Caco-2 Monolayer Permeability and Stability of Chamaelirium luteum

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Article Cite This: ACS Omega 2019, 4, 7658−7666

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Caco‑2 Monolayer Permeability and Stability of Chamaelirium luteum (False Unicorn) Open-Chain Steroidal Saponins Tegan P. Stockdale,† Victoria L. Challinor,† Reginald P. Lehmann,‡ James J. De Voss,† and Joanne T. Blanchfield*,† †

School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4068, Australia Integria Healthcare, Eight Mile Plains, Queensland 4113, Australia

‡

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S Supporting Information *

ABSTRACT: Herbal medicines, although significant components of health care for much of the global populace, are still far less stringently regulated than conventional pharmaceuticals. Safety and efficacy concerns arise because of the limited information regarding many of the phytochemicals present in herbal medicines. Chamaelirium luteum (false unicorn) is an indigenous American herb marketed for “women’s issues”. Recently, the potential bioactives, a new class of open-chain steroidal saponins, have been fully characterized and preliminary bioactivity assays have been performed. The present study utilized the Caco-2 cell monolayer model to evaluate metabolic vulnerability and potential bioavailability of eight saponins and sapogenins from false unicorn. No compound metabolism was detected in Caco-2 cell homogenate. One sapogenin, helogenin, exhibited low-to-moderate permeability across the monolayers. Three saponinschamaeliroside A, heloside A, and 6-dehydrochamaeliroside Awere found to have moderate permeability. Transport studies indicated active transport of these saponins. In contrast, all saponins with more than two sugar units exhibited low permeability.

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INTRODUCTION Plant-based medicines have been used for thousands of years, with evidence seen in ancient Egyptian scrolls and compendiums from Bronze Age China.1 Herbal medicines remain the primary form of health care for 70−95% of people in developing nations.2 Traditional medicines continue to play a role in conventional health care as many of the compounds isolated form the basis of conventional pharmaceuticals, with 26% of approved drugs worldwide, from 1981 to 2014, being either natural products or their derivatives, and a further 21% of drugs being synthetic natural product mimics.3 In developed nations, the public acceptance and usage of complementary and alternative medicines (CAM) has grown significantly over recent years. In 2008, the global market for CAM was reportedly US$83 billion annually and is growing rapidly.2 CAM products are commonly perceived as a safe and effective way to pursue holistic health and well-being and are often used to complement conventional medicine.4−6 For example, American National Institute of Health statistics from 2012 indicate that a third of adults had used complementary health approaches in the last 12 months, and the most commonly used approach was non-vitamin, non-mineral, dietary supplements.7 Although specific regulations surrounding CAM vary between countries, they are generally less stringent than those for conventional pharmaceuticals. Recently, however, there has been a great deal of unease regarding the control and © 2019 American Chemical Society

regulation of CAM because of reports of adulteration of herbal medicine components and of adverse reactions following CAM usage.8−10 Factors such as growth and harvest conditions, transport, storage, and extraction and compounding methods have all been shown to dramatically affect the quantities and relative abundance of constituents present in herbal extracts.11,12 Although numerous studies have assessed the bioactivity of key phytochemicals, evaluation of the bioavailability of bioactives is often limited or nonexistent. The present study addresses this by utilizing the Caco-2 cell monolayer model of the human small intestine to evaluate the relative bioavailability of selected open-chain steroidal saponins and sapogenins, previously isolated from Chamaelirium luteum (false unicorn). First reported in 1989,13 the now widely-accepted Caco-2 model provides apparent permeability (Papp) values, by assessing the rate at which a compound crosses a fully differentiated monolayer of human epithelial cells.14,15 Caco-2 assay results have shown good correlation with in vivo human oral absorbance for a range of passively diffused drug molecules.14 Differentiated cells have been shown to express all the major digestive enzymes and transport proteins present in the human small intestine, allowing metabolism as well as Received: February 21, 2019 Accepted: April 12, 2019 Published: April 26, 2019 7658

