Using high performance thin layer chromatography for the detection of pharmacologically active secondary metabolites in Empetrum nigrum L.

Introduction. The article presents the results of the detection of pharmacologically active secondary metabolites in black crowberry Empetrum nigrum L. using the method of high performance thin layer chromatography (HPTLC).Aim. To show the efficiency of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites in promising medicinal plant species.Materials and methods. HPTLC analysis was carried out on a CAMAG device (Switzerland), using MERCK HPTLC silica gel 60 F154, 20 × 10 cm plates. For the evaporation of the samples, a Heidolph vacuum rotary evaporator (Germany) was used. The aerial parts (shoots) of Empetrum nigrum were harvested next to St. Petersburg State Chemical and Pharmaceutical University (SPCPU) nursery garden of medicinal plants (Leningrad Region, Vsevolozhsky District, Priozerskoe Highway, 38 km) in August 2019.Results and discussion. In the course of the research, four fractions from the aerial parts of Empetrum nigrum were obtained: hexane, dichloromethane, butanol, and water. Then, these fractions were investigated by HPTLC in two solvent systems – n-butanol : acetic acid : water (BAW) (4 : 1 : 2) and hexane : dichloromethane : methanol (HDM) (1 : 2 : 0.5). After scanning densitometric analysis of the plates eluted in the HDM system, it was revealed, that the hexane and dichloromethane fractions have a similar composition and contain the greatest amount of compounds, compared to the butanol and water fractions, and in the BAW system, it was found, that the butanol fraction contains the greatest variety of metabolites. As a result of UV spectroscopy, it was found, that the main groups of compounds contained in the hexane and dichloromethane fractions are derivatives of chalcones, dihydrochalcones, bibenzyls and 9,10-dihydrophenanthrenes. While in the butanol fraction, the main groups of secondary metabolites were derivatives of flavonoids and tanninsConclusion. The data obtained allow us to note the efficiency, speed and simplicity of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites of promising medicinal plant species.

Comparative phytochemical analysis of the aerial parts of Empetrum nigrum L. samples, collected in various regions of the Russian Federation

Introduction. A number of studies have shown that various genetic and environmental factors can affect the biosynthesis and accumulation of secondary metabolites. In particular, it is known that the local geoclimate, seasonal changes, external conditions such as light, temperature, moisture and soil fertility can affect the chemical composition and, as a result, the therapeutic properties of plants used in the pharmaceutical and food industries. Empetrum nigrum L. is a rich source of various pharmacologically active secondary metabolites – chalcones, dihydrochalcones, bibenzyls, 9,10-dihydrophenanthrenes, flavonoids, and proanthocyanidins. In the scientific literature, there is no data on the variation in the chemical composition of E. nigrum depending on the growing area. The obtained data are necessary for a reasonable choice of the collecting location for the plant, with the aim of its further chemical and pharmacological research for the development of promising drug candidates.Aim. To carry out a comparative analysis of secondary metabolites composition in the aerial parts of Empetrum nigrum growing in different regions of the Russian Federation.Materials and methods. Samples collected in three different areas were used to compare HPLC profiles: sample 1 was collected next to SPCPU nursery garden of medicinal plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km) in July 2020, sample 2 was collected on the Kola Peninsula, around the Khibiny mountains in July 2020, sample 3 was collected on the Kamchatka Peninsula, next to Khalaktyrsky beach (Pacific Ocean coast) in July 2020. Extracts were analyzed by analytical high performance liquid chromatography (HPLC) using a Prominence LC-20 device (Shimadzu, Japan) equipped with a diode array detector.Results and discussion. As a result of the research, for the first time, a significant variation in the qualitative chemical composition in the aerial parts of Empetrum nigrum growing in different regions of Russian Federation was established. Sample 3, collected on the Kamchatka Peninsula, in comparison with samples 1 and 2, contain the greatest variety of polar secondary metabolites belonging to the classes of flavonoids, tannins, and phenol carboxylic acids, while in the shoots collected in the Leningrad region, the major metabolites were weakly polar compounds belonging to the classes of chalcones, dihydrochalcones, bibenzyls, and 9,10-dihydrophenanthrenes, and in sample 2, collected in the Khibiny mountains, the lowest qualitative content of secondary metabolites was found. This variation may be caused by various environmental factors (biotic and abiotic).Conclusion. For the first time, the comparison of HPLC profiles of aerial part samples of E. nigrum, collected in different regions of the Russia Federation has been carried out. As a result, significant variations in the secondary metabolites composition of the studied samples were established, depending on the regions and growing conditions of the plants. The data obtained can be used for a reasonable choice of the collection location for the plant, with the aim of its further chemical and pharmacological research for the development of promising drug candidates.

