In the last decade the need of plant-derived cytotoxic compounds exceeds the possibilities for obtaining them from naturally grown sources. In vitro plant biotechnology offers a unique and often invaluable alternative for production of complex biologically active substances without harming the flora. Astragalus glycyphyllos L. (Fabaceae) is a plant native to Bulgaria that has been reported to contain triterpenoid saponins and flavonoids. It is used in folk medicine as an antihypertensive, diuretic, anti-inflammatory, anti-tumour, laxative, expectorant agent, etc. The aim was to study the saponin content of A. glycyphyllos cultures grown in vitro and their antiproliferative activity. Three types of cultures were developed: callus, shoot and suspension cultures. Murashige and Skoog's, as well as other media, supplemented with various concentrations and combinations of plant growth regulators and different photoperiods were used. In all cultures the saponin content was determined by a novel liquid chromatography-mass spectrometry (LC-MS) method. Compared to the wild grown species, in vitro shoot cultures accumulated double the amount of the main saponin (225.00 ng/mg dw) found in the plant. Saponin-rich fractions obtained from shoot cultures were tested for cytotoxicity in a panel of various malignant human cells and half-maximal inhibitory concentration (IC 50) values were determined. Interestingly, the saponin-rich fractions showed higher efficacy against urinary bladder cancer cells with constitutive high expression level of the xenobiotic pump gp170 (MDR1). In vitro cultures of A. glycyphyllos could serve as an alternative way for production of saponins, with promising antineoplastic activity, which deserves further detailed characterization.

This paper discusses the biotechnological process affected by means of plant suspension cultures, for production of colchicoside, the 3- O-glucosyl derivative of 3- O-demethylcolchicine. Colchicoside can be considered as an antitumoural prodrug which is activated after oral administration and may have more beneficial effects and a better toxicity profile (because of a slow-release effect) than colchicine. We have developed a green and efficient biotechnological method using colchicine, as a precursor, derived from its natural source G. superba seeds. Plant suspension cultures of Astragalus vesicarius were used to design a practical biotechnological platform to replace a methyl group at C-3 regiospecifically by a glycosyl moiety in colchicine. Using different concentrations of a colchicine-rich extract, the maximum enzymatic potential of Astragalus vesicarius suspension cells was achieved. According to quantitative HPLC-UV analysis, levels of 9.35 μmol/g DW colchicoside were achieved. This is the first report of region-specific glycosylation at C-3 of the aromatic ring A of the colchicine using plant suspension cultures.

Five isoflavonoids, i.e. 5-hydroxy-7-methoxy-2’, 5’-dihydroxyisoflavone (AV4), 5, 7-dihydroxy-4’-methoxyisoflavone (AV6), 7-methoxy-5-hydroxy-4’-methoxy-2’-hydroxyisoflavone (AV7), 8-pregnyl genistein (AV9), 5,7-dihydroxy-8-pregnyl-4’-methoxy-2’-hydroxyisoflavone (AV10) and one coumarochromone – sophorophenolone (AV8) were isolated from EtOAc of in vitro callus cultures of Astragalus vesicarius ssp. carniolicus, after enzymatic hydrolysis with β-glucosidase. Their structures were tentatively elucidated by spectroscopic mean (1H NMR and HR-ESI-MS spectra). Antiproliferative activity of EtOAc extract and isolated aglycones against chemosensitive human promyelocyte cell line HL-60 and its multidrug-resistant variant HL-60/Dox was assessed in vitro. Despite the strong activity of EtOAc (IC50 8.8 µg/mL (HL-6, 72 h) to 11.8 µg/mL (HL-60/Dox, 72 h)), prenylated compound AV9 showed also antiproliferative activity – 36.1 µg/mL (HL-60 and HL-60/Dox, 72 h).

