Flavonoid glycosides from the leaves of Michelia champaca.

Michelia champaca L. (Magnoliaceae) was cultivated in large scale for flowers as cosmetic raw materials, whereas the value of its leaves remains to be discovered. Our chemical study on the leaves yielded four new flavonol diglycosides, champaflavosides A-D (1-4), together with twenty-three known flavonoid glycosides (5-27). Their structures were determined by spectroscopic and chemical methods. Compounds 5-21 and 23-27 were not previously reported from the genus Michelia, and kaempferol 3-O-rutinoside (22) was obtained from this species for the first time. All the compounds were evaluated for antioxidant activity by four in vitro assays. Compounds 3-12 and 20 showed more potent 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than l-ascorbic acid (l-AA). Compounds 2-23, 25, and 27 exhibited 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation scavenging activity superior to l-AA. The ferric reducing antioxidant powers (FRAP) of compounds 2-13, 17, and 19 were higher than l-AA. Further, eighteen compounds demonstrated cellular reactive oxygen species (ROS) scavenging activity, of which champaflavoside D (4), rhamnetin 3-O-neohesperidoside (8), quercetin 3-O-(6-O-E-p-coumaroyl)-neohesperidoside (9), and liquiritin (27) were more potent than curcumin. The results revealed that the renewable leaves of M. champaca are a rich source of flavonoids and antioxidants.

Fermentation of Robinia pseudoacacia flower for improving the antioxidation: optimized conditions, active composition, mechanism, and biotransformation process.

Robinia pseudoacacia flower is a natural product with many biological activities, including antioxidation. To further develop its antioxidation, the extract was fermented by Aspergillus niger FFCC 3112 in the medium with carbon to nitrogen ratio of 1.4:1 and initial pH of 4.2 for 3.5 days to form the best antioxidant activity of the fermentation product by strain screening, single factor optimization, and response surface methodology. Further analysis, isolation and activity determination showed that a main chemical component, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-ß-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, in the extract was completely hydrolyzed to kaempferol-7-O-α-L-rhamnopyranoside and kaempferol with better antioxidant activity through biotransformation, which was the basis for improving the antioxidant activity of fermentation products. Moreover, the mechanism of antioxidant and the contribution of phenolic hydroxyl groups were investigated by density functional theory. The result indicated that the antioxidant capacity of kaempferol-7-O-α-L-rhamnopyranoside and kaempferol increased with the increase of solvent polarity. In high-polarity solvents, they mainly scavenge free radicals through single electron transfer followed by proton transfer.

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves.

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.

Molecular networking-guided strategy for the pharmacokinetic study of herbal medicines: Cudrania tricuspidata leaf extracts.

In this study, the pharmacokinetic profiles of the bioactive components in the leaf extract of the medicinal herb, Cudrania tricuspidate, were investigated using an MS/MS-based molecular networking system. To identify the major active components of the C. tricuspidate leaf extract (CLE), HPLC-DAD analysis was conducted with a standard mixture of six flavonoids (rutin, isoquercitrin, nicotiflorin, kaempferol 3-O-glucoside, quercetin, and kaempferol). The unknown peaks were determined via molecular networking analysis using the mass dataset obtained by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). For the subsequent pharmacokinetic study, CLE (1 g/kg) was orally administered to rats, and plasma samples were collected. The product ion mass data of plasma samples using LC-QTOF/MS were obtained and subjected to molecular networking analysis. The resulting molecular networking map indicated that the glucuronide metabolites of quercetin and kaempferol were the major circulating species. Accordingly, quercetin and kaempferol were determined following ß-glucuronidase treatment, and their pharmacokinetic parameters were calculated. These findings indicate that the proposed molecular network-based approaches are potential and efficient methods for the pharmacokinetic study of herbal medicines.

Flavonoid Composition and Antibacterial Properties of Crocus sativus L. Petal Extracts.

