Bioaccessibility and Absorption Mechanism of Phenylethanoid

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Bioactive Constituents, Metabolites, and Functions

Bioaccessibility and absorption mechanism of phenylethanoid glycosides using simulated digestion/Caco-2 intestinal cell models Fei Zhou, Weisu Huang, Maiquan Li, Yongheng Zhong, Mengmeng Wang, and Baiyi Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01307 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Journal of Agricultural and Food Chemistry

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Title

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Bioaccessibility and absorption mechanism of phenylethanoid glycosides using

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simulated digestion/Caco-2 intestinal cell models

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Fei Zhou†, Weisu Huang‡, Maiquan Li†, Yongheng Zhong†, Mengmeng Wang†, Baiyi

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Lu†*

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†

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Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture,

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Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture，

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Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science,

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College of Biosystems Engineering and Food Science, Zhejiang University,

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Hangzhou, 310058, China

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‡

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Hangzhou 310018, China

National Engineering Laboratory of Intelligent Food Technology and Equipment,

Department of Applied Technology, Zhejiang Economic & Trade Polytechnic,

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* Corresponding author

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Tel./fax: +86-0571-89882665.

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E-mail address: [email protected].

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Address: Yuhangtang Road 866#, Hangzhou 310058, Zhejiang, P. R. China.

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ACS Paragon Plus Environment


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ABSTRACT

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Acteoside and salidroside are major phenylethanoid glycosides (PhGs) in

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Osmanthus fragrans Lour. flowers with extensive pharmacological activities and poor

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oral bioavailability. The absorption mechanisms of these two compounds remain

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unclear. This study aimed to investigate the bioaccessibility of these compounds using

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an in vitro gastro–intestinal digestion model, and to examine the absorption and

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transport mechanisms of PhGs using the Caco-2 cell model. The in vitro digestion

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model revealed that the bioaccessibility of salidroside (98.7±1.35%) was higher than

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that of acteoside (50.1±3.04%), and the superior bioaccessibility of salidroside can be

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attributed to its stability. The absorption percentages of total phenylethanoid glycoside,

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salidroside and acteoside were 1.42–1.54%, 2.10–2.68% and 0.461–0.698% in the

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Caco-2 model, respectively. Salidroside permeated Caco-2 cell monolayers through

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passive diffusion. At the concentration of 200 µg/mL, the apparent permeability (Papp)

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of salidroside in the basolateral (BL)-to-apical (AP) direction was 23.7±1.33 × 10−7

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cm/s, which was 1.09-fold of that in the AP-to-BL direction (21.7±1.38 × 10−7 cm/s).

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Acteoside was poorly absorbed with low Papp (AP to BL) (4.75±0.251 × 10−7 cm/s),

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and its permeation mechanism was passive diffusion with active efflux mediated by

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P-glycoprotein (P-gp). This study clarified the bioaccessibility, absorption and

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transport mechanisms of PhGs. It also demonstrated that the low bioavailability of

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acteoside might be attributed to its poor bioaccessibility, low absorption and P-gp

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efflux transporter. 2


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KEYWORDS

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Total phenylethanoid glycoside; Acteoside; Salidroside; Bioaccessibility; Absorption

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mechanism.

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INTRODUCTION

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Acteoside (verbascoside) and salidroside (Fig. 1) are phenylethanoid glycosides

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(PhGs) belonging to water-soluble polyphenolic compounds. The two compounds

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have been detected in food and traditional Chinese medicine, such as Olea europaea

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L. fruit, Osmanthus fragrans flower, Rhodiola rosea L., Cistanche deserticola Ma,

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and Striga asiatica.1, 2 The total phenylethanoid glycoside (TPG) are abundant in O.

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fragrans flowers3 with the contents of 92.66–130.57 milligrams of acteoside

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equivalents (AE) per gram of dry weight (mg AE /g DW), and in particular 32.78–

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71.79 mg/g DW for acteoside and 4.72–16.08 mg/g DW for salidroside,4 respectively.

