Research Note
Antifungal activity of Rubus ulmifolius Schott standardized in vitro culture

https://doi.org/10.1016/j.lwt.2007.05.012Get rights and content

Abstract

Vegetables of Rubus species are known to have antimicrobial properties mainly due to its high content in phenolic compounds. Among the 37 potentially pathogenic fungi screened with crude methanolic extract from micropropagated shoots of Rubus ulmifolius Schott, we show that the 70% of the species resulted variably sensitive to methanolic extract. Of the phenolic fractions obtained from the crude methanolic extract by hydrophobic interaction chromatography, those rich in tannins resulted active against the fungal strains tested, whereas those containing chlorogenic, caftaric acid and caffeoyl derivatives resulted partially effective. Our results suggest a potential utilization of the in vitro plant cultures of R. ulmifolius to provide standardized extracts from plant material grown in uniform conditions and available year round, which could be useful as natural antifungal agents.

Introduction

Rubus species (family Rosaceae) have been used traditionally for therapeutic purposes (Patel, Rojas-Vera, & Dacke, 2004). Leaves and young shoots of R. ulmifolius Schott are used in Italian folk medicine for their anti-inflammatory, anti-odontalgic and gastrointestinal spasmolytic properties; crushed young shoots are applied to wounds, infected insect bites and pimples (Guarrera, 2005; Uncini Manganelli & Tomei, 1999). It has also been reported that crude methanolic extracts of closely related species of R. ulmifolius Schott exhibit antimicrobial properties on bacteria and fungi (McCutcheon, Ellis, Hancock, & Towers, 1994; Rauha et al., 2000). The antimicrobial activity of extracts of R. ulmifolius of increasing polarity has been reported and crude methanolic extract has shown a wide range of activity against Gram+ and Gram− bacteria and yeasts, while n-hexane, chloroform, chloroform/methanol 9:1 extracts were less active. The maximum activity was exhibited by phenolic and tannin fractions (McCutcheon et al., 1994; Panizzi, Caponi, Catalano, Cioni, & Morelli, 2002).

Aerial parts of some Rubus species containg flavonoids (quercetin, kaempferol, caffeic acid, chlorogenic acid), saturated or unsaturated fatty acids (Tzouwara-Karayanni & Philianos, 1981), have shown high antifungal activity (McCutcheon et al., 1994). Thus, to overcome the difficulties caused by environmental and intrinsic factors which can affect the phytochemical content and availability in plants, we developed micropropagation of R. ulmifolius shoots and investigated the in vitro activity of methanolic extract and its fractions against several human pathogenic fungi. The potential value of these agents could be considered as well as natural antifungal agents for use in food as “secondary preservatives” (Holley & Patel, 2005; Naidu, 2000).

Section snippets

In vitro plant culture

Shoots were induced from nodal explants taken from young branches of Rubus ulmifolius Schott cultured in MS medium (Murashige & Skoog, 1962) supplemented with 3 mg l−1 N6-benzyladenine (BA) and 0.2 mg l−1 naphthaleneacetic acid (NAA) (multiplication phase). Explants were incubated in a growth chamber at 25 °C±2 in the light (cool white fluorescent light at 50 μmol m−2 s−1) under a 16 h photoperiod. The newly formed shoots were dissected and transferred to the same medium supplemented with 0.1 mg l−1 BA to

Results

The susceptibility of the fungi towards various concentrations of the crude extract of Rubus is summarized in Table 1. Results of the disk assay showed that 67.6% of the species (25/37) was variably sensitive in the interval 12.5–50 μl of methanolic extract. In particular, 18.9% of the species showed high sensitivity (IZ>25.0 mm) to 50 μl of the extract; a good sensitivity (IZ between 20.1 and 25.0 mm) was shown by 2.7% of the species at 12.5 μl of extract, by 13.5% at 25 μl and by 24.3% at 50 μl;

Discussion

In this work, we produced micropropagated plantlets of R. ulmifolius Schott and evaluated the antimycotic properties of its methanolic extract. The filamentous fungi tested with the disk diffusion assay showed variable sensitivity to the methanolic extract of R. ulmifolius. The most inhibited species were 5 filamentous fungi as Beauveria sp., Fusarium solani, Microsporum canis, Phialophora verrucosa and Scopulariopsis brevicaulis which are reported to cause mycotic keratitis, endophthalmitis,

Acknowledgements

This work was supported by funds from the Consorzio Interuniversitario per le Biotecnologie. Authors wish to thank Prof. Luisa Pistelli, Università di Pisa for the support in HPLC analysis.

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