HPLC-HESI-MS/MS Analysis of Phenolic Compounds from Cynoglossum tubiflorus Leaf Extracts: An Assessment of Their Cytotoxic, Antioxidant, and Antibacterial Properties.

The current study aimed to investigate the chemical composition, antioxidant, antibacterial, and cytotoxic properties of three extracts (hexane, dichloromethane, and methanol) from Cynoglossum tubiflorus. The composition of the methanolic extract was elucidated using HPLC-HESI-MS/MS analysis. The antioxidant effect was examined using NO, DPPH, FRAP, and TAC assays. Antimicrobial activity was evaluated by broth microdilution using various bacterial strains such as S. aureus, S. epidermidis, P. aeruginosa, E. coli, and K. pneumoniae. Structural disruptions in Gram-positive bacteria were visualized using scanning electron microscopy (SEM). Cytotoxic effects were evaluated on human MRC-5 in culture according to the MTT assay. The outcomes suggest that methanol extract contained a high amount of phenolic compounds (254.35 ± 0.360 mg GAE/g DE and 211.59 ± 0.939 mg QE/g DE). By applying the HPLC-HESI-MS/MS analysis, 32 compounds were identified, including phenolic acids, flavonoids, lignans, and fatty acids. This extract showed strong antioxidant (IC50 = 0.043 ± 0.001 mg/mL) and antimicrobial (MIC = 156 µg/mL) activities. The SEM suggests that cells exhibited membrane distortions characterized by surface depressions and alterations in bacterial shape, including dents, when compared to untreated cells. The in vitro cytotoxicity effect on human MRC-5 cells showed no toxicity effects at a concentration of 600 µg/mL. In silico analysis predicted low toxicity for all tested compounds across four different administration routes. This research indicates that this plant could be explored as a powerful source of natural drugs to target pathogens, with applications in the food, pharmaceutical, and medical industries.

Fructooligosaccharides from Cynoglossum tubiflorus: Effect of the molecular size on their antidiabetic activity in high-fat diet and alloxan induced diabetic rats.

The use of acetylation followed by silica gel column purification allowed the isolation of eight fructooligosaccharides (FOS) from the ethanol extract of Cynoglossum tubiflorus roots. Each FOS was identified by analyzing its FT-IR, HRMS/MS and NMR data, including 1H, 13C and 2D NMR HH COSY, HMBC and NOESY. In diabetic rats treated with a series of FOS from Glc-(Fru)3 to Glc-(Fru)7, a significant inhibition of intestinal α-amylase was observed. This activity increases proportionally with the FOS molecular size. It was found that they delay the absorption of total cholesterol (TC), ldl-cholesterol (LDL-C) and increase HDL-cholesterol (HDL-C) in a molecular size-dependent manner. This inhibitory effect on the activity of the digestive enzyme causes a significant (p < 0.05) reduction in the level of glucose in the blood as an anti-diabetic action. The ethanolic extract (E.E) exerts a significant effect against α-amylase as well as antihyperglycemic and antihyperlipidemic actions, while its acetylation suppresses these effects. Therefore, this study demonstrates for the first time that pure FOS act as an efficient agent in preventing hyperglycemia and hyperlipidemia and that this action evolves in the same manner with their molecular size.

The sweet and embellishing Lycium arabicum Schweinf. ex Boiss. fruit oil: a potential source of essential ω-6 and ω-9 fatty acids, phytosterols, and carotenoids.

In our current phytochemical investigation on the Tunisian plant Lycium arabicum Schweinf. ex Boiss., we attempted to explore the oil obtained from its fruits. This oil was extracted by cold maceration with ethanol and was analyzed to estimate its sterol and fatty acid content. The physicochemical parameters of the oil were also examined. They were specified in terms of acid value (0.8 ± 0.01 mg KOH g-1 oil), peroxide value (10.9 ± 0.02 meq O2 kg-1 oil), saponification value (162.97 ± 0.03 mg KOH g-1 oil), chlorophylls (1.011 ± 0.03 mg kg-1 oil) and carotenoids (42.1 ± 0.05mg kg-1 oil). Gas chromatography analysis demonstrated that oleic (33.5%), palmitic (26.81%) and linoleic (21.51%) acids were the dominant fatty acids. Furthermore, the analysis of this oil with HPLC equipped with a differential refractometer detector (DRD) showed that Palmitic-Oleic-Oleic (21.10%) and Oleic-Oleic-Oleic/Palmitoleic-Palmitic-Palmitic (17.11%) are the main triacylglycerols in this oil. It was also found that this oil contains high levels of ß-sitosterol (31.51%), Δ-5-Avenasterol (25.17%), and Δ-7-Avenasterol (15.22%). Analysis of its spectroscopic features allowed us to deduce that this oil contains some sugars like glucose, sucrose, and fructose as well as several carotenoid pigments. From this perspective, Lycium arabicum fruits oil (LAFO) maybe regarded as a worthwhile product that deserves supplementary consideration and investigation as a potentially new multi-purpose candidate for agro-food as a sweetener and a beautifier, in addition to its extreme usefulness for cosmetic and medicinal applications owing to its richness in phytosterols and conservative pigments.

