Phytochemical Profile and In Vitro Bioactivities of Wild Asparagus stipularis.

In this study, Asparagus stipularis was characterized concerning its phytochemical composition, antioxidant potential, cytotoxicity, and pancreatic lipase inhibitory activities. Twenty-seven compounds were identified and quantified by HPLC-DAD-MS in the leaf, stem, pericarp, and rhizome of ethanolic extracts. Seven steroidal saponins were detected, and the highest content was quantified in rhizome and pericap. A. stipularis also contained significant amounts of flavonoids in the aerial part. Isorhamnetin tetra-glycoside, quercetin-3-glucosyl-rutinoside, and rutin were the main flavonoid derivatives in leaf, stem, and pericarp extracts, respectively. In addition, eleven phenolic acids were also detected; among them, caffeic acid, protocatechuic acid, p-hydroxybenzoic acid, and ferulic acid were the predominant phenolics, with these having the highest amounts quantified in the rhizome extracts. All the tested extracts possessed antioxidant capacities, with pericarp and rhizome extracts exhibiting the highest activity in DPPH, ABTS, and FRAP assays. The extracts from pericarp and rhizome were revealed to also be the strongest inhibitors of pancreatic lipase. The rhizome extracts exhibited potent cytotoxic activity against HCT-116 and HepG2 with IC50 values of 30 and 54 µg/mL after 48 h of treatment. The present study demonstrated that A. stipularis can be used as a new source of natural antioxidants and potential anticancer and antiobesity compounds.

Applications of Saponin Extract from Asparagus Roots as Functional Ingredient.

When replanting an asparagus field, the roots of the previous crop are crushed and incorporated into the soil, creating problems of autotoxicity and fungal infections. Asparagus roots can be considered as a valuable byproduct, since they are very rich in saponins (3-6%), compounds currently considered as bio-emulsifiers. The objective is to evaluate the emulsifying and foaming capacity of a saponin extract from asparagus roots (ARS) and compare it with other commercial extracts. ARS was obtained using a process patented by our research group. The results have shown that ARS has activity similar to Quillaja extract. Its critical micellar concentration falls between that of Quillaja and Tribulus extracts (0.064, 0.043, and 0.094 g/100 mL, respectively). Both emulsifying and foaming activities are affected by pH, salt, and sucrose to a similar extent as the other extracts. Additionally, it has demonstrated an inhibitory effect on pancreatic lipase, which is even better than the other two studied extracts, as indicated by its IC50 value (0.7887, 1.6366, and 2.0107 mg/mL for asparagus, Quillaja, and Tribulus, respectively). These results suggest that ARS could serve as a natural emulsifying/foaming agent for healthier and safer food products and as a potential aid in treatments for obesity and hyperlipidemia.

A Sustainable Approach for the Valorization of Underutilized Date Fruits.

Secondary varieties of date fruits are often discarded because they do not have commercial value. However, their phytochemicals are very similar to those of the primary ones and therefore, they can be valorized as a source of compounds of interest, mainly phenols and dietary fiber. Their chemical composition changes with ripening, so their characterization throughout this process is of great significance. Date fruit samples were harvested at Khalal, Rutab, and Tamer stages, and a mixture of fruits from ornamental date trees was also analyzed. Aqueous and ethanolic extracts were studied for their phenolic composition. In aqueous extracts, phenols decreased with ripening, while in the ethanolic ones having higher phenolic content. Chelidonic acid, a γ-pyrone, was the major compound found in all extracts, but in the ethanolic ones, flavonoids were also present in similar amounts. After purification by adsorption chromatography, all extracts were assayed for their antimicrobial activity. Those from the Tamer stage showed the highest activity, especially against Gram-positive bacteria. The fibrous residues after aqueous and ethanolic extractions were also characterized. Their chemical composition suggested that they can be considered as a good source of prebiotic arabinoxylans and antioxidant fiber, whose antiradical activity correlated with their phenolic content. Date fruits from secondary varieties are promising as a worthwhile starting point for obtaining new value-added products.

