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Introduction

8Ruta chalapensis L. (Rutaceae), a perennial herb, widely diffused in the Mediterranean area, with glabrous stem, alternate bi-pinnatisect leaves with narrowly oblong-lanceolate or obovate segments, inflorescence cymose, growing in dry, usually rocky areas, is an ancient medicinal plant still used in the traditional medicine of many countries as a laxative, anti-inflammatory, analgesic, antispasmodic, abortifacient, antiepileptic, emmenagogue and for dermatopathy treatment (Johnson T., 1999).

9Pharmacological investigations clearly indicated that the ethanol extract of the aerial part of R. chalepensis shares the anti-inflam- matory and antipyretic activities of the other common non steroi'dal anti-inflammatory drugs and has significant dose-dependent central nervous system depressant activity (Mansour S. et al., 1990), while toxicity studies have provided basic information about the possible safe use of this medicinal plant (Shah A.H. et ai, 1991).

10Phytochemical screening showed that the aerial parts of R. chalepensis yielded the coumarins chalepensin, chalepin, rutamarin, bergapten, isopimpinellin and xanthotoxin (Ulubelen A. et al., 1986), as well as alkaloids kokusaginine, skimmianine , arbori- nine, γ-fagarine, graveoline, 3'-hydroxygraveoline (Ulubelen A. et al., 1986), taifine, isotaifine and 8-methoxytaifine (Mohr N. et al., 1982).

11As some secondary metabolites contained in plants often have good antibacterial or antimycotic activity against microorganisms that are resistant to drugs in common use, in a program of studies on the antimicrobic activity of vegetable drugs we devoted our attention to the aerial parts of this species; in fact, as some coumarins and alkaloids isolated from R. chalepensis have antimicrobial activity (Wolters B. et al., 1981) and its extractive preparations are topically used in several skin diseases, we assayed the anti-mycotic activity of the ethanol extract of the aerial part of this traditional medicinal plant against fresh clinical isolates of hyphomycetes identified by conventional procedures (De Hoog G.S. et al., 1991).

Materials and methods

Plant material

12R. chalepensis was collected in the Messina area in April 1999. A voucher specimen of the plant was deposited in the herbarium of the Pharmaco-biological Department of the University of Messina (Italy). The fresh material was immediately lyophilized and powdered.

Preparation of extract

13Exhaustive extraction of 10 g of lyophilized and poxdered R. chalepensis aerial part was carried out in a Soxhlet using ethanol as solvent. The mixture was filtered and the organic solvent removed under vacuum.

Strains

14Strains of hyphomycetes isolated from clinical specimens principally made up of dermatophytes: Aspergillus fumigatus , Aspergillus terreus, Microsporum canis, Paecilomyces lilacinus, Scopulariopsis brevicaulis, Trichophyton mentagrophytes, Trichophyton rubrum.

Assay medium

15The composition of the assay medium was the following: case amino acids (Difco) 1.0% (p:v), dextrose (Oxoid) 1.0% (p:v), sodium b-glycerophosphate (Sigma) 0.35% (p:v), agar N.l (Oxoid) 1.5% (p:v), glycine (Sigma) 0.1% (p:v), yeast nitrogen base (YNB) (Difco) 0.067%. All the components with the exception of glycine and YNB were solubilized in 100 ml of distilled water and sterilized in an autoclave. The two components, glycine and YNB, were prepared at a concentration of 1 OX, sterilized for filtration and added in aliquots of 1.0 ml to the medium maintained in a water bath at 50°C after sterilization. 12 ml of medium was poured into 90 mm Petri dishes to a thickness of 2 mm.