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active and passive transport to be investigated.15−20 The assay has become a routine preliminary screen in drug development research and has been successfully used, by our group and others, in evaluating the permeability of natural products, including saponins.10,14,21,22 Saponins are secondary metabolites found in a wide range of species, predominantly plants, but also some marine organisms.23 Steroidal saponins are amphipathic molecules, with a lipophilic cholesterol-derived aglycone (sapogenin) skeleton, often sharing structural similarity with human steroid hormones. Diverse hydrophilic sugar moieties can decorate this central skeleton at a variety of attachment points (Figure 1). These natural products are the proposed active constituents

Figure 2. Structures of selected steroidal saponins from C. luteum. (1) Chamaeliroside A; (2) chamaeliroside B; (3) (23R,24S)-chiograsterol B; (4) heloside A; (5) helogenin; (6) 6-dehydrochamaeliroside A; (7) 6-dehydrochamaeliroside B; (8) chamaeliroside E. Figure 1. Structure of cholesterol and typical aglycone skeletons of spirostanol and furostanol steroidal saponins (R = sugar). Conventional numbering for steroids is indicated.

gastrointestinal tract are significant barriers to oral bioavailability.14 The presence of glycosides may render the saponins susceptible to metabolism by enzymes, such as glycosidases, but may also raise the potential for them to be recognized by sugar transporters. It was considered desirable to explore the metabolic susceptibility, permeability, and active transport of these saponins and determine which compounds or metabolites are able to cross the intestinal tract. A subset (1−8) of the open-chain steroidal compounds found in C. luteum was chosen for this systematic investigation of the effects of various structural features on bioavailability (Figure 2). Inclusion of both sapogenins and saponins allowed the effect of size and hydrophobicity or hydrophilicity to be studied. A monodesmodic saponin has only one sugar attachment point and is amphipathic, whereas a bidesmodic saponin has two sugar attachment points, with polarity at both ends of the aglycone core. Inclusion of mono- and bidesmodic saponins, with varying sugar types, number of units, and attachment positions, permitted exploration of the effect of sugar group number, nature, and attachment point to be considered. In the present study, Caco-2 permeability assays showed that sapogenin 5 has borderline low-to-moderate permeability, indicating the potential for partial human oral bioavailability. Three saponins1, 4, and 6have moderate permeability, indicating that they have the potential for partial to complete human oral bioavailability. Results of Caco-2 transport assays on these three permeable saponins suggest that all three compounds are actively transported and that saponin 1 is also an efflux pump substrate. This is the first bioavailability study of open-chain saponins and allows future bioactivity studies to focus on these bioavailable compounds. By elucidating bioavailable compounds and exploring their transport mechanism, this study contributes valuable insight into the safety and efficacy of this medicinal herb.

in numerous herbal medicines and traditional therapies.24,25 A large range of bioactivities have been attributed to steroidal saponins, including cytotoxic, anti-inflammatory, anti-bacterial, anti-fungal, and haemolytic activities.25−27 The broad range of these bioactivities is likely attributable to their structural diversity, arising from different aglycone skeletons, hydroxylation patterns of the steroidal nucleus and varying identity, number and attachment positions of sugar groups.24 Within steroidal saponins, there are two well-known structural classes: hexacyclic spirostanols, with a bicyclic acetal at C-22, and pentacyclic furostanols, with a hemiacetal at C-22 and a C-26 glycosidic bond (Figure 1). More recently, a third class has been added. These are open-chain steroidal saponins. These compounds lack additional rings in the C-17 side chain and constitute the major class of saponins present in C. luteum (Figure 2). This third class of steroidal saponins has been recently surveyed by Challinor and De Voss.24,28 C. luteum, also known as false unicorn, was traditionally used by Native Americans to strengthen the heart and treat nausea, coughs, and female reproductive issues, including miscarriage prevention and prolapse.29,30 The herbal extract has continued to be used for regulating menses, promoting fertility, and preventing miscarriage.31,32 It was noted in 2012 that surprisingly limited phytochemical characterization of this herb had been performed, especially given its commercial medicinal use and inclusion in herbal mixtures in both a mouse model study and a human clinical trial.33 When investigating the open-chain saponins present in C. luteum, Challinor and De Voss reported detailed structural and stereochemical features, in addition to preliminary bioactivity data.24,28,33−35 Of the 15 steroidal saponins isolated from false unicorn, eight were novel compounds. Chamaelirosides A and B (1 and 2), which are based on the same aglycone skeleton, (23R,24S)chiograsterol B (3), were predominant.35 Given the oral administration of false unicorn herbal extracts, it is important to evaluate bioavailability to identify which of these compounds, if any, are able to reach the bloodstream. Both metabolism and permeability across the

