Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture.

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

Prevention of DNA damage and anticarcinogenic activity of Activia® in a preclinical model.

Colorectal cancer is a global public health issue. Studies have pointed to the protective effect of probiotics on colorectal carcinogenesis. Activia® is a lacto probiotic product that is widely consumed all over the world and its beneficial properties are related, mainly, to the lineage of traditional yoghurt bacteria combined with a specific bacillus, DanRegularis, which gives the product a proven capacity to intestinal regulation in humans. The aim of this study was to evaluate the antigenotoxic, antimutagenic, and anticarcinogenic proprieties of the Activia product, in response to damage caused by 1,2-dimethylhydrazine (DMH) in Swiss mice. Activia does not have shown antigenotoxic activity. However, the percent of DNA damage reduction, evaluated by the antimutagenicity assay, ranged from 69.23 to 96.15% indicating effective chemopreventive action. Activia reduced up to 79.82% the induction of aberrant crypt foci by DMH. Facing the results, it is inferred that Activia facilitates the weight loss, prevents DNA damage and pre-cancerous lesions in the intestinal mucosa.

A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.

INTRODUCTION: The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone). OBJECTIVE: To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers. METHODOLOGY: The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C18-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)3NH2 (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min. RESULTS: The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits. CONCLUSION: The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.

Intracellular calcium mobilization as a target for the spasmolytic action of scopoletin.

The coumarin scopoletin was isolated in a pure form from the roots of Brunfelsia hopeana Benth. (Solanaceae). In isolated rat aortic rings, scopoletin (26-520 microM) inhibited to approximately the same extent the contractions induced by a variety of substances, including phenylephrine, potassium chloride, serotonin and PGF(2) (alpha). The effect of the coumarin on phenylephrine-induced contractions was not affected by endothelium removal or NO-synthase blockade by L-NAME (100 microM). Scopoletin (78 - 590 microM) antagonized in a concentration-dependent manner (IC(50) = 300 +/- 20 microM, n = 5), transient contractions in Ca(2+)-free media induced by noradrenaline, but not those induced by caffeine. Also, scopoletin did not interfere with the refilling of noradrenaline-sensitive intracellular calcium stores. It is suggested that the non-specific spasmolytic action of scopoletin can be attributed, at least in part, to its ability to inhibit the intracellular calcium mobilization from the noradrenaline-sensitive stores.

Chromatographic techniques for the determination of putative dietary anticancer compounds in biological fluids.

Although a great number of papers demonstrate an association between high intake of fruits and vegetables and reduced risk of certain types of cancer, the epidemiological evidence is not conclusive. The identification and quantification of specific dietary anticancer compounds in plasma, urine and tissues is an important aspect of this research. We surveyed the recent literature for original papers which involved the use of separation techniques for the detection and quantification in biological fluids and tissues of putative anticancer compounds which are present in the diet. The compounds included in this review are flavonoids, phytoestrogens, carotenoids, retinoids, vitamin E and ascorbic acid. The review covers papers published in the last 3 years. For each class of compounds we discuss the sample preparation, chromatographic conditions, and validation of the methods used, in order to identify current trends in the bioanalysis of each class of these substances.

In vitro glucuronidation of kaempferol and quercetin by human UGT-1A9 microsomes.

Flavonoids are important polyphenolic substances with widespread occurrence in plants and therefore in the human diet. Although considerable work has been done on the pharmacology of flavonoids, the understanding of their metabolism is still incomplete. In this work, the in vitro glucuronidation of the common dietary flavonoids quercetin and kaempferol by human UDP-glucuronosyltransferase microsomes (UGT-1A9) was investigated using HPLC and LC-MS. The two flavonoids were extensively metabolised by this enzyme with four monoglucuronides of quercetin and two of kaempferol being detected after incubation. The presence of a quercetin monoglucuronide in the urine of a volunteer after consumption of Ginkgo biloba tablets was demonstrated.

Solid-phase extraction and gas chromatography-mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets.

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography-mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was +/-9.40% for kaempferol and +/-7.34% for quercetin (n = 6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with beta-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.

Hypotensive and spasmolytic effects of normacusine B from Strychnos atlantica root.

Normacusine B, a tertiary indole alkaloid, was isolated in pure form from the root bark of Strychnos atlantica Krukoff & Barneby. In conscious unrestrained rats, normacusine B (1 mg/kg) decreased the mean arterial blood pressure (27.6 ± 8.4 mmHg, n = 6), followed by a significant increase in heart rate (115.0 ± 12.7 bpm, n = 6). The alkaloid failed to induce tachycardia directly in isolated perfused rat heart. In isolated rat aortic rings, normacusine B antagonized phenylephrine and serotonin-induced contractions. Schild plot analysis of individual cumulative concentration-response curves was compatible with a competitive type of antagonism against phenylephrine (pA(2) = 7.05 ± 0.11) and of a non-competitive nature against 5-hydroxytryptamine (apparent pA(2) = 7.02 ± 0.08). Normacusine B was found inactive against KCl and PGF(2α) induced contractions.