The effect of the well-characterized callus extract of Chaenomeles japonica on viability, morphology, and proliferation of normal human skin fibroblasts was investigated. The phytochemical analysis was performed using the ultra-high performance liquid chromatography method. The total phenolic, phenolic acid, and flavonoid contents were determined spectrophotometrically. The antioxidant activity was investigated using the DPPH (1,1-Diphenyl-1-picrylhydrazyl Radical Scavenging), FRAP (Ferric Reducing Antioxidant Power), and CUPRAC (CUPric Reducing Antioxidant Capacity) assays. The callus growth index during passages was high as well as the content of pentacyclic triterpenoids. The microscopic observations of the fibroblast viability, morphology and the evaluation of the proliferation ratio (xCELLigence system) proved that the influence of callus extract on the fibroblasts was dose-dependent. The evaluated level of fibroblasts proliferation rate after 72 h of incubation with callus extract at concentration 12.5 µg L−1 was the highest compared to all the analyzed ligands. Moreover, callus extract administrated for 72 h caused a significant increase in the proliferation rate in comparison with the control group (5.7 ± 0.1 vs. 4.4 ± 0.9; p < 0.01). The preliminary studies carried out may suggest that the callus extract rich in triterpenoids may be a potential source of cosmetic ingredients with a beneficial effect on human skin.

A protocol for C. japonica micropropagation with a confirmation of genome size stability of the in vitro-propagated plantlets was developed. The highest number of shoots multiplied in vitro was obtained on Murashige & Skoog medium (MS) with 1.0 mg L−1 N6-benzyladenine plus 1.0 mg L−1 indole-3-acetic acid. The highest number of roots was observed for the shoots on MS with 15 g L−1 sucrose plus 1.0 mg L−1 indole-3-acetic acid. The acclimatization rate was significantly high. The qualitative HPLC analyses confirmed the presence of phenolic acids and flavonoids in the extracts. The extracts from both shoot cultures and the leaves from field-grown plants revealed antioxidant activity and they exhibited moderate antimicrobial activity. The conducted research confirmed the regeneration potential of genetically-stable plants of C. japonica under in vitro conditions, the ability of the plantlets to produce polyphenols as those present in field-grown plants, as well as their antioxidant potential.

A new series of hydroxycoumarin derivatives has been synthesized using conventional synthesis. The syntheses were accelerated by microwave assistance. Yields in both cases were comparable (59–69 %). The structures were established by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry. Five compounds (5-hydroxy-4,7-dimethylcoumarin, 6-acetyl-5-hydroxy-4,7-dimethylcoumarin, 4-(cyanomethoxy)chromen-2-one, 5-(cyanomethoxy)-4,7-dimethylchromen-2-one, and 6-acetyl-5-(cyanomethoxy)-4,7-dimethylchromen-2-one) were assayed for anti-cancer activity. For all presented coumarin derivatives, lipophilicity was measured using reversed-phase TLC in different eluent systems with standardization. In addition, the crystal structure of 6-acetyl-5-hydroxy-4,7-dimethylcoumarin has been solved by X-ray structure analysis of single crystals.Graphical abstract

Summary Introduction: Callus and cell suspension cultures are widely applied in investigation of production of high-value secondary metabolites, which may be used as cosmeceuticals, nutraceuticals and pharmaceuticals. Plant cell cultures are promising alternative to intact plant sources for the production of plant-derived drugs of industrial importance. Objective: The aim of the study was to (i) initiate the cell suspension culture of Chaenomeles japonica from homogenous and uniform callus, (ii) stabilize the selected line and (iii) verify its ability to produce the desired groups of secondary metabolites – pentacyclic triterpenoids and polyphenols. Methods: To establish a cell suspension culture, stabilized and homogeneous callus was selected. Cell cultures were systematically passaged every 2 weeks to fresh liquid medium with the same composition. Biomass from cultures at the growth phase and stationary phase was designated for phytochemical research. UHPLC-DAD-MS analyzes were performed. At the same time, their macroscopic and microscopic observations were carried out. Results: Cells of suspension culture line A2 were characterized by the intense divisions. Cell culture extracts (both from the growth phase and stationary phase) contained pentacyclic triterpenoids. In addition, phe-nolic compounds (chlorogenic acid and proanthocyanidins type B) and in a small amount also epicatechin are present in the extract of the cells harvested from the growth phase. In the present studies, three pentacyclic triterpenoids were detected and quantified in the extracts of cell suspensions and callus line A2. Ursolic and oleanolic acids were the main triterpenoids in the studied extracts. The cell suspension culture from the growth phase exhibited the highest content of ursolic, oleanolic, and betulinic acid (separately and together). Conclusion: The cell suspension culture of Chaenomeles japonica is a promising source of pentacyclic triterpenoids.

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The free-living Acanthamoeba sp. causes various diseases. Treatment of them is very difficult and not always effective because of encystation, making it highly resistant to antiamoebic drugs. Gram-positive bacteria staphylococcus aureus, Gram-negative bacteria Escherichia coli, and an yeast candida albicans also exhibit outstanding resistance to antimicrobial substances. The search for new natural amoebicidal and antimicrobial agents of plant origin is still of current interest. The aim of the study was to investigate the amoebicidal activity of the extracts obtained from tissue culture and a field-grown plant of chaenomeles japonica against pathogenic trophozoites of Acanthamoeba spp. and antimicrobial effect against s. aureus, E. coli, and c. albicans. The extracts of c. japonica had an inhibitory effect on the proliferation of Acanthamoeba trophozoites as compared to the non-treated control. among the crude extracts tested, the extract of leaves, from both shoot culture and the field-grown plant had remarkable amoebicidal action against the trophozoites but also antibacterial activity against Gram-positive bacteria staphylococcus aureus. The extract from leaves from shoot culture, already on the second and third days of treatment, showed an antiamoebicidal effect at a concentration of 1 mg ml -1 (inhibition of trophozoites 87.5% and 91.8%, respectively). In addition to leaves from shoot culture (a conc. 5 mg ml -1 , 2 nd day inhibition of trophozoites 85.7% and 3 rd day 97.2%), leaves from a field-grown plant (a conc. 5 mg ml -1 , 2 nd day 91.0% and 3 rd day 94.4%) and callus (a conc. 5 mg ml -1 , 2 nd day 90.0% and 3 rd day -95.4%) also exhibited a good antiamoebicidal activity. Out of the four extracts, the extracts from leaves from both shoot culture and a field-grown plant were reported to be the most active against Gram-positive s. aureus, which was determined by the values of MIC = 5.0 mg ml -1 and MIC = 2.5 mg ml -1 , respectively. The inhibitory potential depends on the yield and composition of mainly bioactive compounds: pentacyclic terpenoids (mainly betulinic, ursolic, and oleanolic acids) and polyphenols (mainly chlorogenic acid and its isomers, epicatechin, dimeric, and trimeric proanthocyanidins, quercetin and kaempferol derivatives).