Saffron petals, which are the main by-products of Crocus sativus L. (Iridaceae family), are produced in large quantities and are known for their many beneficial properties. In this regard, this study aims to investigate the phenolic composition and antibacterial properties of hydroethanolic extracts from Crocus sativus L. petals collected from Serghina (province of Boulmane) in Morocco. The phenolic profiles were characterized using high-performance liquid chromatography coupled to a photodiode array and electrospray ionization mass spectrometry (HPLC-PDA-ESI/MS). The antibacterial potential was evaluated against four bacterial strains potentially causing food-borne disease (Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, and Listeria monocytogenes) using disc diffusion and broth micro-dilution assays. Results showed that a total of 27 phenolic compounds was detected in the Crocus sativus L. petal extracts, which were assigned to flavonoids (kaempferol, quercetin, isorhamnetin, and myricetin derivatives). The most abundant compound was represented by kaempferol-sophoroside isomer (20.82 mg/g ± 0.152), followed by kaempferol-sophoroside-hexoside (2.63 mg/g ± 0.001). The hydroethanolic extracts of Crocus sativus L. petals demonstrated bactericidal effects against Staphylococcus aureus and Listeria monocetogenes and bacteriostatic effects against Escherichia coli and Salmonella typhimurium. Therefore, the by-product Crocus sativus L. petal extracts might be considered as valuable sources of natural antibacterial agents with potential applications in the food and pharmaceutical industries.

Screening the Key Region of Sunlight Regulating the Flavonoid Profiles of Young Shoots in Tea Plants (Camellia sinensis L.) Based on a Field Experiment.

Both UV and blue light have been reported to regulate the biosynthesis of flavonoids in tea plants; however, the respective contributions of the corresponding regions of sunlight are unclear. Additionally, different tea cultivars may respond differently to altered light conditions. We investigated the responses of different cultivars ('Longjing 43', 'Zhongming 192', 'Wanghai 1', 'Jingning 1' and 'Zhonghuang 2') to the shade treatments (black and colored nets) regarding the biosynthesis of flavonoids. For all cultivars, flavonol glycosides showed higher sensitivity to light conditions compared with catechins. The levels of total flavonol glycosides in the young shoots of different tea cultivars decreased with the shade percentages of polyethylene nets increasing from 70% to 95%. Myricetin glycosides and quercetin glycosides were more sensitive to light conditions than kaempferol glycosides. The principal component analysis (PCA) result indicated that shade treatment greatly impacted the profiles of flavonoids in different tea samples based on the cultivar characteristics. UV is the crucial region of sunlight enhancing flavonol glycoside biosynthesis in tea shoots, which is also slight impacted by light quality according to the results of the weighted correlation network analysis (WGCNA). This study clarified the contributions of different wavelength regions of sunlight in a field experiment, providing a potential direction for slightly bitter and astringent tea cultivar breeding and instructive guidance for practical field production of premium teas based on light regimes.

Phytochemical composition and In-vitro activity of ethanolic extract of Eucalyptus globulus leaves against multidrug resistant poultry pathogens.

Aim of the present study was to determine the In-vitro antibacterial activity of ethanolic extract of E. globulus leaves against common multidrug resistant poultry pathogens. Phytochemical analysis through HPLC revealed that kaempeferol (7.315min) followed by querecetin (6.655min) and myrecetin (3.655min). Percent area of kaempeferol (6826.88%) was highest, followed by myrecetin (5516.22%) and querecetin (163.748%). Phytochemical investigation of ethanolic extract of E. globulus leaves through GCMS showed highest retention time (min) α-pinene (20.43) and α-terpineol (20.15) accompanied by spathulenol (11.97), piperitone (11.04). The ethanolic extracts of E. globulus leaves showed a highest zone of inhibition against S. pullorum SP6; 20.64± 2.08, E. coli SE 12; 19.75± 2.83, C. perfringens type A (CPM38-01); 19.46± 2.02. The highest level of MIC of E. globulus noted were against S. gallinarum S22; 133.37±53.294, S. gallinarum S1; 130.20±45.10, S. gallinarum S4; 129.47±24.182, S. gallinarum S3; 126.83±72.392. In conclusion, the study confirmed that the ethanolic extract of E. globulus is composed of active ingredients having antibacterial activity and can be referred as an alternate to antibiotics.

Kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside from Nectandra oppositifolia releases Ca2+ from intracellular pools of Trypanosoma cruzi affecting the bioenergetics system.

Phytochemical analysis of EtOH extract from leaves of Nectandra oppositifolia afforded three flavonoids: kaempferol (1), kaempferol-3-O-α-rhamnopyranoside (2) and kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside (3), which were characterized by NMR and ESI-HRMS. When tested against the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, flavonoids 1 and 3 were effective to kill the trypomastigotes with IC50 values of 32.0 and 6.7 µM, respectively, while flavonoid 2 was inactive. Isolated flavonoids 1-3 were also tested in mammalian fibroblasts and showed CC50 values of 24.8, 48.7 and 153.1 µM, respectively. Chemically, these results suggested that the free aglycone plays an important role in the bioactivity while the presence of p-coumaroyl unities linked in the rhamnoside unity is important to enhance the antitrypanosomal activity and reduce the mammalian cytotoxicity. The mechanism of cellular death was investigated for the most potent flavonoid 3 in the trypomastigotes using fluorescent and luminescent-based assays. It indicated that this compound induced neither permeabilization of the plasma membrane nor depolarization of the membrane electric potential. However, early time incubation (20 min) with flavonoid 3 resulted in a constant elevation of the Ca2+ levels inside the parasite. This effect was followed by a mitochondrial imbalance, leading to a hyperpolarization and depolarization of the mitochondrial membrane potential, with reduction of the ATP levels. During this time, the levels of reactive oxygen species levels (ROS) were unaltered. The leakage of Ca2+ from the intracellular pools can affect the bioenergetics system of T. cruzi, leading to the parasite death. Therefore, flavonoid 3 can be a useful tool for future studies against T. cruzi parasites.

Comparative characterization of cinnamon, cinnamaldehyde and kaempferol for phytochemical, antioxidant and pharmacological properties using acetaminophen-induced oxidative stress mouse model / Caracterización comparativa de canela, cinamaldehído y kaempferol para propiedades fitoquímicas, antioxidantes y farmacológicas utilizando el modelo de ratón con estrés oxidativo inducido por acetaminofeno

This study was aimed to explore the comparative efficacy of cinnamon bark extract, cinnamaldehyde and kaempferol against acetaminophen (APAP)-induced oxidative stress. Cinnamon bark extract, cinnamaldehyde and kaempferol were utilized or in-vivo analysis. From the results of in-vitro screening tests, cinnamon ethanolic extract was selected for in-vivo study in mouse model. For this, Balb/c albino mice were treated with cinnamon ethanolic extract (200 mg/kg), cinnamaldehyde (10 mg/kg) and kaempferol (10 mg/kg) orally for 14 days followed by single intraperitoneal administration of APAP during 8 hours. Blood and organ samples were collected for biochemical and histopathological analysis. The results showed that cinnamon bark ethanolic extract, cinnamaldehyde and kaempferol ameliorated APAP-induced oxidative stress and organ toxicity in mice. In conclusion, cinnamaldehyde and kaempferol possess comparable antioxidant potential even at 20-times less dose as compared to cinnamon bark ethanolic extract suggesting therapeutic potential in oxidative stress-related disorders.

Este estudio tuvo como objetivo explorar la eficacia comparativa del extracto de corteza de canela, cinamaldehído y kaempferol contra el estrés oxidativo inducido por acetaminofén (APAP). Se utilizaron extracto de corteza de canela, cinamaldehído y kaempferol para el análisis in vivo. De los resultados de las pruebas de detección in vitro, se seleccionó el extracto etanólico de canela para estudio in vivo en modelo de ratón. Para ello, los ratones albinos Balb/c fueron tratados con extracto etanólico de canela (200 mg/kg), cinamaldehído (10 mg/kg) y kaempferol (10 mg/kg) por vía oral durante 14 días, seguido de la administración intraperitoneal única de APAP durante 8 horas. Se recogieron muestras de sangre y órganos para análisis bioquímicos e histopatológicos. Los resultados mostraron que el extracto etanólico de la corteza de canela, el cinamaldehído y el kaempferol mejoraron el estrés oxidativo inducido por APAP y la toxicidad orgánica en ratones. En conclusión, el cinamaldehído y el kaempferol poseen un potencial antioxidante comparable, incluso a una dosis 20 veces menor en comparación con el extracto etanólico de la corteza de canela, lo que sugiere un potencial terapéutico en los trastornos relacionados con el estrés oxidativo.

