Comparative study of antioxidant and anti-inflammatory activities and genotoxicity of alcoholic and aqueous extracts of four Fabiana species that grow in mountainous area of Argentina.

ETHNOPHARMACOLOGICAL RELEVANCE: Fabiana species (Solanaceae family) extracts have long been used in Argentinean traditional medicine as anti-inflammatories, antiseptic, bone fractures and others diseases, but there is no scientific evidence which supports their use. AIM OF THE STUDY: The present study was conducted to evaluate the ability of aqueous and ethanolic extracts of four Fabiana species (Fabiana bryoides Phil., Fabiana punensis A.C. Arroyo, Fabiana densa J. Rèmy and Fabiana patagonica Speg.) to inhibit key enzymes in inflammatory processes, free radical scavenging properties and genotoxic effects. MATERIALS AND METHODS: HPLC-DAD of aqueous and ethanolic extracts from four Fabiana species was established. All Fabiana extracts were evaluated on their ability to inhibit hyaluronidase and lipoxygenase enzymes to assess their activity against inflammatory mediators. Antioxidant capacity was determined using the 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assays and ß-carotene-linolenic acid assay. Genotoxicity was evaluated by the Ames assay. RESULTS: The results indicated that the chromatographic patterns of four Fabiana species were different in quantity and absorption intensity of peaks. The alcoholic extract of Fabiana punensis was the most active scavenger of DPPH and ABTS(+) radicals (SC(50) values of 3.85 ± 0.24 and 2.56 ± 0.10 µgGAE/mL, respectively). Fabiana patagonica extracts exhibited the highest peroxyl radical scavenging activity compared with the other three taxa (IC(50) values between 1.00 ± 0.04 and 4.46 ± 0.40 µg GAE/mL for all extracts) and anti-lipoxygenase activity with IC(50) values between 12.5 and 15.5 µg GAE/mL. The absence of mutagenicity indicates that the DNA does not seem to be a relevant target for these extracts. Fabiana bryoides ethanolic extract showed an interesting effect: it inhibited spontaneous mutagenesis, which could be considered as an antimutagenic effect in the TA98 (+S9) and TA100 (+S9/-S9) strains. The potency differences found between the species could be consequence of the different phytochemical pattern observed by HPLC. CONCLUSIONS: The inhibitory effects on lipoxygenase and hyaluronidase, free radical scavenging activities and lack of genotoxicity of Fabiana extracts may support the folk use of Fabiana punensis, Fabiana patagonica, Fabiana bryoides and Fabiana densa as inhibitor of inflammatory mediators.

Potential application of Northern Argentine propolis to control some phytopathogenic bacteria.

The antimicrobial activity of samples of Northern Argentine propolis (Tucumán, Santiago del Estero and Chaco) against phytopathogenic bacteria was assessed and the most active samples were identified. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. Strong antibacterial activity was detected against Erwinia carotovora spp carotovora CECT 225, Pseudomonas syringae pvar tomato CECT 126, Pseudomonas corrugata CECT 124 and Xanthomonas campestris pvar vesicatoria CECT 792. The most active propolis extract (Tucumán, T1) was selected to bioguide isolation and identified for antimicrobial compound (2',4'-dihydroxychalcone). The antibacterial chalcone was more active than the propolis ethanolic extract (MIC values of 0.5-1 µg ml(-1) and 9.5-15 µg ml(-1), respectively). Phytotoxicity assays were realized and the propolis extracts did not retard germination of lettuce seeds or the growth of onion roots. Propolis solutions applied as sprays on tomato fruits infected with P. syringae reduced the severity of disease. Application of the Argentine propolis extracts diluted with water may be promising for the management of post harvest diseases of fruits.

Antimicrobial activity of selected plant species from "the Argentine Puna" against sensitive and multi-resistant bacteria.

