Mechanism of Enhanced Oral Absorption of Morin by Phospholipid

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Mechanism of enhanced oral absorption of morin by phospholipid complex based self-nanoemulsifying drug delivery system Zhirong Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5005806 • Publication Date (Web): 23 Dec 2014 Downloaded from http://pubs.acs.org on January 1, 2015

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Mechanism of enhanced oral absorption of morin by phospholipid complex based self-nanoemulsifying drug delivery system

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Molecular Pharmaceutics mp-2014-005806.R1 Article 27-Oct-2014 Zhang, Jinjie; Sichuan University, Key Laboratory of Drug Targeting, Ministry of Education Li, Jianbo; Sichuan University, Key Laboratory of Drug Targeting, Ministry of Education Ju, Yuan; Sichuan University, Key Laboratory of Drug Targeting, Ministry of Education Fu, Yao; Sichuan University, Gong, Tao; Sichuan University, Key Laboratory of Drug Targeting, Ministry of Education Zhang, Zhirong; WEST CHINA SCHOOL OF PHARMACY, KEY LAB OF DRUG TARGETING & DELIVERY

ACS Paragon Plus Environment

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Jinjie Zhang, Jianbo Li, Yuan Ju, Yao Fu, Tao Gong, Zhirong Zhang Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3, Southern Renmin Road, Chengdu 610041, Pepole’s Republic of China.

Correspondence: Tao Gong, Zhirong Zhang. Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3, Southern Renmin Road, Chengdu 610041, Pepole’s Republic of China. Email [email protected] (T. Gong) [email protected] (ZR. Zhang) Tel + 18 028 8550 1615 (T. Gong)

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For Table of Contents Use Only


Jinjie Zhang, Jianbo Li, Yuan Ju, Yao Fu, Tao Gong, Zhirong Zhang Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3, Southern Renmin Road, Chengdu 610041, Pepole’s Republic of China.

Correspondence: Tao Gong, Zhirong Zhang. Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3, Southern Renmin Road, Chengdu 610041, Pepole’s Republic of China. Email [email protected] (T. Gong)

[email protected] (ZR. Zhang)

Tel + 18 028 8550 1615 (T. Gong)

Table of contents graphic

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Abstract: Phospholipid complex (PLC) based self-nanoemulsifying drug delivery system (PLC-SNEDDS) has been developed for efficient delivery of drugs with poor solubility and low permeability. In the present study, a BCS class IV drug and a P-glycoprotein (P-gp) substrate, morin was selected as the model drug to elucidate the oral absorption mechanism of PLC-SNEDDS. PLC-SNEDDS was superior than PLC in protecting morin from degradation by intestinal enzymes in vitro. In situ perfusion study showed increased intestinal permeability by PLC was duodenum-specific. In contrast, PLC-SNEDDS increased morin permeability in all intestinal segments, and induced a change in the main absorption site of morin from colon to ileum. Moreover, ileum conducted the lymphatic transport of PLC-SNEDDS which was proven by microscopic intestinal visualization of Nile red labeled PLC-SNEDDS and lymph fluids in vivo. Low cytotoxicity and increased Caco-2 cell uptake suggested a safe and efficient delivery of PLC-SNEDDS. The increased membrane fluidity and disrupted actin filaments were closely associated with the increased cell uptake of PLC-SNEDDS. PLC-SNEDDS could be internalized into enterocytes as an intact form in a cholesterol-dependent manner via clathrin-mediated endocytosis and macropinocytosis. The enhanced oral absorption of morin was attributed to the P-gp inhibition by Cremophor RH and the intact internalization of M-PLC-SNEDDS into Caco-2 cells bypassing P-gp recognition. Our findings thus provide new insights into the development of novel nanoemulsions for poorly absorbed drugs.

