Radical scavenging, antioxidant, and cytotoxic activities of the methanolic extracts from different organs of Ternstroemia pringlei.

Ternstroemia pringlei (Rose) Standl. (Theaceae) is widely used in Mexican traditional medicine to treat a diverse array of illnesses including rheumatoid pains, and is listed as one of the most consumed medicinal plants in the country. We selected T. pringlei given the strong relationship between oxidative stress and arthritis pathology, and investigated antioxidant potential of leaf, petal, fruit and seed methanolic extracts. Our method included assessing the in vitro free radical scavenger activity using the 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) test, as well as the in vivo antioxidant action in the H2O2 protection model with Saccharomyces cerevisiae. Leaves and fruits afforded the most active extract in the ABTS assay, with antiradical activity of IC50=33.91 and 38.09µg/mL, respectively; while fruit extracts at 250µg/mL proved the most protective action against H2O2 oxidative stress. All extracts were non-cytotoxic against HF-6 (colon), PC-3 (prostate), MCF-7 (breast), SiHa (cervical) cancer cell lines and also toward the HFS-30 fibroblast normal skin cell line (IC50&>20µg/mL). Leaf methanolic extracts afforded ternstroside B, a known phenylethanoid glycoside with a strong free radical scavenging action. The presence of this kind of metabolites opens new research perspectives for the plant.

Development of antibodies against secoisolariciresinol--application to the immunolocalization of lignans in Linum usitatissimum seeds.

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC(50) value of 94 µg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed (Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.

Extraction of lignans from flaxseed and evaluation of their biological effects on breast cancer MCF-7 and MDA-MB-231 cell lines.

Over the last decade, there has been an increasing interest in using flaxseed (Linum usitatissimum) in diet in order to improve nutritional and health status. Lignans are major components of flaxseed. Therefore an extraction procedure for lignans from flaxseed has been optimized. The influence of some parameters was investigated: first the preliminary extraction step with alcoholic solvent, and then the solvent polarity and pH of the extract. All these conditions affected the total lignan content, but the most critical variables were preliminary extraction and solvent polarity. The optimized procedure, consisting of a direct hydrolysis in hydrochloric acid (1 M) at 100 degrees C for 1 hour followed by an extraction with a mixture of ethyl acetate/hexane (90:10 vol/vol), was applied to 340 g of defatted flaxseed and resulted in the isolation of secoisolariciresinol and anhydrosecoisolariciresinol with a purity of 97% and 98%, respectively, as determined by high-performance liquid chromatography. The ability of these two compounds and that of secoisolariciresinol diglucoside to modulate the growth of human breast cancer MCF-7 and MDA-MB-231 cell lines was assessed. Our results show that lignans modulate development of breast cancer cells. The most intense effect was observed for anhydrosecoisolariciresinol, which significantly decreased cell growth at 50 and 100 microM.

Updated biotechnological approaches developed for 2,7'-cyclolignan production1.

2,7'-Cyclolignans constitute a group of compounds regularly employed in clinical contexts due to their antiviral and anticancer properties as expressed in a number of already available designed drugs. Possibilities for other therapeutic developments are indicated. All commercial preparations actually originate from a single compound, podophyllotoxin, currently extracted from a few limited-in-abundance species of Podophyllum. This supply problem has stimulated considerable interest in the development of alternative strategies offering greater sustainable availability of the compound at affordable costs. Approaches such as plant breeding and micropropagation of the high-producing Podophyllum species have been explored with the aim of establishing production of these compounds. Since 20 years of exploration of the total chemical synthesis of podophyllotoxin has not proved economically viable, extensive research has now been undertaken to find methods for stimulating the accumulation of 2,7'-cyclolignans using tissue cultures. Both undifferentiated and differentiated cell cultures, mainly from Linum, Podophyllum, Juniperus, Callitris, Anthriscus and Forsynthia genera, have been reported. Although considerable progress has been made concerning the concentration and productivity of certain strains of Linum album, no economically viable production system has been established. Exploration and development of biosynthetical pathways leading to 2,7'-cyclolignans may open paths for future metabolic engineering, such as the bioconversion of deoxypodophyllotoxin to epipodophyllotoxin employing a human hepatic enzyme heterologously expressed in Escherichia coli.

Isolation of jacaranone, a sedative constituent extracted from the flowers of the Mexican tree Ternstroemia pringlei.

ETHNOPHARMACOLOGICAL RELEVANCE: Ternstroemia pringlei represents one of the most widely employed and commercially exploited medicinal plant in Mexico, used popularly as a tranquilizer and for the treatment of insomnia. AIM OF THE STUDY: To investigate the sedative constituents of the plant through a bio-guided fractionation of extracts derived from calyx and fruits. MATERIALS AND METHODS: Crude extracts with different polarities (CHCl(3), AcOEt, MeOH, aqueous) were prepared and subjected to chromatographic fractionation, leading to the isolation of the sedative compound (1) from the MeOH crude extract. The identity of 1 was unequivocally established by means of 1D and 2D NMR spectroscopic analysis. The sleeping time induced by sodium pentobarbital and the elevated plus-maze models were performed on mice to determine the sedative and anxiolytic activities, respectively. Bioactivity was also investigated though in vitro GABA release experiments using mice brain slices. RESULTS: The sedative compound was established as jacaranone (1), and its effect was clearly demonstrated through a dose-dependent response analysis (ED(50) = 25 mg/kg mouse weight). When tested in the elevated plus-maze model, none of the extracts from Ternstroemia pringlei displayed anxiolytic activity. GABA release experiments showed that the MeOH and aqueous crude extracts released this neurotransmitter at a ratio of 217 and 179 pmol/g protein, respectively, evidencing the presence of other bioactive constituents in the extracts apart of 1, whose activity was absent in this model. CONCLUSIONS: Although 1 has been isolated and identified in a number of plant species, this is the first time that its sedative effect has been demonstrated. No previous record exists of other sedative compounds having been isolated from Ternstroemia pringlei.

