Search Results (39)

Background/Objectives: Interindividual variability in polyphenol metabolism may help explain the inconsistent effects of polyphenol intake on health outcomes. This study compared, for the first time, (i) polyphenol-related gut microbiota metabotypes (urolithins: UM0, UMA, UMB; equol: EP, ENP; lunularin: LP, LNP) and their clusters (MCs) in non-medicated premenopausal (Pre-M) and postmenopausal (Post-M) women and (ii) the impact of an 8-week intake of a polyphenol-rich plant extract mixture (PPs) on the quality of life (QoL) of Post-M. Methods: Polyphenol metabotypes were determined in urine via UPLC-QTOF-MS after a 3-day intake of PPs containing resveratrol, pomegranate (ellagitannins and ellagic acid), and red clover (isoflavones) in Pre-M (n = 120) and Post-M (n = 90) women. QoL was assessed with the short-form Cervantes Scale in a randomized, placebo-controlled crossover trial (8-week PPs vs. placebo), completed by 78 Post-M participants. Results: At baseline, Pre-M and Post-M women showed only minor differences in metabotype and MC distributions linked to menopausal status. MC3 (UMA+EP+LP) predominated in Pre-M, while MC7 (UMA+EP+LNP) was most frequent in Post-M. PPs intake in Post-M women led to modest shifts in metabotype and MC distributions toward Pre-M patterns. Quantitative metabolite production was comparable between groups, except for equol, which showed a median 2.8-fold increase after PPs intake in EP Post-M women. Clinically meaningful improvements (score reduction ≥ 6.7 points) in QoL were observed in the Psychic domain in EP women (28%, p = 0.039) and in the Menopause and Health domain, specifically in EP (24.1%, p = 0.004), MC3 (22.5%, p = 0.043), and MC4 (UMB+EP+LP; 41.3%, p = 0.022), were mainly driven by a reduction in hot flashes (p = 0.001). Conclusions: These findings support metabotyping as a tool to guide targeted dietary strategies and enhance QoL through precision health in Post-M women. Full article

Iodine has an essential role in the human body; however, its insufficiency is still a challenge. Therefore, the search for new strategies to increase iodine intake in the daily diet is fully justified, with sprouts as a preferred and interesting candidates for biofortification. The aim of this pilot study was to investigate the effect of different methods of iodine enrichment of legume sprouts (yellow lupine, lentil, red and white clover, and common vetch) as well as to identify the most promising species for iodine bioaccumulation. The iodine content in mineralized sprout extracts was determined using the Sandell–Kolthoff method. Watering seeds, previously soaked in water (1 day), with a 6.5 mg/L potassium iodide solution (7 days) revealed to be the most effective fortification model, achieving the highest iodine concentrations in all tested species, with white clover being the best accumulator (1026.7 ± 60.4 µg I/100 g fresh weight) of this component. In turn, the greatest changes in biomass were observed in red clover (even up to 250% of the control). Iodine biofortification of legume sprouts could be effective; nevertheless, further research in this area is required. Full article

Phenylalanine ammonia lyase (PAL) was first purified using affinity chromatography from the leaves of red-flowered clover, a highly antioxidant source. The characterization results of the PAL enzyme were determined, including the concentration of its activity buffer solution, pH, and temperature, which were 0.1 M, 7, and 25 °C, respectively. The V_max and K_M values of the enzyme were calculated to be 0.97 EU and 0.68 mM, respectively. L-phenylalanine was used as the substrate. All kinetic studies were performed spectrophotometrically with a wavelength of 283 nm. Sepharose-4B–L-tyrosine–4-aminocinnamic acid (S-4B-TACA) was also synthesized for the first time and used as an affinity gel. The activity of the PAL extract was measured as 267.9 (millienzyme unit) mU per mL. The yield % and purification fold in the purification step of affinity chromatography were determined to be 3.8% and 19.4, respectively. The experimental results indicate that the PAL enzyme was successfully purified using affinity chromatography. The purity of the enzyme was controlled via SDS-PAGE analysis, which indicated that PAL gave a clear, single band at the line of 45 kDa, while the PAL homogenate gave two bands at around 35 and 45 kDa. Enzyme stabilization was also investigated using PAL stored at 4 °C, which retained completely protected activity for the first 3 weeks. The synthesis of the S-4B-TACA affinity gel, the purification of PAL from red clover leaves using affinity chromatography, and its characterization and statistical analysis have not been previously investigated or reported in the literature. Full article

