Iridoid- and flavonoid-enriched fractions of Cornus sanguinea and Cornus mas exert antioxidant and anti-inflammatory effects and inhibit key enzymes in the treatment of metabolic disorders.

Background: Berry fruits are recognized as a "superfood" due to their high content of bioactive compounds and health benefits. Scope and approach: Herein, extracts of Cornus sanguinea and Cornus mas fresh and dried fruits obtained by different extraction procedures (ethanolic and hydroalcoholic maceration, ultrasound-assisted extraction, and Soxhlet apparatus) were analysed using liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) and compared to identify the main healthy compounds and their impact on the inhibition of key enzymes (pancreatic lipase, α-glucosidase, and α-amylase) associated with metabolic disorders. The antioxidant activity and inhibition of nitric oxide (NO) and NF-κB pathway were also investigated. Key findings and conclusions: Flavonoids, iridoids, and phenolic acids were the main classes of identified compounds. Herein, kaempferol 3-O-galactoside, kaempferol 3-O-glucoside, quercetin, quercetin 3-O-xyloside, and myricetin 3-O-galactoside were detected for the first time in C. sanguinea. Remarkable antioxidant effects and promising α-glucosidase and lipase inhibitory activity were observed with extracts obtained by hydroalcoholic maceration of both Cornus dried fruits. Consequently, these extracts were subjected to fractionation using Amberlite XAD-16 resin. The most promising biological activities, which are attributed to the presence of some flavonoids and iridoids, were detected with the C. sanguinea fractions, in particular SD2(II). The results of this study offer new insights into the potential development of functional foods, nutraceuticals, and food supplements using the Cornus species.

New Insights into the Antioxidant and Anti-Inflammatory Effects of Italian Salvia officinalis Leaf and Flower Extracts in Lipopolysaccharide and Tumor-Mediated Inflammation Models.

This work aimed to investigate and compare the in vitro antioxidant and anti-inflammatory effects of Salvia officinalis L. (sage) from Italy, with the aim of raising its current knowledge in this field. Leaves and flowers (S1-S8), harvested in two areas of Southern Italy, were extracted with methanol as a solvent by maceration or ultrasound-assisted extraction. Sage extracts, analysed by high pressure liquid chromatography-diode-array detection-electrospray ionization-quadrupole-mass spectroscopy (HPLC-DAD-ESI-Q-MS), exerted a promising antioxidant activity investigated using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ß-carotene bleaching tests, and elicited a significant decrease in reactive oxygen species (ROS) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. The anti-inflammatory activity was analysed in the same in vitro model. All the extracts did not affect cell viability although they showed anti-inflammatory activity, as they induced a decrease in nitrite levels that was greater than 50%, when employed at 50 µg/mL. Furthermore, they elicited a decrease in nitrite levels, as well as a decline in pro-inflammatory cytokine expression. The NF-κB transcription factor proved to be involved in the mechanisms that underlie such effects. Interestingly, sage extracts were able to interfere with the inflammatory activity induced by breast cancer cell-conditioned media (nitrite levels were significantly decreased, p < 0.05; p < 0.01), highlighting for the first time the important role of S. officinalis in controlling inflammation processes related to neoplastic progression.

Chemical Profile, Antioxidant, Anti-Inflammatory, and Anti-Cancer Effects of Italian Salvia rosmarinus Spenn. Methanol Leaves Extracts.