AIM: The plant species reported here are traditionally used in the "Puna" or "Altiplano" of Argentina for ailments related to bacterial infections. The aim of this study was to evaluate their antimicrobial properties against a panel of sensitive and multi-resistant gram-positive and gram-negative bacteria. MATERIALS AND METHODS: The antimicrobial activity of tinctures and aqueous extracts (Baccharis boliviensis, Chiliotrichiopsis keidelii, Chuquiraga atacamensis, Fabiana bryoides, Fabiana densa, Fabiana punensis, Frankenia triandra, Parastrephia lucida, Parastrephia lepidophylla, Parastrephia phyliciformis, Tetraglochin cristatum) was determined using the agar macrodilution and broth microdilution methods recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). The antibiotic resistant clinical strains were isolated from nosocomial infection in human lesions of skin and soft parts. RESULTS: The ethanolic extracts of 11 plant species inhibited the growth of one or more of the following strains: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Morganella morganii, Pseudomonas aeruginosa. Ethanol extracts (tinctures) of aerial parts of Baccharis, Fabiana and Parastrephia showed the highest levels of antibacterial activity on methicillin, oxacillin and gentamicin resistant Staphylococcus with MIC values from 20 to 150 microg/ml. Baccharis boliviensis and Fabiana bryoides were more active than the other plant species on Enterococcus faecalis with different phenotype. The most interesting activity on multi-resistant gram-negative strains was obtained from Chuquiraga atacamensis. Parastrephia species showed activity against Enterobacter cloacae, Pseudomonas aeruginosa and Proteus mirabilis. The ethanolic extracts exhibited stronger activity and broader spectrum of action than aqueous extracts. The extracts were bactericidal in most cases. CONCLUSIONS: The presence of antibacterial activity in Puna plant extracts against multi-resistant bacteria give support to their traditional use for treating conditions associated with microorganisms in humans and animals and consequently seems promising for the treatment of multi-resistant bacteria.

Acid invertase localization in leaves of the fern Pteris deflexa link.

The localization of invertase, a key enzyme in plant carbohydrate metabolism, has been established in several higher plants, but there are no reports of it in ferns. The aim of the present work was to establish the localization of the previously reported acid invertase activity of Pteris deflexa in fronds tissues and to compare the findings with invertase localization in higher plants. Acid invertase, localized by immuno-histochemical and histochemical techniques on fresh tissues, was evident in vascular tissue, mainly in phloem. It was also detected in parenchymatic, sclerenchymatic and epidermic cells of petiole, rachis and rachis branches as well as in veins of leaf blades. Our results demonstrate that P. deflexa acid invertase localization is the same to that of higher plants. Hence, potential roles of the fern enzyme in relation to the storage and utilization of sucrose and to control carbon flux could be the same of those proposed to higher plants.

mRNA expression of MAGE-A3 gene in leukemia cells.

Leukemia-associated antigens such as proteins encoded by MAGE genes might provide tools for immunotherapy of leukemia. Positive and negative results of MAGE-A gene expression in hematological malignancies have been reported. This led us to study MAGE-A gene expression in human leukemias using RT-PCR. Among 115 leukemias from various subtypes, 14/34 (41.17%) AML were positive for one of the three genes analyzed (MAGE-A1 1/32; MAGE-A3 10/32; MAGE-B2 3/12). Expression was also detected in 23/76 (30.26%) B-cell ALL patients (MAGE-A1 2/53; MAGE-A3 20/53; MAGE-B2 1/32). One of these patients expressed both MAGE-A1 (weak signal) and -A3 (strong signal) genes. Other patient with CML were positive for MAGE-B2 (1/5, 20%). MAGE-A3 expression data were corroborated by real time RT-PCR through determination of MAGE-A3 transcript levels. We concluded that the MAGE-A3 gene is expressed at the mRNA level in a proportion of human leukemias.

Asian-American variants of human papillomavirus 16 and risk for cervical cancer: a case-control study.

