Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species.

The Balkans endemic species Alkanna primuliflora Griseb., A. stribrnyi Velen., and A. graeca Boiss. & Spruner have limited distribution in the Balkan Peninsula and a large variation in the morphological characteristics. The populations of the three Alkanna species in the Bulgarian flora are small and fragmented. There are no previous reports on the chemical profile or on the embryology of these species. The hypothesis was that the limited distribution of A. primuliflora, A. stribrnyi, and A. graeca was due to their reproductive capacity and genetic diversity. Furthermore, we hypothesized that the three species will contain pyrrolizidine alkaloids (PAs), as other species of the genus Alkanna (Boraginaceae), but they would have differential alkaloids composition. The population genetic structure and differentiation showed a clear distinction between species and revealed average levels of genetic diversity among the natural populations of the three Alkanna species. The embryological investigation observed stability of the processes in the male and female generative spheres and high viability of mature pollen and embryo of the three species. The normal formation of male and female gametophytes without deviations or degenerative processes, and observed levels of genetic diversity between Alkanna individuals are important in maintaining the size and resilience of the Alkanna populations. Eight alkaloids were identified by GC-MS in A. primuliflora and A. graeca and six alkaloids in A. stribrnyi. The main pyrrolizidine alkaloids (PAs) in all investigated species was triangularine. A. primuliflora and A. graeca showed similar chemical composition that comprised 9-angeloylretronecine, 7-tigloylretronecine, 9-tigloylretronecine, triangularicine, dihydroxytriangularine, dihydroxytriangularicine, whereas, in A. stribrnyi 9-tigloylretronecine, triangularicine and dihydroxytriangularicine were not found. This is the first report on the presence of PAs in A. primuliflora, A. stribrnyi and A. graeca. Besides, this is the first report on the embryology of these endemic species. The results contribute to the knowledge of the three endemic Alkanna species and will facilitate policy-making and defining new strategies for their conservation.

Prooxidant and antimicrobic effects of iron and titanium oxide nanoparticles and thalicarpine.

The aim is to evaluate the prooxidant and antimicrobial effects of Fe3O4 and TiO2 nanoparticles and thalicarpine by luminescent and standard microbiological assays. Their effect on the kinetics of free-radical oxidation reactions (at pH 7.4 and pH 8.5) is studied in the following model systems, using activated chemiluminescence: chemical, with Fenton's reagent (H2O2-FeSO4)-for the generation of hydroxyl radicals (.OH); chemical, with oxidant hydrogen peroxide (H2O2); chemical (NAD.H-PhMS), for the generation of superoxide radicals (O2.-). Fe3O4 nanoparticles exhibit highly pronounced antioxidant properties; TiO2 nanoparticles exhibit mild to moderate prooxidant properties at neutral and alkaline conditions. Those properties are tested by the chemiluminescent method for the first time. Thalicarpine and its combination with TiO2 nanoparticles exhibit pronounced antioxidant activities at pH 8.5 which are lost and transformed into well-presented prooxidant effects at pH 7.4. That is a result-supported proof on the observed typical properties of thalicarpine and TiO2, namely antibacterial, organic-preserving and anti-pathogenic activities. The antimicrobial effect is tested on Gram-positive and Gram-negative bacteria: two strains of Escherichia coli, Bacillus cereus 1095 and Staphylococcus aureus. All bacteria are destroyed after the application of TiO2, but not Fe3O4 nanoparticles, showing their antibacterial effect. Thalicarpine, in combination with TiO2, showed even synergetic antibacterial effect.

Hypepontine, a new quaternary alkaloid with antimicrobial properties.

New isoquinoline alkaloid hypepontine (1) together with a five known compounds, were identified in Hypecoum ponticum Velen, the partial synonym of Hypecoum procumbens L. The structure of the new substance was elucidated based on spectroscopic evidence. The tertiary and quaternary alkaloid mixtures as well as the isolated alkaloids were evaluated for their antibacterial and antifungal activity. The result revealed that the crude alkaloid mixture containing quaternary isoquinoline alkaloids showed potent antifungal and antibacterial activity.

