Kaempferol Research Update:
Absorption of kaempferol from endive a source of kaempferol-3-glucuronide in humans.
OBJECTIVE: To determine the absorption excretion and metabolism of kaempferol in humans. DESIGN: A pharmacokinetic study of kaempferol from endive over 24 h. SUBJECTS: Four healthy males and four healthy females. RESULTS: Kaempferol from a relatively low dose (9 mg) was absorbed from endive with a mean maximum plasma concentration of 0.1 microM at a time of 5.8 h indicating absorption from the distal section of the small intestine and/or the colon. Although a 7.5-fold interindividual variation between the highest and lowest maximum plasma concentration was observed most individuals showed remarkably consistent pharmacokinetic profiles. This contrasts with profiles for other flavonoids that are absorbed predominantly from the large intestine (eg rutin). An average of 1.9% of the kaempferol dose was excreted in 24 h. Most subjects also showed an early absorption peak probably corresponding to kaempferol-3-glucoside present at a level of 14% in the endive. Kaempferol-3-glucuronide was the major compound detected in plasma and urine. Quercetin was not detected in plasma or urine indicating a lack of phase I hydroxylation of kaempferol. CONCLUSIONS: Kaempferol is absorbed more efficiently than quercetin in humans even at low oral doses. The predominant form in plasma is a 3-glucuronide conjugate and interindividual variation in absorption and excretion is low suggesting that urinary kaempferol could be used as a biomarker for exposure.
Content of the flavonols quercetin myricetin and kaempferol in 25 edible berries.
The amounts of quercetin myricetin and kaempferol aglycons in 25 edible berries were analyzed by an optimized RP-HPLC method with UV detection and identified with diode array and electrospray ionization mass spectrometry detection. Sixteen species of cultivated berries and nine species of wild berries were collected in Finland in 1997. Quercetin was found in all berries the contents being highest in bog whortleberry (158 mg/kg fresh weight) lingonberry (74 and 146 mg/kg) cranberry (83 and 121 mg/kg) chokeberry (89 mg/kg) sweet rowan (85 mg/kg) rowanberry (63 mg/kg) sea buckthorn berry (62 mg/kg) and crowberry (53 and 56 mg/kg). Amounts between 14 and 142 mg/kg of myricetin were detected in cranberry black currant crowberry bog whortleberry blueberries and bilberry. Kaempferol was detected only in gooseberries (16 and 19 mg/kg) and strawberries (5 and 8 mg/kg). Total contents of these flavonols (100-263 mg/kg) in cranberry bog whortleberry lingonberry black currant and crowberry were higher than those in the commonly consumed fruits or vegetables except for onion kale and broccoli.
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Kaempferol was tested for carcinogenicity in one experiment in rats by administration in the diet. The data are inadequate to make an evaluation.
Kaempferol was mutagenic in bacteria and insects and in mammalian cells in vitro; it induced micronuclei in mice. There is limited evidence that kaempferol is active in short-term tests.
No data were available to evaluate the teratogenicity of kaempferol to experimental animals.
The natural occurrence of kaempferol an aglycone widely distributed in fruit and other edible plants results in wide human exposure to this compound.
No data were available to evaluate the teratogenicity or chromosomal effects of this compound in humans.
No case report or epidemiological study of the carcinogenicity of kaempferol was available to the Working Group.
The available data were inadequate to evaluate the carcinogenicity of kaempferol to experimental animals. In the absence of epidemiological data no evaluation of the carcinogenicity of kaempferol to humans could be made.
For definition of the italicized terms.
Effect of kaempferol on the production and gene expression of monocyte chemoattractant protein-1 in J774.2 macrophages:
Monocyte chemoattractant protein-1 (MCP-1) is produced by activated macrophages and is involved in pathogenesis of
cardiovascular and neurodegenerative disorders. There is a need to develop drugs that inhibit excessive infiltration of monocytes and
lymphocytes to the arterial wall and central nervous system. The aim of this study was to evaluate the effect of kaempferol on the
(MCP-1) gene expression and MCP-1 protein release by J774.2 macrophage cultures in vitro. Kaempferol given both before and
after lipopolysaccharide (LPS) administration reduced secretion of MCP-1.
Kaempferol administered before LPS stimulation
significantly decreased the number of copies of MCP-1 mRNA. The results suggest that kaempferol inhibits MCP-1 production at
the transcriptional level and that this is an additional anti-inflammatory mechanism of action of this flavonoid.
kaempferol J774.2 macrophages MCP-1.
Bioactive compounds are extranutritional constituents that typically occur in small quantities in foods. They are being intensively studied to evaluate their effects on health. The results of many epidemiologic studies have shown protective effects of plant-based diets on cardiovascular and cancer disease [2 11 14].
are present in all plants. They have been studied in apple cocoa onion cereals legumes nuts olive oil fruits tea and red wine. One of the most important flavonoids is kaempferol. There is evidence to suggest that kaempferol inhibits both oxidative susceptibility of low density lipoprotein (LDL) in vitro and eicosanoid synthesis as well as platelet aggregation[7 17 25]. Several in vitro experiments have shown that kaempferol functions as chemopreventive agent as well. It has been shown to inhibit cellular events associated with initiation promotion and progression of carcinogenesis [18 28].