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Acteoside has anti-inflammatory, antioxidant and neuroprotective properties.1 It can PC12

cells

from

CoCl2-induced

damage3

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protect

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1-methyl-4-phenylpyridinium ion-induced apoptosis or necrosis.5 Salidroside has

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anti-inflammation,6 antioxidation,7 antistress,8 anticancer9 and neuroprotective

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effects.10-12 However, the oral bioavailability of acteoside is as low as 0.12%,13 and

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32.1% for salidroside.14 Although acteoside and salidroside exhibit excellent

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pharmacological activities, their bioavailability limits their wide application. This

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poor bioavailability could be linked to the influence of several factors, such as

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degradation in gastrointestinal tract, potential substrate for efflux transporters and

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potential metabolism by microbiome. It has reported that the acteoside and salidroside

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were unstable at high temperature, high pH and light exposure conditions in the

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previous study.3 The degradation of acteoside and salidroside may occur in the

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hypoxia

and

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gastrointestinal tract. Cardinali et al.15 found that verbascoside is remained at 53% in

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vitro digestive conditions, and has low absorption of 0.1%.16 There is no more

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information about the bioaccessibility, absorption and transport mechanism of TPG,

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acteoside and salidroside.

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In vitro digestion and Caco-2 cell monolayer models have been utilized to clarify

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the effects of digestion and absorption on the bioaccessibility and bioavailability of

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bioactive compounds, such as phenolic compounds.15, 17-19 Bioaccessibility is defined

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as the relative amount of a food constituent, which released from the food matrix

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during digestion and might pass through the intestinal barrier to be absorbed.20 In

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general, the bioavailability of dietary compounds, such as phytochemicals, depends

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on the digestive stability and efficiency of the transepithelial passage. Therefore,

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bioaccessibility must be considered in bioavailability studies. The Caco-2 cell line,

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which is derived from human colorectal carcinoma, expresses nutrient and drug

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transporters, and thus is an appropriate model for use in the study of carrier-mediated

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uptake and efflux mechanisms.21 Caco-2 cells can express ATP-binding cassette (ABC)

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transporters, including P-glycoprotein (P-gp), multidrug resistance protein (MRP),

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and breast cancer resistance protein (BCRP).22 These proteins reduce the

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bioavailability by refluxing absorbed substrates into the intestinal lumen.23

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This study aimed to investigate the bioaccessibility of TPG, acteoside and

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salidroside using an in vitro gastro–intestinal digestion model, and to determine their

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absorption and transport mechanisms using the Caco-2 cell monolayer model.

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MATERIALS AND METHODS

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Materials and Chemicals. Acteoside (purity = 99%), salidroside (purity = 98%),

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verapamil hydrochloride (purity = 99%), Ko 143 (purity = 98%), and the chemicals

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used in the in vitro digestion model (including α-amylase, pepsin, pancreatin, lipase,

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bile salts, and uric acid) were purchased from Aladdin (Shanghai, China). MK 571

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(purity = 98%) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Formic

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acid and HPLC-grade acetonitrile were obtained from Merck (Shanghai, China).

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Other chemicals and reagents (analytical grade) were purchased from Sinopharm

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Chemical Reagent Co. (Shanghai, China).

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The human colon adenocarcinoma cell line Caco-2 was obtained from the Cell

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Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified

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Eagle’s medium (DMEM) and porous polycarbonate cell culture Transwell® inserts

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(pore size, 0.4 µm; diameter, 12 mm) were purchased from Coster (Corning

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Incorporated, USA). Hank’s balanced salt solution (HBSS), 0.25% trypsin–

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ethylenediaminetetraacetic acid (EDTA) solution and penicillin–streptomycin (10 000

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IU/mL penicillin, 10 000 µg/mL streptomycin) were purchased from Solarbio (Beijing

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Solarbio Science & Technology Co. Ltd., China). Fetal bovine serum (FBS) and Cell

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Counting Kit-8 (CCK-8) were obtained from Gibco (Life Technologies Inc., USA)

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and Nanjing Jiancheng Bioengineering Institute (Nanjing, China), respectively.

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Total Phenylethanoid Glycoside Extraction. Dried O. fragrans var. thunbergii

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flowers (Guilin, Guangxi, China) were extracted with 95% ethanol for 12 h at 20°C in

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a material-to-solvent ratio of 1:10 (g:mL).3 The mixture was filtered by vacuum

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pump (YuKang, Shanghai, China), and the filtrate was evaporated under a

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vacuum (YaRong, Shanghai, China) at 40°C to dryness.

