Antibacterial activity of the enniatin B, produced by Fusarium tricinctum in liquid culture, and cytotoxic effects on Caco-2 cells

The enniatins (ENs) are bioactive compounds of hexadepsipeptidic structure produced by several strains of Fusarium sp. The EN B was purified from extracts of Fusarium tricinctum growth on liquid culture of potato dextrose broth (PDB), using a semipreparative liquid chromatography (LC) followed by an analytical LC. The purity and the structure of the isolated compound were confirmed by the determination of the extinction coefficient and with electrospray ionization-mass spectrometry (ESI-MS) study. The pure fraction of EN B was utilized to determine the antibiotic effects on several bacterial strains that are considered normally pathogens of the intestinal tract: Escherichia coli, Enterococcus faecium, Salmonella enterica, Shigella dysenteriae, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Pseudomonas aeruginosa, and Staphylococcus aureus, and to study the cytotoxic effects on Caco-2 differentiated and undifferentiated cells. The results obtained demonstrated that in several antibiograms, EN B induced the inhibition of the grown microorganisms tested and no significant differences over control were detected when Caco-2 cells were exposed to EN B, at any of the concentrations used.     

Isolation and purification of enniatins A, A1, B, B1, produced by Fusarium tricinctum in solid culture, and cytotoxicity effects on Caco-2 cells

Enniatins (ENs) are antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The ENs A, A₁, B, B₁ were purified from extracts of Fusarium tricinctum grown on a solid medium of corn, by a low pressure liquid chromatography (LPLC) on reverse phase of Amberlite XAD-7 followed by semipreparative LC. The purity and the structure of the isolated compounds were confirmed by LC-MS/MS.The technique of the purification of the fungal extract enabled complete separation of the ENs A, A₁, B, B₁ with a mean purity of 97% for all the compounds.The cytoxicity of the ENs was tested in the cell lines of human origin (epithelial colorectal adenocarcinoma cells, Caco-2) by MTT assays. Only EN A₁ and B₁ evoked toxicity at the tested concentrations. The inhibitory concentration (IC₅₀) for EN A₁ on Caco-2 cells was 12.3 μM, whereas the IC₅₀ produced by the EN B₁ was 19.5 μM.This study indicates that ENs, fungal metabolites that are commonly found in corn and in general in product composed by corn, may have a toxic potential for human health.     

Study of the potential toxicity of enniatins A, A1, B, B1 by evaluation of duodenal and colonic bioavailability applying an in vitro method by Caco-2 cells

The bioavailability of the minor Fusarium mycotoxins enniatins (ENs) utilizing an in vitro method which allows the simulation of the small and large intestine tracts has been studied. This method, based on the application of the Caco-2 cells grown alone or in symbiosis with several strains characteristics of the gastrointestinal tract, has permitted to simulate the duodenal and colonic intestinal compartments, respectively.The duodenal bioavailability expressed as absorption value after 4 h of exposure, ranged from 57.7 to 76.8% for EN A, from 68.8 to 70.2% for EN A₁, from 65.0 to 67.0% for EN B, and from 62.2 to 65.1% for EN B₁. Colonic bioavailability after 48 h of incubation ranged from 17.3 to 33.3% for EN A, from 40.8 to 50.0% for EN A₁, from 47.7 to 55.0% for EN B, and from 52.4 to 57.4% for En B₁. The highest duodenal and colonic bioavailability was observed after Caco-2 cells exposed to EN A, ENs B and B₁, respectively.     

DNA damage and oxidative stress induced at low doses by the fungicide hexachlorobenzene in human intestinal Caco-2 cells

Abstract

Context: Hexachlorobenzene (HCB), a persistent chlorinated organic chemical, could be detected in human tissues in several countries of the world. Human exposure to Persistent Organic Pollutants (POPs) occurring primarily through diet, HCB and its metabolites are therefore supposed to interact directly with intestinal mucosa.

Objective: The aim of this study was to investigate the possible effects of low doses of HCB on DNA integrity, cellular viability, differentiation and oxidative status in vitro in human colonic carcinoma cell line Caco-2.

