Search Results (76)

Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely applied analytical techniques in the development and quality control testing of finished pharmaceutical products. The combination of gradient chromatographic methods with diode-array detection (DAD) enhances selectivity, ensuring accuracy and reliability when testing drugs with diverse chemical properties in a single dosage form (i.e., fixed-dose combination (FDC) products). In this study, an RP-DAD-HPLC method was developed for the quantitative analysis of clofazimine (CFZ) and pyrazinamide (PZA) for inclusion in an FDC topical drug delivery system. Chromatographic separation was achieved using a C18 column (4.6 mm × 150 mm, 5 µm particle size) with gradient elution at 1 mL/min, employing 0.1% aqueous formic acid and acetonitrile (mobile phases). PZA and CFZ were detected at 254 nm and 284 nm, respectively. The method was validated in accordance with ICH Q2 guidelines, assessing specificity (considering interference from solvents, product matrix, and degradation products), linearity (7.8–500.0 µg/mL, r² = 0.9999), system repeatability (%RSD ≤ 2.7%), and intermediate precision (25–500 µg/mL, %RSD ≤ 0.85%). Method robustness was evaluated using a three-level Box–Behnken design (BBD) with response surface methodology (RSM) to assess the effects of variations in detection wavelength, mobile phase flow rate, and column temperature. Full article

Background: The in vivo accumulation of Advanced Glycation End products (AGEs) is associated with the development of several chronic aging-related and degenerative diseases, as they alter protein structures and activate oxidative and inflammatory processes through interactions with the receptor for AGEs (RAGE). Plant secondary metabolites play a key role in counteracting the glycation process through various mechanisms of action. Therefore, Aloysia citrodora leaf polyphenolic extract could represent a source of anti-glycative compounds. Methods: The methanolic extract was characterized by RP-HPLC-DAD-MSⁿ, and its anti-glycative properties were investigated using several in vitro assays mimicking the different steps of the glycation reaction. In parallel, molecular docking studies were carried out to evaluate potential interactions between the identified metabolites and RAGE. Furthermore, A. citrodora metabolites’ stability under simulated in vitro digestion was assessed, and the anti-glycative activity of the bioaccessible fraction was investigated. Results:A. citrodora extract, rich in iridoid glycosides, phenylethanoid glycosides, and flavones, strongly inhibited AGE formation (from 10% to 100%) in both the middle and end step of the reaction and had high methylglyoxal and glyoxal trapping capacity. However, the digestion process affected extract stability, particularly under intestinal conditions, yielding an overall bioaccessibility of about 40% and leading to a subsequent reduction in anti-glycative properties. Finally, molecular modeling analysis highlighted the ability of the studied metabolites to bind RAGE. Conclusions:A. citrodora represents a promising source of natural anti-glycative agents with potential applications as food ingredients. However, it is essential to improve the extract bioaccessibility and to preserve its anti-glycative properties by developing a suitable formulation. Full article

High concentrations of anthocyanin pigments and vitamin C are the principal health-promoting constituents in black currant (Ribes spp.) berries. This study aimed to develop a rapid greener protocol—reducing the analytical workload, application of toxic solvents, and processing time—for analysis of anthocyanins in black currants. The influence of several laboratory factors on anthocyanin extraction was assessed (berry size, disintegration method). A rapid and robust RP-HPLC-DAD method was developed and validated for the separation and quantification of anthocyanins across 123 black currant genotypes. The optimized method employing a Kinetex C18 column demonstrated excellent sensitivity and reproducibility, achieving complete separation within 11 min. Extraction using 50% (v/v) ethanol acidified with 0.36 N HCl proved superior to methanol-based solvents. Single extraction produced similar extract concentration to triple extraction; cryogenic grinding enhanced anthocyanin extractability by approximately 25%. Smaller berries within the genotype contained higher anthocyanin levels than larger berries. On average, delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside contributed 12.5%, 43.7%, 6.2%, and 35.0% of total anthocyanin content, respectively, and 97.5% of total anthocyanins, ranging from 90.5% to 98.8%. The developed greener chemistry-compliant protocols facilitate rapid, safe, and reliable high-throughput screening of anthocyanin profiles in black currant, useful for breeding programs and analytical workflows. Full article

