Analysis of the of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. by PBET digestion in vitro/Caco-2 cell model and their cumulative risk assessment / 药学学报

Inductively coupled plasma mass spectrometry (ICP-MS) was applied to determine the concentrations of lead (Pb), cadmium (Cd) and arsenic (As) in Lindera aggregata (Sims) Kosterm. The physiologically based extraction test (PBET) digestion in vitro/Caco-2 cell model was established to investigate the bioaccessible contents of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. The target-organ toxicity dose modification of HI method (TTD) was used to evaluate the cumulative risk caused by the combined exposure of the total levels of Pb, Cd and As in Lindera aggregata (Sims) Kosterm. and the bioaccessible contents in the decoction. The results showed that the total contents of Pb, Cd and As in 4 batches of samples were in the range of 2.901-3.872, 1.299-1.800 and 0.062-0.216 mg·kg-1, respectively. After transportation by Cacco-2 cells, the bioaccessible contents of Pb, Cd, and As in the decoction were in the range of 0.045-0.080, 0.070-0.112 and 0.004-0.018 mg·kg-1. The results of risk assessment showed that calculated by the total amounts of heavy metals in the Lindera aggregata (Sims) Kosterm., for the end points of nervous system, the cumulative risks of co-exposure of heavy metals in 3 batches of samples were of concern. After decoction and transportation by Caco-2 cells, for the end points of cardiovascular system, blood, nervous system, kidney and testis, the TTD modification of HI values of all batches of samples were less than 1, and the health risks were acceptable. The study provided methodology basis for a more objective assessment of the health risks of heavy metals and harmful elements in traditional Chinese medicine and for a more scientific limit standard of heavy metals and harmful elements.

Effects of HPMCAS MF on absorption of silybin from supersaturable self-nanoemulsifying drug delivery system / 中国中药杂志

To evaluate the effects of Hydroxypropyl methylcellulose acetate succinate(HPMCAS MF) on absorption of silybin(SLB) from supersaturable self-nanoemulsifying drug delivery system which was pre-prepared at the early stage experiment. The cell toxicity of self-emulsifying preparation was evaluated by the MTT method, and the in vitro membrane permeability and absorption promoting effect of the self-emulsifying preparation were evaluated by establishing a Caco-2 cell monolayer model. The in vivo and in vitro supersaturation correlation was evaluated via the blood concentration of SLB. The results of MTT showed that the concentration of the preparation below 2 mg·mL~(-1)(C_(SLB) 100 μg·mL~(-1)) was not toxic to Caco-2 cells, and the addition of polymer had no significant effect on Caco-2 cells viability. As compared with the solution group, the transport results showed that the P_(app)(AP→BL) of the self-emulsifying preparation had a very significant increase; the transport rate of silybin can be reduced by polymer in 0-30 min; however, there was no difference in supersaturated transport between supersaturated SLB self-nanoemulsion drug delivery system(SLB-SSNEDDS) and SLB self-nanoemulsion drug delivery system(SLB-SNEDDS) within 2 hours. As compared with SLB suspension, pharmacokinetic parameters showed that the blood concentration of both SLB-SNEDDS and SLB-SSNEDDS groups were significantly increased, and C_(max) was 5.25 times and 9.69 times respectively of that in SLB suspension group, with a relative bioavailability of 578.45% and 1 139.44% respectively. C_(max) and relative bioavailability of SLB-SSNEDDS were 1.85 times and 197% of those of SLB-SNEDDS, respectively. Therefore, on the one hand, SSNEDDS can increase the solubility of SLB in gastrointestinal tract by maintaining stability of SLB supersaturation state; on the other hand, the osmotic transport process of SLB was regulated through the composition of its preparations, and both of them could jointly promote the transport and absorption of SLB to improve the oral bioavailability of SLB.

