Trifolium pratense leaves (red clover) has been used in Oriental and European folk medicine for the treatment of whooping cough, asthma, and eczema, and is now being used to treat and alleviate the symptoms, such as hot flushes, cardiovascular health effects that occur in postmenopausal women. However, relatively little scientific data is available on the physiological activity of this plant. Therefore, in this study, we investigated the anti-cancer activity of T. pratense leaves using methanol extract of T. pratense leaves (MeTP) on human FaDu hypopharyngeal squamous carcinoma cells. MeTP inhibited the viability of FaDu cells by inducing apoptosis through the cleavage of procaspase-3,
Apoptosis ; Asthma ; Blotting, Western ; Carcinoma, Squamous Cell ; Caspase 3 ; Down-Regulation ; Eczema ; Female ; Flow Cytometry ; Humans ; Hypopharynx ; Medicine, Traditional ; Methanol ; Mouth Neoplasms ; Plants ; Trifolium ; Up-Regulation ; Whooping Cough

Magnetic molecularly imprinted polymers(MMIPs) were prepared with ZL006 as template, acrylamide(AA) as the functional monomer, and acetonitrile as pore-forming agent; then Fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy(SEM) were used to characterize their forms and structures. Simultaneously, the MMIPs prepared previously were used as sorbents for dispersive magnetic solid phase extraction(DSPE) to capture and identify potential nNOS-PSD-95 uncouplers from extracts of Trifolium pratense and the the activities of the screened compounds were evaluated by the neuroprotective effect and co-immunoprecipitation test. The experiment revealed that the successfully synthesized MMIPs showed good dispersiveness, suitable particle size and good adsorption properties. Formononetin, prunetin and biochanin A were separated and enriched from Trifolium pratense by using the MMIPs as artificial antibodies and finally biochanin A was found to have higher cytoprotective action and uncoupling action according to the neuroprotective effect and co-immunoprecipitation test.
Adsorption ; Genistein ; chemistry ; Molecular Imprinting ; Phytochemicals ; chemistry ; Polymers ; chemistry ; Solid Phase Extraction ; Spectroscopy, Fourier Transform Infrared ; Trifolium ; chemistry

OBJECTIVES: Some conventional medications used to treat insomnia, such as benzodiazepine, non-benzodiazepine, and hormone therapy, are associated with side effects. Therefore, there is a growing interest in the use of herbal medicine. The aim of this systematic review is to assess the effects herbal medicines have on sleep disorders of peri- and postmenopausal women. METHODS: Three databases were searched including MEDLINE, Scopus, and the Cochrane Library from inception to March 2018. RESULTS: Six trials assessed the effect of soy on sleep disorders. Forrest plot for 2 studies showed that the standardized mean difference (SMD) of the sleep problems was lower in the soy group than the placebo group (SMD = −0.996; P < 0.001; 95% confidence interval, −1.42 to −0.563). Three trials showed soy had no significant effect on sleep disorders and one study showed a positive effect on sleep disorders. Accordingly, it is difficult to reach a definite conclusion regarding the effects of soy on sleep disorders based on these findings. According to a trial, significant beneficial effect associated with taking Schisandra chinensis. Findings suggested that some herbal medicines like red clover, lavender combined with bitter orange and pin bark extract had attenuating effects on sleep disorders. After intake of 530 mg of valerian twice a day for one month, statistically significant effects were observed regarding the quality of sleep of postmenopausal women. CONCLUSIONS: The present systematic review and meta-analysis found inadequate evidence to draw conclusions regarding the effectiveness of herbal medications, especially soy.
Benzodiazepines ; Citrus sinensis ; Female ; Herbal Medicine ; Humans ; Lavandula ; Postmenopause ; Schisandra ; Sleep Initiation and Maintenance Disorders ; Sleep Wake Disorders ; Trifolium ; Valerian

OBJECTIVES: In this systematic review, the effectiveness of herbal medicines in improving depression and anxiety in menopausal women was assessed. METHODS: Three following databases were individually searched: MEDLINE (1966-March 2017), SCOPUS (1990-March 2017), and the Cochrane Library (Cochrane Central Register of Controlled Trials; 2017). RESULTS: A total of 9 trials were included in this systematic review. Overall, soy was found to have a beneficial effect. Also, fennel had a significant positive effect on menopausal women with depression and anxiety disorder, but not on healthy women. Red clover showed varying effects ranging from significant to non-significant on depression and anxiety. Moreover, kava was found to have a significant beneficial effect on depression and anxiety at dose of 200 mg/days. CONCLUSIONS: Our study demonstrated that herbal medicines could improve anxiety and depression in among menopausal women. However, the beneficial effect still remains indefinite due to the poor methodology.
Anxiety Disorders ; Anxiety* ; Depression* ; Female ; Foeniculum ; Humans ; Kava ; Menopause ; Phytoestrogens* ; Trifolium

AIM: The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis. METHODS: Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels. RESULTS: Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected. CONCLUSION Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα.
Animals ; Cholesterol ; metabolism ; Choline Deficiency ; complications ; Diet ; adverse effects ; Disease Models, Animal ; Inflammation ; drug therapy ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Methionine ; deficiency ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease ; drug therapy ; etiology ; metabolism ; PPAR gamma ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; metabolism ; Trifolium ; Triglycerides ; metabolism