Geum aleppicum Jacq. (yellow avens), a species traditionally used in folk medicine, remains understudied in the ethnopharmacological aspects. In this study, we comprehensively evaluated the phytochemical composition and biological activity of a hydroethanolic (50:50, v/v) extract from the aerial parts of G. aleppicum collected in Kazakhstan. Using the high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS), we identified 24 compounds, predominantly phenolic acids, flavonoids, tannins, and triterpenoids. The major compound was ellagic acid (2.28 mg/g dry extract) as revealed by the reverse phase high-performance liquid chromatography–diode array detector (RP-HPLC-DAD). The extract exhibited a high polyphenol content (131.45 mg GAE/g) and strong antioxidant activity in Ferric Reducing Antioxidant Power (FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay (3.82 ± 0.07 mmol Fe²⁺/g and 106.61 ± 0.89 mg GAE/g, respectively). Antimicrobial assay of the extract revealed notable antifungal activity against Candida spp., especially against C. glabrata and C. tropicalis with minimum inhibitory concentration (MIC) of as low as 0.125 mg/mL, showing fungistatic effect. Although the extract inhibited the cytopathic effect induced by Human Herpesvirus 1 (HHV-1) in VERO cells, it did not significantly reduce viral replication. Moreover, among human cancer cell lines studied, the extract exerted moderate and selective cytotoxicity against A549 lung cancer cells (CC₅₀ = 75.51 µg/mL, SI = 9). These findings highlight G. aleppicum as a rich source of bioactive compounds, especially phenolics, supporting its potential for development of pharmaceutical and cosmetic applications. Full article

Grape pomace (GP), a significant by-product of winemaking, is gaining increasing recognition for its potential as a source of bioactive compounds with antioxidant and cardioprotective properties. This study aimed to characterize the polyphenolic profile, fatty acid composition, and antioxidant activity of 17 GP samples from Transylvanian cultivars. Polyphenolic content was determined using the Folin–Ciocalteu method and high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC–DAD–ESI MS) analysis. Fatty acid composition was analyzed using gas chromatography with flame ionization detection (GC–FID). Antioxidant capacity was assessed using five methods, which included the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2′-azino-bis (3-ethylbenzothialzoline-6-sulfonic acid) (ABTS) radical scavenging, ferric-reducing antioxidant power (FRAP), cupric ion reducing antioxidant capacity (CUPRAC), and reducing power (RP) assays. Additionally, all extracts were analyzed by Fourier transform infrared (FTIR) spectroscopy to identify the presence of functional groups and chemical bonds associated with bioactive compounds. The results showed that Neuburger (NE), Radames (RA), and Regent (RE) cultivars had the highest phenolic concentrations, particularly of catechin, epicatechin, and procyanidin dimers. NE and Feteascǎ Regalǎ (FR) exhibited the greatest radical scavenging and electron transfer activities across multiple antioxidant assays. Rose Blaj (RB) and Astra (AS) displayed the most favorable fatty acid profiles, with high unsaturated-to-saturated fatty acid (UFA/SFA) and hypocholesterolemic-to-hypercholesterolemic fatty acid (H/H) ratios, as well as low atherogenicity (AI) and thrombogenicity (TI) indices, suggesting cardioprotective potential. Additionally, RB and NE cultivars also demonstrated a strong chelation of Cu²⁺ and Fe²⁺ ions, enhancing their antioxidant efficacy by mitigating metal-catalyzed oxidative stress. These findings underscore the potential of GP, particularly from NE, RB, RA, and AS cultivars, the last three of which were homologated in Transylvania at SCDVV Blaj, as valuable sources of health-promoting compounds for use in food, nutraceuticals, and other health-related applications. Full article

The quality of Crocus sativus L. flowers, beyond their stigmas, is influenced by the presence of bioactive metabolites also in their floral bio-residues. Given the effect of climatic and soil variables on these bioactive compounds, the aim of this research was to develop an agroecological zoning of saffron crop areas within the Protected Designation of Origin (PDO) La Mancha region (Castilla-La Mancha, Spain) by integrating the floral metabolite content with climatic and soil variables. To achieve this, a total of 173 samples were collected during the 2022 and 2023 harvests and analyzed via RP-HPLC-DAD to determine crocins, picrocrocin, kaempferols, and anthocyanins. Two new indices, Cropi (crocins + picrocrocin) and Kaeman (kaempferols + anthocyanins), were defined to classify flowers into four quality categories (A–D). High-quality classifications (A and B) were consistently associated with plots grouped in the meteorological stations of Ontur, El Sanchón, and Bolaños, indicating favorable edaphoclimatic conditions and climatic parameters, such as moderate temperatures and reduced humidity, for metabolite biosynthesis. In contrast, plots included in the meteorological stations of Tarazona and Pedernoso were mostly assigned to lower categories (C and D). Spatial analysis using thematic maps revealed that areas with an intermediate carbonate content, less calcareous soils, and higher organic matter levels were linked to higher flower quality. These findings highlight the influence of soil characteristics and climate, with distinct seasonal contrasts, that positively influence metabolite synthesis and flower quality. Full article

