Research Progress on the Extraction and Purification of Anthocyanins and Their Interactions with Proteins.

Anthocyanins, as the most critical water-soluble pigments in nature, are widely present in roots, stems, leaves, flowers, fruits, and fruit peels. Many studies have indicated that anthocyanins exhibit various biological activities including antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, vision protection, and anti-aging. Hence, anthocyanins are widely used in food, medicine, and cosmetics. The green and efficient extraction and purification of anthocyanins are an important prerequisite for their further development and utilization. However, the poor stability and low bioavailability of anthocyanins limit their application. Protein, one of the three essential nutrients for the human body, has good biocompatibility and biodegradability. Proteins are commonly used in food processing, but their functional properties need to be improved. Notably, anthocyanins can interact with proteins through covalent and non-covalent means during food processing, which can effectively improve the stability of anthocyanins and enhance their bioavailability. Moreover, the interactions between proteins and anthocyanins can also improve the functional characteristics and enhance the nutritional quality of proteins. Hence, this article systematically reviews the extraction and purification methods for anthocyanins. Moreover, this review also systematically summarizes the effect of the interactions between anthocyanins and proteins on the bioavailability of anthocyanins and their impact on protein properties. Furthermore, we also introduce the application of the interaction between anthocyanins and proteins. The findings can provide a theoretical reference for the application of anthocyanins and proteins in food deep processing.

Extraction, purification, structural characteristics, bioactivity and potential applications of polysaccharides from Semen Coicis: A review.

Semen Coicis (S. Coicis) has been regarded as a valuable source of traditional herbal medicine in China for thousands of years. S. Coicis polysaccharides (SCPs) are one of the most important bioactive ingredients of S. Coicis, which have attracted worldwide attention, because of their great marketing potential and development prospects. Hot water extraction is currently the most commonly used method to isolate SCPs. The structural characteristics of SCPs have been extensively investigated through various advanced modern analytical techniques to dissect the structure-activity relationships. SCPs are mainly composed of diverse monosaccharides, from which Rha and Ara are the most prevalent glycosyl groups. In addition, the structures of SCPs are found to be closely related to their multiple biological activities, including antioxidant activity, immunomodulatory function, antitumor activity, hypoglycemic effect, intestinal microbiota regulatory activity, anti-inflammatory activity, among others. In view of this, this review aimed to provide systematic and current information on the isolation, structural characteristics, and bioactivities of SCPs to support their future applications as therapeutic agents and functional foods.

Frontiers of Plant-derived Exosomes from Research Methods to Pharmaceutical Applications in Plant-based Therapeutics

Exosomes have emerged as critical mediators of intercellular communication and various physiological processes between cells and their environment. These nano-sized vesicles have been extensively investigated and confirmed to exhibit multifunctionality in animal systems. In particular, they participate in intercellular signaling, influence disease progression, and exhibit biological activity. However, Plant-Derived Exosomes (PDEs), especially therapeutic PDEs, have received relatively limited attention in the past few decades. Recent studies have demonstrated that PDEs are involved in signaling molecule transport in addition to intercellular communication, as they serve as functional molecules in the cellular microenvironment. This characteristic highlights the immense potential of PDEs for a wide array of applications, including antioxidation, anti-inflammation, tumour cell elimination, immune modulation, and tissue regeneration. In addition, PDEs hold substantial promise as efficient drug carriers, enhancing the stability and bioavailability of therapeutic agents and consequently enabling targeted delivery to specific cells or tissues. Therefore, PDEs may serve as effective tools for drug delivery and the treatment of various diseases. This comprehensive review provides an overview of recent studies on therapeutic PDEs, focusing on their extraction, isolation, characterization methods, biological activities, and application prospects. It summarises the research progress of exosome-like nanovesicles derived from medicinal plants, with a specific emphasis on traditional Chinese medicine, and highlights their importance in disease treatment and nanoparticle delivery. The main objective is to accelerate the clinical translation of these nanovesicles while providing novel approaches and methodologies for the research and development of innovative drugs.

Phycocyanin from microalgae: A comprehensive review covering microalgal culture, phycocyanin sources and stability.