BACKGROUND: Human papillomavirus 16 (HPV16) has a number of variants, each with a different geographic distribution and some that are associated more often with invasive neoplasias. We investigated whether the high incidence of cervical cancer in Mexico (50 cases per 100 000 women) may be associated with a high prevalence of oncogenic HPV16 variants. METHODS: Cervical samples were collected from 181 case patients with cervical cancer and from 181 age-matched control subjects, all from Mexico City. HPV16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV classes and subclasses were identified by sequencing regions of the E6 and L1/MY genes. Clinical data and data on tumor characteristics were also collected. All statistical tests were two-sided. RESULTS: HPV16 was detected in cervical scrapes from 50.8% (92 of 181) of case patients and from 11% (20 of 181) of control subjects. All HPV16-positive samples, except one, contained European (E) or Asian-American (AA) variants. AA and E variants were found statistically significantly more often in case patients (AA = 23.2% [42 of 181]; E = 27.1% [49 of 181]) than in control subjects (AA = 1.1% [two of 181]; E = 10% [18 of 181]) (P<.001 for case versus control subjects for either E or AA variants, chi2 test). However, the frequency of AA variants was 21 times higher in cancer patients than in control subjects, whereas that ratio for E variants was only 2.7 (P =.006, chi2 test). The odds ratio (OR) for cervical cancer associated with AA variants (OR = 27.0; 95% confidence interval [CI] = 6.4 to 113.7) was higher than that associated with E variants (OR = 3.4; 95% CI = 1.9 to 6.0). AA-positive case patients (46.2 +/- 12.5 years [mean +/- standard deviation]) were 7.7 years younger than E-positive case patients (53.9 +/- 12.2 years) (P =.004, Student's t test). AA variants were associated with squamous cell carcinomas and adenocarcinomas, but E variants were associated with only squamous cell carcinomas (P =.014, Fisher's exact test). CONCLUSIONS: The high frequency of HPV16 AA variants, which appear to be more oncogenic than E variants, might contribute to the high incidence of cervical cancer in Mexico.

Invertase proteinaceous inhibitor of Cyphomandra betacea Sendt fruits.

This work describes a new invertase proteinaceous inhibitor from Cyphomandra betacea Sendt. (tomate de arbol) fruits. The proteinaceous inhibitor was isolated and purified from a cell wall preparation. The pH stability, kinetics of the inhibition of the C. betacea invertase, inhibition of several higher plant invertases and lectin nature of the inhibitor were studied. The inhibitor structure involves a single polypeptide (Mr = 19000), as shown by gel filtration and SDS-PAGE determinations. N-terminal aminoacid sequence was determined. The properties and some structural features of the inhibitor are compared with the proteinaceous inhibitors from several plant species (Beta vulgaris L., Ipomoea batatas L. and Lycopersicon esculentum Mill.). All these inhibitors share lectinic properties, some common epitopes, some aminoacid sequences and a certain lack of specificity towards invertases of different species, genera and even plant family. In consequence, the inhibitors appear to belong to the same lectin family. It is now known that some lectins are part of the defence mechanism of higher plants against fungi and bacteria and this is a probable role of the proteinaceous inhibitors.

Asian-american variants of human papillomavirus type 16 have extensive mutations in the E2 gene and are highly amplified in cervical carcinomas.

Human-papillomavirus (HPV)-E2 protein is involved in gene-expression regulation and replication of HPV genome. Disruption of the E2 gene during viral integration has been proposed as a mechanism of tumoral progression, since the expression of E6/E7 viral oncogenes is allowed. However, retention of E1/E2 genes and high viral amplification are frequently found in HPV16-positive carcinomas of some populations. In this study, we investigated whether retention of E1/E2 and viral amplification are associated with particular HPV16 E2 variants in cervical carcinomas. HPV16 detection, E1/E2 integrity and viral amplification were explored by Southern blot in 123 cervical carcinomas. HPV16 variants were identified by Southern blot and by sequencing E6, L1/MY and E2 regions. Of 46 HPV16-positive tumors, 34 were positive for E1/E2 and 14 of them showed a variant restriction pattern by mutations in E2. All 14 were Asian-American (AA) variants and, of 11 sub-classified, 6 were AA-a and 5 AA-c. Two E1/E2-negative tumors also contained the AA-c variant, while the remaining HPV16-positive tumors contained only European variants. The E2 gene of AA variants showed 24 mutations, 19 identical in both sub-classes. The 24 mutations were distributed throughout the entire gene and 19 result in 18 amino-acid changes. The AA variants were associated with E1/E2-positive carcinomas with more than 50 viral copies/cell (p = 0.035). The association of Asian-American E2 variants with retention of E1/E2 suggests that E2 variation may be an alternative mechanism de-regulating the expression of viral oncogenes.

Invertase activity associated with the walls of Solanum tuberosum tubers.

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.