The selection of plant-based substrates for mushroom cultivation is a key factor influencing their growth and metabolism. The aim of this study was to demonstrate, in an innovative approach, differences in the content of biologically active compounds, bioelements, and antioxidant properties of Hericium erinaceus (Bull.) Pers. cultivated on various plant-based substrates derived from waste materials, specifically hemp straw and beech sawdust. Another objective was to compare various extraction methods in terms of their impact on the concentration of these compounds. Elemental analysis was performed using the TXRF method, while bioactive constituents were determined using the DAD/UV RP-HPLC technique. The plant-based substrate and extraction method influenced the levels of obtained metabolites. Dual extraction with moderate ethanol concentrations was most effective for isolating key bioactive compounds from H. erinaceus—notably ergothioneine, lovastatin, L-phenylalanine, and ergosterol—while antioxidant activity did not correlate with the concentration of the solvent used. Although dual extracts enhanced certain antioxidants and metabolites, whole fruiting bodies contained higher levels of bioelements. Overall, fruiting bodies grown on beech sawdust had greater amounts of most bioactive compounds compared to those cultivated on hemp straw, emphasizing that both substrate choice and extraction method critically influence the mushroom’s bioactive profile and its potential health benefits. Full article

This study aimed to evaluate the anti-inflammatory properties of Carthamus caeruleus L. root juice (CRJ), which is used in the traditional medicine of Algeria. The product was characterized by colorimetric assays (total polyphenols, flavonoids, and tannins) and by RP-HPLC-DAD analysis. Experiments were conducted in vitro to assess the ability of CRJ to stabilize human erythrocyte membranes under various stress conditions and inhibit albumin denaturation, a process linked to inflammation. An in silico study was also performed to investigate the inhibitory effects on cyclooxygenase-2 (COX-2) and assess the phenolic constituents with the highest activity. Moderate levels of polyphenols, flavonoids, and tannins were assessed; among these, 22 compounds were identified via chromatographic analysis. While present at low concentrations, some of these compounds, including myricetin, luteolin, and quercetin, are known to exhibit bioactivity at micromolar levels. CRJ provided erythrocyte membranes with notable protection against disruption caused by hypotonic NaCl solutions (protection levels of 90.51%, 87.46%, and 76.87% at NaCl concentrations of 0.7%, 0.5%, and 0.3%, respectively), heat stress (81.54%), and oxidative damage from HClO (75.43%). Additionally, a protection of 61.5% was observed against albumin denaturation. Docking analysis indicated favorable COX-2 binding for myricetin, luteolin, and quercetin. In conclusion, the root juice derived from C. caeruleus demonstrated potential anti-inflammatory activity in vitro and in silico. However, further studies, including in vivo investigations, are necessary to confirm efficacy and fully elucidate the mechanisms of action. Full article

The Touriga Nacional grape variety is renowned in Portuguese red wines for its intense color and aromatic complexity, largely attributed to its rich phenolic composition. Several factors influence the phenolic profile of wines, including edapho-climatic conditions, grape variety, and winemaking techniques such as fermentation, maceration, barrel aging, and maturation. In this study, the technique for winemaking was the only controlled variable, allowing for a specific evaluation of its impact on phenolic composition and antioxidant capacity. Ten single-varietal Touriga Nacional wine samples from the 2019 vintage, produced in the Cima Corgo sub-region of the Douro by different wineries, were analyzed. The phenolic composition was determined using colorimetric methods to quantify total phenols, ortho-diphenols, flavonoids, anthocyanins, and tannins. Antioxidant capacity was assessed through the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and Ferric Reducing Antioxidant Power (FRAP) assays. Since all wines shared the same grape variety, region, and harvest year, the fermentation technique was the main differentiating factor, enabling a direct comparison of its influence on phenolic extraction and antioxidant properties. Additionally, Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array Detection coupled with Mass Spectrometry (RP-HPLC-DAD-ESI-MS/MS) was employed to identify and quantify individual phenolic compounds. This study highlights the key role of winemaking techniques in modulating the phenolic composition and antioxidant potential of Touriga Nacional wines. Full article