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Keywords: Mechanism, Morin, phospholipid complex, self-nanoemulsifying, intestinal absorption, Caco-2 cells

Abbreviations: PLC: phospholipid complex; SNEDDS: self-nanoemulsifying drug delivery system; PLC-SNEDDS: phosphoipid complex based self-nanoemulsifying drug delivery system; M-PLC: morin phospholipid complex; M-PLC-SNEDDS: morin phospholipid complex based self-nanoemulsifying drug delivery system.

Introduction: Oral administration is by far the most commonly used route which has high levels of patient acceptance and long-term compliance. Many conventional drugs are of poor solubility or low permeability thus presenting great challenges on the development of oral formulations1. In the past decade, successful attempts have been made to address this problem via nanocarriers including micelles2, polymeric nanoparticles3, solid lipid nanoparticles4 and nanoemulsions5. Among them, nanoemulsions offer following advantages such as increased drug solubility by providing both hydrophilic and hydrophobic environments, protection of drugs from premature degradation and

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enhanced mucosal permeability5, 6. Nevertheless, its application has been limited by low patient compliance resulted from poor palatability and large volume7. Recently, the advent of self-nanoemulsifying drug delivery systems (SNEDDS) has spurred a resurgence of interest in nanoemulsions. SNEDDS is anhydrous homogenous mixtures of drugs, oils, surfactants and cosurfactants, which spontaneously form fine oil-in-water nanoemulsions when exposed to gastrointestinal fluids7. As anhydrous forms of nanoemulsions, SNEDDS not only overcome the limitations of conventional nanoemulsions but also circumvent the stability problems encountered in solid lipid nanoparticles and liposomes8. However, drugs without sufficient liposolubility may result in the failure of the SNEDDS formation. Several strategies have been employed to improve the liposolubility of drugs such as chemical modification9, phospolipid complex10, 11 and oleic acid ionic complex12. Among them, phospholipid complex (PLC) exhibits superior ability to improve the liposolubility of drugs10. Unlike other strategies, PLC has been proven to improve the oral bioavailability and retain pharmacological actions of the drugs10, 13 PLC is a cell membrane like structure with the bioactive agents bounded by the polar portion of phospholipid while the non polar tail of phospholipids wrap over the complex. The lipophilic outer layer thereby renders drug lipophilic and hence increases oral absorption of drugs14. Combined use of SNEDDS with PLC (PLC-SNEDDS) may offer a novel approach to deliver drugs that are previously prohibited from oral formulation screening. Previously, our group employed the PLC-SNEDDS approach to deliver a water

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soluble drug, matrine15. As an antitumor agent, its oral formulations were limited by its low therapeutic efficacy owing to poor mucosa permeability. PLC-SNEDDS loaded matrine showed a 4-fold increase in oral bioavailability which exhibited great potential for further clinical application. More recently, we attempted to develop an oral formulation of morin (3, 5, 7, 2′, 4′-pentahydroxyflavone), which is an efficient inhibitor of xanthine oxidase and uricosuric agent16, a P-gp substrate and a BCS Class IV drug17. Due to the very low oral bioavailability of morin, a high dose was needed to achieve the therapeutic effect. In addition, the oral formulation of morin has never been reported. SNEDDS loaded with morin could not be prepared directly due to the poor liposolubility of morin. After formation of morin-phospholipid complex (M-PLC, w/w 1:1.5), M-PLC was successfully incorporated into SNEDDS (M-PLC-SNEDDS) with a high drug loading efficiency of 10%. M-PLC-SNEDDS increased the oral bioavailabiity of morin to 623%. As a promising strategy for oral drug delivery, no mechanistic study has been reported on the oral absorption of PLC-SNEDDS. It would also be interesting to compare the effects of PLC and SNEDDS on the oral absorption behavior of morin. The present work thus aimed to elucidate the possible mechanisms involved in the oral absorption of PLC-SNEDDS using morin as a model drug via a systematic study.