The biological role of the fluorescence of flowers is a matter of debate. Anthocyanins are a group of compounds that are weakly fluorescent; their fluorescence in flowers has been rarely studied. This study aimed to compare the absorption and fluorescence spectra of anthocyanins extracted from several anthocyanin-containing autumn flowers and examine changes in these spectra during the storage of petals at cold-room and room temperatures and during the storage of dried petals. Petals of red clover Trifolium pratense, pink petunia Petunia × hybrida, Pelargonium horatum, Pelargonium. zonale, Pelargonium. peltatum, red and pink Begonia semperflorens, Buddleja japonica, and purple Chrysanthemum were studied. The results demonstrate that it is possible to distinguish between petals of various flowers based on the absorption spectra of petal extracts and the fluorescence spectra of petal extracts and intact petals. Spectral changes during storage were not always unidirectional and progressive; the most common one was the increase in the intensity of the fluorescence band at 500–560 nm at the excitation wavelength of 460 nm. These results point to the possibility of using fluorescence measurements to identify and estimate the freshness of petal-based material in herbalism, forensic analysis, and the food industry. The measurement of the spectra of whole petals or their fragments by front-face fluorimetry, including common plate readers, may be especially useful due to its simplicity and rapidity. Full article

Isoflavone components extracted from red clover have anti-inflammatory, antioxidant and immune boosting effects. We hypothesize that red clover isoflavones (RCIs) achieve health-promoting effects via altering the gut microbiota. A total of 48 mice (20 ± 2 g) were randomly divided into a control group, low-dose group (0.05% RCIs in feed), middle-dose group (0.1% RCIs in feed), and high-dose group (0.2% RCIs in feed) with 12 mice per group. The feeding period was 20 d. The results showed that RCIs can increase the daily gain and decrease the ratio of feed to gain in mice. The organ indexes and blood biochemical indexes of the mice in each RCI group were in the normal range, indicating that RCIs do not damage liver or kidney function. RCI supplementation increased serum immunity and altered the microbial community structure in the cecum of the mice. RCIs can increase the diversity of beneficial bacteria such as Bacteroidaceae, Muribaculaceae, and Akkermansiaceae, and reduced the pathogenic Staphylococcaceae. Therefore, supplementing the diet with RCIs results in improved growth performance and notable alterations in the cecal microbiota in mice, and has potential applications as a feed additive to improve livestock production. Full article

The increasing popularity of herbal supplements emphasizes the need of scientific data regarding their health benefits and possible toxicological concerns. The complexity of botanical extracts, which include thousands of distinct compounds, contributes to the challenging nature of this endeavor. In this study, we explored the hormetic effects of two Trifolium pratense extracts on breast cell lines. Using a wide range of concentrations (0.1 to 3.33 mg/mL), we analyzed how extracts modulate cellular processes such as viability, proliferation, and oxidative stress on breast adenocarcinoma highly invasive estrogen receptor negative (ER-) and noninvasive ER+ cells, as well as on non-tumorigenic ER- normal cells. The cytotoxicity and real-time cell analysis (RTCA) assays showed that both extracts exercised a biphasic dose effect on adenocarcinoma ER+ and normal ER- cell proliferation and oxidative stress. We report a monotonic dose-dependent cytotoxicity on highly invasive adenocarcinoma ER- cells; the induced apoptosis was based on the pro-oxidant activity of extracts. The reactive oxygen species (ROS) generation by high-dose ethanolic extract was observed in all cells, followed by mitochondria dysfunction. Oxidative stress parameters, such as malondialdehyde (MDA) and reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity were affected. Our study demonstrates that T. pratense extracts have chemoprevention potential in normal and tumorigenic breast cells by modulating cellular proliferation and oxidative stress. Full article

