Metabolite profiling and histochemical localization of alkaloids in Hippeastrum papilio (Ravena) van Scheepen.

Hippeastrum papilio (Amaryllidaceae) is a promising new source of galanthamine - an alkaloid used for the cognitive treatment of Alzheimer's disease. The biosynthesis and accumulation of alkaloids are tissue - and organ-specific. In the present study, histochemical localization of alkaloids in H. papilio's plant organs with Dragendorff's reagent, revealed their presence in all studied samples. Alkaloids were observed in vascular bundles, vacuoles, and intracellular spaces, while in other plant tissues and structures depended on the plant organ. The leaf parenchyma and the vascular bundles were indicated as alkaloid-rich structures which together with the high proportion of alkaloids in the phloem sap (49.3% of the Total Ion Current - TIC, measured by GC-MS) indicates the green tissues as a possible site of galanthamine biosynthesis. The bulbs and roots showed higher alkaloid content compared to the leaf parts. The highest alkaloid content was found in the inner bulb part. GC-MS metabolite profiling of H. papilio's root, bulb, and leaves revealed about 82 metabolites (>0.01% of TIC) in the apolar, polar, and phenolic acid fractions, including organic acids, fatty acids, sterols, sugars, amino acids, free phenolic acids, and conjugated phenolic acids. The most of organic and fatty acids were in the peak part of the root, while the outermost leaf was enriched with sterols. The outer and middle parts of the bulb had the highest amount of saccharides, while the peak part of the middle leaf had most of the amino acids, free and conjugated phenolic acids.

Phytochemical and biological investigations on Centranthus kellereri (Stoj., Stef. & T. Georgiev) Stoj. & Stef. and C. ruber (L.) DC. and their potential as new medicinal and ornamental plants.

INTRODUCTION: Centranthus kellereri is a Bulgarian endemic plant species, found only in two locations in the world: The Balkans Mountains (Stara Planina), above the town of Vratsa, and The Pirin Mountains, above the town of Bansko, Bulgaria. Being endemic and endangered species precluded any significant research on it. The hypothesis of this study was that the populations of C. kellereri may represent genetically, phytochemically, and morphologically distinct forms and these will differentiate from C. ruber. Furthermore, C. kellereri possibly imperfect embryology may preclude its more widespread distribution under natural conditions. RESULTS: This study revealed the phytochemical profile, antioxidant activity, embryology, surface microstructural morphological traits, and genetic differences between the C. kellereri plants from the only two natural populations and compares them to the ones of the related and better-known plant C. ruber. The essential oil (EO) content in aboveground plant parts and in roots was generally low and the EO composition varied significantly as a function of plant part, year of sampling, location, and species. Methylvaleric acid was a major EO constituent in the C. kellereri EO, ranging between 60.2% and 71.7% of the total EO. The EO included monoterpenes, sequiterpenes, long-chain alkanes and fatty acids. Phytochemical analyses of plant tissue revealed the occurrence of 32 compounds that were tentatively identified as 6 simple phenolics, 18 flavonoids, 1 quinone, 1 lipid, 1 alkaloid, 2 diterpenes, and 3 triterpenes. There were differences in detected compounds between the C. kellereri plants at the two locations and between the roots and shoots in both species. The total phenols and flavonoids varied between the two species but were also dissimilar between the plants from the two populations of C. kelleri. Free radical scavenging activity, measured with ABTS and DPPH in aqueous and methanol extracts, had similar values; however, overall, C. kellereri from Vratsa showed the highest antioxidant activity while C. ruber had the lowest activity. Genetic analyses showed a clear differentiation between C. kellereri and C. ruber, and between the two populations of C. kellereri. Embryological studies revealed the peculiarities of the male and female generative spheres of the two species that were defined as being sexually reproducing. The pollen had high viability; however, the low viability of seeds demonstrated possible high sensitivity of C. kellereri to the environmental conditions, perhaps the main factor modifying and restricting the population sizes. The SEM analyses exposed differences in surface microstructural traits between the species (C. kellereri and C. ruber) but also between the two populations of C. kellereri. The observed dissimilarities in genetic makeup, micromorphological characteristics, and phytochemical composition strongly indicate that the two populations can be classified as distinct subspecies or varieties of C. kellereri; var. pirinensis and var. balkanensis. Further research is needed to introduce C. kellereri into culture and develop it as a high-value specialty crop or ornamental in order to conserve C. kellereri natural populations. C. kellereri may be utilized as a source for phytochemicals of interest and as an ornamental plant like C. ruber; however, it may have a greater environmental plasticity and adaptation as evidenced by its current locations.

