What Are We Eating? Surveying the Presence of Toxic Molecules in the Food Supply Chain Using Chromatographic Approaches.

Consumers in developed and Western European countries are becoming more aware of the impact of food on their health, and they demand clear, transparent, and reliable information from the food industry about the products they consume. They recognise that food safety risks are often due to the unexpected presence of contaminants throughout the food supply chain. Among these, mycotoxins produced by food-infecting fungi, endogenous toxins from certain plants and organisms, pesticides, and other drugs used excessively during farming and food production, which lead to their contamination and accumulation in foodstuffs, are the main causes of concern. In this context, the goals of this review are to provide a comprehensive overview of the presence of toxic molecules reported in foodstuffs since 2020 through the Rapid Alert System for Food and Feed (RASFF) portal and use chromatography to address this challenge. Overall, natural toxins, environmental pollutants, and food-processing contaminants are the most frequently reported toxic molecules, and liquid chromatography and gas chromatography are the most reliable approaches for their control. However, faster, simpler, and more powerful analytical procedures are necessary to cope with the growing pressures on the food chain supply.

Urinary volatomic profile of traditional tobacco smokers and electronic cigarettes users as a strategy to unveil potential health issues.

Many harmful and potentially harmful constituents are present in tobacco products. Cigarette smoke is known to cause different forms of cancer and trigger the development of chronic diseases. In the last decade, electronic cigarettes have emerged as a healthier alternative associated to less harmful effects in comparison to traditional tobacco. However, the lack of standardization of electronic cigarettes products makes it difficult to establish and compare the real effects on health of products from different manufacturers. To better understand the impact of smoking and vaping, the volatomic composition of urine samples from traditional tobacco smokers and electronic cigarette users was established and compared with nonsmokers (control group), using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry. A total of 45 urinary volatile organic metabolites belonging to different chemical families were identified in the urine of the studied groups. Benzene derivatives, terpenes, and aromatics were the chemical families that contributed the most to the urinary profile of smokers. The vapers urinary volatomic pattern was also dominated by terpenes and aromatics, in addition to alcohols. The orthogonal partial least squares-discriminant analysis of the data obtained indicated that the urinary profile of vapers is more closely related to the control group, reinforcing the hypothesis of the lowest harmfulness of electronic cigarettes. Further studies recruiting a higher number of subjects are therefore necessary to consolidate the data obtained.

Chromatographic-Based Platforms as New Avenues for Scientific Progress and Sustainability.

Chromatography was born approximately one century ago and has undergone outstanding technological improvements in innovation, research, and development since then that has made it fundamental to advances in knowledge at different levels, with a relevant impact on the well-being and health of individuals. Chromatography boosted a comprehensive and deeper understanding of the complexity and diversity of human-environment interactions and systems, how these interactions affect our life, and the several societal challenges we are currently facing, namely those related to the sustainability of our planet and the future generations. From the life sciences, which allowed us to identify endogenous metabolites relevant to disease mechanisms, to the OMICS field, nanotechnology, clinical and forensic analysis, drug discovery, environment, and "foodprint", among others, the wide range of applications of today's chromatographic techniques is impressive. This is fueled by a great variability of powerful chromatographic instruments currently available, with very high sensitivity, resolution, and identification capacity, that provide a strong basis for an analytical platform able to support the challenging demands of the postgenomic and post COVID-19 eras. Within this context, this review aims to address the great utility of chromatography in helping to cope with several societal-based challenges, such as the characterization of disease and/or physiological status, and the response to current agri-food industry challenges of food safety and sustainability, or the monitoring of environmental contamination. These are increasingly important challenges considering the climate changes, the tons of food waste produced every day, and the exponential growth of the human population. In this context, the principles governing the separation mechanisms in chromatography as well the different types and chromatographic techniques will be described. In addition, the major achievements and the most important technological advances will be also highlighted. Finally, a set of studies was selected in order to evince the importance of different chromatographic analyses to understand processes or create fundamental information in the response to current societal challenges.