Red clover (Trifolium pratense L.) is found in southeast Europe and Anatolia. Its primary traditional medicinal use includes the treatment of various conditions of the upper respiratory tract. In recent years, its isoflavones have become the focus of research aimed at developing treatments to alleviate menopausal symptoms. Reduced levels of circulating estrogen due to reduced ovarian function can cause short-term symptoms such as hot flashes, palpitations, difficulty sleeping, headaches, fatigue, mood disorders and reduced concentration but also long-term chronic conditions, such as cardiovascular disease, accelerated weight and bone mass loss, atrophic vaginitis, osteoporosis, and cognitive impairment. The aim of this narrative review was to analyze the effects of commercially available and standardized red clover extracts on menopausal women. Eight randomized controlled trials on a total of 8769 menopausal women (aged 40 to 65 years) evaluated the effect of red clover isoflavone extract on menopausal symptoms. In all studies, isoflavone extract treatment showed improvement in all menopausal symptoms, including some common comorbidities, namely, hot flashes (1487 women, 25%), blood lipids (1155 women, 19%), atherosclerosis (6938 women, 79%), risk of breast cancer and endometrial cancer (428 women, 5%), osteoporosis and osteopenia (555 women, 6%), and menopause-related cognitive impairment (3530 women, 40%). Full article

Encapsulating antioxidant-rich plant extracts, such as those found in red clover, within microcapsules helps protect them from degradation, thus improving stability, shelf life, and effectiveness. This study aimed to develop a microencapsulation delivery system using chitosan and alginate for microcapsules that dissolve in both the stomach and intestines, with the use of natural and synthetic emulsifiers. The microcapsules were formed using the extrusion method and employing alginate or chitosan as shell-forming material. In this study, all selected emulsifiers formed Pickering (β-CD) and traditional (white mustard extract, polysorbate 80) stable emulsions. Alginate-based emulsions resulted in microemulsions, while chitosan-based emulsions formed macroemulsions, distinguishable by oil droplet size. Although chitosan formulations with higher red clover extract (C1) concentrations showed potential, they exhibited slightly reduced firmness compared to other formulations (C2). Additionally, both alginate and chitosan formulations containing β-CD released bioactive compounds more effectively. The combined use of alginate and chitosan microcapsules in a single pill offers an innovative way to ensure dual solubility in both stomach and intestinal environments, increasing versatility for biomedical and pharmaceutical applications. Full article

The purpose of this study was to analyze the effect of the type of extraction solution (water, different concentrations of ethanol), temperature and time on the polyphenol content and antioxidant properties of red clover extracts and the effect of the addition of selected extracts on the antioxidant properties of enriched blackcurrant beverages. In both the extractions carried out under different conditions and in the enriched beverages, the content of selected polyphenols was determined by HPLC. This study confirmed the significant effect of the alcohol content of the extract, extraction time and temperature on the antioxidant properties of clover extracts. Ethanolic extracts had better antioxidant properties than aqueous extracts. The addition of ethanol extracts had a significant effect on the antioxidant properties of the fortified beverages. Increasing the temperature, time or ethanol content in the extracts mostly resulted in an increase in the total polyphenol content in the obtained extracts. Based on the analysis of the response surface, it was found that for the DPPH radical, the best activity was obtained by extraction for 20 min with a solution of approximately 65% at low temperatures. In the case of the ABTS radical, the best antiradical activity was obtained after extraction for 60 min at 80 °C with a solution of approximately 50% ethanol. It was also found that the use of a solution of approximately 60% ethanol after extraction for 60 min at 80 °C would provide an extract with high antiradical activity against both radicals. Full article

