Gregorio Peron

Phytochemical investigations and antiproliferative secondary metabolites from Thymus alternans growing in Slovakia

Abstract Context: Thymus alternans Klokov (Lamiaceae) is a neglected species of the genus Thymus (Sect. Serpyllum) endemic to Carpathian area, where it is used as a flavouring agent and for medicinal purposes. Objective: The aim of the work was to identify antiproliferative constituents from the flowering aerial parts of this plant. Materials and methods: Thymus alternans extracts were analyzed by HPLC-MSn and subjected to extensive chromatographic separations. The isolated compounds (phenolics and triterpenes) were structurally elucidated by MS and 1D and 2D NMR experiments. Essential oil (EO) composition was determined by GC-FID and GC-MS. Six purified triterpenes and EO were assayed for in vitro antiproliferative activity against a panel of human cancer cells, namely, breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810), pancreatic (BxPC3), melanoma (A375) and cervical carcinoma (A431) cells. Results: The structures of the isolated compounds were achieved on the basis of H-NMR and MS experiments. Luteolin-4′-O-β-d-glucopyranoside (P1), chrysoeriol-7-O-β-d-glucopyranoside (P2), chrysoeriol-5-O-β-d-glucopyranoside (P3), apigenin-7-O-β-d-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic acid-3′-O-β-d-glucopyranoside (P6), caffeic acid-3-O-β-d-glucopyranoside (P7), 3α-hydroxy-urs-12,15-diene (T1), α-amyrin (T2), β-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6). GC-MS analysis revealed that the EO of T. alternans was devoid of phenols and belonged to the nerolidol-chemotype, that is typical of the Sect. Serpyllum. The six purified triterpenes (T1-T6) were active with IC50 ranging from 0.5 to 5 μM being comparable or better than those of reference compounds betulinic acid and cisplatin. The EO exhibited significant effects on A375, MDA-MB 231 and HCT116 cell lines with IC50 in the range of 5–8 μg/mL. Conclusion: The reported results suggest that T. alternans can be considered as a good source of phytoconstituents with possible importance in the pharmaceutical field.

Phytochemical investigations on Artemisia alba Turra growing in the North-East of Italy

Abstract Artemisia alba Turra (Asteraceae) is an Euro-Mediterranean plant used in Veneto (North-East of Italy) as traditional medicine for the treatment of various diseases. A. alba is a taxonomically problematic species, characterised by common polymorphism leading to a quite high variability in secondary metabolites content. Nonetheless, the phytochemical knowledge on its phytoconstituents, especially non-volatile components, is limited. In the present paper, the phytochemical composition of a tincture obtained from the aerial parts of A. alba growing in Veneto is presented. Extensive chromatographic separations led to the isolation of three new sesquiterpene derivatives, whose structures were elucidated by 1D and 2D NMR experiments and mass spectrometry. Furthermore, flavonoid composition and volatile constituents of the tincture of A. alba were preliminary studied by HPLC–MSn and GC–MS, respectively.

Natural Deep Eutectic Solvents (NADES) to Enhance Berberine Absorption: An In Vivo Pharmacokinetic Study

In the present study results related to the in vivo administration of Natural Deep Eutectic Solvents (NADES)-solubilized berberine are reported for the first time. NADES are mixtures of small natural compounds having a melting point significantly lower than that of any individual component. Such solvents have gained much attention of the scientific community in the green chemistry area, being considered useful alternatives to common organic solvents. NADES can be used also as administration vehicles, and this can be attractive for nutraceutical products when eutectics are formed with food grade ingredients. In this work, different NADES were prepared using mainly food grade constituents and were tested as solvents for the alkaloid berberine. Three selected NADES/berberine solutions and an aqueous suspension were orally administered to mice with in dose of 50 mg/Kg. Blood levels of berberine were measured by a LC-MS/MS method. The pharmacokinetic analysis revealed a 2–20 fold increase in blood concentration of NADES/berberine with significant changes in pharmacokinetic profile. Natural Deep Eutectic Solvents may thus be considered attractive solubilizing agents and may also play a role in the increase of absorption of poorly bioavailable natural products such as berberine.