Monocyte chemoattractant protein (MCP-1) is the most important chemotactic factor involved in this process. It is produced in response to the action of lipopolysaccharide (LPS) oxidized LDL and such cytokines as interleukin-1_ (IL-1_) tumor necrosis factor- _ (TNF-_) and interferon-_ (IFN-_) [22 23]. It has been shown that MCP-1 is a very important mole- cule in the initial steps of atherosclerotic plaque formation[4 12]. It seems valuable for developing drugs that inhibit inflammatory process thus preventing for example cardiovascular and autoimmune disease.Such a role would be played by drugs that can inhibit MCP-1 release from macrophages especially those located in the arterial wall. At present there is no literature data on the effect of kaempferol on macrophage MCP-1 synthesis and secretion. The abovementioned data prompted us to evaluate the effect of kaempferol on MCP-1 gene expression and release in J.774.2 macrophage cell line.
Materials and Methods Chemicals
The mouse macrophage cell line J.774.2 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig Germany).Cells were maintained in an atmosphere of 5% CO2 37°C in DMEM supplemented with 10% FCS 100 U/ml of fungizone (Gibco BRL Life Technologies Paisleypenicillin100 _g/ml of streptomycin and 10 _g/ml of UK). The cells were cultured in 75 cm2 plastic flasks(Nunc A/S Roskilde Denmark) and passed three times a week. For the MCP-1 secretion experiments cells were detached by vigorous pipeting and after
centrifugation plated using fresh medium. Macrophagesat a density of 5 × 104 cells/ml were plated in96-well plates (Becton Dickinson and CompanyFranklin Lakes USA) and incubated 24 h before experiment.Then the culture medium was replaced with a fresh medium. In one series of experiments dilutions of kaempferol (0.3 1 3 10 30 _M) were added to cell cultures 20 min before LPS (1 _g/ml) in other experiment single concentration of kaempferol(30 _M) was introduced to the cultures 20 min after LPS stimulation. The concentrations of kaempferol
used in this investigation were similar to those previously used by other authors [3 20]. Stimulation was used by other authors [3 20]. Stimulation was conducted for 24 h then supernatants were harvestedcentrifuged at 2000 rpm for 5 min and assayed for MCP-1. For the study of gene expression macrophages were plated in 35 mm Petri dishes and
incubated for 24 h. Then the culture medium was replaced
with fresh medium and 30 _M of kaempferol was added to cultures before LPS administration. After 24 h total RNA from such cultures was extracted. Cells not treated with LPS or kaempferol were used as control. Kaempferol was dissolved in DMSO and diluted in complete cell culture medium in order to obtain appropriate concentrations. The final concentration of DMSO was adjusted to 0.1% (v/v). The control cells received the same amount of DMSO. The effect of kaempferol on cell viability was assessed by rypan blue exclusion test. Cell viability was greater than 95% in all performed experiments.
Effect of kaempferol on LPS-induced MCP-1 release:
In the first study kaempferol was added to cultures20 min before administration of LPS. Twenty-four hour exposure to the higher concentrations of kaempferol (30 and 10 _M) caused a dose-dependent decrease in MCP-1 secretion while 3 1 and 0.3 _M doses were ineffective (Fig. 1). In the next experiments kaempferol was investigated at a single 30 _M concentration using one time point. Similarly MCP-1release decreased after 24-h exposure to 30 _M of kaempferol added to cultures 20 min after LPS treatment.
The discovery of molecules released by activated macrophages in the course of inflammatory autoimmune and neurodegenerative disorders has prompted the search for agents inhibiting the release of these molecules [8 9 13]. Kaempferol used in this study
decreased both MCP-1 gene expression and protein secretion by LPS-activated macrophages. As MCP-1 strongly induces inflammatory process the results of
this study suggest that kaempferol has an anti-inflammatory potential. In the available literature there is no data concerning direct effects of kaempferol on MCP-1 synthesis and production. It is not surprising that kaempferol inhibits macrophage activity. A number of reports have indicated that flavonoids are immunosuppressive for lymphocytes
and macrophages. Blonska et al.  found that kaempferol and other flavone derivatives such as chrysin and quercetin supressed IL-1_ nitric oxide
(NO) release and gene expresion in J774A.1 macrophages.Krol et al.  observed diminution of free radical and nitrite production in neutrophils and
macrophages by flavonoids. Many authors documented inhibitory effect of kaempferol on the inducible nitric oxide synthase expression in J774.2.
