Acta Poloniae Pharmaceutica Drug Research, Vol. 63 No. 4 pp.

277280, 2006

ISSN 0001-6837
Polish Pharmaceutical Society

NATURAL DRUGS

ANTI-INFLAMMATORY ACTIVITY OF ACHILLEA

AND RUSCUS TOPICAL GEL ON CARRAGEENAN-INDUCED
PAW EDEMA IN RATS
HAMZAH M. MASWADEH*, MOHAMMAD H. SEMREEN and AHMAD R. NADDAF
School of Pharmacy, Department of Pharmaceutical Technology, Al-Isra University,
P.O. Box 961582, Code No. 11196 Amman, Jordan
Abstract: The anti-inflammatory activity of Achillea and Ruscus extracts was studied in comparison with
diclofenac sodium topical gel (diclosal Emulgel), using the carrageenan induced paw edema model in Albino
rats. Gel formulation was prepared containing 6% of each extract in gel base, namely sodium
carboxymethylcellulose (NaCMC). The kinetics of drug release from the prepared formulation was studied
separately in each case. Results showed that the release follows the Higuchi square root equation. The
pharmacological screening revealed that the percent reduction of edema for Achillea extract and Ruscus extract
were 48.1% and 18.8%, respectively, while diclosal Emulgel produced 47% reduction of edema.
Keywords: Achillea, Ruscus, carrageenan-induced paw edema

topical administration and comparison of the

prepared gels with a standard gel in the market by
using the carrageenan-induced edema model.
The partition coefficient was determined for
the two extracts as a measurement of a drugs
lipophilicity. The in vitro release study through
cellophane membrane of each prepared gel was
studied using a stainless steel diffusion cell.

Achillea fragrantissima (Asteraceae) is a common plant in the Mediterranean region and easily
found growing in fields and on roadsides. It contains
high percentage of flavonoids, tannins, volatile oils,
sterols and triterpenes. Also, it contains unsaturated
amides, and sesquiterpene lactones (1). Achillea was
highly valued as a medicinal plant for its antiseptic
properties. It was used to cover cuts and sores and
hasten scar tissue formation, but till now no clinical
uses for Achillea fragrantissima is described (2).
Ruscus aculeatus (Liliaceae) is growing wildly
in the forests. It contains steroidal saponins derived
from ruscogenin and neoroscogenin in addition to
essential oils, flavonoids, resin and minerals. Ruscus
is used in supportive therapy for venous insufficiencies such as circulatory disorders, edema,
thrombophlebits, swelling and also used as diuretic.
Since it may cause vascular contraction, caution
should be used in individuals with hypertension (3).
In very rare cases it may cause gastrointestinal
upset.
This work was suggested on the basis of
presence of flavonoidal and ruscosides constituent
in the two investigated parts of plants, respectively,
namely Achillea fragrantissima Linne, Asteraceae
(dried flowers) and Ruscus aculeatus, Liliaceae
(leaves). The aim of this study was to study their
possible anti-inflammatory effect by formulation of
the two extracts in a suitable gel formulation for

MATERIALS AND METHODS

Sodium carboxymethylcellulose (Na-CMC),
normal saline and diclofenac sodium emulgel
(diclosal Emulgel) were obtained from Dar Al
Dawa, Naur, Jordan. Carageenan (0.1% solution in
normal saline) was obtained from Sigma Chemical
Co. Steinheim, Germany. Ruscus (leaves) and
Achillea (flowers) were obtained from the Jordanian
market in March 2004. Organic solvents i.e. chloroform, methanol, acetone and octanol were of
spectroscopic grade.
Male Albino rats (weighing 200-250 g) of local
strain were used for the anti-inflammatory study by
carrageenan induced rat paw edema method. The
animals were kept for one week in the animal house
before the experiment to be acclimatized, and they
were maintained on unrestricted supplies of food
and water.

* Correspondence: e-mail: [email protected]

277

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HAMZAH M. MASWADEH et al.