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RESULTS AND DISCUSSION On the basis of chemical properties, such as size and localized or overall hydrophobicity and hydrophilicity, initial qualitative predictions were made regarding the likely permeability of the compounds in this work. It was predicted that the smaller, 7659

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provided some updated guidelines as to how permeability values might correlate to human oral absorption: low permeability (0−20% human fraction absorbed (Fa)) is correlated to Papp values of 10 × 10−6 cm/ s.52 Although it is acknowledged that the actual permeability values may vary in Caco-2 monolayer permeability assays, the assay results are most reliably interpreted in comparison to compounds evaluated in the same assay, relative to standard compounds representing upper and lower bounds.22,54 Propranolol has >90% human absorption, is considered to be completely orally available, and exhibits high permeability values (Papp > 20 × 10−6 cm/s) in Caco-2 cell permeability assays.14 In contrast, fluorescein is a low molecular weight, very poorly orally available, hydrophilic molecule, with low permeability values (Papp < 1 × 10−6 cm/s) in the assay.58 In this study, propranolol and fluorescein were chosen as high and low permeability standards, respectively. Fluorescein additionally provides a way to verify monolayer integrity independent of TEER values, which may not be sufficient on its own. Fluorescein’s high molar absorptivity (ε490nm = 76 900 M−1 cm−1) allows for efficient analysis by UV−vis spectrophotometry.59 The propranolol and fluorescein standards included in the assay gave permeability values within the expected ranges, providing further evidence of good monolayer integrity (Table 1, Figure 3).

more hydrophobic aglycones would be likely to be most permeable, via passive transcellular or paracellular diffusion, whereas the larger, more polar, amphipathic saponins would be unlikely to have good permeability via these routes. Scrutiny of the literature in the area of Caco-2 transport studies shows that many triterpenoid36,37 and steroidal38,39 sapogenins are moderately to highly permeable. The literature appears to support the hypothesis that, when absorption occurs by passive diffusion, sapogenins are more permeable than their saponin counterparts.36−38,40,41 When saponins are actively transported, they may be rendered more permeable than their passively transported sapogenin analogues.42 However, when the number of sugars incorporated into a saponin exceeds two, the permeability tends to decrease.41,43−46 The clinically significant cardiac glycoside digoxin is an obvious exception to this trend. Digoxin is a steroidal saponin bearing a trisaccharide at C3 and is known to have an oral bioavailability that varies between 50 and 80%.47 There are many factors that contribute to this variability in the absorption of digoxin. One of these is the fact that the glycoside is a P-glycoprotein substrate and so factors that can alter the expression or activity of this efflux pump can result in varying levels of digoxin absorbed from the small intestine.47,48 We first evaluated the metabolic vulnerability of the false unicorn sapogenins, 3 and 5, and saponins, 1, 2, 4, 6−8 (Figure 2). The expression of human digestive enzymes, including glycosidases, by Caco-2 cells renders them an appropriate model for such a study.20 The cells were cultured and maintained according to standard protocols,10,49,50 then seeded into 96-well plates. They were allowed to grow to between 21 and 28 days old50,51 as this not only allows cells to become confluent, but also provides the necessary time for the cells to fully mature and differentiate to express functional enzymes.13,52,53 The compounds were exposed to a homogenate of differentiated Caco-2 cells and then analyzed by LCMS. Endomorphin-1, a swiftly metabolized neuroactive tetrapeptide, with a half-life of 5−9 min in Caco-2 homogenate, was chosen as the positive control.50 Although the expected metabolism of endomorphin-1 did occur (t1/2 = 8.5 min), no metabolism of the steroidal saponins or sapogenins was detected in this initial stability assay. In addition, no metabolites were detected during the later permeability and transport assays. This suggests that the false unicorn saponins and sapogenins are not susceptible to metabolic breakdown under the conditions of the Caco-2 model. However, it should be noted that this does not account for variable enzyme expression in Caco-2 cells or possible in vivo metabolism by intestinal microflora.54−56 The Caco-2 cells for the permeability assay were seeded into Transwells and maintained according to standard protocols.10,49,50 The pre-assay integrity of the monolayers was assessed by measuring the transepithelial electrical resistance (TEER). TEER values of 300−600 Ω cm2 have been shown to strongly indicate the development of good monolayer integrity, with strong tight junctions between cells.54 Wells that were used in this work displayed values in the acceptable range of 300−500 Ω cm2. Variability between Caco-2 permeability studies, because of differences in culturing and experimental conditions and evolved differences in the cell line, has led to reporting of different permeability values for the same compounds.54−57 In an effort to understand this variability, a recent study has

