Cultivar based selection and genetic analysis of strawberry fruits with high levels of health promoting compounds.

Twenty different strawberry genotypes from phenolic compound content and genetic diversity have been investigated. Twelve phenolic derivatives in the strawberry fruit extracts, their total phenolic content (TPC) and their radical scavenging activity have been quantified. In order to study the influence of the genetic basis of each cultivar (cv) on the chemical composition of fruits, Principal Component Analysis of the obtained data was also used. Significant differences in the content of individual anthocyanins among the 20cvs have been found. Pelargonidin 3-O-glucoside was the predominant anthocyanin in the strawberry extracts with 61.0% of the total anthocyanins in Salva cv, followed by cyanidin 3-O-glucoside. TPC values ranged from 129,96 (Laica cv) to 269,04 (Naiad cv) mg of gallic acid equivalent per 100g of fresh weight and it was congruent previous studies. Moreover RAPD markers have been applied in order to describe their genetic relationships. A total of 32decamer primers were used in RAPD analysis; 19 of them provided at least one polymorphic band, the remaining primers were monomorphic. A total of 124 bands were detected with the mean number of 11.53 accountable fragments per primer and 59.98% were polymorphic. The results of the present study highlighted the health-promoting compound content of strawberry fruits, and provided a good prospect for discriminating strawberries by phenolic content and genetic analysis.

Chemical proteomics reveals HSP70 1A as a target for the anticancer diterpene oridonin in Jurkat cells.

Oridonin, an ent-kaurane diterpene isolated from well known Chinese medicinal plant Isodon rubescens, has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. The chemopreventive and antitumor effects of oridonin have been related to its ability to interfere with several pathways which are involved in cell proliferation, cell cycle arrest, apoptosis and/or autophagy. Despite the number of studies performed on this diterpene, the molecular mechanism underlying its cellular activity remains to be elucidated. Hence, we tried to mine target protein(s) of oridonin by employing a mass spectrometry-based chemical proteomics approach, providing evidences that oridonin is able to directly bind the multifunctional, stress-inducible heat shock protein 70 1A (HSP70 1A). Oridonin/HSP70 complex formation was confirmed in leukemia-derived Jurkat cells. The characterization of HSP70 inhibition by oridonin was performed using chemical and biological approaches. Moreover, the binding site of oridonin on the chaperone was identified by a mass-based approach combined with Molecular Dynamics simulations. BIOLOGICAL SIGNIFICANCE: Although natural products showed high efficiency and several of these agents have now entered in clinical trials, information concerning the mechanisms of action at a molecular level of many of them is very poor or completely missed. Nevertheless, the identification of the molecular target of a drug candidate has several advantages. The most significant is the ability to set up target-based assays and to allow structure-activity relationship studies to guide medicinal chemistry efforts towards lead optimization. The knowledge of drug targets can also facilitate the identification of potential toxicities or side effects, if there is any precedent of toxicities for the identified target. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. In the present study, we used a chemical proteomic approach aimed to define the possible protein target of the ent-kaurane diterpene oridonin. This natural compound has drawn a rising attention for cancer biologists due to its remarkable anti-tumor activities: accumulating evidence has suggested that oridonin is able to hamper the progression of tumor, mitigate tumor burden and alleviate cancer syndrome, which may improve greatly the survival rates of cancer patients; however molecular mechanisms by which this compound exerts its anti-tumor activities still remained to be discovered. We identified the molecular chaperone HSP70 1A as an oridonin target in Jurkat cells, thus suggesting a mechanism of action for the diterpene consistent with the multiple biological activities described for it. HSP70 inhibition by oridonin might indeed simultaneously result in the impairment of some of client proteins, thus in turn affecting several molecular pathways. Shedding light on the molecular basis of the biological activity of oridonin, our findings may be relevant for possible therapeutic applications of oridonin, such as its use in combination and the design of new therapeutic approaches. In addition, this research demonstrates the effectiveness of chemical proteomic approaches in drug discovery studies and in orphan drug molecular target identification.

Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun.

The main aim of this work was to study the leaf secondary metabolite profiles of artificially induced tetraploids (2n=4x=48) of Solanum commersonii, a diploid (2n=2x=24) wild potato species. The tetraploid genotypes of S. commersonii were produced by oryzalin treatment. Both HPLC-UV and LC/MS analyses revealed that there were no qualitative differences in the metabolite profiles between the diploid S. commersonii and its tetraploids. By contrast, the results showed that the phenylpropanoid content was generally significantly higher in the tetraploids than in the diploid S. commersonii. Concerning the glycoalkaloids (GAs), the results provided evidence that the content of minor GAs (solanidenediol triose, solanidadienol lycotetraose, and solanidenol lycotetraose) was higher in tetraploids than in the diploid progenitor, while the content of major GAs (dehydrodemissine and dehydrocommersonine) was significantly higher in diploid S. commersonii than in its tetraploid genotypes. The results are discussed from the practical perspective of potato biodiversity enhancement.

Anti-angiogenic activity evaluation of secondary metabolites from Calycolpus moritzianus leaves.

Angiogenesis is a crucial step in many pathological conditions like cancer, inflammation and metastasis formation; on these basis the search for antiangiogenic agents has widened. In order to identify new compounds able to interfere in the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1, also known as Flt-1) recognition by VEGFs family members, we screened Calycolpus moritzianus (O. Berg) Burret leaves extracts by a competitive ELISA-based assay. MeOH and CHCl3 extracts and several their fractions demonstrated to be able to prevent VEGF or PlGF interaction with Flt-1, with an inhibition about 50% at concentration of 100 microg/mL. Phytochemical and pharmacological investigation of the active fractions led to the isolation of flavonoids, and terpenes.