Materials and methods: Cells were exposed to increasing doses of HCB for 14 days to assess the cytotoxic, genotoxic and oxidative properties of this compound. The involvement of oxidative stress in the observed effects was evaluated by co exposure of Caco-2 cells with HCB and α-tocopherol.

Results: Exposure of Caco-2 cells to HCB resulted in a dose-dependent cytotoxicity, DNA damages and alterations of the cell layer integrity and the barrier function. Moreover, exposure of Caco-2 cells to HCB led to an enhancement of H₂O₂ production and to an increased activity of antioxidant enzymes. In addition, Co exposure of Caco-2 cells to HCB and α-tocopherol reversed the effects observed in cells exposed to HCB alone.

Conclusion: These results suggested that HCB effects on Caco-2 cells could be linked, at least in part, to its pro-oxidative potential.     

Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A,A1, B and B1

The cytotoxic effects, the generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) as well as the cell cycle disruption, the induction of apoptosis and changes in mitochondrial membrane potential (ΔΨm) as a function of increasing time have been determined in human colorectal adenocarcinoma (Caco-2) cells after exposure to enniatins (ENs) A, A₁, B and B₁. IC₅₀ values obtained by the MTT and Neutral Red assay, after 24, 48 and 72 h of exposure ranged from 0.5 ± 0.1 to >15 μM. A significant increase (p ≤ 0.05) in ROS generation and LPO production, as determined by the fluorescent probe H₂-DCFDA and TBARS method respectively, was observed for all mycotoxins tested at 3.0 μM concentration. The highest increase in ROS generation (2.6 fold higher than control) and LPO production (111%, as compared to control) was observed with EN A. Cell cycle was significantly arrested at G2/M phase after 24 h of exposure to EN A, A₁, B₁, whereas after 72 h of exposure an arrest in S phase was observed almost for all mycotoxins tested. Moreover, after 24 and 48 h of exposure, ENs increased the early apoptotic cells, whereas after 72 h of exposure necrosis was observed. In addition the loss of ΔΨm was produced on Caco-2 cells after ENs exposure. ENs A, A₁, B and B₁ cytotoxicity involved early ROS generation that induced LPO oxidative damage, apoptosis and necrosis via the mitochondrial pathway. ENs A, A₁ and B₁ induced DNA damage. However the same effects cannot be proposed for EN B. Further studies on the toxicological effects induced by ENs A, A₁, B and B₁ are needed.     

Interaction effects of Fusarium enniatins (A,A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells

Foodstuff is usually contaminated by more than one mycotoxin, however toxicological data are lacking as regards the effects in combinations compared to their individual effect. This study investigated the in vitro effects of enniatins (ENs) A, A₁, B and B₁, alone and in combinations, on Caco-2 cells viability by MTT assay after 24 h of exposure. Cells were treated with concentrations ranging from 0.9 to 15.0 μM, individually and in combination of two, three and four mycotoxins. Dose–response curves were generated for each mycotoxin and the isobologram method was used to determine the interactive effects of tested mixtures. Tested ENs produced significant cytotoxic effects both individually and in combination in a dose-dependent manner. IC₅₀ values obtained for all individually tested mycotoxins ranged from 1.3 to >15 μM. In ENs combination tests, synergistic effect in Caco-2 viability are observed for EN B + EN A₁, EN B₁ + EN A₁ and EN A + EN A₁ + EN B (CI = 0.33–0.52). All other combinations showed additive effect at medium and high affected fraction with exception of lower fraction affected and the EN B + EN B₁ mixture that produced antagonistic effect (CI = 1.76–10.36). The use of combination index-isobole method could help to better understand the potential interaction between co-occurring mycotoxins and may contribute to their risk assessment.     