In this study, we evaluated and compared the chemical composition, the antioxidant, anti-inflammatory, and anti-proliferative effects of four methanol extracts (R1-R4), of Salvia rosmarinus Spenn. in two different sites of Southern Italy obtained by maceration or ultrasound-assisted extraction. Extracts of S. rosmarinus collected on the Ionian coast are indicated with the abbreviations R1 (maceration) and R2 (ultrasound-assisted extraction). Extracts of S. rosmarinus collected on the Tyrrhenian coast are indicated with the abbreviations R3 (maceration) and R4 (ultrasound-assisted extraction). The chemical composition was analyzed using High Pressure liquid chromatography-Diod-Array detection-Electrospray ionization-Quadrupole-Mass Spectroscopy (HPLC-DAD-ESI-Q-MS). The antioxidant activity was analyzed by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 2,2-diphenyl-1-picrylhydrazyl (DPPH), ß-carotene bleaching, and Ferric Reducing Antioxidant Power (FRAP) assays. Antioxidant features were also assessed in lipopolysaccharide (LPS)-stimulated RAW-264.7 murine macrophages, evaluating Reactive Oxygen Species (ROS) production; in the same experimental model, the anti-inflammatory activity of the extracts was investigated. Interestingly, all extracts displayed antioxidant and anti-inflammatory properties. They exhibited significative nitrite production inhibitory activity, whith IC50 values ranging from 3.46 to 5.53 µg/mL, without impairing cell viability. The anti-inflammatory activity was also investigated by Western Blotting and immunofluorescence assay, highlighting the R3 and R4 extracts ability to reduce NF-κB translocation, as well as to disrupt the MAPKs signaling pathway. Extracts exhibited both potential anti-proliferative activity on breast cancer cells, inducing apoptosis, without affecting non-tumorigenic cells, and the ability to inhibit MDA-MB-231 cells' motility. Finally, the rosemary extracts treatment significantly reduced the power of conditioned media, from MCF-7 or MDA-MB-231 cells to induce nitrite production on RAW 264.7 cells, confirming their promising anti-inflammatory activity.

Citrus × Clementina Hort. Juice Enriched with Its By-Products (Peels and Leaves): Chemical Composition, In Vitro Bioactivity, and Impact of Processing.

This work investigated a model for the reuse of Citrus × clementina Hort. by-products for the development of a functional drink able to exert antioxidant, hypoglycaemic, and hypolipidemic effects. Juice obtained from fruits collected in three different areas of Calabria (Italy) was analysed. C. × clementina juice from Corigliano Calabro (JF), characterized by the highest content of bioactive compounds and bioactivity, was chosen as a matrix to be enrichment with hydroalcoholic ultrasound-assisted maceration of C. × clementina leaf from Corigliano Calabro (CO2) and ethanol ultrasound-assisted maceration of C. × clementina peel from Cetraro (BC3) extracts at different concentrations. The highest phytochemical content and bioactivities were found in juice enriched with leaf and leaf + peel extracts, with particular reference to antioxidant activity. In order to estimate the effects of pasteurization, 20% (mg/100 mL) enriched juice was subjected to this process. Based on obtained data of bioactivity and sensorial analysis, C. × clementina by-products could be proposed as a promising source of bioactive compounds useful for the formulation of a functional drink for preventing diseases associated with oxidative stress such as type 2 diabetes and obesity.

Contribution of Flavonoids and Iridoids to the Hypoglycaemic, Antioxidant, and Nitric Oxide (NO) Inhibitory Activities of Arbutus unedo L.

This study aims at investigating the contribution of two classes of compounds, flavonoids and iridoids, to the bioactivity of Arbutus unedo L. leaves and fruits. The impact of different extraction procedures on phytochemicals content and hypoglycemic, antioxidant, and nitric oxide (NO) inhibitory activities of A. unedo fresh and dried plant materials was investigated. Ellagic acid 4-O-ß-D-glucopyranoside, kaempferol 3-O-glucoside, and norbergenin were identified for the first time in this genus by using liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS). Three iridoids (gardenoside, geniposide, unedoside) are specifically identified in the leaves. Interestingly, asperuloside was extracted only from dried fruits by ethanol with Soxhlet apparatus. Extracts were screened for their potential antioxidant activities by using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferric Reducing Activity Power (FRAP), and ß-carotene bleaching tests. Based on the Global Antioxidant Score (GAS) calculation, the most promising antioxidant extract was obtained by hydroalcoholic maceration of dried leaves that showed half maximal inhibitory concentration (IC50) of 0.42 and 0.98 µg/mL in ABTS and DPPH assays, respectively. The hypoglycaemic activity was investigated by α-amylase and α-glucosidase inhibition tests. Extracts obtained by ethanol ultrasound extraction of fresh leaves and hydroalcoholic maceration of fresh fruits (IC50 of 19.56 and 28.42 µg/mL, respectively) are more active against α-glucosidase than the positive control acarbose (IC50 of 35.50 µg/mL). Fruit extracts exhibited the highest anti-inflammatory activity.

