Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages.

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells.

Studies on pharmacokinetic properties and absorption mechanism of phloretin: In vivo and in vitro.

Phloretin is a natural dihydrochalcone flavonoid that is mainly distributed in apple, pear and other juicy fruit peels or root peels. Phloretin exhibits several pharmacological properties, such as antidiabetic, antioxidant, anti-inflammatory, and antitumor activities. However, the poor water solubility of phloretin limits its application in the treatment of numerous diseases. To date, the underlying mechanisms of phloretin absorption have not been investigated. In this study, the pharmacokinetics of phloretin orally administered to Sprague-Dawley (SD) rats were examined, and the absorption mechanisms of phloretin were investigated in a Caco-2 cell monolayer and single-pass intestinal perfusion in SD rat. The effects measured by basic parameters, such as compound concentration, time, temperature, paracellular pathway, in different intestinal segments were analyzed, and various inhibitors, such as the P-glycoprotein (P-gp) inhibitor verapamil, the multidrug resistance protein 2 (MRP2) inhibitor indomethacin, the breast cancer resistance protein (BCRP) inhibitor reserpine, and the closely related regulator EDTA, were evaluated to determine their effects on the absorption of phloretin. The pharmacokinetics of phloretin was studied by oral and intravenous injection in rats. The bioavailability was 8.676 %.The SPIP experiments showed that P-gp, MRP2, BCRP protein inhibitor and closely related regulator, could significantly increase the apparent permeability coefficient (Papp) of phloretin. Monolayer transport experiments in Caco-2 cells showed that P-gp, MRP2 protein inhibitor and closely related regulator EDTA, significantly increased the Papp value of phloretin. In conclusion, phloretin is a substrate of P-gp and MRP2, and its modes of transport include active transport, efflux protein transport and cell bypass.

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1.

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 µM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.

Translocation of transfected GLUT2 to the apical membrane in rat intestinal IEC-6 cells.

AIM: In this study, we transfected the full length cDNA of glucose transporter 2 (GLUT2) into IEC-6 cells (which lack GLUT2 expression) to investigate GLUT2 translocation in enterocytes. The purpose of this study was to investigate cellular mechanisms of GLUT2 translocation and its signaling pathway. METHODS: Rat GLUT2 cDNA was transfected into IEC-6 cells. Glucose uptake was measured by incubating cell monolayers with glucose (0.5-50 mM), containing (14)C-D-glucose and (3)H-L-glucose, to measure stereospecific, carrier-mediated and passive uptake. We imaged GLUT2 immunoreactivity by confocal fluorescence microscopy. We evaluated the GLUT2 inhibitor (1 mM phloretin), SGLT1 inhibitor (0.5 mM phlorizin), disrupting microtubular integrity (2 µM nocodazole and 0.5 µM cytochalasin B), protein kinase C (PKC) inhibitors (50 nM calphostin C and 10 µM chelerythrine), and PKC activator (50 nM phorbol 12-myristate 13-acetate: PMA). RESULTS: In GLUT2-IEC cells, the K(m) (54.5 mM) increased compared with non-transfected IEC-6 cells (7.8 mM); phloretin (GLUT2 inhibitor) inhibited glucose uptake to that of non-transfected IEC-6 cells (P < 0.05). Nocodazole and cytochalasin B (microtubule disrupters) inhibited uptake by 43-58% only at glucose concentrations ≥25 and 50 mM and the 10-min incubations. Calphostin C (PKC inhibitor) reproduced the inhibition of nocodazole; PMA (a PKC activator) enhanced glucose uptake by 69%. Exposure to glucose increased the GFP signal at the apical membrane of GLUT-1EC cells. CONCLUSION: IEC-6 cells lacking GLUT2 translocate GLUT2 apically when transfected to express GLUT2. Translocation of GLUT2 occurs through glucose stimulation via a PKC-dependent signaling pathway and requires integrity of the microtubular skeletal structure.

Influence of phloretin on the skin permeation of lidocaine from semisolid preparations.

