Determination of 18 Intact Glucosinolates in Brassicaceae Vegetables by UHPLC-MS/MS: Comparing Tissue Disruption Methods for Sample Preparation

Glucosinolates (GSLs) are important precursor compounds with anticancer activities in Brassicaceae vegetables and are readily hydrolyzed by myrosinase. Given the diversity of these species, establishing an accurate and universal method to quantify intact GSLs in different plant tissues is necessary. Here, we compared and optimized three tissue disruption methods for sample preparation. After microwave treatment for 90 s, 13 GSLs in homogenized Chinese cabbage samples were recovered at 73–124%. However, a limitation of this method was that different tissues could not be processed under the same microwave conditions. Regarding universality, GSLs in Brassicaceae vegetables could be extracted from freeze-dried sample powder with 70% methanol (v/v) or frozen-fresh sample powder with 80% methanol (v/v). Moreover, heating extraction is necessary for GSLs extracted from frozen-fresh sample powder. Average recoveries of the two optimized methods were 74–119% with relative standard deviations ≤15%, with the limits of quantification 5.72–17.40 nmol/g dry weight and 0.80–1.43 nmol/g fresh weight, respectively. Notably, the method for analyzing intact GSLs was more efficient than that for desulfo-GSLs regarding operational complexity, detection speed and quantification accuracy. The developed method was applied to identify the characteristic GSLs in 15 Brassicaceae vegetables, providing a foundation for further research on GSLs.

Neglected Potential of Wild Garlic (Allium ursinum L.)—Specialized Metabolites Content and Antioxidant Capacity of Wild Populations in Relation to Location and Plant Phenophase

Wild garlic (Allium ursinum L.) is one of the species widely distributed in Europe and Asia and is often nutritionally neglected, characterized by a high content of various phytochemicals with high therapeutic potential and a range of biological activities. The aim of this study was to determine the content of bioactive compounds in the leaves of wild garlic populations collected from different micro-locations, and to determine the differences in the content of phytochemicals in the vegetative and generative phases. A significant content of different specialized metabolites was detected in all analyzed leaves of wild garlic populations regardless of the different factors (location and phenophase): vitamin C content with the highest determined value of 63.31 mg/100 g fw; total phenolic content with the highest determined value of 186.18 mg GAE/100 g fw (according to gallic acid in fresh sample); and antioxidant capacity with the highest determined value of 2230.66 µmol TE/L (according to Trolox). Significant differences in all the phytochemicals analyzed were observed depending on both the location and phenophase of the plants, with the most pronounced differences depending on the phenophase. Thus, lower levels of polyphenolic compounds and vitamin C were generally observed before the flowering phase, while the trend toward higher levels of pigment compounds was observed during the flowering phase of the plants. The results suggest that the leaves of wild garlic can be considered a valuable source of a variety of specialized metabolites with high antioxidant capacity, and thus have high production potential for various functional products and food supplements of natural origin, which are important for the promotion of human health.

Response Surface Approach to Optimize the Conditions of Foam Mat Drying of Plum in relation to the Physical-Chemical and Antioxidant Properties of Plum Powder

This research was done to optimize the influence of various egg albumin (EA) concentrations of 2, 4, and 6% as a foaming agent and whipping times of 5, 10, and 15 minutes, on physicochemical and antioxidant properties of plum powder produced using response surface methodology (RSM). Physical properties of the foam such as density, porosity, and expansion were determined. After drying and powder manufacturing, physical properties, namely, the water absorption index (WAI) and water solubility index (WSI), as well as chemical characteristics such as pH, titratable acidity, and browning index, were assessed. Finally, antioxidant capabilities such as the total phenol content (TPC), DPPH scavenging activity, beta carotene, and total flavonoid content (TFC) were measured. According to the findings, both whipping duration and EA concentration had a substantial effect on the foam forming characteristics. Foam expansion increased significantly with EA concentration and whipping time increase, but foam density exhibited an inverse relationship as expected. Increases in EA concentration and whipping duration both raised pH values whereas titratable acidity exhibited an inverse tendency as variable quantity rose. The browning index dropped as EA concentration increased. Antioxidant qualities were retained in dried sample powder as compared with the fresh sample, and they were also altered by variable changes. Overall, a 4% EA concentration for 10 to 15 minutes produced the best dehydration effects with the most antioxidant retention.

