Functional Analysis of Genes GlaDFR1 and GlaDFR2 Encoding Dihydroflavonol 4-Reductase (DFR) in Gentiana lutea L. Var. Aurantiaca (M. Laínz) M. Laínz

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

The assessment of the current status of Gentiana lutea L. populations of the Ukrainian Carpathians: Ecological and genetic approaches

Based on the analysis of the genetic polymorphisms (RGAP- and CDDP-PCR) among populations and comparison of these results with ecological characteristics (density, area, spatial and vitality structure, age, ability of renewal and self-maintenance), the status of five natural (Lemska, Gutyn Tomnatyk, Sheshul-Pavlyk, Krachuneska, Troyaska-Tataruka) and one man-made (Pozhyzhevska) populations of Gentiana lutea from the Ukrainian Carpathians was assessed. The results of the complex ecological and genetic analysis have revealed that three populations are unstable (Krachuneska, Troyaska-Tataruka, Gutyn Tomnatyk), two are relatively stable (Lemska, Pozhyzhevska) and only one is stable (Sheshul-Pavlyk). The research results can be used for stabilizing the number of violations and restoring the endangered natural populations. Based on these data, the recommendations for the conservation and protection of G. lutea populations have been developed.

Cuckoo male bumblebees perform slower and longer flower visits than free-living male and worker bumblebees

Cuckoo bumblebees are a monophyletic group within the genus Bombus and social parasites of free-living bumblebees, upon which they rely to rear their offspring. Cuckoo bumblebees lack the worker caste and visit flowers primarily for their own sustenance and do not collect pollen. Although different flower-visiting behaviours can be expected between cuckoo and free-living bumblebees due to different biological constraints, no study has yet quantified such differences. Here, we provide the first empirical evidence of different flower-visiting behaviours between cuckoo and free-living bumblebees. We recorded the flower-visiting behaviour of 350 individual bumblebees over two years in a wild population of the entomophilous plant Gentiana lutea, of which they are among the main pollinators. In cuckoo bumblebees (28.9% of the total), we only found males, while we found both workers and males in free-living bumblebees. Cuckoo bumblebees visited significantly more flowers for longer time periods than both free-living bumblebee workers and males within whorls, while differences at the whole-plant level were less marked. Free-living bumblebee males visited more flowers and performed slightly longer flower visits than workers. Behavioural differences between cuckoo male bumblebees and free-living bumblebee workers are likely related to different foraging needs, while differences between cuckoo and free-living bumblebee males may be caused by differences in colony development and a delayed mating period of free-living bumblebees. The longer visits made by cuckoo male bumblebees will likely negatively affect plant reproductive success through increased within-plant pollen flow.

Biological Profile of Two Gentiana lutea L. Metabolites Using Computational Approaches and In Vitro Tests

Natural products have been the main source of bioactive molecules for centuries. We tested the biological profile of two metabolites extracted from Gentiana lutea L. by means of computational techniques and in vitro assays. The two molecules (loganic acid and gentiopicroside) were tested in silico using an innovative technique, named Inverse Virtual Screening (IVS), to highlight putative partners among a panel of proteins involved in inflammation and cancer events. A positive binding with cyclooxygenase-2 (COX-2), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein emerged from the computational experiments and the outcomes from the promising interaction with COX-2 were confirmed by Western blot, highlighting the reliability of IVS in the field of the natural products.

Antioxidative properties and antigenotoxic potential of Gentiana lutea extracts against the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine, PhIP

Lipid oxidation that occurs in different types of food can cause alterations in nutritional qualities, flavour, texture and shelf life of foods. Furthermore, high temperature cooking of protein-rich food can lead to formation of heterocyclic aromatic amines capable of compromising the integrity of DNA molecules. To reduce these harmful effects, research has been focused on investigating plants as a source of potential natural food additives and preservatives. Thus, the aim of this study was to estimate antioxidant and antigenotoxic activities of 50% ethanolic-aqueous root and leaf extracts of the medicinal plant, Gentiana lutea. Antioxidative effect was investigated using the DPPH assay, while antigenotoxicity against the mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined using Salmonella Typhimurium TA 1535 in the SOS/umuC assay. Leaf extract showed high antioxidative effect with the ability to neutralize up to 87% of free radicals at 400 µg mL-1. Antigenotoxicity testing revealed that both extracts exhibited remarkable genoprotective activity against PhIP-induced DNA damage, with the highest inhibition levels being 70% and 85% for root and leaf extracts, respectively. Results obtained are encouraging and suggest further research of G. lutea extracts as potential food preservatives and additives in improving food quality and human health.

