Gianluca Gilardoni

Anti-secretory, anti-inflammatory and anti-Helicobacter pylori activities of several fractions isolated from Piper carpunya Ruiz & Pav.

ETHNOPHARMACOLOGICAL RELEVANCE The leaves of Piper carpunya Ruiz & Pav. (syn Piper lenticellosum C.D.C.) (Piperaceae), are widely used in folk medicine in tropical and subtropical countries of South America as an anti-inflammatory, anti-ulcer, anti-diarrheal and anti-parasitical remedy as well as an ailment for skin irritations. AIMS OF THE STUDY To study the anti-inflammatory, anti-secretory and anti-Helicobacter pylori activities of different fractions isolated from an ethanolic extract of the leaves of Piper carpunya, in order to provide evidence for the use of this plant as an anti-ulcer remedy. Moreover, to isolate the main compounds of the extract and relate their biological activity to the experimental results obtained with the fractions. MATERIALS AND METHODS Sixteen fractions were obtained from the ethanolic extract (F I-XVI) and 16 pure compounds were isolated and identified from these fractions. We studied the effects of the fractions (0.1-400microg/mL) on the release of myeloperoxidase (MPO) enzyme from rat peritoneal leukocytes, on rabbit gastric microsomal H(+), K(+)-ATPase activity and anti-Helicobacter pylori anti-microbial activity using the microdilution method (MM). The main compounds contained in the fractions were isolated and identified by (1)H- and (13)C NMR spectra analysis and comparison with the literature data. RESULTS Eight fractions showed inhibition of MPO enzyme (F I-IV, X, XII, XIV and XV). The highest inhibition was observed with F XIV (50microg/mL, 60.9%, p<0.001). F X and XII were the most active ones, inhibiting the gastric H(+), K(+)-ATPase activity with IC(50) values equal to 22.3microg/mL and 28.1microg/mL, respectively. All fractions, except F XV, presented detectable anti-Helicobacter pylori activity, with a diameter of inhibition zones ranging from 11mm up to 50mm. The best anti-Helicobacter pylori activity was obtained with F III and V. Both fractions killed Helicobacter pylori with lowest concentration values, about 6.25mug/mL. Sixteen pure compounds were isolated, five of them are flavonoids that possess strong anti-oxidant and free radical scavenging activity, e.g. vitexin, isovitexin, and rhamnopyranosylvitexin. Terpenoids like sitosterol, stigmasterol and phytol, which have shown gastroprotective activity, and dihydrochalcones, like asebogenin, with anti-bacterial activity, were also isolated. Furthermore, the rare neolignan 1, that is a DNA polymerase beta lyase inhibitor, and (6S, 9S)-roseoside, that shows strong anti-bacterial activity, were isolated, for the first time, from the genus Piper. CONCLUSIONS We suggest that the flavonoids isolated from F I and II (vitexin, isovitexin, rhamnopyranosylvitexin and isoembigenin) contribute to the anti-MPO activity, as well as to their anti-Helicobacter pylori activity. These flavonoids may also be responsible for the important inhibition of H(+), K(+)-ATPase activity. Also the phytosterols and phytol obtained from F XIV and XV could be involved in these gastroprotective activities. These results encourage us to continue phytochemical studies on these fractions in order to obtain full scientific validation for this species.

Chemical Constituents of Muehlenbeckia tamnifolia (Kunth) Meisn (Polygonaceae) and Its In Vitro α-Amilase and α-Glucosidase Inhibitory Activities

The phytochemical investigation of Muehlenbeckia tamnifolia, collected in Loja-Ecuador, led to the isolation of nine known compounds identified as: lupeol acetate (1); cis-p-coumaric acid (2); lupeol (3); β-sitosterol (4) trans-p-coumaric acid (5); linoleic acid (6) (+)-catechin (7); afzelin (8) and quercitrin (9). The structures of the isolated compounds were determined based on analysis of NMR and MS data, as well as comparison with the literature. The hypoglycemic activity of crude extracts and isolated compounds was assessed by the ability to inhibit α-amylase and α-glucosidase enzymes. The hexane extract showed weak inhibitory activity on α-amylase, with an IC50 value of 625 µg·mL−1, while the other extracts and isolated compounds were inactive at the maximum dose tested. The results on α-glucosidase showed more favorable effects; the hexanic and methanolic extracts exhibited a strong inhibitory activity with IC50 values of 48.22 µg·mL−1 and 19.22 µg·mL−1, respectively. Four of the nine isolated compounds exhibited strong inhibitory activity with IC50 values below 8 µM, much higher than acarbose (377 uM). Linoleic acid was the most potent compound (IC50 = 0.42 µM) followed by afzelin, (+)-catechin and quercitrin.