Table 1. Papp Values in the Apical to Basolateral Direction (A−B) for Compounds Isolated from C. luteuma

a

compound

Papp(A−B) (cm/s) (±SD) × 10−6

propranolol fluorescein chamaeliroside A (1) chamaeliroside B (2) chiograsterol B (3) heloside A (4) helogenin (5) 6-dehydrochamaeliroside A (6) 6-dehydrochamaeliroside B (7) chamaeliroside E (8)

36.5 (±2.96) 0.606 (±0.203) 2.64 (±0.872) 0.262 (±0.099) 7.61 (±1.47) 1.45 (±0.355) 0.711 (±0.148) 13.6 (±2.99) 0.067 (±0.049) 0.250 (±0.090)

Replicates: n = 3−4.

The permeability assay was conducted by placing compound and standard solutions into the apical side of the wells and buffer into the basal chamber. Samples were periodically taken and replaced with buffer. The wells used to evaluate the natural products were washed and a post-run fluorescein permeability assay similarly conducted. The post-run fluorescein assay results allowed data from failed wells to be excluded and drew attention to the drop in monolayer integrity for cells exposed to sapogenin 3. This result was unsurprising, given that 3 has previously been shown to exhibit cytotoxic activity.33 Consequently, little weight can be given to the seemingly moderate-to-high permeability value calculated for aglycone 3 (Papp = 7.61 (±1.47) cm/s (±SD) × 10−6). Sapogenin 5 displayed low-to-moderate permeability (Papp = 0.711 (±0.148) × 10−6 cm/s); however, 5 was not easily soluble in the assay buffer, and so this result may be influenced by this poor solubility and possible precipitation during the assay. If these solubility issues are addressed, the observed permeability 7660

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Figure 3. Apparent permeability (A−B) for compounds isolated from C. luteum and fluorescein (propranalol control not shown Papp = 36.5 (±2.96) × 10−6). The Papp values are color coded as follows: green for good permeability; light blue for moderate permeability; dark blue for low permeability; and orange indicates caution as 3 disrupted monolayer integrity.

which evaluates the ratio of apical to basal permeability compared to basal to apical permeability, was conducted to explore whether these compounds are actively influxed or effluxed across the cell monolayer. Caco-2 cells are known to express active transporters, including sugar transporters, such as SGLT1, GLUT2, and GLUT5, and efflux pumps, including members of the ATP-binding cassette, such as P-gp and MRP2.19,43,53 Once again, transport assay monolayer TEER values fell within the acceptable range, and the propranolol and fluorescein standards gave permeability values within the expected ranges, providing evidence of good monolayer integrity (Table 2, Figure 4). Notably, the propranolol transport assay value was within a standard deviation of the permeability assay value. The fluorescein permeability values, both during and after the assay, were consistently low, indicating that none of the wells were damaged during the assay. Saponins 1, 4, and 6 all gave permeability values consistent with moderate permeability. The Papp of 6 was lower

may increase. Thus, sapogenin 5 arguably shows potential for low-to-moderate human oral bioavailability. Saponins 1 and 6 are both monodesmodic, with two sugars. In the initial permeability assays, they showed moderate-tohigh permeability (Papp = 2.64 (±0.872) and 13.6 (±2.99) × 10−6 cm/s, respectively). Upon repetition (vide infra), the permeability value for 1 was approximately the same (Papp = 1.85 (±0.599)) but that of 6 was lower (1.21 (±0.092) × 10−6 cm/s); however, in all cases, at least moderate permeability was found for these saponins. These results indicate that 1 and 6 have the potential for moderate oral absorption in vivo. These saponins have very similar sapogenin cores, differing only in the oxidation state at C-6, with 1 having a C-6 hydroxyl and 6 having a C-6 ketone. Saponin 4, which has sapogenin 5 as a core, is also moderately permeable (Papp = 1.45 (±0.355) × 10−6 cm/s). This saponin also has only two sugar units, but is bidesmodic. Saponins 2, 8, and 7 are all bidesmodic, each with three sugar units, and showed low-to-negligible permeability (Papp = 0.262 (±0.0991), 0.250 (±0.0901), and 0.0667 (±0.0493) × 10−6 cm/s, respectively), indicating they are unlikely to be orally bioavailable. Comparing the C-6 hydroxy saponins 1, 2, 7, and 8, a trend is observed. Saponin 1, with two sugar units, is moderately permeable, whereas saponins 2, 8, and 7, all with three sugar units, are poorly permeable. This trend continues when comparing the C-6 keto saponins, 6 and 7. Although 6, with two sugar units, exhibits moderate permeability, saponin 7, with three sugar units, is almost 10 times less permeable than fluorescein. Thus, it appears that all tested false unicorn saponins with two sugar units are, at least, moderately permeable. However, when the number of sugar units exceeds two, regardless of the position of the attachment or identity of the sugar groups, the Papp measured drops to a level corresponding to poor human oral bioavailability. The moderate permeability of saponins 1, 4, and 6 suggests that, despite their size and amphipathic nature, these compounds may be actively transported. A transport assay,

Table 2. Apparent Permeability of Compounds 1, 4, and 6 and Control Compounds in Both the Apical to Basolateral (A−B) and Basolateral to Apical (B−A) Directions and the Efflux Ratios for Each Compound (Papp(A−B)/Papp(B−A)) sample propranolol (A−B) propranolol (B−A) chamaeliroside A (1) (A−B) chamaeliroside A (1) (B−A) heloside A (4) (A−B) heloside A (4) (B−A) 6-dehydrochamaeliroside A (6) (A−B) 6-dehydrochamaeliroside A (6) (B−A) fluorescein (A−B) 7661

Papp (cm/s) (±SD) × 10−6 (0−150 min)

efflux ratio

35.3 (±4.00) 37.9 (±4.30) 1.85 (±0.599) 5.87 (±3.09) 1.52 (±0.809) 0.576 (±0.100) 1.21 (±0.092)

0.93 0.31 2.64 2.64

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Figure 4. Apparent permeability of compounds 1, 4, and 6 in the apical to basolateral (A−B) and basolateral to apical (B−A) directions and the Papp(A−B) for fluorescein (propranalol control not shown). Light green = 1, light blue = 4, dark blue = 6, purple = fluorescein.

