Intestinal Absorption Mechanisms of Five Flavonoids from Malus hupehensis (Pamp.) Rehd. Extracts in Situ Single-Pass Intestinal Perfusion and in Vitro Caco-2 Cell Model

Background: Previous studies have shown that Malus hupehensis (Pamp.) Rehd. extracts have antioxidant, anti-aging and other effects, its bioavailability is low, however its absorption mechanism is still unclear. To investigate the absorption properties of hyperin, quercitrin, phloridzin, quercetin, and phloretin in total avonoids of Malus hupehensis (Pamp.) Rehd. Extracts. Methods: In situ single-pass intestinal perfusion model and in vitro Caco-2 cell model were used in this study. The effects of concentration of the extract, administration time, temperature, different intestinal segments, paracellular pathway were analyzed, and the effect of eux inhibitors, such as the P-gp inhibitor verapamil, the multidrug resistance protein2 (MRP2) inhibitor indomethacin, the breast cancer resistance protein (BCRP) inhibitor reserpine, on the transport were evaluated. As well as EDTA, a tight junction regulator, was studied. Results: The results indicated that the jejunum was the optimal absorption intestine segment of quercitrin, phloridzin, and phloretin. And the greatest absorption intestine segment of quercetin was ileum. Furthermore, it was found that the absorption mechanisms of phloridzin in extract was involved in passive diffusion and the mediation of P-gp and MRP2 should not be neglected. The absorption mechanisms of quercetin and phloretin from extract involved active transport and were accompanied by the participation of eux transporters, such as P-gp, MRP2 and BCRP. And also the paracellular pathway was involved in hyperin and quercitrin. Conclusion: absorption mechanisms ve from eux

The total avonoids were extracted and puri ed from the leaves of Malus hupehensis (Pamp.) Rehd. according to the previous method of our research group [9,29]. In brief, leaves were extracted twice by re uxing for 150 min with 70% ethanol (1:8, w/v). The supernatant was collected and concentrated after centrifugation. Subsequently, total avonoids were puri ed by using HPD 100 macroporous resin.

In situ single-pass intestinal perfusion of Malus hupehensis (Pamp.) Rehd. extracts
Male Sprague-Dawley rats (220 ± 30 g) were used and supplied by the Experimental Animal Center of Xi'an Jiaotong University School of Medicine. The rats were raised under standard temperature, humidity, and light conditions, and had access to a standard rodent diet and water. The study was approved by the Ethical Committee of Shaanxi University of Chinese Medicine. Before surgery, animals were fasted for approximately 12 h, but with free access to water. Rats were anesthetized by intraperitoneal injection of 10% urethane (0.01 mL·g -1 ), and xed on the operating table. Then the abdominal cavity was opened. The duodenum, jejunum, ileum and colon, which were approximately 10cm, inserted the silicone tube and ligation carefully. Washed the contents of the intestines with physiological saline, and then equilibrated with a pre-warmed K-R solution and a drug-containing perfusate at 37 ℃ for 30 min at a ow rate of 0.2 mL·min -1 , respectively, to make the intestinal absorption stabilized. Subsequently, the e uent was collected every 15 min until 120min. The perfusate was collected and weighed. At last, cut off the intestines to measure the length and then sacri ced the rats. The effects of various concentrations (12.52, 25.04, and 50.08 mg·mL -1 ) of Malus hupehensis (Pamp.) Rehd. extracts, different intestinal segments (duodenum, jejunum, ileum, and colon), various transporter inhibitors, including the P-gp inhibitor verapamil hydrochloride (100 μmol·L −1 ), MRP2 inhibitor indomethacin (50 μmol·L −1 ) and BCRP inhibitor reserpine (50 μmol·L −1 ), and the tight junction regulator EDTA (20 μmol·L −1 ) on the absorption of extracts were studied.
Intestinal perfusion samples were taken 200μL precisely, then added 800 μL of methanol to vortex for 60 s. After that, centrifuged at 13500 r·min -1 for 10 min, then ltered the supernatant through a 0.22 μm microporous membrane and injected 10 μL to analysis by HPLC.