Effect of bile on intestinal alkaline phosphatase in intestinal mucosa / 中华肝胆外科杂志

Objective:To investigate the expression of intestinal alkaline phosphatase (IAP) in intestinal mucosa with bile deficiency and the effect of bile on the expression of IAP in intestinal epithelial Caco-2 cell model.Methods:Thirty healthy male SD rats were randomly divided into control group (Ctrl, n=10), external drainage group (ED, n=10) and obstructive jaundice group (OJ, n=10). Ileum specimens were collected on the 7th day after modeling. Western blot and immunohistochemical staining were used to determine the expression of IAP in rat intestinal mucosa. Different concentrations of human bile were used to treat on Caco-2 cells, and Western blot was used to detect the changes in IAP expression in Caco-2 cells. Results:Rat models were successfully established. The expression level of IAP in the intestinal mucosa of ED group [(9.19±1.67)%] was significantly lower than that of the Ctrl group [(15.09±0.61)%, P<0.05]; the expression of IAP in the intestinal mucosa of OJ group [(6.86±1.07)%] was significantly lower than that of the Ctrl group ( P<0.05). Through in vitro cell experiments, expression of IAP in Caco-2 cells was increased in a time and dose-dependent manner when treated with human bile. Conclusions:Bile deficiency in the intestine can cause inhibition of IAP in the intestinal mucosa. Bile can promote the expression of IAP in intestinal mucosal epithelial cells.

Studies on absorption and transportation of coumarins in Angelica dahurica 'Yubaizhi' across human intestinal epithelial by using human Caco-2 cell monolayers / 中国中药杂志

The absorption is the key to the resulted efficacy of orally administered drugs and the small intestine is the main site to absorb the orally administered drug. In this paper, internationally recognized human colon adenocarcinoma cell line(Caco-2) monola-yer model which can simulate small intestinal epithelial cell was used to comparatively study the absorption and transportation diffe-rences of total coumarins and main individual coumarin in Angelica dahurica 'Yubaizhi' by separately using 6-and 12-well plates. It was found that apparent permeability coefficient(P_(app)) values of oxypeucedanin hydrate, byakangelicin and phellopterin were at the quantitative degree of 1 × 10~(-5) cm·s~(-1) when the individual administration was conducted independently, indicating that they were well-absorbed compounds. P_(app) ratio of their bi-directional transportation was close to 1, indicating that they can be absorbed across Caco-2 monolayer by passive diffusion mechanism without carrier mediation during the transportation. The similar trend of transportation was also observed for imperatorin, isoimperatorin and bergapten. The P_(app) values of oxypeucedanin hydrate, byakangelicin and bergapten were at quantitative degree of 1 × 10~(-5) cm·s~(-1) when the administration of total coumarins in Angelica dahurica 'Yubaizhi' was conducted, indicating that they were well-absorbed compounds. The results were consistent with those of independent administration of individual coumarins. Whereas, the P_(app) values of imperatorin, phellopterin and isoimperatorin in the total coumarins decreased, indicating that the interaction between compounds may exist although the P_(app) value ratio of bi-directional transportation was between 0.5 and 1.5. The results laid the foundation for intestinal absorption study of Angelica dahurica 'Yubaizhi' coumarins in compound Chinese medicine.

Study on oral absorption mechanisms of puerarin in nanocrystals self-stabilized pickering emulsion / 中国中药杂志

Nanocrystals self-stabilized Pickering emulsion(NSSPE) is a new kind of emulsion where only nanocrystals of poorly soluble drugs are used as stabilizers. Our previous study showed that NSSPE with Ligusticum chuanxiong oil as the main oil phase can significantly promote oral absorption of puerarin. The present study aimed to explore its absorption mechanism in oral administration. The in vitro dissolution test was carried out to study the effect of NSSPE on release of puerarin. The effects and mechanism of NSSPE on uptake and transport of puerarin across Caco-2 cell were investigated. The results showed that the drug release rate of NSSPE was similar to that of nanocrystals, with their cumulative dissolution of puerarin not affected by pH of releasing mediums, both significantly higher than that of crude material. The uptake of puerarin in NSSPE was concentration-dependent and significantly higher than that of solution or surfactant stabilized emulsion. Genistein and indomethacin, inhibitors of lipid rafts/caveolin, could significantly reduce the uptake of puerarin in NSSPE. Compared with solution, NSSPE and surfactants stabilized emulsion obviously increased transport rate K_a and apparent permeability coefficient P_(app) of puerarin in AP → BL direction, but there was no significant difference in BL → AP direction. It could be inferred that there were both passive and active transport mechanisms, as well as lipid raft/caveolin mediated endocytosis for absorption of NSSPE. The promoted oral absorption of puerarin in NSSPE was mainly related to the existing nanocrystal form which could promote dissolution, puerarin as well as Ligusticum chuanxiong oil which could promote drug transmembrane transport and inhibit drug efflux. It is the unique structure and composition of the compound NSSPE that promoted the oral absorption of puerarin.