Effect of extraction procedures on the chemical structure, antitumor and anticoagulant properties of ulvan from Ulva lactuca of Tunisia coast.

The effect of extraction procedures on chemical composition, structural, antitumor and anticoagulant properties of the sulphated polysaccharide 'ulvan' from the green seaweed Ulva lactuca were investigated. The structural features of ulvans were carried out by FTIR and by one- and two- dimensional 1H and 13C NMR spectroscopic. The ulvans were mainly composed of rhamnose, xylose, and uronic acid. Chemical and spectroscopic analyses demonstrated that ulvans were constituted of (1→4)-ß-glucuronic acid, (1→3,4)-α-L-rhamnose-3-sulphate and (1→4)-α-xylose. The extraction procedures effect were observed in chemical structure, Mw and biological activities. Cytotoxic activity of enzymatic-chemical extract on cervical cancer cells (HeLa) (IC50 = 1000 µg/mL) was higher than on normal peripheral blood lymphocytes cells (PBL). Acid extracts promoted to reduce HeLa cells and to grow PBL cells. At high concentrations, acid extracts showed the highest APTT and TT clotting time. Antitumoral and anticoagulant activities of ulvans from Ulva lactuca promote their use as effective therapeutic agent.

The saltern-derived Paludifilum halophilum DSM 102817T is a new high-yield ectoines producer in minimal medium and under salt stress conditions.

In the present study, the growth conditions and accumulation of ectoines (ectoine and hydroxyectoine) by Paludifilum halophilum DSM 102817T under salt stress conditions have been investigated. The productivity assay of this strain for ectoines revealed that the highest cellular content was reached in the minimal glucose sea water medium (SW-15) within 15% salinity. The addition of 0.1% (w/v) aspartic acid to the medium allowed an average of four times higher biomass production, and a dry mycelial biomass of 1.76 g L-1 was obtained after 6 days of growth in shake flasks at 40 °C and 200 rpm. Among the inorganic cations supplemented to the glucose SW-15 medium, the addition of 1 mM Fe2+ yielded the highest amount of mycelial biomass (3.45 g L-1) and total ectoines content (119 mg g-1), resulting in about 410 mg L-1 of products at the end of exponential growth phase. After 1 h of incubation in an osmotic downshock solution containing 2% NaCl, 70% of this content was released by the mycelium, and recovering cells maintained a high survival, with a maximal growth rate (µ max) of about 93% of the control population exposed to 15% NaCl. During growth at optimal salinity and temperature (15% NaCl and 40 °C), P. halophilum developed a compact and circular pellets that were easy to separate by simple decantation from both fermentation media and after hypoosmotic shock. Overall, the ectoines excreting P. halophilum could be a promising resource for ectoines production in a commercially valuable culture medium and at a large-scale fermentation process.

Polysaccharide from Lycium arabicum: Structural Features, in Vitro Antioxidant Activities and Protective Effect against Oxidative Damage in Human Erythrocytes.

In this research work, a water-soluble polysaccharide (LAP) isolated from the fruits of Lycium arabicum was investigated. LAP contains carbohydrates (82.45±1.23 %), protein (1.56±0.21 %), and uronic acids (3.56±0.34 %). The analysis of the monosaccharide composition revealed the presence of rhamnose, arabinose, galactose, glucose and mannose in a molar ratio of 4.7 : 1.5 : 1 : 8.7 : 16.4 : 5.6. The extracted polysaccharide (PS) was considered as heterogeneous and highly branched by interpreting its GC/MS, FT-IR and NMR data. Crystallinity of LAP was inferred from its X-ray diffractometry (XRD) and Scanning Electron Microscopy (SEM) analysis. LAP exhibited an interesting stability at high temperatures (∼254 °C) and in a wide range of pH (3-9) deduced, respectively, from its DSC and zeta potential analysis. LAP displayed a strong antioxidant activity at low concentrations evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging, ferric reducing activity power (FRAP), free radical scavenging ability, superoxide radical-scavenging and hydroxyl radical-scavenging abilities. Inhibition of erythrocyte hemolysis and lipid peroxidation was also assessed. In 5 h, LAP treatment allowed the protection of the damaged erythrocytes caused by AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), to reduce the level of malondialdehyde (MDA) as well as to increase the reduced glutathione (GSH) level.

Water-Soluble Polysaccharides from Ephedra alata Stems: Structural Characterization, Functional Properties, and Antioxidant Activity.