Extraction
Achillea flowers (500 g) were extracted by
70% ethanol till exhaustion. The combined extracts
were evaporated to dryness to give 51 g of solid
residue.
Ruscus leaves (100 g) were extracted by 70%
ethanol till exhaustion. The combined extracts were
evaporated to dryness to give 9 g of solid residue.
Determination of partition coefficient (Ko/w)
10 mg of each extract was added to 50 mL of
distilled water in a stoppered bottle and shaked at
37OC in water bath shaker (GFL 1083, Germany)
overnight. Octanol (50 mL) was added to the bottle
with shaking at the same temperature for 24 h. The
two layers were then separated using a separatory
funnel, and the absorbance of drug in the aqueous
layer was determined, at 338.6 nm for Achillea
extract and at 236 nm for Ruscus extract. The drug
concentration in aqueous layer was determined by
a calibration curve for each extract. The concentration
of drug in octanol was determined by difference. The
partition coefficient of drug between octanol and
water (KO/W) was calculated from the equation:
Ko/w = Co / Cw

Table 1. The content of gels formulations.

Material
Extract
Sodium carboxymethylcellulose
Glycerol
Propylene glycol
Water up to

Concentration
(w/w) %
6
5
10
5
100

where: Ko/w is the partition coefficient, Co is the

concentration of drug in octanol and Cw is the
concentration of drug in water.
Preparation of topical extract gel
Achillea and Ruscus gels were separately
prepared according to the formula in Table 1, by
dissolving weighed amount of extract in the needed
volume of water, and then added to 5% of the base.
The specified amounts of glycerol and propylene
glycol were added with continuous stirring at room
temperature for 15 min using mechanical stirrer.
Each gel was kept separately in dark cool place
overnight (10-15OC).
In vitro release study through cellophane
membrane
The release of extract from its prepared gel was
studied using a stainless steel diffusion cell. More
specific, 2 g sample of each formulation was
accurately weighed and placed in the hollow bottom
of the diffusion cell (donor part), the Fisher 27/30
standard membrane was adjusted between the two
joints and the tow screw was fitted, and the cell was
then placed in a beaker containing 600 mL of
phosphate buffer (pH = 7.4), which was adjusted to
the water bath of the dissolution apparatus
(ERWEKA GmbH, Heusentamm, Germany)
operating at 37OC and 100 rpm.
5 mL samples were withdrawn at predetermined
time intervals and immediately replaced with equal
volumes of phosphate buffer, then analyzed
spectrophotometrically at mentioned above max,
using UV/Vis apparatus (model 7800 Jasco, Japan).
The kinetics of the release process was studied
by analyzing the release data using two kinetic

Table 2. Rats groups used in the carrageenan-induced edema model (each comprised of 4 rats) with the type of extract composition and
dose received for each group.

Group
1- Control group
2- Treated group (1)
3- Treated group (2)
4- Treated group (3)

Extract composition and dose received

Received 2 g of sodium carboxymethylcellulose (gel base only).
Received 2 g of commercial diclofenac gel (Diclosal Emulgel).
Received 2 g of 6% of NaCMC-Achillea extract gel.
Received 2 g of 6% of NaCMC-Ruscus extract gel.

Table 3. Partition coefficient, release rate constant and r2 values for Achillea and Ruscus extracts.

Formula

Ko/w

Achillea extract
Ruscus extract

0.828
0.638

Zero-order rate
constant (K0)
0.085 (R2 = 0.86)
0.122 (R2 = 0.94)

Higuchi square root rate

constant (K1)
1.184 (R2 = 0.95)
1.627 (R2 = 0.96)

Anti-inflammatory activity of achillea and ruscus topical gel...

279

Table 4. Effect of topical administration of extract gel and diclofenac gel on carrageenan-induced paws edema in rats.

Treatment
Gel bases pure (control)
Diclosal Emulgel (diclofenac sodium)
Extract of Achillea
Extract of Ruscus

Mean % increase
in paw weight
34.5 1.1
18.3 1.5
17.9 1.3
28.0 1.8

% reduction
of edema
0.0
47.0 0.8
48.1 1.1
18.8 1.6

Figure 1. A linear plot of amount released versus time for the

release data in accordance with the zero-order equation. Achillea
extract (- -) and Ruscus extracts (--).