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In Vitro Digestion. The in vitro digestion model was described in previous

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reports with some modifications.4,

24, 25

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described by Versantvoort et al.25 The digestion of samples was initiated through

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the addition of 1 mL of PhGs (TPG, 10 mg/mL; acteoside and salidroside standard

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solution, 1mg/mL) and 3 mL of saliva (mixture pH adjusted to 6.8), and incubation

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for 5 min. Then, 6 mL of gastric juice was added, and gastric digestion was

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simulated for 2 h (mixture pH adjusted to 2.0). Finally, intestinal digestion was

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simulated for 2 h with 6 mL of duodenal juice and 3 mL of bile juice (mixture pH

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adjusted to 6.8). All incubations were performed at 37°C in a shaking water bath.

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The gastric and intestinal digestion samples were collected, respectively, and ethanol

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was added to ensure enzyme inactivation.

Digestive juices were prepared as

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The digestion mixtures were filtered using a vacuum pump by filter paper (30-50

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µm), and the filtrates were concentrated to dryness and then diluted to 5 mL with

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distilled methanol. A controlled trial without the prepared samples was conducted to

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improve accuracy. All the processes were performed in triplicate. PhGs with high

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retention rates were stable in gastrointestinal conditions, and the retention rate was

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calculated as follows: (%) =

ℎ ℎ × 100% ℎ ℎ 7



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To evaluate the bioaccessibility of PhGs, the intestinal digestion samples were

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centrifuged at 10000 rpm for 30 min at 4 °C (H1850R centrifuge, Hunan, China) to

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obtain the supernatants.26 The supernatants were concentrated to dryness and then

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diluted to 5 mL with distilled methanol. The bioaccessibility was calculated as

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follows: (%) =

ℎ ℎ × 100% ℎ ℎ

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Cell Culture and Cell Viability Assay. Caco-2 cells between passages 40 and

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60 were cultured in DMEM, containing 100 U/mL penicillin, 100 µg/mL

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streptomycin and 10% FBS in a humidified incubator with 5% CO2 at 37°C.27

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Caco-2 cells at 80% confluence were treated with 0.25% trypsin–EDTA and seeded

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on Transwell® inserts (1.12 cm2) at a density of 1 × 105 cells/cm2. The culture

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medium was replaced every day, and Caco-2 cell monolayers were obtained for

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experiments at least 21 days after seeding.28 The integrity of the cell monolayer was

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checked before each experiment on the basis of transepithelial electrical resistance

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(TEER), which was measured with a Millicell ERS electrode (Millipore Corp,

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Billerica, MA, USA). Only cell monolayers with a TEER value of more than 300

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Ω·cm2 were considered intact and were used for transport experiments.29

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To identify the appropriate TPG, acteoside and salidroside concentrations that can

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be used in transport experiments, cytotoxicity was analyzed through a cell viability

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experiment. Cell viability was determined through Cell counting kit-8 (CCK-8)

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method.30, 31 Caco-2 cells were seeded in 96-well plates at a density of 1 × 104

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cells per well and a volume of l80 µL. The plates were cultured for 24 h, and 20

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µL of sample was added to the experimental groups, whereas 20 µL of culture

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medium was added to control groups. After 24 h of incubation, the culture

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medium was replaced with 100 µL of medium containing 10 µL of CCK-8

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solution, and the plates were incubated at 37°C for an additional 2 h.

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Absorbance was measured at 450 nm using a Biotek microplate reader

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(Winooski, VT, USA), and background absorbance was excluded by performing

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blank corrections. Cell viability was expressed as the percentage of the

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untreated group (control = 100%). In the cell viability assay, every sample was

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tested with five replicates.

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Transepithelial Transport Experiments. Experiments on the transport of TPG,

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acteoside and salidroside across Caco-2 monolayers were performed in

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accordance with previously reported method with some modifications.29 In

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brief, cell monolayers were gently rinsed twice with HBSS (pH 6.8, 37°C) prior

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to the experiments, and incubated with transport buffer for 30 min at 37°C. The

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incubation medium was then aspirated.