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in this assay than the initial experiment, but the sensitivity of Caco-2 cells to inter-assay transporter level variability may explain the reduced permeability of 6 in the transport assay.19,57 The permeability was different in the apical-to-basal compared to the basal-to-apical direction for saponins 1 (p = 0.003), 4 (p = 0.01) and 6 (p = 0.002). A transport ratio favoring one direction by a factor of, at least, 1.5−2.0 is considered indicative of active transport.22 All ratios in the transport study exceeded this threshold, except for propranolol, which acts as a negative control for active transport. The data for saponin 1 gave an efflux ratio, defined as the quotient of Papp in the A-to-B direction divided by the Papp in the B-to-A direction, of 0.31, indicating that 1 is a substrate for an efflux pump.22 This is a moderate efflux ratio,39,60 representing much lower efflux levels than some other saponins, which have been reported to exhibit efflux rates 10 to >50 times higher than the rate of absorption.21,36,61 This moderate efflux ratio may suggest that 1 is a comparatively poor efflux pump substrate. Alternatively, given that 1 still displays overall moderate permeability in the apical−to-basal direction, it may suggest that 1 is a substrate for both an active transporter and an efflux pump. In contrast, the data for saponins 4 and 6 gave efflux ratios of 2.6, indicating active transport in the apical-tobasolateral direction.22 These are moderate transport rates, comparable to those reported for other saponins.43,45 The literature on the Caco-2 permeability of saponins suggests that sapogenins are generally more permeable than saponins, except where significant active transport occurs. Increasing glycosylation tends to further reduce permeability. Consistent with this trend, sapogenin 5 had low-to-moderate permeability, whereas all false unicorn saponins with three sugar units had low-to-negligible permeability. Only actively transported saponins 1, 4, and 6 were more permeable than sapogenin 5.

CONCLUSIONS

This study demonstrated that a number of steroidal saponins and sapogenins in C. luteum exhibit moderate permeability across Caco-2 cell monolayers. On the basis of this in vitro experiment, sapogenin 5 is predicted to have moderate oral bioavailability. Despite their larger size and amphipathic nature, the permeability assay results indicated that saponins 1, 4, and 6, each with two sugar units, had moderate permeability, whereas all saponins with three sugar units had poor-to-negligible permeability. This raised the possibility that active transport processes account for the observed permeability values. Bidirectional transport assays showed that 1, 4, and 6 have transport ratios indicative of active uptake for 4 and 6 or, in the case of 1, efflux. The transport study described in this paper does not implicate the involvement of specific uptake transporters or efflux pumps. In future, transport inhibition studies should be undertaken to confirm active transport and identify the particular transporters involved. These studies involve assessing the effect of adding known transporter substrates to act as inhibitors, such as cyclosporine A to inhibit P-gp, or altering ion levels necessary for transporter function, such as using sodium-free medium to inhibit SGLT1.43 This may be particularly interesting in the case of saponin 1, to ascertain whether both active uptake and efflux transport is occurring. The results presented in this paper are consistent with the hypothesis that sapogenins are generally more permeable than saponins, except where significant active transport occurs and that saponins with more than two sugar units are generally impermeable. These structural relationships should be further explored. This was the first bioavailability study of open-chain steroidal saponins, and the results will be helpful in addressing safety and efficacy concerns surrounding herbal medicines as well as allowing future bioactivity studies to focus on putatively bioavailable compounds. 7662