Figure 2. A linear plot of amount released versus square root of

time for the release data in accordance with the Higuchi square
root equation. Achillea extract (- -) and Ruscus extracts (--).

equations, the zero-order equation and the Higuchi

square root equation.

The mean percentage of inflammation

reduction was measured from the difference in %
swelling between treated groups and the control
group by the following equation:
% reduction of edema = (C T / C) x 100
where: C = % swelling of control group (untreated)
and T = % swelling of treated group.

Screening of anti-inflammatory activity of

extracts gel
The anti-inflammatory activities of the agent
under study were evaluated by using the
carrageenan-induced edema model. More specific,
rats were divided into 4 groups, each comprised of
four rats as shown in Table 2.
The gel was applied to the planter surface of
the left hind paw by gently rubbing 50 times with
the index finger. Three hours after the dose, 0.1 mL
of 1% carrageenan solution in normal saline was
injected subplantarly into the treated paw. Three
hours after the carrageenan injection, the right and
the left paw were cut at the tibiotarsal articulation
under chloroform anesthesia and weighed (4-6).
The percentage increase in the weight of the
left paw in comparison with the right one of each rat,
as an indication of the inflammation produced, was
calculated by the following equation:
% increase in paw weight = (L R / L) x 100
where: R is the weight of right leg and L: is the
weight of left leg.

RESULTS AND DISCUSSION

Achillea contains volatile oils, mainly azulenes
which are anti-inflammatory and could be lost
during evaporation. However, it contains also
several known anti-inflammatory compounds as
flavonoids. Also Ruscus contains ruscosides which
are known as anti-inflammatory. In the UV
spectrum of these extract, fortunately, the only very
clear peaks were found at 338.6 nm for flavonoids in
Achillea and 236 nm for ruscosides in Ruscus. No
other significant peaks were found.
The partition coefficient of Achillea extract
and Ruscus extract were 0.828 and 0.638,
respectively, indicating that both extracts can be
applied topically depending on their lipid solubility.

280

HAMZAH M. MASWADEH et al.

The prepared gel formulations were found to

have acceptable rheological properties. Figure 1
shows that the Ruscus gel release was about 20 mg
within 120 min, whereas only 14 mg was released
from Achillea extract at the same time. This may be
attributed to the lower aqueous solubility of Achillea
extract.
In order to describe the kinetics of the release
process of drug through cellophane membrane in the
two gel formulations, two equations were used,
namely, the zero-order rate equation and the
Higuchi square root equation (7, 8). The release data
obtained from the two formulations were plotted in
accordance with the zero-order equation i. e. amount
released as a function of time (Figure 1).
The release data obtained from the two gel
formulations were plotted in accordance with the
Higuchi square root equation, i.e. the amount
released as a function of the square root of time
(Figure 2). A linear relationship was obtained with r2
value close to unity as shown in Table 3, indicating
that the release kinetics follows the Higuchi square
root equation and that the release process is
diffusion-controlled (9, 10).
The pharmacological screening was carried out
by using the carrageenan-induced edema model to
evaluate the possible anti-inflammatory activity of
the two extracts. As shown in Table 4, the reduction
of edema was 48% for Achillea extract, nearly
equally effective to that produced by the standard
(Diclosal Emulgel) 47%, indicating an
anti-inflammatory effect. This good result was not
achieved by using Ruscus extracts, the reduction of
edema was only 18%, which indicates that the
anti-inflammatory effect of Ruscus extract was very

low compared to that obtained for Achillea extract

as well as that obtained from the standard.
From this study it can be concluded that
NaCMC can be successfully used as gel base and
that the kinetic of drug release from this formulation
can be described by Higuchi square root equation.
Also it can be concluded that both extracts have an
anti-inflammatory effect, with Achillea extract to be
nearly equally effective as anti-inflammatory agent
at the market diclofenac sodium.
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Received: 26.03.2006