As food safety continues to gain prominence, phycocyanin (PC) is increasingly favored by consumers as a natural blue pigment, which is extracted from microalgae and serves the dual function of promoting health and providing coloration. Spirulina-derived PC demonstrates exceptional stability within temperature ranges below 45 °C and under pH conditions between 5.5 and 6.0. However, its application is limited in scenarios involving high-temperature processing due to its sensitivity to heat and light. This comprehensive review provides insights into the efficient production of PC from microalgae, covers the metabolic engineering of microalgae to increase PC yields and discusses various strategies for enhancing its stability in food applications. In addition to the most widely used Spirulina, some red algae and Thermosynechococcus can serve as good source of PC. The genetic and metabolic manipulation of microalgae strains has shown promise in increasing PC yield and improving its quality. Delivery systems including nanoparticles, hydrogels, emulsions, and microcapsules offer a promising solution to protect and extend the shelf life of PC in food products, ensuring its vibrant color and health-promoting properties are preserved. This review highlights the importance of metabolic engineering, multi-omics applications, and innovative delivery systems in unlocking the full potential of this natural blue pigment in the realm of food applications, provides a complete overview of the entire process from production to commercialization of PC, including the extraction and purification.

Polysaccharides from Platycodon grandiflorum: A review of their extraction, structures, modifications, and bioactivities.

Platycodon grandiflorum (P. grandiflorum) has long been used as a food and traditional herbal medicine. As a food, P. grandiflorum is often transformed into pickles for consumption, and as a traditional Chinese medicine, P. grandiflorum clears the lung, nourishes the pharynx, dispels phlegm, and discharges pus. Polysaccharides are among the main active components of P. grandiflorum. Recent literature has described the preparation, identification, and pharmacological activity of these polysaccharides. Studies have shown that these polysaccharides exhibit a variety of significant biological effects in vitro and in vivo, such as immune stimulation and antioxidant, anti-liver injury, anti-apoptosis and antitumour effects. However, there is no systematic summary of the related research articles on P. grandiflorum polysaccharide, which undoubtedly brings some difficulties to the future research. The purpose of this review is to comprehensively describe research progress on the extraction, purification, structural characterization, modification, and biological activity of P. grandiflorum polysaccharides. The shortcomings of recent research are summarized, further research on their biological activity is proposed to provide new reference value for the application of P. grandiflorum polysaccharides in drugs and health products in the future.

Future proofing of chondroitin sulphate production: Importance of sustainability and quality for the end-applications.

Chondroitin sulphates (CSs) are the most well-known glycosaminoglycans (GAGs) found in any living organism, from microorganisms to invertebrates and vertebrates (including humans), and provide several health benefits. The applications of CSs are numerous including tissue engineering, osteoarthritis treatment, antiviral, cosmetics, and skincare applications. The current commercial production of CSs mostly uses animal, bovine, porcine, and avian tissues as well as marine organisms, marine mammals, sharks, and other fish. The production process consists of tissue hydrolysis, protein removal, and purification using various methods. Mostly, these are chemical-dependent and are complex, multi-step processes. There is a developing trend for abandonment of harsh extraction chemicals and their substitution with different green-extraction technologies, however, these are still in their infancy. The quality of CSs is the first and foremost requirement for end-applications and is dependent on the extraction and purification methodologies used. The final products will show different bio-functional properties, depending on their origin and production methodology. This is a comprehensive review of the characteristics, properties, uses, sources, and extraction methods of CSs. This review emphasises the need for extraction and purification processes to be environmentally friendly and gentle, followed by product analysis and quality control to ensure the expected bioactivity of CSs.