The biflavonoid amentoflavone inhibits neovascularization preventing the activity of proangiogenic vascular endothelial growth factors.

The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low µm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.

Isolation and structural characterization of glycosides from an anti-angiogenic extract of Monnina obtusifolia H.B.K.

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. Substantial data over the last decade has implicated uncontrolled angiogenesis with various pathological states. Vascular endothelial growth factors (VEGFs) play a critical role in its regulation, and have become one of the most interesting anti-angiogenesis targets. We have investigated the anti-angiogenic potential of plant extracts in a preliminary ELISA screening. The n-BuOH extract obtained from the leaves of Monnina obtusifolia (Polygalaceae) demonstrated an inhibition of VEGF-A or Placental Growth Factor interaction with Flt-1 (VEGF receptor 1), with an inhibition over 50% in particular for VEGF-A/Flt-1 interaction at a concentration of 500 µg/mL. Successively fractionation of the bioactive n-BuOH extracts of M. obtusifolia aerial parts led to the isolation of six new compounds, 1-O-(4-hydroxy-2-methylene-butanoic acid)-6-O-ß-D-(4-hydroxy-2-methylene-butanoyl)-glucopyranose (1), 1-O-(isopentenyl)-6-O-ß-D-(4-hydroxy-2-methylene-butanoyl)-glucopyranose (2), 1-O-(4-hydroxy-2-methylene-butanoic acid)-6-O-ß-D-(isovaleroyl)-glucopyranose (3), 1-O-(3-methylbut-3enyl)-6-O-ß-D-(isovaleroyl)-glucopyranose (4), two new sucrose esters, 3,4-O-ß-D-di-feruloyl-fructofuranosyl-6-O-α-D-(p-coumaroyl)-glucopyranoside (5), and 3,4-O-ß-D-di-feruloyl-fructofuranosyl-6-O-α-D-(caffeoyl)-glucopyranoside (6), together with known flavonoids. Their structures were established on the basis of detailed spectral analysis. Since none of the isolated compounds showed a relevant inhibition of VEGFs, the biological activity observed for the butanolic extract might be due to the presence of a combination of compounds acting synergistically.

A fast and efficient LC-MS/MS method for detection, identification and quantitative analysis of bioactive sesterterpenes in Salvia dominica crude extracts.

Sesterterpenes are a small group of terpenoids showing a number of interesting pharmacological properties, including cytotoxicity, anti-inflammatory, anti-microbial and anti-angiogenic activities and platelet aggregation inhibition. Recently, some sesterterpene lactones isolated from Salvia dominica have been shown to modulate enzymatic activity of tubulin tyrosine ligase (TTL), a promising target for new anticancer therapeutic strategies. However, to allow a direct use of S. dominica extracts as a source of TTL inhibitors, analytical method aimed to their fast qualitative and quantitative characterization is required. Despite the structural features and diverse biological activities of sesterterpenoids, actually no analytical method for their quantization into complex mixtures has been published. Here we describe an LC-MS/MS method aimed to qualitative and quantitative analysis of sesterterpenes lactones in the crude extracts obtained from different parts of S. dominica. This approach allowed us to characterize all the sesterterpenes by a single step analysis and also to identify two unknown compounds. Moreover, a quantitative comparison of the composition in sesterterpenes of extracts obtained from S. dominica leaves, roots and leaf galls was performed, leading to the definition of both leaves and leaf galls as suitable sources of TTL inhibitors.

beta-Glycosyl azides as substrates for alpha-glycosynthases: preparation of efficient alpha-L-fucosynthases.

Fucose-containing oligosaccharides play a central role in physio-pathological events, and fucosylated oligosaccharides have interesting potential applications in biomedicine. No methods for the large-scale production of oligosaccharides are currently available, but the chemo-enzymatic approach is very promising. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, have been demonstrated to be an interesting alternative. However, examples of glycosynthases available so far are restricted to a limited number of glycosidases families and to only one retaining alpha-glycosynthase. We show here that new mutants of two alpha-L-fucosidases are efficient alpha-L-fucosynthases. The approach shown utilized beta-L-fucopyranosyl azide as donor substrate leading to transglycosylation yields up to 91%. This is the first method exploiting a beta-glycosyl azide donor for alpha-glycosynthases; its applicability to the glycosynthetic methodology in a wider perspective is presented.

Sesterterpenes as tubulin tyrosine ligase inhibitors. First insight of structure-activity relationships and discovery of new lead.

Twenty-four new sesterterpenes, compounds 1-24, were isolated from the aerial parts of Salvia dominica. Their structures were elucidated by 1D and 2D NMR experiments as well as ESIMS analysis and chemical methods. The evaluation of the biological activity of Salvia dominica sesterterpenes by means of a panel of chemical and biological approaches, including chemical proteomics, surface plasmon resonance (SPR) measurements, and biochemical assays were realized. Obtained results showed that 18 out of the 24 sesterterpene lactones isolated from Salvia dominica interact with tubulin-tyrosine ligase (TTL) an enzyme involved in the tyrosination cycle of the C-terminal of tubulin, and inhibit TTL activity in cancer cells. Besides, results of our studies provided an activity/structure relationship that can be used to design effective TTL inhibitors.