Pyrrolizidine alkaloids from Cynoglossum columnae Ten. (Boraginaceae)

Seven secondary metabolites were isolated from the methanolic extracts of the roots and the aerial part of Cynoglossum columnae Ten. β-Arbutin and three pyrrolizidine alkaloids, namely 2′-epi-heliosupine N-oxide, rinderine N-oxide and 3′-O-acetylrinderine N-oxide were isolated from the aerial part of the plant. Echinatine N-oxide, rosmarinic acid and 9″-methyl lithospermate were isolated from the root extract. © 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved

Bioactive rinderol and cynoglosol isolated from Cynoglossum columnae Ten. in vitro root culture

Plant in vitro cultures can effectively reduce the overexploitation of natural resources for the efficient production of natural compounds with therapeutic potential, as in case of Boraginaceae family which is well studied for its shikonin/alkannin derivatives production in biotechnological systems. In this study two naphthoquinone derivatives were isolated for the first time from in vitro root cultures of Cynoglossum columnae Ten. The structures of 2-methoxy-5O, 6-(isohex-1-ene-1,2-diyl)-5,8-dihydroxynaphthalene-1,4-dione (1)and 2-[(2S)-5-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-5,8-dihydroxynaphthalene-1,4-dione (2)were identified by spectroscopic methods and named as rinderol and cynoglosol, respectively. Both naphthoquinones were examined for their cytotoxicity towards HL-60, HeLa and HCT-116 human cancer cell lines. Rinderol (1)showed the highest activity towards HL-60 (IC50=2.0 μg/mL), while cynoglosol (2)was selectively active against HL-60 (IC50=4.3 μg/mL). Antibacterial activity of rinderol (1)was noted for Staphylococcus epidermidis ATCC 12228 as MIC=3.91 μg/mL and Staphylococcus epidermidis MRSE 456 as MIC=7.81 μg/mL. These results present Cynoglossum columnae Ten. in vitro root culture as a new biotechnological source of natural compounds with a potential for therapeutic applications. © 2019 Elsevier B.V

Enhanced production of antitumour naphthoquinones in transgenic hairy root lines of Lithospermum canescens

The effects of medium exchange and methyl jasmonate addition on growth and production of shikonin and its derivatives acetylshikonin and isobutyrylshikonin in hairy root cultures of Lithospermum canescens were investigated. Responses varied depending on the transgenic line and stage of growth at which these lines were subjected to treatment. Shikonin itself was not detected, irrespective of the transgenic line and culture treatment used. A eightfold increase in acetylshikonin and isobutyrylshikonin accumulation was achieved when 32-day-old transgenic roots of Lc1D line were transferred from LS to M9 medium for the subsequent 3 weeks of culture. Methyl jasmonate exerted a detrimental effect on red naphthoquinones production. The extracts obtained from roots cultivated in M9 medium for 3 weeks were subjected to a cytotoxicity assay and displayed cytotoxic activity against human promyelocytic leukemia cells (HL-60) at the dose of 4 μg ml -1. © 2011 The Author(s)

Chemical profile and screening of bioactive metabolites of rindera graeca (A. dc.) bois. & heldr. (boraginaceae) in vitro cultures

Rindera graeca is a rare endemic plant where in vitro culture has been used in order to investigate bioactive metabolites. Phytochemical study of the in vitro shoots and hairy roots led to the isolation of seven phenolic derivatives and the unusual furano‐naphthoquinone rinderol. R. graeca was also analyzed for its pyrrolizidine alkaloids content by LC‐MS, and it was found to contain echinatine together with echinatine and rinderine N‐oxides. Rinderol, isolated only from in vitro hairy root culture for the first time in the genus, revealed promising bioactivities. It was evaluated in vitro against a panel of microorganisms, showing very strong activity specifically against Gram‐positive bacteria (MIC values 0.98 × 10−2–1.18 μg/mL) as well as very interesting antiproliferative effect against the human non‐small‐cell bronchopulmonary carcinoma cell line NSCLC‐N6‐L16 and the epidermoid lung cancer cell line A549. These findings were compared with the chemical profile of the plant from nature, while this study is the first to report on the effects of R. graeca extracts obtained from in vitro culture, providing a valuable contribution to the scientific community towards this sustainable method of production of potential bioactive molecules. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1 mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1 mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5 mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1 mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8 μg ml -1 for the HL-60, HeLa, and MCF-7 cells, respectively. © 2012 The Author(s)

Polyurethane foam rafts supported in vitro cultures of rindera graeca roots for enhanced production of rinderol, potent proapoptotic naphthoquinone compound

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaph-thoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 ± 10.47 µg/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity. © 2021 by the authors. Licensee MDPI, Basel, Switzerland