Effects of low molecular weight chitosan and its nanoparticles on intestinal permeability of Panax notoginseng saponins / 中草药

Objective: To evaluate the effect of low molecular weight chitosan (LMW-CTS) and its nanoparticles (LMW-CTS-NPs) on the intestinal permeability of Panax notoginseng saponins (PNS) by using Caco-2 cell model. Methods: LMW-CTS was prepared by combining chitosanase hydrolysis combined with ultrafiltration separation technology, and molecular weight of LMW-CTS was determined by using permeation gel chromatography (GPC). LMW-CTS-NPs were prepared by ionic gel method, and characterized by scanning electron microscopy, nano particle sizer, and flourier transformation infrared spectroscopy. Caco-2 cell model was established and validated to evaluate the effects of LMW-CTS and LMW-CTS-NPs on the intestinal permeability of PNS. Results: LMW-CTS has a molecular weight of 5 760 and a polydispersity coefficient of 1.42. LMW-CTS-NPs have a round shape and narrow particle size distribution, with an average particle size of 115.5 nm and zeta potential of +37.1 mV. The apparent permeability coefficients (Papp, AB→BL) of PNS was less than 1 × 10-6 cm/s, indicating a poor permeability. In LMW-CTS group, the Papp of R1 and Rg1 was increased by 17.83% and 20.29%, respectively, but no significant effect of promotion was observed on other components. However, the Papp of R1, Rg1, Re, Rb1, and Rd in LMW-CTS-NPs group was increased by 35.66%, 23.28%, 29.41%, 37.99%, and 36.00%, respectively, compared tothe control group. Conclusion: LMW-CTS can significantly promote the intestinal mucosal permeability of R1 and Rg1 in PNS, but has no significant effect on Re, Rb1, and Rd. LMW-CTS-NPs significantly increased the permeability of the major monomer saponin components in PNS. Namely, the intestinal permeability of PNS can be further improved by transforming LMW-CTS into LMW-CTS-NPs.

Preparation of silybin self-nanomicroemulsion containing functional oil and its in vitro evaluation / 中草药

Objective: In order to improve the bioavailability of the insoluble drug silybin, silybin supersaturated self- nanoemulsifying drug delivery systems (SLB-S-SNEDDS) containing functional oil were prepared, its characterization and in vitro evaluation were also performed. Methods: Functional oils were screened by performing potassium ferrohydride reduction and 1,1- diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiments. The pseudo-ternary phase diagram was drawn to investigate the emulsifying ability of emulsifier. The proportion of mixed oil phase and drug loading capacity were explored by analyzing particle size, polydispersity index (PDI), zeta potential, etc. The type and concentration of supersaturated substance in SLB-S-SNEDDS were obtained by conducting the compatibility and dissolution experiments. SLB-S-SNEDDS was characterized with appearance, particle size distribution, self-emulsification efficiency, and morphology, and its in vitro dissolution, antioxidant capacity, and cytotoxicity were also evaluated. Results: The prescriptions of SLB-S-SNEDDS were as follows: (1) wheatgerm oil/Capryol 90- Cremophor ELP-Transcutol HP; (2) seabuckthorn seed oil/Capryol 90-Cremophor ELP-Transcutol HP. One g S-SNEDDS matrix contained 0.043 g of wheatgerm oil or sea-buckthorn seed oil, 0.387 g of Capryol 90, 0.380 g of Cremophor ELP, and 0.190 g of Transcutol HP. The adding amount of silybin in S-SNEDDS prescription was 20% of the sum of the equilibrium solubility of silybin in each component, and the adding amount of Soluplus was 0.1% of the total mass described above. The two obtained SLB-S-SNEDDS were transparent homogeneous liquid with light yellow (wheat germ oil) and bright yellow (seabuckthorn seed oil) color, respectively. After being dispersed, SLB-S-SNEDDS turned into subspherical white flat emulsion droplets with the particle size of about 50 nm, and the emulsification time was 65 s. Compared with raw materials and SLB-SNEDDS, the cumulative dissolution of silybin in SLB-S-SNEDDS was maintained between 85% and 110% within 8 h, indicating that the two systems can significantly improve the dissolution of silybin. The absorbance of SLB-S-SNEDDS after reaction with potassium ferricyanide (0.452-0.782, 0.488-0.765) and the DPPH free radical clearance of SLB-S-SNEDDS (39.09%-96.02%, 30.54%-89.20%) were all higher than those of raw silybin (0.411-0.760, 22.89%-63.21%), which suggested that the two systems can enhance the antioxidant capacity of silybin. Cytotoxicity test results showed that the cell survival rate in silybin raw material group, combination of silybin and S-SNEDDS group, and blank S-SNEDDS group were greater than 90% at 5 µmol/L and 10 µmol/L drug concentration, indicating that SLB-S-SNEDDS and its auxiliary materials were safe and less toxic to human cloned colorectal adenocarcinoma cell line (Caco-2). Conclusion: The SLB-S-SNEDDS containing functional oil prepared in this paper can not only increase the cumulative dissolution of silybin, but also enhance its antioxidant capacity, which provides a useful reference for supersaturated self-nanoemulsifying drug delivery systems (S-SNEDDS) to improve the water-solubility and bioactivity of insoluble drugs.

