基于Caco-2细胞单层与大鼠小肠模型的大豆皂苷Ⅰ和Ⅱ经上皮传递的变化研究

光翠娥，王世强，桑尚源，张海玲，杨红飞，程水源（.江南大学 食品科学与技术国家重点实验室，江苏 无锡 4；.黄冈师范学院 经济林木种质改良与资源综合利用湖北省重点实验室，湖北 黄冈 438000）

基于Caco-2细胞单层与大鼠小肠模型的大豆皂苷Ⅰ和Ⅱ经上皮传递的变化研究

光翠娥1，王世强1，桑尚源1，张海玲1，杨红飞1，程水源2
（1.江南大学 食品科学与技术国家重点实验室，江苏 无锡 214122；2.黄冈师范学院 经济林木种质改良与资源综合利用湖北省重点实验室，湖北 黄冈 438000）

利用Caco-2细胞单层与大鼠小肠模型研究大豆皂苷Ⅰ和Ⅱ的吸收变化与机制。在Caco-2细胞单层中，大豆皂苷Ⅰ和Ⅱ从肠腔侧到基底侧的表观渗透系数（apparent permeability coefficients，Papp）随时间的延长趋向平稳，前120 min近似线性，且随浓度增大，斜率减小，Papp值分别为（1.02×10－6～3.41×10－6）cm/s和（0.9×10－6～3.05×10－6） cm/s；传递的饱和性、双侧Papp比率＞1.5以及线粒体呼吸链抑制剂叠氮化钠的抑制作用表明了两者的主动转运机制。抑制剂维拉帕米没有提高大豆皂苷Ⅰ和Ⅱ的吸收，排除了p-糖蛋白介导的外排；吸收促进剂按照冰片＞脱氧胆酸钠＞卡波姆934P＞聚山梨酯80的强弱提高两者的吸收，壳聚糖则未能加强渗透。跨膜转运也表现出组织差异性：两者在大鼠空肠的Papp是十二指肠和回肠的2倍多。因此，控制的传递应能提高大豆皂苷Ⅰ和Ⅱ的小肠吸收以便两者实施它们的生理功能。

大豆皂苷；Caco-2；叠氮化钠；p-糖蛋白；吸收促进剂

GUANG Cuie, WANG Shiqiang, SANG Shangyuan, et al. Variability of transepithelial transport of soyasaponins I and II using a Caco-2 cell monolayer and a rat intestinal model[J]. Food Science, 2016, 37(11): 174-179. (in English with Chinese abstract) DOI:10.7506/spkx1002-6630-201611030. http://www.spkx.net.cn

Soyasaponins Ⅰ and Ⅱ are naturally occurring oleanane triterpenoid glycosides and primarily found in soybean (Glycine max). Their contents vary according to soybean variety, culture year, location grown and degree of maturity with an average of 0.24 and 0.1 mmol/g, respectively[1]. SoyasaponinⅠmainly exists in soybean germ whereas soybean cotyledon contains a higher content of soyasaponinⅡthan germ[2]. SoyasaponinsⅠandⅡare both amphiphilic molecules, with polar sugar moieties attached to a nonpolar pentacyclic ring (soyasapogenol B) at the C-3 position[3]. The structures of soyasaponins I and Ⅱ have been elucidated to be 3-O-[α-L-rhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→2)-β-D- glucuronopyranosyl]-soyasapogenol B and 3-O-[α-L-rhamnopyranosyl(1→2)-α-L- arabinopyranosyl(1→2)-β-D-glucuronopyranosyl]-soyasapogenol B, respectively. Soyasaponin I has been reported to have anti-inflammatory[4], anti-carcinogenic[5], anti-microbial[6], antioxidative[4], adjuvant[7], hepato-[8], cardiovascular[9]and kidney[10]protective functions; soyasaponinⅡalso displays anti-viral[11], adjuvant[7], hepato-[8]and cardiovascular[9]protective effects[12].

The human colonic carcinoma Caco-2 cells form monolayers that allow absorption to occur simultaneously with food digestion under conditions similar to those found along the surface of the intestinal tract. Preluding the human trials, the Caco-2 cell monolayer model is generally used to screen bioactives with high productivity and thereafter predict their permeation in human intestine[13]. Excellent correlation exists between in vivo absorption and in vitro apparent permeability coefficient (Papp) for compounds including transcellular, paracellular and carrier-mediated mechanisms[14]. An end-point mode of experiment showed the mucosal transfer of soyasaponinⅠacross the Caco-2 cell monolayer with an Pappvalue of (0.9 × 10-6-3.6 × 10-6) cm/s[15]. Herein a detailed experiment would be conducted to investigate the time- and concentration-dependent permeability of soyasaponinsⅠandⅡand therefore confirm their transport mechanism.

