Red Clover (Trifolium pratense) and Inflammation (general): evidence and sources

Biochanin A (BCA) is a natural organic compound of the class of phytochemicals known as flavonoids and isoflavone subclass predominantly found in red clover ( Trifolium pratense ). It has anti-inflammatory activity and some pro-resolving actions, such as neutrophil apoptosis. However, the effect of BCA in the resolution of inflammation is still poorly understood. In this study, we investigated the effects of BCA on the neutrophilic inflammatory response and its resolution in a model of antigen-induced arthritis. Male wild-type BALB/c mice were treated with BCA at the peak of the inflammatory process (12 h). BCA decreased the accumulation of migrated neutrophils, and this effect was associated with reduction of myeloperoxidase activity, IL-1β and CXCL1 levels, and the histological score in periarticular tissues. Joint dysfunction, as seen by mechanical hypernociception, was improved by treatment with BCA. The resolution interval (Ri) was also quantified, defining profiles of acute inflammatory parameters that include the amplitude and duration of the inflammatory response monitored by the neutrophil infiltration. BCA treatment shortened Ri from ∼23 h observed in vehicle-treated mice to ∼5.5 h, associated with an increase in apoptotic events and efferocytosis, both key steps for the resolution of inflammation. These effects of BCA were prevented by H89, an inhibitor of protein kinase A (PKA) and G15, a selective G protein–coupled receptor 30 (GPR30) antagonist. In line with the in vivo data, BCA also increased the efferocytic ability of murine bone marrow–derived macrophages. Collectively, these data indicate for the first time that BCA resolves neutrophilic inflammation acting in key steps of the resolution of inflammation, requiring activation of GPR30 and via stimulation of cAMP-dependent signaling.

PURPOSE: Myocardial ischemia/reperfusion (Ml/R) injury is a leading cause of damage in cardiac tissues, with high rates of mortality and disability. Biochanin A (BCA) is a main constituent of Trifolium pratense L. This study was intended to explore the effect of BCA on Ml/R injury and explore the potential mechanism. METHODS: In vivo MI/R injury was established by transient coronary ligation in Sprague-Dawley rats. Triphenyltetrazolium chloride staining (TTC) was used to measure myocardial infarct size. ELISA assay was employed to evaluate the levels of myocardial enzyme and inflammatory cytokines. Western blot assay was conducted to detect related protein levels in myocardial tissues. RESULTS: BCA significantly ameliorated myocardial infarction area, reduced the release of myocardial enzyme levels including aspartate transaminase (AST), creatine kinase (CK-MB) and lactic dehydrogenase (LDH). It also decreased the production of inflammatory cytokines (IL-1β, IL-18, IL-6 and TNF-α) in serum of Ml/R rats. Further mechanism studies demonstrated that BCA inhibited inflammatory reaction through blocking TLR4/NF-kB/NLRP3 signaling pathway. CONCLUSION: The present study is the first evidence demonstrating that BCA attenuated Ml/R injury through suppressing TLR4/NF-kB/NLRP3 signaling pathway-mediated anti-inflammation pathway.

Nowadays, the tendency in pharmaceutical and food industries is to replace synthetic antioxidants with the natural ones. For this reason, there is a growing interest in analyzing natural, healthy and non-toxic additives as potential antioxidants. Some plants, which contain high levels of phenolic compounds, present an increasing interest for medicine due to their ability to scavenge free radicals, along with other pharmacological activities, such as antibacterial activity, wound healing and anti-inflammatory effect, to mention only a few. The aim of this review is to explore the therapeutic potential ofandin relation with their phytochemical profile and to highlight the pharmacological activity of aqueous or ethanol extracts. Special attention was devoted to the dermal pathology and wound healing effects, in the context of multiple skin conditions such as acne, eczema boils, psoriasis and rashes. Additionally, both extracts (sp. andsp.) are characterized by high content of antioxidant compounds, which are responsible for the radiance and resistance of the skin and slowing down of the aging process by maintaining estrogen levels. Moreover, the potential combined effect of the mixed extract is pointed out in terms of future applications for wound healing, based on some preliminary results obtained from a "scratch tests" assay performed with respect to human dermal fibroblasts.

Red clover (Trifolium pratense L.) is a traditional Chinese medicine and use as herbal medicine which has the effects of regulating menopausal symptoms, heart problem, inflammatory disease, psoriasis and cognitive deficits. In previous reported, the studies of red clover were mainly focused on clinical practice. the pharmacological functions of red clover not fully elucidated. To identify the molecules that regulate ferroptosis, we examined whether red clover (Trifolium pratense L.) extracts (RCE) affected ferroptosis induced by chemical treatment or cystine/glutamate antiporter (xCT) deficiency. Cellular models for ferroptosis were induced by erastin/Ras-selectiv lethal 3 (RSL3) treatment or xCT deficiency in mouse embryonic fibroblasts (MEFs). Intracellular iron and peroxidized lipid levels were determined using Calcein-AM and BODIPY-Cfluorescence dyes, respectively. Protein and mRNA were quantified by Western blot and real-time polymerase chain reaction, respectively. RNA sequencing analysis was performed on xCTMEFs. RCE significantly suppressed ferroptosis induced by both erastin/RSL3 treatment and xCT deficiency. The anti-ferroptotic effects of RCE correlated to ferroptotic phenotypic changes such as cellular iron accumulation and lipid peroxidation in cellular ferroptosis models. Importantly, RCE affected levels of iron metabolism-related proteins including iron regulatory protein 1, ferroportin 1 (FPN1), divalent metal transporter 1, and transferrin receptor. RNA sequencing analysis of xCTMEFs identified that expression of cellular defense genes was upregulated, while expression of cell death-related genes was downregulated, by RCE. RCE potently suppressed ferroptosis triggered both by erastin/RSL3 treatment and xCT deficiency by modulating cellular iron homeostasis. This is the first report that RCE has therapeutic potential in diseases associated with ferroptotic cell death, particularly ferroptosis induced by dysregulation of cellular iron metabolism.

Red clover is a raw material of interest primarily due to its isoflavone content. However, other groups of compounds may affect the pleiotropic biological effects of this raw material. It is used to alleviate menopausal symptoms, but the fact that there are many varieties of this plant that can be grown makes it necessary to compare the biological activity and phytochemical composition of this plant. Also of interest are the differences between the leaves and flowers of the plant. The aim of this study was to evaluate the properties of the leaves and flowers of six clover varieties-'Tenia', 'Atlantis', 'Milena', 'Magellan', 'Lemmon' and 'Lucrum'-with respect to their ability to inhibit α-glucosidase, lipase, collagenase and antioxidant activity. Therefore, the contents of polyphenols and the four main isoflavones-genistein, daidzein, biochanin and formononetin-were assessed. The study was complemented by testing for permeability through a model membrane system (PAMPA). Principal component analysis (PCA) identified a relationship between activity and the content of active compounds. It was concluded that antioxidant activity, inhibition of glucosidase, collagenase and lipase are not correlated with isoflavone content. A higher content of total polyphenols (TPC) was determined in the flowers of red clover while a higher content of isoflavones was determined in the leaves of almost every variety. The exception is the 'Lemmon' variety, characterized by high isoflavone content and high activity in the tests conducted.