Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

Insulin secretion, insulin sensitivity, and hepatic insulin extraction in first-degree relatives of type 2 diabetic patients

To identify early metabolic abnormalities in type 2 diabetes mellitus, we measured insulin secretion, sensitivity to insulin, and hepatic insulin extraction in 48 healthy normal glucose-tolerant Brazilians, first-degree relatives of type 2 diabetic patients (FH+). Each individual was matched for sex, age, weight, and body fat distribution with a person without history of type 2 diabetes (FH-). Both groups were submitted to a hyperglycemic clamp procedure (180 mg/dl). Insulin release was evaluated in its two phases. The first was calculated as the sum of plasma insulin at 2.5, 5.0, 7.5, and 10.0 min after the beginning of glucose infusion, and the second as the mean plasma insulin level in the third hour of the clamp procedure. Insulin sensitivity index (ISI) was the mean glucose infusion rate in the third hour of the clamp experiment divided by the mean plasma insulin concentration during the same period of time. Hepatic insulin extraction was determined under fasting conditions and in the third hour of the clamp procedure as the ratio between C-peptide and plasma insulin levels. FH+ individuals did not differ from FH- individuals in terms of the following parameters [median (range)]: a) first-phase insulin secretion, 174 (116-221) vs 207 (108-277) µU/ml, b) second-phase insulin secretion, 64 (41-86) vs 53 (37-83) µU/ml, and c) ISI, 14.8 (9.0-20.8) vs 16.8 (9.0-27.0) mg kg-1 min-1/µU ml-1. Hepatic insulin extraction in FH+ subjects was similar to that of FH- ones at basal conditions (median, 0.27 vs 0.27 ng/µU) and during glucose infusion (0.15 vs 0.15 ng/µU). Normal glucose-tolerant Brazilian FH+ individuals well-matched with FH- ones did not show defects of insulin secretion, insulin sensitivity, or hepatic insulin extraction as tested by hyperglycemic clamp procedures.

Inotropic effects of extracts of Psidium guajava L. (guava) leaves on the guinea pig atrium

Many pharmacological effects have been ascribed to extracts of Psidium guajava L. (guava) leaves. However, in spite of its widespread use in Brazilian folk medicine and a reasonable number of scientific reports about it, we could not find any study dealing with its action on the mammalian myocardium. In the present study, by measuring isometric force, we observed that the crude extract of P. guajava (water-alcohol extract obtained by macerating dry leaves) depresses the guinea pig atrial contractility in a concentration-dependent fashion (N = 8 hearts, 15 trials). The compound with cardiac activity was concentrated by extraction in a Soxhlet apparatus using 17 M glacial acetic acid after removing the less polar fractions (hexane, chloroform, acetone, ethanol and methanol), suggesting that this compound is a highly polar substance. In the isolated guinea pig left atrium the acetic acid fraction (10-800 mg/l) of P. guajava 1) reversibly decreased myocardial force in a concentration-dependent fashion (EC50 = 0.07g/l, N = 5 hearts, 9 trials, P<0.05), 2) increased the atrial relaxation time measured at 20% of the force amplitude up to 35% (91 ± 15 to 123 ± 30 ms, N = 3 hearts, 6 trials, P<0.05), 3) abolished the positive staircase effect (Bowditch phenomenon) in a concentration-dependent fashion suggesting a decrease of the cellular inward calcium current (N = 4 hearts, 8 trials, P<0.05), and 4) its inotropic effect was abolished by cholinergic receptor blockade with 1.5 mM atropine sulfate, indicating a cholinergic involvement in the mechanism of action of the extract (N = 7 hearts, 15 trials, P<0.05). The acetic acid extract was 20 times more potent than crude extract (EC50 = 1.4 g/l). The results showed that extracts from P. guajava leaves depress myocardial inotropism.

Antioxidant status of dog aqueous humor after extracapsular lens extraction

We determined the antioxidant status of the aqueous humor after extracapsular lens extraction in 14 mongrel dogs weighing about 10 kg. The animals were examined by slit lamp biomicroscopy, applanation tonometry and indirect ophthalmoscopy. One eye was submitted to conventional extracapsular lens extraction and the other was used as control. Samples of aqueous humor were obtained by anterior chamber paracentesis before and at days 1, 2, 3, 7 and 15 after surgery. Total antioxidant status was determined as the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis(2-amidopropane) chlorine. Ascorbic acid concentration was measured by HPLC with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test. Protein concentration increased from 0.61 to 22 mg/ml 24 h after surgery. These levels were maintained and returned to normal at day 7. Total antioxidant capacity was reduced from 50 to about 30 min until day 3 and at day 7 it was equal to control. Ascorbic acid levels were reduced from 252 to about 110 µM and then returned to control values at day 15. Considering the importance of ascorbic acid concentration in aqueous humor for the maintenance of the antioxidant status of the anterior segment of the eye, the decrease of antioxidant defenses suggests that the surgical procedures promote an oxidative stress condition in the eye.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard® genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 µg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

