Water-soluble chitooligosaccharides (COS) elicit xanthone biosynthesis in Hypericum perforatum root cultures

Hypericum perforatum L. (Hypericaceae), popularly known as St. John’s wort, is a medicinal plant widely used in folk medicine since ancient times, with a long list of medicinal uses. Most of the research on St. John’s wort has been focused on the aerial organs of the plant, and only recently the root has been recognized as an attractive source of secondary metabolites of pharmacological interest [1]. The root of H. perforatum contains xanthones, although at very low levels [2;3]. Xanthones are a wide and structurally diverse group of polyphenol with several pharmacological properties [4]. We have recently demonstrated that H. perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. Among elicitors, CHIT is one of the most used to increase the biosynthesis of plant phytoalexins, including xanthones. However, CHIT is insoluble in neutral water as well as in most organic solvents, therefore, it is commonly dissolved in water acidulated with acetic acid [5], which greatly limits its application. Furthermore we recently demonstrated that acetic acid strongly affect xanthone biosynthesis, altering the effect of CHIT [5]. To overcome these problems, in this study, water-soluble oligosaccharides obtained through enzymatic digestion and deacetylation of chitosan have been tested. Initially, chitooligosaccharides (COS) have been administered to the root cultures following the same protocols used for CHIT elicitation. Fifteen days after the addition with COS a 100% increase in the production of total xanthones was obtained (from 4.4 to 9.8 mg/g DW). The most represented xanthones were paxanthone and 5-O-methyl-2 deprenylrheediaxanthone B. Additional experiments were carried out to optimize the elicitation protocol with COS. In particular, it was tested the effect of different concentrations of COS (from 50 to 800 mg/l) and the residence time of the elicitor in the culture medium (from 5 to 25 days). The best results were obtained with 400 mg/l COS and with 25 days of residence time (530% xanthone increase). The antifungal activity of the methanol extracts of roots treated with COS was evaluated on several human pathogenic fungi, including Candida albicans, Microsporum gypseum and Trichophyton mentagrophytes. Several extracts showed interesting MIC values (16-64), suggesting a possible use as antifungal agents. Gene expression studies are in progress to evaluate the contribution of some key genes in polyphenol biosynthesis, such as phenylalanine ammonia lyase (PAL, involved in the biosynthesis of all phenols), chalcone synthase (CHS, involved in both xanthones and flavonoids) and benzefenone synthase (BPS, specifically involved in xanthone biosynthesis). Preliminary results on COS-elicited roots shown a sub-expression of PAL and an overexpression of BPS, while the CHS expression does not shown significant alterations with respect to the control roots.