Cassia fistula ameliorates chronic toxicity of cypermethrin in Catla catla.

Protective effects of Cassia fistula, in Catla catla exposed to synthetic pyrethroid cypermethrin were evaluated. Fish, after chronic exposure to environmentally relevant sub-lethal concentration 0.41 µg/l of the pesticide were assessed for antioxidant activity, histopathological and ultrastructural alterations. Significant (p < 0.05) decrease in the activities of antioxidants such as CAT, SOD, GST, GSH was registered, whereas LPO level got elevated. Histological damage depicted necrosis, epithelial hypertrophy, hyperplasia and fusion of secondary lamellae and changes in gill vasculature. Histopathological alteration index was employed for the semi quantitative evaluation of the degree of tissue change (DTC). Transmission electron microscopy displayed swollen and distorted mitochondria, damaged chloride cells and necrosis. Dietary supplementation of Cassia fistula bark extract significantly (p < 0.05) improved the antioxidant activity, reduced lipid peroxidation and prevented histopathological alterations. The findings suggest that sub-lethal concentration of cypermethrin is toxic to fish. The study also draws attention towards potential of plant derived antioxidants in mitigating pesticide induced toxic effects.

Bioassay-Guided Identification of the Antiproliferative Compounds of Cissus trifoliata and the Transcriptomic Effect of Resveratrol in Prostate Cancer Pc3 Cells.

The bioassay-guided fractionation of a CHCl3-MeOH extract from the stems of Cissus trifoliata identified an active fraction against PC3 prostate cancer cells. The treatment for 24 h showed an 80% reduction in cell viability (p ≤ 0.05) by a WST-1 assay at a concentration of 100 µg/mL. The HPLC-QTOF-MS analysis of the fraction showed the presence of coumaric and isoferulic acids, apigenin, kaempferol, chrysoeriol, naringenin, ursolic and betulinic acids, hexadecadienoic and octadecadienoic fatty acids, and the stilbene resveratrol. The exposure of PC3 cells to resveratrol (IC25 = 23 µg/mL) for 24 h induced significant changes in 847 genes (Z-score ≥ ±2). The functional classification tool of the DAVID v6.8 platform indicates that the underlying molecular mechanisms against the proliferation of PC3 cells were associated (p ≤ 0.05) with the process of differentiation and metabolism. These findings provide experimental evidence suggesting the potential of C. trifoliata as a promising natural source of anticancer compounds.

Elucidation of glycosylation sites of kaempferol di-O-glycosides from methanolic extract of the leaves of Prunus domestica subsp. syriaca.

RATIONALE: Flavonol glycosides containing the glycosylation patterns 3,4'-di-O and 4',7-di-O are rare in nature and they have not yet been studied in detail by electrospray ionization mass spectrometry (ESI-MS(+/-), in contrast to the flavonol glycosides containing the glycosylation pattern 3,7-di-O. METHOD: The leaves from Prunus domestica L. subsp. syriaca were extracted with pure methanol or, in order to perform hydrolysis and extraction simultaneously, with a 5% methanolic solution of hydrochloric acid. The high-performance liquid chromatography (HPLC)/ESI-MS(+/-) analyses were performed using a Waters model 2690 HPLC pump and a Waters/Micromass ZQ2000 mass spectrometer. RESULTS: Three kinds of kaempferol di-O-glycosides have been identified, namely kaempferol-3-O-hexoside-7-O-rhamnosides, kaempferol-3-O-pentoside-4'-O-rhamnosides and kaempferol 4',7-di-O-rhamnoside. The identification was performed on the basis of the abundances of the respective Y-type product ions. CONCLUSIONS: The abundances of [Yn 0 - H]-· , Yn 0 - and Yn 0 + product ions were of crucial importance for the determination of glycosylation patterns. The obtained results can be useful for HPLC/ESI-MS identification of rare flavonol-di-O-glycosides.