EXTRACTION OF OIL FROM PRESSED PALM OIL (Elaes guineensis) FIBERS USING SUPERCRITICAL CO2

Residual fibers from palm oil production are a good source of carotene, since they contain more than 5% of the original oil, with about 5000 ppm of carotenoids. As carotenoids are thermosensitive molecules, supercritical CO2 can be used for oil recovery, because this technique employs low temperatures. In this work results of oil extraction experiments from pressed palm oil fibers are shown. Fibers were from AGROPALMA, an industry which is located in Tailândia (Pará, Brazil). Extractions were carried out at 200, 250 and 300 bar and at temperatures of 45 and 55oC. Oil was analyzed by UV/vis spectrophotometry for total carotene determination. Results showed a large increase in extraction rate from 200 to 250 bar and a small variation from 250 to 300 bar. The total amount of carotenes did not increase in the course of extraction at 300 bar, but it showed a large increase at 200 and at 250 bar. Free fatty acids are present in amounts larger than those found in commercial oils.

Effect of blanching time on selective mineral elements extraction from the spinach substitute (Tetragonia expansa) commonly used in Brazil

The true spinach (Spinacia oleracea) does not grow well in warm climates and for that reason is not commercialized in Brazil. Instead, a spinach substitute (Tetragonia expansa), originally from New Zealand, is widely used in the country. There is scant information on the mineral profile and none on the soluble mineral fraction of this vegetable. The solubility of a mineral is one of the important factors for its absorption. For this reason, the calcium, magnesium, iron, manganese, copper, zinc, potassium, and sodium soluble fractions in the raw spinach substitute were determined and the effect of blanching times on the solubility of these minerals was investigated. Blanching times of 1, 5, and 15 minutes were employed. The magnesium, manganese, potassium, and sodium soluble fractions increased sizably with shorter blanching time. Longer blanching time (15 minutes) caused large losses of minerals. The soluble mineral fractions can contribute poorly to diet in terms of potassium, magnesium, manganese, and zinc. The spinach substitute cannot be considered a dietary source of calcium, iron and copper due to the insolubility of these minerals in the vegetable, possibly caused by the large oxalate content.

Head Space Solid Phase Micro-Extraction (HS - SPME) of volatile organic compounds produced by Sporidiobolus salmonicolor (CBS 2636)

The aim of the present study was the assessment of volatile organic compounds produced by Sporidiobolus salmonicolor (CBS 2636) using methyl and ethyl ricinoleate, ricinoleic acid and castor oil as precursors. The analysis of the volatile organic compounds was carried out using Head Space Solid Phase Micro-Extraction (HS - SPME). Factorial experimental design was used for investigating extraction conditions, verifying stirring rate (0-400 rpm), temperature (25-60 ºC), extraction time (10-30 minutes), and sample volume (2-3 mL). The identification of volatile organic compounds was carried out by Gas Chromatography with Mass Spectrum Detector (GC/MSD). The conditions that resulted in maximum extraction were: 60 ºC, 10 minutes extraction, no stirring, sample volume of 2.0 mL, and addition of saturated KCl (1:10 v/v). In the bio-production of volatile organic compounds the effect of stirring rate (120-200 rpm), temperature (23-33 ºC), pH (4.0-8.0), precursor concentration (0.02-0.1%), mannitol (0-6%), and asparagine concentration (0-0.2%) was investigated. The bio-production at 28 ºC, 160 rpm, pH 6,0 and with the addition of 0.02% ricinoleic acid to the medium yielded the highest production of VOCs, identified as 1,4-butanediol, 1,2,2-trimethylciclopropilamine, beta-ionone; 2,3-butanodione, pentanal, tetradecane, 2-isononenal, 4-octen-3-one, propanoic acid, and octadecane.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

Antioxidant and fatty acid profile of gabiroba seed (Campomanesisa Xanthocarpa Berg)

This study aimed to evaluate the antioxidant potential and fatty acid profile of gabiroba (Campomanesia xanthocarpa Berg) seeds. In order to obtain the extract, the seeds were dried, crushed, and subjected to sequential extraction by maceration and percolation in a modified soxhlet extractor using solvent polarity gradient composed of hexane, chloroform, ethyl acetate, and alcohol, respectively. The extraction time was six hours. The ethanol extract showed the highest antioxidant potential, given by the EC50 value and the amount of total phenolic compounds. High amounts of unsaturated fatty acids were found in the oil studied, especially the oleic acid.

