[P-117]
TOPICAL ANTI-INFLAMMATORY ACTIVITY
OF Rosmarinus officinalis L. EXTRACTS

Silvio SOSA1, Dejan BRKIĆ2, Aurelia TUBARO1
and Roberto DELLA LOGGIA1
1DEMREP, Universitŕ di Trieste, Via A. Valerio, 6, 34127 Trieste, Italy
2Institute for Medicinal Plant Research "Dr Josif Pančić", Tadeuša Košćuška, 1, 11000 Belgrade, FR Yugoslavia

ABSTRACT

The leaves of Rosmarinus officinalis L. (Labiatae) were investigated for their topical anti-inflammatory properties. The hexane and the chloroform extracts dose-dependently inhibited the Croton oil-induced ear oedema in mice, whereas the methanolic one showed a low effect. The chloroform extract was the most active and gave the highest contribution to the activity of rosemary. Its potency was higher than that of indomethacin: the ID50 (dose giving 50 % oedema inhibition) of the two compounds were 83 and 93 µg/cm2, respectively. Chemical and pharmacological investigations of the chloroform extract revealed ursolic acid as its main anti-inflammatory principle.


INTRODUCTION

Rosmarinus officinalis L. (Labiatae) is a common, evergreen shrub spontaneous in the Mediterranean area. The leaves of the plant are commonly used as a spice, but also for their antioxidant activity, for their wound healing properties as well as for the treatment of several inflammatory-based disorders (1, 2). Most of the biological studies on R. officinalis were carried out considering its antioxidant properties, due to diterpenoids and polyphenols, or its antimicrobial activity, due to volatile constituents (3, 4). On the contrary, the component(s) responsible for the anti-phlogistic activity of this species are not clearly defined. Since in the therapeutically related plant Salvia officinalis L. non volatile constituents, such as ursolic acid, were identified as the main anti-inflammatory principles (5), a study on the topical anti-inflammatory activity of a rosemary commercial sample was carried out. To this aim, R. officinalis leaves was extracted with increasing polarity solvents and the obtained extracts were evaluated for their ability to reduce the Croton oil-induced oedema in the mouse ear (6). The anti-inflammatory effect of the extracts was compared to that of the non-steroidal anti-inflammatory drug indomethacin.


MATERIALS AND METHODS

Materials

The commercial sample of rosemary, originary from Macedonia, was supplied by the Institute for Medicinal Plant Research "Dr. Josif Pančić" Belgrade (Yugoslavia). Croton oil and indomethacin were Sigma products (St. Louis, Missouri, USA). Ursolic acid was supplied by Indena S.p.A. (Milano, Italy). Ketamine hydrochloride and TLC plates were purchased from Virbac S.r.l. (Milano, Italy) and Merck (Darmstadt, Germany).The animals used were male CD-1 mice (28-32 g weight) supplied by Harlan Italy (Udine, Italy).

Methods

Plant extraction: Dried and pulverised leaves (45 g) were submitted to successive extractions with 600 ml of n-hexane, chloroform or methanol in Soxhlet apparatus for 6 hours each (Figure 1).

 Figure 1. Extraction scheme of Rosmarinus officinalis leaves

The obtained extracts were dried under vacuum at 30°C.

Thin layer chromatography (TLC): TLC analysis was performed using benzene:ethyl acetate:formic acid (36:12:5) as eluent. The detection was in UV radiation (366 nm) after spraying with anisaldehyde/sulphuric acid reagent. Anti-inflammatory activity: Inflammation was induced on the right ear (surface: about 1 cm2) of anaesthetised mice (145 mg/kg ketamine hydrochloride, intraperitoneally) by application of 80 µg of Croton oil. The tested substances were dissolved in the irritant solution. After 6 hours, mice were sacrificed and a punch (6 mm Ć) was excised from both the ears (6). Inflammation, measured as oedema formation, was quantified by the weight difference between the treated and the untreated (opposite) ear samples. Statistical analysis: The pharmacological data were analysed by the Student's t-test, considering a probability level lower than 0.05 as statistically significant. The doses inhibiting the oedematous response by 50 % (ID50) were calculated by graphic interpolation of the dose-effect curves.


RESULTS AND DISCUSSION

The extraction yields of Rosmarinus officinalis leaves, reported in Table 1, show that the chloroform extract was obtained in the highest amount, followed by the methanolic extract and by the hexane one.