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For the experiment on transport from the apical (AP) side to the basolateral (BL)

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side, 0.5 mL of HBSS containing TPG, acteoside or salidroside (100, 200, 300,

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400 and 500 µg/mL) was added to the AP side, and 1.5 mL of HBSS was added

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to the BL side. After 30, 60, 90, 120, 150 and 180 min of incubation at 37°C,

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samples (0.4 mL) were collected from the BL side and replaced with the same

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volume of HBSS. For the experiment on transport from BL to AP, 1.5 mL of

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sample was added to the BL side, and 0.5 mL of HBSS was added to the AP

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side. At the above time intervals, 0.4 mL of samples was collected from the AP

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side and replaced with the same volume of HBSS. The acteoside and salidroside

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concentrations in the samples were determined through the UHPLC–DAD analytical

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methods. All incubations were performed in triplicate.

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Inhibition studies were conducted using 100 µM verapamil (P-gp inhibitor), 100

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µM MK571 (MRP2 inhibitor)32 or 10 µM Ko143 (BCRP inhibitor)33. The inhibitors

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were added in the sample solution in the AP and BL sides. The transport study was

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then conducted as described above.

177 178

Apparent permeability coefficients (Papp) were calculated using the following equatioin: !"" (⁄) = ($ ⁄ )(1⁄%&' )

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Where, dQ/dt is the transport rate on the receiver side (µg/s); A is the membrane

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surface area of the insert (1.12 cm2); and C0 is the initial drug concentration in

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the donor compartment (µg/mL).

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The efflux ratio (ER), which is the ratio of Papp (BL to AP) to Papp (AP to BL), was determined using the following equation: ( =

!"" () %) !"" (% ))

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Total Phenylethanoid Glycoside Content Determination. The TPG content was

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determined using a method described by Zhou et al.3 The OFE was diluted with

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methanol to a suitable concentration and added 200 µL per well to 96-well plates.

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The absorbance of OFE was measured at 334 nm using a Biotek microplate reader

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(Winooski, VT, USA), and the TPG content was expressed as micrograms of

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acteoside equivalents (AE) per milliliters. The acteoside concentration range of the

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calibration series was 0.5 to 200 µg/mL.

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UHPLC–DAD Analysis. Samples were filtered through a 0.22 µm nylon syringe

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filter (ANPEL, Shanghai, China) and were analyzed following a previously described

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method34 with modifications using an Agilent 1290 UHPLC instrument (Agilent,

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Waldbronn, Germany) equipped with autosampler, binary pump, column thermostat

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and diode-array detector. Samples were separated on an Agilent ZORBAX

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Eclipse XDB-C18 column (3.5 µm, 2.1 mm × 150 mm) at 25°C. The mobile

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phase consisted of acetonitrile (solvent A) and water (containing 0.1% formic

198

acid, solvent B). A gradient program was used with the following profiles: 0–1

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min, 6% A; 1–4 min, from 6% to 15% A; 4–8 min, from 15% to 20% A; 8–10 min,

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from 20% to 30% A; 10–12 min, from 30% to 100% A; and 12–12.5 min, from 100%

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to 6% A; 12.5–15 min, 6% A. The flow rate was 0.2 mL/min, and the injection

202

volume was 4 µL. The DAD detector was set from 190 nm to 400 nm.

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Statistical Analysis. Values were reported as mean ± SD. Statistical analysis was

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performed using SPSS 20.0. One-way analysis of variance was used to determine the

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level of significance (p < 0.05).

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RESULTS AND DISCUSSION

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Digestive Stability and Bioaccessibility of TPG, Salidroside and Acteoside in

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the In Vitro Digestion Model. The stability of PhGs after gastric and intestinal

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digestion phases was evaluated on the basis of the remaining PhGs content. In the

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gastric digestion phase, the retention rates of salidroside and acteoside (Fig. 2)

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standards were 99.6% and 102%, respectively, indicating that PhGs are stable under

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gastric digestion conditions. However, the retention rate of acteoside remarkably

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dropped to 51.5%, and that of salidroside decreased to 99.8% in intestinal digestion.

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Salidroside

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phenylethanoid disaccharide with an ester linkage, which is easily destroyed.

