Sulforaphane Treatment in Children with Autism: A Prospective Randomized Double-Blind Study.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder with repetitive behaviour which affects interaction and communication. Sulforaphane (SFN), an isothiocyanate abundant in the seeds and sprouts of cruciferous vegetables, has been shown to be effective in alleviating autistic behaviour. We performed a prospective double-blind placebo-controlled study to examine the possible effect of SFN in a paediatric cohort aged three to seven years based on measurements of the Autism Diagnostic Observation Schedule-2 (ADOS-2), the Social Responsiveness Scale-2 (SRS-2), and the Aberrant Behaviour Checklist (ABC). The study consisted of three visits over the duration of 36 weeks (baseline, 18 weeks, and 36 weeks). Twenty-eight of the 40 randomized children completed the study. The mean total raw scores on ABC and SRS-2 improved in both groups, but none of the changes reached statistical significance (ABC: 0 weeks p = 0.2742, 18 weeks p = 0.4352, and 36 weeks 0.576; SRS-2: 0 weeks p = 0.5235, 18 weeks p = 0.9176, and 36 weeks 0.7435). Changes in the assessment of the ADOS-2 subscale scores also did not differ between the two study cohorts (ADOS-2: 0 weeks p = 0.8782, 18 weeks p = 0.4788, and 36 weeks 0.9414). We found no significant clinical improvement in the behavioural outcome measures evaluated in children with ASD aged 3-7 years that were treated with sulforaphane.

The Use of a Thermal Process to Produce Black Garlic: Differences in the Physicochemical and Sensory Characteristics Using Seven Varieties of Fresh Garlic.

Black garlic (BG) is a product originating from fresh garlic (FG) and substantially differs in many aspects from FG due to the process called ageing. During this thermal process, the health-promoting properties of FG are enhanced, and the sensory traits are altered. However, very little is known about how the physicochemical properties of different FG varieties affect these properties of BG. Thus, the aim of this study was to investigate the influence of seven FG varieties subjected to the thermal process on the physicochemical parameters of BG. To prepare the BG samples, a fifteen-day ageing process involving a temperature gradient ranging from 30 to 82 °C was used. It was found that the antioxidant activity, the total polyphenol content, and the total soluble solids increased during ageing, while the pH level, moisture content, and lightness decreased in all the garlic varieties. The varieties of garlic differed in the studied traits significantly, both before (FG) and after ageing (BG). In the sensory analysis, significant differences between the BG varieties were observed only in the pleasantness of texture, while the remaining sensory descriptors (pleasantness of color, odor, taste and intensity of the garlic aroma, and overall acceptability) were not affected by variety. The correlations suggest that most of the FG's studied parameters in this study do not correlate with the properties of BG and cannot be used for the prediction of the quality of BG. Additionally, HPLC-MS/MS analysis revealed substantial changes in the composition of low molecular compounds.

Antifungal and Insecticidal Potential of the Essential Oil from Ocimum sanctum L. against Dangerous Fungal and Insect Species and Its Safety for Non-Target Useful Soil Species Eisenia fetida (Savigny, 1826).

The antifungal and insecticidal effect of the essential oil from Ocimum sanctum L. was evaluated using a model set of harmful organisms hazardous for health and the economy. Toxigenic and plant pathogenic filamentous fungi, including causal agents of human infections, were chosen as exemplary fungal groups-Fusarium verticillioides, Penicillium expansum and Aspergillus flavus. Spodoptera littoralis (African cotton leafworm), Culex quinquefasciatus (Southern house mosquito), the lymphatic filariasis vector and potential Zika virus vector, and the common housefly, Musca domestica were chosen as model insects. Major and minor active substances were detected and quantified using GC/MS analysis. Environmental safety was verified using the non-target useful organism Eisenia fetida. Significant antifungal and insecticidal activity, as well as environmental safety, were confirmed. The essential oil showed the highest efficacy against A. flavus according to MIC50/90, and against S. littoralis larvae according to LD50/90. The monoterpenoid alcohol linalool, t-methyl cinnamate, and estragole as phenylpropanoids were detected as effective major components (85.4%). The essential oil from Ocimum sanctum L. was evaluated as universal and significantly efficient, providing a high potential for use in environmentally safe botanical pesticides.

Factors Influencing Sulforaphane Content in Broccoli Sprouts and Subsequent Sulforaphane Extraction.