The effect of the shape of nanocarriers on their transmembrane across Caco-2 cell monolayer / 药学学报

Cerasomes with different shapes were constructed to investigate the effect of the nanocarriers' shape on the cellular uptake and transmembrane capacity. Cerasome-forming lipid (CFL) was synthesized via halogenation, nucleophilic addition and acylation reaction and detected by mass spectrometry and nuclear magnetic resonance spectroscopy. CFL and short chain 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were employed to prepare organic-inorganic hybrid bicelles in discal shapes (nanodisc) by the thin-film hydration method, and CFL was also used to prepare spherical cerasomes (nanosphere). The particle size and zeta potential of nanocarriers were measured by dynamic light scattering analysis, and the morphology was observed by transmission electron microscopy. With human colon cancer cell line Caco-2 as the model, the effect of the shape of nanocarriers on cellular uptake and transmembrane capacity was investigated qualitatively by confocal laser scanning microscope (CLSM), and the transmembrane capacity was analyzed quantitatively by high performance liquid chromatography (HPLC). The results showed that nanosphere and nanodisc had similar particle diameters around 110 nm and similar zeta potential around -25 mV, with regular morphology under transmission electron microscope. The cellular uptake rate of nanodisc was significantly higher than that of nanosphere in 20 minutes. Further research on Caco-2 cell monolayer demonstrated that nanodisc with faster uptake had less accumulation in the monolayer, which means it had a higher transmembrane rate on Caco-2 cell monolayer and the transmembrane capacity of the nanodisc was better than that of nanosphere within 2 h. These results suggest that rational design of the shape of nanocarriers is expected to regulate nano-bio interactions, promote the transmembrane transport of nanocarriers, and improve the drug absorption.

Effects of sildenafil on the proliferation of Caco-2 cells and inflammatory response in NCM460 cells / 中国药科大学学报

@#To investigate the inhibitory effect of sildenafil on Caco-2 cell proliferation and its anti-inflammatory effect on menadione-induced NCM460 cell inflammation model, MTT assay was used to determine cell proliferation. Intracellular reactive oxygen species(ROS)and nitric oxide(NO)levels were detected by fluorescent probe. Western blot was used to detect the expression of eNOS/ERK/JNK pathway related proteins in Caco-2 cells and correlated inflammatory cytokines in NCM460 cells. The effect of sildenafil on the growth of two probiotics was determined by spectrophotometry. Results showed that sildenafil signi-ficantly inhibited the proliferation of Caco-2 cells and enhanced the expression levels of eNOS, p-eNOS, p-JNK1/2 and p-ERK1/2 proteins in Caco-2 cells; while after adding NG-nitro-L-arginine methyl ester(L-NAME), the expression levels of eNOS, p-eNOS, p-JNK1/2 and p-ERK1/2 proteins were significantly lower than those of the sildenafil group. Compared with the menadione group, sildenafil significantly reduced ROS levels in NCM460 cells and inhibited the expression levels of IL-6, IL-1β, p62, and TNF-α. Moreover, high concentrations of sildenafil had no obvious toxic effects on Lactobacillus casei and Lactobacillus rhamnosus. Thus, the results indicated that sildenafil could effectively inhibit the intestinal inflammatory response without affecting the balance of the intestinal flora, and prevent colorectal cancer by reducing the oxidative stress responses in the intestinal cells.