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Acteoside destabilizes under increasing pH.3,

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likely decreased during intestinal digestion because of the elevated pH. The acteoside

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retention rate in intestinal digestion is similar to that (53%) in olive mill wastewater.15

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The OFE was analyzed by UHPLC–DAD (Fig. 3), and the TPG, salidroside and

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acteoside contents in OFE were 117.23 µg AE/mL, 7.46 µg/mL and 76.61 µg/mL,

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respectively.3 The retention rates of TPG, salidroside and acteoside in OFE (Fig. 2)

222

were 102%, 100% and 102% in gastric digestion phase, respectively, and the retention

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rates of TPG, salidroside and acteoside in OFE dropped to 82.3%, 97.3% and 49.4%

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in intestinal digestion, respectively. It indicated that TPG and acteoside in OFE were

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less stable in intestinal digestion, and the retention rates of salidroside and acteoside

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in OFE showed no significant differences with those of salidroside and acteoside

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standards during digestion. In previous study, Jiang et al.4 reported that the retention

is

a phenylethanoid

monosaccharide,

35

whereas acteoside

is a

Therefore, acteoside concentration

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rates of TPG, salidroside and acteoside in O. fragrans var. thunbergii were 35.47%,

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97.41% and 5.11% after intestinal digestion, respectively. The total phenylethanoid

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glycoside extraction method and digestion samples were different from this study.

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Also Jiang et al. did not adjust the pH of mixture during digestion. Those might lead to

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lower retention rates of TPG and acteoside than those in this study.

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Bioaccessibility includes digestive recovery, aqueous solubility in the intestinal

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digesta and necessary degradation before absorption.15 The bioaccessibilities of TPG,

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salidroside standard and acteoside standard were 80.9±2.92%, 98.7±1.35% and

236

50.1±3.04%, respectively, and had no significant difference with the retention rates in

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intestinal digestion. This result indicated that higher amounts of salidroside than of

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acteoside are available for absorption in the intestinal tract. Salidroside has better

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bioaccessibility than acteoside because of its stability, and the bioaccessibilities of

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TPG and acteoside were most impacted by poor stability rather than limited solubility

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in the intestine.

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Cytotoxicities of TPG, Salidroside and Acteoside to Caco-2 Cells. Cytotoxicities

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of TPG in OFE, salidroside standard and acteoside standard (100, 200, 400, 600 and

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800 µg/mL) were measured using the CCK-8 assay on Caco-2 cells. As shown in Fig.

245

4, the cell viabilities of salidroside treated cells were 105%, 108%, 109%, 102% and

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97.1% corresponding to 100, 200, 400, 600 and 800 µg/mL. Cell viability higher than

247

90% indicates that compounds were nontoxic to cells at the indicated concentration.36

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It suggested that salidroside was nontoxic to Caco-2 cells from 100 to 800 µg/mL. For

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TPG and acteoside, the cell viabilities were more than 100% at the concentrations

250

lower than 600 µg/mL. However, the cell viabilities of TPG and acteoside decreased

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to 79.4% and 80.0% at 800 µg/mL, respectively. TPG and acteoside showed

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inhibitory effects on Caco-2 cells at 800 µg/mL. Therefore, the concentrations of TPG,

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salidroside and acteoside less than 600 µg/mL were used in the following experiment.

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Transport of TPG, Salidroside and Acteoside across Caco-2 Cells. TPG in OFE,

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salidroside standard and acteoside standard absorption was investigated using the

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Caco-2 cell model, and the TEER value of the Caco-2 cell monolayers was 357±22

257

Ω·cm2. PhGs transport in the AP-to-BL and the BL-to-AP directions was studied, and

258

absorptive Papp (AP to BL) and secretory Papp (BL to AP) permeabilities were

259

estimated. The transported amounts of 200 µg/mL TPG, salidroside and acteoside

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linearly increased with time (Fig. 5). The Papp (AP to BL) of acteoside (Table 1) was

261

determined as 4.75 × 10−7 cm/s in 180 min, indicating poor permeability and

262

absorption.36 This result is slightly higher than the Papp (AP to BL) of acteoside (1.15 ×

263

10−7) from Cistanche deserticola across Caco-2 cells,37 but is considerably lower than

264

that of acteoside (1.67 × 10−6) from olive mill wastewater using the Ussing chamber

265

model,16 which is different from the model used in the present study. In this work, the

266

Papp (BL to AP) of acteoside (9.17 × 10−7 cm/s, Table 1) was 1.93-fold greater than its

267

Papp (AP to BL) value.

