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Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011
2To whom correspondence should be addressed. E-mail: pmurphy{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • soyasaponin I • soyasapogenol B • Caco-2 cell monolayer • human bioavailability
Soyasaponins are major phytochemicals present in legume seeds (1,2). The basic structure of soyasaponins is an oleanene-type triterpenoid aglycone with one or more polysaccharide chains attached, resulting in the amphiphilic nature of the molecules. Substantial concentrations of soyasaponins, 0.5–114 µmol/g, are found in soybeans and soy products (3). Soyasaponins have drawn interest in recent years due to their potential multiple health-promoting properties including plasma cholesterol lowering (4,5), anticarcinogenic (6,7), hepatoprotective (8,9), and anti-viral activities (10,11). Studies investigating soyasaponin biological activities have been limited to in vitro experiments and a few animal studies. The relevance of these findings to humans is not clear because little is known about how and to what extent dietary soyasaponins may enter systemic circulation after ingestion.
The bioavailability of soyasaponins and soyasaponin aglycones (soyasapogenols) is not well understood. Saponins have been considered poorly absorbed in the intestine. The sugars of saponins could be hydrolyzed to liberate the aglycones by the bacterial enzymes in the intestine. The degradation of soyasaponins by gut microbes was reported (12,13). Soyasaponins could be metabolized by intestinal microflora to release sugars and aglycones as the metabolites in animals and humans. Soyasapogenols were shown to be more effective than their various glycosides in suppression of 2-acetoxyacetylaminofluorene (2-AAAF)3 -induced genotoxicity in Chinese hamster ovary cells (14). Soyasapogenol B (10 µmol/L) was growth inhibitory to MDA-MB-231 human breast cancer cells in vitro (15).
Soyasaponin absorption was studied in rats, chickens, and mice after oral dosing (12). In that study, neither soyasaponins nor soyasapogenols were found in the urine or blood based on TLC and hemolysis assays, suggesting that dietary soyasaponins might not be absorbed. However, no direct evidence demonstrates the absorbability and pharmacokinetics of soyasaponins in humans.
The present study investigated the bioavailability of Group B soyasaponins and soyasapogenols in humans. Soyasaponins are divided into Groups A, B, and E in plants (1–3,16). Soyasaponin I, a group B soyasaponin (Fig. 1), was used as a representative soyasaponin because it is a main form of soyasaponins in heat-treated soy products (3,16). Soyasapogenol B, a major gut microbial metabolite of soyasaponin I, was used to evaluate the absorbability of soyasaponin aglycones. A human feeding study of a single dose evaluated dietary soyasaponin bioavailability in vivo. The mucosal absorption and transepithelial kinetics of these compounds were evaluated using the human colon adenocarcinoma cell line, Caco-2 cells, a well-established in vitro model, to understand nutrient absorption (17). The results obtained from this study will help in understanding the bioavailability of dietary soyasaponins and absorption of their gut metabolites in humans, and thus in predicting their potential beneficial effects.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Human feeding study.
The 8 subjects were healthy, nonsmoking women, 25–34 y old, with a body mass index of 21.0 ± 2.6 kg/m2, who had not taken any medication for 3 mo prior to and during the study. The study protocol was approved by the Iowa State University Human Subjects in Research Committee in 2001. Informed consent was obtained from each subject before the experiment began. The subjects were given a list of soyasaponin-containing foods and instructed to avoid these foods for 4 d. Then the subjects ingested 4 g of Prevastein (Central Soya) containing 108.9 µmol group B soyasaponins/g composed of 4.89 µmol soyasaponin V/g, 58.4 µmol soyasaponin I/g, 25.8 µmol soyasaponin II/g, 0.5 µmol soyasaponin 
g/g, 13.96 µmol soyasaponin ßg/g, and 4.5 µmol soyasaponin ßa/g at breakfast following an overnight fast. According to the manufacturer’s product specification sheet, Prevastein contains 58–81 µmol genistin/g, 30–54 µmol daidzin/g, and 1–6 µmol glycitin/g. Breakfast consisted of free choice of orange juice, skim or 2% milk, bagels, low-fat cream cheese, strawberry jam, bananas, apples, or pancakes and syrup. The subjects took a carmine red dye marker capsule (500 mg, University of Iowa Pharmacy) to determine gut transit time. A urine sample was collected from each subject just before feeding and for 24 h after dosing. Stools were collected from the time of feeding until the red dye marker disappeared from the feces. Urine (50-mL aliquots) and fecal samples were freeze-dried and stored at –20°C until analysis.