Polygonum cuspidatum polysaccharide: A review of its extraction and purification, structure analysis, and biological activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum cuspidatum Sieb. et Zucc. is mainly distributed in Shanxi, Gansu, and Sichuan province of China. It is also found in Korea and Japan. Its dried roots and rhizomes are used as medicinal herbs and have been used to treat hyperglycemia and various inflammatory disorders. AIM OF THE REVIEW: This paper aims to provide an up-to-date review of the developments in the studies involving the extraction and purification, structure analysis, pharmacological effects, and potential applications of polysaccharides obtained from Polygonum cuspidatum. Additionally, the possible future research directions of this plant are discussed. MATERIALS AND METHODS: This article used "Polygonum cuspidatum polysaccharide (PCP)" and "Polygonum cuspidatum" as the keywords and gathered relevant data on Polygonum cuspidatum using electronic databases (Elsevier, PubMed, ACS, CNKI, Google Scholar, Baidu Scholar, Web of Science), relevant books, and classic literature about Chinese herb. RESULTS: Excluding irrelevant and repetitive documents, 278 documents were finally included, of which 88 were in Chinese and 190 were in English. The CiteSpace software was used to visualize the trends and keywords in this research field. We concluded that the main extraction methods for Polygonum cuspidatum polysaccharide are water extraction and alcohol precipitation, microwave-assisted extraction, ultrasound-assisted extraction, and microjet extraction. High-performance liquid chromatography and column chromatography are also commonly used in the separation and purification of PCP. PCP has antitumor, immunomodulatory, hypoglycemic, and antioxidant effects. This paper provides an updated and deeper understanding of PCP, serving as a theoretical foundation for the further optimization of polysaccharide structures and the development of PCP as a novel functional material for clinical application.

An Eco-Friendly Extraction and Purification Approach for Obtaining Active Ingredients for Cosmetics from Two Marine Brown Seaweeds.

Brown seaweeds are attracting attention due to their richness in bioactive compounds, in particular, their phlorotannins. We present here a case study of two Fucales, Ascophyllum nodosum and Halidrys siliquosa, sustainably collected, to produce active polyphenols for the cosmetics sector. Phenolic contents of crude extracts, obtained by Accelerated Solvent Extraction (ASE), were more elevated in H. siliquosa at 100.05 mg/g dry weight (DW) than in A. nodosum (29.51 mg/g DW), considering 3 cycles with cell inversion. The temperature of extraction for a high phenolic content and high associated antioxidant activities close to positive controls was 150 °C for both algae and the use of only one cycle was enough. A semi-purification process using Solid-phase Extraction (SPE) was carried out on both ASE crude extracts (one per species). The majority of phlorotannins were found in the ethanolic SPE fraction for A. nodosum and the hydroethanolic one for H. siliquosa. The SPE process allowed us to obtain more concentrated fractions of active phenolic compounds (×1.8 and 2 in A. nodosum and H. siliquosa, respectively). Results are discussed in regard to the exploitation of seaweeds in Brittany and to the research of sustainable processes to produce active natural ingredients for cosmetics.

Research progress in the preparation, structural characterization, and biological activities of polysaccharides from traditional Chinese medicine.

Traditional Chinese medicines are tremendous sources of polysaccharides, which are of great interest in the human welfare system as natural medicines, food, and cosmetics. This review aims to highlight the recent trends in extraction (conventional and non-conventional), purification and analytic techniques of traditional Chinese medicine polysaccharides (TCMPs), and the chemical structure, biological activities (anti-tumor, hypoglycemic, antioxidant, intestinal flora regulation, immunomodulatory, anti-inflammatory, anti-aging, hypolipidemic, hepatoprotective, and other activities), and the underlying mechanisms of polysaccharides extracted from 76 diverse traditional Chinese medicines were compared and discussed. With this wide coverage, a total of 164 scientific articles were searched from the database including Google Scholar, PubMed, Web of Science, and China Knowledge Network. This comprehensive survey from previous reports indicates that TCMPs are non-toxic, highly biocompatible, and good biodegradability. Besides, this review highlights that TCMPs may be excellent functional factors and effective therapeutic drugs. Finally, the current problems and future research advances of TCMPs are also introduced. New valuable insights for the future researches regarding TCMPs are also proposed in the fields of therapeutic agents and functional foods.