Impact of extraction processes on phytochemicals content and biological activity of Citrus × clementina Hort. Ex Tan. leaves: New opportunity for under-utilized food by-products.

Plants are a rich source of natural bioactive compounds with a wide range of applications in the food and pharmaceutical industries. Citrus × clementina leaves extracts and essential oils may be a potential candidate for formulation of products characterized by hypoglycaemic, antioxidant and anti-browning properties. C. × clementina leaves collected in three different areas in Calabria (South Italy) were extracted by Soxhlet apparatus, maceration, Ultrasound assisted maceration, and hydrodistillation. Hesperidin, tangeritin, and sinensetin were identified by HPLC-DAD analysis as dominant constituents of the extracts. The absence of coumarins and furanocoumarins was demonstrated. GC-MS analyses of essential oils evidenced the presence of sabinene, linalool, and (E)-ß-ocimene as main compounds. Based on RACI and GAS values calculated by the integration of data obtained by DPPH, ABTS, FRAP and ß-carotene bleaching methods, hydroalcoholic extract obtained by Ultrasound assisted maceration of the leaves collected in Corigliano Calabro showed the highest antioxidant activity. Ultrasound assisted hydroalcoholic extract of Cetraro leaves revealed the highest α-amylase inhibitory activity (IC50 of 64.37 µg/ml). Promising results of C. × clementina extracts as tyrosinase inhibitors were also obtained. To evaluate the relationship between identified compounds and bioactivity PCA was performed. Taking into account results obtained by this study, C. × clementina leaves that were considered Citrus-by-products could be utilized for formulation of food additives, nutraceuticals and functional foods.

Arbutus species (Ericaceae) as source of valuable bioactive products.

In addition to nutrients, plant foods contain compounds that may provide additional health benefits improving the quality of life. Species from Arbutus genus (Ericaceae) represent a promising source of healthy phytochemicals. Bioactive compounds including such as anthocyanins, iridoids, phenols, triterpenes, sterols, and fatty acids are reported from Arbutus species. Some Arbutus species revealed promising biological activities including antioxidant, anti-inflammatory, anti-proliferative, anti-diabetic, and antimicrobial activities, and deserve for that reason further consideration for new drug discovery. However, only few species are investigated scientifically for their chemical profile and biological activities. The aim of this article is to summarize the current knowledge of the components and biological properties of Arbutus species common in Mediterranean area, as well as the future prospects on their applications as potentially valuable products.

Clerodane furanoditerpenoids as the probable cause of toxic hepatitis induced by Tinospora crispa.

Tinospora crispa is a popular traditional herbal plant commonly used throughout the world for treatment of various diseases, in particular type 2 diabetes mellitus. We report here a new case of toxic hepatitis in a 57-year old male patient in the French West Indies following the consumption of two aqueous extracts of fresh Tinospora crispa stems. It thus differs from two previously reported cases that concerned the chronic intake of powdered dry stems delivered in solid oral dosage forms (i.e. pellets and tablets). Liquid Chromatography-Diode Array Detection-Mass Spectrometry (LC/DAD/MS) analyses were performed on an aqueous extract of the offending sample that mimics the swallowed preparation. They revealed the presence of species-specific molecular marker borapetoside C (1) and thus enabled an unambiguous phytochemical identification. The exploration of tandem MS/MS data obtained by ultra-high performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-HRMS) allowed the identification of 17 additional cis-clerodane-type furanoditerpenoid lactones, analogues of 1. These results support the hypothesis that the mechanisms underlying hepatotoxicity of Tinospora crispa are the same as those encountered with furanoditerpenoids-containing plants such as Teucrium chamaedrys or Dioscorea bulbifera. In the context of type 2 diabetes treatment, we recommend that Tinospora crispa intake should be more closely monitored for signs of hepatotoxicity.