Antibacterial effects of enniatins J1 and J3 on pathogenic and lactic acid bacteria

Enniatins (ENs) are N-methylated cyclohexadepsipeptides, secondary metabolites produced by various species of the genus Fusarium. They are known to act as antifungal, antiyeast and antibacterial and to possess antiinsecticidal and phytotoxic properties. In this study we evaluated for the first time the antibiotic effect of pure fractions of EN J₁ and J₃ on several pathogenic strains and lactic acid bacteria.The ENs J₁ and J₃ were purified from the fermentation extract of Fusarium solani growth on solid medium of wheat kamut, using the technique of the low pressure liquid chromatography (LPLC) followed by a semipreparative liquid chromatography (LC). The purity and the structure of the isolated compound were confirmed by electrospray ionization-mass spectrometry study-linear ion trap (ESI-MS-LIT).The use of both chromatographic techniques have permitted to produce and purify 47 mg of the En J₁ and 50 mg of the EN J₃ with a mean purity of 98% completely characterized with the technique of the ESI-MS-LIT.Microbial bioassay analyses were carried out by incubation in MRSA and TSA for acid lactic and pathogenic bacteria, respectively during 24 h at 37 °C. None of the tested strains were inhibited by a 1 ng dose of EN J₁ and J₃. These compounds were only not effective against Listeria monocytogenes, Pseudomonas aeruginosa and Salmonella enteric. This study highlight ENs J₁ and J₃ could be potentially effective antibacterial agents against several pathogenic and lactic acid bacteria.     

Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29

Enniatins (ENs) are ionophoric, phytotoxic, antihelminthic, and antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The cytotoxicity effect of the ENs A, A₁, A₂, B, B₁, B₄ and J₃ was compared on three tumor cell lines, the human epithelial colorectal adenocarcinoma (Caco-2), the human colon carcinoma (HT-29), and the human liver carcinoma (Hep-G2). The endpoint evaluated was the mitochondrial integrity by using the MTT assays, after 24 and 48 h of incubation. The IC₅₀ value for EN A₂ on Caco-2 cells, after 24 h exposure, was 18.7 ± 4.5 μM and decrease to 2.6 ± 0.7 μM at 48 h of incubation. However, ENs A, A₁, B₁ and B₄ exert pronounced cytotoxic effects in all the cell lines tested by the MTT assay after 24 and 48 h of incubation. The EN A₁ demonstrated to be the most cytotoxic ENs tested. Moreover, no statistical differences were found between the IC₅₀ values obtained for EN A₁ on Caco-2, HT-29 and Hep-G2, with IC₅₀ values ranging from 9.1 ± 2.2 μM to 12.3 ± 4.3 μM at 24 h and decreasing in a range variable from 1.4 ± 0.7 μM to 2.7 ± 0.8 μM at 48 h. On the other hand, EN A, B₁ and B₄ showed lower cytotoxicity, but in a similar range as the IC₅₀ values reported on HT-29 (IC₅₀ values (24 h): 16.8 ± 4.3-26.2 ± 6.7 μM), Caco-2 (IC₅₀ values (24 h): 19.5 ± 4.1 μM) and Hep-G2 (IC₅₀ values (24 h): 23.4 ± 5.6-26.2 ± 7.6 μM) cells. Cytotoxic effect with a 48 h of incubation revealed also a significant toxicity of ENs A (IC₅₀ values ranged from 8.2 ± 1.8 to 11.4 ± 4.6 μM), B₁ (IC₅₀ values variables from 3.7 ± 0.7 to 11.5 ± 5.3 μM) and B₄ (IC₅₀ of 4.5 ± 2.9-15.0 ± 4.0 μM). In summary, this study demonstrated that ENs can exert toxic activity at low micromolar concentrations in mammalian cells.     

Effects of dietary fruits,vegetables and a herbal tea on the in vitro transport of cimetidine: Comparing the Caco-2 model with porcine jejunum tissue

Context: Dietary botanicals are often consumed together with allopathic medicines, which may give rise to pharmacokinetic interactions. In vitro intestinal models are useful to identify botanical-drug interactions, but they may exhibit different expressions of transporters or enzymes.

Objective: To compare the effects of selected dietary botanical extracts on cimetidine transport across two in vitro intestinal models.

Materials and Methods: Bi-directional transport of cimetidine was measured across Caco-2 cell monolayers and excised porcine jejunum tissue in the absence (control) as well as the presence of verapamil (positive control) and selected plant extracts.