Extraction, purification, structural characterization and pharmacological activities of polysaccharides from sea buckthorn (Hippophae rhamnoides L.): A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides L.) is an edible fruit with a long history in China as a medicinal plant. The fruits of H. rhamnoides are rich in a variety of nutrients and pharmacological active compounds. As one of the most important active ingredients in sea buckthorn, polysaccharides have attracted the attention of researchers due to their antioxidant, anti-fatigue, and liver protective qualities. AIM OF THE REVIEW: This review summarizes recent studies on extraction, purification, structural characterization and pharmacological activities of polysaccharides from sea buckthorn. In addition, the relationship between the structure and the activities of sea buckthorn polysaccharides (SBPS) were discussed. This review would provide important research bases and up-to-date information for the future in-depth development and application of sea buckthorn polysaccharides in the field of pharmaceuticals and functional foods. MATERIALS AND METHODS: By inputting the search term "Sea buckthorn polysaccharides", relevant research information was obtained from databases such as Web of Science, Google Scholar, PubMed, China Knowledge Network (CNKI), China Master Theses Full-text Database, and China Doctoral Dissertations Full-text Database. RESULTS: The main extraction methods of SBPS include hot water extraction (HWE), ultrasonic assisted extraction (UAE), microwave-assisted extraction (MAE), flash extraction (FE), and ethanol extraction. More than 20 polysaccharides have been isolated from sea buckthorn fruits. The chemical structures of sea buckthorn polysaccharides obtained by different extraction, isolation, and purification methods are diverse. Polysaccharides from sea buckthorn display a variety of pharmacological properties, including antioxidant, anti-fatigue, liver protection, anti-obesity, regulation of intestinal flora, immunoregulation, anti-tumor, anti-inflammatory, and hypoglycemic activities. CONCLUSIONS: Sea buckthorn has a long medicinal history and characteristics of an ethnic medicine and food. Polysaccharides are one of the main active components of sea buckthorn, and they have received increasing attention from researchers. Sea buckthorn polysaccharides have remarkable pharmacological activities, health benefits, and broad application prospects. In addition, further exploration of the chemical structure of SBPS, in-depth study of their pharmacological activities, identification of their material basis, characterization of disease resistance mechanisms, and potential health functions are still directions of future research. With the accumulation of research on the extraction and purification processes, chemical structure, pharmacological effects, molecular mechanisms, and structure-activity relationships, sea buckthorn polysaccharides derived from natural resources will ultimately make significant contributions to human health.

Review of isolation, purification, structural characteristics and bioactivities of polysaccharides from Portulaca oleracea L.

Portulaca oleracea L., also known as purslane, affiliates to the Portulacaceae family. It is an herbaceous succulent annual plant distributed worldwide. P. oleracea L. is renowned for its nutritional value and medicinal value, which has been utilized for thousands of years as Traditional Chinese Medicine (TCM). The extract derived from P. oleracea L. has shown efficacy in treating various diseases, including intestinal dysfunction and inflammation. Polysaccharides from P. oleracea L. (POP) are the primary constituents of the crude extract which have been found to have various biological activities, including antioxidant, antitumor, immune-stimulating, and intestinal protective effects. While many publications have highlighted on the structural identification and bioactivity evaluation of POP, the underlying structure-activity relationship of POP still remains unclear. In view of this, this review aims to focus on the extraction, purification, structural features and bioactivities of POP. In addition, the potential structure-activity relationship and the developmental perspective for future research of POP were also explored and discussed. The current review would provide a valuable research foundation and the up-to-date information for the future development and application of POP in the field of the functional foods and medicine.

A Blood Drying Process for DNA Amplification.

The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid-phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano-porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin-resistant Staphylococcus aureus, Methicillin-Sensitive Staphylococcus aureus using loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.

Isolation, structural properties and bioactivities of polysaccharides from Crataegus pinnatifida.

