Matteo Marieschi

Quality control of saffron (Crocus sativus L.): development of SCAR markers for the detection of plant adulterants used as bulking agents.

A method based on sequence-characterized amplified regions (SCARs) was developed from random amplified polymorphic DNA markers (RAPDs) specific for Arnica montana L., Bixa orellana L., Calendula officinalis L., Carthamus tinctorius L., Crocus vernus L. (Hill), Curcuma longa L., and Hemerocallis sp. to detect these common bulking agents in commercial saffron (Crocus sativus). The method enabled the unequivocal detection of low amounts (up to 1%) of each adulterant, allowing the preemptive rejection of suspect samples. Its enforcement limits the number of samples to be subjected to further evaluation with pharmacognostic or phytochemical analyses, especially when multiple batches have to be evaluated in a short time. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from dried, stored, processed, and finely ground commercial material. Proper SCAR markers may represent a fast, sensitive, reliable, and low-cost screening method for the authentication of dried commercial saffron material.

RAPD-Based Method for the Quality Control of Mediterranean Oregano and Its Contribution to Pharmacognostic Techniques

A pharmacognostic survey of 84 commercial samples of Mediterranean oregano, obtained from wholesale traders between 2001 and 2007, pinpointed the presence of extraneous plant material in 90.5% of the samples. In 59% of them extraneous material of plant origin was above 20%. Two major groups of botanical foreign matter were identified: oregano-like flavored plants ( Satureja montana L., Origanum majorana L.) and plants lacking a clearly detectable essential oil profile ( Rubus sp., Cistus incanus L., Rhus coriaria L.), added as bulk extraneous material. A random amplified polymorphic DNA (RAPD) method was developed to make the detection of the second group of adulterants easier and speed pharmacognostic analysis of large batches of samples. Thirteen primers discriminating between Origanum spp. and Rubus caesius , R.coriaria, and C. incanus were individuated, allowing their detection in oregano samples with a limit of detection of 1%. The utilization of RAPD as a reliable test to probe the authenticity of Mediterranean oregano or previously screen the presence of specific contaminants is proposed as a complementary approach to pharmacognostic and phytochemical screening.

In vitro micropropagation of the aquatic fern Marsilea quadrifolia L. and genetic stability assessment by RAPD markers

In order to conserve and multiply the aquatic fern Marsilea quadrifolia L., in a long-term in vitro procedure, the effects of different cytokinins, i.e., 6-benzylaminopurine, zeatine riboside, and N6-(2-isopentenyl)adenine, were investigated, varying their concentration and period of supplementation. No clear stimulatory effect on the de novo nodes produced per explant was detected when compared with hormone-free (HF) condition. On the contrary, the rhizome explant micropropagation was inhibited, the inhibition decreasing with the decreasing strength of cytokinins, though without reaching any significant enhancement. Since, as a consequence of the tissue culture procedure, the occurrence of somaclonal variation may introduce genomic alterations, genetic stability was assessed by random amplified polymorphic DNA (RAPD) analysis by comparing eight randomly selected micropropagated plants derived from repeated subcultures, with donor plant. Eighteen different primers generated 189 bands ranging from 100 to 3250 bp, and the same banding profiles were exhibited. No genomic alterations were evidenced in any of the micropropagated plants. Well-developed micropropagated plants were also successfully acclimatized under greenhouse condition. These positive results suggest that the in vitro HF micropropagation could be useful in the development of ex situ conservation programs of M. quadrifolia, even in order to possibly reintroduce the plants in their natural environment.

Phytochemical profile and bioactivity of traditional ayurvedic decoctions and hydro-alcoholic macerations of Boerhaavia diffusa L. and Curculigo orchioides Gaertn.

Decoctions (DECs) and hydro-alcoholic extracts (HEs) prepared from roots of Boerhaavia diffusa L. (Nyctaginaceae) and Curculigo orchioides Gaertn. (Hypoxidaceae) were phytochemically characterised by HPLC-DAD and profiled for their antioxidant, antigenotoxic and cytotoxic activities. B. diffusa DEC was rich in ferulic acid and vanillin, while the HE also contained boeravinone B and eupalitin. Both C. orchioides HE and DEC displayed the main occurrence of orcinol-β-d-glucoside and curculigoside A. Antioxidant activity was assayed through spectrophotometric DPPH, ABTS and β-carotene bleaching test, and using (HP)TLC bioautographic strategies. For both crude drugs, HE was the best performing preparation. Properly modified SOS-Chromotest evidenced a 10% inhibition by phytocomplexes against 4-nitroquinoline-N-oxide, and a higher bioactivity for vanillin (36.60 ± 1.68%) and ferulic acid (35.09 ± 1.53%). C. orchioides HE was the preparation which showed higher cytotoxicity against drug-sensitive human T-lymphoblastoid cell line (CCRF-CEM) and multidrug-resistant leukaemia cell line (CEM/ADR5000), and eupalitin was the only pure compound to exhibit an IC50 value.