Materials and methods Materials Morin phospholipid complex loaded self-nanoemulsifying delivery system was

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prepared and characterized according to our previous work17. Morin, chlorpromazine, amiloride, M-β-CD, simvastatin, filipin, 1, 6-diphenyl-1, 3, 5-hexatriene (DPH), nile red, verapamil were purchased from Sigma-Aldrich Co. (Shanghai, China). Dulbecco’s modified Eagle medium, fetal bovine serum, Hank’s balanced solution were purchased from KeyGEN Biotech, Co. (Nanjing, China). All other chemicals were of analytical or high performance liquid chromatography grade.

Animals and Cell cultures: Male Wistar rats were provided by the Laboratory Animal Center of Sichuan University (Chengdu, P. R. China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University. Rats were acclimated to the enviroment 5 days and fasted overnight with free access to water before experiments. Caco-2 cells (Passage: 40-50) were cultured under the same condition as previously reported5. For celluar uptake studies, cells were seeded in 6-well plates at a density of 5×105 cells/well and cultivated for 14 days. For transport studies, Caco-2 cells were seeded on Polyester membrane Tranwell-clear inserts (0.4-µm pore size) (Corning Costar, Cambridge, U.K.) at a density of 1×105 cells/well in 12-well plates and cultivated for 21 days.

Preparation and in vitro release of M-PLC-SNEDDS M-PLC-SNEDDS was prepared according to our previous study17. Labrafil M

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1944CS (oleoyl macrogolglycerides) was used as an oil phase with Cremophor RH (PEG-40 hydrogenated castor oil) and Transcutol HP (purified diethylene glycol monoethyl ether) as emulsifier and coemulsifier, respectively. Briefly, accurately weighed Labrafil M 1944CS, Cremophor RH, and Transcutol HP (3:5:3, w/w) were mixed together and heated at 40 ℃ to form blank-SNEDDS. M-PLC-SNEDDS with a drug loading effficiency of 10% (w/w) was prepared by dissolving certain amount of M-PLC in blank-SNEDDS. Then certain volume of suitable medium was added and gently agitated to obtain M-PLC-SNEDDS dispersion. To mimic the in vivo absorption of M-PLC-SNEDDS, free morin was not removed from the dispersion during the study. To investigate the effects of PLC and PLC-SNEDDS on morin release behavior, in vitro release studies were performed using a dialysis method at 37 ℃. The dissolution media used were artificial gastric juice (0.1 M HCl) and phosphate buffer solutions (PBS with pH 6.8 and 7.4). The dialysis bags (MWCO of 3500) were first soaked in medium for 12 h before use. 1 mL of freshly prepared morin suspension, M-PLC suspension or M-PLC-SNEDDS dispersion (diluted with release medium, 1 mg/mL) was added to the bags. Morin suspension and M-PLC suspension were prepared by dispersing morin and M-PLC in 0.5% CMC-Na solution (1 mg/mL), respectively. Thereafter, the bags were placed in antibiotic bottles containing 50 mL of the release medium and shaken in a horizontal shaker at 100 rpm. At predetermined time intervals, 1 mL of the release medium was withdrawn, and an equal volume of fresh media was added into the antibiotic bottles. The samples collected were

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analyzed using HPLC as decribed earlier17. Morin concentration for HPLC assay was linear over the range of 1.212–102.12 µg/mL with the regression equation of concentration (C) to area (A) : C=0.0448A−0.2427 (r=0.9998). The intra- and inter-day coefficients of variation were less than 10%. The quantification limit was 100 ng/mL. The cumulative drug percentage released from morin suspension or M-PLC-SNEDDS dispersion was calculated as the ratio of the amount of drug released at time (t) to the initial amount used. Herein, 100% means that all the amount of drug was released from the morin suspension or M-PLC-SNEDDS.