ETHNOPHARMACOLOGIC RELEVANCE: Crataegus pinnatifida, commonly known as hawthorn, is a plant species with a long history of medicinal use in traditional Chinese medicine. Hawthorn polysaccharides (HP) have gained worldwide attention due to their decent biological activities and potential health benefits. Their excellent antioxidant activity, antitumor activity, immunomodulatory activity, hypoglycemic effect and hypolipidemic effects, intestinal microbiota modulatory activity makes them valuable in the field of ethnopharmacological research. AIM OF THE STUDY: The purpose of the current review is to provide a systematic and comprehensive summary of the latest literatures and put forward the future perspectives on hawthorn polysaccharides in the context of its extraction, purification, structural characteristics and bioactivities. Furthermore, the underlying structure-bioactivity relationship of hawthorn polysaccharides was also explored and discussed. The current review would provide the important research underpinnings and the update the information for future development and application of hawthorn polysaccharides in the pharmaceutical and functional food industries. MATERIALS AND METHODS: We use Google Scholar, CNKI, PubMed, Springer, Elsevier, Wiley, Web of Science and other online databases to search and obtain the literature on extraction, isolation, structural analysis and the biological activity of hawthorn polysaccharides published before October 2023. The key words are "extraction", "isolation and purification", "bioactivities", and "Crataegus pinnatifida polysaccharides ". RESULTS: Crataegus pinnatifida has been widely used for the treatment of cardiovascular diseases, digestive disorders, inflammatory and oxidative stress in traditional Chinese medicine. Polysaccharides are the key active components of Crataegus pinnatifida which have gained widespread attention. The structure and bioactivity of polysaccharides from Crataegus pinnatifida varies in terms of raw materials, extraction methods and purification techniques. Crataegus pinnatifida polysaccharides possess diverse bioactivities, including antitumor, immunomodulatory, hypoglycemic activity, cardioprotective and antioxidant activities, among others. These biological properties can not only lay firm foundation for the treatment of diverse diseases, but also provide a theoretical basis for the in-depth study of the structure-activity relationship. In addition, the underlying structure-activity relationship is also explored and discussed, and further research and development of hawthorn polysaccharides are also prospected. CONCLUSION: As a natural compound, hawthorn polysaccharides has garnered significant attention and held immense research potential. Hawthorn polysaccharides can be obtained through different extraction methods, including hot water extraction method, ultrasonic extraction method and enzymatic extraction method etc. The structures of hawthorn polysaccharides have also been characterized and reported in numerous studies. Moreover, hawthorn polysaccharides exhibit a wide range of bioactivities, such as the antioxidant activity, the antitumor activity, the immunomodulatory activity, the hypoglycemic effect and the hypolipidemic effect, as well as the intestinal microbiota modulatory activity. These diverse bioactivities contribute to the growing interest in hawthorn polysaccharides and its potential applications. Hawthorn polysaccharides has promising application prospects in various industries, including functional food, pharmaceuticals and biomedical research. Therefore, it is imperative to fully explore and harness the potential of hawthorn polysaccharides in the food and medicine fields.

Research progress in extraction, purification, structure of fruit and vegetable polysaccharides and their interaction with anthocyanins/starch.

Fruits and vegetables contain polysaccharides, polyphenols, antioxidant enzymes, and various vitamins, etc. Fruits and vegetables polysaccharides (FVPs), as an important functional factor in health food, have various biological activities such as lowering blood sugar, blood lipids, blood pressure, inhibiting tumors, and delaying aging, etc. In addition, FVPs exhibit good physicochemical properties including low toxicity, biodegradability, biocompatibility. Increasing research has confirmed that FVPs could enhance the stability and biological activities of anthocyanins, affecting their bioavailability to improve food quality. Simultaneously, the addition of FVPs in natural starch suspension could improve the physicochemical properties of natural starch such as viscosity, gelling property, water binding capacity, and lotion stability. Hence, FVPs are widely used in the modification of natural anthocyanins/starch. A systematic review of the latest research progress and future development prospects of FVPs is very necessary to better understand them. This paper systematically reviews the latest progress in the extraction, purification, structure, and analysis techniques of FVPs. Moreover, the review also introduces the potential mechanisms, evaluation methods, and applications of the interaction between polysaccharides and anthocyanins/starch. The findings can provide important references for the further in-depth development and utilization of FVPs.

A Review of Extraction and Purification, Biological Properties, Structure-Activity Relationships and Future Prospects of Schisandrin C: A Major Active Constituent of Schisandra Chinensis.

Since ancient times, China has used natural medicine as the primary way to combat diseases and has a rich arsenal of natural medicines. With the progress of the times, the extraction of bioactive molecules from natural drugs has become the new development direction for natural medicines. Among the numerous natural drugs, Schisandrin C (Sch C), derived from Schisandra Chinensis (Turcz.) Baill. It has excellent potential for development and has been shown to possess various pharmacological properties, including hepatoprotective, antitumor and anti-inflammatory activities. Based on the biological properties of hepatoprotection, scholars have explored Sch C and its synthetic products in depth; some studies have shown that pentosidine has the effect of improving the symptoms of liver fibrosis and reducing the concentration of alanine transaminase (ALT) and aspartate aminotransferase (AST) in the serum of rats, which is an essential inspiration for the development of anti-liver fibrosis drugs. But more in vivo and ex vivo studies still need to be included. This paper focuses on Sch C's extraction and synthesis, biological activities and drug development progress. The future application prospects of Sch C are discussed to perfect its development work further.