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EXPERIMENTAL SECTION General Procedures. All solvents and chemicals were used as purchased without further purification, except solutions to be utilized in cell culture, which were filtered through a 0.22 μm filter to ensure sterility. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance 500 (500.13 MHz for 1H) using pyridine-d5 solvent. Chemical shifts are given in parts per million on the δ scale, with internal reference to calibrated residual solvent peak (δH 8.71). Solvents used in sample analysis were LCMS grade (Fischer Scientific). All C. luteum compounds were pure compounds extracted, purified by HPLC and previously characterized by 1H and 13C NMR.33,35 Further 1H NMR spectra were collected immediately prior to performing the assays to confirm compound purity. Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD). Transwells (24 well, 6.5 mm polycarbonate inserts, 0.4 μm pore) were from Costar (Cambridge, MA), and cell culture reagents were purchased from Gibco (Grand Island, NY). Cell Cultures. Cells were maintained, at 37 °C, 95% humidity, and 5% CO2, in flask media composed of Dulbecco’s modified Eagle’s medium, 4 mM L-glutamine, 25 mM Dglucose, supplemented with 10% fetal bovine serum (FBS), and 1% non-essential amino acids (NEAA). Flask media was changed on alternate days. Cells were passaged, upon reaching 80% confluence, using 0.05% trypsin−EDTA. Metabolism Assay. Approximately 5 × 104 cells (passage 41) were seeded onto 96-well plates (Nucleon, Polysorb). Plated cells were fed with flask media supplemented with 1% 5000 unit/mL penicillin\streptomycin antibiotic mixture (plate media). On days 1−8, all media was refreshed (100 μL) on alternate days. Plate media was increased (300 μL) and refreshed daily as cells matured. On day 25, media was removed and wells washed with ice-cold phosphate-buffered saline (PBS; 2 × 200 μL). Fresh PBS (100 μL) was added and the cells scraped from the bottom of the wells, centrifuged (2000 rpm, 5 min), and the supernatant discarded. Cells were resuspended in ice-cold Hank’s buffered salt solution (HBSS)25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4, 6 mL), lysed via sonication, the mixture centrifuged (2000 rpm, 5 min), and the supernatant collected. In fresh 96-well plates, cell homogenate (30 μL) was mixed, in quadruplicate, with an equal volume of compound solution or endomorphin-1 as a positive control (2 mg/mL). Plates were placed into a shaking incubator (37 °C, 55 rpm) and, at 10, 20, 30, 60, 120 min, and 12 h, samples (10 μL) were taken and deproteinized with water acidified with formic acid (pH = 1−2, 90 μL). Samples were refrigerated until analysis. Samples were analyzed by LCMS according to gradient elution methods optimized for each compound. Further optimization was conducted by running single ion monitoring (SIM) mode scans for predicted metabolite ions. Protein Content Determination. Standard solutions of bovine serum albumin (0−1 mg/mL) were prepared. Each of the standard solutions and quadruplicate cell homogenate samples (20 μL) was added to cuvettes with distilled water (800 μL). Biorad dye (200 μL) was then added to each cuvette, mixed thoroughly and left to stand (15 min) before UV−vis absorbance was measured (595 nm). The protein content of the homogenate was determined to be 0.22 mg/mL. Permeability Assay. Approximately 1 × 105 cells (passage 39) were seeded onto Transwell inserts. On days 1−8, cells