Moreover, the absorption of bioactives in human intestine is influenced by diverse factors. Passive intestinal permeability depends on molecular size, lipophilicity, hydrogen bonding capacity and so on[16]; the active transport needs carriers and energy; efflux mechanisms, absorption enhancers and food matrix can retard or promote the permeability of bioactives. Therefore, the effects of inhibitors, including sodium azide and verapamil, and absorption enhancers, including borneol, sodium deoxycholate (SDC), polysorbate (Tween) 80, crosslinked poly(acrylate) derivative carbomer 934P and poly(2-deoxy-2-amino glucan) polymer chitosan on the permeability of soyasaponins Ⅰ and Ⅱ, would be predicted using the Caco-2 cell monolyer model. The uptake of bioactives also displays tissue difference. Herein the optimal intestine regions for absorption of soyasaponins Ⅰ and Ⅱ would be determined.

1 Materials and Methods

1.1 Materials and animals

Human colon adenocarcinoma Caco-2 cell line Cell Bank of Chinese Academy of Sciences (Shanghai, China); alkaline phosphatase assay kit, penicillin and streptomycin Beyotime Institute of Biotechnology (Shanghai, China); carbomer 934P Xinhenglong Technology (Wuhan, China); soluble chitosan (50% deacetylation degree) Hecreat Biotech (Qingdao, China); Hank’s balanced salt solution (HBSS, pH 7.4), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and non-essential amino acids (NEAA) GibcoBRL (New York, USA); soyasaponins I and Ⅱ ChromaDex (Irvine, USA); TranswellTMplates of 6 wells (24 mm diameter, 3 mm pore size) Corning Costar (New York, USA); atenolol and propranolol Sigma (St. Louis, USA). Male Sprague-Dawley (SD) rats with a body mass of approximately 250 g Shanghai Super-B&K Laboratory Animal Corporation (Shanghai Laboratory Animal Center, China). The animals had free access to food and water in the room maintained at about 25 ℃ with a 12 h light/dark cycle.

1.2 Preparation of Caco-2 cell monolayers

Caco-2 cells were cultured in DMEM containing 4.5 g/L glucose and supplemented with 10% (V/V) FBS, 1% (V/V) L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 1%(V/V) NEAA, and maintained at 37 ℃ in a controlled atmosphere of 5% CO2and 90% relative humidity. Medium was replaced every two days until the confluence reached 80%-90%. After 32-40 passages by trypsinization with 0.25% trypsin and 0.02% of ethylenediaminetetraacetic acid (EDTA) in PBS, Caco-2 cells were inoculated at a density of 1.5×105cells/cm2on Transwell membrane inserts. Medium was renewed every 2 days for the 5 weeks and every day for the next 8-21 day s[13]. Differentiation of Caco-2 cells was examined by determining the activity of alkaline phosphatase with an assay kit; the integrity was checked by measuring the transepithelial electrical resistance (TEER)with an Evom resistance voltohmmeter (World Precision Instruments, Sarasota, USA) after monitoring for 60 min; and the transportation ability was tested by running standard assays using atenolol and propranolol as paracellular fl ux and transcellular fl ux markers, respectively[16].

1.3 Transport of soyasaponinsⅠandⅡacross Caco-2 cell monolayers

After the integral cell monolayers were washed twice with prewarmed Hank’s balanced salt solution (HBSS) medium, 0.5 mL aliquots of HBSS containing different concentrations of soyasaponins Ⅰ and Ⅱ (0.5, 1, 3 mmol/L) were added to the apical side and 1.5 mL of fresh HBSS to the basolateral side, or 0.5 mL HBSS to the apical side and 1.5 mL samples to the basolateral side. The monolayers were incubated at 37 ℃ on a vibrax shaker at 60 r/min. At the time intervals of 30, 45, 60, 90, 120, 150, 180 and 240 min, 0.5 or 0.25 mL aliquots were drawn from the receiving side for analysis and replaced with an equal volume of fresh buffer. In order to investigate the transport variation, a 0.5 mL aliquot of HBSS containing 1mmol/L soyasaponin Ⅰ and 1 mmol/L soyasaponin Ⅱ was added to the apical side, or 0.5 mL aliquots of HBSS containing 1 mmol/L soyasaponin Ⅰor soyasaponin Ⅱ and sodium azide (0.5 mmol/L) or verapamil (0.1 mmol/L) or borneol (0.5 g/100 mL) or SDC (0.5 g/100 mL) or polysorbate 80 (0.5 g/100 mL) or carbomer 934P (0.5 g/100 mL) or chitosan (0.5 g/100 mL) were added to the apical side, and 0.5 mL aliquots were removed from the basolateral side over a period of 180 min. The collected samples were immediately frozen, lyophilized and stored below －20 ℃ for subsequent high-performance liquid chromatography (HPLC) analysis[13]. The Pappwas calculated according to the following equation.