Phytochemicals in Chinese Chive (Allium tuberosum) Induce the Skeletal Muscle Cell Proliferation via PI3K/Akt/mTOR and Smad Pathways in C2C12 Cells.

Chinese chive (Allium tuberosum) is a medicinal food that is cultivated and consumed mainly in Asian countries. Its various phytochemicals and physiological effects have been reported, but only a few phytochemicals are available for skeletal muscle cell proliferation. Herein, we isolated a new compound, kaempferol-3-O-(6″-feruloyl)-sophoroside (1), along with one known flavonoid glycoside (2) and six amino acid (3-8) compounds from the water-soluble fraction of the shoot of the Chinese chive. The isolated compounds were identified using extensive spectroscopic methods, including 1D and 2D NMR, and evaluated for their proliferation activity on skeletal muscle cells. Among the tested compounds, newly isolated flavonoid (1) and 5-aminouridine (7) up-regulated PI3K/Akt/mTOR pathways, which implies a positive effect on skeletal muscle growth and differentiation. In particular, compound 1 down-regulated the Smad pathways, which are negative regulators of skeletal muscle growth. Collectively, we suggest that major constituents of Chinese chive, flavonoids and amino acids, might be used in dietary supplements that aid skeletal muscle growth.

Antioxidant and Antiproliferative Activity of The Ethanolic Extract of Equisetum Myriochaetum and Molecular Docking of Its Main Metabolites (Apigenin, Kaempferol, and Quercetin) on ß-Tubulin.

Equisetum myriochaetum is a semi-aquatic plant found on riverbanks that is commonly used in traditional medicine as a diuretic agent. Additionally, the genus Equisetum stands out for its content of the flavonoid kaempferol, a well-known antiproliferative agent. Therefore, in this study, E. myriochaetum ethanolic extract was tested in vitro against a cervical cancer cell line (SiHa). Additionally, the antioxidative activity was evaluated through a 2,2-diphenyl-1-picrilhidrazil (DPPH) assay. Finally, a molecular docking analysis of apigenin, kaempferol, and quercetin on the active site of ß-tubulin was performed to investigate their potential mechanism of action. All fractions of E. myriochaetum ethanolic extract showed antioxidative activity. Fraction 14 displayed an antiproliferative capacity with a half maximal inhibitory concentration (IC50) value of 6.78 µg/mL against SiHa cells.

Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Pharmacological Potential and Chemical Composition of Crocus sativus Leaf Extracts.

Crocus sativus L. (saffron) has been traditionally used as a food coloring or flavoring agent, but recent research has shown its potent pharmacological activity to tackle several health-related conditions. Crocus sp. leaves, and petals are the by-products of saffron production and are not usually used in the medicine or food industries. The present study was designed to determine the chemical composition of the water and ethanolic extracts of C. sativus leaves and test their cytotoxic activity against melanoma (IGR39) and triple-negative breast cancer (MDA-MB-231) cell lines by MTT assay. We also determined their anti-allergic, anti-inflammatory, and anti-viral activities. HPLC fingerprint analysis showed the presence of 16 compounds, including hydroxycinnamic acids, xanthones, flavonoids, and isoflavonoids, which could contribute to the extracts' biological activities. For the first time, compounds such as tectoridin, iristectorigenin B, nigricin, and irigenin were identified in Crocus leaf extracts. The results showed that mangiferin (up to 2 mg/g dry weight) and isoorientin (8.5 mg/g dry weight) were the major active ingredients in the leaf extracts. The ethanolic extract reduced the viability of IGR39 and MDA-MB-231 cancer cells with EC50 = 410 ± 100 and 330 ± 40 µg/mL, respectively. It was more active than the aqueous extract. Kaempferol and quercetin were identified as the most active compounds. Our results showed that Crocus leaves contain secondary metabolites with potent cytotoxic and antioxidant activities.

