Micromorphological and phytochemical survey of Ballota acetabulosa (L.) Benth.

Within the Open Science project entitled 'Botanic Garden, factory of molecules', a multidisciplinary study approach was applied to Ballota acetabulosa (L.) Benth., at the Ghirardi Botanic Garden (Toscolano Maderno, BS, Italy). Micromorphological and histochemical investigations were performed on the secreting structures of the vegetative and reproductive organs under light, fuorescence and electronic microscopy. Concurrently the characterization of the volatiles spontaneously emitted from leaves and flowers were examined. Four trichome morphotypes were identified: peltate and short-stalked, medium-stalked and long-stalked capitate trichomes, each with a specific distribution pattern. The histochemical analysis was confirmed using ultrastructural observations, with the peltates and long-stalked capitates as the main sites responsible for terpene production. The head-space characterization revealed that sesquiterpene hydrocarbons dominated both in leaves and flowers, with γ-muurolene, ß-caryophyllene and (E)-nerolidol as the most abundant compounds. Moreover, a comparison with literature data concerning the ecological roles of the main compounds suggested their dominant roles in defence, both at the leaf and flower level. Hence, we correlated the trichome morphotypes with the production of secondary metabolites in an attempt to link these data to their potential ecological roles. Finally, we made the obtained scientific knowledge available to visitors of the Botanic Garden through the realization of new labelling dedicated to B. acetabulosa that highlights the 'invisible', microscopic features of the plant.

A novel study approach on Scutellaria altissima L. cultivated at the Ghirardi Botanic Garden (Lombardy, Italy).

Within an Open Science project, research was carried out to describe to the public of the Ghirardi Botanic Garden (BS, Lombardy, Italy) the invisible features of plants. This work is dedicated to Scutellaria altissima L. (Lamiaceae). Micromorphological, histochemical and phytochemical investigations were conducted on the vegetative and reproductive organs to correlate the structures involved in the emission of substances and their unique productivity. This work reports volatile organic compound (VOC) profiles of leaves and flowers and the composition of essential oil (EO) obtained from aerial parts of plants cultivated in Italy that have never been described before. Three morphotypes of glandular trichomes were observed: peltate, short-stalked capitate and long-stalked capitate. Peltate trichomes were the main producers of terpenes, short-stalked capitates of polysaccharides and long-stalked capitates of terpenes and polyphenols. The leaf VOC profile showed heterogeneous composition, with non-terpene derivatives as the major chemical class (71.04%), while monoterpene hydrocarbons represented almost the totality of the flower (99.73%). The leaf presented a higher number of total (37 versus 11) and exclusive (33 versus 7) compounds. (Z)-3-Hexenol acetate was most abundant in the leaf and (E)-ß-ocimene in the flower. Four common compounds were detected: ß-pinene, ß-caryophyllene, γ-muurolene and germacrene-D. The EO contaied 21 compounds, dominated by ß-caryophyllene, linalool and hexahydrofarnesyl acetone. This research allowed us to correlate morphotypes of the secretory structures with the production of secondary metabolites, with the aim of providing the public of the Ghirardi Botanic Garden with a dedicated iconographic approach, which accounts for olfactory perception linked to S. altissima.

Author Correction: Essential oils sensory quality and their bioactivity against the mosquito Aedes albopictus.

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Essential oils sensory quality and their bioactivity against the mosquito Aedes albopictus.

Repellents are a main tool to prevent the outbreak of mosquito-borne diseases that represents a threat for millions of people worldwide. Plant-based products are very promising, low-toxic and eco-friendly alternative to synthetic repellents. Here, we performed an olfactory screening of the essential oils (EOs) of Artemisia verlotiorum Lamotte (Asteraceae), Lavandula dentata L. (Lamiaceae), and Ruta chalepensis L. (Rutaceae) for their possible use as ingredients in topical repellents. The EOs smell profiles were then matched with their repellence against the mosquito Aedes albopictus (Skuse) (Diptera Culicidae). To obtain a more complete bioactivity description, we also tested the EOs oviposition deterrence and the larvicidal activity. The best smell profile was associated with A. verlotiorum EO, while R. chalepensis EO showed the lowest overall pleasantness. All the EOs had a significant activity as skin repellent against Ae. albopictus, deterred the oviposition in the field, and exerted a clear larvicidal activity. Beside the best smell profile, A. verlotiorum EO showed also the longest lasting repellent effect, assuring the complete protection of the treated skin against Ae. albopictus for a time 60% longer than the synthetic repellent DEET.

Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L.

This study describes the chemical composition and evaluates the antibacterial and the cytotoxic effects of the essential oil from the flowers of Convolvulus althaeoides. Its chemical composition, determined by GC and GC-MS, is reported for the first time. A total of 24 compounds, accounting for 95.5% of the total oil, have been identified. The oil was characterised by a high proportion of sesquiterpene hydrocarbons (36.3%), followed by oxygenated sesquiterpenes (34.7%) and oxygenated monoterpenes (24.5%). The main compounds were germacrene D (12.5%), T-cadinol (11.8%) and verbenone (6.9%). The essential oil was tested for its antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and the clinical strain Acinetobacter sp. as well as facts cytotoxic activity towards the human breast cancer cells MCF-7. This oil did not exhibit significant antibacterial activity against the tested bacteria; however, it exerted a significant cytotoxic activity against the tested cell line (IC50 = 8.16 µg/mL).