Green Extraction Techniques as Advanced Sample Preparation Approaches in Biological, Food, and Environmental Matrices: A Review.

Green extraction techniques (GreETs) emerged in the last decade as greener and sustainable alternatives to classical sample preparation procedures aiming to improve the selectivity and sensitivity of analytical methods, simultaneously reducing the deleterious side effects of classical extraction techniques (CETs) for both the operator and the environment. The implementation of improved processes that overcome the main constraints of classical methods in terms of efficiency and ability to minimize or eliminate the use and generation of harmful substances will promote more efficient use of energy and resources in close association with the principles supporting the concept of green chemistry. The current review aims to update the state of the art of some cutting-edge GreETs developed and implemented in recent years focusing on the improvement of the main analytical features, practical aspects, and relevant applications in the biological, food, and environmental fields. Approaches to improve and accelerate the extraction efficiency and to lower solvent consumption, including sorbent-based techniques, such as solid-phase microextraction (SPME) and fabric-phase sorbent extraction (FPSE), and solvent-based techniques (µQuEChERS; micro quick, easy, cheap, effective, rugged, and safe), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), in addition to supercritical fluid extraction (SFE) and pressurized solvent extraction (PSE), are highlighted.

Synthetic Routes Towards the Synthesis of Geminal α-Difunctionalized Ketones.

The importance of gem-difunctionalized ketones is represented by their broad applications across chemical boundaries over recent years. The interesting reactivities that this class of compounds possess have made them ideal building blocks to access high-value organic molecules. Furthermore, the gem-difunctionalized ketone moiety has featured in numerous bioactive molecules. For these reasons, a plethora of routes to access such significant molecules have been developed by research groups worldwide - this account looks at delineating the synthesis of gem-difunctionalized ketones from carbonyl substrates, diazo compounds, sulfur ylides and alkynyl reactants.

Evaluation of the Health-Promoting Properties of Selected Fruits.

In this study, the health-promoting benefits of different fruits grown in Madeira Island, namely lemon (Citrus limon var. eureka), tangerine (Citrus reticulata var. setubalense), pitanga (Eugenia uniflora var. red), tomato (Solanum lycopersicum var. gordal) and uva-da-serra, an endemic blueberry (Vaccinium padifolium Sm.), were investigated. The phenolic composition (total phenolics and total flavonoids content) and antioxidant capacity (assessed through ABTS and DPPH assays) were measured revealing a high phenolic potential for all fruits, except tomato, while uva-da-serra is particularly rich in flavonoids. In relation to the antioxidant capacity, the highest values were obtained for pitanga and uva-da-serra extracts. The bioactive potential was also assessed through the ability of the extracts to inhibit digestive enzymes linked to diabetes (α-amylase, α- and ß-glucosidases) and hypertension (angiotensin-converting enzyme, ACE). The results obtained point to a very high bioactive potential with the selected samples exhibiting very important ACE anti-enzymatic capacities. A statistical analysis of the obtained data reveals a very strong correlation between ABTS and TPC, and a strong contribution of the fruit polyphenols for enzyme inhibition, and thus, presenting high antihypertensive and antidiabetic capacities. Overall, the results obtained clearly show a high bioactive potential of the selected fruits that should be further studied, in terms of specific phenolic composition. Moreover, these results strongly support the valorisation of pitanga seeds usually discarded as a waste, and uva-da-serra, an endemic and wild bush, as potential bioresources of bioactive compounds with impact in human diet.

Extracellular volatilomic alterations induced by hypoxia in breast cancer cells.

