HPLC analysis of alizarin and purpurin produced by Rubia tinctorum L. hairy root cultures

We have determined the quantities of the anthraquinones alizarin and purpurin, with especial regard to their effective antigenotoxic activity, in genetically transformed hairy root cultures of Rubia tinctorum L. Hairy roots were cultured on solid and in liquid Gamborg B5 and 1/2 NMS media in a shaking cabinet and in a bioreactor. Methanolic extracts of lyophilized hairy roots were hydrolysed, and then purified by solid phase extraction with good recovery. A new HPLC method was developed for the determination of alizarin and purpurin. The analysis was performed on a Luna C8 RP column using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate-formic acid buffer (pH 3.00) as eluent. Peaks were identified by addition of standards and by diode-array detection. External standardization allowed the determination of alizarin and purpurin with good sensitivity and reliability. The maximum purpurin content was observed in cultures cultivated on solid B5 medium (5.94 mg g(-1)). The highest alizarin content was measured in cultures cultivated on solid 1/2 NMS medium (2.14 mg g(-1))

Flavones in genetically transformed Scutellaria baicalensis roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and H-1 and C-13 NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species. i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones

New terpenoids in cultivated and wild chamomile (in vivo and in vitro)

The effect of Chamomilla recutita (L.) Rauschert is made up by several groups of active substances, among which terpenoids in the inflorescences are of greatest importance. Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its (-)-alpha-bisabolol. Both components have important antiinflammatory activities. Among wild chamomile populations in Hungary, a population was found in the area of Szabadkigyos containing significant amounts-on average 48%-of (-)-alpha-bisabolol in its inflorescence oil. In vitro cultures were made from this population to obtain propagation material containing a high number of active substances. The intact roots contained no (-)-alpha-bisabolol but the sesquiterpene alcohol beta-eudesmol as new compound was identified by our group. Sterile plantlets, cultured in vitro, were multiplied for phytochernical investigations. Pharmacologically important compounds of the essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. These volatile compounds were identified by comparing their retention times with those of authentic standards, essential oils of known composition and peak enrichment. The confirmation of identity was done by comparison of their mass spectra with those reported in the literature and reference compounds. The percentage evaluation of each component was made by area normalisation. Gas chromatography (GC) and mass spectrometry (MS) showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristic of the parent plant. We identified germacrene-D, berkheyaradulene, 4-(2', 4, 4'-trimethyl-bicyclo[4.1.0]hept-2'-en-3'-yl)-3-buten-2-one, geranyl-isovalerate and cedrol as new components in these sterile cultures. (C) 2003 Elsevier B.V. All rights reserved

GC and GC-MS studies on the essential oil and thiophenes from Tagetes patula L.

The production and the composition of the compounds (mono-, sesquiterpenes and acetylenic thiophenes) obtained by the steam distillation of Tagetes patula L. have been investigated. The volatile oil was produced by steam distillation. GC was carried out on three types of stationary phase using flame ionization and mass selective detection. Percentage data were calculated by the area normalization method with very good repeatability (RSD below 5%). Oils from flowerheads were rich in beta-caryophyllene (53.5%) and the leaves contained terpinolene in high concentration (21.1%). The main volatile component of the hairy roots and intact roots was 5(3-buten-1-ynyl)-2,2'-bithienyl (BBT) yielding 28.5% and 44.0% in the oils. Three new minor constituents were identified as alpha-guriunene, beta-caryophyllene and (E)-beta-farnesene. A flash chromatographic method was developed for the isolation of thiophenes from a solvent extract of intact roots. The collected fractions were screened by TLC and analyzed by GC-MS. Three thiophene fractions were obtained containing BBT, 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc) and 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH)