Broad bean (Vicia faba L.) pods: a rich source of bioactive ingredients with antimicrobial, antioxidant, enzyme inhibitory, anti-diabetic and health-promoting properties.

This study was aimed at investigating the chemical composition (proximate, minerals, fatty acids and phenolic compounds) and the in vitro (antimicrobial, radical scavenging, anti-acetylcholinesterase and protein denaturing activities) and in vivo (anti-diabetic and histo-protective effects in alloxan-induced diabetic mice) biological activities of broad bean pods (BBPs), a food waste by-product material. The results showed that BBPs have high dietary fiber (57.46%), carbohydrate (18.93%) and protein (13.81%) content versus low fat content (<1%) contributing to a low energy value of 139.24 kcal per 100 g. Profiling of fatty acids showed an abundance of the essential polyunsaturated α-linolenic and linoleic acids, exhibiting an excellent nutritional quality as revealed by their low atherogenic and thrombogenic indices and their hypocholesterolemic properties. The methanol extract which exhibited the highest total phenolic, flavonoid and tannin contents was found to be the most active extract in terms of antimicrobial and anti-radical activities. In alloxan-induced diabetic mice, the oral administration of a methanol extract (500 mg per kg bw) attenuated the elevated levels of serum alanine aminotransferase (ALA), aspartate aminotransferase (AST), and alkaline phosphatase activities, and urea, uric acid, and creatinine. It effectively normalized the status of lipid profiles, mitigated oxidative stress through the activation of antioxidant enzymes (CAT, GPx and SOD), and alleviated oxidative stress-mediated histopathological changes in the pancreas, liver, kidney and testis. Compositional analysis by HPLC-PDA-ESI-MS/MS revealed the presence of flavan-3-ols (catechin, epicatechin and their derivatives), flavones (apigenin derivatives) and flavonols (glycosides of quercetin and kaempferol), among others. These findings suggest that BBPs may be an effective functional food for the management of diabetes and its complications.

Unlocking the in vitro anti-inflammatory and antidiabetic potential of Polygonum maritimum.

CONTEXT: Several Polygonum species (Polygonaceae) are used in traditional medicine in Asia, Europe and Africa to treat inflammation and diabetes. OBJECTIVE: Evaluate the in vitro antioxidant, anti-inflammatory and antidiabetic potential of methanol and dichloromethane extracts of leaves and roots of the halophyte Polygonum maritimum L. MATERIAL AND METHODS: Antioxidant activity was determined (up to 1 mg/mL) as radical-scavenging activity (RSA) of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), copper (CCA) and iron (ICA) chelating activities and iron reducing power (FRAP). NO production was measured in lipopolysaccharide (LPS)-stimulated macrophages for 24 h at concentrations up to 100 µg/mL and antidiabetic potential was assessed by α-amylase and α-glucosidase inhibition (up to 10 mg/mL) assays. The phytochemical composition of the extracts was determined by gas chromatography-mass spectrometry (GC-MS). RESULTS: The methanol leaf extract had the highest activity against DPPH• (IC50 = 26 µg/mL) and ABTS+• (IC50 = 140 µg/mL), FRAP (IC50 = 48 µg/mL) and CCA (IC50 = 770 µg/mL). Only the dichloromethane leaf extract (LDCM) showed anti-inflammatory activity (IC50 = 48 µg/mL). The methanol root (IC50 = 19 µg/mL) and leaf (IC50 = 29 µg/mL) extracts strongly inhibited baker's yeast α-glucosidase, but LDCM had higher rat's α-glucosidase inhibition (IC50 = 2527 µg/mL) than acarbose (IC50 = 4638 µg/mL). GC-MS analysis identified ß-sitosterol, stigmasterol, 1-octacosanol and linolenic acid as possible molecules responsible for the observed bioactivities. CONCLUSIONS: Our findings suggest P. maritimum as a source of high-value health promoting commodities for alleviating symptoms associated with oxidative and inflammatory diseases, including diabetes.

Isololiolide, a carotenoid metabolite isolated from the brown alga Cystoseira tamariscifolia, is cytotoxic and able to induce apoptosis in hepatocarcinoma cells through caspase-3 activation, decreased Bcl-2 levels, increased p53 expression and PARP cleavage.

BACKGROUND: Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. Inthis study we isolated isololiolide from Cystoseira tamariscifolia for the first time. PURPOSE: To examine the therapeutical potential of isololiolide against tumor cell lines. METHODS/STUDY DESIGN: The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting. RESULTS: Isololiolidewas isolated for the first time from the brown macroalga C.tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels. CONCLUSION: Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins.

In vitro antioxidant and anti-inflammatory properties of Limonium algarvense flowers' infusions and decoctions: A comparison with green tea (Camellia sinensis).

This work reports the in vitro antioxidant and anti-inflammatory activities and toxicity of infusions and decoctions of Limonium algarvense flowers, and green tea. The total contents in different phenolic groups and the quantification of individual phenolics by HPLC are also reported. L. algarvense and green tea had similar antioxidant properties, except for hydroxyl radical-scavenging activity, higher on green tea, and iron chelating potential, higher on L. algarvense. The later species also had the uppermost anti-inflammatory potential. Green tea decoction had the highest content of phenolic groups, but the infusion of L. algarvense had higher amounts of salicylic, gallic and coumaric acids. L. algarvense was not toxic, whereas green tea was toxic for S17 cells. Under our experimental conditions, infusions and decoctions of L. algarvense flowers had similar or higher antioxidant and anti-inflammatory properties than green tea, and thus, may be useful for alleviating symptoms associated with oxidative and inflammatory-related diseases.

Digestibility and Bioavailability of the Active Components of Erica australis L. Aqueous Extracts and Their Therapeutic Potential as Acetylcholinesterase Inhibitors.

Erica australis L. (Ericaceae) is used in traditional medicine to treat many free-radical related ailments. In the present work, the stability and biological activity of the plant aqueous extracts submitted to an in vitro digestive process were investigated. Chemical stability was monitored by HPLC-DAD and LC-MS/MS, while the bioactivities were evaluated through the inhibition of acetylcholinesterase (AChE) and DPPH radical scavenging activity. Both extracts, whose main components were flavonol glycosides, inhibited AChE, showing IC50 values of 257.9 ± 6.2 µg/mL and 296.8 ± 8.8 µg/mL for the decoction and for the infusion, respectively. Significant radical scavenging activities were also revealed by both extracts, as denoted by the IC50 values for the decoction, 6.7 ± 0.1 µg/mL, and for the infusion, 10.5 ± 0.3 µg/mL. After submission to gastric and pancreatic juices, no remarkable alterations in the composition or in the bioactivities were observed, suggesting that the extracts may pass through the gastrointestinal tract, keeping their composition and therefore their biological properties. Moreover, the bioavailability of the components of both extracts, as studied in a Caco-2 cell model, showed that compounds can permeate the membrane, which is a condition to exert their biological activities. Our results add further support to the potential of E. australis for its antioxidant and neuroprotective properties.

In vitro antitumoral activity of compounds isolated from Artemisia gorgonum Webb.

Artemisia gorgonum (Asteraceae) is an endemic plant to the Cape Verde islands and plays an important role in traditional medicine. The chloroform extract of the plant aerial parts afforded six sesquiterpene lactones, two methoxylated flavonoids, two lignans, and one tetracyclic triterpene, which were isolated by chromatographic methods and their structure established by physical and spectroscopic techniques. The cytotoxic activity of the three major constituents, namely, arborescin, artemetin, and sesamin, was evaluated on neuroblastoma (SH-SY5Y), hepatocarcinoma (HepG2), and nontumoral bone marrow stromal (S17) cell lines. The application of different concentrations of the compounds significantly decreased tumor cells viability at different extents, especially at the highest concentrations tested. Arborescin is the most promising compound as it was able to reduce tumoral cell viability with an IC50 significantly lower (229-233 µM; p < 0.01) than that of S17 cells (445 µM). Arborescin and artemetin were less toxic to nontumoral cells than the antitumoral drug tested, etoposide. Our results indicate that arborescin has a significant cytotoxic activity in vitro, more pronounced on the cancer cell lines, confirming A. gorgonum as a source of potential antitumoral molecules.

Molecular recognition of rosmarinic acid from Salvia†sclareoides extracts by acetylcholinesterase: a new binding site detected by NMR spectroscopy.

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer's disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC-MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4'-O-glucoside, luteolin 3',7-di-O-glucoside and luteolin 7-O-(6''-O-acetylglucoside) were identified by MS(2) and MS(3) data acquired during the LC-MS(n) runs. Quantification of rosmarinic acid by HPLC with diode-array detection (DAD) showed that the butanol extract is the richest one in this component (134 µg mg(-1) extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE-bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD-based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.

Extraction of volatile oil from aromatic plants with supercritical carbon dioxide: experiments and modeling.

An overview of the studies carried out in our laboratories on supercritical fluid extraction (SFE) of volatile oils from seven aromatic plants: pennyroyal (Mentha pulegium L.), fennel seeds (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), savory (Satureja fruticosa Béguinot), winter savory (Satureja montana L.), cotton lavender (Santolina chamaecyparisus) and thyme (Thymus vulgaris), is presented. A flow apparatus with a 1 L extractor and two 0.27 L separators was built to perform studies at temperatures ranging from 298 to 353 K and pressures up to 30.0 MPa. The best compromise between yield and composition compared with hydrodistillation (HD) was achieved selecting the optimum experimental conditions of extraction and fractionation. The major differences between HD and SFE oils is the presence of a small percentage of cuticular waxes and the relative amount of thymoquinone, an oxygenated monoterpene with important biological properties, which is present in the oils from thyme and winter savory. On the other hand, the modeling of our data on supercritical extraction of volatile oil from pennyroyal is discussed using Sovová's models. These models have been applied successfully to the other volatile oil extractions. Furthermore, other experimental studies involving supercritical CO(2) carried out in our laboratories are also mentioned.

Synthesis of sugars embodying conjugated carbonyl systems and related triazole derivatives from carboxymethyl glycoside lactones. Evaluation of their antimicrobial activity and toxicity.

The synthesis of a series of pyranoid derivatives comprising a conjugated carbonyl function and related triazole derivatives, structurally suitable for bioactivity evaluation, was achieved in few steps starting from readily available carboxymethyl glycoside lactones (CMGL). 3-Enopyranosid-2-uloses were generated by oxidation/elimination of tri-O-acylated 2-hydroxy pyranosides. Subsequent Wittig olefination provided stereoselectively 2-C-branched-chain conjugated dienepyranosides with (E)-configuration around the exocyclic double bond. A heterogeneous CuI/Amberlyst-catalyzed 'click' chemistry protocol was used to convert glycosides bearing a propargyl moiety into the corresponding 1,2,3-triazoles. These new molecules were screened for their in vitro antibacterial and antifungal activities and those containing conjugated carbonyl systems demonstrated the best efficacy. (N-Dodecylcarbamoyl)methyl enone glycerosides were the most active ones among the enones tested. The α-anomer displayed very strong activities against Bacillus cereus and Bacillus subtilis and strong activity toward Enterococcus faecalis and the fungal pathogen Penicillium aurantiogriseum. The corresponding ß-anomer presented a very strong inhibitory effect against two fungal species (Aspergillus niger and P. aurantiogriseum). (N-Dodecyl-/N-propargyl/or N-benzylcarbamoyl)methyl dienepyranosides exhibited selectively a strong activity toward E. faecalis. Further acute toxicity evaluation indicated low toxic effect of the (N-dodecylcarbamoyl)methyl enone glyceroside α-anomer and of the carbamoylmethyl dienepyranosides N-protected with propargyl or benzyl groups.

Antihyperglycaemic and protective effects of flavonoids on streptozotocin-induced diabetic rats.

The antihyperglycaemic effect of eight standard flavonoids, previously identified in the ethanol extract of the claimed antidiabetic plant Genista tenera, was evaluated on streptozotocin (STZ)-induced diabetic Wistar rats. The aglycones apigenin, chrysoeriol and genistein, the monoglucosides apigenin 7-O-glucoside, luteolin 7-O-glucoside and genistein 7-O-glucoside and the diglycosides rutin and luteolin 7,3'-di-O-glucoside were administered i.p. for 7 days (4 mg/kg b.w./day). The protective effect of these compounds over liver and kidneys of STZ-diabetic models was also evaluated by the determination of seric AST, ALT and urea levels. After 7 days of treatment, apigenin, chrysoeriol and genistein significantly lowered the blood glucose levels of diabetic animals; this effect was more pronounced (P < 0.01) in the oral glucose tolerance test. Glucose tolerance was also significantly improved in the rutin (P < 0.01) and in the genistein 7-O-glucoside (P < 0.05) treated groups. In addition, almost all the tested compounds effectively protected the liver and kidneys against STZ-induced damage in rats.

Phytochemical profile and anticholinesterase and antimicrobial activities of supercritical versus conventional extracts of Satureja montana.

Winter savory Satureja montana is a medicinal herb used in traditional gastronomy for seasoning meats and salads. This study reports a comparison between conventional (hydrodistillation, HD, and Soxhlet extraction, SE) and alternative (supercritical fluid extraction, SFE) extraction methods to assess the best option to obtain bioactive compounds. Two different types of extracts were tested, the volatile (SFE-90 bar, second separator vs HD) and the nonvolatile fractions (SFE-250 bar, first and second separator vs SE). The inhibitory activity over acetyl- and butyrylcholinesterase by S. montana extracts was assessed as a potential indicator for the control of Alzheimer's disease. The supercritical nonvolatile fractions, which showed the highest content of (+)-catechin, chlorogenic, vanillic, and protocatechuic acids, also inhibited selectively and significantly butyrylcholinesterase, whereas the nonvolatile conventional extract did not affect this enzyme. Microbial susceptibility tests revealed the great potential of S. montana volatile supercritical fluid extract for the growth control and inactivation of Bacillus subtilis and Bacillus cereus, showing some activity against Botrytis spp. and Pyricularia oryzae. Although some studies were carried out on S. montana, the phytochemical analysis together with the biological properties, namely, the anticholinesterase and antimicrobial activities of the plant nonvolatile and volatile supercritical fluid extracts, are described herein for the first time.

Bioactivity studies and chemical profile of the antidiabetic plant Genista tenera.

AIM OF THE STUDY: Genista tenera is a plant endemic to the island of Madeira and is used in folk medicine to control diabetes. In the present work we evaluate the antihyperglycaemic activity of its n-butanol extract and determine its chromatographic profile. In addition, this extract, the ethyl acetate and diethyl ether plant extracts were studied in order to assess the plant antioxidant and acetylcholinesterase inhibitory activities, as well as its cyto- and genotoxicities. MATERIALS AND METHODS: HPLC-DAD-ESI-MS was used to analyze the flavonoid profile of the n-butanol extract. The antihyperglycaemic activity of this extract was performed over streptozotocin induced diabetic Wistar rats (200 mg/kg, bw/day), for 15 days. Antioxidant activity (DPPH assay) and acetylcholinesterase inhibitory effect (Ellman method) were also performed. Acute cytotoxicity and genotoxicity were assessed by proliferative index quantification and the short-term chromosomal aberration technique, after exposure of lymphocytes to the extracts. RESULTS AND CONCLUSIONS: The n-butanol extract, where 21 monoglycosyl and 12 diglycosyl flavonoids were detected, significantly lowered blood glucose levels, bringing them to normal values after 15 days of treatment. The best radical scavenging activity was observed for the ethyl acetate extract (48.7% at 139.1 microg/mL), which was also the most effective one at the minimal concentration tested. The highest acetylcholinesterase inhibitory activity (77.0% at 70.0 microg/mL) was also obtained with the ethyl acetate extract. In vitro toxicity studies showed no evidence for acute cytotoxicity or genotoxicity. This is the first report on antidiabetic activity of genus Genista.

