Valentina Petitto

Chemical fingerprinting of Equisetum arvense L. using HPTLC densitometry and HPLC

Equisetum arvense L. is a herbaceous medicinal plant, commonly known as horsetail, whose extracts have been reported to possess diuretic and haemostatic properties. The aim of this study was to evaluate the use of fingerprint chromatographic methods on commercially available raw materials or preparations of E. arvense L. in order to ascertain their quality and identify possible adulterants using HPLC and HPTLC densitometry. Two chromatographic methods were used to determine the chemical fingerprints of E. arvense and other allied species. The first was based on HPTLC identification followed by densitometric measurement at 350 nm. The second was based on HPLC separation. The ease of sample preparation and the possibility of simultaneous analysis of several samples in a short time make HPTLC a method of choice for the comprehensive quality evaluation of herbal products.

The Modern Analytical Determination of Botanicals and Similar Novel Natural Products by the HPTLC Fingerprint Approach

Abstract Efficient, useful and sensitive analytical devices for the quality control of complex mixtures of natural products are urgently needed. Controls should be based on simple, viable, comprehensible and low cost methods. High Performance Thin Layer Chromatography (HPTLC) the last evolution of planar chromatography, is a reliable candidate for analysis of botanicals. Although HPLC remains the best choice for organic substances, HPTLC can solve several problems in a quicker, simpler and easier method. Furthermore, the visualization and the analysis of similar products performed exactly in the same conditions, allow an evident, good and safe result of comparison. The paper is dedicated to HPTLC application on selected market products, in order to evidence benefits and limits of the HPTLC fingerprint approach.

Natural daucane sesquiterpenes with antiproliferative and proapoptotic activity against human tumor cells.

Plants of the genera Ferula and Ferulago are known for their complex content in bioactive secondary metabolites such as coumarins, phenylpropanoids, and sesquiterpenes. We used the ground parts of Ferula communis subsp. communis, Ferula glauca subsp. glauca and Ferulago campestris as natural sources for the isolation of four coumarins (CU-1 to CU-4), two phenylpropanoids (PE-1 and PE-2), one polyacetylene (PA-1) and 16 daucane esters (DE-1 to DE-16). The cytotoxic activity of the isolated compounds was evaluated against a panel of seven human tumor cell lines. Fourteen of the daucane derivatives showed antiproliferative activity at least against one of the human tumor cell lines tested, four compounds (DE-5, DE-8, DE-11, and DE-16) were active against all the tested cell lines. Among them DE-11 was the most cytotoxic compound against HeLa (4.4 ± 0.7 μM), A549 (2.8 ± 1.4 μM), HL-60 (2.6 ± 0.4 μM), K562 (26.5 ± 6.0 μM) RS 4;11 (1.7 ± 0.3 μM) and SEM (2.4 ± 0.1 μM) cell lines, while DE-8 was the most active against Jurkat (3.3 ± 0.8 μM). Preliminary structure-activity relationship suggests that the most active compounds in the daucane series present the trans fusion of the penta- and hepta-atomic cycles, and lipophylic ester groups linked to position 6. Isomeric derivatives such as DE-8 and DE-9 or DE-3, DE-4, and DE-5 exhibited significant differences in their IC(50) supporting that the β orientation for the ester group in the position 2 enhances the cytotoxic activity. Furthermore, the pro-apoptotic effect of the most active compounds evaluated in Jurkat cell line showed that these compounds are able to induce apoptosis in a time and concentration-dependent manner. Our findings suggest the potential role of daucane derivatives as models for the development of proapoptotic compounds.

Alkaloids from Glaucium flavum from Sardinia

Glaucium flavum collected in Sardinia was studied using a phytochemical approach in order to evaluate its alkaloid composition and obtain a comparison with the alkaloid contents of the same species in populations of other geographic proveniences. In fact, different chemoecotypes of G. flavum have been identified, on the basis of their particular content and composition in alkaloids, in accordance with the different distribution areas. The analysis showed that Sardinian G. flavum contains a homogeneous alkaloid pattern of aporphyne type, significantly different from those reported for populations from other parts of Europe.

Bearberry identification by a multidisciplinary study on commercial raw materials.

Herbal species different from the official bearberry, Arctostaphylos uva-ursi, are sold through conventional markets and also through non-controlled Internet websites, putting consumer safety at risk owing to the lack of quality control. Recently, Arctostaphylos pungens has become one of the most used species as a raw material for herbal medicines and dietary supplements in the place of official bearberry, a plant used for the treatment of various urinary disorders. A fingerprint identification based on an integrated application of different analytical techniques (HPTLC, NMR, HPLC-DAD and LC-ESI-MS) is here described to distinguish A. uva-ursi from A. pungens. The HPTLC and HPLC-DAD fingerprints resulted the simplest methods to differentiate the two species, whereas LC-ESI-MS was more useful to quantify arbutin, the main component of bearberry, and to evaluate its different content in the two species. This multidisciplinary study showed for the first time a specific phytochemical fingerprint of the new species A. pungens.

Contaminations of herbal products determined by NMR fingerprint.

