Two new clerodane diterpenoids and a new pyran-2-one derivative with anti-neuroinflammatory activities from Croton crassifolius.

Two new clerodane diterpenoids (1 and 2), a new pyran-2-one derivative (3), along with five known compounds (4‒8), were isolated from Croton crassifolius. Notably, crassifolin X (1) is a novel clerodane diterpenoid, characterized with a peculiar δ-lactone core being formed between C-1 and C-4. Their structures, including absolute configurations, were established on the basis of spectroscopic methods (UV, IR, HRESIMS and NMR), and circular dichroism experiments. In addition, all compounds were evaluated for their anti-neuroinflammatory activities based on the expression of TNF-α and IL-6 levels on LPS-induced BV2 cells, and compounds 1‒3 and 5 showed potential anti-neuroinflammatory activity.

Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis.

BACKGROUND: Angoroside C, cinnamic acid, and harpagoside are bioactive constituents in Scrophularia ningpoensis. Currently, an infrared-assisted extraction (IRAE) method coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the analysis of bioactive constituents in this plant is lacking. METHODS: A method based on HPLC following IRAE has been developed for quantifying angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis. Four main factors, namely, extraction solvent, solid/liquid ratio, illumination time, and distance between the infrared lamp and the round-bottom flask, were optimized for extraction. Furthermore, conventional ultrasonic extraction (USE) and microwave-assisted extraction (MAE) were also investigated to validate the developed method. RESULTS: The optimal extraction conditions were as follows: ethanol concentration, 37.5%; solid/liquid ratio, 1:25; illumination time, 10 min; and distance between infrared lamp and round-bottom flask, 3 cm. The results of method validation demonstrated that the developed method meets the requirement of analysis. CONCLUSION: The results show that the IRAE-HPLC is a simple, accurate, and green analytical preparatory method for the potential extraction and quantification of angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis.

Effect of casein on pure lecithin liposome: Mixed biomacromolecular system for providing superior stabilization to hydrophobic molecules.

Formulation of new liposoma-based systems can always be in the spotlight for their unique utilization as carriers. Some changes in the composition of lipids may give rise to new mixed liposomes exhibiting modified size and physico-chemical characteristics. Consequently, these display different encapsulating properties toward various molecules. In this work, we have explored the variations in the model lecithin liposomes with casein additive from their size, shape, microenvironment and dynamics in solution. It is observed that with introduction of casein, the size of the liposome is substantially reduced due to incorporation of the additive in its bilayer. Strong interaction between the hydrophobic side chains of casein and lipid bilayer, and electrostatic repulsion of head groups of lipid are responsible to result small casein-mixed liposomes. Spectral properties of coumarin-153 disclose that the microenvironment of the bi-layer of mixed system is predominantly hydrophobic in nature and much rigid too. Fluorescence-lifetime-imaging-microscopy indicates that casein mixed systems exhibit wider lifetime distribution than pure liposomes with predominance of longer lifetimes, entirely arising from the outer bilayer. This indicates that casein gets incorporated in the bilayer of the vesicle to cater rigid and more hydrophobic microenvironment with an effective decrease in size. It is found that such mixed system is very efficient to stabilize the hydrophobic drugs (curcumin and ß-carotene) for prolonged period than the pure liposome or casein-only. The findings are indeed important from the perspective of drug stabilization by liposomes, and is suggestive of possible utilization in oral drug delivery applications involving such size-reduced nano-encapsulates.

The involvement of phenolic-rich extracts from Galician autochthonous extra-virgin olive oils against the α-glucosidase and α-amylase inhibition.