Polyphenol oxidase dominates the conversions of flavonol glycosides in tea leaves.

Flavonol glycosides are associated with astringency and bitterness of teas. To clarify the dominant enzymatic reaction of flavonol glycosides in tea leaves, the catalytic effects of polyphenol oxidase (PPO), peroxidase (POD) and ß-glucosidase were studied, with the maintaining rates of total flavonol glycosides (TFG) being 73.0%, 99.8% and 94.3%. PPO was selected for further investigations, including the effects of pH value (3.5 ~ 6.5), temperature (25 °C ~ 55 °C) and dosage (39 ~ 72 U/mL PPO and 36 U/mL PPO, 3 ~ 36 U/mL POD). The oxidation of flavonol glycosides were intensified at pH 6.5, with 51.8% and 15.4% of TFG maintained after PPO and PPO + POD treatments, suggesting an enhancement from POD. The sensitivity ranking to PPO was: myricetin glycosides > quercetin glycosides > kaempferol glycosides. The inhibitor treatment testified the leading role of PPO in catalyzing flavonol glycosides in tea leaves. Sugar moiety enhanced the docking affinity of flavonol glycosides for PPO. PPO shows the potential of modifying flavonol glycoside composition.

The binding of kaempferol-3-O-rutinoside to vascular endothelial growth factor potentiates anti-inflammatory efficiencies in lipopolysaccharide-treated mouse macrophage RAW264.7 cells.

BACKGROUND: Vascular Endothelial Growth Factors (VEGFs) are a group of growth factor in regulating development and maintenance of blood capillary. The VEGF family members include VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. VEGF receptor activation leads to multiple complex signaling pathways, particularly in inducing angiogenesis. Besides, VEGF is produced by macrophages and T cells, which is playing roles in inflammation. In macrophages, VEGF receptor-3 (VEGFR-3) and its ligand VEGF-C are known to attenuate the release of pro-inflammatory cytokines. METHODS: Immunoprecipitation and molecular docking assays showed the binding interaction of kaempferol-3-O-rutinoside and VEGF-C. Western blotting and qRT-PCR methods were applied to explore the potentiating effect of kaempferol-3-O-rutinoside in VEGF-C-mediated expressions of proteins and genes in endothelial cells and LPS-induced macrophages. Enzyme-linked immunosorbent assay (ELISA) was employed to reveal the release of proinflammatory cytokines in LPS-induced macrophages. Immunofluorescence assay was performed to determine the effect of kaempferol-3-O-rutinoside in regulating nuclear translocation of NF-κB p65 subunit in the VEGF-C-treated cultures. In addition, Transwell® motility assay was applied to detect the ability of cell migration after drug treatment in LPS-induced macrophages. RESULTS: We identified kaempferol-3-O-rutinoside, a flavonoid commonly found in vegetable and fruit, was able to act on cultured macrophages in inhibiting inflammatory response, and the inhibition was mediated by its specific binding to VEGF-C. The kaempferol-3-O-rutinoside-bound VEGF-C showed high potency to trigger the receptor activation. In LPS-treated cultured macrophages, applied kaempferol-3-O-rutinoside potentiated inhibitory effects of exogenous applied VEGF-C on the secretions of pro-inflammatory cytokines, i.e. IL-6 and TNF-α, as well as expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This inhibition was in parallel to transcription and translocation of NF-κB. Moreover, the binding of kaempferol-3-O-rutinoside with VEGF-C suppressed the LPS-induced migration of macrophage. CONCLUSION: Taken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.

Effect of kaempferol on IgE-mediated anaphylaxis in C57BL/6 mice and LAD2 cells.