The utilisation of NMR fingerprinting is proposed as a rapid, available and reliable method to determine the contamination of herbal products. The presence of nimesulide has been reported recently as the contaminant of P.C. 28 Plus, a product based on herbal drugs marketed by the Italian company Cosval. The presence of the substance, as well as its relevant concentration (5%), was first reported by HPLC/MS analysis by other authors. The use of an NMR fingerprint confirmed the previous contamination with nimesulide in P.C. 28 Plus. The same contaminant was also found in P.C. 28 Pink. Furthermore, an analysis of Alergix Plus, another product of the same factory, evidenced the presence of bromhexin.

Endemic Plants of Italy and Their Peculiar Molecular Pattern

Abstract Chemistry of natural organic products regards studies of molecular structural characters devoted to the identification of new molecular architectures and searching new compounds present in natural matrices characterized by biological useful activity. More recently, particular attention has been directed to phytochemical studies of plants, the so-called endemic species, growing in particular areas, characterized by narrow limits. The hypothesis is that a wide variety of climates, environmental, geological, geographical, and biological conditions has led to the diversification of species. Endemism is the condition of being restricted to a particular area with a prescribed extent and describes species that are native to a particular geographic territory, and usually occurs in areas that are in some way isolated. The term “endemism” derives from the medical language and it was used for the first time, referred to plants, in 1820 by De Candolle. It is possible to distinguish several categories of endemisms. In 1926 Herzog sets the differences between neoendemics and paleoendemics species. Favarger & Contandriopoulos in 1961 and Favarger in 1964 proposed a new classification based on the mode of genesis of the species: schizoendemism, apoendemism, patroendemism, and paleoendemism. Paleoendemism and patroendemism represent the conservative component of the flora of a territory while schizoendemism and apoendemism represent the innovating component of it. Like other Mediterranean-type ecosystems, the Mediterranean Basin has high levels of plant diversity and endemism. The floral diversity of the Mediterranean Basin is dramatic. Its 25,000 vascular plants species (13,000 endemic species) are more than four times the number found in all the rest of Europe. The Mediterranean region constitutes both a refuge area and one that encourages floral exchange and active plant speciation. Many of the endemic species are concentrated on islands, peninsulas, cliffs, and peaks. In this review, studies on the molecular pattern of endemic plants of restricted areals (Sardinia Island and Central Italy with Monti Sibillini National Park and Majella National Park) are reported, with particular attention toward plants containing active compounds and plants with an ethnopharmacologic history and tradition. These two areas were selected as representative sites of insular and peninsular areas of Italy.

Iridoids from Euphrasia genargentea, a rare Sardinian endemism

The phytochemical study of Euphrasia genargentea, a rare species only present in Sardinia, led to the identification of iridoid glucosides, i.e. aucubin, catalpol, mussaenosidic acid and melampyroside, which allowed chemotaxonomic considerations on the genus. On the basis of iridoid distribution in the genus, E. genargentea does not show any particular analogy with other Italian Euphrasia spp. This study is also important considering the severe risk of extinction of E. genargentea.

Pharmacological activities on Ephedra nebrodensis Tineo

As a part of our endeavour to screen Mediterranean medicinal plants for various pharmacological activities, we evaluated antihistaminic, adaptogenic, anti-inflammatory, antinociceptive, hypotensive and locomotor properties, and antioxidant potential of Ephedra nebrodensis. 1H-NMR spectroscopy was carried out to identify the plant metabolites, which confirmed the presence of ephedrinic skeleton alkaloids. The ethanol : acetone (1 : 1) extract exhibited dose-related antihistaminic, anti-inflammatory, antinociceptive, hypotensive, antioxidant and locomotor stimulant activity. The plant bears potential for further studies.

Solid-Phase Microextraction (SPME) Analysis of Six Italian Populations of Ephedra nebrodensisTineo ex Guss. subsp. nebrodensis

Headspace solid‐phase microextraction (HS‐SPME) coupled with GC/FID and GC/MS was applied for the first time in the analysis of the volatile fraction of an Ephedra species. Notably, six Italian populations (Marche, Abruzzo, and Sardinia) of Ephedra nebrodensis subsp. nebrodensis, covering almost the entire Italian area, were investigated to examine the chemical variability and to support the taxonomy of the species. A fiber screening with polymethylsiloxane (PDMS), CarboxenTM/polymethylsiloxane (CAR/PDMS), and polymethylsiloxane/divinylbenzene (PDMS/DVB) coatings, together with an optimization of the extraction conditions were carried out before analysis of the six populations. A total of 119 volatiles were identified in the headspace of different samples, accounting for 63.35–100.00% of the total volatiles. A great variability was found in the qualitative composition of different samples, since only 18 components were in common among all populations. The headspace composition was dominated by sesquiterpene hydrocarbons (52.30–88.32%), with β‐maaliene (traces–7.49%), β‐patchoulene (traces–1.29%), β‐panasinsene (traces–6.85%), α‐isocomene (traces–31.25%), α‐trans‐bergamotene (traces–6.95%), alloaromadendrene (traces–33.20%), α‐acoradiene (traces–9.41%), and γ‐muurolene (0.61–16.33%) being the most abundant constituents. Noteworthy is the occurrence in a sample of two major unknown sesquiterpenes, one hydrocarbon (24.49%, RI: 1396) and one oxygenated compound (10.37%, RI: 1591), whose mass spectra were reported for the first time. Multivariate chemometric techniques, such as cluster analysis (CA) and principal component analysis (PCA), were used to characterize the samples according to the geographical origin.