Data analysis
Using the gravimetric formula, the apparent absorption coe cient ( ) of ve avonoids in the extract of Malus hupehensis (Pamp.) Rehd. extract was calculated. The formula is as follows: where V in and V out are the volumes of the perfusate (mL) entering and leaving the intestine, respectively; C in and C out are concentrations of the ve avonoids from extracts in the perfusate at the inlet and outlet, respectively; Q in is ow rate of the perfusion; l and r are the length (cm) and radius (cm) of the perfused intestinal segment, respectively. All data were analyzed using SPSS 19.0 and expressed as the mean ± SD (n = 3). Differences between groups were calculated using unpaired t-test. When p < 0.05, it was represented statistically signi cant.

Establishment of monolayer cell model
Caco-2 cells were cultured with DMEM containing 10% fetal bovine serum, 1% 100 IU·mL −1 penicillin and 100μg·mL −1 streptomycin at 37 ℃ in a humidi ed atmosphere of 5% CO 2 . Then, cells were seeded into 12-well plates with Transwell culture inserts at a density of 1×10 5 cells per insert and cultured for 21 days. The medium was changed once every two days in the rst week, and replaced with fresh medium every day after one week. The TEER value was used to monitor the integrity of a monolayer, that was used for transport studies with a TEER value above 200Ω·cm 2 . Differentiation of Caco-2 cells was checked on the 3rd, 7th, 14th, and 21st days by observing cell morphology with light microscopy and determining the activity of alkaline phosphates with an assay kit.

Transport studies
Warmed HBSS (37℃) was used as the transport buffer for bidirectional transport studies including absorption transport from the AP to the BL side and secretion transport from the BL to AP side. Before transport studies, the cell monolayer was washed twice with warm (37℃) HBSS and then the monolayers were incubated with the HBSS for 30 min, after pre-incubation the incubation medium was aspirated, the concentration of Malus hupehensis (Pamp.) Rehd. extracts, which was dissolved in HBSS at 50, 100, and 150 μg·mL -1 , was loaded onto the AP (0.5 mL) or BL side (1.5 mL) and blank HBSS was added to the other side, respectively. After loading, a sample aliquot of 0.2 mL was taken from the BL or the AP receiver chamber at different time points (30, 60, 90, 120, 150 and 180 min). After each sampling, an equal volume of HBSS was added to the receiver chamber to keep a volume constant. In this study, we explored the transportation of extract on the different concentration, different temperatures (4℃ and 37℃), different e ux inhibitors, including the P-gp inhibitors verapamil (100 μmol·L −1 ), the MRP2 inhibitor indomethacin (50 μmol·L −1 ) and the BCRP inhibitor reserpine (50 μmol·L −1 ), and the tight junction regulator EDTA (20 μmol·L −1 ). All samples were stored at −20 °C until assayed. Mass spectrometry detection conditions ESI source was used as the source, the detection mode was negative ion detection with the multiple reaction-monitoring (MRM). The optimized instrument parameters were as follows: desolvation temperature was 550℃; nebulizer was 50 psi; both nebulizer and blowback gas and collision gas were nitrogen; the declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) of the ve avonoids from Malus hupehensis (Pamp.) Rehd. extracts and bergenin (IS) are presented in Table 1. at 13500 r·min -1 for 15 min, and the supernatant was ltered through a 0.22 μm microporous membrane. 5 μL sample was injected to analysis and determine the concentrations of hyperin, quercitrin, phloridzin, quercetin and phloretin by LC-MS/MS.