INTRODUCTION: The metabolic shift induced by hypoxia in cancer cells has not been explored at volatilomic level so far. The volatile organic metabolites (VOMs) constitute an important part of the metabolome and their investigation could provide us crucial aspects of hypoxia driven metabolic reconfiguration in cancer cells. OBJECTIVE: To identify the altered volatilomic response induced by hypoxia in metastatic/aggressive breast cancer (BC) cells. METHODS: BC cells were cultured under normoxic and hypoxic conditions and VOMs were extracted using HS-SPME approach and profiled by standard GC-MS system. Univariate and multivariate statistical approaches (p < 0.05, Log2 FC ≥ 0.58/≤ - 0.58, PC1 > 0.13/< - 0.13) were applied to select the VOMs differentially altered after hypoxic treatment. Metabolic pathway analysis was also carried out in order to identify altered metabolic pathways induced by the hypoxia in the selected BC cells. RESULTS: Overall, 20 VOMs were found to be significantly altered (p < 0.05, PC1 > 0.13/< - 0.13) upon hypoxic exposure to BC cells. Further, cell line specific volatilomic alterations were extracted by comparative metabolic analysis of aggressive (MDA-MB-231) vs. non-aggressive (MCF-7) cells incubated under hypoxia and normoxia. In this case, 15 and 12 VOMs each were found to be significantly altered in aggressive cells when exposed to hypoxic and normoxic condition respectively. Out of these, 9 VOMs were found to be uniquely associated with hypoxia, 6 were specific to normoxia and 6 were found common to both the conditions. Formic acid was identified as the most prominent molecule with higher abundance levels in aggressive as compared to non-aggressive cells in both conditions. Furthermore, metabolic pathway analyses revealed that fatty acid biosynthesis and nicotinate and nicotinamide metabolism were significantly altered in aggressive as compared to non-aggressive cells in normoxia and hypoxia respectively. CONCLUSIONS: Higher formate overflow was observed in aggressive cells compared to non-aggressive cells incubated under both the conditions, reinforcing its correlation with aggressive and invasive cancer type. Moreover, under hypoxia, aggressive cells preferred to be bioenergetically more efficient whereas, under normoxia, fatty acid biosynthesis was favoured when compared to non-aggressive cells.

Unravelling the Potential of Salivary Volatile Metabolites in Oral Diseases. A Review.

Fostered by the advances in the instrumental and analytical fields, in recent years the analysis of volatile organic compounds (VOCs) has emerged as a new frontier in medical diagnostics. VOCs analysis is a non-invasive, rapid and inexpensive strategy with promising potential in clinical diagnostic procedures. Since cellular metabolism is altered by diseases, the resulting metabolic effects on VOCs may serve as biomarkers for any given pathophysiologic condition. Human VOCs are released from biomatrices such as saliva, urine, skin emanations and exhaled breath and are derived from many metabolic pathways. In this review, the potential of VOCs present in saliva will be explored as a monitoring tool for several oral diseases, including gingivitis and periodontal disease, dental caries, and oral cancer. Moreover, the analytical state-of-the-art for salivary volatomics, e.g., the most common extraction techniques along with the current challenges and future perspectives will be addressed unequivocally.

Current trends on microextraction by packed sorbent - fundamentals, application fields, innovative improvements and future applications.

MEPS, the acronym of microextraction by packed sorbent, is a simple, fast and user- and environmentally-friendly miniaturization of the popular solid-phase extraction technique (SPE). In fact, it has been widely shown that MEPS can easily replace SPE for most, if not all, previous applications. It can attain this with obvious gains in sample and solvent usage, which is greatly reduced without compromising the extraction efficiency. Furthermore, MEPS can be operated with semiautomatic electronic syringes, making it very reliable and versatile, particularly to handle very low and very high sample volumes. This review will focus on the strengths and weaknesses of this technique and the different MEPS architectures commercially available in the context of the MEPS applications reported in the last five years. Additionally, innovative improvements will be highlighted, particularly those related with new applications and recent MEPS configurations and sorbents, such as the controlled directional flow or the innovative µSPEed variant.

Expanding Knowledge of the Host Range of Tomato chlorosis virus and Host Plant Preference of Bemisia tabaci MEAM1.