macrophages [17 20] and cyclooxygenase-12 expression in RAW264.7 cells . Okamoto et al.  found that kaempferol inhibited release of Th1 cytokine IFN-_
and IL-2 in T lymphocyte cultures and shifted TH1/TH2 to TH2 activation. Such flavonoids as kaempferol and quercetin inhibited release of hydrogen peroxide (H2O2) from neutrophils .The mechanism by which kaempferol inhibits MCP-1 release and gene expression is unknown. The production and release of various cytokines (TNF-_IL-1_ MCP-1) by LPS-activated macrophages is mediated by the induction of such transcriptional factors as nuclear factor-kappa B (NF-_B) and activator protein-1 (AP-1) [10 21]. NF-_B is sequestered in the cytoplasm as an inactive complex with the inhibitory subunit is phosphorylated and degraded. Then the active
NF-_B is translocated to the nucleus . It was found that flavones. (chrysin luteolin oroxylin A and myricetin) inhibited NF-_B activation .Thus it is likely that kaempferol inhibits MCP-1 production and synthesis by inhibiting NF-_B activation.
The role of MCP-1 in atherogenesis is well documented.On the other hand some epidemiologic studies have reported association between protective action of flavonoids and cardiovascular disease. The results of the present study may support the view about health benefits of flavonoids and flavonoid-rich diet.Summing up in our study we demonstrate for the first time that kaempferol is an inhibitor of LPSinduced MCP-1 release in J774.2 macrophages.
Epidemiological studies have suggested beneficial effects of dietary polyphenols in reducing the risk of chronic diseases. This study was performed to investigate the effects of polyphenol-depleted and polyphenol-rich diets on blood oxidative stress markers and urinary excretions of major phenols.
Nineteen healthy female non-smokers 19 to 21 years of age took part in the study which consisted of two dietary intervention periods separated by three days. Experimental diets were composed of common foods selected to comply with low contents of polyphenols for phenol-depleted intervention and high contents of polyphenols for phenol-rich diets. Blood and urine samples were collected on day 0 3 and 6 of each intervention. Duplicate portions of foods provided to the subjects were also collected. Blood oxidative stress markers included plasma antioxidant vitamins erythrocyte superoxide dismutase (SOD) activity and lymphocyte DNA damage. Urinary excretions of major phenols were measured to affirm bioavailability of dietary phenols.
Plasma -tocopherol and ß-carotene concentrations were slightly decreased on day 3 and 6 of the phenol-depleted dietary intervention period although no change was observed with phenol-rich diets. The erythrocyte SOD activity was also slightly decreased during phenol-depleted dietary intervention. However at day 6 of the phenol-rich intervention the activity of SOD was significantly increased by 41%. Tail moment and tail length of lymphocyte DNA as markers of DNA damage were higher on day 6 of phenol-depleted intervention although only tail moment showed a statistical significance. The average intakes of quercetin and kaempferol during the phenol-rich intervention were 21 mg/day and 9 mg/day respectively. The average urinary excretion rates during phenol-rich intervention were 2.06% for quercetin and 0.46% for kaempferol. There were positive correlations between erythrocyte SOD activity and urinary concentration of quercetin or kaempferol.
These results suggest that polyphenol-rich diets may decrease the risk of chronic diseases by reducing oxidative stress.
Analysis of Dietary and Urinary Quercetin and Kaempferol
Dietary quercetin and kaempferol were determined based on the methods of Arai et al.
Briefly a 0.25 g freeze-dried food sample was extracted with 25 mL of 50% methanol containing 1.2 mol/L HCl and 1.6 g/L tert-butylhydroquinone for 2 hours at 90°C. The extract was diluted to 100 mL with methanol. After centrifugation (1000 x g 5 minutes 4°C) a 2 mL aliquot was dried by evaporation under nitrogen gas flow. The residue was dissolved in 100 µL methanol of which 10 µL was used for HPLC analysis. Mobile phase was acetonitrile/phosphate buffer (pH 2.4 40/60) and flow rate was 0.6 ml/min. Quantification was made using a UV detector at 370 nm.
kaempferolAnti-inflammation Anti-bacteria Restrain cough
Antioxidant，Anti-spasmodic or Spasmolytic，AntiulcerCholeretic，Diuretic，Relieve coughing.
The vacuoles of lower epidermal strips from Vicia faba exhibit an intrinsic green fluorescence when incubated in alkaline buffers. Using an alkaline-induced absorbance change as a spectrophotometric assay the major pigment responsible for this fluorescence was isolated and identified as the flavonoid: kaempferol 3-O-galactoside 7-O-rhamnoside. The aqueous absorption maxima were 394 and 341 nanometers at pH 10.0 and 6.0 respectively with a pKa of 8.3 and the fluorescence emission maximum was 494 nanometers at pH 10.0. The in vivo concentration was estimated to be between 3 and 10 micromolar. The absorption spectrum of this flavonoid is different from the action spectrum for stomatal opening indicating that this compound is not the photoreceptor pigment for the blue light response of Vicia faba guard cells