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

Abstract

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.

Effect of varying NaCl doses on flavonoid production in suspension cells of Ginkgo biloba: relationship to chlorophyll fluorescence, ion homeostasis, antioxidant system and ultrastructure

“…Two peaks of PAL activity were observed for Salvia miltiorrhiza cells (Dong et al 2010) with elicitor induction time, but the first one coincided with the peak of a phenolic compound while the second one coincided with the shikonin peak. However, there was no direct correlation between the peaks of PAL activity and the shikonin production peak under L-phenylalanine treatments in Arnebia euchroma (Sykłowska-Baranek et al 2012). In our study, the peak of PAL activity was observed at 25 mM (low dose), 150 and 175 mM (high dose) NaCl treatments and coincided with the peak of flavonoid production (Table 1), while the lower PAL activity and flavonoid content was observed at 75 mM NaCl.…”

Two new dimeric naphthoquinones with neuraminidase inhibitory activity fromLithospermum erythrorhizon

“…It exhibits biological activities such as wound healing (Yin et al 2013), antioxidant (Weng et al 2000), antifungal and antitumour (Han et al 2008;Yang et al 2009;Lu et al 2011;Andujar et al 2013). Previous phytochemical studies reported that the red pigments of L. erythrorhizon were mainly composed of naphthoquinone compounds, such as b-hydroxyisovalerylshikonin, acetylshikonin (Gwon et al 2012), propionylshikonin (Cho et al 1999), isobutyrylshikonin (Syklowska-Baranek et al 2012), b,b-dimethylacryloylshikonin (Li et al 2012, isovalerylshikonin, isobutyrylshikonin (Zhao et al 2011) and a-methylbutyrylshikonin.…”

Study of the Antifungal Activity of Shikonin and Alcoholic – Oily Extracts of Iranian Arnebia euchroma L

“…16,17 Baranek et al showed that shikonin had positive effects on inhibition of cells. 18 Haghbeen et al prepared 2 different cultures for A. euchroma which had high amounts of pigments. However, antimicrobial experiments showed that they had no effects on gram-negative bacteria and fungi, but optimal effects were observed on gram-positive bacteria.…”