Metals in Calluna vulgaris, Empetrum nigrum, Festuca vivipara and Thymus praecox ssp. arcticus in the geothermal areas of Iceland

This investigation was conducted to identify the content of metals in Calluna vulgaris (family Ericaceae), Empetrum nigrum (family Ericaceae), Festuca vivipara (family Poaceae) and Thymus praecox subsp. arcticus (family Lamiaceae), as well as in the soils where they were growing in eight geothermal heathlands in Iceland. Investigation into the vegetation of geothermal areas is crucial and may contribute to their proper protection in the future and bring more understanding under what conditions the plants respond to an ecologically more extreme situation. Plants from geothermally active sites were enriched with metals as compared to the same species from non-geothermal control sites (at an average from about 150 m from geothermal activity). The enriched metals consisted of Cd, Co, Cu, Fe and Ni in C. vulgaris; Cd, Mn and Ti in E. nigrum; Hg and Pb in F. vivipara; and Cd, Fe and Hg in T. praecox. Notably, C. vulgaris, E. nigrum, F. vivipara and T. praecox had remarkably high concentrations of Ti at levels typical of toxicity thresholds. Cd and Pb (except for C. vulgaris and F. vivipara) were not accumulated in the shoots of geothermal plants. C. vulgaris from geothermal and control sites was characterised by the highest bioaccumulation factor (BF) of Ti and Mn; E. nigrum and F. vivipara by the highest BF of Ti and Cr; and T. praecox by the highest BF of Ti and Zn compared to the other elements. In comparison with the other examined species, F. vivipara from geothermal sites had the highest concentration of Ti in above-ground parts at any concentration of plant-available Ti in soil.

Isolation and Structure Elucidation of Three Dimeric A-type Proanthocyanidins from Empetrum Nigrum L.

Introduction. Urinary tract infections are a common group of diseases worldwide, affecting more than 150 million people every year. In about 30 % of patients with initial infection, UTI becomes chronic. Herbal medicines, along with synthetic diuretics and antibiotics, are widely used for the prevention and treatment of UTIs, which makes the search and isolation of various substances from plant materials an important task. The present study is devoted to the isolation of compounds belonging to the class of proanthocyanidins from the aerial part of the black crowberry (Empetrum nigrum L.).Aim. Method development for the isolation of individual dimeric type A proanthocyanidins from the aerial part of Empetrum nigrum and the elucidation of their chemical structure using modern physicochemical methods of analysis.Materials and methods. Shoots of Empetrum nigrum were collected next to the Saint Petersburg State Chemical-Pharmaceutical University Nursery Garden of Medicinal Plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km) in August 2019. Fraction analysis was performed through analytical high-performance liquid chromatography (HPLC) using a Prominence LC-20 (Shimadzu corp., Japan) equipped with a SPD-M20A diode-array detector, as well as by high performance thin layer chromatography (HPTLC) using a CAMAG HPTLC system (Switzerland). The isolation of compounds was carried out by open column chromatography using sorbents with different selectivity, as well as by preparative HPLC using a Smartline system (Knauer, Germany) equipped with a spectrophotometric detector. The structures of the isolated compounds were established by 1D and 2D NMR experiments (Bruker Avance III 400 MHz, Germany), as well as high-resolution mass spectrometry (HR-ESI-MS) (Bruker Micromass Q-TOF, Germany).Results and discussion. Using the developed methods, from the Empetrum nigrum shoots we managed to isolate and characterised three individual compounds belonging to the class of A-type proanthocyanidins. According to NMR and mass spectrometry data, compound 1 is epicatechin-(2β → O → 5, 4β → 6)-epicatechin, with an extremely rare type of intermonomer bond (2β → O → 5, 4β → 6). Compounds 2 and 3 are epicatechin-(2β → O → 7, 4β → 8)-epicatechin (procyanidin A2) and epicatechin-(2β → O → 7, 4β → 8)-catechin (procyanidin A1), respectively. All individual compounds (1-3) were found and isolated from Empetrum nigrum for the first time.Conclusion. As a result of the research, three individual compounds (A-type proanthocyanidins) were isolated from the aerial part of Empetrum nigrum. All individual compounds (1-3) were found and isolated from Empetrum nigrum for the first time. Future assessment of the isolated compounds biological activity is presumed.