Artemisia santonicum L. and Artemisia lerchiana Web. Essential Oils and Exudates as Sources of Compounds with Pesticidal Action.

The application of natural products for pest control is important in modern farming. In the present study, Artemisia santonicum L. and Artemisia lerchiana Weber essential oil and exudate profiles were determined, and their potential as inhibitors of seed germination, acetylcholinesterase, and phytopathogenic mycelium growth were evaluated. Essential oils (EO) were obtained via hydrodistillation and exudates (AE) by washing aerial parts of the species with acetone. EO and AE's composition was identified using GC/MS. Eucalyptol (1,8-cineole) and camphor were found to be the main components of A. lerchiana EO, while ß-pinene, trans-pinocarveol, α-pinene, α-terpineol, and spathulenol were established as major compounds of A. santonicum EO. Strong inhibition on Lolium perenne seed germination was found at 2 µL/mL and 5 mg/mL using aqueous solutions of EO and AE, respectively. An inhibitory effect on acetylcholinesterase was established, with an IC50 value of 64.42 and 14.60 µg/mL for EO and 0.961, >1 mg/mL for the AE of A. lerchiana and A. santonicum, respectively. The low inhibition on the mycelium growth of studied phytopathogenic fungi was established by applying 2 µL of EO and 15 µL of 100 mg/mL of AE, with the exception of A. lerchiana AE against Botrytis cinerea. These results show that the studied EO and AE exhibited strong phytotoxic and AChE inhibitory activities, providing new data for these species.

Reproductive Capacity and Scanning Electron Microscopy (SEM) Analyses of the Micromorphological Surfaces of Three Endemic Satureja Species from Bulgaria.

Satureja pilosa Velen., S. coerulea Janka and S. kitaibelii Wierzb. ex Heuff. are Balkan endemic species, and they are distributed in restricted territories, primarily found in dry grasslands, rocky slopes, and stony habitats. This study presents the results of the first embryological and micromorphological analyses of three Satureja species (S. pilosa, S. kitaibelii, and S. coerulea) from the Bulgarian flora. The aim of this study was to establish the features of the male and female reproductive sphere, as well as surface characteristics of leaves, stem, and calyx in order to understand the mode of reproduction, character, size and state of species populations and delimitation. For the embryological study, flowers and flower buds in different developmental stages were collected from plants of natural populations and treated with the classic paraffin method. Reproductive capacity was assessed using the following approaches: (1) acetocarmine test for pollen viability, (2) tetrazolium test (TTZ) for seed viability, and (3) germination test. The surfaces characteristics of leaves, stem, and calyx were analyzed by scanning electron microscopy (SEM). As a result, the study revealed the structures of the male (M) and female (F) generative spheres as well as the processes of gamete development, pollination, and endosperm and embryo formation. It was found that the three Satureja species exhibited a high pollen viability but low seed viability and germination. The SEM analysis showed both common and distinct micromorphology features regarding epidermis, calyx and stem surfaces among the three Satureja species. Notably, the S. coerulea surfaces (leaves, calyx, stem) were clearly distinguishable from the other two Satureja species. Regarding the nutlet surfaces, it was observed that the exocarp surfaces of S. coerulea and S. kitaibelii have a reticulate convex type surface and comprise two types of cells: (1) highly bulging, bubble-like cells; and (2) flat cells with numerous striations. On the other hand, the exocarp of S. pilosa displayed rectangular or polygonal shapes without bubble-like cells, and it had a tabular to slightly convex type surface. Additionally, nutlets (seeds) of both S. coerulea and S. pilosa exhibited distinct papilla formations resembling non-glandular trichomes seen on the ends of the nutlets for S. coerulea and over the entire surface for S. pilosa. The characteristics of the embryological structures and processes, along with the absence of apomixis, characterized the three studied Satureja species as sexually reproducing. The established balanced processes and stable structures contribute to their high reproductive potential and population stability. However, these traits may also decrease their adaptability to environmental changes.

The Establishment of an Ex Situ Collection of Primula veris in Bulgaria.