The crinivirus Tomato chlorosis virus (ToCV) is often found infecting tomato crops in Brazil, with variable incidence, but associated with prevalence of its primary vector, Bemisia tabaci MEAM1. ToCV control is difficult because there are no resistant commercial tomato varieties or hybrids available and chemical spray for control of the whitefly vector has not been effective. The present study evaluated the partial host range of a Brazilian isolate of ToCV and the preference of B. tabaci MEAM1 for oviposition on those species identified as susceptible to the virus. Subsequently, transmission tests were performed using plants of each ToCV host species as sources of inoculum to elucidate the epidemiological importance of nontomato sources of inoculum for infection of tomato. Among 80 species experimentally inoculated, 25 were susceptible, including 6 previously not known to be hosts (Jaltomata procumbens, Physalis pruinosa, Solanum aculeatissimum, S. viarum, Beta vulgaris var. cicla, and Chenopodium quinoa). Preference of whitefly for oviposition and infection by ToCV under free-choice transmission tests varied among the susceptible species. When ToCV-infected tomato, eggplant, and C. quinoa were used separately as sources of inoculum for virus transmission to tomato plants, mean percentages of infected plants were 76.6, 3, and 0%, respectively. Average oviposition of Bemisia tabaci on these three hosts were 2.7, 10.6, and 0.0 eggs/cm2, respectively. Additional studies will be necessary to evaluate the importance of ToCV host plants under field conditions and their efficiency as sources of inoculum for virus acquisition and transmission to tomato crops.

Screening of salivary volatiles for putative breast cancer discrimination: an exploratory study involving geographically distant populations.

Saliva is possibly the easiest biofluid to analyse and, despite its simple composition, contains relevant metabolic information. In this work, we explored the potential of the volatile composition of saliva samples as biosignatures for breast cancer (BC) non-invasive diagnosis. To achieve this, 106 saliva samples of BC patients and controls in two distinct geographic regions in Portugal and India were extracted and analysed using optimised headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME/GC-MS, 2 mL acidified saliva containing 10% NaCl, stirred (800 rpm) for 45 min at 38 °C and using the CAR/PDMS SPME fibre) followed by multivariate statistical analysis (MVSA). Over 120 volatiles from distinct chemical classes, with significant variations among the groups, were identified. MVSA retrieved a limited number of volatiles, viz. 3-methyl-pentanoic acid, 4-methyl-pentanoic acid, phenol and p-tert-butyl-phenol (Portuguese samples) and acetic, propanoic, benzoic acids, 1,2-decanediol, 2-decanone, and decanal (Indian samples), statistically relevant for the discrimination of BC patients in the populations analysed. This work defines an experimental layout, HS-SPME/GC-MS followed by MVSA, suitable to characterise volatile fingerprints for saliva as putative biosignatures for BC non-invasive diagnosis. Here, it was applied to BC samples from geographically distant populations and good disease separation was obtained. Further studies using larger cohorts are therefore very pertinent to challenge and strengthen this proof-of-concept study. Graphical abstract ᅟ.

Biological and molecular characterization of a putative new potexvirus infecting Senna occidentalis.

In this work, we report the complete genome sequence of, production of polyclonal antibodies against, and development of biological assays for a putative new potexvirus, named senna mosaic virus (SenMV), found infecting Senna occidentalis in the state of São Paulo, Brazil. The complete genome sequence of SenMV comprises 6775 nucleotides excluding the poly(A) tail. The genome organization is similar to those of other potexviruses, with five open reading frames coding for RNA-dependent RNA polymerase (RdRp), the triple gene block (TGB 1, 2, and 3) proteins, and coat protein (CP). The virus was transmitted to S. occidentalis by mechanical inoculation and trimming scissors, but not by seeds.

Genome organization and host range of a Brazilian isolate of johnsongrass mosaic virus.

This work reports the complete genome sequence, production of a polyclonal antiserum, and host range of a Brazilian strain of johnsongrass mosaic virus (JGMV) found infecting Panicum maximum in the state of São Paulo, Brazil. The complete genome sequence of this potyvirus, comprising 9874 nucleotides, showed 82 % amino acid sequence identity in the polyprotein to that of an isolate of JGMV from Australia. The experimental host range of this virus included mainly fodder species. Cultivated species such as rice, oats, sugarcane, rye, corn and wheat were not infected, suggesting that current isolates of this potyvirus do not represent a threat to these crops in Brazil.