268

The Papp values (Table 1) of TPG and salidroside were higher than those of acteoside

269

in both the AP-to-BL and the BL-to-AP direction. The Papp (AP to BL) values of TPG

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and salidroside were higher than that of acteoside, which were 13.2 × 10–7 and 21.7 ×

271

10–7 cm/s, respectively. This result indicated that TPG and salidroside are more easily

272

absorbed than acteoside. The ER of TPG and salidroside were 1.78 and 1.09,

273

respectively.

274

The effect of concentration on the transport of TPG, salidroside and acteoside

275

(100-500 µg/mL) was determined. As the concentration of acteoside increased from

276

100 µg/mL to 500 µg/mL, the transported amount of acteoside in the AP-to-BL

277

direction increased in a concentration-dependent manner without saturation (Fig. 6 c),

278

indicating that acteoside transport in the AP-to-BL direction mainly occurred through

279

passive diffusion. The Papp (AP to BL) values of acteoside at concentrations of 100

280

µg/mL to 500 µg/mL ranged from 4.26 × 10−7 cm/s to 6.48 × 10−7 cm/s (Table 1).

281

However, acteoside transport in the BL-to-AP direction was considerably faster than

282

that in the AP-to-BL direction, with saturation at concentrations higher than 400 µg/mL.

283

The Papp (BL to AP) values of acteoside were greater than its Papp (AP to BL) values at

284

different concentrations, with ER values of 2.00, 1.93, 1.94, 1.85 and 1.54 at 100, 200,

285

300, 400 and 500 µg/mL (Table 1), respectively. The Papp (BL to AP) of acteoside

286

decreased at the concentration of 500 µg/mL, suggesting the saturation of the

287

transported amount of acteoside in the BL-to-AP direction. Generally, ER values of

288

more than 1.5 indicate active efflux.38 The present result suggested that the permeation

289

mechanism of acteoside is passive diffusion with active efflux in the BL-to-AP

290

direction. Acteoside might be the substrate of one or more efflux transporters (P-gp,

15



291

MRP2 or BCRP).

292

Results of TPG transport were similar to that of acteoside. The Papp (AP to BL) values

293

were in a small range from 13.2 × 10–7 cm/s to 14.3 × 10–7 cm/s over 100 µg/mL to 500

294

µg/mL, and the ER values calculated as 1.85, 1.78, 1.77, 1.73 and 1.52 at 100, 200, 300,

295

400 and 500 µg/mL (Table 1), respectively. The transport mechanism of TPG is also

296

passive diffusion with active efflux. The transported amounts and Papp values of

297

salidroside in the AP-to-BL and BL-to-AP directions were similar over 100 µg/mL to

298

500 µg/mL (Fig. 6 b) with ER values of 1.07–1.15. Thus, the permeation mechanism

299

for salidroside may be passive diffusion without active efflux.

300

In addition, the absorption percentages of TPG, salidroside and acteoside were

301

1.42–1.54%, 2.10–2.68% and 0.461–0.698% across Caco-2 monolayers, respectively

302

(Table 2). In a previous study, approximately 0.1% of acteoside in olive mill

303

wastewater was absorbed.16 These conflicting results may be attributed to the

304

different absorption model (Ussing chamber), acteoside concentrations (100 µM) and

305

incubation times (60 min) used in the previous study.