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi.

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P<0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.

Efficacy of an essential oil of Cinnamomum zeylanicum against Psoroptes cuniculi.

The aim of the present study was to investigate the in vitro and in vivo acaricidal effects of an essential oil of Cinnamomum zeylanicun leaves on Psoroptes cuniculi, a mange mite. In vitro, 2.5 ml of the essential oil diluted at different concentrations, from 10% to 0.03%, in paraffin oil were added to Petri dishes containing all motile stages of P. cuniculi. Mites mortality observed in these dishes was compared with that observed in untreated and treated (AcaCerulen R) control plates. In vivo, one group of six P. cuniculi infected rabbits was topically treated two times at seven days interval with two ml of the essential oil at the concentration of 2.5% in paraffin oil and compared with untreated and treated (AcaCerulen R) control groups of six rabbits each. After 24 h of contact, all concentrations of essential oil between 0.10 and 10% showed a good in vitro acaricidal efficacy if compared with the untreated controls (p<0.01), but only the concentrations between 0.16 and 10% turned out as active as the drug. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed with the treated control group.

Efficacy of mangiferin against Cryptosporidium parvum in a neonatal mouse model.

The inhibitory activity of mangiferin (50 mg/kg/die and 100 mg/kg/die) on Cryptosporidium parvum was evaluated in a neonatal mouse model and its activity was compared with that of paromomycin (100 mg/kg/die). At 4 days of age, neonatal Swiss conventional outbred mice were experimentally infected by oral administration of 10(4) oocysts/animal of C. parvum and treated orally for 10 consecutive days, starting 7 days after the experimental infection. One group of mice was left untreated. To evaluate the efficacy of mangiferin, from euthanised mice, 3-mum-thick tissue sections of the intestine were stained with haematoxylin-eosin and periodic acid Schiff. Immunohistochemistry was also used by employing a monoclonal anti-C. parvum antibody. Oocysts were counted and results were expressed as mean oocysts number/intestine. Results obtained show that mangiferin at 100 mg/kg/die has a significant anticryptosporidial activity and that its activity is similar to that showed by the same dose (100 mg/kg/die) of paromomycin. However, both mangiferin and paromomycin were not able to completely inhibit intestinal colonization of C. parvum but only to reduce it. This reduction was calculated at over 80% for both mangiferin and paromomycin with respect to the untreated control. A significant activity was found also for mangiferin at 50 mg/kg/die only after the end of treatment.

Biological screening of Nigella damascena for antimicrobial and molluscicidal activities.

The essential oil, various extracts at different polarity, fractions, and pure compounds obtained from Nigella damascena plants and seeds were screened for biological activity. Antimicrobial tests showed the essential oil to be active only against Gram positive bacteria; among the extracts, the BuOH was active against Pseudomonas aeruginosa and Staphylococcus aureus. Molluscicidal activity was absent.

Acaricidal activity of pine essential oils and their main components against Tyrophagus putrescentiae, a stored food mite.

Some essential oils obtained from the branches of four Pinus species (P. pinea L., P. halepensis Mill., P. pinaster Soil in Ait., and P. nigra Arnold) have been evaluated for their acaricidal activity by aerial diffusion against the stored food mite Tyrophagus putrescentiae (L.). All the essential oils showed a good efficacy, but P. pinea oil and its two constituents 1,8-cineole and limonene were the most effective compounds, showing 100% acaricidal activity at 8 microL; 1,8-cineole showed the same activity at 6 microL.

A flavonoid sulphate and other compounds from the roots of Centaurea bracteata.

The roots of Centaurea bracteata Scop. (Asteraceae) have been studied for the first time. Twenty-three compounds were isolated and identified, namely a sterol glucoside, two quinic acid derivatives, one sugar, and 19 flavonoids (five sulphates), one of which, centaradixin, resulted in a new natural product. Structural elucidation was performed mainly by means of FABMS, 1D and 2D NMR spectroscopy. NMR data of some sulphate flavonoids are reported for the first time.

Chemical composition of some traditional herbal drug preparations: essential oil and aromatic water of costmary (Balsamita suaveolens Pers.).

The compositions of the essential oil and the aromatic water of costmary (Balsamita suaveolens Pers.) cultivated in Tuscany were investigated. They represent the main ingredients of some traditional preparations sold commercially. The essential oil as such and the n-hexane extract of the aromatic water were analyzed by GC and GC-MS. Both samples were found to be rich in monoterpenes. Eighty-five compounds were identified, accounting for 95.1 and 95.4% of the essential oil and n-hexane extract of aromatic water, respectively. Carvone was the main compound (43.5% in the essential oil and 74.9% in the n-hexane extract of aromatic water). In addition, solid phase microextraction was used to sample the volatile organic compounds emitted from the fresh plant and from the aromatic water, and carvone was again the main component, amounting to 46.2 and 41.3%, respectively.