Idea of Rapid Preparation of Fatty Acid Methyl Ester Using In Situ Derivatization from Fresh Horse Mussel

The analysis of the fatty acid (FA) profile requires multiple preparation steps, which are lipid extraction followed by derivatization of the FA into a fatty acid methyl ester (FAME). The procedures are time-consuming, and generally require large volumes of sample sizes and solvents. This report proposes a technique for the preparation of FAME from fresh horse mussels without a step of lipid extraction. A rapid in situ derivatization using N,N-dimethylformamide dimethyl acetal (DMF-DMA) methylation followed by alkali-transesterification was examined. In this method, acylglycerols and free fatty acids (medium to long-chain FA) of the sample are targeted to convert into FAME. Direct alkali-transesterification of the fresh sample gave only 58.7% FAME with 12.4% triglyceride and 21.1% FFA. The alkali in situ method showed low conversion efficiency due to the initial sample containing high contents of moisture and FFA (75.11% of the fresh sample and 14.3% of total oil, respectively). The reaction was developed by using two steps in situ derivatization. A 50 mg sample was methylated with 1 mL of DMF-DMA (100 °C, 15 min), followed by transesterified with 10 mL of 1% (w/v) NaOH in methanol (60 °C, 3 min). The conversion into FAME was monitored using size-exclusion HPLC with evaporative light-scattering detection. The column was a 100 Å Phenogel with toluene and 0.25% acetic acid as a mobile phase. The FAME yield of 79.9% with 7.8% triglyceride and 8.5% FFA was obtained. The two steps in situ derivatization gave a promising result with the higher conversion with lower FFA. It is a simple and rapid (less than 20 min) method that requires a low volume of sample and solvent for FAME preparation. However, increasing the conversion efficiency as well as the variety of samples should be further studied.

Soluble fluoride in Na2FPO3/CaCO3-based toothpaste as an indicator of systemically bioavailable fluoride

Fluoride chemically soluble in toothpaste is an indicator of fluoride bioavailability when the teeth are brushed and the same should be expected systemically when toothpaste is ingested. A four-phases study was conducted, in which eight participants were subjected in each phase to one of the assigned treatment groups: Group I: fresh sample of a Na2FPO3/CaCO3 toothpaste with 1,334 μg F/g of total soluble fluoride (TSF); groups II–IV: aged samples of toothpaste presenting TSF concentrations of 1,128, 808, and 687 μg F/g, respectively. In all phases, the participants ingested an amount of toothpaste equivalent to 70.0 µg F/kg body weight, as total fluoride (TF). Blood was collected before (baseline) and up to 180 min after toothpaste ingestion as indicator of fluoride bioavailability. Total urine (24 h before and 24 h after ingestion) was collected as indicator of absorbed fluoride that was excreted. F concentration in blood plasma and urine was determined with a fluoride ion-specific electrode. The areas under the curve of F concentration vs. time (AUC=ng F/ml x min) and the peaks of fluoride concentration in blood plasma (Cmax) were calculated. The net amount of fluoride excreted (mg/day) was calculated by subtraction. A significant correlation of the amount (mg) of TSF ingested was found between the AUC (r= 0.76; p<0.01) and Cmax (r= 0.86; p<0.01) in plasma, and the fluoride excreted (r= 0.65; p<0.01). For TF no statistical correlations were found (p>0.05). Data suggest that the concentration of TSF found in Na2FPO3/CaCO3-based toothpastes is a useful predictor of how much fluoride will be systemically bioavailable when this type of formulation is ingested.

Effects of sun-drying on the antioxidant potentials of pepper (Capsicum) varieties