306

Effect of Inhibitors on TPG, Salidroside and Acteoside Absorption. Three ABC

307

transporter inhibitors, namely verapamil (P-gp inhibitor), MK571 (MRP2 inhibitor)

308

and Ko143 (BCRP inhibitor) were used to identify the transporters involved in the

309

transport of TPG in OFE, salidroside standard and acteoside standard. Treatment with

310

verapamil, MK571 or Ko143 did not significantly affect on the Papp (AP to BL) and Papp

311

(BL to AP) values of salidroside (Fig. 7 b and Table 3). Similarly, MK571 and Ko143

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did not significantly affect the Papp values of TPG and acteoside in both transport

313

directions. The addition of verapamil significantly decreased the Papp (BL to AP)

314

values of TPG and acteoside to 17.5 × 10–7 cm/s and 6.52 × 10−7 cm/s, respectively,

315

and significantly increased Papp (AP to BL) to18.8 × 10–7 cm/s and 6.76 × 10−7 cm/s

316

(Fig. 7 a, Fig. 7 c and Table 3), respectively. Verapamil inhibited the BL-to-AP efflux of

317

TPG and acteoside, and significantly increased their AP-to-BL influx. The ER values

318

of TPG and acteoside decreased to 0.932 and 0.964, respectively, indicating that TPG

319

and acteoside transport is mediated by P-gp. In other words, the transport mechanism

320

of TPG and acteoside is passive diffusion with active efflux mediated by P-gp.

321

In conclusion, the bioaccessibility, absorption and transport mechanisms of PhGs

322

were investigated. TPG, salidroside and acteoside exhibited bioaccessibilities of

323

80.9%, 98.7% and 50.1%, respectively, and absorption percentages of 1.42–1.54%,

324

2.10–2.68% and 0.461–0.698%, respectively. The transport experiment demonstrated

325

that the intrinsic permeability of salidroside is better than that of acteoside. The

326

permeation mechanism of salidroside is passive diffusion without active efflux, while

327

acteoside is the substrate of P-gp. This study demonstrated that the low bioavailability

328

of acteoside might be attributed to its poor bioaccessibility, low absorption and P-gp

329

efflux transporter.

330

AUTHOR INFORMATION

331

Corresponding author

332

* (B. L.) Tel./fax: +86-0571-89882665. E-mail: [email protected].

17



333

Funding

334

This study was supported by the National Major R & D Program of China (No.

335

2017YFD0400200), the Zhejiang Provincial Natural Science Foundation of China

336

(No. R15C200002), and the Special Project of Agricultural Product Quality Safety

337

Risk Assessment (No. GJFP2018015), Ministry of Agriculture, China.

338

Notes

339

The authors declare no competing financial interest.

340

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Figure Captions Figure 1. Chemical structures of (a) salidroside and (b) acteoside. Figure 2. Retention rates of TPG in OFE, salidroside in OFE, acteoside in OFE, salidroside standard and acteoside standard in the in vitro digestion model during the gastric and intestinal digestion stages. Retention rate is expressed as the percentage of the group before digestion (before digestion = 100%). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; *, p < 0.05 compared with the group before digestion. Figure 3. UHPLC–DAD spectrum of O. fragrans extracts at 280nm. Figure 4. Cytotoxicity of TPG in OFE, salidroside standard and acteoside standard on Caco-2 cells as determined using the CCK-8 assay. Data are presented as mean value ± SD (n = 5). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; *, p < 0.05 compared with the control group at the concentration of 0 µg/mL. Figure 5. Bidirectionally transported amounts (µg/cm2) of 200 µg/mL (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard from 0 min to 180 min. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side. Figure 6. Effect of concentration on the bidirectionally transported amounts (µg/cm2) of (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side.

25



Figure 7. Effect of inhibitors on the apparent permeability coefficients (Papp) of (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard during transportation across Caco-2 cell monolayers. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side; *, p < 0.05 compared with the control group.

26


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Table 1. Effect of concentration on the apparent permeability coefficients (Papp) and efflux ratio (ER) of TPG in OFE, salidroside standard and acteoside standard across Caco-2 cell monolayers. Concentration (µg/mL) 100 200 300 400 500

TPG Papp (× 10-7 cm/s) AP to BL BL to AP a 13.2±2.46 24.3±3.69a* 13.2±0.259a 23.4±1.10a* 13.5±1.43a 23.9±2.70a* 13.5±1.52a 23.3±2.41a* 14.3±2.25a 21.7±2.21b*

ER 1.85 1.78 1.77 1.73 1.52

Salidroside Papp (× 10-7 cm/s) AP to BL BL to AP a 24.8±1.01 28.2±0.268a 21.7±1.38b 23.7±1.33b 20.1±2.46b 22.0±1.05b 19.4±1.60b 20.8±2.26b 19.4±1.72b 22.3±2.26b