Adding certain excipients during the extraction process can enhance the concentration of target compounds, leading to potentially increased biological properties of the plant extract. This study explores the impact of PVP/VAC and SSG excipients on red clover bud extracts, aiming to enhance their concentration of target compounds and, consequently, their biological properties. The antioxidative potential was evaluated using DPPH, ABTS, and FRAP methods, and the chemical profile was determined using mass spectrometry. Antibacterial activity against various strains was determined through the minimal inhibitory concentration (MIC) method. The results revealed that the excipient-enriched samples exhibited significantly elevated antioxidant activities as well as phenolic and flavonoid contents compared to control samples. Notably, sample V1E3 demonstrated the highest antioxidant potential, with 52.48 ± 0.24 mg GAE/g dw (phenolic content), 463 ± 6.46 μg TE/g dw (ABTS), 12.81 ± 0.05 μg TE/g dw (DPPH), and 29.04 ± 1.16 mg TE/g dw (post-column ABTS). The highest flavonoid content was found in the S1E3 sample—24.25 ± 0.17 mg RU/g dw. Despite the increased antioxidant potential, no significant variance in antimicrobial activity was noted between the test samples and controls. This implies that excipients may hold the potential to enhance the biological properties of red clover extracts for pharmaceutical applications. These findings contribute valuable insights into optimizing extraction processes for improved functionality and application of plant-derived compounds in therapeutic formulations. Full article

Red clover (Trifolium pratense L.) is an important legume that is also known as a rich source of isoflavones, which are compounds with mild estrogenic activity. Therefore, this plant is often used as a raw material in the production of dietary supplements recommended in menopause. Many factors can influence isoflavone content, but those genetically related are considered to be the most important. Therefore, the aim of this study was to evaluate the phytochemical profile of different plant parts of 30 red clover genotypes grouped according to ploidy and country of seed origin by analyzing the content of dominant isoflavones, total phenolic content (TPC) and antioxidant activity. It was found that there are significant differences in the examined traits among plant parts. Red clover leaves had the highest total isoflavone content, with biochanin A as the dominant compound, while flower extracts had the highest TPC and antioxidant activity. Diploid and tetraploid genotypes were significantly different concerning the content of daidzein, genistein, formononetin and TPC with higher quantities in tetraploid samples. On the other hand, seed origin was not a useful separating factor for the analyzed samples. The results of this research indicate that ploidy, as a previously poorly studied factor, could influence isoflavone content in red clover. Full article

Postmenopausal women are at risk of developing an overactive bladder (OAB). Conventional vaginal estrogen has shown promise for symptom relief. Isoflavones have proven effective as an alternative to estrogen treatment against menopause-related symptoms. However, its effect on OAB symptoms has not been studied. This study investigates if fermented red clover isoflavones reduce OAB symptoms in postmenopausal women. In this randomized, double-blinded, placebo-controlled trial, women were administered red clover extract (RCE) or a placebo twice daily for three months. Women filled out the International Consultation on Incontinence Questionnaire Overactive Bladder (ICIQ-OAB) and Urinary Incontinence Short Form (ICIQ-UI-SF), together with a fluid intake and voiding diary. A total of 33 women (16 in the RCE group and 17 in the placebo group) were included in the analysis. Baseline demographics and OAB characteristics were comparable across groups. Intake of RCE did not lead to significant relief in most urinary bladder symptom measures, although a significant reduction in the bother of urinary urgency (p = 0.033) and a tendency towards a decreased ICIQ-OAB score were observed (p = 0.056). In contrast, the placebo exhibited a significant decrease in the ICIQ-OAB score (p = 0.021) and in some diary outcomes. We found that an intake of isoflavones did not relieve OAB symptoms in postmenopausal women. Full article