Role of intracellular Ca2+ in regulating NHE3 expression in Caco-2 cells during rotavirus infection / 中华微生物学和免疫学杂志

Objective To investigate the effects of rotavirus ( RV) on the expression and bioactiv-ity of Na+-H+ exchanger 3 ( NHE3 ) in Caco-2 cells and the possible regulatory mechanism. Methods Caco-2 cells expressing NHE3 were constructed and divided into four groups as follows: control ( CTL ) group, RV group, BAPTA-AM ( a Ca2+ chelator) group and BAPTA-AM+RV group. Na+-H+ exchanger ac-tivity and NHE3 expression on cell surface were determined using BCECF-AM and biotinylation assay, re-spectively. Expression of Cdc42 at protein level was measured by Western blot. Results Compared with the control group, RV infection significantly decreased the activity of NHE3 and its expression on cell surface. BATPA-AM antagonized the inhibitory effects on NHE3. Moreover, the expression of Cdc42 at protein level was increased following RV infection, which was also antagonized by BATPA-AM. Conclusions Intracellu-lar Ca2+-mediated Cdc42-dependent endocytosis pathway might be involved in regulating the expression and bioactivity of NHE3 during RV infection.

Intestinal uptake of barley protein-based nanoparticles for -carotene delivery

Our previous study introduced a barley protein microparticle for encapsulation of hydrophobic drug/nutraceutical, which could release nanoparticles upon gastric digestion and deliver encapsulated compound to a simulated intestinal environment intact. This work focused on evaluating the potential of liberated nanoparticles to improve the absorption of encapsulated compounds (., -carotene) using Caco-2 cell and small intestine models. Nanoparticles obtained from gastric digestion of barley protein microparticles had a spherical shape and an average size of 351 nm. Nanoparticles showed low cytotoxicity in Caco-2 cells and their cellular uptake was dependent on time, concentration and temperature. In a Caco-2 cell monolayer model, significantly greater uptake and transport of -carotene were observed when it was delivered by nanoparticles (15%), compared to free -carotene suspension (2.6%). In an rat jejunum model, nanoparticles showed the capacity to retain in small intestinal tissue. Approximately 2.24 and 6.04 μg nanoparticle were able to permeate through each cm intestinal tissue and translocate to the serosal side after 60 and 90 min, respectively. Results from this study demonstrated the absorption improving effect of the barley protein nanoparticles and suggested their potential as vehicles for hydrophobic compounds.

Effect of bioadhesive excipients on absorption of total flavonids from Puerariae Lobatae Radix transporting across Caco-2 cell monolayer / 中草药·英文版

Objective: Pueraria total flavonids (PTF) can treat cardiovascular and cerebrovascular diseases, but it has poor membrane permeability and oral bioavailability. Some excipients, such as carbomer, chitosan, and hydroxypropyl methylcellulose, can improve the oral bioavailability. Traditional in vitro evaluation techniques, including the rat intestinal perfusion and cell line models, cannot evaluate PTF absorption and holistic transporters. Methods: This study evaluated excipients’ adhesiveness and effect on PTF transport across Caco-2 cell monolayer. cDNA microarrays identified gene expression changes in Caco-2 cells exposed to PTF and PTF with excipients, and revealed the mechanism underlying the effect of excipients on PTF absorption. Results: In vitro adhesion and transport experiments across Caco-2 showed that excipients had higher adhesiveness to gastric mucosa and transport efficiency across Caco-2 cells than PTF alone. The interaction of PTF with excipients significantly changed the expression of some genes, which might influence the absorption rate of PTF. Conclusion: Different bioadhesive polymers can improve intestinal absorption of PTF, which was related to some genes affiliated to the ATP-binding cassette (ABC) and solute carrier transporter (SLC) to some extent.