Freeze-dried urine was dissolved in 25 mL of 0.2 mol/L sodium acetate buffer at pH 5.5 and incubated at 37°C for 8 h with or without 100 µL of 86.9 MU ß-glucuronidase/sulfatase/L (Sigma-Aldrich) to hydrolyze possible metabolites of soyasaponins. Then the sample was applied to a preconditioned Sep-Pak cartridge (classic short-body C18, Waters) and washed with 5 mL distilled water followed by 5 mL of 5% methanol. Soyasaponins were eluted with 2.0 mL HPLC-grade methanol. After filtration through a 0.45-µm PTFE filter (Alltech Associates) TLC and HPLC were performed.
Three grams of freeze-dried fecal samples were extracted with 100 mL 70% ethanol at room temperature (RT) for 2 h. The extract was filtered through No. 42 filter paper (Whatman) and evaporated at RT (Buchi Rotovap). The residue was suspended in 5 mL of 20% methanol, loaded onto a preconditioned C18 Sep-Pak cartridge, and washed as above, and soyasaponins were eluted with 3.0 mL HPLC-grade methanol and filtered as above before TLC and HPLC analysis. All urine and fecal samples were extracted and analyzed in duplicate.
TLC was performed on silica gel LK6F plates (Whatman). Urine and fecal samples were analyzed with 2 different mobile phases: butanol-ethanol-aqueous ammonia (5:5:4, v:v) and hexane-ethyl acetate (2:1, v:v). Soyasaponins were detected by spraying acetic acid-sulfuric acid-anisaldehyde (100:2:1, v:v) and heating at 120°C for 10 min, giving a blue-purple color.
Soyasaponin I concentration was determined by HPLC as previously reported (3). Soyasapogenol B concentration was measured using a different HPLC gradient program: solvent B increased from 73 to 100% linearly in 35 min, and then solvent B recycled back to 73% in 4 min.
Mucosal uptake and transepithelial kinetics of soyasaponin in the Caco-2 cell model. Caco-2 cells were purchased at passage 18 from American Type Culture Collection and experiments were conducted at passages 35–45. The cells were grown in DMEM (Sigma) with 16% fetal bovine serum (Sigma), 1% nonessential amino acids (Gibco BRL), and 1% antibiotic-antimyotic solution (Gibco BRL) at 37°C in an incubator with 5% CO2/95% air. At 80–100% confluency, cells were trypsinized and seeded on collagen-coated polytetrafluroethylene membrane inserts (0.45 µm) fitted in bicameral chambers (Transwell-COL, 24 mm ID, Corning Costar) at 5.5 x 104 cells/cm2. At 14–16 d postseeding (90–100% confluence), a phenol red test (18) measured cell monolayer integrity before and after the transport assay. A serum-free medium (1% antibiotic-antimyotic solution, 4 mg hydrocortisone/L, 10 mmol Pipes/L, 5 µg selenium/L, and 34 µg triiodothyronine/L in DMEM medium) was used to perform the transport assay.