Establishment of in vitro cultures from Astragalus glycyphyllos, determination of biomass and analysis of total flavonoids, rutin and camelliaside A were performed. To increase flavonoid production various combinations of plant hormones and light/dark regimen were investigated. Suspension cultures with exogenous quercetin were evaluated for possible increase in flavonoid production. Shoots, calli and suspensions were successfully established. Rutin and camelliaside A were proved in highest amount in shoots. Calli, cultivated on modified G48 medium, with double amount of Ca2+ and Mg2+, achieved higher total flavonoid content (2.37 and 2.03 mg/g DW). Suspensions cultures, cultivated on modified G48 medium with 10, 20 and 30 mg/mL quercetin achieved higher total flavonoid content (0.09, 0.10 and 0.13 mg/mg DW). Biotransformation of quercetin to isoquercitrin was achieved. The highest concentration of isoquercitrin (56.73 ng/mg DW) was observed on suspensions cultures cultivated on modified G48 medium, with 20 mg/mL quercetin.

Biotransformation of exogenous substrates quercetin, kaempferol and apigenin by suspension cultures of Astragalus vesicarius ssp. carniolicus to their monoglycosylated derivatives was performed. The maximal enzymatic potential of cells of A. vesicarius ssp. carniolicus was evaluated by different concentrations of substrate exposure. According to quantitative ultra-high performance liquid chromatography-high resolution electrospray ionization mass spectrometry (UHPLC-HR-ESI-MS) analysis, the highest concentration of kaempferol O-glycoside (14.88 nmol/g dry weight, DW), apigenin O-glycoside (10.55 nmol/g DW) and quercetin O-glycoside (150.83 nmol/g DW) was achieved, when suspension cultures were treated with 4 mg/mL kaempferol, 4 mg/mL apigenin and 3 mg/mL quercetin, respectively. The glycosidic products of biotransformation were not detected in the untreated control.

Effects of increased concentration of calcium chloride on growth and production of flavonoids in newly established shoot and callus Gypsophila glomerata cultures were studied. The highest impact of CaCl2 on the growth index was determined in callus cultures (GI = 0.92), while in shoot cultures calcium treatment reduced the amount of biomass (GI = 0.38). Total flavonoids in shoot cultures grown on MS medium and MS medium supplemented with double amount of CaCl2 were 0.36 mg/g d. w. In both callus cultures, 2 mg/g d. w. total flavonoids were quantified. Shoots and callus grown on non-modified media accumulated 0.02 mg/g d. w. quercetin derivatives. Unlike these, both shoots and callus grown on calcium-enriched media accumulated 0.03 and 0.05 mg/g d. w. of isorhamnetin derivatives. In vitro shoot cultures grown on MS medium enriched in twice the amount of CaCl2 accumulated the highest amount of saponarin (0.138 mg/mg d. w.).

Introduction: Gloriosa superba L. is a promising antitumoural plant species as a source of colchicinoids. Ethnobotanical applications of G. superba are associated with different plant parts such as leaves, seeds, fruits, tuber and the whole plant. Objectives: A comparative phytochemical study of purified extracts from in vitro cultures and native tubers of G. superba was carried out by ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS) in combination with the mass defect filtering (MDF) technique. Material and methods: The individual compounds were tentatively annotated using database correlations, retention time (Rt), accurate m/z data obtained by electrospray ionisation (ESI) (+)-HR-MS, proposed elemental composition, ring double bond equivalent (RDBeq) values and HR-MS/MS fragmentation patterns. Moreover, the identification was based on transforming the exact mass ratio (m/z) for the protonated molecular ions [М + Н] + of the observed metabolites in Kendrick nominal masses (NKMs) and calculation of the Kendrick mass defect (KMD), which made it possible to graphically present the ion peaks in Kendrick plots.Results: Building Kendrick plots allows easy differentiation of small structural differences such as methylation or demethylation of compounds from the same homologous series. In this way, a wide range of tropolone alkaloids was characterised. A greater variety was observed in in vitro cultures, compared to native sources. Conclusion:This LC-MS analysis unambiguously demonstrated the presence of tropolone alkaloids in in vitro cultures of G. superba. This approach of LC-MS data interpretation can be used to understand complex mass spectra such as those of plant extracts.

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