Anna Rita Santamaria

Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum cv. Topas

Abstract Methanolic extracts from cell suspension cultures, calli, and in vitro regenerated shoots and roots of Hypericum perforatum cv. Topas have been evaluated for their ability to produce active metabolites (hypericins, hyperforins and flavonoids). Biosynthesis of hypericins is connected with the formation of secretory structures (black globules) in regenerated vegetative buds. A further degree of leaf development is necessary to stimulate the production of hyperforins and flavonoids. Xanthones are the main metabolic products in suspension cells, undifferentiated calli and roots regenerated from plantlets or formed by callus. No xanthones are detected in the aerial parts of regenerated plantlets accumulating hypericins, hyperforins and flavonoids.

Effects of elicitors on the production of resveratrol and viniferins in cell cultures of Vitis vinifera L. cv Italia.

Methyl jasmonate, jasmonic acid and chitosan were tested as elicitors on cell suspension cultures obtained from Vitis vinifera cv Italia to investigate their effect on stilbene production. Stilbene accumulation in the callus, grown under nonelicited conditions, was also investigated. Calli and cell suspensions were obtained in a B5 culture medium supplemented with 0.2 mg L(-1) NAA and 1 mg L(-1) KIN. Stilbene determination was achieved by HPLC/DAD/MS. Whereas callus biosynthesized only piceid, cell suspensions elicited with jasmonates produced several stilbenes, mainly viniferins. In suspended cells, methyl jasmonate and jasmonic acid were the most effective in stimulating stilbene biosynthesis, whereas chitosan was less effective; in fact, the amount of stilbenes obtained with this elicitor was not significantly different from that obtained for the control cells. The maximum production of total stilbenes was at day 20 of culture with 0.970 and 1.023 mg g(-1) DW for MeJA and JA, respectively.

Apoptosis-inducing factor and caspase-dependent apoptotic pathways triggered by different grape seed extracts on human colon cancer cell line Caco-2

Consumption of grape seed extract (GSE) is widely marketed as a dietary supplement and is considered safe for human health. Nevertheless, the analytical composition of GSE from different grape cultivars, growing in special agronomic constraints, differs greatly in flavan-3-ols content. The major concern with GSE studies is a lack of availability of uniformly standardised preparations, which raises an important question whether different GSE samples have comparable activity and trigger the same mechanisms of action on a given biological system. Therefore, it is tempting to speculate that GSE, obtained from different cultivars, could exert differentiated anticancer effects. The focus of the present study is to determine the selective biological efficacy of GSE obtained from three different sources on the human colon cancer cell line Caco-2. Irrespective of its source, high doses of GSE induced a significant inhibition on Caco-2 cell growth. Moreover, apoptosis was enhanced through both caspase-dependent and caspase-independent mechanisms, leading to an early apoptosis-inducing factor release and, further, to a dramatic increase in caspase 7 and 3 activity. However, a significant difference in apoptotic rates induced by the three grape sources clearly emerged when treating cancer cells with low and intermediate GSE concentrations (25 and 50 microg/ml).

Anthocyanins and xanthones in the calli and regenerated shoots of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh.

The present paper reports on the production of anthocyanins and xanthones in different in vitro systems of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh. Undifferentiated calli and regenerated shoots at different developmental stages were analyzed by applying an extractive and an analytical procedure capable of detecting and quantifying anthocyanins. The findings revealed, for the first time, the co-presence of hypericins and anthocyanins in shoots at initial and more developed stages of H. perforatum var. angustifolium L. Moreover, a high production of xanthones was found in the undifferentiated calli.

Enhancement of viniferin production in Vitis vinifera L. cv. alphonse lavallée cell suspensions by low-energy ultrasound alone and in combination with methyl jasmonate

This study examined for the first time the effect of low-energy ultrasound (US), used alone or in combination with methyl jasmonate (MeJA), on viniferin production in cell cultures of Vitis vinifera L. cv Alphonse Lavallée. Cell suspensions were exposed for 2 min to US (power 30, 60, and 90 mW cm(-3)). The highest viniferin production was obtained at 30 mW cm(-3). When sonication was performed twice, the effect on viniferin production was negligible, whereas triple sonication slightly increased production. US treatment at 30 mW cm(-3) for 5 min decreased viniferin production and induced cellular death. The combined use of MeJA and US (2 min) increased the production of δ-viniferin, the dominant stilbene, more than each elicitor used alone. These results suggest that low-energy US, alone and in combination with MeJA, can act as a physical elicitor to stimulate viniferin production in V. vinifera cell cultures.