Data analysis
The apparent absorption coe cient and ER value of ve avonoids from the extracts of Malus hupehensis (Pamp.) Rehd. were calculated. The formula is as follows: where P app (BL-AP) and P app (AP-BL) are the apparent permeability coe cients of the drug from the BL side to the AP side and from the AP side to the BL side, respectively. The value of dQ/dt is the cumulative transport rate of the compound on the receiver side. A is the surface area of the membrane, and C 0 is the initial drug concentration in the donor chamber. ER is e ux rate. All data were analyzed using SPSS 19.0 and presented as the mean ± SD (n=3). Differences between groups were calculated using unpaired t-test. When p<0.05, it was considered statistically signi cant.  and (0.12 ± 0.05) ×10 -5 cm·s −1 , respectively, indicating that EDTA can promote the absorption of hyperin. However, e ux inhibitors did not signi cantly affect the absorption of hyperin. In the Fig. 3b, we found that indomethacin or EDTA existed, the P app values of quercitrin were signi cantly increased in the intestine, suggesting that quercitrin was the substrate of the MRP2 and the mechanism of the paracellular may be involved. As shown in Fig. 3c, when phloridzin were combined with verapamil, a P-gp inhibitor, or indomethacin, a MRP2 inhibitor, the Papp values were signi cantly increased, speculating that P-gp and MRP2 may be the substrate of the phloridzin. The results of the effects of inhibitors on quercetin were shown in Fig. 3d. In the absence of inhibitors, the P app values were (0. 16  The effects of extract on cell viability were examined by MTT assay, and the results are shown in Fig.4.
The cell viability in the presence of 40-180 μg·mL −1 extract was greater than 80%. Therefore, transportation studies were carried out at concentrations of 50, 100, and 150 μg·mL −1 extract.

Results of the establishment of monolayer cell model
Under the microscope, it was found that the Caco-2 cells were round or ovoid on the 3rd day. On the 7th day, the cells were fused into pieces. On the 14th day, the cells were uniform and dense. On the 21st day, the cells have merged into a monolayer. Beyond that, the results of the activity of alkaline phosphates (AKP) were shown in Fig.5. On the 3rd day, the AKP ratio was only 1.58, so the activity was low. When the cell was cultured to 14th day, the ratio of AKP reached to 2.12 which was signi cantly increased; when cultured to 21 days, the ratio reached 2.71. In addition, when the cells were cultured for 21 days, the results of TEER values were shown in Fig.6, which was reached to 380.22 Ω·cm 2 and was greater than 200 Ω·cm 2 . These results indicated that the cell monolayer was established successfully.

The result of transport
The effects of the concentration on the transport of ve avonoids from Malus hupehensis (Pamp.) Rehd. extracts were shown in Fig. 7. It was found that the P app values increased from (0.08±0.01) × 10 -6 cm·s -1 to (0.25±0.03) × 10 -6 cm·s -1 from (1.98±0.12) × 10 -6 cm·s -1 to (4.13±0.12) × 10 -6 cm·s -1 and from (1.52±0.04) × 10 -6 cm·s -1 to (2.39±0.03) × 10 -6 cm·s -1 in the AP-BL direction of hyperin phloridzin and phloretin with the increase of concentration, respectively. However, the P app values of quercitrin were increased with the concentration increase from 50 μg·mL −1 to 100 μg·mL −1 , but the Papp values of quercitrin reduced with the concentration increase from 100 μg·mL −1 to 150 μg·mL −1 . About quercetin, there were no signi cant changes as its concentration increased. The results of the ER about ve avonoids from extract were shown in Table 2. At three concentrations, the ER of hyperin and phloridzin are both less than 1.5, the ER of quercitrin, quercetin, and phloretin are higher than 1.5, suggesting that quercitrin, quercetin, and phloretin may be involved in active transport, and there may be involvement of e ux transporters.
The effect of several inhibitors on the transport of ve avonoids was shown in Fig. 9. Compared with the control group, the Papp values of phloretin were signi cantly increased after adding verapamil (p < 0.05), indicating phloretin is the P-gp substrates; after indomethacin was added, the P app values of quercitrin, quercetin, and phloretin from AP to BL was increased signi cantly (p < 0.05), it was speculated that the quercitrin, quercetin, and phloretin were the substrates of MPR2; for the addition of reserpine, the Papp values of quercetin was increased signi cantly (p < 0.05) in the direction of AP-BL, suggesting that the quercetin was the substrates of BCRP. After adding EDTA, the Papp values of hyperin and quercitrin were signi cantly increased (p < 0.05), indicating that the EDTA can promote the transport of hyperin and quercitrin, so these may involve the transport mechanism of paracellular.