Results: Sclerocarya birrea Hochst. (Anacardiaceae) (marula) and Psidium guajava L. (Myrtaceae) (guava) crude extracts significantly decreased cimetidine efflux in both in vitro models resulting in increased absorptive transport of the drug. On the other hand, Dovyalis caffra Sim. (Flacourtiaceae) (Kei-apple), Prunus persica (L.) Batsch (Rosaceae) (peach), Aspalathus linearis (Burm. f.) R. Dahlgren (Fabaceae) (rooibos tea), Daucus carota L. (Apiaceae) (carrot), Prunus domestica A. Sav. (Rosaceae) (plum), Beta vulgaris L. (Chenopodiaceae) (beetroot) and Fragaria x ananassa (Weston) Duchesne ex Rozier. (Rosaceae) (strawberry) crude extracts exhibited different effects on cimetidine transport between the two models.

Discussion: Caco-2 cells were more sensitive to changes in cimetidine transport by the plant extracts and therefore may overestimate the effects of co-administered plant extracts on drug transport compared to the excised pig tissue model, which is congruent with findings from previous studies.

Conclusions: The excised porcine jejunum model seemed to provide a more realistic estimation of botanical-drug pharmacokinetic interactions than the Caco-2 cell model.     

Study on the pharmacokinetics of deoxyschizandrin and schizandrin in combination with epigallocatechin gallate,a component of green tea,in rats

1.?Green tea is commonly used worldwide due to its potential positive health benefits. We have examined the effects of epigallocatechin gallate (EGCG), the most abundant catechin in green tea, on the pharmacokinetics of deoxyschizandrin (DSD) and schizandrin (SD), which are the representative lignans in popular traditional Chinese medicines Fructus schisandrae, in rats.

2.?The effects on the transport in Caco-2 cells and metabolism in human liver microsomes (HLMs) of DSD and SD by EGCG were determined to analyze their interactions thoroughly.

3.?In pharmacokinetic studies, rats were divided into four groups. Each group was orally treated with DSD alone (Group 1), DSD combined with EGCG (Group 2), SD alone (Group 3) and SD combined with EGCG (Group 4). The pharmacokinetic parameters of DSD and SD in rats were determined by UPLC-MS/MS.

4.?The in vivo results indicated that EGCG had no significant influence on the pharmacokinetic behaviors of DSD or SD in rats, which were in accordance with the in vitro transport and metabolism studies. However, there were marked differences between male and female rats among C_max, AUC_0–t, AUC_0–∞ of DSD and SD. This disparity suggested that gender differences might exist in the pharmacokinetic processes of DSD or SD in rats.     

Characterization of the Uptake Mechanism for a Novel Loop Diuretic,M17055, in Caco-2 Cells: Involvement of Organic Anion Transporting Polypeptide (OATP)-B

Purpose M17055 is under development as a novel loop diuretic for oral administration. To investigate the molecular mechanism of its gastrointestinal absorption, we initially aimed to clarify the mechanism of uptake of M17055 by Caco-2 cells, focusing on possible involvement of OATP-B (SLCO2B1), which is localized in the apical membranes of human intestinal epithelial cells.Materials and Methods The uptake of [¹⁴C]M17055 by Caco-2 cells cultured on multi-well dishes was measured after cultivation for 14 days. Uptake of [¹⁴C]M17055 by HEK293 cells stably expressing OATP-B (HEK293/OATP-B cells) was also examined.Results M17055 uptake by Caco-2 cells was saturable, and was inhibited by various organic anions, including other loop diuretics, and several bile acids. Uptake of M17055 by HEK293/OATP-B cells was much higher than that by mock cells. The inhibitory profiles of various organic anions and the estimated K _m values for M17055 uptake were similar in Caco-2 and HEK293/OATP-B cells. Moreover, the values of inhibition constants of several inhibitors for M17055 uptake were comparable in the two cell lines.Conclusion Our data suggest that OATP-B plays a major role in the uptake of the novel loop diuretic M17055 from apical membranes in Caco-2 cells.     