Epithelial uptake was measured for soyasaponin I and soyasapogenol B at concentrations of 0.5, 1, and 3 mmol/L in triplicate assays. Soyasaponin I or soyasapogenol B was suspended in the transport buffer by sonication for 30 s (Sonic Demembrator, Fisher Scientific). Caco-2 monolayers grown on the membrane inserts were first rinsed with 2 mL of Earle’s balanced salt solution (EBSS) and then bathed in 2 mL transport buffer, 37°C, 15 min before treatment. Then the apical solution was replaced with 1.5 mL transport buffer containing soyasaponin I or soyasapogenol B. A total of 1.0 mL transport buffer was added to the basal chamber. The system was incubated at 37°C for 4 h and samples were taken from the basal chamber at 30 min and 1, 2, and 4 h. The basal chamber buffer was replenished with transport buffer at each time point. Cumulative transport rates were the sum of the amount transported from all time points (19). At the end of the experiment, the buffer in the apical chamber was collected to determine untransported test compound. Samples were stored at –20°C until analysis.
The amount of test compound in the cells was measured to determine uptake. After the transport assay, the apical and basal chambers were rinsed 3 times with 1 mL of ice-cold EBSS buffer. The membrane inserts were then peeled off the membrane holders and placed in 1.5 mL of ice-cold 0.5 mol sodium hydroxide/L to solubilize the cells. The cells were lysed by sonic demembrator for 30 s (20). The total protein content of cells on the inserts was determined using Lowry’s method (21) to ensure that there were comparable numbers of cells on each insert.
The contents of soyasaponin I or soyasapogenol B in the samples were determined as follows. The sample from the basal chamber was directly loaded at RT onto a preconditioned Sep-Pak cartridge (light short-body C18, Waters), washed with 3 mL of 5% methanol and eluted with 0.8 mL HPLC-grade methanol. The sample from the apical chamber was loaded onto a larger size preconditioned Sep-Pak cartridge (classic short-body C18, Waters) and eluted with 2.0 mL HPLC-grade methanol. Then all samples were analyzed by HPLC. Permeability coefficients (Ptrans and Papp) were determined using the following equations (19,22,23):
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where Papp and Pfilter are the apparent permeability coefficients estimated by transport assay in the presence and absence of Caco-2 cells, respectively; Ptrans is the final transport coefficient. 
Q/t is the permeability rate constant (µmol/min); A is the surface area of the membrane (in square centimeters); and C0 is the initial concentration of the compound in the apical chamber (µmol/mL). The rate constants (Q/t) are the slopes of the regression lines for the plot of transferred amount to the basal chambers vs. time. The assay was done before >10% of the compound had been transported to the basal chamber.
The acute toxicity of soyasaponin I and soyasapogenol B to Caco-2 cells was evaluated at the same concentrations used above in triplicate assays. Each treatment was replicated. After a 4-h incubation, the insert was rinsed with 1.5 mL of 1 mol/L PBS. The cells were digested with 0.5 mL of 0.25% trypsin–1 mmol/L EDTA (Gibco BRL) and suspended in the serum-free medium. The viability of the harvested cells was determined by trypan blue dye exclusion (24).
Statistical analysis. All data are expressed as means ± SD. Statistical analyses were performed with SAS (Version 8.1, SAS Institute). The transport kinetics of soyasaponin I and soyasapogenol B across the Caco-2 cell monolayer were analyzed by general linear regression. Differences in cell uptake, transport kinetics, and cytotoxicity of soyasaponin I and soyasapogenol B at different concentrations were compared with ANOVA and Tukey’s multiple comparison test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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When examining the uptake of the test compounds, 5.4 to 12.3% of soyasapogenol B was found in the cells. Unlike soyasaponin I, soyasapogenol B accumulated in the cells in a concentration-dependent manner (Fig. 2). The uptake of soyasapogenol B was higher than that of soyasaponin I at 0.5 and 3 mmol/L (P < 0.05) but was not different at 1 mmol/L.
