Evaluation of the Intestinal Transport of a Phenylethanoid Glycoside-Rich Extract from Cistanche deserticola across the Caco-2 Cell Monolayer Model

February Evaluation of the Intestinal Transport of a Phenylethanoid Glycoside-Rich Extract from Cistanche deserticola across the Caco-2 Cell Monolayer Model Yuan Gao 0 1 2 Chuanjie Zong 0 1 2 Fen Liu 0 1 2 Lei Fang 0 1 2 Runlan Cai 0 1 2 Yue Shi 0 1 2 Xi Chen 0 1 2 Yun Qi 0 1 2 0 1 Department of Research Center for Pharmacology and Toxicology, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , P.R. China , 2 Heilongjiang University of Chinese Medicine , Harbin, Heilongjiang , P.R. China 1 Data Availability Statement: All relevant data are within the paper 2 Academic Editor: Simon Patrick Hogan, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, UNITED STATES Phenylethanoid glycosides (PhGs), a class of polyphenolic compounds, are considered one of major bioactive constituents of Cistanche deserticola Y.C. Ma (CD), whose extract is orally used in traditional Chinese medicine. Although previous pharmacological studies have reported that PhGs exert many activities, their intestinal transport profiles have not been clarified. In this study, we investigated the intestinal permeability of a PhG-rich extract (PRE) from CD as an integrated system in the Caco-2 cell monolayer model using a bioassay system. The results showed that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. We also determined the intestinal permeability of three major PhGs [acteoside (AC), isoacteoside (IS) and echinacoside (EC)] by HLPC. Furthermore, we developed a novel HPLC-fluorescence detection method to accurately determine the flux amount of AC and IS. As expected, the transport characteristics of the three PhGs are consistent with those of PRE, indicating that the present bioassay system is appropriate and reliable for the evaluation of the transport characteristics of active ingredient groups (AIG) in PRE. Moreover, this system may also be suitable for other plant extracts given appropriate bioactivity. - Funding: This work was supported by the National Natural Science Foundation of China (Nos. 81173645 and 81274163) and National S&T Major Project and Scientific Researchers Aiding Enterprise Item (Nos. 2012ZX09301-002-001 and 2014ZX09201022-006) from the Ministry of Science and Technology of the Peoples Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Caco-2 cell line, which was derived from human colon adenocarcinomas, exhibits enterocyte-like characteristics. Under normal conditions, Caco-2 cells spontaneously differentiate from mature cells and form intact monolayers [1]. The adjacent cells adhere via tight junctions formed at the apical side of the monolayer, which can discriminate the passively Competing Interests: The authors have declared that no competing interests exist. and actively transported drugs across the epithelial layer [2]. Due to the morphological and biochemical similarity to normal enterocytes, Caco-2 cell monolayers serve as a well-accepted in vitro model for the study of the intestinal absorption potential and transport characteristics of drugs [3, 4]. In contrast to chemicals, plant extracts (PE) are mixtures whose biological activity and active constituents are often not well identified [5]. Moreover, the intestinal transport properties of PE, as opposed to the properties of its constituents, are closely related to clinical use. Flux measurements for a test sample across a Caco-2 cell monolayer commonly involve chemical methods, such as HPLC, LC/MS, etc. Although these methods are powerful tools, they are complex, time-consuming, expensive, and occasionally require sophisticated equipment. More importantly, neither a single nor a minority component can reflect PE as a whole. Thus, a novel approach independent of the determination of constituents needs to be established to identify and evaluate the transport characteristics of PE. Cistanche deserticola Y.C. Ma (CD), a holoparasitic plant, is a common traditional Chinese medicine mainly used to treat kidney deficiency, body weakness and constipation, and these uses have been officially recorded in the Chinese Pharmacopoeia [6]. Phenylethanoid glycosides (PhGs), including echinacoside (EC), acteoside (AC) and isoacteoside (IS), etc., are a class of polyphenolic compounds [7]. They are considered one of major bioactive constituents of Cistanche species [8]. Pharmacological studies have shown that the bioactivity of PhGs is diverse and includes anti-oxidative [9], anti-fatigue [10], hepatoprotective [11], immunomodulatory [12], anti-inflammatory [7, 13] and neuroprotective effects [14]. However, the intestinal transport characteristics of PhGs have not been investigated. In this study, we explored the intestinal permeability of a PhG-rich extract (PRE) from CD as an integrated system and the permeability of three major PhGs (AC, IS and EC) in differentiated Caco-2 cells. Our results indicated that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. The human intestinal Caco-2 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). AC, IS and EC (>98%) were purchased from Must Biotechnology Co. (Chengdu, China). Dulbeccos modified Eagles medium (DMEM), fetal bovine serum (FBS) and non-essential amino acids (NEAA) were produced by Gibco BRL (Grand Island, NY, USA). 6-well TranswellTM plates (insert membrane growth area 4.67 cm2) were obtained from Corning (Costar) Inc. (Tewksbury, MA, USA). Rat-tail collagen was obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents and chemicals for the HPLC analysis were of analytical grade. Preparation of PRE from CD The air-dried CD material was powdered and extracted by percolation with 70% ethanol. The PhG-rich fraction was prepared as previously described [10] and extracted with water-saturated n-butyl alcohol. The extract liquor was concentrated and dried under reduced pressure. Macroporous resin-UV spectrophotometry [15] measured a PhG content of 78.4%. The final sample represented a 1.75% yield of raw material by dry weight. The obtained sample was stored at 20C until further use. Determination of AC, IS and EC by HPLC A Shimadzu HPLC system equipped with the LC solution software was used to assay the contents of AC, IS and EC in PRE. A reverse phase Intersil C18 column (4.6 mm 250 mm, 5 m) was used and maintained at room temperature. The mobile phases were acetonitrile and water containing 0.1% phosphoric acid (v/v) with a gradient elution (Table 1) at a flow rate of 1.0 ml/min. The UV spectrophotometer detector was set to 334 nm. To a (...truncated)