Studies on the alkaloid production of genetically transformed and non-transformed cultures of Lobelia inflata L.

The investigations of the growth and alkaloid production of cell suspension-, callus-, organized- and hairy root cultures from Lobelia inflata L. proved that these cultures are able to synthesize the characteristic piperidine alkaloids of the intact plant. Alkaloid precursor amino acids (Phe, Lys) and plant growth regulators affect not only the growth and differentiation of tissue cultures but also their secondary metabolism. The synthetic regulator Sz/I I combined with Phe increased the total alkaloid content considerably in callus- and organized cultures; regulator Sz/28 especially increased the lobeline content (in organized cultures in response to Lys, in callus tissues as a result of Phe application). With the aim of optimizing growth and alkaloid production of the genetically transformed hairy root cultures of Lobelia inflata L. we studied the effect of some growth regulators (NAA, IAA, kinetin) and precursor amino acids (Lys, Phe). The kinetin had inhibiting effect on the growth and lobeline production of the hairy roots. The IAA and NAA increased the biomass formation and lobeline production. The highest lobeline level was detected in tissues cultivated on hormone-free medium containing Phe. &nbsp

FACTORS INFLUENCING THE PHARMACEUTICALLY IMPORTANT CHARACTERISTICS OF LOBELIA INFLATA L.

Lobelia inflata L. (Lobeliaceae) contains pharmacologically important secondary metabolites, which consist of more than 20 piperidine alkaloids. Lobeline with its stimulatory effect on the respiratory centre is used in anti-smoking preparations. The pharmaceutical importance could be improved by the polyacetylenes (lobetyol, lobetyolin, lobetyolinin). The main goal of the experiments: to introduce the species into field production in Hungary and to identify the factors influencing its pharmaceutically important characteristics. Plantlets were propagated and raised both in vitro and in vivo. After acclimatization in a glass-house they were transplanted to irrigated open field conditions at 4 Hungarian locations. In vitro organized cultures produced intensive growth and considerable secondary metabolites. In contrast to lobetyolin (polyacetylene), the alkaloid (lobeline) content decreased in the open field. The effect of nutrient supply was also considered. Phytochemical analyses revealed differences in the total alkaloid content, both in view of the geographic region of cultures and the propagation technology. The highest alkaloid and polyacetylene contents were detected in the population grown at Mosonmagyaróvár. Lobetyolin content exceeded in all samples the content of lobetyolinin. No similar differences were observed with lobeline. Field grown plants contained sufficient amounts of active principles. The nearly 1 t/ha biomass yield and active substance production could be increased by higher plant density and fertilization. N-fertilization increased the above ground phytomass, due to the increased number of side-branches. Dry mass was highest under the influence of MgSO4 (2%) foliar spraying. A similarly positive influence was detected both in the total alkaloid production and the lobeline content. As a contrast, a decrease in the total alkaloid content was observed with the doubling of both polyacetylenes. Acknowledgements: GVOP 3.1.1.-0309/

Improved RP-HPLC method for analysis of isoquinoline alkaloids in extracts of Chelidonium majus

A new high-performance liquid chromatographic method has been developed for analysis of isoquinoline alkaloids in extracts of Chelidonium majus L. Samples were extracted with acidic methanol and the extracts were purified by solid-phase extraction on Supelclean LC-18 cartridges. Optimized conditions resulted in high recovery and reproducibility. Simultaneous determination of protopine, chelidonine, coptisine, sanguinarine, and berberine was performed by HPLC on a C-18 reversed-phase column. Use of the Luna C18(2) new-generation silica-based stationary phase and 14.7:18:67.3 (v/v) acetonitrile-methanol-30 mM ammonium formate, pH 2.80, as mobile phase resulted in excellent peak shapes. Validation proved the repeatability of the method was good and recovery was satisfactory. Lower limits of detection were 0.2 ng for coptisine, 0.4 ng for sanguinarine, and 0.5 ng, for protopine, chelidonine, and berberine

Antioxidant activity of medicinal plants used in phytotherapy

Oxygen free radicals play an important role in the development of different disorders like inflammatory-immune injury, carcinogenesis, hepatic toxicity and artherosclerosis. The antioxydant role of a wide spectrum of natural products has been established. Flavonoids and other phenolic compounds (proanthocyanidins, rosmarinic acid, hydroxicinnamic derivatives, catechines, etc.) of plant origin have been reported as scavengers and inhibitors of lipid peroxidation. We have studied the antioxidant activity as well as content and composition of natural phenolics in a series of medicinal plants with phytotherapeutical significance. Thus we determined the total phenol contents and studied the composition of flavonoids, polyphenols, phenolic acids of different vegetative and reproductive organs of medicinal plants: Anthriscus cerefolium (L.) Hoffm., Petroselinum crispum L., Cichorium intybus L., Helichrysum arenarium D.C.„cempervivum tectorum L., Taravacum officinale Web. Characteristic constituents in the various crude drugs were determined by chromatographic (TLC, HPLC) and spectroscopic (UV, UV-VIS) methods. The non specific scavenger activities of the medicinal plant extracts were studied by the chemiluminometric technique. The changes of chemiluminescence intensity of the H,G,•0H-luminol system at increasing concentrations of the H702/ -OH were measured. Inhibitory effects of selected standardized fractions from plants were tested on ascorbic acid induced lipid peroxidation in rat liver and homogenates. The best correlation were established with total phenolics in some medicinal plants (S. tectorum, T. officinale) while activities in other cases seem to be influenced by flavonoids (P. crispum, H. arenarium, A. cerefolium) and by hydroxicinnamic derivatives (C. intybus). &nbsp

Antioxidant activity of medicinal plants used in phytotherapy

Oxygen free radicals play an important role in the development of different disorders like inflammatory-immune injury, carcinogenesis, hepatic toxicity and artherosclerosis. The antioxydant role of a wide spectrum of natural products has been established. Flavonoids and other phenolic compounds (proanthocyanidins, rosmarinic acid, hydroxicinnamic derivatives, catechines, etc.) of plant origin have been reported as scavengers and inhibitors of lipid peroxidation. We have studied the antioxidant activity as well as content and composition of natural phenolics in a series of medicinal plants with phytotherapeutical significance. Thus we determined the total phenol contents and studied the composition of flavonoids, polyphenols, phenolic acids of different vegetative and reproductive organs of medicinal plants: Anthriscus cerefolium (L.) Hoffm., Petroselinum crispum L., Cichorium intybus L., Helichrysum arenarium D.C.„cempervivum tectorum L., Taravacum officinale Web. Characteristic constituents in the various crude drugs were determined by chromatographic (TLC, HPLC) and spectroscopic (UV, UV-VIS) methods. The non specific scavenger activities of the medicinal plant extracts were studied by the chemiluminometric technique. The changes of chemiluminescence intensity of the H,G,•0H-luminol system at increasing concentrations of the H702/ -OH were measured. Inhibitory effects of selected standardized fractions from plants were tested on ascorbic acid induced lipid peroxidation in rat liver and homogenates. The best correlation were established with total phenolics in some medicinal plants (S. tectorum, T. officinale) while activities in other cases seem to be influenced by flavonoids (P. crispum, H. arenarium, A. cerefolium) and by hydroxicinnamic derivatives (C. intybus). &nbsp

Investigation of tropane alkaloids in genetically transformed Atropa belladonna L. cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform - methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile - 30 mM phosphate buffer - methanol 12.2:79.7:8.1 (v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K-5 (0,223 m/m%) and in hairy root clone #K-4 (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants