
Argentina for providing pure silibinin powder
as a gift.
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