Planta Med 2015; 81 - PM_235
DOI: 10.1055/s-0035-1565612

LC and LC-MS studies on genetically transformed cultures of Rubia tinctorum cultivated in bioreactor

P Bányai 1, IN Nikolaevna Kuzovkina 2, R Pálkovács 1, M László 3, L Kursinszki 1, É Szőke 1
  • 1Department of Pharmacognosy, Semmelweis University, Budapest, Hungary
  • 2Timiryazev Insitute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia
  • 3Eötvös Loránd University, Faculty of Sciences, Department of Plant Anatomy, Budapest, Hungary

Rubia tinctorum L. (european madder) is a perennial plant from the Rubiaceae family. It is a source of a natural dye; it produces a variety of anthraquinone pigments in its roots and rhizomes. The main components are di- and trihydroxy-anthraquinones, alizarin, and purpurin and their derivatives, ruberythric acid and pseudopurpurin. These substances show antimicrobial and spasmolytic activity and facilitate the loosening of kidney stones. Recent studies indicated that alizarin and purpurin have strong inhibitory effect on the genotoxicity of several carcinogens.

We have investigated the anthraquinone composition of the genetically modified hairy root cultures. Transformed root cultures of R. tinctorum were obtained by their inoculation with Agrobacterium rhizogenes (strain R-1601). After the elimination of bacteria, the hairy roots were cultured on liquid or solid Gamborg B5 and ½NMS media. In order to increase the biomass formation, we cultivated the hairy roots in a Braun Biostat S bioreactor (24 °C, pH 5.7).

For qualitative investigation of the anthraquinones produced by the hairy root cultures, various extracts were analyzed by LC-MS/MS (Agilent Triple Quadropole) method using electrospray ionisation in negative mode. A total of 11 anthraquinones were identified including ruberythric acid, lucidin-primeveroside and pseudopurpurin. For determination of anthraquinone aglycones, MeOH extracts were hydrolysed by refluxing with HCl. The hydrolysates were purified by SPE then investigated by HPLC method (Surveyor System) on Luna C-8 column [1], using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate (pH 3.00) as eluent. The cultures cultivated in bioreactor showed intensive biomass formation (fw: 175 g/3 L medium) and significant biosynthetic capacity (purpurin content: 5.76 mg g-1).

References:

[1] Bányai P, Kuzovkina IN, Kursinszki L, Szőke É. HPLC Analysis of alizarin and purpurin produced by Rubia tinctorum L. Hairy Root Cultures. Chromatographia 2006; 63: 111 – 114