Broccoli sprouts contain 10-100 times higher levels of sulforaphane than mature plants, something that has been well known since 1997. Sulforaphane has a whole range of unique biological properties, and it is especially an inducer of phase 2 detoxication enzymes. Therefore, its use has been intensively studied in the field of health and nutrition. The formation of sulforaphane is controlled by the epithiospecifier protein, a myrosinase co-factor, which is temperature-specific. This paper studies the influence of temperature, heating time, the addition of myrosinase in the form of Raphanus sativus sprouts in constant ratio to broccoli sprouts, and other technological steps on the final sulforaphane content in broccoli sprout homogenates. These technological steps are very important for preserving sulforaphane in broccoli sprouts, but there are some limitations concerning the amount of sulforaphane. We focused, therefore, on the extraction process, using suitable ß-cyclodextrin, hexane and ethanol, with the goal of increasing the amount of sulforaphane in the final extract, thus stabilizing it and reducing the required amount sulforaphane needed, e.g., as a dietary supplement.

Beer and beer-based beverage contain lignans.

Lignans are members of a broad group of plant phenols that can positively affect human health. They occur in negligible quantities in processed foodstuffs such as lager beer. The aim of this work was to utilize the high levels of lignans in the knots of spruce trees (Picea abies) to increase the lignans content in beer, without negatively impacting the natural taste and aroma. By means of lignans addition in the forms of spruce knot chips or different extracts made from spruce knots during the wort boiling were produced beer and beer-based beverages with lignans content ranging from 34 to 174 mg/L.

Thermal stability and bioavailability of bioactive compounds after baking of bread enriched with different onion by-products.

This study investigated the effects of the addition of onion waste fractions into gluten-free (GF) bread to promote its health benefits. 5% of the control (C) GF flour blend was replaced with three waste fractions in the form of: fried onion (FO), dried onion (DO) and onion peel (OP). Antioxidant activity, content of flavonols and total polyphenols of breads increased in the following order: C < FO < DO < OP. No differences were observed in sensory analysis. We found that quercetin glycosides, dimers and trimer in OP-bread, determined according to their mass spectra, decomposed during baking and released free quercetin, which points to their thermal instability. Cross-over study revealed that consumption of OP-bread significantly increased (p < 0.05) antioxidant activity of consumers' blood compared to control bread consumption, indicating good bioavailability of flavonols. Results suggest incorporation of OP into GF bread can increase its biological value with satisfactory sensory acceptance.

Xanthones Content in Swertia multicaulis D. Don from Nepal.

The medicinal plant Swertia multicaulis D. Don was collected in Rasuwa District (Nepal) and the xanthone content of its ethyl acetate extracts was studied. The total amount of xanthones in S. multicaulis determined by HPLC reaches almost 13 g of xanthones per 1 kg of dry matter. The identification of xanthones in S. multicaulis was achieved by a combination of HPLC, LC⁻MS and LC⁻NMR. The final assignment of the individual chemical structures was provided by NMR, supported by preparative HPLC. In eight chromatographic peaks, four major xanthones were identified­1,3-dihydroxy-5,8-dimethoxyxanthone, 1-hydroxy-3,5,8-trimethoxyxanthone, bellidifolin (1,5,8-tri-hydroxy-3-methoxyxanthone), and decussatin (1-hydroxy-3,7,8-trimethoxyxanthone).

Contribution to the study of rutin stability in the achenes of Tartary buckwheat (Fagopyrum tataricum).

Tartary buckwheat achenes are rich in rutin. However, its utilisation as a food is low due to the bitter taste of the products because of the transformation of rutin to quercetin. The aim of this study was to determine the stability of rutin and selected flavonoids, which are influenced by the different sample preparations exposing the achenes to higher temperatures (60 °C, 100 °C, and 140 °C), and also to determine the specificity of the rutin degrading enzymes in Tartary buckwheat. The achenes were directly extracted with methanol pre-extracted with hexane, or lyophilised before extraction. The rutin and quercetin contents were determined by HPLC. The rutin-degrading enzymes in Tartary buckwheat achenes were not inactivated after a heat treatment up to 140 °C acting on them for three hours; neither to lyophilisation nor pre-extraction with hexane. The rutin-degrading enzymes probably caused the deglycosylation of kaempferol-3-O-rutinoside, kaempferol-3-O-ß-d-glucoside and piceid. Rutin-degrading enzymes were inactivated with 100% methanol.

