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.

This is the author's translation of the Polish-language article: Spiewak R, Szostak W, Jurzysta M, Bialy Z, Maleszka R, Rzepecka B, Mazurek M. Wplyw lucerny na wzrost Trichophyton mentagrophytes w mikrohodowli. Wiad Parazytol 2001; 47 (4): 839-844.


Abstract: The study aimed at assessing the effect of dried root and aerial parts of Medicago spp. on growth of Trichophtyton mentagrophytes. Fungus strains were inoculated onto microcultures with Sabouraud agar supplemented each with 1 g/100 ml of dried and pulverised roots or aerial parts of 3 species: Medicago arabica, M. sativa, and M. murex. The strongest inhibitory effect on T. mentagrophytes growth was recorded for aerial parts of M. arabica (median diameter 6 mm compared to 13 mm of control), followed by root of M. arabica (10 mm) and root of M. murex (10.5 mm) - in all cases p < 0.001. Slight inhibitory effect was also found in the case of aerial parts of M. murex (median diameter 12 mm, p = 0.03). In contrast, M. sativa has shown stimulating effect on growth of T. mentagrophytes (15 mm for root and 16.5 mm for aerial parts, p < 0.001).

Key words: saponins, lucerne, alfalfa, Medicago arabica, Medicago sativa, Medicago murex, antifungal effect, mycoses, fungi, Trichophtyton mentagrophytes, human dermatophyte infections.

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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.

Table 1. Effect of addition of 1 g of pulverised Medicago per 100 ml of Sabouraud medium on growth of dermatophyte Trichophyton mentagrophytes. Numerical values represent diameter of colonies at the end of observation.
  Control Medicago arabica Medicago sativa Medicago murex
root aerial root aerial root aerial
N 44 44 44 44 44 44 44
Max 20.0 12.0 10.0 21.0 23.0 14.0 16.0
Q3 15.0 11.0 7.0 16.0 18.0 12.0 13.0
Median 13.0 10.0 6.0 15.0 16.5 10.5 12.0
Q1 12.0 8.0 6.0 13.0 14.5 9.0 11.0
Min 6.0 7.0 5.0 11.0 12.0 7.0 8.0
p - < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 = 0,03

N - number of cultures, Min - the minimal value observed, Q1 - the 25th percentile, Q3 - the 75th percentile, Max - the maximal value observed. The significance of differences between diameters of colonies grown on Sabouraud agar supplemented with the plant material tested and of control colonies was calculated using Wilcoxon rank test for pairs.


Table 2. Compilation of own results for aerial parts of the 3 Medicago species (first row) with data on inhibition of Trichoderma viride, sapogenin content, and hemolytic activity from the work by JURZYSTA and WALLER (1996).
  M. arabica M. murex M. sativa
Effect on T. mentagrophytes strong inhibition inhibition stimulation
Effect on Trichoderma viride strong inhibition strong inhibition no effect
Medicagenic acid - - ++
Hederagenin ++++ ++++ -
Haemolytic index 1464 1778 150

Semiquantitative scale: not detectable (-), low (+), medium (++), high (+++), and very high (++++) concentration.



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