Discussion
In this study, the intestinal absorption mechanisms of hyperin, quercitrin, phloridzin, quercetin and phloretin in Malus hupehensis (Pamp.) Rehd. extracts were investigated by using the Caco-2 cell model and single-pass intestinal perfusion model. It was known that the bioavailability and pharmacological effects are related to the interaction of avonoids or other ingredients in the extract in the intestinal absorption, in which the ABC transporters and related enzyme systems played an important role [28].
In situ single-pass intestinal perfusion model, it could be inferred that site-dependence occurred in the intestinal absorption process of ve avonoids from the extract. Such as the best intestinal absorption of quercitrin, phloridzin, and phloretin was jejunum, and the ileum may be the best absorption intestine segment of quercetin. These results were similar to the result of related literatures, those reported that quercetin, from Hippophaë rhamnoides extracts, has the best absorption effect in the ileum [14], and the best absorption intestinal segments of the phloridzin monomer is the jejunum [30]. These results were possibly related to the pK a of the drug, the degree of dissociation, pH of the intestinal uid, the surface area of intestine mucosa, the thickness of mucous layers, the activities of enzyme, the ow rate of capillary blood, the components of membrane, the resistance of tight junction, and the distribution of e ux transporters and uptake transporters in the different intestinal [31,32]. In addition, we also found the P app values of the hyperin quercitrin, and quercetin were lower than 3 × 10 -6 cm·s −1 in the four segments, however the P app values of the phloridzin and phloretin were between 3 × 10 -6 cm·s −1 and 2×10 -5 cm·s −1 in jejunum. It has been reported in the literature that at a P app value of < 3 × 10 -6 cm·s −1 , the drug is not well absorbed, whereas at a P app value of > 2×10 -5 cm·s −1 , the drug is well absorbed [33,34]. Therefore, our data showed that hyperin quercitrin, and quercetin absorbed poorly, but the phloridzin and phloretin absorbed better in jejunum. Other than these, P-gp, MRP2, and BCRP, as ABC transporters, are distributed in the intestine. These e ux proteins located in the apical membrane may drive compounds back into the intestinal lumen from inside the cell, preventing their absorption into blood. This e ux mechanism plays a key role in limiting the absorption and accumulation of drugs [35]. By studying different e ux protein inhibitors, it was found that the e ux proteins involved in the single-pass intestinal perfusion of the ve avonoids, which means quercitrin is the substrate for MRP2, phloridzin is the substrate for P-gp and MRP2, as well as quercetin and phloretin are the substrate for P-gp, MRP2, and BCRP. EDTA, as a regulator to open tight junctions, can react with Ca 2+ and Mg 2+ ions in the mucus layer to change the viscosity of the mucus, further to improve the permeability of the drug and enhance the absorption and bioavailability during paracellelar transport of drugs [36,37]. In this study, it was found that paracellular pathway was involved in hyperin and quercitrin.
In the Caco-2 cell model presented similar results from single-pass intestinal perfusion model. In the whole transmembrane transport experiments, the integrity is extremely critical. Hence, prior to the transport experiments, we measured the TEER values of Caco-2 cell monolayers and the activity of alkaline phosphates. The TEER value is one of the indicators for detecting the integrity of the Caco-2 cell monolayer model. The more complete the monolayer model, the greater the TEER value formed. It is generally believed that when TEER > 200 Ω·cm 2 , a cell monolayer model has been formed. Alkaline phosphatase is a marker enzyme of the brush border of the small intestine epithelium, which can differentiate during the formation of Caco-2 cell monolayer. By measuring the alkaline phosphatase activity of Caco-2 cells at various stages, the biochemical characteristics and cell polarity of Caco-2 cells can be re ected [38]. In this study, The TEER values were above 300 Ω·cm 2 , indicating that the cells formed a dense monolayer. Besides, when the cells monolayer cultured to 21 days, the AKP ratio of AP/BL reached 2.71, which was proved that the distribution of alkaline phosphatase is asymmetric and obvious polarization phenomenon was occurred. In the transport studies, it was found that there was a signi cant difference between the high and low concentrations of P app about the hyperin, phloridzin, and phloretin (p < 0.05), and its ER 1.0, so it can be speculated that the absorption mechanism of hyperin and phloridzin