'Brava' and 'Mansa de Figueiredo' extra-virgin olive oils (EVOOs) are two varieties identified from north-western Spain. A systematic phenolic characterization of the studied oils was undertaken by LC-ESI-IT-MS. In addition, the role of dietary polyphenols from these EVOOs has been evaluated against the inhibition of key enzymes (α-glucosidase and α-amylase) in the management of diabetes mellitus (DM). Oleuropein and ligstroside derivatives comprised 83% and 67% of the total phenolic compounds in 'Brava' and 'Mansa de Figueiredo' EVOOs, respectively. The main secoiridoids from oleuropein were DOA (3,4-DHPEA-EDA, 59 and 22 mg kg-1, respectively) and the main isomer of OlAgl (3,4-DHPEA-EA, 74 and 23 mg kg-1). The main secoiridoids from ligstroside were D-LigAgl (p-HPEA-EDA or oleocanthal, 23 and 167 mg kg-1) and the main isomer of LigAgl (p-HPEA-EA, 214 and 114 mg kg-1). For α-glucosidase, both EVOO extracts displayed stronger inhibitory activity (IC50 values of 60 ±â€¯8 and 118 ±â€¯9 µg mL-1, respectively) than the commercial inhibitor acarbose (IC50 = 356 ±â€¯21 µg mL-1). Nevertheless, for α-amylase, only 'Brava' extracts showed anti-α-amylase capacity. A daily VOO intake lower than the requirements of EFSA seem to be enough to reach both 50% for α-glucosidase and 25% for α-amylase inhibition. These findings support the potential health benefits derived from Galician EVOOs that might be probably linked to the outstanding high concentration levels of phenolic acids and flavonoids.

Preparative Separation of Phenylethanoid and Secoiridoid Glycosides from Ligustri Lucidi Fructus by High-Speed Counter-Current Chromatography Coupled with Ultrahigh Pressure Extraction.

Three phenylethanoid glycosides, echinacoside (1), salidroside (3), and acteoside (6), and three secoiridoid glycosides, isonuezhenide (2), nuezhenoside G13 (4), and specnuezhenide (5), have been extracted and separated by a combined method of ultrahigh pressure extraction (UPE) and high-speed counter-current chromatography (HSCCC) from Ligustri Lucidi Fructus. For the UPE, the optimal extraction was developed with conditions including solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of compounds 4 and 5 were 15.0 and 78.0 mg/g, respectively. For the HSCCC separation, the strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition for 2.7 mg of compound 1, 4.5 mg of compound 2, 6.8 mg of compound 3, 5.9 mg of compound 4, 11.2 mg of compound 5, and 2.2 mg of compound 6 in one separation run under the solvent system of ethyl acetate:n-butanol:water (2:1:3, v/v) from 200 mg of the UPE extract. The structures of these phenylethanoid and secoiridoid glycosides were elucidated by extensive spectroscopic methods.

A Comprehensive Quality Evaluation Method Based on C30-HPLC and an Analytic Hierarchy Process for the Chinese Herbal Formula, Erzhiwan.

The quantitative analysis of multiple indexes remains an important quality evaluation method of traditional Chinese medicine (TCM) herbal formulas. The Chinese Pharmacopoeia 2015 only stipulates the content of a single component, specnuezhenide, in Erzhiwan composed of the Fructus Ligustri Lucidi (FLL) powder and aqueous extracts of Herba Ecliptae (HE). To generalize the intrinsic quality of Erzhiwan, a novel C30-HPLC method with good precision, accuracy, and reproducibility was developed for the simultaneous determination of six compounds, including two isomers, and then an analytic hierarchy process was further applied to integrate and discriminate the quality of four samples prepared via different methods. The results of the analysis were in agreement with the antioxidant tests in vitro. This comprehensive strategy could provide a reference and suggestions for the improvement of the quality evaluation method of TCM herbal formulas.

Single-Lab Validation for Determination of Kavalactones and Flavokavains in Piper methysticum (Kava).