where ΔQ/Δt is the appearance rate of the soyasaponin on the receiving side/(mol/s), A is the membrane surface area /cm2, and C0is the initial concentrationin the donor compartment/ (mol/mL).

Transport enhancement ratio (ER) was calculated from Pappvalues according to the following equation:

1.4 ex vivo transport of soyasaponinsⅠandⅡacross rat intestinal tissues

Rats were anaesthetized via intraperitoneal injection of 15% urethane (10 mL/kg) and then a laparotomy was performed. The intestine was excised and rinsed in ice-cold PBS (pH 7.4). The duodenal segment was the first 10 cm portion from the stomach, the ileal segment was the fi nal 10 cm portion of the small intestine, and the remaining intestine was used as the jejunum. After experimental segments were obtained, the underlying muscularis was removed before mounting in an Ussing chamber, in which a surface area of 0.293 cm2was exposed. PBS (3 mL) was added to the serosal side and an equal volume of sample solution (1 mmol/L) was added to the mucosal side. After the chamber was placed in a water bath at 30 ℃, mixing was performed by bubbling with 95% O2-5% CO2[17]. Samples were taken away from the serosal side over a period of 180 min and were immediately frozen, lyophilized and stored below －20 ℃ for subsequent HPLC analysis.

1.5 Quantifi cation of soyasaponins I and Ⅱ by HPLC

The lyophilized samples were dissolved in 200 mL MeOH and centrifuged at 15 000 × g for 10 min. The resulting supernatant (20 μL) was injected and separated by the reversed phase-HPLC(RP-HPLC) system comprised of a Jupiter 4 μ Proteo 90A C12 reversed-phase column (250 mm × 4.6 mm, Phenomenex, Inc., Torrance, CA), a Waters 2695 Separations Module and a Waters 996 photodiode array detector (Waters Co., Milford, MA) recording absorbance from 190 to 350 nm. Solvent A was 0.05% (V/V) trifluoroacetic acid (TFA) in filtered deionized water, and solvent B was 0.05% (V/V) TFA in acetonitrile. Elution was achieved by a linear gradient from 38% to 48% solvent B within 40 min at a fl ow rate of 1 mL/min[1]. Calibration curves of the peak area versus standard concentration were used to calculate the soyasaponin concentrations.

1.6 Data analysis

All data were expressed as the± s and unpaired Student’s t-test was used to assess the significance of the difference between two mean values at a signifi cant level of P < 0.05.

2 Results and Analysis

2.1 Time- and concentration-dependent transport of soyasaponinsⅠandⅡacross Caco-2 cell monolayers

After Caco-2 cells grew for 14 days, alkaline phosphatase could hydrolyze the substrate para-nitrophenyl phosphate into yellow para-nitrophenol. On the 21thday, TEER measurement showed a value of above 450 Ω/cm2after subtracting the intrinsic resistance of insert alone.Pappvalues of two known model substrates atenolol (poor permeability) and propranolol (high permeability) were (2.37 ± 0.02) × 10-7cm/s and (2.62 ± 0.07) × 10-5cm/s, respectively. These control assays confi rmed the integrity and transportation ability of Caco-2 cell monolayers. Within the test concentration range, soyasaponins Ⅰ and Ⅱ showed no apparent cytotoxicity on Caco-2 cells. The recovery during transport assays was measured as the total amount of soyasaponins in two sides of the insert. A recovery rate of > 95% for both soyasaponins indicated low cell accumulation and supported the experimental reliability.

