Federica Pellati

Analysis of phenolic compounds and radical scavenging activity of Echinacea spp.

The aim of this study was to set up and validate an RP-LC method with DAD-detection to quantify caffeic acid derivatives in various Echinacea spp. Samples were extracted with 80% methanol. The analyses were carried out on a Lichrospher RP-18 column (125 mm x 4 mm i.d., 5 microm), with a mobile phase gradient, which increases the acetonitrile level in a phosphoric acid solution (0.1%). The flow rate was 1.5 ml/min. Detection was set at 330 nm. This method allowed the identification and quantification of caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid in Echinacea roots and derivatives. The total phenolic content was 10.49 mg/g for E. angustifolia, 17.83 mg/g for E. pallida and 23.23 mg/g for E. purpurea. Among Echinacea commercial herbal medicines, a certain variability in the concentrations of phenolic compounds was observed. The radical scavenging activity of Echinacea methanolic extracts was evaluated in vitro with a spectrophotometric method based on the reduction of an alcoholic 2,2-diphenyl-1-picrylhydrazyl (DPPH*) radical solution at 517 nm in the presence of a hydrogen donating antioxidant. As for pure compounds, echinacoside had the highest capacity to quench DPPH* radicals (EC50 = 6.6 microM), while caftaric acid had the lowest (EC50 = 20.5 microM). The average EC50 values for E. purpurea, E. pallida and E. angustifolia were 134, 167 and 231 microg/ml, respectively. The radical scavenging activity of Echinacea root extracts reflected their phenolic composition. The results indicate that Echinacea roots and derivatives are a good source of natural antioxidants and could be used to prevent free-radical-induced deleterious effects.

Determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by LC

The purpose of this study was to set up a HPLC method to separate adrenergic amines (dl-octopamine, dl-synephrine and tyramine) and to determine their content in fruits, extracts and herbal products of Citrus aurantium L. var. amara. A rapid method for the quantitative analysis of these amines is described, based on their separation by RP-HPLC technique with UV detection. The analysis were conducted on a Lichrospher RP-18 column at room temperature, using a mobile phase consisting of 0.02 M citric acid-0.02 M NaH2PO4 (7:3 v/v) and adjusted to a final pH of 3. The detection was at 220 nm. Since some of these amines are chiral compounds and their enantiomers showed different pharmacological activity, the direct separation of synephrine enantiomers was carried out with HPLC on a beta-cyclodextrin stationary phase. The mobile phase consisted of methanol-NaH2PO4 25 mM pH 3.5 (20:80 v/v) and tetrabutylammonium hydrogen sulfate 10 mM in ratio of 30:70 v/v in isocratic condition and the detection was at 220 nm. The two proposed methods were applied to the analysis of fruits, extracts and herbal products of C. aurantium L. var. amara. Taking into account that some authors have reported that l-synephrine may be converted into its d-form by high temperature, this optical isomerization was monitored by the same HPLC method used for the separation of enantiomers.

HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts.

In this study, the composition of polyphenols (phenolic acids and flavonoids) in propolis extracts was investigated by HPLC-DAD and HPLC-ESI-MS/MS by comparing the performance of ion trap and triple quadrupole mass analyzers. The analyses were carried out on an Ascentis C(18) column (250mm×4.6mm I.D., 5μm), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Overall, the UV spectra, the MS and MS/MS data allowed the identification of 40 compounds. In the case of flavonoids, the triple quadrupole mass analyzer provided more collision energy if compared with the ion trap, originating product ions at best sensitivity. The HPLC method was validated in agreement with ICH guidelines: the correlation coefficients were >0.998; the limit of detection was in the range 1.6-4.6μg/ml; the recovery range was 96-105%; the intra- and inter-day %RSD values for retention times and peak areas were found to be <0.3 and 1.9%, respectively. The developed technique was applied to the analysis of hydroalcoholic extracts of propolis available on the Italian market. Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.67mg/ml and the level of total flavonoids from 2.48 to 41.10mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy.