Authentication of Punica granatum L.: development of SCAR markers for the detection of 10 fruits potentially used in economically motivated adulteration

The large commercial success of pomegranate increase the likelihood of economically motivated adulteration (EMA), which has been gradually spotted with the undeclared addition of anthocyanin-rich plants or cheaper fruit juices used as bulking and diluting agents. A method based on Sequence-Characterized Amplified Regions (SCARs) was developed to detect the presence of Aristotelia chilensis, Aronia melanocarpa, Dioscorea alata, Euterpe oleracea, Malus×domestica, Morus nigra, Sambucus nigra, Vaccinium macrocarpon, Vaccinium myrtillus, Vitis vinifera as bulking agents in Punica granatum. The method enabled the unequivocal detection of up to 1% of each adulterant, allowing the preemptive rejection of suspect samples. The recourse to such method may reduce the number of samples to be subjected to further phytochemical analyses when multiple batches have to be evaluated in a short time. Vice versa, it allows the cross-check of suspect batches previously tested only for their anthocyanin profile. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from stored and processed commercial material. Proper SCAR markers may represent a fast, sensitive, reliable and low-cost screening method for the authentication of processed commercial pomegranate material.

Identification of S2-T A63: a cDNA fragment corresponding to a gene differentially expressed in a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus.

The gene expression of the wild type (S2-N) and a Cr-tolerant strain (S2-T) of the unicellular green alga Scenedesmus acutus has been compared in order to get more insight on their different chromium sensitivity. The RNA of the two strains was extracted after 4 days of culture in standard medium without chromium and analyzed by means of RNA differential display. The two strains showed differential gene transcription even in the absence of the heavy metal and six putatively differential amplicons were evidenced in the Cr-tolerant strain. Among the isolated amplicons, S2-T A63 was much more pronouncedly transcribed in the tolerant than in the wild type strain and was further characterized. S2-T A63 corresponding gene is present with the same copy number in the wild type and tolerant genomes and corresponds to an mRNA of about 2000 nt. The corresponding transcript is overexpressed in the Cr-tolerant strain after a 4-day culture and is not up-regulated by chromium exposure. The S2-T A63 sequence, obtained up to now, does not show significant homologies with any known gene. However, the analysis of the putative translation product reveals the presence of an interrupted fasciclin domain. This extracellular domain has been found in proteins from mammals, insects, echinoderms, plants, yeast and bacteria and is usually involved in cell adhesion. This finding suggests that the product for the S2-T A63 translation has an extracellular collocation, maybe as surface or secreted protein involved in external chromium detoxification.

Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation: Chromium uptake and compartmentalization in two strains with different sensitivities to Cr(VI).

In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys. Hence, sulfur availability can modulate the capacity to cope with environmental stresses, a phenomenon known as SIR/SED (Sulfur Induced Resistance or Sulfur Enhanced Defence). Since chromate may compete for sulfate transport into the cells, in this study chromium accumulation and tolerance were investigated in relation to sulfur availability in two strains of the unicellular green alga Scenedesmus acutus with different Cr-sensitivities. Paradoxically, sulfur deprivation has been demonstrated to induce a transient increase of Cr-tolerance in both strains. Sulfur deprivation is known to enhance the sulfate uptake/assimilation pathway leading to important consequences on Cr-tolerance: (i) reduced chromate uptake due to the induction of high affinity sulfate transporters (ii) higher production of cysteine and GSH which can play a role both through the formation of unsoluble complexes and their sequestration in inert compartments. To investigate the role of the above mentioned mechanisms, Cr accumulation in total cells and in different cell compartments (cell wall, membranes, soluble and miscellaneous fractions) was analyzed in both sulfur-starved and unstarved cells. Both strains mainly accumulated chromium in the soluble fraction, but the uptake was higher in the wild-type. In this type a short period of sulfur starvation before Cr(VI) treatment lowered chromium accumulation to the level observed in the unstarved Cr-tolerant strain, in which Cr uptake seems instead less influenced by S-starvation, since no significant decrease was observed. The increase in Cr-tolerance following S-starvation seems thus to rely on different mechanisms in the two strains, suggesting the induction of a mechanism constitutively active in the Cr-tolerant strain, maybe a high affinity sulfate transporter also in the wild-type. Changes observed in the cell wall and membrane fractions suggest a strong involvement of these compartments in Cr-tolerance increase following S-starvation.