Piper methysticum (Kava) is a plant whose roots are used in the preparation of traditional beverages with spiritual, medicinal, and social importance for the Pacific Islanders. Kava is also sold as a herbal supplement or recreational beverage consumed for its mild inebriating effect in Europe and North America. With an ongoing interest in the safety and quality of kava products, it is necessary to develop a validated method for determination of kava chemical composition to ensure confidence in quality assessment. Thus, an high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed, optimized, and validated for determining six major kavalactones and three flavokavains in kava raw materials and finished products based on AOAC single-laboratory validation guidelines. This is the first fully validated analytical method for measuring kavalactones and flavokavains in a single run. The separation of the analytes was achieved in 10 min with an Agilent Poroshell C18 column using gradient separation. The sample was extracted with methanol first and then acetone. The signals were detected at 240 nm and 355 nm. The limit of quantification was under 1.2 µg/mL (0.3 mg/g) for kavalactones and under 0.35 µg/mL (0.01 mg/g) for flavokavains. The Horwitz ratio values described ranged from 0.3 to 1.82. The spike recovery experiments showed an accuracy between 92 and 105% for all analytes. The results of the study demonstrate that the method is fit for the purpose of determining methysticin, dihydromethysticin, kavain, dihydrokavain, yangonin, desmethoxyyangonin, flavokavain A, flavokavain B, and flavokavain C in kava raw material and finished products (dry-filled capsule, liquid phytocaps, and tincture).

Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC-IT-TOF-MSn.

Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese medicine Scrophulariae Radix. High-performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-ESI-IT-TOF-MSn ) was used to profile and identify the metabolites of harpagoside in rats in vivo and to study the distribution of these metabolites in rats for the first time. A total of 45 metabolites were identified, 37 of which were postulated to be new compounds. The number of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitution, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites were determined to have bioactivities based on the literature and 'PharmMapper' analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological effects of harpagoside. Moreover, these findings provide a reference for studying the metabolism and distribution of iridoid compounds.

Chemical metabolome assay by high-resolution Orbitrap mass spectrometry and assessment of associated antitumoral activity of Actinocephalus divaricatus.

RATIONALE: Actinocephalus divaricatus (Eriocaulaceae) is an important source of income for rural communities as it is sold as an ornamental plant. To date, no investigation has been conducted concerning the chemical composition and biological studies of the aerial parts of A. divaricatus. METHODS: The methanolic extract of the aerial parts of this species was chemically characterized. We applied an analytical dereplication approach based on Liquid Chromatography coupled to High-Resolution Orbitrap Mass Spectrometry in order to develop, identify and define rapidly the metabolite fingerprint of the aerial parts of A. divaricatus. Biological in vitro antitumor tests were undertaken using breast and lung cell lines of mice and humans. RESULTS: High-Resolution Mass Spectrometry (HRMS) allowed the fast determination of 30 compounds, which comprised three different classes of compounds: naphthopyranones, flavonoids and saponins. Chromatographic fractionation of the crude methanolic extract validated these results, since it led to the isolation of compounds belonging to the aforementioned classes of compounds, including new acyl glycosylated flavonoids (6-hydroxy-7-methoxyquercetin-3-O-(2"-O-acetyl)-ß-D-glucopyranoside and 6-hydroxy-7-methoxyquercetin-3-O-(6"-O-acetyl)-ß-D-glucopyranoside), which were fully characterized by Nuclear Magnetic Resonance and Mass Spectrometry experiments, and a known triterpenic saponin (3-O-ß-D-glucuronopyranosyl-30-norolean-12,20(29)-dien-28-O-ß-D-glucopyranosyl ester). Biological assays indicated that the methanolic extract of the capitula exhibited the best in vitro cytotoxicity against MCF7 cells (human breast cancer). CONCLUSIONS: The HRMS technique enabled us to identify several classes of compounds. In addition, saponins were identified for the first time in plants belonging to the Eriocaulaceae family. Thus, the essential contribution of this work lies in the new elements it brings to the taxonomic discussion which the Actinocephalus genus as a distinct genus of the Paepalanthus. The results obtained show that the methanolic extract of the capitula could be a promising source of bioactive fractions and/or compounds that may contribute towards breast cancer treatment.

Use of Innovative (Micro)Extraction Techniques to Characterise Harpagophytum procumbens Root and its Commercial Food Supplements.