Variability in the Content of Trans-Resveratrol, Trans-ε-Viniferin and R2-Viniferin in Grape Cane of Seven Vitis vinifera L. Varieties during a Three-Year Study.

Grape canes are a waste product from viticulture that show potential as an industrially extractable source of stilbenes, which are valuable for medical and other purposes. In this work, grape canes collected in three consecutive years (2014-2016) at six different places in South Moravia, Czech Republic were extracted, and the contents of trans-resveratrol, trans-ε-viniferin, and r2-viniferin were determined by high-performance liquid chromatography. The study included three blue grape varieties of Vitis vinifera L. (Cabernet Moravia, Blaufränkisch, and Piwi variety Laurot) and four white grape varieties (Chardonnay, Green Veltliner, Piwi variety Hibernal, and Piwi variety Malverina). From the viewpoint of producing extracts with high stilbenes content, the Hibernal variety is clearly the best. The mean amounts of the stilbenes for this variety at all localities and for all three years were 4.99 g/kg for trans-resveratrol, 3.24 g/kg for trans-ε-viniferin, and 1.73 g/kg for r2-viniferin. The influence of vintage, locality, and variety on the amounts of stilbenes was studied using PCA analysis. In contrast to expectations, there was no strong impact of locality on stilbenes content. The differences were varietal for most varieties, regardless of the area of cultivation. Laurot and Hibernal varieties did differ significantly in that respect, however, as they exhibited clear dependence on location.

Effect of variety on content of bioactive phenolic compounds in common elder (Sambucus nigra L.).

The inflorescence of common elder (Sambucus nigra L., Adoxaceae) is known to be rich in phenolic compounds. The content of five selected phenolic compounds (rutin, chlorogenic acid, isoquercitrin, isorhamnetin-3-O- rutinoside and dicaffeoylquinic acid) was determined in methanolic extracts from flowers and floral stems by HPLC in samples obtained from 20 varieties of S. nigra cultivated in Czech Republic. In all samples, there were determined rutin (11-54 mg/g), chlorogenic acid (23-46 mg/g), isoquercitrin (0.6-18 mg/g), isorhamnetin-3-O-rutinoside (3-10 mg/g), calculated on air-dried material. The content of dicaffeoylquinic acid was 0-13 mg/g of air-dried material. The amount of the analysed compounds in floral stems was lower than the flowers. The results are a unique set of information on the content of main phenolics in the inflorescence of cultured elderberry varieties.

Larvicidal activity of extracts from Ammi visnaga Linn. (Apiaceae) seeds against Culex quinquefasciatus Say. (Diptera: Culicidae).

Efficacies of the Ammi visnaga seeds extract and a majority of substances on larval Culex quinquefasciatus mortality in various development stages including pupae were studied. The effect of exposure time on larval mortality was also studied. The effect of sublethal concentrations or short exposure times on further larval development and subsequent fecundity in adults were studied as well. Lethal doses of the extract were estimated for the 2nd, 3rd and 4th instar of C. quinquefasciatus (LC50 for 18, 23 and 180 mg L(-1), respectively). The majority of furanochromenes, khellin and visnagin, were identified by analysing the extract. Khellin was significantly more effective compared to visnagin, whose LC50 was estimated at 8, 10 and 41 mg L(-1) for the 2nd, 3rd and 4th instar larvae. Khellin showed very fast efficacy on mortality for the 3rd instar larvae in a concentration of 100 mg L(-1). Fifty percent mortality was determined 30 min after application, a time which was considerably shorter compared to the extract (113 min) or visnagin (169 min). The effect of the application of lethal concentrations on C. quinquefasciatus larval mortality was studied. The least number of adults were hatched after application of the extract and khellin (41.8% and 37.9%, respectively), less than after visnagin application (46.7%) or in the control (94.2%). LC50 application caused lower fecundity in the hatched adults, lower hatchability of the eggs, and also very low natality, more than 77% lower for khellin compared to the control. A short exposure, corresponding to our estimated LT30, caused no significant acute toxicity in the larvae (until 24 h) for the extract or visnagin (4.3% and 11.5%, respectively); however, 18 min of action from khellin caused a 54.3% mortality rate of the larvae within 24 h.