Phytotoxic Effects and Phytochemical Fingerprinting of Hydrodistilled Oil, Enriched Fractions, and Isolated Compounds Obtained from Cryptocarya massoy (Oken) Kosterm. Bark.

The hydrodistilled oil of Cryptocarya massoy bark was characterized by GC‐FID and GC/MS analyses, allowing the identification of unusual C10 massoia lactone (3, 56.2%), C12 massoia lactone (4, 16.5%), benzyl benzoate (1, 12.7%), C8 massoia lactone (3.4%), δ‐decalactone (5, 1.5%), and benzyl salicylate (2, 1.8%) as main constituents. The phytotoxic activities of the oil, three enriched fractions (lactone‐rich, ester‐rich, and sesquiterpene‐rich), and four constituents (compounds 1, 2, 5, and δ‐dodecalactone (6)) against Lycopersicon esculentum and Cucumis sativus seeds and seedlings were screened. At a concentration of 1000 μl/l, the essential oil and the massoia lactone‐rich fraction caused a complete inhibition of the germination of both seeds, and, when applied on tomato plantlets, they induced an 85 and 100% dieback, respectively. These performances exceeded those of the well‐known phytotoxic essential oils of Syzygium aromaticum and Cymbopogon citratus, already used in commercial products for the weed and pest management. The same substances were also evaluated against four phytopathogenic bacteria and ten phytopathogenic fungi, providing EC50 values against the most susceptible strains in the 100–500 μl/l range for the essential oil and in the 10–50 μl/l range for compound 6 and the lactone‐rich fraction. The phytotoxic behavior was related mainly to massoia lactones and benzyl esters, while a greater amount of 6 may infer a good activity against some phytopathogenic fungi. Further investigations of these secondary metabolites are warranted, to evaluate their use as natural herbicides.

Identification and genetic structure of wild Italian Humulus lupulus L. and comparison with European and American hop cultivars using nuclear microsatellite markers

Nine genic SSR loci were used to evaluate the genetic diversity and identify accessions in wild Italian Humulus lupulus L., in comparison with widely cultivated European and U.S. commercial cultivars. A collection of 80 wild hop samples from Italy and 43 hop cultivars from Europe and U.S., were characterized. Allelic frequency analysis revealed 65 distinct Italian genotypes and differentiated all the commercial cultivars; moreover, specific alleles were observed for wild and cultivated hops. The number of alleles identified in the wild population were 104 and 123 within all the accessions. The maximum polymorphic information content was evidenced for locus HlGA23 in the Italian wild population and in the whole set of accessions (0.905 and 0.902 respectively). The dendrogram constructed from Euclidean distance with the UPGMA method showed two main clusters, one including commercial American and European accessions and one mostly composed by wild Italian accessions. Model-based clustering (Bayesian method) placed the accessions into five germplasm groups, one of which was characterized by Italian genotypes only. The study showed for the first time the great biodiversity present in Italy, and the remarkable differences with European and American hops. It was also found that within the population of north-central Italy a large genetic variability is present, suited to be studied and exploited; this genetic wealth could be used in future breeding programs in order to develop new hop varieties carrying characteristics useful for brewers.

Ultrastructural features, chromium content and in situ immunodetection of 5-methyl-cytosine following Cr (VI) treatment in two strains of Scenedesmus acutus M. (Chlorophyceae) with different chromium sensitivity

Abstract Two strains of the unicellular green alga Scenedesmus acutus (F.J.F. Meyen) with different sensitivity to chromium (VI) were compared to evaluate their ultrastructural morphology in chromium-free and -supplemented medium with a sub-lethal concentration of Cr(VI) for 72 hours. The ultrastructural alteration in different cytological compartments indicated that Cr(VI) induced earlier and stronger alterations in the wild type (wt) compared with the chromium-tolerant strain (Cr-t). After Cr treatments, ICP-MS (Inductively Coupled Plasma Mass Spectrometry) showed a higher Cr accumulation in the wild type than in the Cr-tolerant strain, suggesting a more efficient chromium-exclusion mechanism in the latter. The Cr treatment induced an increase in the nuclear area and a rearrangement in the eu-heterochromatic fraction, suggesting that chromatin remodelling could be at the basis of differential gene expression and metal tolerance. To gain additional information on the remodelling of the nuclear chromatin, we analysed DNA methylation by immunolocalization of 5-methyl-cytosine, before and after Cr exposure. Significant differences in the quantification of the immunolabelling of DNA cytosine-rich zones between the two strains were observed. These data suggest that an epigenetic mechanism could be at the basis of the Cr tolerance in S. acutus, as supported by previous data reporting that the acquired tolerance was inherited and maintained through the progeny.