Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 µM NAA + 5 µM kinetin (ODK3) or 5 µM NAA + 0.5 µM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 µM IBA + 2.5 µM kinetin (ODY1) or 5 µM NAA + 0.5 µM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

Extensive analysis of the cultivated medicinal herbal drug Origanum dictamnus L. and antimicrobial activity of its constituents.

Phytochemical investigations of the methanol extract from Origanum dictamnus L. (Lamiaceae) resulted in the isolation of forty compounds belonging to the classes of terpenes, resorcinol derivatives, flavonoids, depsides, neolignans and jasmonates. Chromatographic isolations were targeted by using two analytical platforms, NMR and HPLC-PDA-MS. In parallel, HPLC-PDA-MS of individual fractions enabled the unambiguous identification of additionally eight components. In total 48 constituents were isolated/identified. Among the isolated constituents are four undescribed compounds, one resorcinol derivative, one monoterpene, one diterpene and one acylated flavonoid glycoside. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, and HPLC-ESI-MS and HRMS experiments. Representative compounds were tested for their antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. I4-II7-dicarvacrol was the most potent constituent.

A Validated Method for the Determination of Carnosic Acid and Carnosol in the Fresh Foliage of Salvia rosmarinus and Salvia officinalis from Greece

In the framework of a project aiming at identifying genotypes of Greek rosemary and sage producing high amounts of carnosic acid, an HPLC-PDA method was developed for the determination of the main antioxidant in the fresh leaves. To this end, an effective and repeatable extraction process of the labile diterpene was developed to ensure a good extraction yield. A fast RP-HPLC protocol was developed and optimized to allow for a short and reliable analysis of the unstable target constituent. The HPLC-PDA method was validated for precision and accuracy according to ICH guidelines. Finally, the overall method was validated for precision and accuracy at three concentration levels. The precision was acceptable with % RSD values ranging between 1.42 and 4.35. The recovery ranged between 85.1% and 104.6% with RSD values < 5%, within the acceptable limits. The developed assay was fast and simple and allowed for the fast and accurate determination of carnosic acid and carnosol in the fresh herbs. The methodology was applied to the quantitative analysis of several cultivated samples of S. rosmarinus and S. officinalis, and some of them were revealed to be promising starting materials for the development of Greek genotypes rich in carnosic acid.

Phenols from Origanum dictamnus L. and Thymus vulgaris L. and their activity against Malassezia globosa carbonic anhydrase.

Malassezia spp. are lipophilic fungi that are part of the normal flora of the human skin and are the etiological agents of dandruff and seborrheic dermatitis. ß-Carbonic Anhydrases (CAs; EC 4.2.1.1) expressed from the pathogenic fungi are an alternative/complementary drug target. Previous work by our groups demonstrated that flavonoids and depsides can effectively inhibit Malassezia globosa ß-CA (MgCA). In continuation of this study herein we report the inhibitory activity of a variety of phenols from Origanum dictamnus L. and Thymus vulgaris L. against ß-MgCA, among them I4-II7-di-carvacrol, a new natural product. Structure elucidation of the compounds was performed by 1 D, 2 D NMR and spectrometric analyses. Xanthomicrol and rosmarinic acid were active in the (sub)micromolar range (KIS 0.6 and 2.2 µM, respectively vs 40.0 µM of the standard inhibitor acetazolamide). Finally, the compounds were not cytotoxic, but showed in vitro no activity against Malassezia furfur.