The mixotrophic dinoflagellate Akashiwo sanguinea is known to have acute toxic effects on multiple marine organisms, while the composition and chemical properties of its toxins remain unclear. In this study, we established a method for separation and purification of A. sanguinea toxins using chromatographic techniques. The acetone extract of A. sanguinea exhibited higher hemolytic activity and shorter incubation time compared to methanol and ethyl acetate extracts. Five fractions were obtained by solid-phase extraction (SPE), of which SPE3 (acetone/water ratio 3:2) and SPE4 (acetone/water ratio 4:1) exhibited the highest hemolytic activities and allelopathic effects. Further purification on SPE3 and SPE4 using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD) resulted in 11 subfractions, among which Fr4-5 displayed the strongest hemolytic activity. Nearly all active subfractions exhibited higher hemolytic activities incubated under light than those in the dark (p < 0.05), suggesting that A. sanguinea can produce both photosensitive and non-photosensitive toxins, with the former being the primary contributors to its hemolytic activity. Molecular characterization by UV-Vis, FTIR, and HRMS/MS analysis revealed that the structural features of Fr4-5 were highly consistent with porphyrin analogs and could be derived from chlorophyll c-related precursors. These findings highlight that the photosensitive toxins in A. sanguinea may serve dual roles in stress adaptation and ecological competition, potentially contributing to the formation of the blooms. Full article

The members of the genus Helichrysum Mill. are notable for producing a diverse range of structurally intricate secondary metabolites, being the focus of current phytochemical research. Their importance is recognized as several species hold significant ethnopharmacological value, being traditionally used to address ailments in human systems, such as respiratory, gastrointestinal, and urinary conditions, among others. This study used liquid chromatography coupled with high-resolution mass spectrometry results to present the phytochemical composition of non-volatile secondary metabolites in 11 Greek Helichrysum taxa (species and subspecies). For the first time, their total polyphenol content is comparatively assessed and an overview of the non-volatile compounds for five Endangered or Critically Endangered Greek Endemic Helichrysum taxa are presented herein. Almost all of the studied Helichrysum taxa differed significantly in the mean values of their polyphenolic content, except for H. doerfleri and H. heldreichii. A particular emphasis was placed on extracting polyphenols from a cultivated genotype of H. amorginum using aqueous pressurized liquid extraction as an alternative to the traditional organic solvent extraction method. Analysis by high-performance liquid chromatography revealed that this method increased the concentration of neochlorogenic acid and astragalin, compared to other extraction conditions. These findings highlight the potential of alternative extraction techniques for obtaining natural products from sustainably managed phytogenetic sources. Full article

The genus Inula has been used in folk medicine for centuries; however, the data concerning Inula britannica L. are scarce. This study aimed at investigating the chemical composition of methanolic and ethanolic extracts from the aerial parts of I. britannica collected in Kazakhstan and evaluating their antimicrobial and antioxidant properties, with special attention being paid to polyphenols. The total content of polyphenols and flavonoids in the extracts was determined colorimetrically, while their qualitative and quantitative analyses were conducted using HPLC/ESI-QTOF-MS and RP-HPLC/DAD. Their antioxidant potential was determined using the FRAP and DPPH methods, whereas their antimicrobial activity was determined by the microdilution method towards a panel of reference microorganisms, including pathogens of the human gastrointestinal tract. Chemical analysis demonstrated that the methanolic extract had a higher content of polyphenols (58.02 vs. 43.44 mg GAE/g) and flavonoids (21.69 vs. 13.91 mg QUE/g) than the ethanolic extract. In both extracts, 15 compounds were identified, with the highest contents being those of cynarine (13.96 and 11.68 mg/g) and chlorogenic acid (9.22 and 5.09 mg/g). The DPPH assay showed a higher antioxidant activity of the methanolic extract (19.78 ± 0.12 mg GAE/g) in comparison to that of the ethanolic extract (15.56 ± 0.24 mg GAE/g). Similarly, the FRAP method showed that the methanolic extract exerted a much higher antioxidant activity (5.07 ± 0.18 mmol Fe²⁺/g) than the ethanolic extract (0.39 ± 0.01 mmol Fe²⁺/g). In contrast, both extracts showed similar antimicrobial properties, with the highest activity being that against Helicobacter pylori ATCC 43504 (MIC = 0.125–0.25 mg/mL). This paper presents novel data on I. britannica L., implying its significance as a source of valuable active compounds and being a prerequisite for further biological studies. Full article