INTRODUCTION: For the determination of harpagoside and the wide phenolic pattern in Harpagophytum procumbens root and its commercial food supplements, dispersive liquid-liquid microextraction (DLLME), ultrasound-assisted DLLME (UA-DLLME), and sugaring-out liquid-liquid extraction (SULLE) were tested and compared. OBJECTIVES: In order to optimise the extraction efficiency, DLLME and UA-DLLME were performed in different solvents (water and aqueous solutions of glucose, ß-cyclodextrin, (2-hydroxypropyl)-ß-cyclodextrin, sodium chloride, natural deep eutectic solvent, and ionic liquid). MATERIAL AND METHODS: The plant material was ground and sieved to obtain a uniform granulometry before extraction. Commercial food supplements, containing H. procumbens are commercially available in Italy. RESULTS: The most effective sodium chloride-aided-DLLME was then optimised and applied for analyses followed by HPLC-PDA. For comparison, microwave-assisted extraction was performed using the same solvents and the best results were obtained using 1% of ß-cyclodextrin or 15% of sodium chloride. CONCLUSION: All commercial samples respected the European Pharmacopoeia monograph for this plant material, showing a harpagoside content ≥ 1.2%. Copyright © 2017 John Wiley & Sons, Ltd.

Iridoid glycosides and polyphenolic compounds from Teucrium chamaedrys L.

In this work, the phytochemical analysis of Teucrium chamaedrys L. collected in Italy was reported. Eight compounds were isolated and identified by means of classical column chromatography and spectroscopic techniques, such as NMR and MS. In detail, these compounds were: verbascoside (1), forsythoside b (2), samioside (3), alyssonoside (4), harpagide (5), 8-O-acetyl-harpagide (6), cirsiliol (7) and ß-arbutin (8). The presence of these compounds, in particular iridoids and phenyl-ethanoid glycosides, has a chemotaxonomic relevance and results to be in perfect accordance with the current botanical classification of the species. In addition, it provides a phytochemical rationale for the use of this particular plant in the ethno-pharmacological field. Conversely, it is worth of mention the absence of potentially toxic components, unlike to what observed in other species of the genus which can no longer be used for ethno-medicinal purposes.

Quantitative Determination of Lactones in Piper methysticum (Kava-Kava) by Supercritical Fluid Chromatography.

A fast and validated supercritical fluid chromatography method for the quantitative determination of major lactones in Piper methysticum, a plant used against nervous anxiety, stress, and restlessness, was developed. The baseline separation of dihydrokavain, demethoxyyangonin, kavain, yangonin, dihydromethysticin, and methysticin was possible in less than 4 min on an Aquity UPC2 BEH 1.7 µm column, in combination with a mobile phase comprising CO2 and methanol with diethylamine. The column temperature had a great impact on the results because only at 70 °C could kavain and yangonin be fully resolved. With correlation coefficients above 0.998, recovery rates between 95.9 and 104.1 % as well as limit of detection values below 1.5 ng on-column, the procedure fulfilled all validation requirements and was well suited for the quantitative analysis of commercial products containing P. methysticum root powder and/or extract. All of them contained the target analytes, however, the absolute content of lactones was quite variable. Accordingly, depending on the product, the total daily intake of lactones varied from 56 to 312 mg. Concerning speed, selectivity, and environmental friendly operation, this supercritical fluid chromatography approach surpasses all previously reported ones.

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

INTRODUCTION: Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. OBJECTIVE: To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). METHODOLOGY: Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. RESULTS: HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. CONCLUSION: HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Monoterpenoids from the traditional North Italian vegetable Aruncus dioicus (Walter) Fernald var. vulgaris (Maxim.) H.Hara (Rosaceae).