Quality of antiepileptic drugs in sub-Saharan Africa: A study in Gabon, Kenya, and Madagascar.

OBJECTIVE: Epilepsy is a major public health issue in low- and middle-income countries, where the availability and accessibility of quality treatment remain important issues, the severity of which may be aggravated by poor quality antiepileptic drugs (AEDs). The primary objective of this study was to measure the quality of AEDs in rural and urban areas in 3 African countries. METHODS: This cross-sectional study was carried out in Gabon, Kenya, and Madagascar. Both official and unofficial supply chains in urban and rural areas were investigated. Samples of oral AEDs were collected in areas where a patient could buy or obtain them. Pharmacological analytical procedures and Medicine Quality Assessment Reporting Guidelines were used to assess quality. RESULTS: In total, 102 batches, representing 3782 units of AEDs, were sampled. Overall, 32.3% of the tablets were of poor quality, but no significant difference was observed across sites: 26.5% in Gabon, 37.0% in Kenya, and 34.1% in Madagascar (P = .7). The highest proportions of substandard medications were found in the carbamazepine (38.7%; 95% confidence interval [CI] 21.8-57.8) and phenytoin (83.3%; 95% CI 35.8-99.5) batches, which were mainly flawed by their failure to dissolve. Sodium valproate was the AED with the poorest quality (32.1%; 95% CI 15.8-42.3). The phenobarbital (94.1%; 95% CI 80.3-99.2) and diazepam (100.0%) batches were of better quality. The prevalence of substandard quality medications increased in samples supplied by public facilities (odds ratio [OR] 9.9; 95% CI 1.2-84.1; P < .04) and manufacturers located in China (OR 119.8; 95% CI 8.7-1651.9; P < .001). The prevalence of AEDs of bad quality increased when they were stored improperly (OR 5.4; 95% CI 1.2-24.1; P < .03). SIGNIFICANCE: No counterfeiting was observed. However, inadequate AED storage conditions are likely to lead to ineffective and possibly dangerous AEDs, even when good-quality AEDs are initially imported.

Degradation pathways study of the natriuretic and ß-adrenoceptor antagonist tienoxolol using liquid chromatography-electrospray ionization multistage mass spectrometry.

Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to combine a diuretic and a ß-adrenoreceptor antagonist into a single molecule. Its degradation products generated in several stress media have been determined by high-pressure liquid chromatography (HPLC) coupled to a hybrid mass spectrometer with a triple quadrupole-linear trap. A Polaris(®) column with a C18-A stationary phase and a linear gradient mobile phase composed of a mixture of trifluoroacetic acid 1% (v/v) and acetonitrile allowed for optimal separation. Structural elucidation of the degradation products has been based on MS/MS techniques, by comparing their fragmentation patterns to the precursor's data. Up to seven degradation products of the active ingredient, resulting from hydrolysis, oxidation, dehydration and transamidation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics have been studied to assess the molecule's shelf life and to identify the most important degradation factor.

Chemical composition and antimicrobial activity of the essential oils from New Caledonian Citrus macroptera and Citrus hystrix.