The present study investigated the effects of sun-drying on the antioxidant potential of three pepper varieties: Capsicum annuum var, Capsicum chinense and Capsicum annuum. Fresh fruits of the pepper varieties were collected, washed under distilled water and were divided into two parts: one for fresh sample and the other for the dried sample. Dried and fresh samples of the pepper varieties were homogenized and extracted with methanol. The concentrations of total phenolics and flavonoids were evaluated; DPPH-radical scavenging activity and the FRAP potential of the extracts were also determined. The results revealed that sun-drying process significantly reduced the total phenolic content of C. annuum var, C. chinense and C. annuum from 5.91 ± 0.22 mg/g GAE, 6.9 ± 0.23 mg/g GAE, 6.67 ± 0.99 mg/g GAE to 3.31 ± 0.72 mg/g GAE, 3.59 ± 0.89 mg/g GAE, 3.01 ± 0.17 mg/g GAE respectively and flavonoid content from 3.80 ± 0.02 mg/g QE, 3.91 + 0.08 mg/g QE, 3.84 ± 0.08 mg/g QE to 1.26 ± 0.90 mg/g QE, 1.95 ± 0.07 mg/g QE, 1.23 ± 0.04 mg/g QE respectively. The result also revealed that the fresh samples of C. annuum var, C. chinense and C. annuum exhibited higher percentage inhibition of DPPH-radical at 59.4 ± 0.5%, 61.2 ± 0.6%, 58.9 ± 0.2% respectively and were significantly different from the percentage inhibition by the dried samples: 39.2 ± 0.5%, 42.4 ± 0.4%, 38.6 ± 0.6% respectively.The FRAP potential of the fresh samples of C. annuum var, C. chinense and C. annuum: 588.56 ± 29.4 ìmol Fe(II)/g, 691.34 ± 20.46 ìmol Fe(II)/g and 598.9 ± 23. 82 ìmol Fe(II)/g respectively were significantly different from the dried samples: 370.22 ± 14.75 ìmol Fe(II)/g, 392.34 ± 45.74 ìmol Fe(II)/g and 358.6 ± 30.08 ìmol Fe(II)/g respectively. The three Capsicum species are very rich in antioxidants. However, the sun drying method reduced the antioxidant capacities of the peppers, thus further studies should be carried out on the best method for the preservation of Capsicum species. Key Words: Capsicum. annuum var, C. chinense, C. annuum, Antioxidant, Sun-drying, methanolic extract

Vitamins Composition in Clarias gariepinus Fish Body Parts (Liver, Muscle, Head): Reporting on Samples on Fresh, Smoked-Dried and Dry Extract Bases

An investigation into the vitamins composition levels in Clariasgariepinus fish was carried out and reported in dry extract/fresh; dry extract / smoked-dried on individual vitamins and the sum of the whole vitamins. Parts investigated were liver, muscle and head. Whereas fresh and smoked-dried data were laboratory results, the dry extract portions were calculated and reported as dry extract /fresh sample, dry extract / smoked-dried sample for liver, muscle and head. Results obtained ran thus and all values were in mg/100g vitamin where d = difference, CV% = coefficient of variation and % difference = % value that shows what made dry extract value greater than its reported comparison: dry extract/fresh, % d = 74.5 (all), CV% = 84.0 (all), in liver; dry extract/smoked, % d =24.5 (all), CV% = 19.7 (all), in liver; dry extract/fresh, % d = 74.3 (all), CV% = 83.6 (all), in muscle; ndry extract/smoked, % d = 10.2 (all), CV% =7.60 (all), in muscle;m dry extract/fresh, % d = 68.5 (all), CV% = 73.7 (all), in head; dry extract/smoked, % d = 9.10 (all), CV% = 6.74 (all), in head; dry extract/fresh, % d = 71.9- 74.5, CV% = 79.4 - 82.4 in total vitamin body load; dry extract/smoked, % d = 9. 69- 24.5, CV% = 7.20 - 19.7 in total vitamin body load; dry extract (fresh) – dry extract (smoked), %d = 69.6 - 82.0 in liver; dry extract (fresh) – dry extract (smoked), %d = 72.3 - 76.3 in muscle; dry extract (fresh) – dry extract (smoked), %d = 62.9 - 75.2 in head; dry extract (fresh) – dry extract (smoked), %d = 69.7- 79.0 in total vitamins body load.Among the dry extract values calculated from fresh samples and subjected to chi-square (χ2) values, significant values were observed in vitamins B6, C, A, B1, D, E and total at α=0.05. In the dry extract values from smoked samples, only three significant χ2 values in vitamins A, E and total were observed. In reflection to vitamin concentration levels, percentage higher levels in dry extracts (from fresh) had these trends: liver (74.5%) > muscle (74.3%) > head (68.5%) whereas from smoked, we had liver (24.5%) > muscle (10.2%) > head (9.10%). Also total vitamin body load from dry extract (fresh) was 71.9-74.5% difference and dry extract (smoked) was 9.69 -24.5% difference. It should be noted that liver occupied the higher part of the range in the two comparisons, like 74.5% (fresh) and 24.5% (smoked).

Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients

Background Cryopreservation of human spermatozoa has been identified as an efficient procedure to preserve fertility in men before any cancer therapy or surgical infertility treatment. Despite the benefits of the procedure, the deleterious effects of cryopreservation have been proven on sperm structure and function. This study aimed to evaluate seminal plasma effects on human sperm characteristics after cryopreservation, and compare the addition of normozoospermic and oligozoospermic seminal plasma in the prepared oligozoospermic samples. Semen samples were collected from fifty-five oligozoospermic men and the twenty fertile individuals who referred to the infertility center. At first, a semen analysis was carried out on each neat ejaculate, and then some were cryopreserved. The remainder of the semen was divided into two, one for seminal plasma removal and the other for sperm preparation. Then, the prepared spermatozoa were cryopreserved in three groups: one with, and another without the addition of oligozoospermic seminal plasma, and still another with the addition of normal seminal plasma. After thawing, sperm DNA integrity, viability, motility, and morphology were determined. Results The percentages of all parameters were significantly lower after cryopreservation in all groups compared to the fresh sample. However, this reduction was lower in the oligozoospermic samples cryopreserved with normal seminal plasma. Conclusion The results indicated that seminal plasma in oligozoospermic patients could not support sperm against cryo-injuries, an indication likely due to insufficient antioxidants and other protective components in oligozoospermic patients. However, normal seminal plasma could slightly preserve sperm characteristics after cryopreservation in oligozoospermic patients.

Effect of Freeze-Thaw Cycles on Juice Properties, Volatile Compounds and Hot-Air Drying Kinetics of Blueberry

This paper studied the effects of freeze-thaw (FT) cycles on the juice properties and aroma profiles, and the hot-air drying kinetics of frozen blueberry. After FT treatment, the juice yield increased while pH and total soluble solids of the juice keep unchanged. The total anthocyanins contents and DPPH antioxidant activities of the juice decreased by FT treatments. The electronic nose shows that FT treatments significantly change the aroma profiles of the juice. The four main volatile substances in the fresh juice are (E)-2-hexenal, α-terpineol, hexanal and linalyl formate, which account for 48.5 ± 0.1%, 17.6 ± 0.2%, 14.0 ± 1.5% and 7.8 ± 2.7% of relative proportions based on total ion chromatogram (TIC) peak areas. In the FT-treated samples, the amount of (E)-2-hexenal and hexanal decreased significantly while α-terpineol and linalyl formate remained almost unchanged. Repeated FT cycles increased the ethanol content and destroyed the original green leafy flavor. Finally, the drying kinetics of FT-treated blueberries was tested. One FT treatment can shorten the drying time by about 30% to achieve the same water content. The Deff values of the FT-treated sample are similar, which are about twice as large as the value of the fresh sample. The results will be beneficial for the processing of frozen blueberry into juice or dried fruits.

Goat’s Milk as a Potential Anti-proliferative against Colon Cancer Cell Lines

Aim: To investigate anti-proliferative effect of fresh and pasteurized goat milk against colon cancer cell lines (HT-29, HCT-116, CT-26). Study Design: Experimental study. Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between January 2020 and April 2020. Methodology: Samples comprised of two types goat milk, which were fresh and pasteurized in powder form. The samples were analysed for the anti-proliferative effect by MTT assay, and IC50 value was determined. Then, cell apoptotic changes were observed by light inverted microscope by 24, 48 and 72 hours. Results: Experimental data showed that the fresh sample produce the highest yield (9.40%) than the pasteurized sample (7.17%). The fresh sample yielded the most potent cytotoxic value (0.28 ± 0.03), followed by pasteurized sample with value IC50 0.32 ± 0.02 against HCT-116 cells. Then, the anti-proliferative effect was observed on cell apoptotic changes by reduction of cell volume, cell densed, and presence of fragmentation and apoptotic bodies at 24, 48 and 72 hours treatment. Conclusion: In conclusion, the fresh sample of goat milk yielded the potent anti-proliferative effect than pasteurized sample.