Chemical composition and antimicrobial activity of Phyllanthus muellerianus (Kuntze) Excel essential oil.

ETHNOPHARMACOLOGICAL RELEVANCE Phyllanthus muellerianus (Kuntze) Excel (family Euphorbiaceae) stem bark is used in Cameroon by Baka pygmies as a remedy for wound healing and tetanus. AIM OF THE STUDY To characterize the chemical composition and to evaluate the antimicrobial properties of the essential oil of the plant. MATERIALS AND METHODS The essential oil was extracted from the stem bark by dynamic head space and by hydrodistillation and characterized by GC and GC-MS analyses. The antimicrobial activity was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal-fungicidal concentration (MBC-MFC) by the micro and macrodilution methods. The following bacteria and fungi were used: Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, Candida albicans LM 450, Trichophyton mentagrophytes LM 230, Trichophyton rubrum LM 237, Microsporum canis LM 324. RESULTS The hydrodistillation afforded 0.06% (dry weight basis) of pale yellow oil. Thirty-eight compounds representing 90.69% were identified. The major component (36.40%) was found to be (E)-isoelemicin, identified by comparison of its (1)H-NMR experimental data, with literature data. The oil showed good antibacterial activity against Clostridium Sporogenes, Streptococcus mutans and Streptococcus pyogenes with MIC ranging from 13.5 to 126 μg/ml. A weak antifungal activity (MIC 250 μg/ml) was found against Trichophyton rubrum, only. CONCLUSIONS The antimicrobial activity and the chemical composition of Phyllanthus muellerianus stem bark essential oil are reported for the first time.

Chemical Composition and Antimicrobial Activity of the Volatile Fractions from Leaves and Flowers of the Wild Iraqi Kurdish Plant Prangos peucedanifolia Fenzl

The volatile fractions isolated from Prangos peucedanifolia Fenzl leaves and flowers were investigated for their phytochemical composition and biological properties. Flower and leaf hydrodistillation afforded 3.14 and 0.49 g of yellowish oils in 1.25 and 0.41% yields, respectively, from dry vegetable materials. According to the GC‐FID and GC/MS analyses, 36 (99.35% of the total oil composition) and 26 compounds (89.12%) were identified in the two oils, respectively. The major constituents in the flower volatile fraction were β‐pinene (35.58%), α‐pinene (22.13%), and β‐phellandrene (12.54%), while m‐cresol (50.38%) was the main constituent of the leaf volatile fraction. The antimicrobial activity was evaluated against several bacterial and fungal strains, on the basis of the minimum inhibitory concentration (MIC) by the micro‐ and macrodilution methods. The two volatile fractions showed moderate antifungal and antibacterial activities, especially against Trichophyton rubrum (MIC of 2×103 μg/ml), Streptococcus mutans, Streptococcus pyogenes, and Staphylococcus aureus (MIC≤1.9×103 μg/ml for all).

Chemical composition, enantiomeric analysis, AEDA sensorial evaluation and antifungal activity of the essential oil from the Ecuadorian plant Lepechinia mutica Benth (Lamiaceae)

This study describes the GC‐FID, GC/MS, GC‐O, and enantioselective GC analysis of the essential oil hydrodistilled from leaves of Lepechinica mutica (Lamiaceae), collected in Ecuador. GC‐FID and GC/MS analyses allowed the characterization and quantification of 79 components, representing 97.3% of the total sample. Sesquiterpene hydrocarbons (38.50%) and monoterpene hydrocarbons (30.59%) were found to be the most abundant volatiles, while oxygenated sesquiterpenes (16.20%) and oxygenated monoterpenes (2.10%) were the minor components. In order to better characterize the oil aroma, the most important odorants, from the sensorial point of view, were identified by Aroma Extract Dilution Analysis (AEDA) GC‐O. They were α‐Pinene, β‐Phellandrene, and Dauca‐5,8‐diene, exhibiting the characteristic woody, herbaceus, and earthy odors, respectively. Enantioselective GC analysis of L. mutica essential oil revealed the presence of twelve couples and two enantiomerically pure chiral monoterpenoids. Their enantiomeric excesses were from a few percent units to 100%. Moreover, the essential oil exhibited moderate in vitro activity against five fungal strains, being especially effective against M. canis, which is a severe zoophilic dermatophyte causal agent of pet and human infections.