Stability of M-PLC-SNEDDS in gastrointestinal (GI) luminal contents and homogenates The procedures for the stability study were performed according to previous studies with slight modifications18. Briefly, rats were sacrificed and GI segment were immediately isolated. The content of each segment was washed out with cold normal saline and then collected separately. Afterwards, the mucosa of each segment was scraped gently followed by homogenization and centrifugation. The supernatant was removed and centrifuged for another 10 min. The final protein concentration in the supernatant was determined using a BCA protein assay kit and then was adjusted to 1 mg/mL. Thereafter, morin solution (dissolved in DMSO, 10 mg/mL), M-PLC solution (dissolved in Transcutol HP, 10 mg/mL) and M-PLC-SNEDDS dispersion (diluted by PBS, pH 6.8, 10mg/mL) were directly added to make a final concentration of morin of 10 µg/mL. At predetermined time intervals, samples were immediatly diluted by equal

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amout of methanol to precipitate protein, vortexed for 5 min and centrifuged at 13225 g for 10 min, and then subjected to HPLC. Unless otherwise stated, the samples obtained in the following studies were treated as described above if protein precipitation was needed.

In situ evaluation of M-PLC-SNEDDS across intestinal barrier using single-pass intestinal perfusion model (SPIP) The in situ single-pass perfusion studies were performed according to previously published reports19, 20. Briefly, rats were anesthetized with 1% sodium pentobarbital (45 mg/kg, ip). After abdomen was opened, bile duct was ligated to eliminate the influence of bile. Then duodeum and ileum were cannulated at both ends with glass tubing. The selected segments were gently rinsed with physiological saline and were emptied by a perfusion pump (BT00-100M, Baoding Longer Precision Pump Co.Ltd., China). Studied drugs (Morin, M-PLC, M-PLC-SNEDDS), dissolved or dispersed in 15 mL of Krebs-Rings perfusate solution with morin concentration of 300 µg/mL, were perfused through the segments at a flow rate of 0.2 mL/min for 1 h. Afterwards, the remaining perfusate solution was collected and combined with a certain volume of the Krebs-Ringer perfusate solution which was used to rinse the segments twice. The mixed solution was then diluted by methanol to 25 mL. After protein precipitation, the sample was injected into HPLC. For another group of rats, about 10 cm long jejunum and 2 cm long colon of each rat were cannulated and perfused as described above. The absorption rate constant (Ka) and apparent permeability coefficients (Papp) were

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calculated using the following equations: Ka =

( X 0 − X t ) Ct0V

Papp = Q ⋅ ( X in X out ) 2πrl Where X0 was the amount of drug in the initial perfusate solution, Xt was the amount of drug in the remaining perfusate solution. C0 was the initial drug concentration, t was the perfusion time, V was the volume of the perfused intestinal segment; Q was the flow rate, Xin and Xout were the inlet and outlet drug amount, and r and l were the radius and length of the intestinal segment.

Visualizing intestinal absorption and lymph transport of M-PLC-SNEDDS To

visualize

the

absorption

of

M-PLC-SNEDDS

in

the

intestine,

M-PLC-SNEDDS was fluorescently labeled with hydrophobic Nile red and orally administered to rats at 200 mg/kg morin with 5 mg/kg of Nile red

5, 21

. At different

time points, rats were sacrificed, and different intestinal segments (duodenum, jejunum and ileum) were isolated. The segments were gently washed by cold PBS (pH 7.4), frozen at -30 °C in cryoembedding media (Zeta, SAKURA, Japan) and sectioned at 16 µm (MEV, SLEE, Germany). After fixed with 4% buffered paraformaldehyde, and sequentially stained with FITC-phalloidin (Sigma) and DAPI (1 µg/mL, Sigma), they were imaged under LCSM (Olympus, Japan). To visualize lymph transport of M-PLC-SNEDDS, the surgical procedure was performed as previously reported19. Briefly, after being anesthetized, rats were orally given M-PLC-SNEDDS at 200 mg/kg. A midlined incision was made immediately

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and then mensentric lymphatic duct was exposed by blunt dissection. Afterwards, the lymphatic duct was cannulated by plyetheylene tubing. At predetermined time point, lymph fluid were collected and observed under microscope. (Axiovert 40 CFL, Carl Zeiss, Germany).