The stem bark of black locust (Robinia pseudoacacia L.) was extracted, and nine antioxidant compounds (R1–R9) were detected by high-performance thin-layer chromatography combined with the radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, multi-detection, and heated electrospray high-resolution mass spectrometry. For structure elucidation, the methanolic crude extract was fractionated by solid-phase extraction, and the compounds were isolated by reversed-phase high-performance liquid chromatography with diode array detection. The structures of isolated compounds were elucidated by nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy as well as gas chromatography-mass spectrometry to determine the double bond position. 3-O-Caffeoyl oleanolic acid (R1), oleyl (R2), octadecyl (R3), gadoleyl (R4), eicosanyl (R5), (Z)-9-docosenyl (R6), docosyl (R7), tetracosyl (R8), and hexacosanyl (R9) caffeates were identified. While R1 has been reported in R. pseudoacacia stem bark, the known R3, R5, R7, R8, and R9 are described for the first time in this species, and the R2, R4, and R6 are new natural compounds. All nine caffeates demonstrated antioxidant activity. The antioxidant effects of the isolated compounds R1–R8 were quantified by a microplate DPPH• assay, with values ranging from 0.29 to 1.20 mol of caffeic acid equivalents per mole of isolate. Full article

The yeast Lachancea thermotolerans has the ability to produce notable amounts of lactic acid and reduce alcoholic strength in fermentation, so it has a considerable potential for mitigating negative impacts of climate changes in winemaking. In this study, a treatment with L. thermotolerans and Saccharomyces cerevisiae in sequential inoculation was compared to a control S. cerevisiae monoculture fermentation of Malvazija istarska (aka Malvasia Istriana) white grape must. Standard physico-chemical parameters of the obtained wines were determined by the OIV methods. Targeted (GC/FID and GC/MS) and untargeted (GC×GC/TOF-MS) gas chromatographic techniques were combined for the analysis of volatile compounds. Phenolic compounds were analyzed by UPLC/QqQ-MS/MS, and proteins by RP-HPLC-DAD, while a sensory analysis of wines was performed by a panel of trained and certified tasters. L. thermotolerans co-fermentation treatment increased the concentration of lactic acid and decreased alcoholic strength. L. thermotolerans increased the concentrations of geraniol, β-ionone, isobutanol, isobutyric acid, ethyl isobutyrate, several major acetates, ethyl lactate, and diethyl succinate, followed by many minor compounds. This wine also contained more hydroxycinnamoyl tartrates, while control S. cerevisiae wine had higher levels of free hydroxycinnamates. The effects on PR proteins were minor. L. thermotolerans co-fermentation slightly enhanced the sensory perception of tropical fruit, herbaceous, tobacco, and buttery odor notes, as well as fullness of body. With the largest number of identified volatile compounds up to date and other results obtained, this study contributes to the better understanding of oenological and especially aromatic potential of L. thermotolerans in white wine production. Full article

The extraction of bioactive compounds from food by-products is one of the most important research areas for the nutraceutical, pharmaceutical, and food industries. This research aimed to evaluate the efficiency of Ultrasound-Assisted Extraction (UAE) and Microwave-Assisted Extraction (MAE), either alone or in combination, of phenolic compounds from cocoa bean shells (CBSs). These extraction techniques were compared with conventional methods, such as under simple magnetic stirring and the Soxhlet apparatus. After the preliminary characterization of the gross composition of CBSs, the total polyphenol content and radical scavenging of extracts obtained from both raw and defatted cocoa bean shells were investigated. Quantification of the main polyphenolic compounds was then performed by RP-HPLC-DAD, identifying flavonoids and phenolic acids, as well as clovamide. The application of MAE and UAE resulted in a similar or superior extraction of polyphenols when compared with traditional methods; the concentration of individual polyphenols was variously influenced by the extraction methods employed. Combining MAE and UAE at 90 °C yielded the highest antiradical activity of the extract. Spectrophotometric analysis confirmed the presence of high-molecular-weight melanoidins, which were present in higher concentrations in the extracts obtained using MAE and UAE, especially starting from raw material. In conclusion, these results emphasize the efficiency of MAE and UAE techniques in obtaining polyphenol-rich extracts from CBS and confirm this cocoa by-product as a valuable biomass for the recovery of antioxidant compounds, with a view to possible industrial scale-up. Full article