Structures and biological significance of lactarane sesquiterpenes from the European mushroom Russula nobilis

The results of the first investigation on secondary metabolites occurring in intact and damaged fruiting bodies of Russula nobilis growing in Europe, are reported. Intact mushrooms contain fatty acid esters of velutinal, while three new sesquiterpenoids, russulanobilines A–C, along with eight known ones were isolated from extracts of injured fruiting bodies. The structures of the new compounds were elucidated on the basis of spectral data, molecular modeling, and 13C NMR spectra calculation. This pattern of sesquiterpenes constitutes a new variant of a chemical defense machinery which protects mushrooms against predators, parasites, and microorganisms.

Cadinane sesquiterpenes from the mushroom Lyophyllum transforme

Two rare cadinane-type sesquiterpenes, lyophyllone A (1) and lyophyllanetriol A (2), were isolated from the mushroom Lyophyllum transforme. The structures were elucidated on the basis of exhaustive NMR techniques, together with MS, UV-Vis and molecular modelling. The absolute configuration of lyophyllone A was determined by ab initio theoretical CD calculation performed by Density Functional Theory (DFT) using the B3PW91/6-31G(d,p) basis set. The experimental CD were found to be in good agreement with the corresponding population-weighted theoretical CD spectra, allowing for the determination of the absolute stereochemistry of the compound.

Phytochemical researches and antimicrobial activity of Clinopodium nubigenum Kunth (Kuntze) raw extracts

The essential oil of the species Clinopodium nubigenum (Kunth) Kuntze, Lamiaceae, was analyzed by GC-MS and GC-FID, taking into account the more recent literature. Among the seventy compounds identified, the majority are oxygenated monoterpenoids. The essential oil, tested for antimicrobial activity, resulted effective in vitro against Candida albicans. From the aqueous MeOH extract of the aerial parts of the plant two nonvolatile compounds, named schizonepetoside A and schizonepedoside C, have been isolated. They are rare glycosyl terpenoids, which were previously isolated from only one plant, but never found before in the genus Clinopodium.

Acorenone B: AChE and BChE Inhibitor as a Major Compound of the Essential Oil Distilled from the Ecuadorian Species Niphogeton dissecta (Benth.) J.F. Macbr

This study investigated the chemical composition, physical proprieties, biological activity, and enantiomeric analysis of the essential oil from the aerial parts of Niphogeton dissecta (culantrillo del cerro) from Ecuador, obtained by steam distillation. The qualitative and quantitative analysis of the essential oil was realized by gas chromatographic and spectroscopic techniques (GC-MS and GC-FID). Acorenone B was identified by GC-MS and NMR experiments. The enantiomeric distribution of some constituents has been assessed by enantio-GC through the use of a chiral cyclodextrin-based capillary column. We identified 41 components that accounted for 96.46% of the total analyzed, the major components were acorenone B (41.01%) and (E)-β-ocimene (29.64%). The enantiomeric ratio of (+)/(−)-β-pinene was 86.9:13.1, while the one of (+)/(−)-sabinene was 80.9:19.1. The essential oil showed a weak inhibitory activity, expressed as Minimal Inhibitory Concentration (MIC), against Enterococcus faecalis (MIC 10 mg/mL) and Staphylococcus aureus (MIC 5 mg/mL). Furthermore, it inhibited butyrylcholinesterase with an IC50 value of 11.5 μg/mL. Pure acorenone B showed inhibitory activity against both acetylcholinesterase and butyrylcholinesterase, with IC50 values of 40.8 μg/mL and 10.9 μg/mL, respectively.

New 3,4‐Secocycloartane and 3,4‐Secodammarane Triterpenes from the Ecuadorian Plant Coussarea macrophylla

Coussarea macrophylla (Mart.) Müll.Arg. (Rubiaceae) was collected in Ecuador, and the bark was extracted with AcOEt. Chromatographic separation afforded six novel 3,4‐secocycloartane and 3,4‐secodammarane triterpenes, named macrocoussaric acids A–F, together with the known metabolites secaubryenol and 3,4‐secodammara‐4(28),20,24‐triene‐3,26‐dioic acid. The structures of the new compounds were determined on the basis of their spectroscopic data. This is the first report of 3,4‐secocycloartane and 3,4‐secodammarane triterpenes occurring in a Coussarea species. Macrocoussaric acids A and B (2 and 3, resp.) were found to be moderately cytotoxic against five different tumor cell lines.