Effects of six kinds of Chinese herb extracts, including Folium Crataegi extract, Herba Epimedii extract, Folium Acanthopanacis Senticosi extract, Trifolium pratense L. extract, Folium Ginkgo extract and Radix Puerariae extract, on the activities of CYP450 isozymes (CYP1A2, CYP2C, CYP2E1, CYP2D, CYP3A) in rat hepatic microsomals were studied by using a UPLC-MS/MS (MRM) and cocktail probe substrates method. The results showed that effects of six kinds of Chinese herb extracts on each CYP450 isozyme activity were inhibitory. The IC50 of Folium Crataegi extract for the inhibition of rat microsomal CYP2D activity was only for 4.04 microg x mL(-1), which showed the highest inhibition; Trifolium pratense L. extract had strong inhibitory action to CYP2D, the IC50 value was 5.73 microg x mL(-1); Folium Crataegi extract also had strong inhibitory action on CYP2E1, the IC50 value was 10.91 microg x mL(-1). Furthermore, the IC50 of Folium Ginkgo extract for the inhibition of rat microsomal CYP3A, 2D, 2E1 activities were 45.12, 35.45 and 22.41 microg x mL(-1), respectively, and the IC50 of Folium Acanthopanacis Senticosi extract on the inhibition of rat microsomal CYP2E1 activity was 32.89 microg x mL(-1). In addition, mechanism of inhibition experimental results showed that the inhibiting abilities of Folium Crataegi extract and Radix Puerariae extract on each CYP450 isozyme increased with the increasing of the preincubation time, therefore, the inhibitory effects were a mechanism-based inhibition.
Animals ; Chromatography, High Pressure Liquid ; Crataegus ; chemistry ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Eleutherococcus ; chemistry ; Epimedium ; chemistry ; Ginkgo biloba ; chemistry ; Inhibitory Concentration 50 ; Male ; Microsomes, Liver ; enzymology ; Plants, Medicinal ; chemistry ; Pueraria ; chemistry ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Trifolium ; chemistry

This study was designed to investigate the potential estrogenic effects of perinatal dietary phytoestrogens on the rat uterus. Pregnant rats were divided to three groups provided the following diets: (1) rat chow, (2) rat chow with 7.5% Trifolium (T.) pratense, or (3) rat chow supplemented with 17beta-estradiol (0.5 mg/kg). The dams in each group were kept on the same diet during pregnancy and lactation. Female offspring were euthanized on day 21 at which time body and organ weights were recorded and tissue samples were taken for histology. Immunohistochemistry was performed to detect estrogen receptor alpha (ERalpha) and progesterone receptor (PR) levels. Our results revealed estrogen-like biological effects of perinatal T. pratense exposure. Relative uterus and ovary weights in the experimental groups were increased compared to control. The number of uterine glands and luminal epithelium heights were also increased. However, there were no statistically significant changes detected in the immunostaining intensity of ERalpha and PR between the groups.
Animals ; Animals, Suckling ; Body Weight/drug effects ; Estrogen Receptor alpha/*metabolism ; Female ; Immunohistochemistry ; Isoflavones/*pharmacology ; Lactation ; Maternal Exposure ; Organ Size/drug effects ; Phytoestrogens/*pharmacology ; Plant Components, Aerial/chemistry ; Pregnancy ; Rats ; Rats, Wistar ; Receptors, Progesterone/*metabolism ; Trifolium/*chemistry ; Uterus/*drug effects

OBJECTIVETo evaluate the effects of red clover isoflavones on the proliferation and apoptosis of human benign prostatic hyperplasia (BPH) stromal cells.

METHODSWe treated human prostate stromal cells with red clover isoflavones at the concentration of 12.5, 25, 50 and 100 microg/ml, and established a PBS blank control, a dimethyl sulphoxide (DMSO) negative control and four finasteride positive control groups (at the concentration of 12.5, 25.0, 50.0 and 100.0 microg/ml). We determined the effects of different concentrations of red clover isoflavones on the proliferation of the cells by MTT assay and on their apoptosis by Annexin V/PI double staining flow cytometry.

RESULTSRed clover isoflavones inhibited the proliferation of the BPH stromal cells by 18.86% at 25.0 microg/ml, compared with 5.17% in the blank control group (P < 0.05), and more obviously at a higher concentration. At 50.0 microg/ml, red clover isoflavones exhibited a weaker inhibitory effect than finasteride (28% vs 69.88% , P < 0.05). Annexin V/PI double staining flow cytometry showed that red clover isoflavones at 25.0 microg/ml induced the apoptosis of the prostate stromal cells by (18.54 +/- 2.5)%, with significant differences from the negative control and blank control (P < 0.01).

CONCLUSIONRed clover isoflavones can inhibit the proliferation and promote the apoptosis of human BPH stromal cells.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Isoflavones ; pharmacology ; therapeutic use ; Male ; Plant Extracts ; pharmacology ; therapeutic use ; Prostate ; cytology ; drug effects ; Prostatic Hyperplasia ; drug therapy ; pathology ; Stromal Cells ; drug effects ; Trifolium ; chemistry

The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; analysis ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; Lymphocyte Activation ; drug effects ; Macrophages ; cytology ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Plants, Medicinal ; chemistry ; Receptors, Transferrin ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Trifolium ; chemistry

OBJECTIVETo establish a high performance liquid chromatography method with spectrofluorescence detector for the quantitative analysis of benzopyrene in red clover extract.

METHODAnalysis was performed on Hypersil C18 column (4. 6 mm x 200 mm, 5 microm) with acetonitrile and water (70: 30) as mobile phase; The excitation wavelength and emission wavelengh were 368 nm and 405 nm, respectively; Flow rate was 1.0 mL x min(-1).

RESULTThe linear range was 0.93-37.2 ng x mL(-1), and r = 0.9992 (n = 5). The average recovery of benzopyrene was 85.2% (n = 9).

CONCLUSIONThe method was convenient and accurate with good recovery.

Benzo(a)pyrene ; analysis ; isolation & purification ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Fluorescence ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Trifolium ; chemistry