Absorption Mechanism of Baicalin and Its Solid Lipid Nanoparticles on Caco-2 Cells / 中国药学杂志

OBJECTIVE: To study the absorption mechanism of baicalin (BA) and baicalin solid lipid nanoparticles (BA SLN) by establishing an in vitro Caco-2 cell model. METHODS: A Caco-2 cell model was established. The appropriate concentration of BA and BA SLN in Caco-2 cell monolayer model was screened by CCK-8 method and LDH method. The content of BA was determined by high performance liquid chromatography, and the effects of time, concentration, temperature and endocytosis inhibitor on the uptake of BA and BA SLN were studied by this model. The transport of BA and BA SLN in the presence or absence of efflux inhibitors was also investigated. The expression of efflux protein was detected by Western blot. RESULTS: At 50-150 μg·mL-1, the uptake of BA and BA SLN was concentration-dependent; at 4 to 37 ℃, the uptake of BA and BA SLN increased with increasing temperature; endocytosis inhibitor influenced the cellular uptake of BA SLN; multidrug resistance-associated protein 2 (MRP2) inhibitors and breast cancer resistance protein (BCRP) inhibitors significantly reduced BA efflux, but did not affect BA SLN efflux; blank SLN and BA SLN can reduce the expression of MRP2 and BCRP in cells. CONCLUSION: BA is taken up and transported by small intestinal epithelial cells in a passive manner, and may be accompanied by energy dependence, which is related to MRP2 and BCRP efflux. BA SLN can significantly increase the uptake of drugs in the Caco-2 cell model, which may be related to the increase of endocytic pathway uptake and inhibition of extracellular repressor expression inhibition drug efflux.

Transmembrane absorption and methylation metabolism of quercetin on a caco-2 monolayer model / 中国药理学与毒理学杂志

OBJECTIVE To study transmembrane transport and methylation metabolism of quercetin based on the human intestinal absorption model. METHODS Caco-2 cell monolayer was used as the human intestinal absorption model. The incubation concentration of quercetin was 9.0 and 18.0 mg-L'1. Samples were collected at 30, 60, 90, 120 and 150 min, and the contents of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side were determined by high performance liquid chromatography-mass spectrometry (LC-MS) method. Bupropion was used as the internal standard, and the injection volume was 20 pL. The specific inhibitors of P-glycoprotein (P-pg) or multidrug resistant protein 2 (MRP2), cyclosporins (CysA) 10 mmol-L-1 or MK571 1 mmol • L-1 (final concentration), were added to the apical side. After 15 min incubation, quercetin 9.0 or 18.0 mg-L-1 (final concentration) was added to the apical side, respectively. The quercetin content on the receiver side was determined by the same method. RESULTS During bi-directional transport, the dynamic change of quercetin residues on the loading side showed a continuous decline within 150 min (P<0.05 between adjacent time points), while the amount of quercetin on the receiver side tended to increase before decreasing, reaching the peak at 120 min and falling at 150 min (P<0.05 between adjacent time points). Isorhamnetin and tamarixetin could be detected on both the loading side and receiver side. But the difference was that when quercetin was loaded on the apical side or the basolateral side, there was much more isorhamnetin and tamarixetin on the apical side than on the basolateral side after 30-60 min. Analysis of the percentage of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side found that when incubated for 30 min, the residual quercetin on the loading side was less than 20%-25%, and quercetin on the receiver side was only about 1% of the loading amount. At 150 min, the residual quercetin decreased to <10%, while quercetin in the receiver side was only 6%-7% of the loading amount, and isorhamnetin and tamarixetinin on both sides were only 0.1%-0.3% of loading quercetin. Compared with the quercetin control group, the addition of CysA or MK571 in advance significantly increased the transport of quercetin from the apical side to the basolateral side (P<0.05). CONCLUSION The transport of quercetin on the Caco-2 monolayer cell model shows a trend of rise and fall, accompanied by methylation metabolism. The efflux of P-pg and MRP2 may have an effect on the transmembrane transport of quercetin.