Effect of polyphenols on enniatins-induced cytotoxic effects in mammalian cells

Enniatins (ENs) are fungal secondary metabolites produced by genus Fusarium. The ENs exert antimicrobial and insecticidal effect, and has also been demonstrated cytotoxic effects on several mammalian cell lines. On the other hands, it has been proved that natural polyphenols have antioxidant effect. In this study, cell effects at low levels of exposure of four ENs (A, A₁, B and B₁) and five polyphenols (quercetin, quercetin-3-β-D-glucoside, rutin, myricetin and t-pterostilbene) present in wine; and the cytoprotective effect of these polyphenols exposed simultaneously with ENs in Chinese Hamster Ovary (CHO-K1) cells, were studied. Cell effects were determined by the MTT test after 24?h of exposure. All ENs showed cytotoxic effect. The IC₅₀ obtained ranged from 4.5?±?1.2 to 11.0?±?2.7 µM. The concentration of polyphenols tested ranged from 5 to 50 µM. Polyphenols did not show cytotoxicity and the cytoprotective effect of polyphenols varies depending on the EN tested. The cytoprotective effect of polyphenols in CHO-K1 cells exposed to ENs was as follow: quercetin, from 24 to 84%; quercetin-3-β-D-glucoside, from 12 to 76%; rutin, from 17 to 83%; myricetin, from 16 to 92% and pterostilbene from 25 to 100%. All polyphenols protected CHO-K1 cells against EN A₁ exposure.     

Cyclodextrin modulates the cytotoxic effects of chlorhexidine on microrganisms and cells in vitro

Abstract

Although several studies have shown that chlorhexidine (Cx) has bactericidal activity and exerts toxic effects on periodontal tissues a few studies evaluated mechanisms to reduce its adverse effects maintaining the antimicrobial properties. Therefore, the aim of the present study was to investigate the in vitro antimicrobial activity and cellular cytotoxicity of Cx included on cyclodextrins (Cd), α, β or Hp-β-cyclodextrins (Hp-β-Cd). The influence of Cds was determined by increasing its molar rate 1:1 to 1:4 in relation with free Cx. The minimal inhibitory concentrations (MICs) for Candida albicans, Aggregatibacter actinomycetemcomitans actinomycemcomitans and Streptococcus mutans were determined. An ergosterol solubilization assay was carried out using the C. albicans model and osteoblasts, fibroblasts and tumoral Caco-2 cells for cytotoxicity assay. The antimicrobial activity results in a significant growth inhibition of C. albicans when it was treated with Cx:α-Cd complexes, whereas Cx:β-Cd was more effective for A. actinomycetemcomitans, and Cx:Hp-β-Cd complexes was for S. mutans when compared to the other complexes. The cytotoxicity for fibroblasts and osteoblasts decreased in relation with each kind of Cd been β-Cd?≤?Hp-β-Cd?≤?α-Cd. Although the Hp-β-Cd inclusion complexes had more severe effects on Caco-2 cells, all complexes exhibited less cytotoxicity than free Cx. The α-Cd, β-Cd and Hp-β-Cd increase the antimicrobial activity of Cx, but decrease its cytotoxic effects on mammalian cells. Taken together these findings suggest that cyclodextrins are a tool for modulation of effects of Cx. It could be useful to design Cx/Cd delivery systems with high efficacy and minimum cytotoxic effects.     

Luffa cylindrica suppresses development of Dermatophagoides farinae-induced atopic dermatitis-like skin lesions in Nc/Nga mice

Abstract

Context: The fruit pulp of Luffa cylindrica Roemer (Cucurbitaceae) (LC) has been used to induce hemostasis, resolve phlegm and clear fever in traditional Korean medicine. However, the efficacy of LC has not been examined in atopic dermatitis (AD).

Objective: A 70% ethanol extract of LC was evaluated to determine anti-inflammation and anti-AD effects in vitro and in vivo.

Materials and methods: The inhibitory effects of LC on the production of PGE₂ and histamine were respectively measured in lipopolysaccharide-treated (1?μg/mL) RAW264.7 macrophages and phorbol-12 myristate 13-acetate (50?nM) and A23187 (1?µM)-stimulated HMC-1 mast cells. The production of AD-related chemokines (RANTES, TARC, and MDC) were evaluated in IFN-γ and TNF-α-stimulated (10?ng/mL, each) HaCaT keratinocytes. LC (10?mg/mouse/d) was topically applied to the dorsal skin and ears of Dermatophagoides farina (Pyroglyphidae)-sensitized Nc/Nga mice for 4?weeks.