Investigations of young shoots of Aruncus dioicus (Walter) Fernald var. vulgaris (Maxim.) H.Hara (Rosaceae), collected from the wild and used as vegetables in alpine provinces of Italy, yielded eight monoterpenoids. Besides known compounds, aruncin A, aruncide A, and cimicifugolide, five previously undescribed substances, aruncins C, D, and E, and aruncides D and E, were identified. Based on results from the full synthesis of aruncin B, structures of aruncin A and aruncide A were revised. Structures were established by HR mass spectrometry and extensive 1D and 2D NMR spectroscopy and based on data from synthetic aruncin B. An HPLC-DAD-ESI-MS method was developed to investigate the distribution of the monoterpenoids in different organs of Aruncus dioicus var. vulgaris and in aerial parts of A. dioicus var. aethusifolius (H.Lév.) H.Hara [Syn.: Aruncus aethusifolius (H.Lév.) Nakai]. Preliminary bioactivity studies moreover indicated weak cytotoxicity for some of the compounds against human prostrate adenocarcinoma cells.

Karrikins Identified in Biochars Indicate Post-Fire Chemical Cues Can Influence Community Diversity and Plant Development.

BACKGROUND: Karrikins are smoke-derived compounds that provide strong chemical cues to stimulate seed germination and seedling growth. The recent discovery in Arabidopsis that the karrikin perception system may be present throughout angiosperms implies a fundamental plant function. Here, we identify the most potent karrikin, karrikinolide (KAR1), in biochars and determine its role in species unique plant responses. METHODS: Biochars were prepared by three distinct commercial-scale pyrolysis technologies using systematically selected source material and their chemical properties, including karrikinolide, were quantified. Dose-response assays determined the effects of biochar on seed germination for two model species that require karrikinolide to break dormancy (Solanum orbiculatum, Brassica tourneforttii) and on seedling growth using two species that display plasticity to karrikins, biochar and phytotoxins (Lactuca sativa, Lycopersicon esculentum). Multivariate analysis examined relationships between biochar properties and the plant phenotype. FINDINGS AND CONCLUSIONS: Results showed that karrikin abundant biochars stimulated dormant seed germination and seedling growth via mechanisms analogous to post-fire chemical cues. The individual species response was associated with its sensitivity to karrikinolide and inhibitory compounds within the biochars. These findings are critical for understanding why biochar influences community composition and plant physiology uniquely for different species and reaffirms that future pyrolysis technologies promise by-products that concomitantly sequester carbon and enhance plant growth for ecological and broader plant related applications.

Reassessment of Melittis melissophyllum L. subsp. melissophyllum iridoidic fraction.

The analysis of the polar fraction of Melittis melissophyllum L. subsp. melissophyllum led to the identification of several iridoid glycosides: monomelittoside (1), melittoside (2), harpagide (3), acetyl-harpagide (4) and ajugoside (5). Compounds 3 and 4 are considered marker compounds for the genus and, as well as compounds 1, 2 and 5, were already evidenced in a previous study on the nominal species. It was noteworthy of the presence of allobetonicoside (6) which was never reported for this genus. The isolation of 6 is very relevant because of its allose residue on the structure. Allose has been often found in the species of the subfamily Lamioideae even if it mostly regarded flavonoids considered of chemotaxonomical relevance for some correlated genera of Lamiaceae. Same as allosyl-glycosidic flavonoids, the presence of allosyl-glycosidic iridoids may also be an additional chemosystematic evidence of botanical relationships among Lamiaceae species and genera.

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae).

INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.

Simultaneous determination of alizarin and rubimaillin in Rubia cordifolia by ultrasound-assisted ionic liquid-reversed phase liquid chromatography.