Primula veris is a valuable medicinal plant species with declining populations, protected in Bulgaria by the Biodiversity Act. The present study aimed to increase its extremely low seed germination rate, starting with seeds originating from two Bulgarian populations, and to set up an ex situ field collection. The stimulation effect of three factors was tested in in vivo and in vitro experiments: seeds treated with gibberellic acid (in different concentrations and exposure time), light quality (white, infrared, red, and blue or dark), and cold stratification. The combination of factors resulted in 36 treatment variants in vivo and 8 treatment variants in vitro. No germination was observed in control treatment variants. The highest germinating rate (95%) was noticed in vivo under blue monochromatic light after seed soaking into 0.2% GA3 for 10 h; however, the best results (55% of well-developed seedlings) were observed with a combination of blue light and 0.3% GA3 for 5 h. Seedlings were successfully strengthened in vermiculite in a phytotron, potted in soil and grown in a greenhouse, and then 75 plants were transferred to the field plot, where most of them bloomed at the first vegetation season. These results are intended to serve as a basis for establishing a pilot agriculture of the species.

Chemical Profile and Antimicrobial Activity of the Essential Oils of Helichrysum arenarium (L.) Moench. and Helichrysum italicum (Roth.) G. Don.

This study compared the essential oils (EO) composition of Helichrysum arenarium (Bulgarian populations) with that of the cultivated species H. italicum. The EO composition of H. arenarium and H. italicum were analyzed via gas chromatography. In general, 75 components were identified in H. arenarium EO and 79 in H. italicum EO. The predominant constituents in H. arenarium EO were α-pinene (34.64-44.35%) and sabinene (10.63-11.1%), which affirmed the examined population as a new chemical type. Overall, the main EO constituents of H. italicum originating in France, Bosnia and Corsica were neryl acetate (4.04-14.87%) and ß-himachalene (9.9-10.99%). However, the EOs profile of H. italicum introduced from the above three countries differed to some extent. D-limonene (5.23%), italicene, α-guaiene and neryl acetate (14.87%) predominated in the H. italicum introduced from France, while α-pinene (13.74%), δ-cadinene (5.51%), α-cadinene (3.3%), ß-caryophyllene (3.65%) and α-calacorene (1.63%) predominated in plants introduced from Bosnia. The EOs of the plants introduced from France and Corsica had similar chemical composition and antimicrobiological activity.

Metabolic Profiles, Genetic Diversity, and Genome Size of Bulgarian Population of Alkanna tinctoria.

Alkanna tinctoria (L.) Tausch Boraginaceae is a medicinal plant whose root is used for its antimicrobial and anti-inflammatory properties. A. tinctoria roots have been subject to numerous studies. However, the aerial parts have been explored less. The objective of the present study was to compare the chemical profile of aerial parts and roots as well as the total alkannin content in roots of 11 populations of the species from different floristic regions of Bulgaria. Methanolic extracts from 22 samples were analyzed by GC/MS. Phenolic, fatty, and organic acids, sterols, polyols, fatty alcohols, and sugars were identified. Ononitol (4-O-methyl-myo-inositol) was found as the main compound in the aerial parts. The total alkannin content in the roots was evaluated by the spectrophotometric method and compared with that of the commercial product. Populations with high alkannin content and rich in other bioactive compounds were identified. A relatively low genetic diversity in the studied populations was observed. The present study is the first comprehensive study on metabolite profiles and genetic diversity of the Bulgarian populations of A. tinctoria. The occurrence of ononitol in the aerial parts of the species is reported for the first time, as well as the phenolic acid profiles of the species in both aerial parts and roots. The results showed that aerial parts of the plant are also promising for use as a source of valuable biologically active substances.

On the Reproductive Potential in Primula veris L. (Primulaceae): Embryological Features, Pollen and Seed Viability, Genetic Diversity.

Primula veris (Primulaceae) is a valuable medicinal plant. The main characteristics for assessing the reproductive potential (embryological features; mode of reproduction; pollen and seed viability) and the genetic diversity of populations of the species from Bulgaria were studied. The anthers are tetrasporangiate. Their wall development follows the Dicotyledonous-type and consists of: epidermis, a fibrous endothecium, an ephemeral middle layer and a secretory (glandular) tapetum. After meiosis in pollen mother cells and simultaneous microsporogenesis tetrahedral tetrads are formed predominantly in the anthers. Many ovules (approximately 20) develop in the unilocular ovary and are anatropous, tenuinucellate and bitegmic. The embryo sac (ES) develops after Polygonum (monosporic)-type from the chalazal cell of linear megaspore tetrad in the ovule. After double fertilization, a Caryophyllad-type embryo and initially nuclear endosperm form. In the studied populations, high pollen viability of more than 95% was established. Extremely low viability (about 4%) of the seeds obtained from natural populations was established. The results reveal P. veris to be a predominantly amphimictic (sexually reproducing) species, although rare vegetative propagation is also observed. As a result of this study, essential data were obtained about the reproductive structures and processes and for assessing the reproductive potential of P. veris.