Effect of six bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) on P-glycoprotein and its herb-drug interaction with digoxin / 药学学报

This study was aimed to investigate the effects of six Schisandra lignans of Wuzhi tablet (WZ, a preparation of ethanol extract of Schisandra sphenanthera) on the pharmacokinetic process of digoxin (DG, a classical P-gp substrate) after intravenous and oral administration in rats. The effect of Schisandra lignans on the transportion of DG in Caco-2 cells was further elucidated. Our data showed that the plasma concentrations of DG were increased to different extent following co-administration of schisandrin A, schisandrin B, schisandrol B and schisantherin A, respectively. Schisandrol B showed the most potent effect among the six lignans. However, schisandrin C and schisandrol A showed little effect on pharmacokinetic of DG. Schisandrol B led to 99.0% (P < 0.05) and 109.2% (P < 0.05) increase in the AUC after orally or intravenously administered of DG, suggesting that co-administration of schisandrol B induced a more potent effect on increasing hepatic bioavailability of DG than that of intestinal. Furthermore, in vitro transport experiment showed that schisandrin A, schisandrin B, schisandrol B and schisantherin A inhibited P-gp-mediated efflux of DG, suggested that these lignans inhibited the P-gp-mediated efflux of DG. In conclusion, the exposure of DG in rats was increased when co-administered with Schisandra lignans, and schisandrol B showed the strongest effect. The dramatic increase in oral bioavailability of digoxin in the presence of schisandrol B may be due to the inhibition of hepatic/renal P-gp activity.

Effect of different concentrations of Gegen Qinlian decoction on expression of P-glycoprotein and multi-drug resistance protein transporter in Caco-2 cells / 中国中药杂志

To investigate the effects of Gegen Qinlian decoction on the expression of P-glycoprotein (P-gp) and multi-drug resistance protein (MRP) in epithelial cells of human colon adenocarcinoma Caco-2 cells．The effects of different concentrations of Gegen Qinlian decoction on the expression levels of p-gp and MRP1-6 mRNA in Caco-2 cells were detected by real time quantitative reverse transcriptase PCR (qRT-PCR).12 h after drug treatment (5.00 g·L⁻¹), the expression levels of MDR1 and MRP1-6 were significantly down-regulated at concentration of 5.00 g·L⁻¹; the mRNA expression levels of MDR1，MRP1，MRP2，MRP4，MRP5 and MRP6 were significantly down-regulated at concentration of 2.50 g·L⁻¹; only the expression levels of MRP2 and MRP5 were significantly affected at concentration of 1.00 g·L⁻¹. The results showed that the expression levels of MDR1 and MRP1-6 mRNA in Caco-2 cells could be down-regulated in a dose-dependent manner. Gegen Qinlian decoction may reduce drug efflux by down-regulating the mRNA expression of cell transporters in Caco-2 cell, and increase the time of drug action, thereby enhancing the bioavailability of chemotherapeutic drugs.

Active components from Andrographis paniculata with affinity to Caco-2 cells screened by UPLC-Q-TOF-MS / 中草药

Objective To identify the active components of Andrographis paniculata coupled with Caco-2 cells screened by UPLC-Q-TOF-MS. Methods The cell-combining compounds from A. paniculata were screened based on drug uptake and transport absorption experiment using Caco-2 cells, then the cell dissociation solution was detected before and after treating with A. paniculata by using UPLC-Q-TOF-MS method. The cell-combining compounds from the ethanol extracts of A. paniculata were identified based on the retention time and mass spectrometry information of each chemical composition combined with comparing the extracted ion chromatograms and mass spectrometry datas of reference substances and the related articles. Results A total of 10 compounds combined with Caco-2 cells in ethanol extract of A. paniculata, which were detected and identified as 12,13- dihydroandrographolide (1), skullcapflavone I-2'-O-glucoside (2), andrographolide (3), 14-deoxy-11,12-didehydro andrographiside (4), 7-hydroxy dehydroandrographolide (5), neoandrographolide (6), 3-deoxy dehydroandrographiside (7), deoxyandrographolide (8), dehydroandrographolide (9), and one unidentified component. Conclusion This research establishes a method of screening the active components of A. paniculata coupled with Caco-2 cells using UPLC-Q-TOF-MS analysis, which can be used to screen out the active components of the complex system on traditional Chinese medicines, as well as to lay the foundation for further study of the synergetic compatibility effects among active ingredients.

