The effect of Medicago spp. on growth of Trichophyton mentagrophytes in microculture

Radoslaw Spiewak¹, Wieslawa Szostak¹, Marian Jurzysta², Zbigniew Bialy², Romuald Maleszka³, Bozena Rzepecka³, Maria Mazurek³

¹Institute of Agricultural Medicine, Lublin, Poland
²Institute of Soil Science and Plant Cultivation, Pulawy, Poland
³Department of Dermatology, Police Hospital in Poznan, Poland.

Introduction

Saponins are substances that defend plants against fungal infections. Activity of some saponins against plant pathogens is well-documented (LEVY et al. 1989). Their fungitoxic effects is due to their interactions with sterols of the fungal cell (GESTETNER et al. 1972). These substances attract attention as possible therapeutics for fungal infections in humans. Antifungal effects of alfalfa saponins have been demonstrated in vitro on yeast pathogenic to humans (POLACHECK et al. 1986a, b). Previously, we have shown that purified alfalfa saponin - medicagenic acid glucoside - inhibits growth in vitro of the human pathogenic filamentous fungi Trichophyton mentagrophytes (SPIEWAK et al. 1999) and Scopulariopsis brevicaulis (SPIEWAK et al. 2000). It is not certain whether medicagenic acid is the only alfalfa saponin with antifungal properties. Moreover, introduction of pure medicagenic acid into human therapy would be very difficult due to high costs of its purification from the raw plant material. Therefore, this study was aimed at the assessment of antifungal properties of dried and ground roots and aerial parts of alfalfa. This material is much less expensive and also contains other saponins that possibly have antifungal potential.

Material and methods

Seeds of crop alfalfa (Medicago sativa L.) cv. Radius (Polish variant), M. murex Willd. cv. Zodiak (Australian variant) and M. arabica SA 7746 (Australian Medicago Genetic Resource Centre, Adelaide) were planted on 17 May 1999 on an experimental field in Pulawy (Poland). In the early blossoming phase, the plants were harvested, dried at the temperature of 60°C and ground.

The biological effects of the material were tested on the Trichophyton mentagrophytes dermatophyte strains, which were isolated from people with skin mycoses. The microcultures were made of punches of 3 mm diameter from fungal colonies. Altogether 44 microcolonies were set up, each on 7 different media: pure Sabouraud agar (control), and agars with each 1 g of ground roots or aerial parts of Medicago arabica, M. sativa, and M. murex per 100 ml Sabouraud agar. The microcultures were incubated at 27°C. Incubation time depended on the speed of fungal growth: The colonies were measured when the border of a fastest growing one was close to the edge of the agar block. Typically, this was after 6 - 7 days. Colony diameters at this time point were subject to further statistical analyses.

Results and discussion

The results are shown in Table 1. The strongest inhibition of T. mentagrophytes growth was achieved by addition of aerial parts of the species M. arabica (median 6 mm versus 13 mm for control microcultures), followed by the roots of M. arabica (10 mm), and roots of M. murex (10.5 mm). These differences were statistically significant at p < 0.001. A minor inhibitory activity was also observed of aerial parts of M. murex (median 12 mm, p = 0.03). In contrast to the above, addition of dried M. sativa to the culture caused a faster growth of T. mentagrophytes. The median diameter of colonies cultured with dried root of this species was 15 mm, and with aerial part - 16.5 mm (in both cases p < 0,001).

It is intriguing, which substances contained in the tested plant material are responsible for the observed inhibition or stimulation of the dermatophyte T. mentagrophytes. Some light might be shed by comparing the present results with data by JURZYSTA and WALLER (1996) on the biological effect and chemical characteristics of saponin in the aerial parts of various alfalfa species (Table 2). Both species found in the present study to possess antifungal properies also have a high haemolytic index (M. murex: 1778, M. arabica: 1464) and a strong inhibitory effect on the fungus Trichoderma viride (the growth inhibition by 0.1 g plant extract/100 ml of agar was 69% for both M. murex and M. arabica). In contrast to this, the species M. sativa, which in this study stimulated growth of T. mentagrophytes has a low haemolytic index (150) and no inhibitory effect on T. viride. Even more interesting is the comparison of the effects on T. mentagrophytes by these 3 species with the concentrations of saponins. M. murex and M. arabica contain very high levels of hederagenin glucosides but no detectable glucosides of medicagenic acid and soyasopogenol A. Aerial parts of M. sativa, which stimulated growth of T. mentagrophytes, do not contain hederagenine glucosides but medium levels of medicagenic acid glucosides and soyasopogenol A. The presence of glucosides of medicagenic acid in an alfalfa species that stimulates growth of T. mentagrophytes is somewhat surprising in the light of our previous study, which showed clearly an inhibitory action of this saponin (as pure substance) on T. mentagrophytes (SPIEWAK et al. 1999). A possible explanation for this might be that M. sativa contains other substances that stimulate the dermatophyte growth to such extent that they would compensate the inhibitory effect of medicagenic acid. It is also possible that other species contain substances that have even stronger antifungal properties. According to the above-discussed data, hederagenin seems a good candidate for this.

In conclusion, particular alfalfa species differ regarding the influence on the growth of the fungus Trichophyton mentagrophytes: Medicago arabica has the strongest inhibitory effect, M. murex a moderate one, whereas addition of M. sativa stimulates growth of the dermatophyte.

References

1. GESTETNER B, ASSA Y, HENIS Y, TENCER Y, ROTMAN M, BIRK Y, BONDI A. 1972. Interaction of lucerne saponins with sterols. Biochim. Biophys. Acta 270: 181-187.
2. JURZYSTA M, WALLER GR. 1996. Antifungal and hemolytic activity of aerial parts of alfalfa (Medicago) species in relation to saponin composition. W: Waller GR, Yamasaki H (Red). Saponins Used in Traditional and Modern Medicine. New York: Plenum Press: 565-274.
3. LEVY M, ZEHAVI U, NAIM M, POLACHECK I, EVRON R. 1989. Structure-biological activity relationships in alfalfa antimycotic saponins: the relative activity in medicagenic acid and synthetic derivatives thereof against plant pathogenic fungi. J. Phytopatol. 125: 209-216.
4. POLACHECK I, ZEHAVI U, NAIM M, LEVY M, EVRON R. 1986 a. Activity of compound G2 isolated from alfalfa roots against medically important yeasts. Antimicr. Agents Chemother. 30: 290-294.
5. POLACHECK I, ZEHAVI U, NAIM M, LEVY M, EVRON R. 1986 b. The susceptibility of Cryptococcus neoformans to an antimycotic agents (G2) from alfalfa. Zbl. Bakt. Hyg. A 261: 481-486.
6. SPIEWAK R, SZOSTAK W, JURZYSTA M, BIALY Z. 1999. The effect of medicagenic acid 3-O-ß glucopyranoside on different strains of pathogenic fungus Trichophyton mentagrophytes obtained from skin lesions in humans - preliminary results. In: Saponins in Food, Feedstuffs and Medicinal Plants. Book of Abstracts. Pulawy: Institute of Soil Science and Plant Cultivation: 106.
7. SPIEWAK R, SZOSTAK W, JURZYSTA M, BIALY Z. 2000. Hamujace dzialanie 3-glukozydu kwasu medikagonowego na wzrost Scopulariopis brevicaulis in vitro. Mikol. Lek. 7(Supl. 1):112-113.

© Radoslaw Spiewak