Phytochemical Investigation and Reproductive Capacity of the Bulgarian Endemic Plant Species Marrubium friwaldskyanum Boiss. (Lamiaceae).

Marrubium friwaldskyanum Boiss (Lamiaceae) is a Bulgarian endemic species. Overall, the essential oil (EO) composition of M. friwaldskyanum was different from that of the other Marrubium species reported in the literature. The main EO constituents of M. friwaldskyanum were (E)-caryophyllene, germacrene D, and caryophyllene oxide. The effect of the harvest stage was significant only on α-copaene, (E)-caryophyllene, caryophyllene oxide, and τ-muurolol. The concentration of α-copaene (1.26-1.83% range of the total oil), (E)-caryophyllene (31-41%), caryophyllene oxide (6.4-11.8%), and τ-muurolol (1.3-2.8%) were the highest at 2-3 pair of leaves or before flowering and lower at flowering. The harvest stage did not significantly affect the concentrations of the other six identified EO compounds ß-bourbonene (1.1%), α-humulene (2.8%), germacrene D (23.3%), bicyclogermacrene (2.85%), δ-cadinene (1.1%), and spathulenol (2.8%). In a separate experiment, grinding of the biomass prior to EO extraction had a significant effect only on the concentrations of D-limonene (0.24-3.3%) and bicyclogermacrene (3.6-9.1%). Grinding in water or without water, maceration, and addition of Tween®20 had rather small effects on the EO profile. The identified EO constituents and their mean concentrations in this experiment were (E)-caryophyllene (25.4%), germacrene D (17.6%), caryophyllene oxide (9.1%), spathulenol (6.5%), τ-muurolol (5.0%), carvacrol (3.9%), α-copaene (2.5%), ß-bourbonene (2.5%), δ-cadinene (2.4%), α-humulene (1.8%), and Z-ß-farnesene (1.3%). Embryological studies observed anther and the development of the male gametophyte and ovule and development of the female gametophyte of M. friwaldskyanum. Furthermore, pollen and seed viability assays were conducted, and mass spectrometry-based metabolomics analysis of an extract from shoots revealed the presence of 45 natural products, identified as flavonoids, phenolic acids, and (tri)terpenoids. Overall, the phytochemistry and some of the microscopic analyses distinguished this endemic species from other species in Marrubium.

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.

Study on Galanthus species in the Bulgarian flora.

Genus Galanthus (Amaryllidaceae) includes 19 species in Europe and the Middle East. The Flora of Bulgaria recognizes two species: G. nivalis L. and G. elwesii Hook. Galanthus elwesii is characterized by relatively high morphological variability, leading some authors to identify some populations as G. gracilis Celak. However, the occurrence of G. gracilis in the Bulgarian flora is disputed. The hypothesis was that populations previously identified as G. gracilis belong indeed to a separate species. Therefore, the objective of this study was to compare G. nivalis and G. elwesii with plants from populations previously identified as G. gracilis. Morphological, DNA, embryological and anatomical analyzes were conducted to meet the objective. The morphological characteristics and DNA dendrogram revealed that G. gracilis and G. elwesii were situated in the same cluster and had significant morphological similarity, whereas plants from populations identified as G. nivalis were dissimilar in morphology and situated in a separate cluster. The revealed features of the generative sphere showed similarities across the species. Scanning electron microscopy (SEM) analyses of the surface revealed that the anticillinal walls of G. elwesii and G. gracilis were straight, while those of G. nivalis were wavy. This research demonstrated that the plants of G. elwesii and those from populations identified as G. gracilis are morphologically, embryologically and genetically similar, thus refuting the hypothesis. This study did not provide sufficient evidence to support the claim of the existence of G. gracilis in the Bulgarian flora; the populations identified as G. gracilis in Bulgaria may be forms of G. elwesii.