Studying the effects of natural extracts with metabolomics: A longitudinal study on the supplementation of healthy rats with Polygonum cuspidatum Sieb. et Zucc.

Background: A longitudinal study was performed to evaluate the effects of Polygonum cuspidatum extract (standardized at 20% resveratrol) supplementation on healthy rats. The effects were explored by monitoring urinary metabolome changes using UPLC‐HRMS and 1H NMR‐based approaches. The aim of the study was to explore the effects of P. cuspidatum supplementation on a healthy animal model using metabolomics, in order to determine possible modes of action and obtain information on bioactivity. Methods: Healthy Sprague‐Dawley rats were orally supplemented with 100 mg/kg of dried P. cuspidatum extract for 49 days and 24‐h urinary outputs were collected. Samples were analysed by untargeted UPLC‐HRMS and 1H NMR approaches and the obtained data sets were modelled by an adaptation of post‐transformation of PLS2 to longitudinal studies. Putative markers were discovered by a stability selection procedure and specific oxidative stress markers were monitored by a targeted HPLC–MS/MS analysis to assess the in vivo antioxidant activity of P. cuspidatum extract. Results: UPLC‐HRMS and 1H NMR platforms showed two different but complementary patterns of metabolites describing the changes ascribable to P. cuspidatum supplementation and using both approaches, a comprehensive resveratrol metabolism and urinary excretion could be observed. Markers of P. cuspidatum supplementation effects identified by UPLC‐HRMS were mainly related to its antioxidant activity and to a possible “adaptogenic” activity. Urinary changes observed by 1H NMR were mainly related to energy metabolism. UPLC‐HRMS and 1H NMR metabolomics approaches allowed the effects of a prolonged supplementation with P. cuspidatum on healthy rats to be observed. The statistical models built from both data sets showed metabolic changes in urines related to rat aging. HighlightsP. cuspidatum consumption caused changes in urine composition revealed by NMR and UPLC–MS based metabolomics.Both approaches showed changes of urine composition related to time passing.Effects of P. cuspidatum on energetic metabolism and oxidative status were observed in treated rats.

Antiadhesive Activity and Metabolomics Analysis of Rat Urine after Cranberry (Vaccinium macrocarpon Aiton) Administration

Cranberry (Vaccinium macrocarpon Aiton) is used to treat noncomplicated urinary tract infections (UTIs). A-type procyanidins (PAC-A) are considered the active constituents able to inhibit bacterial adhesion to the urinary epithelium. However, the role of PAC-A in UTIs is debated, because of their poor bioavailability, extensive metabolism, limited knowledge about urinary excretion, and contradictory clinical trials. The effects of 35-day cranberry supplementation (11 mg/kg PAC-A, 4 mg/kg PAC-B) were studied in healthy rats using a ultra performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics approach. Microbial PAC metabolites, such as valeric acid and valerolactone derivatives, were related to cranberry consumption. An increased urinary excretion of glucuronidated metabolites was also observed. In a further experiment, urine samples were collected at 2, 4, 8, and 24 h after cranberry intake and their antiadhesive properties were tested against uropathogenic Escherichia coli. The 8 h samples showed the highest activity. Changes in urinary composition were studied by ultra performance liquid chromatography-time-of-flight (UPLC-QTOF), observing the presence of PAC metabolites. The PAC-A2 levels were measured in all collected samples, and the highest amounts, on the order of ng/mL, were found in the samples collected after 4 h. Results indicate that the antiadhesive activity against uropathogenic bacteria observed after cranberry consumption is ascribable to PAC-A metabolites rather than to a direct PAC-A effect, as the measured PAC-A levels in urine was lower than those reported as active in the literature.