Fig. 1 Effects of time and concentration on the transport ofsoyasaponins I (A) and Ⅱ (B) across Caco-2 cell monolayers (apical to basolateral, n = 5)

Pappvalues for soyasaponins Ⅰ and Ⅱ across Caco-2 cell monolayers from the apical to basolateral direction were showed in Fig. 1. With a defi ned concentration, Pappincreased linearly until a plateau was reached at 120 min. According to the equation (1), the transport rate (ΔQ/Δt) increased for the fi rst 120 min and afterwards tended to remain constant. When the soyasaponin concentration was elevated, the transported mass was increased with a less magnitude, Pappdecreased and the uptake tended to be saturable probably due to the carrier saturation. The results show that Pappvalues from the apical to basolateral direction were significantly higher (P < 0.05) than those from the basolateral to apical direction with the ratios being larger than 1.5 (Table 1) further indicated the active transport[16]. Therefore, the transport of soyasaponinsⅠand Ⅱ might involve a carriermediated mechanism; the absorption could be enhanced when the soyasaponin concentration is low and could be limited by the capacity of epithelial cells to take up and transfer soyasaponins to the basolateral side when the high concentration is present[15]. The order of magnitude (10-6cm/s) for fi nal Pappvalues indicated an intermediate permeability of two soyasaponins. For comparison, Pappvalues of 36 fl avonoids across Caco-2 monolayers from the apical to basolateral side ranged from less than 5 × 10-7to 2.96 × 10-5cm/s[16]. Compounds of intermediate or low permeability have a lower permeability in Caco-2 model than in vivo. Atenolol, ranitidine, furosemide and chlorothiazide, which are adequately absorbed in humans, showed poor permeability in the standard 21-day Caco-2 cell monolayer. Caco-2 cells originate from the colon and have a tighter paracellular route than in vivo. The average pore radius of tight junctions in the human intestine is around 8-13Å, whereas the corresponding radius in Caco-2 cells is about 5 Å[18].

TTaabble 1 Bilateral apparent permeation coeffi cients (Paapppp)) ooff soyasaponins in the Caco-2 model

2.2 Effects of inhibitors and absorption enhancers on the transport of soyasaponins Ⅰ and Ⅱ across Caco-2 cell monolayers

When soyasaponins Ⅰ and Ⅱ were simultaneously added to the apical side, the individual Pappwas mildly lower than that for a soyasaponin added separately (Fig. 2), which indicated that two soyasaponins might use the same carrier and therefore competitively inhibit the permeation each other and that the interaction in food matrix could regulate their absorption. Sodium azide, a cytochrome c oxidase-respiratory chain complex Ⅳinhibitor due to enhanced cytochrome c holoenzyme dissociation that inhibits the electron transfer between mitochondrial respiratory chain and thus prevents the oxidative ATP production[19], significantly reduced the transport of both soyasaponins (P < 0.05). Competitive inhibition and energy requirement during transport further indicated the carrier-mediated flux of soyasaponins Ⅰand Ⅱ. P-glycoprotein, a transmembrane permeability glycoprotein, is an ATP dependent efflux pump that is strongly expressed by Caco-2 cells and often causes multidrug resistance and poor bioavailability[20]. Its specific inhibitorverapamil did not significantly increase the permeation of both soyasaponins, hence suggesting that the carrier might not involve in the efflux of soyasaponins Ⅰ and Ⅱ in the Caco-2 model. When different enhancers were added, the ranking in terms of absorption enhancing ability was borneol > SDC > carbomer 934P > polysorbate 80 > chitosan. Among them, borneol, SDC, carbomer 934P and polysorbate 80 significantly promoted the permeation of soysaponins (P < 0.05) with ERs being 3.06, 2.98, 2.52, 2.44 for soyasaponin Ⅰ and 3.21, 3.03, 2.73 and 2.62 for soyasaponin Ⅱ, respectively. Chitosan showed no absorption enhancing effect for two soyasaponins and in contrast, suppressed in different degrees. Borneol is an efficacyenhancing ingredient in traditional Chinese medicine; SDC is a type of bile salts that tend to dissolve the extracellular proteins and loosen the tight junctions and also to dissolve the membrane bound cholesterol and increase the fl uidity of the membrane, thereby increasing the transcellular permeability[21]; polysorbate 80 is a nonionic surfactant used in the manufacture of a variety of pharmaceutical products and can induce alternation of biomembranes and therefore increase the permeability[22]; carbomer and chitosan with strong mucoadhesiveness and low toxicity have been proved to function by opening intercellular junctions and thereby enhancing the paracellular permeability[23].