Assay method

16The activity of the extract was evaluated by a method designed by the Department of Microbiological Sciences and Gynaecological Sciences at the University of Catania. It allows the evaluation of the growth of hyphomycetes strains under the action of a concentration gradient of the substance under examination, obtained by the diffusion of three quantities deposited around an inoculum point. In particular, in solid media some pits were created according to the plan in figure 1. Around each pit of 2 mm diameter, into which the inoculum of conidia suspended in sterile distilled water is added, three pits of 3 mm diameter are made: the first along the axis that joins the inoculum pit to the center of the dish, the second and the third on the axis that is perpendicular, passing through the center of the inoculum pit. The distance between the edges of the pits along the two axes is 10 mm. The amounts of both the control chemotherapeutic and the assayed extract were used in the following ratio 1: 1: 0.01. For each test the three amounts of the substance were applied in a clockwise direction starting from the pit nearest the center of the dish. The activity of the extract, solubilized in dimethyl sulfoxide (DMSO) at 20%, was evaluated against that from miconazole, used as a standard, this was also solubilized in dimethyl sulfoxide at 20% . Into the 2 mm diameter pit, 5 (il of the inoculum was poured, for each strain, from a suspension of spores kept in sterile distilled water having a concentration of between 5 x 10⁴ and 1 x 10⁵ ml^-1. In the 3 mm diameter pits 10 µl of the Ruta extract and miconazole were poured. For the Ruta extract doses of 2000, 200 and 20 µg were used, for miconazole doses of 10, 1, 0.1 µg. In each dish two assays were carried out for the Ruta extract, one for miconazole and one for the solvent (DMSO at 20%). The dishes were incubated at 30° C, from 48 to 120 h, depending on the growth rates of the different species.

Evaluation of antifungal activity

17The anti-fungal activity was evaluated by means of the appearance on the surface growth area determined by the image acquisition system Foto/Analyst by Fotodyne Incorporated (Hartland U.S.A.) and the software Collage (Image Analysis Software). For the estimation of the surface growth area in the presence of Ruta extract the mean values of the two tests on each dish were used.

18The evaluation of the activity of the extract of Ruta was carried out with the parameter found from the following formula: I_r = (S_r – S_d) / S_d · S_r, where I is the value of the surface growth area in the presence of the extract, S_d is the value of the surface growth area in the presence of only the solvent. As regards miconazole an analogous formula was used. The value of I varies from -1 to ≥ 0. The value of -1 indicates the maximum activity while a value tending to 0 or just above indicates the absence of activity of the substance in examination. The interval from -1 to 0 was divided into three classes correlated to three levels of activity: low or null (-0.20 < l), intermediate (0.6 ≤ I ≤ -0.2), elevated (-1 ≤ I < -0.6).

Results and discussion

19The activity of Ruta extract is characterized by a marked variability between the strains of the same species. In fact, the range of the parameter I (Fig. 2) varies for M. canis from -0.20 to -0.74, for T. mentagrophytes from 0 to -0.65 and for T. rubrum from 0 to -0.64. From table 1, where the number of strains for each class of parameter I is shown, it can be seen that for M. canis the ethanol extract of Ruta has an elevated activity in 8 strains (57.1%) and intermediate in 6 strains (42.9%); miconazole has an elevated activity in 12 strains (87.5%) and intermediate in 2 strains (12.5%). Against T. mentagrophytes the extract of Ruta has a low activity: in fact, it is elevated in 4 strains (40%), intermediate in 4 strains (40%) and inactive in 2 strains (20%). Miconazole has an elevated activity in 6 strains (60%) and intermediate in 4 strains (40%). Against T. rubrum the extract of Ruta has an elevated activity in 1 strain (12.5%), discreet in 6 strains (75%) and is inactive in 1 strain (12.5%). Miconazole, instead, has an elevated activity in 8 strains (100%).

20For all the other hyphomycetes (Fig. 3), Ruta has an intermediate activity while miconazole has an elevated activity with the exception of one strain of Scopulariopsis brevicaulis. The intervals of the value of the parameter I relative to the extract of Ruta are: from -0.21 to -0.35 for A. fumigatus, from -0.21 to -0.32 for A. terreus, from -28 to -0.29 for P. lilacinus, from -0.33 to -0.44 for S. brevicaulis.

Conclusion

21The ethanol extract of Ruta aerial parts has, in vitro, a good antifungal activity against the strains of dermatophytes assayed and in particular against M. canis even if the doses of the extract assayed in our model are greater than those of miconazole used as an antifungal standard because in this study we used crude extract; this activity seems probably due to the presence of alkaloids in the aerial parts of Ruta chalepensis.

22The results obtained, as a whole, justify the use in traditional medicine of several regions in the world of this medicinal plant for the treatment of cutaneous pathologies in which dermatophytes could be involved.

Table I. Distribution of I values for Ruta extract (A) and miconazole (B) against other hypomycetes