Study on absorption activity of five components of Polygonum orientale L. Flower in Caco-2 cell monolayer / 中国药理学通报

Aim To observe the absorption activity of five components of Polygonum orientale L. Flower ex-tract in Caco-2 cell monolayer. Methods The effects of different concentrations, time, temperature, pH and P-glycoprotein inhibitors on Caco-2 cells were investi-gated by UPLC-MS/MS. Results The absorption of five components of Polygonum orientale L. Flower ex-tract presented a concentration-and time-dependent manner, and the uptake of quercetin was reduced in Caco-2 cells after 90 min. There was a highest intake at 37℃,and the uptake of four components was best in acid environment except for the quercetin at pH 6 , which was best at pH5 . The uptake of quercetin and kaempferol was significantly improved after the addition of P-glycoprotein inhibitors of verapamil. Conclusions The cellular uptake mechanisms of the five compo-nents of Polygonum orientale L. Flower extract is man-inly through passive diffusion. P-glycoprotein is in-volved in the uptake of quercetin and kaempferol.

Establishment and Validation of Caco-2 Cell in Vitro Absorption Model / 医药导报

Objective To establish and validate the Caco-2 cell in vitro absorption model,so as to play a foundation for next study of drug absorption and transport. Methods Caco-2 cells were cultured on the polycarbonate membrane inserts fixed in the 24-well transwell transport chamber with three types cell density of low,medium and high concentration (5×104,1×105,2× 105·mL-1) respectively.After being cultured for 3,6,9,12,15,18,21 d,the integrity of monolayer were assessed and compared by the cell morphology,growth characters and transepithelial electrical resistance (TEER),aiming to determine the inoculum density and culture time.Then the permeability and polarity were validated by the apical-to-basolateral amount of Lucifer Yellow across cell monolayer,the alkaline phosphatase activity in the apical side (AP),the basolateral side (BL) and intracellular activity. Results The cells of low,medium and high concentration group had fusion into a integrate cell monolayer and the maximum absorbance after being cultured for 15,12,9 d respectively.However,conglobated and dead cells were observed at the later growth stage in the medium and high concentration group and the TEER of cell monolayer were smaller than the low concentration group,which could reach 300 Ω·cm2after cultured 15 d and keep a relatively stable value, then cells were cultured with 5×104·mL-1cell density for 21 d.The Lucifer Yellow apparent permeability coefficient (Papp) was 3.57×10-7 cm·s-1which was lower than 5. 0×10-7cm·s-1according to provision. And the intracellular alkaline phosphates’ activity increased,AP/BL increased by 5 times in day 21. Conclusion The integrity,permeability and polarity of the established Caco-2 cell model in our laboratory was validated,and it can be used as an in vitro model similar with small intestinal epithelium for absorption and transport studies.

Transport Study of Andrographolide Sulfonate on Cell Models / 中国药学杂志

OBJECTIVE: To establish a method for determination of the total content of andrographolide sulfonate, transport of two kinds of main components in the injection in Calu-3 and Caco-2 cell model, can be applied to the assessment of lung and oral administration to the safety and effectiveness of drugs. METHODS: Screening of safe concentration of drugs by cytotoxicity test. Injection of andrographolide total sulfonate in safe concentrationwere applied to Calu-3 and Caco-2 cell models. Samples were taken for a certain period of time to determine the influence of concentration and time on the transport, and the apparent osmotic coefficients of the principal components were calculated. RESULTS: When the concentration of the drug was less than 500 μg•mL-1, the survival rate of Calu-3 and Caco-2 cells was close to 100%. The established cell model showed excellent compactness and integrity. In the Calu-3 cell model, the apparent permeability coefficients of andrographolide sulfate C and 9-dehydro-17-hydro-andrographolide were 1 × 10 -5 orders of magnitude. In the Caco-2 cell model, both were 1 × 10-6 orders of magnitude. On two cell models, the efflux ratios were less than 2. CONCLUSION: The principal component of andrographolide total sulfonate injection is not the substrate for P glycoproteins, it has moderate absorption in the intestine, and has good absorption in the respiratory tract. Pulmonary administration is safer and more effective.