Trypan blue exclusion as a measure of cell viability and cytotoxicity of saponin I and sapogenol B showed that the percentage of dye-excluding cells ranged from 89.3 to 96.2% for all treatments (Fig. 3). The percentage of dye-excluding cells after soyasaponin I treatment at all 3 concentrations was not different from that of the control group. However, 1 and 3 mmol soyasapogenol B/L reduced the percentage of dye-excluding cells in the culture compared with the control (P < 0.01), whereas 0.5 mmol/L soyasapogenol B did not have a cytotoxic effect.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In this study, only 8.4% of ingested group B saponins were recovered as sapogenol B in the feces of women. Saponins and sapogenol B were absent from the urine. Sixty to 65% of total ingested soyasaponins was recovered as soyasapogenols in rat feces (12). One explanation for the lesser recovery of sapogenol B in women compared with rats is the possibility that soyasaponins and soyasapogenol B may have been metabolized more extensively by human gut microflora than in rats to additional degradation products not recognized by our analytical methods. Stable isotopically labeled soyasaponin would be useful for tracing the fate of ingested soyasaponins in animal models or humans.
Very limited information is available on the bioavailability of other types of saponins. Glycyrrhizin, a triterpene saponin found in licorice, was reportedly metabolized by human intestinal microflora to its aglycone, glycyrrhetinic acid (25). Absorption of glycyrrhetinic acid has been reported, resulting in an anti-inflammatory effect in mice (26). A metabolite of ginseng saponins was detected in blood after 2 oral administrations of ginseng extract to a human subject (27). The original ginsenosides of Panax ginseng and their metabolites were detected in the blood and urine after oral administration of ginsenosides to rats. However, the doses used in that study were very high compared to dietary levels, such that the permeability of intestinal mucosal cells might have been compromised (28). The urinary ginseng saponins and metabolites may have resulted from a leaky intestinal tract rather than from absorption by enterocytes.
For comparison with soyasaponins, the gastrointestinal absorption of soy sterol and soy stanols in humans is very low (29). After a single meal of 600 mg lecithin-emulsified soy stanols or sterols, the absorption of sitosterol, campesterol, sitostanol, and campestanol was only 0.51, 2.2, 0.044, and 0.26%, respectively. Soyasaponins are larger molecules but of comparable hydrophobicity to these phytosterols. It is likely that soyasaponins have oral bioavailability that is even less than that of phytosterols. Failure to observe the parent compounds in the urine after oral dosing may not necessarily indicate a lack of beneficial health effects. Beneficial health effects of soyasaponins may be due to their or their metabolites’ inhibition of cholesterol absorption (5,30), inhibition of action of potential toxicants such as bile acids (4), and their direct cytotoxicity to preneoplastic enterocytes (31).
The uptake and transport kinetics of soyasaponin I and soyasapogenol B were evaluated in a Caco-2 cell model to understand their bioavailability. The concentrations used in this study were based on the dose of group B soyasaponins given in the human study above, i.e., 436 µmol/person. Assuming soyasaponins and their gut microbial metabolite, soyasapogenol B, remained unabsorbed in the lower intestine and an average subject produced 150 to 300 g feces/d, the resulting concentration of soyasaponins or soyasapogenol B would be 1.5–3 µmol/g feces or 1.5–3 mmol/L gut contents; a slightly broader concentration range of 0.5–3 mmol/L was therefore chosen for in vitro studies.
In our study, the protein content of Caco-2 cell monolayers used in the experiment was 1.21 ± 0.16 mg/insert. Although variation was small (13.4%), data were normalized to protein content. The integrity of Caco-2 cell monolayers was maintained at confluence above 92% during the experiment as determined by phenol red transport. A total of 0.01–0.1% quillaja saponin DS-1 increased the permeability of mannitol and d-decapeptide to a Caco-2 cell monolayer without causing morphological changes of the monolayer (32). In our study, soyasaponin I and soyasapogenol B at up to 3 mmol/L (
0.1–0.3%) in the apical chamber did not affect monolayer confluence, suggesting that soyasaponins were less disruptive to the integrity of Caco-2 cell monolayer than quillaja saponins. Soyasaponins were less able to increase the permeability of rat intestinal mucosal cells in vitro in comparison with gypsophylla saponins and saponaria saponins (33).
