Proanthocyanidins in Rhodiola kirilowii and Rhodiola rosea callus tissues and transformed roots -determination with UPLC-MS/MS method

S u m m a r y Several species of Rhodiola genus (Crassulaceae family) like Rhodiola kirilowii and Rhodiola rosea are used in official or traditional medicine. The aim of this study was to determine qualitative and quantitative content of proanthocyanidins using ultra performance liquid chromatograph connected to a tandem mass spectrometer (UPLC MS/MS method) in the callus tissues and in the transformed roots (infected by Agrobacterium rhizogenes LBA 9402 strain) of R. kirilowii and R. rosea. This validated assay allows to determine the content of five flavan-3-ols: (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG). Our results concerning the material from in vitro cultivation of R. kirilowii and R. rosea indicate that R. rosea callus can be a better source of catechin when compared with other tested materials, especially when the content of (-)-gallate epigallocatechin is taken under consideration (3.429 mg/100 g of dry powdered material). The application of UPLC MS/MS method allowed to determine the content of proanthocyanidins in tested samples with satisfactory precision and can be used in the phytochemical investigations of Rhodiola sp. in vitro cultivated tissues. Key words: Rhodiola kirilowii, Rhodiola rosea, callus tissues, transformed roots, proanthcyanidins, The mentioned proanthocyanidins (flavan-3-ols), like catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, exhibit antioxidative properties and can protect the organism against harmful effect of free radicals and influenced reactive oxygen forms The in vivo and in vitro cultivation of Rhodiola species are carried out in the Institute of Natural Fibres and Medicinal Plants in Poznan and in the Department of Biology and Pharmaceutical Botany, Warsaw Medical University for several years. In some previously published articles we presented the presence of proanthocyanidins in R. rosea and R. kirilowii roots and callus tissues MATERIAL AND METHODS Plant material Investigations were carried out on five kind of plant material: Rhodiola kirilowii and Rhodiola rosea roots from field cultivation, Rhodiola kirilowii callus tissues, Rhodiola rosea callus tissues and Rhodiola kirilowii transformed roots. Roots from field cultivation The roots of R. kirilowii Callus tissues The callus tissues cultured on solid medium were used in the experiments. Plant material originated from the Garden of Medicinal Plants, Institute of Natural Fibres and Medicinal Plants, Poznań. The callus of R. kirilowii was obtained from the cotyledone of sterile seedling; the callus of R. rosea was obtained from hypocotyl of the sterile seedling. Tissues were cultivated on the modificated Murashige-Skoog (MS) medium Hairy root culture Hairy root culture of Rhodiola kirilowii, established in the Department of Biology and Pharmaceutical Botany, Warsaw Medical University, was derived from a single root developed at the wounded site of the internodal shoot segment, as it was described previously [23], and with the addition of 500 mg/l -1 of L-glutamine. The culture was maintained at 25°C in the dark on a GioGyrotorys Shaker (New Brunswick Scientific Co.) at 122 rpm. The used hairy root line has been investigated for integration of the bacteria DNA into the R. kirilowii genome, as described by Zych et al. using PCR reaction [21]. The content of secondary metabolites was determined in powdered lyophilized tissue of transformed roots. Standard substances The following comparison substances were used in the experiment: (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate (ChromaDex) and D-(-)-salicine (SIGMA). Preparation of test samples: extraction of flavan-3-ols (proanthocyanidins) from dry plant materials The method of flavan-3-ol extraction by P. Mammela roots, an exact amount of ca. 0.75 g of dried powdered (0.315) R. rosea roots, an exact amount of ca. 2.5 g of dried powdered (0.315) R. kirilowii or R. rosea callus tissue and an exact amount of ca. 0.25 g of dried powdered (0.315) R. kirilowii transformed roots were weighed out and placed in a 20 ml volumetric flasks. Methanol in the amount of 15.0 ml of 80% (v/v) was added and the solutions were subjected to ultrasounds for 60 min at a room temperature (20-25ºC). Then the solutions were made up to the mark with the same solvent and filtered on a quantitative filter paper. The filtrates were concentrated to evaporate the methanol up to a volume of about 1/5 in a rotary evaporator in vacuum. The residues were extracted with 4 × 16.0 ml of diethyl ether. The combined ether extracts were dried with anhydrous sodium sulphate and evaporated to dryness in a rotary evaporator in vacuum. The dry residues were dissolved in 4.0 ml of 10% (v/v) methanol and then quantitatively transferred to proper volumetric flasks (in the case of callus tissues 2 ml volumetric flasks were used; in the case of roots or transformed roots 5 ml volumetric flask was used). D-(-)-salicine (IS) in amount of 0.023 ml of 0.5 mg/ml was added to every flasks and the solutions were made up to the mark with 10% (v/v) methanol. The samples were filtered through a membrane filter with a diameter of 0.20 μm