Can yellow gentian (Gentiana lutea) be useful in protection against foodborne mutagens and food contaminants?

Gentiana lutea is a wellknown and respected medicinal plant that is used in many pharmacopoeias, mainly against different gastrointestinal disorders. The plant is under protection regimes in its natural habitats and for that reason is grown on plantations. In addition, it could be cultivated in vitro. The plants grown on plantation and in in vitro conditions were used to prepare methanolic and 50% ethanolic extracts of root and leaf/shoot, which were tested for antigenotoxic and antibacterial properties, against foodborne mutagens (heterocyclic aromatic amines PhIP and IQ) and food contaminants, respectively. The results obtained pointed out the excellent genoprotective effect (up to 78% inhibition of PhIP/IQ genotoxicity) based mostly on the antioxidative potential. The antibacterial effect was mainly weak; only the extracts of in vitro grown plant induced moderate activity against Listeria monocytogenes and Staphylococcus aureus (MICs ranged 0.15-5 mg/ml). In addition, the extracts’ potential to prevent biofilm formation by L. monocytogenes was very high (up to 90% inhibition). Taken together, the results obtained encourage further research that would be directed to the formulation of potent antigenotoxic and antibiofilm agents based on G. lutea.

Dynamics of growth parameters of Gentiana lutea L. in vitro plants under different lighting conditions

Aim. Study of the dynamics Gentiana lutea L. plant growth processes in vitro depending on the light regime changes of their cultivation in order to develop a scheme to increase their adaptive potential. Methods. Methods of plant cultivation in vitro, biometric method, as well as ANOVA variance analysis and middle group analysis in pairs using the Tukey test (Tukey test) were used. Results. It is shown that the cultivation of G. lutea plants in vitro using 25 W/m2 light flux intensity in the region of photosynthetically active radiation and the ratio of blue (Eb): green (Eg): red (Er) ranges = 41.8%: 42.7 %: 15.5% triggers non-specific photomorphogenesis reactions for intact plants, which lead to poor root system development, stem elongation, formation of small leaves with a thin leaf blade, overall low productivity and low adaptation potential of G. lutea plants to ex vitro and in situ conditions. Increasing the light flux intensity to 44 W/m2 and increasing the red wave proportion up to 20.3% allows not only to improve the bioproductivity of G. lutea plants which are cultivated in vitro, but also to increase the coefficient of microclonal reproduction without the additional use of exogenous growth regulators. The greatest growth of the aboveground and underground parts, increase in effective leaf surface are observed in vitro plants during cultivation at 135 W/m2 light flux intensity and spectral composition Eb: Eg: Er = 29.5%: 32.5%: 38.0%. Conclusions. It is possible to improve plant bioproductivity by changing the light conditions of plant cultivation in vitro, and, accordingly, to increase the adaptive potential to ex vitro and in situ conditions. Keywords: Gentiana lutea L., in vitro plants, light flux intensity, spectral composition, growth parameters.

A Process of Electrohomeopathic remedy “SLASS” Preparation, Compared with Krauss Method By phytochemical Analysis, TLC and FTIR Studies

Electrohomeopathy medicines have been practicing since the 1850s in European countries and from the 1870s in India. There are various methods of preparation of Electrohomeopathic medicines that have been developed and practice by the different manufacturers. For the safety, efficacy, and stability of the medicines, there is a need for scientific research to standardize the procedure of medical preparations. SLASS is one of the commonly using remedy by the electro homeopathy practitioners for various disorders concern to the digestive system, excretory system and nervous system etc... In the present studies we attempted a comparative study of a Process of Electrohomeopathic remedy “SLASS” Preparation, Compared with the Krauss method by phytochemical analysis, TLC, and FTIR Studies. Our process and Krauss method showed significant differences in phytochemical profile TLC and FTIR spectral peaks. The yield of the extracts of Aloe capensis and Gentiana lutea are more in our method compared to Krauss method. Glycoside is present in Krauss method. These research outcomes with reproducibility will become standard markers or signatures to assess the quality, safety, and efficacy of the electro homeopathy medicine as well as reduce the risk of adulterations helps to develop specifications of the Electrohomeopathy remedies. Future work is essential to explore its therapeutic applications