Biochanin A (BCA), an isoflavone derived from various plants such as chickpea, red clover and soybean, is attracting increasing attention and is considered to have applications in the development of pharmaceuticals and nutraceuticals due to its anti-inflammatory, anti-oxidant, anti-cancer and neuroprotective properties. To design optimised and targeted BCA formulations, on one hand there is a need for more in-depth studies on the biological functions of BCA. On the other hand, further studies on the chemical conformation, metabolic composition and bioavailability of BCA need to be conducted. This review highlights the various biological functions, extraction methods, metabolism, bioavailability, and application prospects of BCA. It is hoped that this review will provide a basis for understanding the mechanism, safety and toxicity of BCA and implementing the development of BCA formulations. Full article

The current study presents the most suitable method for encapsulating nutmeg essential oil with liquorice and red clover. Two widely used methods, spray-drying and freeze-drying, were employed to find the most suitable for essential oil volatile compounds’ protection. Results showed that freeze-dried capsules (LM) had a higher yield (85.34%) compared to the exact formulation of spray-dried microcapsules (SDM)—45.12%. All the antioxidant and total phenolic compounds’ results obtained with the LM sample were significantly higher compared with SDM. LM microcapsules were incorporated in two different bases with no additional sugar (gelatin and pectin) for targeted release. Pectin tablets had firmer and harder texture properties, while gelatin tablets had a more elastic texture. There was a significant impact on texture changes caused by microcapsules. Microencapsulated essential oil with extracts can be used alone or in a gel base (pectin or gelatin, depending on user preferences). It could be an effective product to protect the active volatile compounds and regulate the release of active compounds and give a pleasant taste. Full article

Increasing prospective phytochemical investigations and biological activities on Pueraria lobata and Trifolium pratense flowers exhibited their nutritional (food industry) pharmaceutical (anticancer, anti-inflammatory, anti-microbial, etc.) potentials. Previous studies paid great attention to the screening of isoflavones profile from phytochemicals. This study, in contrast, aimed at identifying the flavonoids from Pueraria lobata flowers or kudzu flower (KF) and Trifolium pratense flowers or red clover (RC) flowers and determining their cytotoxic activities on normal; (HEK-293) and cancer cell lines; human glioblastoma (A-172), osteosarcoma (HOS), embryonic rhabdomyosarcoma (Rd), lung carcinoma (A-549) and liver carcinoma (HepG2). The phytochemical screening using a high-performance liquid chromatography-diode array detector coupled with quadrupole time-of-flight mass spectrometry (HPLC-DAD-Q-TOF/MS) showed that 15 flavonoids, including isoflavones, flavones, flavonols, and flavanones, were identified in KF and 8 flavonoids, including isoflavones and flavonols, were found in RC. Nevertheless, the majority of flavonoid chemical constituents in KF or RC were found to be isoflavones (66.6%) and (62.5%), respectively. HPLC-DAD analysis following eco-friendly extraction of phytochemicals showed that KF contains mainly daidzein and genistein, while RC contains primarily formononetin and biochanin A. Cytotoxic activities evaluated according to IC₅₀ values exhibited the most pronounced dose-dependent antiproliferative effect of KF and RC extracts were against HOS and Rd cancer cell lines, respectively. Accordingly, the morphological observation carried out using acridine orange/ethidium bromide and Giemsa stains revealed apoptotic activities and cell death in HOS and Rd cell lines when subjected to KF or RC extracts. Cytotoxic activities and apoptotic changes were pronounced among all cancer cell lines except for the control cells (HEK-293). Additionally, various polyphenols and flavonoids were identified and quantified in KF and RC extracts and exhibited potent radical scavenging activities. Overall, this study suggests that KF and RC could be valuable edible sources of flavonoids. The comprehensive flavonoid profiles for KF and RC may explain their remarkable biological activities and contribution to inducing antioxidant and cytotoxic activities against cancer cell lines. Full article