The essential oils from the leaves of Citrus macroptera and C. hystrix, collected in New Caledonia, have been analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for their antimicrobial activity. A total of 35 and 38 constituents were identified, representing 99.1 and 89.0% of the essential oils, respectively. Both essential oils were rich in monoterpenes (96.1 and 87.0%, resp.), with beta-pinene as major component (33.3 and 10.9%, resp.), and poor in limonene (2.4 and 4.7%, resp.). Other main components of C. macroptera oil were alpha-pinene (25.3%), p-cimene (17.6%), (E)-beta-ocimene (6.7%), and sabinene (4.8%). The essential oil of C. hystrix was characterized by high contents of terpinen-4-ol (13.0%), alpha-terpineol (7.6%), 1,8-cineole (6.4%), and citronellol (6.0%). The antimicrobial activity was evaluated against five bacteria and five fungi strains. Both oils were inactive against bacteria. However, the C. macroptera leaf oil exhibited a pronounced activity against Trichophyton mentagrophytes var. interdigitale, with a minimal-inhibitory concentration (MIC) of 12.5 microg/ml.

Characterization of monohydroxylated derivatives of the anticancer agent flavone-8-acetic acid by liquid chromatography with on-line UV and mass spectrometry.

The experimental anticancer agent flavone-8-acetic acid (FAA) is metabolized into several monohydroxylated derivatives using mouse microsomes. Because these metabolites could be involved in the biological effects of FAA, the aim of this study was to characterize all its possible monohydroxylated derivatives. To do so, we have developed a methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) to analyze and identify FAA derivatives hydroxylated at the 2', 3', 4', 3, 5, 6, or 7 position. In RP-HPLC, 4'-, 3'-, 2'-, 6-, and 7-OH-FAA eluted before FAA, whereas 3- and 5-OH-FAA eluted after FAA. UV spectra showed a bathochromic shift of band I for all derivatives and of band II for 5- and 6-OH-FAA. In addition, the position of the OH group could be determined by the presence of certain product ions in MS. Ions at m/z 133 and 151 were specific for 2'-, 3'-, 4'-, and 3-OH-FAA, whereas the ion at m/z 177 was specific for 3-OH-FAA only. The ions m/z 133, 151 and 167 were specific for 2'-OH-FAA. Ions at m/z 149 were specific for the presence of the OH group on cycle A only (i.e., 5-, 6- or 7-OH-FAA). The presence of both product ions m/z 149 and 179 were specific for 7-OH-FAA. Finally, ions at m/z 149 and several product ions of even m/z values were specific for 5-OH-FAA. In conclusion, the methodology described can be used to identify all possible monohydroxylated FAA derivatives.

Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes.

Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.

Despite extensive research, the pathogenesis of neonatal necrotizing enterocolitis (NEC) remains elusive. The aim of our work was to investigate the role of bacterial strains involved in NEC in gnotobiotic quails as experimental model. Six groups of germ-free quails that were fed a lactose diet were associated with Klebsiella pneumoniae, Clostridium perfringens, C. difficile, C. paraputrificum, or C. butyricum (two strains). Implantation level, incidence of cecal lesions, production of short-chain fatty acids, and histologic lesions of the cecal wall were investigated. Whatever the strain, the implantation level was high (10(9) UFC/g). Neither K. pneumoniae nor C. difficile induced any cecal lesions. In contrast, the four other clostridial strains led to cecal NEC-like lesions with a variable occurrence: four of 12 quails for C. perfringens, eight of 12 quails for C. paraputrificum, and the same highest value, nine of 12 quails and eight of 10 quails for both C. butyricum strains. Gross aspects of the lesions may be linked to the short-chain fatty acid profiles and/or concentrations: thickening of the cecal wall (C. butyricum and C. perfringens) with high proportion of butyric acid, hemorrhages (C. paraputrificum) with high proportion of iso-butyric acid, and presence of other iso-acids. In addition, C. butyricum was characterized by pneumatosis, linked to a high gas production. Microscopic aspects confirmed the presence of edemas and intramucosa hemorrhages. Clostridia species, whose role is controversial, seem to be strongly implicated in NEC through excessive production of butyric acid as a result of colonic lactose fermentation. These results call for anaerobe detection in feces of infants who have NEC.