Four kinds of ionic liquids were adopted to analyze the content of rubimaillin and alizarin in Rubia cordifolia roots with ultrasonic-assisted extraction coupled with HPLC. The chromatographic column, Purospher star RP-C18 (4.6 mm x 250 mm, 5 microm), was used. Methanol and 0.4% acetic acid-water as mobile phase with flow rate at 0.85 mL min(-1), gradient elution, detection wavelength at 250 nm, chromatographic column temperature was controlled at room temperature. The result showed that rubimaillin and alizarin had the highest extraction yield when the [ HMIM] PF6methanol solution concentration of 0.6 mol x L(-1) as extraction solvent and the conditions were solid-liquid ratio of 1:80 (g x mL(-1)). Under the optimal extraction conditions, the content of alizarin from 0.01 to 0.04 microg showed a good linearity (r = 0.9999), the average recovery was 97.12%, the content of rubimaillin from 0.41 to 1.35 microg showed a good linearity (r = 0.9999), the average recovery was 98.10%. This experiment adopted environmentally friendly reagent as extraction solvent, the extraction efficiency was improved, and the environmental pollution caused by organic solvent was avoided, the harm of human body aslo was reduced. This method was simple and reliable, its repeatability was also very good, which had an important significance in the study of traditional Chinese medicine active ingredient extraction methods.

Secoiridoid type of antiallergic substances in olive waste materials of three Japanese varieties of Olea europaea.

2-Hydroxy-3-ethylidene-5-(methoxycarbonyl)-3,4-dihydro-2H-pyran-4-acetic acid 2-(3,4-dihydroxyphenyl)ethyl ester (3,4-DHPEA-EA) is a kind of secoiridoid first found in three Japanese olive pomaces: Mission, Lucca, and Manzanillo. These varieties showed high activity of 3,4-DHPEA-EA as an antiallergic active substance with IC50 at 33.5 ± 0.6 µg/mL. Because 3,4-DHPEA-EA was the most abundant among the active substances in the pomaces and the activity of 3,4-DHPEA-EA was greater than that of hydroxytyrosol and elenolic acid, 3,4-DHPEA-EA, which has the ester linkage of hydroxytyrosol and elenolic acid, should be essential for antiallergic activity. Although a trace amount (1.04 mg/kg) of luteolin in the pomace showed the highest antiallergic activity with IC50 at 0.752 ± 0.1 µg/mL, we concluded that the entire antiallergic effect derives from the abundance of 3,4-DHPEA-EA, especially in the green olive pomace of the Mission variety in October, which showed the highest level of 3,4-DHPEA-EA (5033 ± 118 mg/kg). Therefore, the Mission variety had the most effective antiallergy property.

Looking beyond sugars: phytochemical profiling and standardization of manna exudates from Sicilian Fraxinus excelsior L.

Different grades of genuine and counterfeit Fraxinus excelsior exudates, marketed as natural sweeteners or mild laxatives, were evaluated for their proximate composition and for saccharidic, organic acids, lipidic and phenolic profile by means of GC-MS and (1)H NMR. Genuine samples contained mannitol (39-48 g/100 g, according to the grade), fructose (9-16 g/100 g), glucose (2-3.7 g/100 g), sorbitol (0,5-0,6 g/100 g), galactose (0.02-0.74 g/100 g), oligosaccharides as mannotriose (13-22 g/100 g) and stachyose (1-11 g/100 g), and traces of myo-inositol, mannose, sucrose. On the contrary, counterfeit samples contained mostly mannitol and sorbitol, with traces of fructose, glucose and mannose. Differences in ash, total polyphenolic content and fatty acid composition allowed a quick identification of counterfeit products, confirmed by a distinct mono-, oligosaccharidic and phenolic pattern. Elenolic acid (63-1628 mg/kg), tyrosol (15-774 mg/kg), homovanillic acid (2,39-52.8 mg/Kg), dopaol (0.8-63 mg/kg), pinoresinol (4.2-18.5 mg/kg) and fraxetin (0.25-11.64 mg/kg), albeit showing a wide concentration range, were the most abundant substances detected in the phenolic fraction of Fraxinus manna, while esculetin, p-hydroxybenzoic acid, 4-hydroxyphenacetic acid, 3,4 hydroxybenzoic acid, hydroxy-pinoresinol, medioresinol and siringaresinol were present in low amounts. The polyphenolic profile may be used as a marker for authentication and should be considered in the evaluation of nutritional and health properties ascribed to Fraxinus manna.