Growth curves of carcass traits obtained by ultrasonography in three lines of Nellore cattle selected for body weight.

The effect of selection for postweaning weight was evaluated within the growth curve parameters for both growth and carcass traits. Records of 2404 Nellore animals from three selection lines were analyzed: two selection lines for high postweaning weight, selection (NeS) and traditional (NeT); and a control line (NeC) in which animals were selected for postweaning weight close to the average. Body weight (BW), hip height (HH), rib eye area (REA), back fat thickness (BFT), and rump fat thickness (RFT) were measured and records collected from animals 8 to 20 (males) and 11 to 26 (females) months of age. The parameters A (asymptotic value) and k (growth rate) were estimated using the nonlinear model procedure of the Statistical Analysis System program, which included fixed effect of line (NeS, NeT, and NeC) in the model, with the objective to evaluate differences in the estimated parameters between lines. Selected animals (NeS and NeT) showed higher growth rates than control line animals (NeC) for all traits. Line effect on curves parameters was significant (P < 0.001) for BW, HH, and REA in males, and for BFT and RFT in females. Selection for postweaning weight was effective in altering growth curves, resulting in animals with higher growth potential.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation.

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM - 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Underlying Features of Prostate Cancer-Statistics, Risk Factors, and Emerging Methods for Its Diagnosis.

Prostate cancer (PCa) is the most frequently occurring type of malignant tumor and a leading cause of oncological death in men. PCa is very heterogeneous in terms of grade, phenotypes, and genetics, displaying complex features. This tumor often has indolent growth, not compromising the patient's quality of life, while its more aggressive forms can manifest rapid growth with progression to adjacent organs and spread to lymph nodes and bones. Nevertheless, the overtreatment of PCa patients leads to important physical, mental, and economic burdens, which can be avoided with careful monitoring. Early detection, even in the cases of locally advanced and metastatic tumors, provides a higher chance of cure, and patients can thus go through less aggressive treatments with fewer side effects. Furthermore, it is important to offer knowledge about how modifiable risk factors can be an effective method for reducing cancer risk. Innovations in PCa diagnostics and therapy are still required to overcome some of the limitations of the current screening techniques, in terms of specificity and sensitivity. In this context, this review provides a brief overview of PCa statistics, reporting its incidence and mortality rates worldwide, risk factors, and emerging screening strategies.

Delving into Agri-Food Waste Composition for Antibacterial Phytochemicals.

The overuse of antibiotics in the healthcare, veterinary, and agricultural industries has led to the development of antimicrobial resistance (AMR), resulting in significant economic losses worldwide and a growing healthcare problem that urgently needs to be solved. Plants produce a variety of secondary metabolites, making them an area of interest in the search for new phytochemicals to cope with AMR. A great part of agri-food waste is of plant origin, constituting a promising source of valuable compounds with different bioactivities, including those against antimicrobial resistance. Many types of phytochemicals, such as carotenoids, tocopherols, glucosinolates, and phenolic compounds, are widely present in plant by-products, such as citrus peels, tomato waste, and wine pomace. Unveiling these and other bioactive compounds is therefore very relevant and could be an important and sustainable form of agri-food waste valorisation, adding profit for local economies and mitigating the negative impact of these wastes' decomposition on the environment. This review will focus on the potential of agri-food waste from a plant origin as a source of phytochemicals with antibacterial activity for global health benefits against AMR.

Competition between lysogenic and sensitive bacteria is determined by the fitness costs of the different emerging phage-resistance strategies.