Antitumoural activity of viniferin-enriched extracts from Vitis vinifera L. cell cultures

The aim of this work was to evaluate the effect of stilbenes from different cultivars of Vitis vinifera on tumour proliferation. Extracts were obtained from elicited V. vinifera cell cultures and characterised by HPLC/DAD/MS. Cell growth was evaluated in four human cancer cell lines and in normal human fibroblasts. The cells were exposed to the extracts or to trans-resveratrol, used as reference molecule, for 48 h, at 1–10 μM concentrations of total stilbenoids. All the extracts exhibited antiproliferative activity, mediated by modulation of the cell cycle and induction of cytotoxicity in cancer but not in normal cell lines, and positively correlated with the content in dimeric stilbenoids. The Alphonse Lavallée extract was the most active, and the obtained stilbenoid fraction resulted 8–10 times more active than trans-resveratrol. Extracts from V. vinifera cell cultures could represent new sources of active stilbenoid compounds to be further assayed in in vivo studies for their antitumoural properties.

Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae)

BackgroundCamptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production.ResultsThe pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase) and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase)]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected.ConclusionsThe finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors.

Chitosan enhances xanthone production in Hypericum perforatum subsp. angustifolium cell cultures

Hypericum perforatum is an important medicinal plant containing numerous biologically active compounds. The effect of chitosan elicitation on xanthone biosynthesis in calli and in cell suspension cultures of H. perforatum subsp. angustifolium was evaluated. Elicited cell cultures showed an increase in xanthone production and a simultaneous decrease in flavonoid production. Chitosan also induced the production of 1,7-dihydroxyxanthone (euxanthone) and cadensin G, which were not detected in either the calli nor the non-elicited cell cultures. 1,7-Dihydroxyxanthone was in part (21%) released in the culture medium.

Stilbene production in cell cultures of Vitis vinifera L. cvs Red Globe and Michele Palieri elicited by methyl jasmonate

Cell cultures obtained from Vitis vinifera cvs Michele Palieri and Red Globe were cultured in order to stimulate stilbene production. In the calli, stilbene production peaked at day 22 of culture for both cultivars; the main compound was trans-piceid, followed by cis-piceid. Methyl jasmonate, which was added to cell suspensions in the first half of the exponential growth phase, enhanced stilbene accumulation, producing mainly trans-piceid and ε-viniferin. Other stilbenoids, though in lower quantities, were identified by liquid chromatography/positive electrospray mass spectrometry. ε-Viniferin and trans-resveratrol were the main compounds released into the culture medium. The total quantity of stilbenes was genotype dependent, with a better response found for the cv Red Globe.

Anthocyanins and flavan-3-ols from grapes and wines of Vitis vinifera cv. Cesanese d’Affile

The objective of the present study was to evaluate the amount of some potential health-promoting phenols in the grape of Vitis vinifera cv. Cesanese d’Affile and in wines made from these grapes. The analyses were performed using HPLC/DAD/MS. The accumulation of anthocyanins in the skin and flavan-3-ols in the seed was determined at different stages of ripening of the grape (i.e. green, veraison, middle stage of ripening, and complete ripening). Thirteen anthocyanins were identified in the skin at all stages of ripening, except the green stage. With regard to flavan-3-ols, (+)-catechin, (−)-epicatechin, and (−)-epicatechin gallate were detected in all of the seed samples. The highest (+)-catechin content was found in the seeds of the green grape (2 mg g−1 DW), whereas in the seeds from the completely ripe grape the content was more than ten times lower. The highest catechin content in the seed was correlated with the lowest anthocyanin content in the skin. The wines produced in the years 2004 and 2005 showed, at wavelengths of 520 and 280 nm, almost identical quali-quantitative chromatographic profiles, with high concentrations of anthocyanin 3-O-glucosides, low concentrations of acylated anthocyanins, and trace amounts of (+)-catechin and (−)-epicatechin.