Molecular Networking and Bioassay-Guided Preparation and Separation of Active Extract and Constituents from Vicia tenuifolia Roth.

Molecular networking drove the selection of material from V. tenuifolia organs that targeted active flavonoid glycosides. To optimize the extraction process, the flowers of V. tenuifolia were used to produce an anti-inflammatory extract. The effects of variables-organic solvent ratio; extraction time; and temperature-were investigated by the response of anti-inflammatory activity. Bioactivities-guided experiments helped identify fractions with high total phenolic and flavonoid content as well as antioxidant potential. Furthermore, one new compound (1), 19 first isolated together, and two known compounds were obtained and identified from the active fraction of this plant. Among them, compounds (15 and 22) were first reported for nuclear magnetic resonance (NMR) data from this study. All the isolates were evaluated for their anti-inflammatory capacity throughout, modulating nitric oxide (NO), interleukin (IL)-1ß, and IL-8 production. Active compounds were further investigated for their regulation and binding affinity to the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins by Western blot and in silico approaches, respectively. The findings of this study suggested that the developed extract method, active fraction, and pure components should be further investigated as promising candidates for treating inflammation and oxidation.

Optimization of preparation method of hepatoprotective active components from Coreopsis tinctoria Nutt. and its action mechanism in vivo.

Capitula of Coreopsis tinctoria are widely used as a flower tea with great health benefits due to rich content of flavonoids and phenolic acids. The hepatoprotective effect of C. tinctoria and its bioactive basis have seldom been investigated until now. In the present study, capitula of C. tinctoria were extracted with a method optimized by response surface methodology (RSM) and BoxBehnken design (BBD) and further purified by macroporous resin HPD-300 to obtain a fraction (CE) enriched with flavonoids and phenolic acids. The contents of the four most abundant compounds, isookanin-7-O-ß-d-glucoside (1), quercetigetin-7-O-ß-d-glucoside (2), okanin (3), and marein (4), were determined by HPLC as 9.98, 5.21, 41.78 and 1.85 mg/g, respectively. Seventy-four compounds including fifity-five flavonoids, fifteen organic acids (twelve of them were phenolic compounds), and three coumarins were tentatively identified in CE by LC-HRMS/MS. In vivo hepatoprotective effect and potential mechanism of CE were studied with a high-fat diet-induced NASH mouse model. CE administration decreased the amount of weight gain, hepatic lipid, and sequentially improved dyslipidemia, inflammation, oxidative stress, and IR in HFD-fed mice. Molecular data revealed that CE inhibited hepatic inflammation by reducing NFκB/iNOS/COX-2/NLRP3/MAPK in the liver tissues and ameliorated oxidative stress by activating the Nrf2/HO-1 pathway. Modulation of inflammation and oxidative stress with CE may represent a promising target for the treatment of NAFLD and provide insight into the mechanism by which CE protects against obesity.

Mitochondrial DNA D-Loop Amplification and Sequencing for Species Differentiation in Milk.

Adulteration of dairy products, mainly through the substitution of high-quality milk for lower-quality milk, results in the production of low-value products, raising health, social, and economic concerns. As such, the development of methods to ensure dairy products' safety and quality is of great concern for governments and consumers. Although several methods have been developed for species differentiation in dairy products, their application and the establishment of reliable molecular markers for authentication purposes still need to be improved. In this chapter, we describe a low-cost, sensitive, fast, and reliable PCR-based method for mitochondrial D-loop DNA amplification for efficient detection of cattle milk in binary mixtures with sheep milk, thereby allowing the authentication of processed dairy products.

[Determination of short- and medium-chain chlorinated paraffins in different components of human blood using gas chromatography-electron capture negative ion-low resolution mass spectrometry].