BACKGROUND: Immunoglobulin E (IgE)-mediated mast cell (MC) activation is crucial in multiple allergic diseases. Parkinson disease protein 7 (DJ-1) and Lyn kinase were reported as the receptor-proximal events in IgE receptor (FcεRI) signals in human MC. Kaempferol, a natural flavonol mainly derived from the rhizome of traditional Chinese herb Kaempferia galanga L. (Zingiberaceae), has been known to inhibit allergic reactions, but it was limited to the receptor-distal signals on rat basophilic leukemia cells. A thorough investigation of the inhibitory effects of kaempferol on human MC has not been done. PURPOSE: To investigate the inhibitory effects of kaempferol on IgE-mediated anaphylaxis in vivo and in human MCs, as well as the mechanism underlying its effects, especially the receptor-proximal signals. METHODS: IgE-mediated passive cutaneous anaphylaxis and systemic anaphylaxis model were applied to elucidate the antiallergic activity of kaempferol in vivo. The degranulation assay, calcium imaging, the release of cytokines and chemokines on the laboratory of allergic disease 2 (LAD2) cells were used to evaluate the antiallergic effect of kaempferol in vitro. Western blot analysis was performed to investigate the DJ-1/Lyn signaling pathway and downstream molecules. Kinase activity assay, immunofluorescence, and molecular docking were conducted to confirm the influence of kaempferol on DJ-1/Lyn molecules. RESULTS: Kaempferol dose-dependently attenuated ovalbumin/IgE-induced mice paw swelling, primary MC activation from paw skin, as well as rehabilitated the hypothermia, and reduced the serum concentrations of histamine, tumor necrosis factor-alpha, interleukin-8, and monocyte chemo-attractant protein-1. Additionally, kaempferol suppressed IgE-mediated LAD2 cell degranulation and calcium fluctuation. Remarkably, kaempferol was found to bind with DJ-1 protein, and initially prevented DJ-1 from translocating to the plasma membrane, thereby inhibited full activation of Lyn, and eventually restrained those receptor-distal signaling molecules, involved Syk, Btk, PLCγ, IP3R, PKC, MAPKs, Akt and NF-κB. CONCLUSION: Kaempferol could be used as a DJ-1 modulator for preventing MC-mediated allergic disorders through attenuating Lyn activation.

Antioxidant compounds from Banisteriopsis argyrophylla leaves as α-amylase, α-glucosidase, lipase, and glycation inhibitors.

Banisteriopsis argyrophylla belongs to the Malpighiaceae family, which is a species from Cerrado, also known as "cipó-prata" or "cipó-folha-de-prata." Several species of this family present biological potential. This work reports the chemical identification of the ethanol extract (EE) and its fractions from B. argyrophylla leaves and shows the analysis of the antioxidant activity and inhibitory effects on activities of α-amylase, α-glucosidase and lipase, and non-enzymatic glycation. The ethyl acetate fraction (EAF) and n-butanol fraction (BF) showed antioxidant activity, with IC50 values of 4.1 ± 0.1 and 4.8 ± 0.1 µg mL-1, respectively, by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, and IC50 values of 6046.3 ± 174.2 and 6264.2 ± 32.2 µmol Trolox eq g-1 by the oxygen radical absorbance capacity (ORAC) method. Furthermore, the DPPH method with these fractions presented electroactive species with antioxidant potential, as shown by the differential pulse voltammetry (DPV) method. The inhibitory effects of the EAF and BF were demonstrated by the following results: IC50 of 5.1 ± 0.3 and 2.5 ± 0.2 µg mL-1 for α-amylase, IC50 of 1093.5 ± 26.0 and 1250.8 ± 21.9 µg mL-1 for α-glucosidase, IC50 of 8.3 ± 4.1 and 4.4 ± 1.0 µg mL-1 for lipase, and IC50 of 1.3 ± 0.1 and 0.9 ± 0.1 µg mL-1 for glycation. Some bioactive compounds were identified by (-)-ESI-MS/MS, such as catechin, procyanidins, glycosylated flavonoids, kaempferol, and megastigmane glucosides. The antidiabetic activity of B.argyrophylla has been reported for the first time.