Alkaloids from the aerial parts of Leptopyrum fumarioides express immunomodulatory activity.

A new type aporphine-benzylisoquinoline alkaloid leptofumarine (1) was isolated from Leptopyrum fumarioides L. The structure of 1 was elucidated based on spectroscopic evidence. Leptofumarine together with the previously isolated leptopyrine (2) were evaluated for their effect on macrophages and bone marrow (BM) cells in vitro. Both alkaloids inhibited cytokine production by LPS-stimulated peritoneal macrophages. Leptopyrine suppressed osteoclast and osteoblast formation, while leptofumarine affected only osteoblastogenesis.

Alkaloids of Seeds, in vitro Cultivated, and ex vitro Adapted Plants of the Bulgarian Endemic Species Papaver degenii (Papaveraceae).

Papaver degenii (Urum. & Jav.) Kuzmanov is a Bulgarian endemic and glacial relict growing in the alpine zone of Pirin Mountains and it is included in the Red Data Book of Bulgaria. Numerous plants were in vitro multiplied in order to study the alkaloid composition of the species and to test the potential for green biotechnology establishment. Percentages of the crude alkaloid mixture in dried in vitro shoots and ex vitro adapted plants to room phytoiron were similar to that in seeds (0.49%) and 5 to 6 times higher than in the aerial parts of wild-growing plants. Amurensine was the only alkaloid detected in the seeds. Four isoquinoline alkaloids were isolated from the regenerated plants, belonging to two alkaloid types: isopavines (amurensine and Ο-methylthalisopavine) and protopines (allocryptopine and protopine), amurensine being the main alkaloid: 63.4% in in vitro shoots, up to 88.1% in the aerial parts of ex vitro adapted plants. Allocryptopine was in higher amount (30%) in the roots of the ex vitro adapted plants.

Comparative Study of Alkaloid Pattern of Four Bulgarian Fumaria species.

The alkaloid pattern of four Fumaria species (Fumaria kralikii, Fumaria rostellata, Fumaria schleicherii, Fumaria thureii) growing in Bulgaria was investigated by GC-MS and twenty isoquinoline alkaloids were determined. Phytochemical investigation of the alkaloid composition on Fumaria thuretii Boiss was made for the first time. The alkaloid profile of the species was compared at two levels, between different species and within two species from different habitats. Two chemotypical groups, based on the types of isoquinoline alkaloids were suggested. To group A belong species F. kralikii, F. rostellata (F. r. 1) and F. thuretii containing more than 50% spirobenzylisoquinoline alkaloids of the crude alkaloid mixtures. To group B belong species F. rostellata (F. r. 2) and F. schleicherii containing more than 40% protopine alkaloids and relatively high percentage phthaldeisoquinoline alkaloids (11-19%). In group A phthaldeisoquinoline alkaloids were not detected.

Leptopyrine, new alkaloid from Leptopyrum fumarioides L. (Ranunculaceae).

A new type of isoquinoline alkaloid leptopyrine was isolated from the aerial parts of Leptopyrum fumarioides L. (Ranunculaceae) of Mongolian origin. The known alkaloids protopine and thalifoline were isolated for the first time from this the species. All structures were established by physical and spectral analyses.

Alkaloid patterns in some varieties of Datura stramonium.

A comparative GC-MS investigation of the alkaloid patterns of three varieties of Datura stramonium vars. stramonium, tatula and godronii, was carried out. Twenty-five tropane alkaloids were identified in the plant organs. Alkaloid patterns of the roots, leaves and seeds of the varieties grown at equal conditions in Bulgaria were very similar. In contrast, alkaloid pattern of D. stramonium var. stramonium, grown in Egypt, showed significant differences indicating that it is influenced more strongly by the environmental factors than genetic ones.