Fig. 2 Pappand transport ER of soyasaponins Ⅰ (A) and Ⅱ (B) in theCaco-2 model in the presence of various inhibitors and enhancers (apical to basolateral)

2.3 Regional difference of the transport of soyasaponinsⅠ and Ⅱ in the intestine

The optimal sites for absorption of soyasaponins Ⅰ andⅡ were determined by ex vivo transport across rat intestinal segments in Ussing chambers. Pappvalues of soyasaponins across rat duodenum, jejunum and ileum were summarized in Fig. 3. The calculated Pappvalues for soyasaponins Ⅰand Ⅱ across the jejunal segment were more than 2-times greater (P < 0.05) than Pappvalues across the duodenal and ileal segments, whereas the Pappvalues across duodenum and ileum did not differ signifi cantly. These results indicated jejunum was the optimum absorption site of soyasaponins I and Ⅱ. The unstirred water layer, differences in the thickness of mucous layers, the tightness and the number of tight junctions and membrane components might have infl uenced the transport of soyasaponins Ⅰ and Ⅱ across the various intestinal membranes[17]. Additionally, Pappvalues obtained through the ex vivo Ussing chambers were higher than those observed in the Caco-2 experiments with permeability ratios ranging from about 3.2 (ileum) up to 6.9 (duodenum), which may be explained by the higher tightness of the Caco-2 cell monolayer compared to intact mammalian intestinal tissue[24].

Fig. 3 Transport of soyasaponins Ⅰ and Ⅱ across the intestinal segments

3 Conclusions

The present study showed the permeability of soyasaponins Ⅰ and Ⅱ as being intermediate in the Caco-2 model. Active transport was suggested to be the major absorption mechanism, which was further supported by the inhibitory effects of sodium azide. Absorption enhancers, borneol, SDC, carbomer 934P and polysorbate 80, did improve the permeability of soyasaponins Ⅰ and Ⅱ in the Caco-2 model. Jejunum was suggested to be the optimal absorption tissue. Thus a manipulated transport would increase the intestinal permeability so that soyasaponinsⅠand Ⅱ could exert their health actions.

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Variability of Transepithelial Transport of Soyasaponins Ⅰ and ⅡUsing a Caco-2 Cell Monolayer and a Rat Intestinal Model

GUANG Cuie1, WANG Shiqiang1, SANG Shangyuan1, ZHANG Hailing1, YANG Hongfei1, CHENG Shuiyuan2
(1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; 2. Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Huanggang Normal University, Huanggang 438000, China)

The absorption mechanism and variability of soyasaponins I and II were investigated using a Caco-2 cell monolayer and a rat intestinal model. Apparent permeability coeffi cients (Papp) across the Caco-2 model increased linearly until plateaus were reached at 120 min with intermediate Pappvalues of (1.02−3.41) × 10-6and (0.9−3.05) × 10-6cm/s for two soyasaponins, respectively. Saturable transport, bilateral Pappratios of more than 1.5 and the inhibitory effect of mitochondrial electron transport chain blocker sodium azi de indicated the active transport mechanisms. The transmembrane permeability glycoprotein (p-glycoprotein) inhibitor verapamil did not increase the permeation of both soyasaponins, excluding the p-glycoprotein-related effl ux. Several absorption enhancers promoted the permeation across the Caco-2 cell monolayers with a rank of borneol > sodiumdeoxycholate > carbomer 934P polysorbate 80; but chitosan did not exhibit such an enhancing ability. The transepithelial transport also showed tissue difference in the intestine with the Pappvalues for soyasaponins I and II across the jejunal segment being more than 2 times greater than those across the duodenal and ileal segments. Therefore, a controlled transport should be able to improve the intestinal absorption so that soyasaponins I and II would exert their health functions. Key words: soya saponin; Caco-2; sodium azide; p-glycoprotein; absorption enhancer

nces:

10.7506/spkx1002-6630-201611030

TS201.4

A

1002-6630（2016）11-0174-06

GUANG Cuie, WANG Shiqiang, SANG Shangyuan, et al. Variability of transepithelial transport of soyasaponins I and II

using a Caco-2 cell monolayer and a rat intestinal model[J]. 食品科学, 2016, 37(11): 174-179. DOI:10.7506/spkx1002-6630-201611030. http://www.spkx.net.cn

2015-03-01

国家自然科学基金青年科学基金项目（31201289）；经济林木种质改良与资源综合利用湖北省重点实验室开放基金资助项目（2011BLKF241）；食品科学与技术国家重点实验室自由探索项目（SKLF-ZZB-201208）

光翠娥（1976—），女，副教授，博士，研究方向为食品营养与功能因子。 E-mail：guang1226@hotmail.com