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.

Melissa officinalis L. as a Sprout Suppressor in Solanum tuberosum L. and an Alternative to Synthetic Pesticides.

The goal of this research was to screen plant essential oils (EOs) as sprout inhibitors or suppressors in potato (Solanum tuberosum L.). Three controlled environment experiments were conducted to screen 18 EOs and several pure compounds as sprout inhibitors. The EOs were applied using the wicked method on potato cv. Gala in 19 L plastic containers. The results indicated that Melissa officinalis L. EO inhibited sprouting, while Coriandrum sativum L. seed oil and the EO blend of Lavandula angustifolia Mill. and Salvia sclarea L. suppressed sprouting. The EOs of interest were analyzed using gas chromatography coupled to mass spectrometry (GC-MS) and/or a flame ionization detector (GC-FID); the detailed chemical profiles are provided. The M. officinalis EO was fractionated into seven fractions, and these were tested on minitubers. We identified two fractions (F and A) that suppressed potato sprouting better than the whole oil. The GC-MS-FID analyses of M. officinalis EO fraction A identified myrcene, Z-ocimene, E-ocimene, trans-caryophyllene, and α-humulene as the main constituents, while the main constituents of fraction F were α-terpineol, ß-citronellol, and geraniol. The pure isolated compounds, together with the major compound in M. officinalis EO (citral), were tested for sprout suppression on three potato cultivars (Ranger Russet, Terra Rosa, and Dakota TrailBlazer), which revealed that ß-citronellol reduced the sprout length and the number of sprouts in all three cultivars, while citral and (+)-α-terpineol reduced the sprout length and the number of sprouts in Ranger Russet relative to the two controls in all three cultivars. Myrcene had a stimulating effect on the number of sprouts in Cv. Terra Rosa. However, none of the pure compounds suppressed sprouting completely or were comparable to the EO of M. officinalis.

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.

Biological Activity of Essential Oils of Four Juniper Species and Their Potential as Biopesticides.

The objective of this study was to assess the biological activity of essential oils (EOs) of four Juniperus species obtained via two different distillation methods and their potential as biopesticides. The studied factors were juniper species (Juniperus communis L., J. oxycedrus L., J. pygmaea C. Koch., and J. sibirica Burgsd), plant sex (male (M) and female (F)), and distillation method (hydrodistillation via a standard Clevenger apparatus (ClevA) and semi-commercial (SCom) steam distillation). The hypothesis was that the EO will have differential antioxidant, antimicrobial, and insecticidal activities as a function of plant species, plant sex, and distillation method. The two distillation methods resulted in similar EO composition within a given species. However, there were differences in the EO content (yield) due to the sex of the plant, and also differences in the proportions of some EO components. The concentration of α-pinene, ß-caryophyllene, δ-cadinene and δ-cadinol was dissimilar between the EO of M and F plants within all four species. Additionally, M and F plants of J. pygmaea, and J. sibirica had significantly different concentrations of sabinene within the respective species. The EOs obtained via ClevA extraction showed higher antioxidant capacity within a species compared with those from SCom extraction. All of the tested EOs had significant repellent and insecticidal activity against the two aphid species Rhopalosiphum padi (bird cherry-oat aphid) and Sitobion avenae (English grain aphid) at concentrations of the EO in the solution of 1%, 2.5%, and 5%. The tested EOs demonstrated moderate activity against selected pathogens Fusarium spp., Botrytis cinerea, Colletotrichum spp., Rhizoctonia solani and Cylindrocarpon pauciseptatum. The results demonstrate that the standard ClevA would provide comparable EO content and composition in comparison with SCom steam distillation; however, even slight differences in the EO composition may translate into differential bioactivity.

Essential Oil Composition and Bioactivity of Two Juniper Species from Bulgaria and Slovakia.

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07-0.32% (w/w), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina. Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

Industrial, CBD, and Wild Hemp: How Different Are Their Essential Oil Profile and Antimicrobial Activity?

Hemp (Cannabis sativa L.) is currently one of the most controversial and promising crops. This study compared nine wild hemp (C. sativa spp. spontanea V.) accessions with 13 registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain belonging to C. sativa L. The first three groups had similar main essential oil (EO) constituents, but in different concentrations; the CBD hemp had a different EO profile. The concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: ß-caryophyllene 11-22% and 15.4-29.6%; α-humulene 4.4-7.6% and 5.3-11.9%; caryophyllene oxide 8.6-13.7% and 0.2-31.2%; and humulene epoxide 2, 2.3-5.6% and 1.2-9.5%, respectively. The concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil while CBD in the EO of the registered cultivars varied from 7.1 to 25%; CBD in the EO of the breeding lines and in the CBD strain varied from 6.4 to 25% and 7.4 to 8.8%, respectively. The concentrations of δ9-tetrahydrocannabinol (THC) in the EO of the three groups of hemp were significantly different, with the highest concentration being 3.5%. The EO of wild hemp had greater antimicrobial activity compared with the EO of registered cultivars. This is the first report to show that significant amounts of CBD could be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. The amount of CBD in the EO can be greater than that in the EO of the USA strain used for commercial production of CBD. Furthermore, this is among the first reports that show greater antimicrobial activity of the EO of wild hemp vs. the EO of registered cultivars. The results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the EO and possibly CBD-rich EO production.

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.

Chemical Composition of the Essential Oil of the Endemic Species Micromeria frivaldszkyana (Degen) Velen.

Micromeria frivaldszkyana is an endemic species found only in Bulgaria. Its essential oil (EO) composition is unknown. This study assessed the EO yield and composition of M. frivaldszkyana as a function of the location and of drying prior to the EO extraction. M. frivaldszkyana was sampled from two natural habitats, Uzana and Shipka in the Balkan Mountains; the EO was extracted via hydrodistillation and analyzed on GC/MS. The plants from the two locations had distinct EO composition. The EO content (in dried material) was 0.18% (Uzana) and 0.26% (Shipka). Monoterpene ketones were the major group of the EO constituents. Also, hydrocarbons predominated in the EO from Shipka, and alcohols predominated in the EO from Uzana. The EO from Uzana had a greater concentration of menthone (56% vs. 17% from Shipka) and neomenthol (7.8% vs. 2.4%). Conversely, the EO from Shipka had greater concentrations of pulegone (50% vs. 20% from Uzana), limonene (10.1% vs. 2.6%), and germacrene D (3.4% vs. 1.1%). Drying prior to the EO extraction altered the concentration of some constituents. This is the first report of M. frivaldszkyana EO yield and composition. The EO showed some similarities with the chemical profile of other Micromeria species, but overall, it has an unique chemical profile and may have distinctive applications.