Cytotoxicity sudies Cell viability was assessed using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method (n = 5). Briefly, the cells were seeded in 96 well plates. Morin was dissolved at 10 mg/mL in dimethyl sulfoxide as stock solution. The working solutions of morin were prepared by diluting the stock solution in fetal bovine free culture medium. After cultured with morin solution or M-PLC-SNEDDS suspension (dispersed in fetal bovine free culture medium) for 4 h, 20 µL MTT PBS solution (5 mg/mL) were added into each well and incubated at 37 °C for another 4 h. Thereafter, the medium was removed, and the cells were mixed with 150 µL DMSO. Cell viability was assessed by measuring the absorbance at 570 nm with a microplate reader. (Thermo, Varioskan Flash). Cells not exposed to samples were used as control with 100% viability. Cell membrane fluidity measurements The cell membrane fluidity experiments were carried out according to a previously reported method22. Fluorescence polarization techniques were applied to evaluate the influence of Blank-SNEDDS and PLC-SNEDDS (containing 15% E-80, w/w) on Caco-2 cell membrane fluidity using DPH (1, 6-diphenyl-1, 3, 5-hexatriene)

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as a fluorescent probe. Here, a Hitachi Fluorescence spectrophotometer (F-7000, Hitachi, Japan) was used to measure the fluorescence intensity. In addition, the effect of M-PLC-SNEDDS on the F-actin filament of Caco-2 cell was visualized under LCSM. After cultured for 14 days, the cells were incubated with 50 µg/mL M-PLC-SNEDDS. At predetermined time intervals, the cell monolayer was rinsed with cold PBS (pH 7.4), fixed with 4% paraformaldehyde solution, stained with FITC-phalloidin and imaged under LCSM. Cellular uptake studies To evaluate the cell uptake efficiency of M-PLC-SNEDDS, cells were incubated with morin or M-PLC-SNEDDS (50 µg/mL) at 37 °C over different durations of time. For p-gp inhibition, cells were incubated with verapamil (100 µg/mL) for 1 h in advance. For energy depletion, the cellular uptake was performed at 4 °C for 1 h. In the end, the cells were subjected to three freeze-thawing cycles following detachment from plates. The cell uptake amount was analyzed using HPLC. To explore the mechanism of M-PLC-SNEDDS mediated internalization, Caco-2 cells were precubated with indicated inhibitors for 1 h and then incubated with M-PLC-SNEDDS for another 1 h. Chlorpromazine (Chlo, 10 µg/mL) was an inhibitor of clathrin-mediated endocytosis23. MßCD (10 mg/mL) with 1 µg/mL simvastatin, an inhibitor of de novo synthesis of cholesterol, inhibited both caveolae and clathrin-mediated pathways by cholesterol depletion24. Filipin (Fip, 5 µg/mL) was an inhibitor of cholesterol disrupted caveolae-mediated endocytosis25. Dimethyl amiloride (DMA, 5 µg/mL) was an indicator of macropinocytosis26. The procedure

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was the same as stated above. Transport studies Trans-epithelial electrical resistance (TEER) values of cell monolayers were evaluated using a Millicell electrical resistance system (Millipore, USA). Monolayers with TEER values exceeded 800 cm·Ω were used in the transport studies. To evaluate the permeability of M-PLC-SNEDDS, the experiment was carried out at 37 °C followed by adding 1.5 mL of fresh HBSS on the basolateral side (BP) and 500 µL of morin solution or M-PLC-SNEDDS suspension (50 µg/mL) on the apical side (AP). Besides, the experiment was performed at 4 °C for energy depletion. To explore the mechanism of M-PLC-SNEDDS medicated transcytosis, the indicated inhibitors were added on the AP at 37 °C for 1 h prior to addition of morin or M-PLC-SNEDDS. To study the effect of PLC-SNEDDS on the enhanced permeability of morin, cells were preincubated with blank-SNEDDS, Labrabil M 1944CS (137 µg/mL), Cremophor RH (226 µg/mL) or Transcutol HP (137 µg/mL) for 1 h before adding morin solution. Thereafter, 200 µL samples was taken from the AP at predetermined time points and complemented with equal amount of fresh HBSS. The