Diagnosis of T1 bladder transitional cell carcinoma by denaturing gradient gel electrophoresis urinalysis.

OBJECTIVE: The diagnosis and follow-up of patients with T1 bladder cancer relies invasive procedures. We developed a non-invasive method for detection of T1 bladder cancer based on a feasible non-radioactive molecular approach. MATERIALS AND METHODS: Urine DNA samples were collected from 24 patients with T1 transitional cell carcinoma (TCC) of the bladder and were processed by denaturing gradient gel electrophoresis (DGGE) analysis. Urine samples obtained from 10 individuals with no clinical evidence of genitourinary malignancy were used as controls. RESULTS: Ten patients out of 24 (41%) had p53 mutations in their tumor samples. Seven of these presented the same mutation in matched urine samples. The p53 mutation pattern found in urine was always identical to that identified in the primary tumor. Decision diagnostic criteria showed that molecular screening by DGGE of amplified DNA from urine sediment had 69.2 % sensitivity, 100% specificity, 95.8 % accuracy, 100% prediction of positive result and 95.4 % prediction of negative result, respectively. No p53 mutation was found in the urine from control subjects. CONCLUSION: DGGE analysis of urine samples could be a useful tool for the early detection of T1 bladder cancer or its recurrence, potentially leading to a reduction in the frequency of invasive procedures used for the management of this disease.

Volatile compounds from three cultivars of Olea europaea from Italy.

The chemical compositions of the volatile fractions from three Olea europaea L. cultivars (Leccino, Frantoio, and Cipressino) were examined by GC and GC-MS. The results showed that the cultivars can be distinguished on the basis of the volatile fraction compositions.

Two flavonoids and other compounds from the aerial parts of Centaurea bracteata from Italy.

The flowering aerial parts of Centaurea bracteata Scop. (Asteraceae) have been studied for the first time. Nineteen compounds were isolated and identified, namely a sterol glucoside, two phenolic acids, three quinic acid derivatives, and 13 flavonoids, two of which, are new natural products. Structural elucidation was performed mainly by mean of FABMS, 1D and 2D NMR spectroscopy.

8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection.

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.

A new flavonoid glycoside from Centaurea horrida.

A new natural compound, horridin (1), was isolated from the aerial parts of Centaurea horrida. Its structure as quercetin 3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-rhamnopyranoside was determined by spectroscopic methods, including 2D NMR.

Detection by denaturant gradient gel electrophoresis of tumor-specific mutations in biopsies and relative bronchoalveolar lavage fluid from resectable non-small cell lung cancer.

A PCR-denaturant gradient gel electrophoresis (DGGE) method was developed for the detection of p53 and K-ras mutations in primary operable tumors and paired BAL samples of non-small cell lung cancer. Among 36 patients, 9 showed p53 exon V mutations in biopsies and in three paired bronchoalveolar lavage (BAL) specimens with a 33% concordance. Five patients presented p53 exon VI mutations in biopsies and in two paired BALs with a 40% concordance. No mutations were found in p53 exon VII either in biopsies or in paired BAL samples with 100% concordance. Exon VIII mutations were found in six primary tumors and in two BALs with a 33% concordance. Of 36 patients, we detected 7 (19.4%) with K-ras exon I mutations on tumor samples. DGGE analysis of DNA from BAL samples revealed three mutations distributed on K-ras exon I with a 42% overall concordance with respect to tumor tissue. Molecular screening by DGGE of p53-amplified DNA from BAL had cumulative 46.6% sensitivity, 100% specificity, and 77.7% accuracy. DGGE K-ras detection showed 43% sensitivity, 100% specificity, and 88.8% test accuracy. The method proposed demonstrated to be specific, accurate, and at relatively low cost but limited by low sensitivity in detecting the presence of neoplastic cells in patients with resectable non-small cell lung cancer.

Evaluation of 8-hydroxydeoxyguanosine in human oral cells: the importance of tobacco smoke and urban environment.

The DNA adduct 8-hydroxydeoxyguanosine (8-OHdG) has been widely used as a sensitive biomarker for oxidative damage. To investigate the role of environmental factors on oxidative DNA damage formation, the level of 8-OHdG was determined in oral cells from 109 healthy volunteers by an immunohistochemical method. A statistically significantly higher content of 8-OHdG was detected in oral cells from smokers (111 +/- 55, n = 38) compared with non smokers (78 +/- 48, n = 71), (p < 0.01). Moreover, subjects living in an urban area showed a higher level of oxidative damage with respect to those living in a countryside-suburban area (99 +/- 53, n = 58 vs. 78 +/- 51, n = 51), (p = 0.03). No significant association was detected between 8-OHdG in oral cells and other variables such as passive smoke, oral infections, alcohol or vitamin intake and grilled food consumption. This work suggests that tobacco smoke and environmental exposure to pollutants lead to a measurable increase of oxidative damage in oral cells and confirms that the immunoperoxidase method is an appropriate approach for epidemiological analyses.