ER 1.13 1.09 1.10 1.07 1.15

Acteoside Papp (× 10-7 cm/s) AP to BL BL to AP a 4.26±0.753 8.52±1.82a* 4.75±0.251a 9.17±0.708a* 4.97±0.676a 9.63±1.73a* 5.60±1.00a 10.4±0.984b* 6.48±1.19b 9.96±1.18b*

ER 2.00 1.93 1.94 1.85 1.54

Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side. Values marked by different superscript letters in a column are statistically different at the level p < 0.05. *, p < 0.05 compared with Papp (AP to BL).

27



Table 2. Absorption percentages of TPG in OFE, salidroside standard and acteoside standard determined by Caco-2 intestinal cell model. Concentration (µg/mL) 100 200 300 400 500

Absorption percentage (%) TPG Salidroside Acteoside a b 1.42±0.112 2.68±0.388 0.461±0.0441c 1.42±0.238a 2.40±0.213b 0.503±0.102c 1.46±0.144a 2.17±0.138b 0.536±0.134c 1.46±0.192a 2.10±0.182b 0.607±0.0912c 1.54±0.256a 2.10±0.201b 0.698±0.113c

Data are presented as mean value ± SD (n = 3). Values marked by different superscript letters in a row are statistically different at the level p < 0.05. TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts.

28


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Table 3. Effect of verapamil, MK571 and Ko143 on the apparent permeability coefficients (Papp) and efflux ratio (ER) of TPG in OFE, salidroside standard and acteoside standard across Caco-2 cell monolayers.

Groups Control Verapamil MK571 Ko143

TPG Papp (× 10 cm/s) AP to BL BL to AP 13.2±0.259 23.4±1.10 18.8±2.05* 17.5±2.63* 12.8±0.526 21.9±0.813 13.5±0.418 22.2±1.75 -7

ER 1.78 0.932 1.71 1.64

Salidroside Papp (× 10-7 cm/s) AP to BL BL to AP 21.7±1.38 23.7±1.33 22.3±0.961 22.6±1.29 23.6±0.770 24.4±0.836 22.5±0.758 23.3±0.929

ER 1.09 1.02 1.03 1.03

Acteoside Papp (× 10-7 cm/s) AP to BL BL to AP 4.75±0.251 9.17±0.708 6.76±0.170* 6.52±0.895* 5.11±0.148 10.2±0.528 4.99±0.118 9.38±0.560

ER 1.93 0.964 1.99 1.88

Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side; *, p < 0.05 compared with the control group.

29



Figure 1. Chemical structures of (a) salidroside and (b) acteoside.

30


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Figure 2. Retention rates of TPG in OFE, salidroside in OFE, acteoside in OFE, salidroside standard and acteoside standard in the in vitro digestion model during the gastric and intestinal digestion stages. Retention rate is expressed as the percentage of the group before digestion (before digestion = 100%). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; *, p < 0.05 compared with the group before digestion.

31



Figure 3. UHPLC–DAD spectrum of O. fragrans extracts at 280nm.

32


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Figure 4. Cytotoxicity of TPG in OFE, salidroside standard and acteoside standard on Caco-2 cells as determined using the CCK-8 assay. Data are presented as mean value ± SD (n = 5). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; *, p < 0.05 compared with the control group at the concentration of 0 µg/mL.

33



Figure 5. Bidirectionally transported amounts (µg/cm2) of 200 µg/mL (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard from 0 min to 180 min. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side.

34


Page 34 of 37

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Figure 6. Effect of concentration on the bidirectionally transported amounts (µg/cm2) of (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side.

35



Figure 7. Effect of inhibitors on the apparent permeability coefficients (Papp) of (a) TPG in OFE, (b) salidroside standard and (c) acteoside standard during transportation across Caco-2 cell monolayers. Data are presented as mean value ± SD (n = 3). TPG, total phenylethanoid glycoside; OFE, O. fragrans extracts; AP, apical side; BL, basolateral side; *, p < 0.05 compared with the control group.

36


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TOC graphic

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Bioaccessibility and Absorption Mechanism of Phenylethanoid

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