Uptake of caprolactam and its influence on growth and oxygen production of Desmodesmus quadricauda algae.

The consumption of polyamides produced from caprolactam is increasing continuously, and for that reason the danger of environmental contamination by this lactam is also rising. This study's aim was to evaluate the influence of caprolactam on the growth and oxygen production of the green alga Desmodesmus quadricauda and on caprolactam uptake by this alga. The presence of caprolactam in water was observed to cause the algae significantly to increase its oxygen production. Caprolactam concentration of 5,000 mg/L stopped algae growth after 6 days and influenced coenobia structure (seen as disappearance of pyrenoids, deformation of cells) but did not decrease the number of cells in the coenobia. Caprolactam uptake is probably passive but relatively rapid. Maximum concentration in the algae was reached after 18-24 h.

Changes in the Content of Biogenic Amines and Fatty Acids in High Pressure-Processed Carp Flesh (Cyprinus carpio).

Biogenic amine and fatty acid contents were determined in vacuum-packed fillets of common carp (Cyprinus carpio). Samples were pressure treated at 300 and 500 MPa and were stored at 3.5 and 12°C for up to 28 days (control, 0 MPa) and 70 days (pressure-treated). The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine, and phenylethylamine) were determined. Putrescine and cadaverine were influenced by all factors (temperature, pressurization level, and time of storage). Tyramine content was the most sensitive indicator of the improper status of sample; levels exceeding 10 mg/kg indicated both the loss of meat freshness and temperature abuse, in spite of persisting good sensory indices. Neither storage temperature nor pressurization level had a statistically important effect on the contents of fatty acids. Only polyunsaturated fatty acids decreased slightly if the storage time exceeded 42 days.

Spread of tetracycline resistance genes at a conventional dairy farm.

The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1-2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1-2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a "core TC-resistome" of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes.

Various extraction methods for obtaining stilbenes from grape cane of Vitis vinifera L.

Grape cane, leaves and grape marc are waste products from viticulture, which can be used to obtain secondary stilbene derivatives with high antioxidant value. The presented work compares several extraction methods: maceration at laboratory temperature, extraction at elevated temperature, fluidized-bed extraction, Soxhlet extraction, microwave-assisted extraction, and accelerated solvent extraction. To obtain trans-resveratrol, trans-ε-viniferin and r2-viniferin from grape cane of the V. vinifera variety Cabernet Moravia, various conditions were studied: different solvents, using powdered versus cut cane material, different extraction times, and one-step or multiple extractions. The largest concentrations found were 6030 ± 680 µg/g dry weight (d.w.) for trans-resveratrol, 2260 ± 90 µg/g d.w. for trans-ε-viniferin, and 510 ± 40 µg/g d.w. for r2-viniferin. The highest amounts of stilbenes (8500 ± 1100 µg/g d.w.) were obtained using accelerated solvent extraction in methanol.

Verification of presence of caprolactam in sprouted achenes of Fagopyrum esculentum Moench and its influence on plant phenolic compound content.

The presence of caprolactam, a precursor of Nylon-6, among those synthetic polymers which are widely-spread throughout the environment, could be the reason for its being found in plants. The aim of this work was to confirm the previously described presence of caprolactam in dry and sprouted achenes, as well as in achene exudates of common buckwheat (Fagopyrum esculentum Moench). When the lyophilized sprouted and dry buckwheat achenes, along with exudates from growth experiments, with caprolactam-free medium were analysed by HPLC, no caprolactam was found. After addition of caprolactam into the growth medium, we confirmed the uptake of caprolactam in the lyophilized sprouted buckwheat achenes. The uptake of caprolactam is also a function of light conditions during the growth experiments. Caprolactam also inhibits the content of phenolic compounds; especially rutin, vitexin, isovitexin, orientin, and homoorientin in buckwheat plants.

Non-cellulosic polysaccharides from the leaves of small balsam (Impatiens parviflora DC.).