[Interest of lignans in prevention and treatment of cancers]. / Intérêt des lignanes dans la prévention et le traitement de cancers.

Lignans are diphenolic compounds widely distributed in the plant kingdom. They are mainly localised in lignified tissues, seeds and roots. These molecules are involved in plant defence mechanisms, but are also interesting for human health. Flax lignans belonging to the phytoestrogens are metabolised after ingestion into enterolignans that may offer a protection against the onset and development of hormono-dependant cancers. In vitro studies based on mammalian cellular models tend to confirm their beneficial effects observed during epidemiological studies and give us insights about their mechanisms of action. The most studied lignan, podophyllotoxin, and its semi-synthetic derivatives (etoposide, teniposide, etoposide phosphate), are particularly interesting at a curative level due to their cytotoxic properties. These semi-synthetic derivatives are used in chemotherapy of lung cancer for example. However, the extensive use of these anticancer drugs will lead to the problem of podophyllotoxin supply. This molecule is currently extracted from the rhizomes and roots of an Indian species Podophyllum hexandrum which has subsequently become endangered. Strategies are investigated to obtain economically viable alternative sources of Podophyllotoxin from plants and in vitro cultures of several species. Among them, north american Podophyllum peltatum, Linum wild species, Hyptis, Anthriscus, Juniperus or Dysosma species which accumulate Podophyllotoxin or closely related derivatives, are good candidates. double dagger.

Coniferin dimerisation in lignan biosynthesis in flax cells.

[(13)C(2)]-Coniferin was provided to a flax (Linum usitatissimum L.) cell suspension to monitor subsequent dimerisation by MS and NMR. The label was mainly incorporated into a 8-8'-linked lignan, lariciresinol diglucoside, a 8-5'-linked neolignan, dehydrodiconiferyl alcohol glucoside and a diastereoisomeric mixture of a 8-O-4'-linked neolignan, guaiacylglycerol-beta-coniferyl alcohol ether glucoside. This latter compound is reported for the first time in flax. The strong and transient increase in these compounds in fed cells was concomitant with the observed peak in coniferin content. These results suggest (i) a rapid metabolisation of coniferin into lignans and neolignans and indicate the capacity of flax cells to operate different types of couplings, and (ii) a continuous synthesis and subsequent metabolisation of coniferin-derived dimers all over the culture period.

Microwave-assisted extraction of the main phenolic compounds in flaxseed.

A microwave-assisted extraction (MAE) method has been applied for the first time to the extraction of the main lignan, secoisolariciresinol diglucoside (SDG), and the two most concentrated hydroxycinnamic acid glucosides in flaxseed. The effects of microwave power, extraction time and alkaline treatment were investigated. It was shown that a 3 min MAE resulted in an SDG content of 16.1+/-0.4 mg/g, a p-coumaric acid glucoside content of 3.7+/-0.2 mg/g and a ferulic acid glucoside content of 4.1+/-0.2 mg/g. These values were compared with those obtained using conventional extraction methods and the results demonstrated that MAE was more effective in terms of both yield and time consumption.

High accumulation of dehydrodiconiferyl alcohol-4-beta-D: -glucoside in free and immobilized Linum usitatissimum cell cultures.

As flaxseed mainly accumulates lignans (secoisolariciresinol diglucoside and matairesinol), these compounds were barely or not detected in plant cell suspensions initiated from Linum usitatissimum. In contrast, these cell suspensions were shown to accumulate substantial amounts of a neolignan identified as dehydrodiconiferyl alcohol-4-beta-D: -glucoside (DCG) (up to 47.7 mg g(-1) DW). The formation of this pharmacologically active compound was evaluated as a function of cell growth and in relation to phytohormone balance of the culture media. After establishment of efficient culture conditions, production of DCG was investigated in immobilized plant cell suspensions initiated from plantlet roots of L. usitatissimum. The results indicate that immobilization enhances the DCG production up to 60.0 mg g(-1) DW but depresses the cell growth resulting in no improvement of the total DCG yield. Nevertheless, with immobilized cell suspensions, a release of DCG into the medium is observed allowing an easier recovery.

Variation in the accumulation levels of N,N-dimethyltryptamine in micropropagated trees and in in vitro cultures of Mimosa tenuiflora.

The present article reports the accumulation of N,N-dimethyltryptamine and its metabolic precursors (tryptophan, tryptamine) in different organs of micropropagated Mimosa tenuiflora trees (leaves, flowers and bark) subjected to seasonal variations (January and June), as well as in in vitro cultures (plantlets and calluses) of this plant species. The accumulation of all the tested compounds varied according to the organ, the month of collection, and age of the plant material. In all cases, the neurotoxic compound N,N-dimethyltryptamine (DMT) was detected with the lowest concentration 0.01% dry weight (DW) in flowers, and the highest 0.33% DW in bark. For the in vitro cultures, DMT was present in high yields in plantlets (0.1-0.2% DW), while in calluses this compound was initially detected but its concentration decreased significantly in the subsequent subcultures.

Altered nitrogen metabolism associated with de-differentiated suspension cultures derived from root cultures of Datura stramonium studied by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy.

De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. (15)N-Labelled precursors [((15)NH(4))(2)SO(4) and K(15)NO(3)] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and gamma-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.