Many bacterial genomes carry prophages whose induction can eliminate competitors. In response, bacteria may become resistant by modifying surface receptors, by lysogenization, or by other poorly known processes. All these mechanisms affect bacterial fitness and population dynamics. To understand the evolution of phage resistance, we co-cultivated a phage-sensitive strain (BJ1) and a polylysogenic Klebsiella pneumoniae strain (ST14) under different phage pressures. The population yield remained stable after 30 days. Surprisingly, the initially sensitive strain remained in all populations and its frequency was highest when phage pressure was strongest. Resistance to phages in these populations emerged initially through mutations preventing capsule biosynthesis. Protection through lysogeny was rarely observed because the lysogens have increased death rates due to prophage induction. Unexpectedly, the adaptation process changed at longer time scales: the frequency of capsulated cells in BJ1 populations increased again because the production of the capsule was fine-tuned, reducing the ability of phage to absorb. Contrary to the lysogens, these capsulated-resistant clones are pan-resistant to a large panel of phages. Intriguingly, some clones exhibited transient non-genetic resistance to phages, suggesting an important role of phenotypic resistance in coevolving populations. Our results show that interactions between lysogens and sensitive strains are shaped by antagonistic co-evolution between phages and bacteria. These processes may involve key physiological traits, such as the capsule, and depend on the time frame of the evolutionary process. At short time scales, simple and costly inactivating mutations are adaptive, but in the long term, changes drawing more favorable trade-offs between resistance to phages and cell fitness become prevalent.

Simultaneous microextraction of pesticides from wastewater using optimized µSPEed and µQuEChERS techniques for food contamination analysis.

Food contamination with pesticides poses significant risks to consumer safety and undermines confidence in food supply chains. Detecting pesticides in food samples is a challenging task that requires efficient extraction techniques. This study aims to compare and validate two microextraction techniques, µSPEed and µQuEChERS-dSPE, for the simultaneous extraction of eight pesticides (paraquat, thiabendazole, asulam, picloram, ametryn, atrazine, linuron, and cymoxanil) from wastewater samples. A good analytical performance was obtained for both methodologies, with selectivity, linearity in the range 0.5-150 mg L-1 with coefficients of determination up to 0.9979, limits of detection (LODs) and limits of quantification (LOQs) ranging from 0.02 to 0.05 mg L-1 and from 0.06 to 0.17 mg L-1, respectively, precision below 14.7 mg L-1, and recoveries from wastewater samples in the range of 66.1-99.9%. The developed methodologies are simpler, faster, and require less sample and solvent volumes than conventional methodologies, having a lower impact on the environment. Nevertheless, the µSPEed approach was found to be more efficient, easier to perform, and with a higher greener profile. This study highlights the potential of microextraction techniques for the analysis of pesticide residues in food and environmental samples. Overall, it presents a fast and efficient method for the analysis of pesticides in wastewater samples, which can be useful for monitoring and controlling pesticide contamination in the environment.

Evaluation of Tropane Alkaloids in Teas and Herbal Infusions: Effect of Brewing Time and Temperature on Atropine and Scopolamine Content.

Atropine and scopolamine belong to the tropane alkaloid (TA) family of natural toxins. They can contaminate teas and herbal teas and appear in infusions. Therefore, this study focused on analyzing atropine and scopolamine in 33 samples of tea and herbal tea infusions purchased in Spain and Portugal to determine the presence of these compounds in infusions brewed at 97 °C for 5 min. A rapid microextraction technique (µSPEed®) followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the selected TAs. The results showed that 64% of the analyzed samples were contaminated by one or both toxins. White and green teas were generally more contaminated than black and other herbal teas. Of the 21 contaminated samples, 15 had concentrations above the maximum limit for liquid herbal infusions (0.2 ng/mL) set by Commission Regulation (EU) 2021/1408. In addition, the effects of heating conditions (time and temperature) on atropine and scopolamine standards and naturally contaminated samples of white, green, and black teas were evaluated. The results showed that at the concentrations studied (0.2 and 4 ng/mL), there was no degradation in the standard solutions. Brewing with boiling water (decoction) for 5 and 10 min allowed for higher extraction of TAs from dry tea to infusion water.