In this study, the physicochemical characterization, functional properties, and antioxidant activity of polysaccharides extracted from Ephedra alata (EAP) were investigated. EAP were extracted in water during 3 h with a liquid/solid ratio of 5 in a water bath at 90 °C. The structure of the extracted EAP was examined by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and gas chromatography-mass spectrometry (GC-MS). The functional properties and biochemical activities of EAP were determined. The chemical analysis revealed that the contents of carbohydrates, uronic acid, and proteins were 73.24% ± 1.24%, 6.82% ± 0.57%, and 6.56% ± 0.36%, respectively. The results showed that the extracted EAP essentially contain three functional groups: C=O, C-H, and O-H. SEM images showed that EAP present numerous high porosity particles. The monosaccharide composition revealed a polymer composed of glucose (43.1%), galactose (36.4%), mannose (14.9%), arabinose (3.7%), and gluconic acid (1.7%). EAP showed interesting functional properties (solubility, oil holding capacity, foaming and emulsion properties). Finally, the results revealed that EAP displayed excellent antihypertensive and antioxidant activities. Overall, EAP present a promising natural source of food additives, antioxidants, and antihypertensive agents.

Enhancement of Antibacterial Activity of Paludifilum halophilum and Identification of N-(1-Carboxy-ethyl)-phthalamic Acid as the Main Bioactive Compound.

The aim of this study was to determine the combined effect of fermentation parameters and enhance the production of cellular biomass and antibacterial compounds from Paludifilum halophilum SMBg3 using the response surface methodology (RSM). Eight variables were screened to assess the effects of fermentation parameters on growth and metabolite production by Taguchi experimental design. Among these, the initial pH, temperature, and the percentage of MgSO4·7H2O in the medium were found to be most influential. The Box-Behnken design was applied to derive a statistical model for the optimization of these three fermentation parameters. The optimal parameters were initial pH: 8.3, temperature growth: 44°C, and MgSO4·7H2O: 1.6%, respectively. The maximum yield of biomass and metabolite production were, respectively, 11 mg/mL dry weight and 15.5 mm inhibition zone diameter against Salmonella enterica, which were in agreement with predicted values. The bioactive compounds were separated by the thick-layer chromatography technique and analyzed by GC/MS, NMR (1D and 2D), and Fourier-transform infrared spectroscopy (FT-IR). In addition to several fatty acids, N-(1-carboxy-ethyl)-phthalamic acid was identified as the main antibacterial compound. This element exhibited a potent activity against the ciprofloxacin-resistant Salmonella enterica CIP 8039 and Pseudomonas aeruginosa ATCC 9027 with a minimum inhibitory concentration (MIC) value range of 12.5-25 µg/mL. Results demonstrated that P. halophilum strain SMBg3 is a promising resource for novel antibacterial production due to its high-level yield potential and the capacity for large-scale fermentation.

UHPLC/HR-ESI-MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti-lipase Activities' Evaluation.

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti-lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and ß-carotene-linoleic acid bleaching assays) while anti-lipase activity was evaluated using 4-methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR-ESI-MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N-caffeoyltyramine, lyciumide A, N-dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.

Enzymatic hydrolysis of pretreated Alfa fibers (Stipa tenacissima) using ß-d-glucosidase and xylanase of Talaromyces thermophilus from solid-state fermentation.

This work aims at realizing an optimal hydrolysis of pretreated Alfa fibers (Stipa tenacissima) through the use of enzymes produced from Talaromyces thermophilus AX4, namely ß-d-glucosidase and xylanase, by a solid state fermentation process of an agro-industrial waste (wheat bran supplemented with lactose). The carbon source was firstly selected and the optimal values of three other parameters were determined: substrate loading (10g), moisture content (85%) and production time (10days); which led to an optimized enzymatic juice. The outcome was then supplemented with cellulases of T. reesei and used to optimize the enzymatic saccharification of alkali-pretreated Alfa fibers (PAF). The maximum saccharification yield of 83.23% was achieved under optimized conditions (substrate concentration 3.7% (w/v), time 144h and enzyme loading of 0.8 FPU, 15U CMCase, 60U ß-d-glucosidase and 125U xylanase).The structural modification of PAF due to enzymatic saccharification was supported by the changes of morphologic and chemical composition observed through macroscopic representation, FTIR and X-Ray analysis.

Agricultural wastes as substrates for ß-glucosidase production by Talaromyces thermophilus: Role of these enzymes in enhancing waste paper saccharification.