A new lupene triterpenetriol and anticholinesterase activity of Salvia sclareoides.

A new lupene triterpenetriol was isolated from the acetone extract of the aerial parts of Salvia sclareoides and characterised as (1beta,3beta)-lup-20(29)-ene-1,3,30-triol (1). In addition, nepetidin (2), nepeticin (3), lupendiol (4), (1beta,11alpha)-dihydroxy-lup-20(29)-en-3-one (5), ursolic acid (6), sumaresinolic acid (7) and hederagenin (8), were identified in this Salvia sp. To the best of our knowledge, the compounds 2 and 7 are new constituents in Salvia spp. The acetone, ethanol, butanol and water extracts of the plant were screened for the in vitro inhibitory activity of acetylcholinesterase (AChE) and butyrilcholinesterase (BChE), enzymes which play a role in the Alzheimer disease. All extracts inhibited acetylcholinesterase activity at 10 microg/ml, a remarkable activity since the standard drug rivastigmine does not inhibit acetylcholinesterase at the same concentration. Regarding the butyrilcholinesterase, the acetone extract at 1000 microg/ml was able to inhibit completely the enzyme activity and the butanol and ethanol extracts, at this concentration, produced a potent inhibition of BchE.

Capillary electrophoresis-mass spectrometry characterisation of secondary metabolites from the antihyperglycaemic plant Genista tenera.

Genista tenera is endemic to the Portuguese island of Madeira, where an infusion of the aerial parts of the plant is used in folk medicine as an antidiabetic agent. Consequently the medicinal properties of the secondary metabolites of this plant have been the subject of an ongoing study. A recently reported LC-MS method using a 100 min separation allowed identification of five flavonoid components in an extract of the aerial parts of this plant. In order to obtain additional information on the range and complexity of the plant's secondary metabolite components a CE-MS method has been developed and applied for the analysis of an extract of G. tenera. Twenty-six different components are distinguished in an analysis time of only 10 min. Results demonstrate that CE-MS/MS rapidly generates data complementary to those obtainable by LC-MS/MS and is particularly suited to the analysis of plant metabolites where concentration is not limiting.

Liquid chromatography-diode array detection-electrospray ionisation mass spectrometry/nuclear magnetic resonance analyses of the anti-hyperglycemic flavonoid extract of Genista tenera. Structure elucidation of a flavonoid-C-glycoside.

The anti-hyperglycemic flavonoid extract obtained from Genista tenera was first studied by liquid chromatography (LC)-diode array detection (DAD) which showed the presence of two major compounds. One of them was identified as genistein-7-O-glucoside. Luteolin-7-O-glucoside was detected as a minor constituent, while luteolin-7,3'-di-O-glucoside and rutin were found in trace amounts. LC-DAD-ESI-MS and NMR were used to confirm the structure of these compounds and allowed the elucidation of the structure of the unknown major compound, which is the flavonoid 5,7,4'-trihydroxyisoflavone-8-C-glucoside.

A new dihydroxysterol from the marine phytoplankton Diacronema sp.

Diacronema sp. was cultured and its sterols were separated by column chromatography on silica gel. The new sterol 24-ethyl-4alpha-methyl-cholestane-3,20-diol (1) was characterised by NMR and MS spectrometry, as well as (22E)-24-ethyl-4alpha-methyl-5alpha-cholest-22-en-3beta-ol (2) and beta-sitosterol, the major components of the sterol fractions. Neither the biosynthetic origin of the new dihydroxysterol nor its role in the biochemistry of Diacronema is known.

A novel pentacyclic triterpene from Leontodon filii.

A novel oleanene triterpenetetrol was isolated from the chloroform extract of the aerial parts of Leontodon filii. Its structure was shown to be 2beta,3beta,15alpha,21beta-olean-12-ene-2,3,15,21-tetrol by chemical and spectroscopic methods. The fungicidal efficacy of the chloroform and methanol extracts of the plant was also evaluated, a protective effect being found against Plasmopara viticola, Botrytis cinerea, particularly powerful against Pyricularia oryzae.