Total recoveries of soyasaponin I and soyasapogenol B from apical and basolateral sides of the cell culture membrane were significantly greater for the glycoside than for the aglycone, 101.3 ± 3.7% vs. 76.7 ± 6.5%, respectively. Sample extraction efficiencies were >92% for both compounds. The 25% loss of soyasapogenol B in terms of mass balance may be because highly hydrophobic soyasapogenol B was adsorbed to the polystyrene chambers during the incubation. Soyasapogenol B might also be further metabolized to compounds dissimilar to saponins by Caco-2 cells.
Our data indicated that the Caco-2 monolayer acted as a barrier against free diffusion of soyasaponin I toward the basolateral side (Table 2). The relationship between Papp values obtained from Caco-2 cell model and human in vivo intestinal absorption for a number of drugs (17) suggests that the observed range of permeability coefficients for soyasaponin I corresponds with some intestinal absorption in humans. For comparison, the apical to basolateral transcellular flux of quercetin over the Caco-2 monolayer had a Papp of 5.8 x 10–6 cm/s (33) and genistein 20 x 10–6 cm/s (34). The human intestinal absorption of soyasaponin I (Papp 1–3 x 10–6 cm/s) is probably less than those compounds. As indicated by T0.1 (Table 2), it would take 11.4 h to transport 10% of 0.5 mmol/L of soyasaponin I from the lumen to the basolateral side of the enterocyte.
The mucosal transport of soyasaponin I might be controlled at the basolateral side of the mucosal cells (Fig. 2), and the uptake of soyasaponin I by Caco-2 cells could be saturable. The uptake of soyasaponin I at 0.5 mmol/L was not saturated, but the output of soyasaponin I at the basolateral membrane had been saturated. At concentrations 
1 mmol/L, the cellular uptake of soyasaponin I at the basolateral side was saturated. Therefore, the accumulation of soyasaponin I in the cells became constant at the 2 higher concentrations and the apical to basolateral transport rate of soyasaponin I was not different at 1 and 3 mmol/L. A concentration-dependent transport rate may be observed if the concentrations of soyasaponin I used in the study were lower than the saturation point of basolateral output. Thus, transport of soyasaponin I by Caco-2 cells may involve a carrier-mediated mechanism; the absorption of soyasaponins in the intestine might be enhanced when the soyasaponin concentration is low. The absorbed amount could be limited by the capacity of epithelial cells to take up and transfer soyasaponins to the basolateral side when high concentrations of soyasaponins are present in the gut.
The very low absorbability obtained in the Caco-2 model studies might not represent the real absorbability of the compounds in this situation. The lack of absorption of soyasapogenol B in the Caco-2 cell model (Table 2) is probably due to low permeability through the polytetrafluroethylene membrane on which the cells grew. The collagenated polycarbonate membrane (0.45 µm) has been commonly used in transport studies and considered a better supporting membrane for Caco-2 cell growth and differentiation with low adsorption of many drugs (22,35). Hydrophobic molecules such as ß-sitosterol and dexamethasone are permeable across Caco-2 cell monolayers growing on 0.45-µm polycarbonate membranes (17,36). The diffusion rate across the cell-free membrane filter of some lipophilic compounds, such as alprenolol and propranolol, was approximately half that of a hydrophilic compound, mannitol (19). In our study, it would have been better to assess the permeability of soyasapogenol B over different filter membranes first to choose the permeable membrane prior to transfer assay.