Sustainable valorization of co-products from asparagus cultivation by obtaining bioactive compounds.

Asparagus cultivation generates every year a significant amount of by-products that consist of root and frond. Leaving these residues on the fields after harvesting negatively affects the following asparagus crops, since they release autotoxic (allelopathic) substances into the soil, whose accumulation causes that asparagus yields gradually decrease over the years, becoming an unprofitable crop in a period of about 10 to 15 years. This phenomenon is known as decay and forces the entire asparagus plantation to be lifted (abandoned). On the other hand, once a certain plantation has been lifted, it is not profitable to immediately re-plant new asparagus plants, since the yields that are achieved are never more than half of normal ones. It is necessary to wait an average of 4 or 5 years before replanting asparagus in these lands. This phenomenon is known as the replanting problem, and causes the need to continually search for new land for growing asparagus. Another added problem for farmers is that the elimination of those plant residues from asparagus cultivation entails significant economic costs. For all these reasons, it is essential to seek alternatives for the management of that waste that improve the sustainability of the crop within the scope of the circular economy. In this context, this work proposes the valorization of asparagus by-products by obtaining bioactive compounds. Main objectives of the present work include: i) phytochemical analyses of asparagus fronds and roots; ii) obtaining bioactive extracts, with distinct technological and nutritional functionalities, by using an environmentally sustainable extraction process, easy to implement in the practice of a food industry and with methods compatible with food use. Characterization of asparagus by-products shown that fronds had an average flavonoid content of 2.637 ± 0.014 g/Kg fresh weight, which is up to 5-6 times higher than that of the spears; and roots contained up to 10 times more saponins (2.25 g/Kg fresh weight), which were accompanied by lower quantities of phenolic acids (368 mg/Kg fresh weight). Statistical analysis revealed that those phytochemical contents were mainly determined by location and phase of the vegetative cycle, whereas genetic factors did not significantly influence them. Based on the results of the present work, the proposal for the recovery and valorization of asparagus by-products is based on obtaining two bioactive extracts, the first being an antioxidant extract enriched in flavonoids, with an average yield of 10.7 g/Kg fresh frond and a flavonoid richness of 17%; and the second, a saponins extract with an average yield of 10.3 g/Kg fresh root and a richness of 51%. These natural extracts have great techno-functional potential in the agri-food industry and some of them are already being tested as additives in the preparation of soups, breads and meat products.

Asparagus Roots: From an Agricultural By-Product to a Valuable Source of Fructans.

Asparagus roots are by-products from asparagus cultivation and they could be considered one of the best sources of fructans. These polymers are interesting food ingredients for their prebiotic and immuno-stimulating characteristics. The aim of this work is to characterize the fructan profile from the roots of several asparagus varieties grown at different locations and pickled at three vegetative statuses in order to valorize these by-products as fructan source. Fructans were extracted with hot water and fractionated into three pools according to their molecular weight (MW). Their average MW was studied by HPSEC and their degree of polymerization by HPAEC. The fructan content was up to 12.5% on fresh weight basis, depending on variety and sampling date. The relative abundance of the three pools also depended on the picking moment as after the spear harvest period their total content and MW increased. The average MW of the three fractions was similar among varieties with 4.8, 8.4 and 9 sugar units, although fructans up to 30 units were identified by HPAEC. These characteristics make them similar to the commercialized Orafti®-GR inulin, a common additive to food products. Therefore, the concept of asparagus roots as cultivation waste must be changed to a new feedstock for sustainable agriculture and industry.

Asparagus Fructans as Emerging Prebiotics.