The leaves of the annual plant Impatiens parviflora DC., a herbal medicine in Asian countries and invasive in managed forests and natural environments in Central Europe, have the potential as a source of bioactive phenolic compounds and polysaccharides. Extractives accounted for ∼22% of the leaves, whereby, the methanolic extract contains mainly caffeic acid, ferulic acid, kaempferol, and quercetin derivatives, and 1,2,4-trihydroxynaphthalene-1-O-glucoside. From the pre-extracted leaves, non-cellulosic polysaccharides were isolated by a five-step extraction procedure using as extractants cold water, 0.05 M EDTA, and DMSO in the first three steps, and 1% and 5% NaOH in the last two steps. The isolated polysaccharide fractions were characterized by chemical, physicochemical, and spectroscopic analyses (FTIR and (1)H NMR). The first three fractions contained mainly pectin and the alkali-extracted ones methylglucuronoxylan and arabinogalactan. The suggested structural features were confirmed using HSQC NMR and COSY experiments for polysaccharides of the EDTA-fraction and the Pronase-treated 5% NaOH-fraction. The EDTA fraction comprised a pectin with low degree of methyl-esterification (DM, 37%) with few rhamnogalacturonan RG I segments bearing ß-1,4-galactan as side chains. The alkali-extracted fraction comprised a degraded methylglucuronoxylan and type II 3,6-arabinogalactan in about equal amounts.

Cow excrements enhance the occurrence of tetracycline resistance genes in soil regardless of their oxytetracycline content.

Fertilizing soils with animal excrements from farms with common antibiotic use represents a risk of disseminating antibiotic resistance genes into the environment. In the case of tetracycline antibiotics, it is not clear, however, whether the presence of antibiotic residues further enhances the gene occurrence in manured soils. We established a microcosm experiment in which 3 farm soils that had no recent history of fertilization with animal excrements were amended on a weekly basis (9 times) with excrements from either an oxytetracycline-treated or an untreated cow. Throughout the study, the concentration of oxytetracycline in excrements from the treated cow was above 500 µg g(-1)dw, whereas no oxytetracycline was detected in excrements from the healthy cow. Both excrements contained tetracycline resistance (TC-r) genes tet(L), tet(M), tet(V), tet(Z), tet(Q) and tet(W). The excrements from the treated cow also contained the tet(B) gene, and a higher abundance of tet(Z), tet(Q) and tet(W). Three weeks after the last excrement addition, the individual TC-r genes differed in their persistence in soil: tet(Q) and tet(B) were not detectable while tet(L), tet(M), tet(Z) and tet(W) were found in all 3 soils. There were, however, no significant differences in the total number, nor in the abundance, of TC-r genes between soil samples amended with each excrement type. The oxytetracycline-rich and the oxytetracycline-free excrement therefore contributed equally to the increase of tetracycline resistome in soil. Our results indicate that other mechanisms than OTC-selection pressure may be involved in the maintenance of TC-r genes in manured soils.

Separation and identification of 1,2,4-trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle.

Methanolic extract from lyophilized roots of Impatiens glandulifera Royle was analyzed by high performance liquid chromatography using DAD and FLD detection and this revealed one dominant highly fluorescent very unstable substance. The stability of this derivative is strongly dependent on the plant material drying procedure and extraction procedure used. The structure of the substance was established as 1,2,4-trihydroxynaphthalene-1-O-glucoside (THNG) according LC-MS and NMR measurements. When lyophilized plant material was extracted with methanol an almost four times higher amount of THNG was found in the extract, compared to the amount of 2-hydroxy-1,4-naphthoquinone obtained, while in the case of the same lyophilized plant material extracted with water there was no THNG in the extract. The main compounds in this case was 2-hydroxy-1,4-naphthoquinone. In the plant material dried at the laboratory temperature and extracted by methanol there are only traces of THNG.

Grass Pollen Pollution from Biofuels Farming.

Based on its low costs and high harvests, red fescue (Festuca rubra ssp. rubra L.) is a promising plant for biofuel production in the subtropics and temperate climate. Once the acreage increases rapidly, its pollen pollution was studied in detail. Robust analysis on the shape, size, and chemical composition, especially the presence of allergens was conducted for 2 successive years in relation to 3 levels of N- and PK-fertilization. Multiparametric statistical analysis showed that there was no significant effect on the pollen grain size nor on the profile and content of the phenolic compounds. On the other hand, intense fertilization naturally increased the number of panicles, and consequently the overall pollen production. In addition, higher levels of fertilization significantly (P0.016, resp. P0.011) influences the content of tryptophan, which is involved in the occurrence and course of the allergic reactions.