Physical and chemical characterization of the pulp of different varieties of avocado targeting oil extraction potential

The aim of this study was to evaluate the physicochemical properties of avocado pulp of four different varieties (Avocado, Guatemala, Dickinson, and Butter pear) and to identify which has the greatest potential for oil extraction. Fresh avocado pulp was characterized by moisture, protein, fat, ash, carbohydrates and energy contents were determined. The carotenoids and chlorophyll contents were determined by the organic solvent extraction method. The results showed significant differences in the composition of the fruit when varieties are compared. However, the striking feature in all varieties is high lipid content; Avocado and Dickinson are the most suitable varieties for oil extraction, taking into account moisture content and the levels of lipids in the pulp. Moreover, it could be said that the variety Dickinson is the most affected by the parameters evaluated in terms of overall quality. Chlorophyll and carotenoids, fat-soluble pigments, showed a negative correlation with respect to lipids since it could be related to its function in the fruit. The varieties Avocado and Dickinson are an alternative to oil extraction having great commercial potential to be exploited thus avoiding waste and increasing farmers’ income.

Influence of the extraction method and storage time on the physicochemical properties and carotenoid levels of pequi (Caryocar brasiliense Camb.) oil

The objective of this study was to analyze the physicochemical properties and carotenoid levels of pequi oil obtained by different extraction methods and to evaluate the preservation of these properties and pigments during storage time. The pequi oil was obtained by solvent extraction, mechanical extraction, and hot water flotation. It was stored for over 180 days in an amber bottle at ambient conditions. Analyses for the determination of the acidity, peroxide, saponification and iodine values, coloration, total carotenoids, and β-carotene levels were conducted. The oil extraction with solvents produced the best yield and carotenoid levels. The oil obtained by mechanical extraction presented higher acidity (5.44 mg KOH.g-1) and peroxide values (1.07 mEq.kg-1). During the storage of pequi oil, there was an increase in the acidity and the peroxide values, darkening of the oil coloration, and a reduction of the carotenoid levels. Mechanical extraction is the less advantageous method for the conservation of the physicochemical properties and carotenoid levels in pequi oil.

Optimization of the extraction of carrageenan from Kappaphycus alvarezii using response surface methodology

This study aims to optimize an alternative method of extraction of carrageenan without previous alkaline treatment and ethanol precipitation using Response Surface Methodology (RSM). In order to introduce an innovation in the isolation step, atomization drying was used reducing the time for obtaining dry carrageenan powder. The effects of extraction time and temperature on yield, gel strength, and viscosity were evaluated. Furthermore, the extracted material was submitted to structural analysis, by infrared spectroscopy and nuclear magnetic resonance spectroscopy (¹H-NMR), and chemical composition analysis. Results showed that the generated regression models adequately explained the data variation. Carrageenan yield and gel viscosity were influenced only by the extraction temperature. However, gel strength was influenced by both, extraction time and extraction temperature. Optimal extraction conditions were 74 ºC and 4 hours. In these conditions, the carrageenan extract properties determined by the polynomial model were 31.17%, 158.27 g.cm-2, and 29.5 cP for yield, gel strength, and viscosity, respectively, while under the experimental conditions they were 35.8 ± 4.68%, 112.50 ± 4.96 g.cm-2, and 16.01 ± 1.03 cP, respectively. The chemical composition, nuclear magnetic resonance spectroscopy, and infrared spectroscopy analyses showed that the crude carrageenan extracted is composed mainly of κ-carrageenan.

Characterization and extraction of volatile compounds from pineapple (Ananas comosus L. Merril) processing residues

The aim of this study was to extract and identify volatile compounds from pineapple residues generated during concentrated juice processing. Distillates of pineapple residues were obtained using the following techniques: simple hydrodistillation and hydrodistillation by passing nitrogen gas. The volatile compounds present in the distillates were captured by the solid-phase microextraction technique. The volatile compounds were identified in a system of high resolution gas chromatography system coupled with mass spectrometry using a polyethylene glycol polar capillary column as stationary phase. The pineapple residues constituted mostly of esters (35%), followed by ketones (26%), alcohols (18%), aldehydes (9%), acids (3%) and other compounds (9%). Odor-active volatile compounds were mainly identified in the distillate obtained using hydrodistillation by passing nitrogen gas, namely decanal, ethyl octanoate, acetic acid, 1-hexanol, and ketones such as γ-hexalactone, γ-octalactone, δ-octalactone, γ-decalactone, and γ-dodecalactone. This suggests that the use of an inert gas and lower temperatures helped maintain higher amounts of flavor compounds. These data indicate that pineapple processing residue contained important volatile compounds which can be extracted and used as aroma enhancing products and have high potential for the production of value-added natural essences.

Extraction of DNA from honey and its amplification by PCR for botanical identification

The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.