Results: The IC₅₀ values of LC on PGE₂ and histamine production were 16.89 and 139.9?μg/mL, individually. The production of TARC and RANTES were inhibited 20% and 12% by LC (50?μg/mL) in HaCaT cells, respectively (p?<?0.05). In sensitized-NC/Nga mice, the plasma levels of IgE and histamine were suppressed 36% and 41% by LC, respectively (p?<?0.05). LC also reduced hemorrhage, hypertrophy, and hyperkeratosis of the epidermis and infiltration of mast cells in the dorsal skin and ear.

Discussion and conclusion: LC can inhibit AD-like skin lesions and reduce the generation of IgE via inhibition of the inflammatory responses. LC has potential as a therapeutic agent to treat allergic diseases, including AD.     

Evidence for Diminished Functional Expression of Intestinal Transporters in Caco-2 Cell Monolayers at High Passages

Purpose. To investigate the effects of passaging on the intrinsic membrane transport parameters of compounds absorbed by means of passive and carrier-mediated processes in the Caco-2 cell line. Methods. Caco-2 cells at low (28–36) and high (93–108) passage numbers were used to evaluate the transport characteristics of model compounds for paracellular diffusion (mannitol), transcellular diffusion (progesterone) and carrier-mediated transport (cephalexin, cephradine, phenylalanine, proline, and taurocholic acid) using side-by-side diffusion chambers. Intrinsic intestinal transport parameters were determined by correcting the effective permeability for potential biases introduced by the microporous filter and aqueous boundary layer. Intrinsic maximal flux (J _max, Michaelis constant (K _m) and carrier permeability (P _c) were determined as a function of passage number. Results. Compared to the low passaged cells, the high passaged Caco-2 cells were characterized by less morphological heterogeneity, higher transepithelial electrical resistance, higher transcellular diffusion, lower paracellular diffusion, lower carrier-mediated transport and lower alkaline phosphatase activity. The use of effective transport parameters overestimated the K _m and underestimated P _c but had no effect on J _max. Conclusions. The current results provide experimental evidence that the passaging process significantly affects the biological characteristics and transport properties of Caco-2 cell monolayers. The effects are consistent with a reduction in the functional expression of a brush border enzyme and several transport proteins as passage number is increased. The underlying basis for this appears to be a selection of fast-growing subpopulations from the original heterogeneous Caco-2 cell line during passaging.     

Phospholipid vesicle–bound lysozyme to enhance permeability in human intestinal cells

Background: Oral peptide and protein drug delivery still remain the area of challenges for pharmaceutical scientists due to their low stability and permeability in gastrointestinal (GI) tract. In this study phospholipid vesicle–bound lysozyme were prepared and assessed for their physicochemical properties, secondary structure, and permeation across Caco-2 cells.

Results: Lysozyme was found to be substantially bound onto negatively charged vesicles via electrostatic interaction as evidenced by zeta potential measurements regardless of cholesterol content. In contrast, the size of phospholipid vesicle–bound lysozyme became larger with the increasing cholesterol content. The secondary structure of vesicle-bound lysozyme examined by FTIR was unchanged compared to that in buffer solution. The apparent permeability of vesicle-bound lysozyme across Caco-2 cells monolayer was significantly enhanced with a size dependent manner compared to that of solution.

Conclusion: The permeation across Caco-2 cell monolayers of phospholipid vesicle–bound lysozyme was demonstrated to be significantly enhanced with a size-dependent manner.     

Intestinal absorption mechanisms of berberine,palmatine, jateorhizine,and coptisine: involvement of P-glycoprotein

1. The absorption and transport mechanisms of berberine, palmatine, jateorhizine, and coptisine were studied using a Caco-2 cells uptake and transport model, with the addition of cyclosporin A and verapamil as P-glycoprotein (P-gp) inhibitors and MK-571 as a multidrug resistance-associated protein 2 (MRP₂) inhibitor.

2. In the uptake experiment, berberine, palmatine, jateorhizine, and coptisine were all taken into Caco-2 cells, and their uptakes were increased in the presence of cyclosporin A or verapamil.