Table 1. Yields of Rosmarini folium extraction
Yield
Hexane
extract
Chloroform
extract
Methanolic
extract
Grams
2.50
4.95
3.50
%
5.6
11.0
7.8

The anti-inflammatory activity assay revealed a strong dose-dependent activity for the hexane and the chloroform extracts whereas the methanolic one was almost inactive. In particular, the hexane extract induced 21% oedema reduction at the dose of 30 µg/cm2, reaching about 70 % reduction at the highest administered dose (1000 µg/cm2). Also the chloroform extract provoked 21% reduction of the oedematous response at the dose of 30 µg/cm2, but already at 300 µg/cm2 it induced more than 90 % inhibition. The methanolic extract revealed low but significant activity (28% oedema reduction) only at the dose of 1000 µg/cm2. As reference, 90 µg/cm2 of indomethacin reduced the oedematous response by 49%. The anti-inflammatory potency of the rosemary extracts was evaluated by calculating their ID50 values (dose giving 50 % oedema inhibition).


Table 2. Anti-inflammatory activity of Rosmarinus officinalis extracts
Substance
Dose
(µg/cm2)
N°. an.
Oedema (mg)
m ± S.E.
% Red.
ID50 (µg/cm2)
Controls
--
30
7.6 ± 0.2
--
--
Hexane extract
30
10
6.0 ± 0.4*
21.1
265
 
100
20
4.5 ± 0.4*
40.8
  
 
300
10
4.1 ± 0.3*
46.1
  
 
1000
10
2.3 ± 0.5*
69.7
  
Chloroform extract
30
10
6.0 ± 0.5*
21.1
83
 
100
20
3.8 ± 0.3*
50.0
  
 
300
10
0.5 ± 0.1*
93.4
  
 
1000
10
0.2 ± 0.1*
97.4
  
Methanolic extract
100
10
7.5 ± 0.5
1.3
>1000
 
1000
10
5.5 ± 0.6*
27.6
  
Indomethacin
90
10
3.9 ± 0.4*
48.7
93
* p<0.005 at the Student's t-test.

The obtained values indicate that the chloroform extract was more potent than the hexane one since only 83 µg/cm2 were sufficient to reduce the oedematous response by 50 % whereas the needed dose of the hexane extract corresponds to 265 µg/cm2. The chloroform extract was also more potent than indomethacin, which ID50 corresponds to 93 µg/cm2 (Table 2). In order to evaluate the contribution of the extracts to the activity of the crude drug, their oedema inhibition was plotted versus the amount of crude drug yielding the administered doses of each extract (drug equivalents; D.E.). As shown in Figure 2, the high anti-inflammatory effect of the chloroform extract was induced by D.E. lower than those corresponding to the hexane and methanolic extracts. It means that the chloroform extract gives the highest contribution to the activity of R. officinalis leaves, followed by the hexane and by the methanolic extracts.


Figure 2. Anti-inflammatory potency of ursolic acid

TLC analysis revealed the presence of a chromatographic band corresponding to that of ursolic acid in the two most active extracts. Furthermore, the band was more evident in the chloroform extract, suggesting a role of this triterpenic acid in its anti-inflammatory activity. Actually, ursolic acid revealed a strong dose-dependent anti-inflammatory activity, showing an ID50 of 64 µg/cm2 that confirms a significant role of this triterpene in the anti-phlogistic properties of Rosmarinus officinalis leaves.


CONCLUSIONS

The obtained results demonstrate that Rosmarinus officinalis leaves possess interesting topical anti-inflammatory activity. In particular, their chloroform extract induced a strong dose-dependent oedema reduction in the mouse ear, demonstrating that the anti-inflammatory principles can be exhaustively extracted by apolar solvents. Among them, the most relevant appears to be the triterpene ursolic acid, which content in Rosmarini folium based preparations, useful for their anti-inflammatory properties, can be proposed as quality control parameter.


ACKNOWLEDGEMENTS

Part of this research was supported by Consorzio per lo Sviluppo Internazionale dell'Universitŕ di Trieste on the basis of the financial support of Regione Friuli-Venezia Giulia (Italy).


LITERATURE

  1. Wichtl M. (1989): In "Teedrogen: ein Handbuch für die Praxis auf wissenschaftlicher Grundlage", Wissenschaftliche Verlagsgesellschaft mbH, Stuttgard.

  2. Schwartz K., Ternes W. (1992): Z. Lebensm. Unters. Forsch. 195, 95-98.

  3. Mangena T., Muyima N.Y. (1999): Lett. Appl. Microbiol 28, 291-296.

  4. Baričevič D., Sosa S., Della Loggia R., Tubaro A., Simonovska B., Krasna A., Zupančič A., submitted to Planta Medica.

  5. Tubaro A., Dri P., Delbello G., Zilli C., Della Loggia R. (1985): Agents Act. 17, 347-349.

[P-117]