Establishment of hairy root cultures of Salvia corrugata Vahl.

Description

Salvia corrugata Vahl. is an ornamental plant that produces two icetexane diterpene quinones with significant antibacterial activity.1,2 The aim of the study was to obtain transformed roots of this species for the evaluation of their phytochemical profile. Two strains of Agrobacterium rhizogenes (wild type ATCC 15834 and hyper virulent LBA 9402) and one of A. tumefaciens (GV 3101) were tested for their ability to induce hairy root formation on wounded leaves. The best response (57 %) was achieved by infection with ATCC 15834 A. rhizogenes thirty days after the infection onto the hormone-free MS basal solid medium. Two hairy-root clones from ATCC 15834 were established. Transformation was confirmed by polymerase chain reaction analysis of rol c and vir c genes. The evaluation of growth in TIBs showed a best biomass production of clone FA8 on MS0 with 30 mg/L of sucrose. In comparison to other tested media, MS0 and half WPM, both additioned with 30 mg/L of sucrose, were found to be the best for biomass production. As there were many different factors that can support or interfere with the biomass production a statistical analysis has been performed to correctly define the various roles of the server agents uses. A Principal Component Analysis (PCA) was performed to identify the most important factors to be considered for an optimal growth. The methanolic extracts of the biomass (16.8 g) was fractionated by Si gel MPLC eluting with n-hexane/CHCl3/CH3OH at concentrations varying from 100:0:0 to 0:0:100 to obtain 16 fractions. The methanolic extract and the semi-purified fractions were tested against several multidrug resistant clinical strains of various bacterial species (Staphylococcus aureus MRSA, S.epidermidis MRSE, Enterococcus faecalis VRE, E.faecium VRE, Micrococcus luteus, E.coli UTI (i.e. obtained from a human urinary tract infection). The total extract was poorly effective while the semi purified fractions displayed variable potency with MIC values ranging from 8 to >128 μg/mL against the Gram-positive species considered. Fractions 3 and fraction 9 were purified by semi-preparative RP HPLC affording horminone (9.1 mg) and ursolic acid (4.3 mg) respectively. Horminone displayed MIC values ranging from 4 to 64 μg/mL.

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