is mainly passive transport and involved active transport. The ER values of quercitrin, quercetin, and phloretin were higher than 1.5, indicating that the transport of quercitrin, quercetin and phloretin may involve active transport or some e ux transporters were participated. Since the temperature can affect the energy supply during active transport, as well as the cell activity, the speed of movement of intestinal villi on the AP side, and the gap between cells [39], P app values were studied at different temperatures. The P app value of phloridzin and phloretin were found to be signi cantly higher at 37°C than at 4°C, suggesting that the absorption process of phloridzin and phloretin may be affected by the temperature, which speculated that an active transport process may be involved. In addition, we observed that P app values of ve avonoids were higher than 0.1 × 10 -6 cm·s -1 at three concentrations. It has been reported that P app values of drugs are above 1.0×10 -6 cm·s -1 that are completely absorbed in the intestine. Drugs whose absorption is more than 1% but less than 100% have P app values of 0.1-1.0×10 -6 cm·s -1 , while drugs whose absorption is less than 1% have P app values of less than or equal to 1.0 × 10 -7 cm·s -1 [40]. Therefore, it can be considered that the absorption of ve avonoids in the extract is between 1% and 100%. When studying the effects of verapamil, a P-gp inhibitor, on the absorption of extracts, it was found that P app values of phloretin were signi cantly higher than those when no inhibitor was added, suggesting that the phloretin was the substrate of the P-gp. It has been reported that quercetin monomer is approved not to be a substrate of P-gp, which is consistent with our results [41]. Meanwhile, it was showed a signi cant increase in the absorption of quercitrin, quercetin and phloretin in the presence of a MRP2 inhibitor, indicating that quercitrin, quercetin and phloretin were the substrate of MRP2, which was in agreement with literatures these reported that quercetin have a comparably strong a nity to MRPs [14,41]. One possible reason was that avonoids aglycone such as isorhamnetin, quercetin, and kaempferol could be transformed into sulfate and glucuronide conjugates after absorption in the intestine and then MRP2 could mediate the transport of their conjugated compounds [14]. However, a strange phenomenon was observed from Fig.9, indomethacin and reserpine reduced the Papp values of phloridzin. This may be due to the bi-directional regulation of e ux protein by other compounds in Malus hupehensis (Pamp.) Rehd. For example, the relative protein and mRNA intensities of MRP2 were down-regulated by 4.5 μg / mL of isorhamnetin, but up-regulated by 18 μg/ml of isorhamnetin. This bi-directional regulation may be related to some key receptors of MRP2, which needs to be explored in future research [28]. Besides, we found EDTA can open the tight junction to promote the absorption of the hyperin and quercitrin, speculating that the paracellular pathway may be involved in hyperin and quercitrin. This is consistent with the results of single-pass intestinal perfusion model.
In short, the absorption mechanisms of ve avonoids from Malus hupehensis (Pamp.) Rehd. extracts are related to the concentration of the drugs, intestinal segments, and e ux protein. In addition, different absorption mechanisms of ve avonoids from extracts may be due to their different structure [42], enterohepatic circulation and intestinal ora [14]. Such as the absorption processes of three avonoids (isorhamnetin, quercetin, and kaempferol) from Hippophaë rhamnoides extracts involves enterohepatic circulation and intestinal ora [14]. Furthermore, the interaction of various components in the extracts may also affect the absorption of the drug. Therefore, further study of the absorption mechanism of extract is not negligible.

Conclusion
In this present study, the effects of the different concentrations, time, temperature, tight junction regulators, and transporter inhibitors on the transport of ve avonoids from Malusa hupehensis (Pamp.) Rehd. extracts were investigated by the Caco-2 cell model in vitro and the single-pass intestinal perfusion in situ. We found that the transport mechanisms of hyperin may involve passive transport and paracellular pathway, quercitrin may involve active transport and paracellular pathway, as well as the MRP2 was involved, phloridzin may involve passive transport and be substrate for P-gp and MRP2, and quercetin and phloretin may involve active transport and the substrate for P-gp, MRP2, and BCRP. In addition, the dependent of the intestinal site is also an indispensable role for the absorption of the ve avonoids.