The uptake of saponins or sapogenols by enterocytes is not known. But the uptake of 0.1 mmol/L sitosterol by Caco-2 cells was 0.5 x 10–3 pmol/insert after 4 h of incubation, about half the uptake of cholesterol (37). The uptake of sitosterol by Caco-2 cells was 25–75% over a concentration range of 4–100 µmol/L (38); in contrast to cholesterol absorption, the low absorbability of sitosterol was due to low intracellular processing and basolateral secretion but not to low uptake at the brush border membrane. We cannot conclude whether soyasapogenol B was transportable across the Caco-2 cell monolayer. Given the extent of accumulation of soyasapogenol B in the Caco-2 cells, the absorption of soyasapogenol B is likely similar to that of soyasaponin I.
Although the Caco-2 cell model has been widely used to evaluate the absorbability and mucosal metabolism of many nutrients and phytochemicals, this is the first report using this model to measure saponin absorbability. In our study, the transport of soyasaponins was evaluated using crystalline soyasaponin material suspended in aqueous transport buffer. The absorption of soyasaponins in the intestine might be more complicated because of the interaction of soyasaponins with other constituents of the gut contents. The uptake and absorption of soy phytosterols was facilitated by partition of bile salt micelles due to the phytosterol’s structural similarity to cholesterol (29,38). The uptake of sitosterol by brush border membrane vesicle and Caco-2 cells was energy-independent and facilitated in a manner analogous to cholesterol uptake (39,40). The absorption of dietary soyasaponins might be enhanced by partitioning of micelles in the intestine due to the amphiphilic nature of soyasaponins and their structural resemblance to cholesterol. Micelle incorporation with cholesterol or phytosterols has been widely used in Caco-2 cell studies of cholesterol absorption (37,41). The effect of bile salt/soyasaponin micelles on saponin transport in the Caco-2 cell model might be of interest, although we did not detect absorption of soyasaponins in women.
It is not surprising that soyasaponin I was not toxic to Caco-2 cells after 4 h of exposure (Fig. 3). One hour of exposure to 600 mg soyasaponins/L did not change the viability of human colon carcinoma HCT-15 cells (31), whereas the same amounts of gypsophilla saponins decreased cell viability significantly. Soyasaponins (50–250 mg/L) had no acute cytotoxic effect on Chinese hamster ovary (CHO) cells (14). Soyasaponins at up to 3 mmol/L seem to be nontoxic to intestinal epithelial cells. Soyasaponin effects over a longer exposure period may be of interest because of the 24 h or longer residence time of digesta in the gut. Inhibition of carcinoma cell proliferation by soyasaponins (31,42,43) and the observed colon cancer inhibitory effect of dietary soyasaponin (6) might not be due to direct cytotoxicity but to the growth inhibitory activity of soyasaponins.
Soyasapogenol B was cytotoxic to Caco-2 cells at concentrations >1 mmol/L. A 2-h treatment of 0.4 mmol/L soyasapogenol B had no effect on CHO cell viability and showed protection of CHO cells against direct DNA damage induced by 2-AAAF (14). Soyasapogenol B (10 µmol/L) was growth inhibitory to human breast cancer cells (15). Unlike soyasaponins, which interact with the outer cell membrane, soyasapogenol B might exert its cytotoxicity within cells. Further research is required to explore the mechanisms and potency of cytotoxicity of soyasapogenol B.
In conclusion, ingested group B soyasaponins are metabolized to soyasapogenol B by human intestinal microorganisms in vivo and excreted in the feces. Soyasaponins may have very low absorbability in the human gut, as seen in the Caco-2 cell transport studies. Soyasaponins are not cytotoxic to enterocytes. Soyasaponin I is taken up by gut epithelial cells in a saturable manner in small amounts; in contrast, the uptake of soyasapogenol B may depend upon its concentration in the lumen. The absorbed soyasaponin or sapogenol B may also contribute to anticancer activity in the colon.
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FOOTNOTES |
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3 Abbreviations used: 2-AAAF, 2-acetoxyacetylaminofluorene; CHO, Chinese hamster ovary; EBSS, Earle’s balanced salt solution; RT, room temperature.
Manuscript received 12 January 2004. Initial review completed 1 March 2004. Revision accepted 29 April 2004.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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