samples

were

diluted

by

methanol

and

analyzed

by

liquid

chromatography–tandem mass spectrometry (LC/MS/MS) according to a previously established method27. The calibration curve was linear from 1 to 25 ng/mL with a limit of detection at 100 pg/mL. The intra- and inter-day coefficients of variation were less than 10%. Papp values of morin were calculated according to the equation: Papp=

(dQ

dt ) A • C0

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where the dQ/dt (µg/s) was the drug permeation rate, A was the surface area of the membrane (cm2) and C0 (µg/mL) was the initial drug concentration.

Statistical analysis Data were analyzed using two-tailed Student’s t-test or one-way ANOVA followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant.

Results and discussion In vitro release studies

Figure 1. In vitro release profile of M-PLC-SNEDDS in artificial gastric juice (0.1M HCl with pH 1.2) and artificial intestinal juice (phosphate buffer solution with pH 6.8 and 7.4) at 37 °C. Results were presented as mean ± SD (n=3)

SNEDDS has been reported to increase oral bioavailability of poor-water soluble 15



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drugs by enhancing drug dissolution rates7. In order to evaluate whether M-PLC-SNEDDS had the same effect with SNEDDS on the improvement of drug dissolution, in vitro release studies were performed in three dissolution media (0.1 M HCl with pH 1.2, PBS with pH 6.8 and 7.4) to mimic drug release in GI tract. As shown in Figure 1, all the three formulations exhibited much higher drug release rates in PBS with pH 6.8 and 7.4 than 0.1M HCl (pH 1.2), and the drug release rates were much faster at pH 7.4 than at 6.8. The drug release rates increased with the increase of pH value probably due to the better solubility of free morin at higher pH values. Regardless of pH values, M-PLC-SNEDDS showed the slowest drug release rate among all the three formulations. The cumulative drug release from morin suspension at pH 6.8 reached 96.12% within 8.5 h, while M-PLC suspension decreased the drug release amount to 72.56%, indicating a more rapid drug release from morin suspension. It was probably because free morin dissolved quickly in PBS with pH values higher than its pKa1 (3.45, determined by our group). However, as a weak acid, morin was prone to lose more than one proton and was presented as anions at pH 7.4. Only the molecular form of morin can be absorbed through the gastrointestinal tract in vivo. Therefore, despite the high release rate at pH 7.4, morin did not show good oral absorption in vivo. Regarding phospholipid complex formation, the phenolic hydroxyl groups of morin may associate with the polar head of the phosphatidylcholine via hydrogen bonding14, and thus hindered the dissolution of morin into PBS (pH 6.8). Compared with morin and M-PLC suspension, M-PLC-SNEDDS showed significantly reduced cumulative drug release amount (56.68%) at pH 6.8 within 8.5 h.