Commercial fructans (inulin and oligofructose) are generally obtained from crops such as chicory, Jerusalem artichoke or agave. However, there are agricultural by-products, namely asparagus roots, which could be considered potential sources of fructans. In this work, the fructans extracted from asparagus roots and three commercial ones from chicory and agave were studied in order to compare their composition, physicochemical characteristics, and potential health effects. Asparagus fructans had similar chemical composition to the others, especially in moisture, simple sugars and total fructan contents. However, its contents of ash, protein and phenolic compounds were higher. FTIR analysis confirmed these differences in composition. Orafti®GR showed the highest degree of polymerization (DP) of up to 40, with asparagus fructans (up to 25) falling between Orafti®GR and the others (DP 10-11). Although asparagus fructan powder had a lower fructan content and lower DP than Orafti®GR, its viscosity was higher, probably due to the presence of proteins. The existence of phenolic compounds lent antioxidant activity to asparagus fructans. The prebiotic activity in vitro of the four samples was similar and, in preliminary assays, asparagus fructan extract presented health effects related to infertility and diabetes diseases. All these characteristics confer a great potential for asparagus fructans to be included in the prebiotics market.

Potential antioxidant effects of Narcissus tazetta phenolic compounds against cadmium chloride-induced hepatotoxicity in Swiss albino mice.

Narcissus tazetta (Amaryllidaceae) is a medicinal plant widely used for cut flowers and potted ornamental plant in Tunisia flora. The current study evaluated the phenolic composition and antioxidant properties of its flower extracts and investigated its potential protective activity against cadmium chloride (CdCl2)-induced hepatotoxicity in mice. Mice were divided into six groups of six each: group 1, serving as negative controls, received by intraperitoneal way only distilled water; group 2 received by intraperitoneal way CdCl2 (0.16 mg/kg bw); groups 3 and 4 received CdCl2 at the same dose of group 2 and 100 or 200 mg/kg bw of Narcissus tazetta flower extracts via oral route; groups 5 and 6, serving as positive controls, received only Narcissus tazetta flower extracts. Polyphenolic compounds of the extract were analyzed by colorimetric and high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. Total antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging potential of the extract were estimated using colorimetric method. Results indicated that ethanolic flower extract contained high levels of total phenolic and flavonoid along with a strong total antioxidant and DPPH free radical scavenging activities. HPLC-MS analysis identified eight phenolic compounds, including rutin, kaempferol glycosides, and chlorogenic acids. The extract also exhibited marked hepatoprotective effects against CdCl2 toxicity by reducing hepatic levels of malondialdehyde, advanced oxidation protein products, hydrogen peroxide, metallothioneins, and DNA degradation. Additionally, co-administration of Narcissus tazetta flower extracts lowered the plasma activities of transaminases, gamma glutamyl transpeptidase, and lactate dehydrogenase and increased hepatic levels of reduced glutathione, nonprotein thiols, vitamin C, and catalase activity. The hepatoprotective effects of the extract were demonstrated by histopathological improvement of liver disorders. The current study provided ethnopharmacological application of Narcissus tazetta flower extracts against CdCl2-induced oxidative stress, suggesting its chemoprevention role of its phenolic compounds as a natural antioxidant.

Phytochemical Characterization and Bioactivity of Asparagus acutifolius: A Focus on Antioxidant, Cytotoxic, Lipase Inhibitory and Antimicrobial Activities.

The phytochemical composition of leaves, stems, pericarps and rhizomes ethanolic extracts of Asparagus acutifolius were characterized by HPLC-DAD-MS. A. acutifolius samples contain at least eleven simple phenolics, one flavonon, two flavonols and six steroidal saponins. The stem extracts showed the highest total phenolic acid and flavonoid contents, where cafeic acid and rutin were the main compounds. No flavonoids were detected in the leaf, pericarp or rhizome while caffeic acid and ferulic acid were the predominant. Steroidal saponins were detected in the different plant parts of A. acutifolius, and the highest contents were found in the rhizome extracts. The stem extracts exhibited the highest antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) and the highest 2,2-azino-bis (3 ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging activity was found in the pericarp extracts. The rhizome and leaf extracts showed a potent cytotoxic activity against HCT-116 and HepG2 cell lines. Moreover, the pericarp and rhizome extracts revealed a moderate lipase inhibitory activity. The leaf and rhizome extracts were screened for their antimicrobial activity against human pathogenic isolates. The leaf extract exhibited a powerful inhibitory activity against all the bacteria and fungi tested.