In the present study, we investigated a potent extracellular ß-glucosidases secreted by the thermophilic fungal strain AX4 of Talaromyces thermophilus, isolated from Tunisian soil samples. This strain was selected referring to the highest thermostability of its ß-glucosidases compared to the other fungal isolates. The ß-glucosidase production was investigated by submerged fermentation. The optimal temperature and initial pH for maximum ß-glucosidase production were 50°C and 7.0, respectively. Several carbon sources were assayed for their effects on ß-glucosidase production, significant yields were obtained in media containing lactose 1% (3.0 ± 0.36 U/ml) and wheat bran 2% (4.0 ± 0.4 U/ml). The combination of wheat bran at 2% and lactose at 0.8% as carbon source enhanced ß-glucosidase production, which reached 8.5 ± 0.28 U/ml. Furthermore, the ß-glucosidase-rich enzymatic juice of T. thermophilus exhibited significant synergism with Trichoderma reesei (Rut C30) cellulases for pretreated waste paper (PWP) hydrolysis. Interestingly, the use of this optimal enzymatic cocktail increased 4.23 fold the glucose yield after saccharification of waste paper. A maximum sugar yield (94%) was reached when using low substrate (2%) and enzyme loading (EC1).

Hexane Extract of Echinops spinosissimus Turra subsp. spinosus from Tunisia: A Potential Source of Acetylated Sterols - Investigation of its Biological Activities.

The hexane extract of Echinops spinosissimus Turra subsp. spinosus flower heads was analyzed for its fatty acid and sterol composition. Its physicochemical characteristics were also studied. The saponification, iodine and peroxide values were determined as 255 mg KOH/g, 42.57 g I2 /100 g and 110 m equiv. O2 /kg of oil, respectively. The oleic (C18:1; 61.14%), palmitic (C16:0; 21.36%) and linoleic (C18:2; 10.45%) acids were the dominant fatty acids. This extract was also found to contain high levels of ß-sitosterol and stigmasterol (44.97% and 34.95% of total sterols, respectively). On the other hand, the identification of terpenoid compounds was investigated by using GC/MS, which revealed fourteen major terpenoids mainly taraxasterol, lupeol, pseudotaraxasterol, lup-22(29)-en-3-yl acetate, taraxasteryl acetate, α-amyrin, ß-amyrin, pseudotaraxasteryl acetate, hop-20(29)-en3-ß-ol, α-amirenone, along with ß-sitosterol and stigmasterol. Moreover, we have evaluated the in vitro antibacterial and antifungal activities of the unsaponifiable matter and a fraction isolated from this extract. These activities were conducted using the diffusion disc methods and broth microdilution assay. The resulted fraction from this extract showed the highest antibacterial activity with significant minimum inhibitory concentrations (MIC) values 125.0 µg/ml against Staphylococcus aureus, Micrococcus luteus and Bacillus cereus. However, it did exhibit no substantial antifungal activity.

Structural characterization and functional properties of antihypertensive Cymodocea nodosa sulfated polysaccharide.

A sulfated polysaccharide was successfully isolated from Cymodocea nodosa (CNSP). This is the first report that indicates the chemical composition, structural characterization, functional and antihypertensive properties of this polysaccharide. The CNSP consisted mainly of sulfate (23.17%), total sugars (54.90%), galactose (44.89%), mannose (17.30%), arabinose (12.05%), xylose (9.18%), maltose (1.07%) and uronic acid (11.03%) with low water activity (0.49). CNSP had an XRD pattern that was typical for a semi-crystalline polymer with homogeneous structure. It also displayed an important anti-hypertensive activity (IC50=0.43mgml) with a dose-dependent manner using a synthetic substrate, N-hippuryl-His-Leu hydrate salt (HHL). Overall, the results indicate that CNSP have attractive chemical, functional and biological properties, with a preliminary structural may have a backbone of branched 6-O-sulfated (1→4) galactosidic linkages, which can be considered in the future as alternative additive in various foods, cosmetic and pharmaceutical preparations.

Structural determination and NMR characterization of a bacterial exopolysaccharide.

A strain of Bacillus licheniformis with high exopolysaccharide (EPS) production ability was isolated and identified. A new type of EPS was isolated from the strain fermentation and its structural characteristics were investigated and elucidated by partial and total acid hydrolysis, Fourier transform infrared, and (1)H and (13)C NMR spectroscopy including 2D (1)H, COSY, NOESY, XHCOR and HMBC experiments. Based on obtained data, the EPS was found to be a levan composed of linear chains of (2→6)-linked ß-d-fructofuranosyl residues with connections ß (2→6).

Sesquineolignans from the flowers of Centaurea furfuracea Coss. et Dur. (Asteraceae).

Two new sesquineolignans, Furfuraceol A (1) and Furfuraceol B (2), have been isolated from the flowers of Centaurea furfuracea as a mixture of two isomers. Their structures have been determined by spectroscopic methods including 2D NMR spectroscopy COSY, HMQC, HMBC and NOESY.