The purpose of the present study was to determine the significance of phloretin on the topical permeation of lidocaine using different semisolid preparations as delivery systems. One hydrophilic and three lipophilic formulations were used. After estimation of the solubility of phloretin and lidocaine in the vehicles and analysis of the viscoelastic properties, standard diffusion experiments with Franz type diffusion cells through porcine skin were performed. Results indicate a general lidocaine enhancement by phloretin in the tested vehicles. The permeation was enhanced 1.39-fold in the hydrophilic formulation and between 1.25- and 1.76-fold in the lipophilic formulations.

Phloretin and 6-ketocholestanol: membrane interactions studied by a phospholipid/polydiacetylene colorimetric assay and differential scanning calorimetry.

The aim of this study was to investigate membrane interactions of phloretin and 6-ketocholestanol using different methods. A previously reported colorimetric assay with phospholipid/polydiacetylene (PDA) vesicles was used to examine a possible interaction of phloretin and 6-ketocholestanol with this target. During this interaction the used aggregates of lipids and conjugated PDA undergo a visible and quantifiable blue to red color transition. A positive result is indicative for a reaction response with membrane lipids of a simplified bilayer structure instead of the complex bilayer system of the stratum corneum. Results of this test confirm previous proposed membrane interactions by skin diffusion studies. Additional differential scanning calorimetry studies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes confirm a membrane interaction and indicates that phloretin and 6-ketocholestanol interact with the lipid layer and change structural parameters. They strongly decrease the lipid phase transition temperature of DMPC and DPPC liposomes by at least about 6.6 degrees C and maximally about 13.9 degrees C which refers to a higher fluidity of the membrane.

Influence of phloretin and 6-ketocholestanol on the skin permeation of sodium-fluorescein.

In the present study, we investigated the feasibility of enhancing the transport of the model drug sodium-fluorescein across rat, porcine and human skin by treating it with phloretin and 6-ketocholestanol. Both 6-ketocholestanol and phloretin were incorporated into unilamellar liposomes and used as a skin pre-treatment. Afterwards standard diffusion experiments with Franz-type diffusion cells were performed. The results indicate a positive effect of 6-ketocholestanol on the sodium-fluorescein diffusion in all skin types, whereas phloretin had no significant influence compared to the control in human skin. In contrast to this, phloretin had a significant positive effect in rat as well as in porcine skin after 30 h of diffusion. In addition to the permeation studies, the human skin samples were analysed by confocal laser scanning microscopy for direct visualisation of the sodium-fluorescein. As expected the results of the diffusion experiments were confirmed by this microscopic method.

The Fluorosome technique for investigating membrane on- and off-loading of drugs by beta-CD and sonicated SUV.

The application of the Fluorosome technique to test drug delivery systems is described. Fluorosomes, egg phosphatidylcholine liposomes with bilayer embedded fluorophores, were employed to investigate the ability of sonicated small unilamellar vesicles (sSUV) and beta-cyclodextrins (beta-CD) to deliver drugs into or extract drugs from the fluorosome's phospholipid bilayer. The addition of phloretin to a fluorosome suspension resulted in fluorescence reduction reflecting phloretin entering the bilayer and quenching fluorophore fluorescence. Subsequent addition of sSUV to phloretin pretreated fluorosomes showed an increase in fluorescence reflecting phloretin extraction from the fluorosome membrane. Sequential additions of beta-estradiol loaded beta-CD to fluorosomes as well as the addition of beta-estradiol alone resulted in fluorescence reduction due to beta-estradiol insertion into the membrane. Further addition of pure beta-CD resulted in a fluorescence increase indicating beta-estradiol extraction from the fluorosome membrane.

Bioavailability of phloretin and phloridzin in rats.

Phloretin is a flavonoid found exclusively in apples and in apple-derived products where it is present as the glucosidic form, namely, phloridzin (phloretin 2'-O-glucose). In the present study, we compared the changes in plasma and urine concentrations of these two compounds in rats fed a single meal containing 0.25% phloridzin or 0.157% phloretin (corresponding to the ingestion of 22 mg of phloretin equivalents). In plasma, phloretin was recovered mainly as the conjugated forms (glucuronided and/or sulfated) but some unconjugated phloretin was also detected. By contrast, no trace of intact phloridzin was detected in plasma of rats fed a phloridzin meal. These compounds presented different kinetics of absorption; phloretin appeared more rapidly in plasma when rats were fed the aglycone than when fed the glucoside. However, whatever compound was administered, no significant difference in the plasma concentrations of total phloretin were observed 10 h after food intake. At 24 h after the beginning of the meal, the plasma concentrations of phloretin were almost back to the baseline, indicating that this compound was excreted rapidly in urine. The total urinary excretion rate of phloretin was not affected by the forms administered, and was estimated to be 8.5 micromol/24 h in rats fed phloretin or phloridzin. Thus, 10.4% of the ingested dose was recovered in urine after 24 h.

Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats.

Absorption and metabolism of quercetin and isoquercitrin (quercetin 3-O-glucose) were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min and compared with those of phloretin and phloridzin (phloretin 2'-O-glucose). After perfusion of the glucosides, the corresponding aglycone forms and conjugated derivatives appeared in the lumen. The conjugated metabolites were similar to those recovered after intestinal perfusion of the aglycone forms. Regardless of the aglycone or glucoside perfused, only conjugated forms were present in the mesenteric vein blood draining the perfused segment showing the importance of intestinal conjugation. The hydrolysis of glucosides was a prerequisite step before their conjugation by intestinal enzymes and their transport towards the mucosal and serosal sides. In contrast to phloridzin, lactase phloridzin hydrolase activity did not seem to be an essential pathway for isoquercitrin hydrolysis. The 3-O-glucosylation of quercetin improved the net absorption of the aglycone (P < 0.05), whereas phloretin absorption decreased when present as 2'-O-glucoside (P < 0.05). Whatever the perfused compound, the efficiency of the absorption seemed to be linked to the intestinal conjugation process and to the luminal secretion of metabolites.

Influence of phloretin and 6-ketocholestanol on the permeation of progesterone through porcine skin.

In this study the effect of phloretin (PH) and 6-ketocholestanol (KC) on the permeation of progesterone through porcine skin has been examined. Both PH and KC were incorporated into unilamellar L-alpha-phosphatidylcholine (PC) liposomes at different concentrations (7.5, 15, 30 and 60 mol%). In diffusion experiments with porcine skin, both substances, to a different degree, enhanced the steady state flux of progesterone. It was increased up to 2.4-fold using 15 mol% KC, and 1.4-fold using 30 mol% PH. The results indicate an interaction of these two compounds with the lipid components of the stratum corneum. In order to visualise the interaction, differential scanning calorimetry (DSC) measurements were performed on porcine skin, which had been impregnated with KC and PH. Both showed a lowering ( approximately 5-6 degrees C) in the lipid phase transition temperature that occurs around 75 degrees C in porcine skin.

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells.

The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 microM) and another component unsaturatable up to 80 microM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of alpha-methyl-glucopyranoside the order was D-glucose=phlorizin>>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes. I. 2,4-dichlorophenoxyacetic acid.

The adsorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as well as of other dipolar molecules to the interface of artificial lipid membranes gives rise to a change of the dipole potential between the membrane interior and water. As a consequence of the adsorption of the neutral species, the conductance of planar membranes, observed in the presence of the macrocyclic ion carriers nonactin or valinomycin, may change by many orders of magnitude. Using this effect in combination with a laser-T-jump technique, the kinetics of the adsorption process were measured and were interpreted on the basis of a Langmuir-isotherm. A partition coefficient (at small concentrations) of beta HA = 4.7 x 10(-4) cm, a rate constant of desorption kHA greater than or equal to 100 s-1 and a maximum surface density ND = 7.7 x 10(13)/cm2 were found. The concentration at half saturation is KHA = 2.7 x 10(-4) M. Using these values the membrane conductance induced by the ion carrier nonactin and the shape of the current-voltage relationship as a function of the ligand concentration in water was analyzed. A maximum dipole potential of VDmax = -239 mV and a contribution of b = 3.1 x 10(-15) V cm2 per single adsorbed 2,4-D molecule was found. 74% of the dipole potential acts on the inner membrane barrier separating the two interfacial adsorption planes of nonactin. The remainder (26%) favours interfacial complex formation between nonactin and K+ from the aqueous phase. The data hold for membranes formed from dioleoyllecithin in n-decane.