Nrf2 controls iron homeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The Nrf2 transcription factor orchestrates cell-intrinsic protective antioxidant responses, and the peptide hormone hepcidin maintains systemic iron homeostasis, but is pathophysiologically decreased in haemochromatosis and beta-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives Bmp6 expression in liver sinusoid endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6-hepcidin response to oral and parenteral iron is impaired and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6-hepcidin axis, improving iron homeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in beta-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to control of systemic iron homeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders.

Does Lycium europaeum leaf have antihyperglycemic, antihyperlipidemic and antioxidant effects

The purpose of the present investigation is to assess, for the first time, the antidiabetic, antihyperlipidemic and antioxidant activities of Lycium europaeum extract in alloxan-induced diabetic rats. Diabetes was induced in adult male Wistar rats via a single subcutaneous alloxan injection (120 mg/kg). Lycium europaeum aqueous extract was orally administered at a dose of 20 mg/kg for 28 consecutive days. Serum concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were assayed at the end of the experimental period in all investigated groups. Antioxidant enzymes such as glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were sought in the serum and pancreas. Lycium europaeum extract significantly increased HDL-C and reduced blood glucose, TC, LDL-C and TG as compared to the alloxan-control group. Lycium europaeum extract was also efficient in reducing oxidative stress in diabetic rats by increasing SOD, CAT and GPx activities both in the pancreas and the plasma of the animals. Moreover, Lycium europaeum extract contained considerable levels of polyphenols and flavonoids. It also exhibited an important antioxidant capacity and a remarkable ability to quench DPPH radicals and reduce irons. The obtained results highlight potentially relevant health beneficial effects of Lycium europaeum extract, reversing hyperglycemic, hyperlipidemic and oxidative stress effects in rats with alloxan-induced diabetes. Therefore, it may be considered as a promising alternative or complementary agent to diabetes treatment.

Inhibition of heme oxygenase ameliorates anemia and reduces iron overload in a ß-thalassemia mouse model.

Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoiesis. In ß-thalassemia, ß-globin synthesis is reduced causing α-globin accumulation. Unpaired globin chains, with heme attached to them, accumulate in thalassemic erythroblasts causing oxidative stress and the premature cell death. We hypothesize that in ß-thalassemia heme oxygenase (HO) 1 could play a pathogenic role in the development of anemia and ineffective erythropoiesis. To test this hypothesis, we exploited a mouse model of ß-thalassemia intermedia, Th3/+ We observed that HO inhibition using tin protoporphyrin IX (SnPP) decreased heme-iron recycling in the liver and ameliorated anemia in the Th3/+ mice. SnPP administration led to a decrease in erythropoietin and increase in hepcidin serum levels, changes that were accompanied by an alleviation of ineffective erythropoiesis in Th3/+ mice. Additionally, the bone marrow from Th3/+ mice treated with SnPP exhibited decreased heme catabolism and diminished iron release as well as reduced apoptosis. Our results indicate that the iron released from heme because of HO activity contributes to the pathophysiology of thalassemia. Therefore, new therapies that suppress heme catabolism may be beneficial in ameliorating the anemia and ineffective erythropoiesis in thalassemias.

The phytochemical and bioactivity profiles of wild Asparagus albus L. plant.