Synergistic immunomopharmacological effects of N-alkylamides in Echinacea purpurea herbal extracts

Echinacea purpurea extracts are used in the production of standardized herbal medicines for the prevention and treatment of upper respiratory infections. Unsaturated N-alkylamide lipids, the main constituent of E. purpurea and E. angustifolia preparations capable of activating the cannabinoid receptor type-2 (CB2) have been suggested to play a role as potential anti-inflammatory and immune-modulatory principles. Here we show that ethanolic E. purpurea radix and herba extracts produce synergistic pharmacological effects on the endocannabinoid system in vitro. Superadditive action of N-alkylamide combinations was seen at the level of intracellular calcium release as a function of CB2 receptor activation. Likewise, synergism of the radix and herba tinctures was observed in experiments measuring LPS-stimulated cytokine expression from human PBMCs. While the expression of the anti-inflammatory cytokine IL-10 was significantly superstimulated, the expression of the pro-inflammatory TNF-alpha protein was inhibited more strongly upon combination of the extracts. We show that N-alkylamides act in concert and exert pleiotropic effects modulating the endocannabinoid system by simultaneously targeting the CB2 receptor, endocannabinoid transport and degradation.

PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid.

There is a broad interest in the development of nanoparticles (NPs) carrying on their surface carbohydrates such as sialic acids. It is known that these carbohydrates influence the biological and physical properties of biopharmaceutical proteins and living cells. Macromolecular compounds containing these carbohydrates showed an anti-recognition effect, exert an antiviral effect and also are able to be recognized by the cell surface of some kind of cancer cells. Thus, in the present research we performed two different approaches in order to obtain polymeric (poly(D,L-lactide-co-glycolide), PLGA) NPs surface decorated with the sialic acid N-acetylneuraminic acid (Neu5Ac). The first strategy that has been followed is based on the derivatization of the polyester PLGA with the thioderivative of Neu5Ac, starting material for the preparation of the NPs; the second is based on the synthesis of compounds potentially able to insert their lipophilic moiety into the underivatized PLGA NPs during their preparation, and to display their hydrophilic moiety (Neu5Ac) on their surface. The first approach allowed the obtainment of NPs surface decorated with Neu5Ac, as evidenced by ESCA spectroscopy and interaction with the lectin Wheat Germ Agglutinin. Moreover, a formulation of these NPs suitable for in vitro assays showed that they are phagocytosed by human monocytes with an apparently different mechanism with respect of those made of underivatized PLGA. The second strategy led to NPs in which their surface appears to be very different with respect to the NPs obtained following the first strategy, with the carboxylic groups of Neu5Ac markedly shielded. Thus, the new Neu5Ac-modified PLGA polyester represent a useful starting material for the preparation of NPs surface decorated with this sialic acid.

Cytotoxic activity of polyacetylenes and polyenes isolated from roots of Echinacea pallida

The n‐hexane extracts of the roots of three medicinally used Echinacea species exhibited cytotoxic activity on human cancer cell lines, with Echinacea pallida found to be the most cytotoxic. Acetylenes are present in E. pallida lipophilic extracts but essentially absent in extracts from the other two species. In the present study, the cytotoxic effects of five compounds, two polyacetylenes (namely, 8‐hydroxy‐pentadeca‐(9E)‐ene‐11,13‐diyn‐2‐one (1) and pentadeca‐(9E)‐ene‐11,13‐diyne‐2,8‐dione (3)) and three polyenes (namely, 8‐hydroxy‐pentadeca‐(9E,13Z)‐dien‐11‐yn‐2‐one (2), pentadeca‐(9E,13Z)‐dien‐11‐yne‐2,8‐dione (4) and pentadeca‐(8Z,13Z)‐dien‐11‐yn‐2‐one (5)), isolated from the n‐hexane extract of E. pallida roots by bioassay‐guided fractionation, were investigated and the potential bioavailability of these compounds in the extract was studied.

An efficient chemical analysis of phenolic acids and flavonoids in raw propolis by microwave-assisted extraction combined with high-performance liquid chromatography using the fused-core technology.

A closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of polyphenols from raw propolis. The results obtained by means of response surface experimental design methodology showed that the best global response was reached when the extraction temperature was set at 106 °C, the solvent composition close to EtOH-H2O 80:20 (v/v), with an extraction time of 15 min. In comparison with other techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm), with a gradient mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by DAD and MS. The method validation indicated that the correlation coefficients were >0.999; the limit of detection was in the range 0.5-0.8 μg/ml for phenolic acids and 1.2-3.0 μg/ml for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in propolis extracts to be used in apitherapy.

Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice

Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mammals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehyde and hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effects of benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. This study examined whether chronic, oral administration of benzylamine could improve glucose tolerance and the circulating lipid profile without increasing oxidative stress in overweight and pre-diabetic mice. The benzylamine diffusion across the intestine was verified using everted gut sacs. Then, glucose handling and metabolic markers were measured in mice rendered insulin-resistant when fed a high-fat diet (HFD) and receiving or not benzylamine in their drinking water (3600micromol/(kgday)) for 17 weeks. HFD-benzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-induced activation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylamine treatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidation markers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, were decreased. In spite of the putative deleterious nature of the hydrogen peroxide generated during amine oxidation, and in agreement with its in vitro insulin-like actions found on adipocytes, the SSAO-substrate benzylamine could be considered as a potential oral agent to treat metabolic syndrome.

P-Glycoprotein inhibitory activity of lipophilic constituents of Echinacea pallida roots in a human proximal tubular cell line.

The N-hexane root extracts from Echinacea pallida, Echinacea angustifolia and Echinacea purpurea were evaluated for inhibition of the multidrug transporter P-glycoprotein (Pgp) activity, the product of the ABCB1 gene, involved in cancer multidrug resistance (MDR) and in herb-drug or drug-drug interactions. The biological assay was performed using the human proximal tubule HK-2 cell line that constitutively expresses ABCB1. The N-hexane extracts of all three species reduced the efflux of the Pgp probe calcein-AM from HK-2 cells two-fold in a concentration-dependent manner, and E. pallida was found to be the most active species. For the first time, two polyacetylenes and three polyenes, isolated from the N-hexane extract of E. pallida roots by a bioassay-guided fractionation, were found to be able to reduce Pgp activity. Pentadeca-(8 Z,13 Z)-dien-11-yn-2-one was the most efficient compound, being able to decrease the calcein-AM efflux about three-fold with respect to the control at 30 microg/mL.

Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity.

ETHNOPHARMACOLOGICAL RELEVANCE The decoction of Terminalia chebula fruit is an ayurvedic remedy whose prolonged oral administration is prized as a generic intestinal and hepatic detoxifying agent. Its administration is suggested also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. AIM OF THE STUDY To evaluate the phytochemical profile and the chemopreventive potential of Terminalia chebula fruit decoction prepared according to the ayurvedic decoction recipe. MATERIALS AND METHODS The quali- and quantitative metabolite profiling of polyphenols was obtained using HPLC-UV/DAD and HPLC-ESI-MS. The crude decoction and purified compounds were tested for their capability to interact with the EphA2-ephrin-A1 system and for their antimutagenic properties against dietary and environmental mutagens (AA, 2-NF, NaN3, and heterocyclic amines IQ, MeIQ, MeIQx, Glu-P1, Glu-P2,) in the Ames-Salmonella/microsome assay, with and without enzymatic induction. RESULTS The decoction was found to contain 3,4,6-tri-O-galloyl-d-glucose (55.87 mg/g), chebulic acid (54.03 mg/g), β-punicalagin (41.25mg/g), corilagin (40.31 mg/g), α-punicalagin (35.55 mg/g), chebulagic acid (29.09 mg/g), gallic acid (27.96 mg/g) 1,3,4,6-tri-O-galloyl-β-d-glucose (24.25mg/g) chebulinic acid (20.23 mg/g), 1,2,3,4,6-penta-O-galloyl-d-glucose (13.53 mg/g), ellagic acid (8.00 mg/g), 1,6-di-O-galloyl-d-glucose (4.16 mg/g). An inhibitory effect was recorded in both Salmonella typhimurium TA98 and TA100 strains against the mutagenic activity of heterocyclic amines (22-61%), promutagen AA (91-97%) and directly acting mutagen 2-NF (52%) with but not against NaN3 (7%). Galloyl derivatives allowed an inhibition of mutagenicity induced by MeIQ above 80% at 0.01 mol/plate. Both decoction and purified compounds were able to modulate the EphA2-ephrinA1 system, suggesting a potential multiple chemopreventive mechanism. CONCLUSIONS The traditional ayurvedic decoction of Terminalia chebula may harbour a potential as a safe and low-cost chemopreventive agent at the intestinal level, if administered according to the ayurvedic specifications. Moreover, its recourse may enhance the presence of some polyphenolic constituents.