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It was probably because when M-PLC-SNEDDS was exposed to GI fluids, the highly lipophilic PLC maintained morin stably entrapped in the oily core of the spontaneously formed O/W nanoemulsions and the diffusion of morin from the nanoemulsions to the release media was hindered. Like wise, this could explain the similar release behavior of morin observed at pH 7.4 from the three formulations. The above data implied that PLC-SNEDDS did not exert its enhanced oral absorption via increasing the drug dissolution rate. This was in accord with our previous study showing a prolonged Tmax from 0.48 h to 0.77 h and 1.0 h after oral administration of morin suspension, M-PLC suspension and M-PLC-SNEDDS, respectively17. Therefore, we speculated that besides the reduced P-gp mediated efflux by Cremophor RH28, the intact M-PLC-SNEDDS could be uptaken into enterocytes as a P-gp-independent way, which contributed to the improved morin absorption as well. Moreover, other mechanisms involved in the enhanced absorption of Class IV drugs by SNEDDS such as increased drug stability in gastrointestinal (GI) tract and promotion

of

intestinal

lymphatic

transport7,

were

also

investigated

for

M-PLC-SNEDDS in our following studies.

M-PLC-SNEDDS improved drug stability in intestine Flavonoids were reported to be very unstable upon transfer from stomach to colon due to enzymatic degradation, leading to a low oral bioavailability29. Incorporating drugs into SNEDDS may offer improvements in the enzymatic stability of drugs7. However, no study has ever been described on the stability of PLC in vitro. To

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compare the protection ability of PLC and PLC-SNEDDS against enzymatic degradation, the stability of morin in GI luminal contents and homogenates were tested. As shown in Figure S1 (supporting information), free morin remained stable in both stomach contents and mucosa homogenates within 2 h of incubation. M-PLC and M-PLC-SNEDDS did not affect the stability of morin during incubation.

Figure 2. The stability of Morin, M-PLC and M-PLC-SNEDDS in intestinal luminal contents (a) and homogenates (b) at a protein concentration of 1mg/mL. Data were presented as mean ± SD

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(n=3).

The stability of morin in the intestinal contents and four different intestinal mucosa homogenates (duodenum, jejunum, ileum and colon) was shown in Figure 2. After 2 h incubation with intestinal contents, the amount of drug decreased by 18.72%, 20.91% and 5.20% for free morin, M-PLC and M-PLC-SNEDDS, respectively. However, the degradation rate for M-PLC-SNEDDS was significantly lower than that of free morin and M-PLC (Figure 2a). In addition, no significant difference was observed between free morin and M-PLC. These results demonstrated that M-PLC-SNEDDS other than M-PLC effectively protected morin from degradation in intestinal contents. Meanwhile, the amount of drug decreased after 2 h incubation in all the four intestinal mucosa homogenates for free morin (Figure 2b). Compared with free morin, M-PLC maintained morin stable in ileum homogenates and significantly decreased the degradation rate of morin in duodenum and jejunum homogenates. For M-PLC-SNEDDS, morin remained stable during 2 h incubation in duodenum and ileum

homogenates.

Meanwhile,

the

drug

was

strongly

protected

by

M-PLC-SNEDDS with a significantly lower degradation rate than that of free morin and M-PLC in jejunum and colon homogenates. Therefore, both PLC and PLC-SNEDDS provided protective effects for drugs against enzymatic degradation in intestine. Moreover, PLC-SNEDDS showed superior ability than PLC alone in protecting drugs against enzymatic degradation in the GI tract. The degradation rate of morin in these three formulations (morin suspension >

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M-PLC > M-PLC-SNEDDS) was positively correlated with their release rate (morin suspension > M-PLC > M-PLC-SNEDDS) in vitro. The decreased release amount of morin implied that more morin was shielded in PLC and PLC-SNEDDS thus escaping from enzymatic degradation in intestine before absorption. Therefore, PLC and PLC-SNEDDS provided better drug protection at a lower drug release rate than morin.

M-PLC-SNEDDS increased intestinal permeability of morin in situ

Figure 3. The apparent permeability coefficients (Papp) (a) and absorption rate constant (Ka) (b) obtained by in situ perfusion in SPIP model comparison among Morin, M-PLC and M-PLC-SNEDDS in different intestinal segments. Data were presented as mean ± SD (n=5). *p

Mechanism of Enhanced Oral Absorption of Morin by Phospholipid

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