The ethanolic extracts from the leaves, pericarps and rhizomes of Asparagus albus L. were investigated for their phytochemical composition, antioxidant (DPPH and FRAP assays), anti-microbial against human pathogenic isolates and cytotoxic (human colon carcinoma HCT-116 cells) activities. The highest flavonoid content was obtained in the leaf extract followed by the pericarp but there were no flavonoids detected in the rhizome. However, the rhizome had a high concentration of saponins. Flavonoid and saponin profiles were similar to those previously described for the triguero Huetor Tajar asparagus landrace. It was found that the pericarp ethanolic extract exhibited higher antioxidant activity than rhizome and leaf extracts. Moreover, the rhizome possessed more evident cytotoxic activity against HCT-116 cells in comparison to leaf and pericarp. All extracts showed varying degrees of antimicrobial activity against most of the human pathogenic isolates. In addition, the leaves showed more powerful inhibitory activities against the maximum number of bacteria and all the fungai isolated and the highest activity was in the pericarp extract against multidrug resistant Pseudomonas aeruginosa (MDR) and Erythromycin resistant Streptococcus agalactiae (ER) with an inhibition zone of 21mm and 19mm, respectively. The results show that A. albus could be a new crop with pharmaceutical interest because its richness in bioactive compounds provides considerable benefits for human health.

Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59Fe by reticulocytes and diminishes 59Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

A small molecule screen in yeast identifies inhibitors targeting protein-protein interactions within the vaccinia virus replication complex.

Genetic and biochemical data have identified at least four viral proteins essential for vaccinia virus (VACV) DNA synthesis: the DNA polymerase E9, its processivity factor (the heterodimer A20/D4) and the primase/helicase D5. These proteins are part of the VACV replication complex in which A20 is a central subunit interacting with E9, D4 and D5. We hypothesised that molecules able to modulate protein-protein interactions within the replication complex may represent a new class of compounds with anti-orthopoxvirus activities. In this study, we adapted a forward duplex yeast two-hybrid assay to screen more than 27,000 molecules in order to identify inhibitors of A20/D4 and/or A20/D5 interactions. We identified two molecules that specifically inhibited both interactions in yeast. Interestingly, we observed that these compounds displayed a similar antiviral activity to cidofovir (CDV) against VACV in cell culture. We further showed that these molecules were able to inhibit the replication of another orthopoxvirus (i.e. cowpox virus), but not the herpes simplex virus type 1 (HSV-1), an unrelated DNA virus. We also demonstrated that the antiviral activity of both compounds correlated with an inhibition of VACV DNA synthesis. Hence, these molecules may represent a starting point for the development of new anti-orthopoxvirus drugs.

Yeast two-hybrid methods and their applications in drug discovery.

The yeast two-hybrid (Y2H) method was first described over 20 years ago. It soon appeared as a major methodological breakthrough in the discovery and analysis of protein interactions, which play a pivotal role in all biological phenomena. Since its inception the Y2H method has constantly evolved and has inspired various assays that have found multiple applications of interest for drug discovery. Y2H methods are used to identify and validate therapeutic targets, discover protein interaction modulators, identify drug targets, and select combinatorial recognition molecules, which themselves find a wide range of applications. We review here the different transcriptional Y2H methods that are directly useful to drug discovery. Most should be increasingly used in the future as they continue to evolve to harness other methodological and conceptual advances.

First BRET-based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors.

The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates.

Incontinentia pigmenti: a rare cause of retinal vasculitis in children.

BACKGROUND: Incontinentia pigmenti is a genetic disease with cutaneous, dental, neurologic and ophthalmologic manifestations. Ocular changes are very varied. Retinal vasculitis is uncommon, but can be observed in this disease. AIM: To report a case of a patient with retinal vasculitis associated to incontinentia pigmenti. REPORT: A seven year old girl was followed for cutaneous lesions with the diagnosis of incontinentia pigmenti confirmed by a skin biopsy. She had delayed eruption of her teeth some of which were conical. She had no neurologic nor developmental abnormalities. Ophthalmological examination and fluorescein angiography was performed and showed retinal neovascularization in the left eye, secondary to retinal ischemia and retinal sequelae vasculitis without inflammation or complications in the right eye. Laser photocoagulation was indicated to the left and a strict surveillance to the right eye. After one month of the beginning of treatment, we noticed an involution of the retinal neovascularisation. CONCLUSION: Early diagnosis and management of retinal changes in incontinentia pigmenti is essential because they may be serious leading to blindness.