3. In the transport experiment, P_app (AP-BL) was between 0.1 and 1.0?×?10⁶ cm/sec for berberine, palmatine, jateorhizine, and coptisine and was lower than P_app (BL-AB). ER values were all >2. Cyclosporin A and verapamil both increased P_app (AP-BL) but decreased P_app (BL-AB) for berberine, palmatine, jateorhizine, and coptisine; ER values were decreased by >50%. MK-571 had no influence on the transmembrane transport of berberine, palmatine, jateorhizine, and coptisine.

4. At a concentration of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no significant effects on the bidirection transport of Rho123.

5. Berberine, palmatine, jateorhizine, and coptisine were all P-gp substrates; and at the range of 1–100 μM, berberine, palmatine, jateorhizine, and coptisine had no inhibitory effects on P-gp.

     

Effects of dietary flavonoids on the transport of cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models

1.?The hypotheses tested were to study cimetidine as a substrate of P-glycoprotein (P-gp) and organic cation transport systems and the modulatory effects of eight flavonoid aglycones and glycosides on these transport systems using Caco-2 and LLC-PK1 cells.

2.?Transport and uptake experiments of (20 µM) ³H-cimetidine were performed with and without co-exposure to quercetin, quercetrin, rutin, naringenin, naringin, genistein, genistin, and xanthohumol. Co-treatment decreased basolateral to apical (B to A) permeability (P_app) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) × 10^?6 cm s^?1 in Caco-2 cells and from 10.76 to 1.65 (quercetin), 2.05 (naringenin), 2.88 (genistein), and 1.95 (xanthohumol) × 10^?6 cm s^?1 in LLC-PK1 cells. Genistin significantly reduced B to A P_app of cimetidine to 1.24 × 10^?6 cm s^?1 in Caco-2 cells. Basolateral intracellular uptake rate of cimetidine was enhanced 145–295% when co-treated with flavonoids. Co-treatment with P-glycoprotein and organic cation transporter inhibitors, verapamil and phenoxybenzamine, resulted in reduced B to A permeability and slower basolateral intracellular uptake rate of cimetidine. Intracellular uptake rate of ¹⁴C-tetraethylammonium (TEA) was reduced in the presence of quercetin, naringenin and genistein in LLC-PK1 cells.

3.?In conclusion, quercetin, naringenin, genistein, and xanthohumol reduced P-gp-mediated transport and increased the basolateral uptake rate of cimetidine. Quercetin, naringenin, genistein, but not xanthohumol, reduced intracellular uptake rate of TEA in LLC-PK1 cells. These results suggest that flavonoids may have potential to alter the disposition profile of cimetidine and possibly other therapeutics that are mediated by P-gp and/or cation transport systems.     

The antitumour effects of eudesmin on lung cancer by inducing apoptosis via mitochondria-mediated pathway in the tumour cells

Context: Limonoids possess broad range of biological activities, including antitumour, antimicrobial and antioxidant activities, etc. Eudesmin (EDN) is a type of limonoid which also possesses various activities. However, there is no report on the antitumour lung cancer (LC) activities of this compound.

Objective: The present study investigates the antitumour effects of EDN and its potential molecular mechanisms.

Materials and methods: The in vitro antitumour effects of EDN on LC A549 cells were evaluated by using MTT assay. The in vivo antitumour effects were investigated on a xenograft athymic nude mouse model. The mice were administered orally with EDN (10, 20 and 40?mg/kg) once daily for 28 days. Effects of EDN on apoptosis-related or signalling proteins (Bcl-2, Bax, caspase-3, caspase-9, P53, Akt and JNK) were assayed by western blot analysis.

Results: EDN showed significant inhibitory effects on the growth of LC A549 cells in vitro with the half maximal inhibitory concentration (IC₅₀) of 18.3?μM. By treating with EDN (10, 20 and 40?μM), expression of caspase-3, caspase-9, Bax, P53 and phosphorylated JNK in A549 cells were significantly upregulated, whereas expression of Bcl-2 and Akt phosphorylation were significantly down-regulated. Interestingly, EDN-induced apoptosis could be attenuated by JNK inhibitor. In addition, in vivo experiments also indicated EDN (10, 20 and 40?mg/kg) had significant antitumour effects (p?Conclusions: Overall, the results indicated that EDN possesses significant antitumour effects on LC and the possible mechanism might be related to induction of mitochondria-mediated apoptosis.