were fed with plate media on alternate days. This pattern was maintained for the basal chamber until the permeability assay. Apical chamber media was changed daily from day 9 onward. The cells were allowed to grow and differentiate for 21 days. On day 21, all plate media was refreshed and TEER values (>300 Ω cm2) measured (Millicell ERS-II voltohmmeter, Millipore Corp. Bedford, MA). Assays were performed on days 22 and 23, in HBSS−HEPES (pH 7.4, 37 °C, 95% humidity, 55 rpm). Prior to the assay the plates were washed with prewarmed PBS and HBSS−HEPES and incubated for 30 min. HBSS−HEPES was removed and fresh HBSS−HEPES (600 μL) placed in the basal wells, and the compound (100 μL, 1000 ppm), propranolol (0.1 mM) or fluorescein (400 ppm), in HBSS−HEPES, was placed onto the apical sides. Samples (200 μL) were taken, from the basal side, every 30 min for 150 min and replaced with HBSS−HEPES. At 150 min, the apical solution was also collected. Post-permeability Study Fluorescein Control Assay. The plates were washed with pre-warmed PBS and HBSS− HEPES. Fresh HBSS−HEPES (600 μL) was added to the basal side and fluorescein (100 μL, 400 ppm) added to the apical side of the compound assay wells (not the propranolol or fluorescein wells). Samples (200 μL) were taken, from the basal side, every 20 min for 60 min and replaced with HBSS− HEPES. Transport Assay. Cells were maintained and plated as for the permeability assays. Transport assays were performed on day 21 (TEER > 300 Ω cm2), with pre-washing and incubation carried out as for the permeability assay. HBSS−HEPES was removed and fresh HBSS−HEPES (100 μL) placed in the apical wells, and the compound (600 μL, 1000 ppm), propranolol (0.1 mM) or fluorescein (400 ppm), in HBSS− HEPES, was placed onto the basal sides. Samples (60 μL) were taken, from the apical side, every 30 min for 150 min and replaced with HBSS−HEPES. At 150 min the basal solution was also collected. Post-transport Study Fluorescein Control Assay. This was performed as for the post-permeability study fluorescein control assay for all apical-to-basal wells, including propranolol and fluorescein wells. For all basal-to-apical wells, following washing with HBSS−HEPES, fresh HBSS−HEPES (100 μL) was added to the apical side and fluorescein (600 μL, 400 ppm) added to the basal side. Samples (60 μL) were taken, from the apical side, every 20 min for 60 min and replaced with HBSS−HEPES. Sample Analysis. Permeability analysis for propranolol and compound samples was performed by gradient elution liquid chromatography mass spectrometry (LCMS) using a Shimadzu LCMS-2020. The column was a Shimadzu Shimpack XR-ODSIII, 2.2 μm particle size (2.0 mm i.d × 150 mm). Eluent A = water; eluent B = acetonitrile (ACN); flow rate = 0.25 mL/min; column temperature 40 °C. Propranolol: 1 μL injection, 5% ACN(aq) for 5 min, 5−80% ACN(aq) over 5 min and held for 4 min, 80−5% ACN(aq) over 1 min and held for 2 min. Initial gradient elution conditions, used for 2 and 8: 50 μL injection, 5% ACN(aq) for 3 min, 5−80% ACN(aq) over 2 min and held for 8 min, 80−5% ACN(aq) over 2 min and held for 5 min. Refined gradient elution conditions, used for remaining compounds: 50 μL injection of basal samples (25 μL for 3), 1 μL injection of apical samples, 5% ACN(aq) for 10 min, 5−80% ACN(aq) over 2 min and held for 8 min, 80−5% ACN(aq) over 2 min and held for 3 min. MS data were collected in the form of both total ion current data, in positive and negative mode, and 7663

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SIM data. Analysis of all fluorescein assay samples was performed by UV−vis spectrophotometry (490 nm, BioRad Microplate Reader). Determination of Permeability Coefficients. Papp values (cm s−1) were calculated according to the following equation Papp =

dC /dT × VR A × C0

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00496. Tables of the 1H NMR data for compounds 1−7 and figures showing the fraction transferred versus time (min) for the replicate wells in all the Caco-2 cell permeability assays performed (PDF) Calculation of the rate of drug transport by linear regression analysis (XLSX)

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REFERENCES

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where VR is the volume in the basal chamber, A is the surface area of the monolayer, C0 is the initial concentration in the apical chamber at 0 min, and dC/dT represents the steadystate linear rate of change of concentration in the basal chamber (μg/s). The rate of drug transport was calculated by linear regression analysis using Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA). Statistical Analysis. All data were presented as mean ± SD in cm/s × 10−6. A two-tailed Student’s t-test for independent variables was performed for each compound to ascertain whether the permeability values were statistically significantly different in the A−B compared to the B−A direction.62 The significance values generated were used in evaluating whether the results indicated that active uptake or efflux was taking place for each compound.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: j.blanchfi[email protected]. ORCID

Joanne T. Blanchfield: 0000-0003-1338-7446 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This research is supported by an Australian Research Council Linkage (LP14) Grant in collaboration with Integria Healthcare entitled “Bioproduction and bioavailability of steroidal saponins, bioactives in herbal medicines”. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS FBS, fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; HBSS, Hank’s buffered salt solution; LCMS, liquid chromatography mass spectrometry; NEAA, non-essential amino acids; PAB, apical-to-basal (absorptive) apparent permeability; Papp, apparent permeability; PBA, basal-to-apical (secretive) apparent permeability 7664

DOI: 10.1021/acsomega.9b00496 ACS Omega 2019, 4, 7658−7666

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DOI: 10.1021/acsomega.9b00496 ACS Omega 2019, 4, 7658−7666