Short- and medium-chain chlorinated paraffins (SCCPs and MCCPs) have attracted significant attention because of their persistence, biotoxicity, bioaccumulation, and long-range migration. Given their worldwide detection in a variety of environmental matrices, concerns related to the high exposure risks of SCCPs and MCCPs to humans have grown. Thus, knowledge of the contamination patterns of SCCPs and MCCPs and their distribution characteristics in the vivo exposure of humans is of great importance. However, little information is available on the contamination of SCCPs and MCCPs in human blood/plasma/serum, mainly because of the difficulty of sample preparation and quantitative analysis. In this study, a new blood sample pretreatment method based on Percoll discontinuous density gradient centrifugation was developed to separate plasma, red blood cells, white blood cells, and platelets from human whole blood. A series of Percoll sodium chloride buffer solutions with mass concentrations of 1.095, 1.077, and 1.060 g/mL were placed in a centrifuge tube from top to bottom to establish discontinuous density gradients. The dosage for each density gradient was 1.5 mL. Human whole blood samples mixed with 0.85% sodium chloride aqueous solution were then added to the top layer of the Percoll sodium chloride solution. After centrifugation, the whole blood was separated into four components. The plasma was located at the top layer of the centrifuge tube, whereas the platelets, white blood cells, and red blood cells were retained at the junction of the various Percoll sodium chloride solutions. The sampling volume of human whole blood and incubation time were optimized, and results indicated that an excessively long incubation time could lead to hemolysis, resulting in a decrease in the recoveries of SCCPs and MCCPs. Therefore, a sampling volume of 1.5 mL and incubation time of 10 min at 4 ℃ were adopted. The cells of the blood components were further broken and extracted by ultrasonic pretreatment, followed by multilayer silica gel column chromatography for lipid removal. The use of 80 mL of n-hexane-dichloromethane (1∶1, v/v) and 50 mL of dichloromethane as the elution solvents (collected together) for the gel column separated the SCCPs and MCCPs from the lipid molecules in the blood samples. Gas chromatography-electron capture negative ion-low resolution mass spectrometry (GC-ECNI-LRMS) was used to determine the SCCPs and MCCPs. Quantification using the corrected total response factor with degrees of chlorination was achieved with linear corrections (R2=0.912 and 0.929 for the SCCPs and MCCPs, respectively). The method detection limits (MDLs) for the SCCPs and MCCPs were 1.57 and 8.29 ng/g wet weight (ww, n=7), respectively. The extraction internal standard recoveries were 67.0%-126.6% for the SCCPs and 69.5%-120.5% for the MCCPs. The developed method was applied to determine SCCPs and MCCPs in actual human whole blood samples. The contents of SCCPs and MCCPs were 10.81-65.23 and 31.82-105.65 ng/g (ww), respectively. Red blood cells exhibited the highest contents of CPs, followed by plasma, white blood cells, and platelets. The proportions of SCCPs and MCCPs in red blood cells and plasma were 70% and 66%, respectively. In all four components, the MCCP contents were higher than the SCCP contents, and the ratios of MCCPs to SCCPs ranged from 1.04 to 3.78. Similar congener patterns of SCCPs and MCCPs were found in the four components of human whole blood. C10-CPs and C14-CPs were predominantly observed in the SCCPs and MCCPs, respectively. In summary, a simple and efficient method was proposed to determine low concentrations of SCCPs and MCCPs in human blood with high sensitivity and selectivity. This method can meet requirements for the quantitative analysis of SCCPs and MCCPs in human blood components, thereby providing technical support for human health risk assessment.

Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer.

Gastric cancer is one of the most frequent types of neoplasms worldwide, usually presenting as aggressive and difficult-to-manage tumors. The search for new structures with anticancer potential encompasses a vast research field in which natural products arise as promising alternatives. In this scenario, piperine, an alkaloid of the Piper species, has received attention due to its biological activity, including anticancer attributes. The present work proposes three heating-independent, reliable, low-cost, and selective methods for obtaining piperine from Piper nigrum L. (Black pepper). Electronic (SEM) and optical microscopies, X-ray diffraction, nuclear magnetic resonance spectroscopies (13C and 1H NMR), and optical spectroscopies (UV-Vis, photoluminescence, and FTIR) confirm the obtention of piperine crystals. The MTT assay reveals that the piperine samples exhibit good cytotoxic activity against primary and metastasis models of gastric cancer cell lines from the Brazilian Amazon. The samples showed selective cytotoxicity on the evaluated models, revealing higher effectiveness in cells bearing a higher degree of aggressiveness. Moreover, the investigated piperine crystals demonstrated the ability to act as a good cytotoxicity enhancer when combined with traditional chemotherapeutics (5-FU and GEM), allowing the drugs to achieve the same cytotoxic effect in cells employing lower concentrations. These results establish piperine as a promising molecule for therapy investigations in aggressive gastric cancer, both in its isolated form or as a bioenhancer.