NMR, HS-SPME-GC/MS, and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by 1H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSn). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. 1H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSn allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.

Development and Validation of an HPLC-ELSD Method for the Quantification of 1-Triacontanol in Solid and Liquid Samples

1-Triacontanol (TRIA) is gaining a lot of interest in agricultural practice due to its use as bio-stimulant and different types of TRIA-containing products have been presented on the market. Up to date, TRIA determination is performed by GC analysis after chemical derivatization, but in aqueous samples containing low amounts of TRIA determination can be problematic and the derivatization step can be troublesome. Hence, there is the need for an analysis method without derivatization. TRIA-based products are in general plant extracts that can be obtained with different extraction procedures. These products can contain different ranges of concentration of TRIA from units to thousands of mg/kg. Thus, there is the need for a method that can be applied to different sample matrices like plant materials and different plant extracts. In this paper we present a HPLC-ELSD method for the analysis of TRIA without derivatization. The method has been fully validated and it has been tested analyzing the content of TRIA in different dried vegetal matrices, plant extracts, and products. The method is characterized by high sensitivity (LOD = 0.2 mg/L, LOQ = 0.6 mg/L) and good precision (intra-day: <11.2%, inter-day: 10.2%) being suitable for routine analysis of this fatty alcohol both for quality control or research purposes.

Supplementation with resveratrol as Polygonum cuspidatum Sieb. et Zucc. extract induces changes in the excretion of urinary markers associated to aging in rats

An UPLC-HR-MS metabolomics approach was used to study the effects of a 49-days oral supplementation with Polygonum cuspidatum extract in healthy rats. Multivariate analysis allowed to observe significant differences in the excretion of several markers between treated animals and control group. Among the others, the amounts of N-methyl-2-pyridone-5-carboxamide (2PY) and phenylacetylglycine (PAG) were reduced in the treated group compared to control. These compounds have been previously considered as markers of aging. Furthermore, the excretion of 3-hydroxysebacic acid and 4,6-dihydroxyquinoline was also changed following supplementation, although not significantly. Despite the relatively short time of treatment (7 weeks), the significant changes in the urinary levels of aging markers observed at day 49 suggests a potential role of this type of studies as a new approach in the evaluation of the anti-aging effects of plant extracts.

Retrospective analysis of a lactose breath test in a gastrointestinal symptomatic population of Northeast Italy: use of (H 2 +2CH 4 ) versus H 2 threshold

Background Lactose malabsorption is normally evaluated by measuring exhaled H2 produced by intestinal flora, from unabsorbed lactose. However, differing microbiome composition can lead to the production of CH4 instead of H2; hence, some authors challenge the H2 method sensitivity and favor the evaluation of both intestinal gases. Aim To compare different approaches to usage of a lactose breath test for lactose malabsorption diagnosis, after medical evaluation of gastrointestinal symptoms. Methods In a retrospective observational study, we compared the 2 approaches in a population of 282 subjects in Northern Italy. Following oral lactose administration, exhaled samples were harvested every 30 minutes for 4 hours and prepared for H2 and CH4 analysis. Basal gas levels were subtracted from H2 and CH4 ppm and values at 4 hours and peaks were considered for analysis. Results Applying the standard methodology, which takes separately into consideration H2 and CH4 produced in the intestinal lumen, the results indicated that 11.7% of the patients were diagnosed “positive” for hypolactasia, differently from what was expected. Conversely, taking into consideration the sum of H2 and CH4, the percentage increased to 62.8%, closer to the expected one. No significant differences were found when comparing the 2 groups for age, gender, or symptoms. The sizable difference between the 2 approaches is likely linked to gut microbiome variability, and consequently the different production of the 2 gases, in the population. Conclusion The threshold normally used for lactose breath test should be reconsidered and changed, merging H2 and CH4 stoichiometric values to increase sensitivity.