Article pubs.acs.org/JAFC
Bioavailability of Ginsenosides from White and Red Ginsengs in the Simulated Digestion Model Eun Ok Kim,† Kwang Hyun Cha,† Eun Ha Lee,† Sang Min Kim,† Sang Won Choi,‡ Cheol-Ho Pan,*,† and Byung-Hun Um*,† †
Biomodulation Team, Natural Products Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Ganwon-do 210-340, Korea ‡ Department of Food Science and Nutrition, Catholic University of Daegu, Gyeongsan, Gyeongbuk 712-702, Korea S Supporting Information *
ABSTRACT: This study aims to investigate the bioavailability of ginsenosides during simulated digestion of white (WG) and red (RG) ginseng powders. Stability, bioaccessibility, and permeability of ginsenosides present in WG and RG were studied in a Caco-2 cell culture model coupled with oral, gastric, and small intestinal simulated digestion. Most ginsenosides in WG and RG were stable (>90%) during the simulated digestion. Bioaccessibilities of total ginsenosides during in vitro digestion of WG and RG were similar at approximately 85%. However, the bioaccessibility of protopanaxatriol type ginsenosides in the early food phase was greater than that of the protopanaxadiol type. The less polar RG ginsenosides were released later following the jejunum phase. Ginsenosides had low permeability ( 0.982) was obtained for each standard curve (data not shown). Statistical Analysis. All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed by a one-way analysis of variance (ANOVA) followed by Tukey’s honestly
significant difference (HSD) test using IBM SPSS statistics 21. P values < 0.05 were considered statistically significant.
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RESULTS AND DISCUSSION Stability of Ginsenosides during Simulated Digestion of WG and RG. Information about the stability and bioaccessibility of ginsenosides from ginseng during transit through the gut is still limited. To assess the stability of ginsenosides during in vitro digestion of WG and RG, the ginsenosides present in the ingredients during simulated oral, gastric, and small intestinal digestion as well as food materials in C
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Table 2. Stability in Each Digestate Generated by Simulated Oral, Gastric, and Small Intestinal Digestion of RGa recovery in digestion phase (%) ginsenoside T-PPD Rb1 Rb2 Rc Rd Rg 3(20S) Rg3 (20R) Rk1 Rg5 T-PPT Re Rf Rg1 Rg2 (20S) Rg2 (20R) Rg6 F4 Rk3 Rh4 T-G
foodb 8.10 3.30 1.14 1.08 0.32 1.10 0.44 0.35 0.36 3.72 0.67 0.49 0.38 1.25 0.44 0.076 0.11 0.11 0.19 11.82
± 0.45 ± 0.27 ± 0.10 ± 0.16 ± 0.0 ± 0.21 ± 0.02 ± 0.05 ± 0.0 ± 0.18 ± 0.05 ± 0.06 ± 0.04 ± 0.17 ± 0.09 ± 0.005 ± 0.02 ± 0.01 ± 0.03 ± 0.46
mouth 95 95 96 96 97 93 92 96 95 97 93 105 97 96 95 98 95 97 95 95
±5 ±1 ±4 ±3 ±3 ±8 ±9 ±6 ±7 ±4 ±8 ±5 ±4 ±3 ±7 ±3 ±6 ±2 ±5 ±5
stomach 98 99 91 96 94 105 100 105 108 100 90 104 93 104 105 101 106 98 102 99
±6 ±3 ±6 ±2 ±6 ±4 ±3 ±3 ±8 ±2 ±8 ±7 ±4 ±5 ±5 ±2 ±4 ±2 ±3 ±4
duodenum 99 96 97 94 96 109 100 114 107 104 104 96 92 107 106 100 102 98 105 101
±3 ±2 ±3 ±2 ±2 ±8 ±4 ± 5* ±5 ±5 ±5 ±8 ±7 ±8 ±6 ±1 ±2 ±1 ±5 ±4
jejunum 95 90 89 89 94 98 101 141 106 97 94 102 97 98 92 103 104 100 97 95
±4 ± 2* ± 3* ± 2* ±4 ±5 ±1 ± 8** ±4 ±5 ±6 ±7 ±2 ±3 ±7 ±4 ±5 ±2 ±3 ±5
ileum 97 92 91 91 97 99 103 162 107 99 96 103 98 100 98 106 109 98 98 98
±4 ± 1* ± 2* ± 2* ±6 ±3 ±2 ± 4** ±6 ±2 ±4 ±5 ±5 ±3 ±3 ±5 ±8 ±2 ±4 ±3
Data represent the mean ± SD from four independent experiments. Mean values in rows for each digestion phase show statistically significant differences in Tukey’s test. *, p < 0.05 compared to food; **, p < 0.01 compared to food. bThe ginsenoside content in food material was expressed as mg/g of dry weight.
a
steaming repetitions for RG,3,27 and by the type of ginseng and its country of origin.8 According to papers by Choi et al.27 and Kim et al.,28 the PPD/PPT ratio of RG ranged from 1.5 to 3.5. Our results are also within this range. However, the contents and profiles of the ginsenosides varied considerably compared to other studies.3,4,8,28 These results suggest that the difference may be due to not only the steaming and drying conditions during RG preparation but also the contents and constituents present in WG itself. Most of the ginsenosides in the WG powders in each digestion phase were stable with a >95% recovery rate (Table 1). In addition, the recoveries of ginsenosides Rb2 and Rc as well as Rg2 (20S) and Rg2 (20R) in the ileum phase were significantly increased to 113−114%. When the WG was incubated with a digestive fluid that did not contain enzymes, the results differed from those shown in Table 1 (data not shown). The increase of the recovery of ginsenosides Rb2, Rc, and Rg2 in the intestinal phase was thought to be due to the increase of extraction rate by intestinal enzymes during digestion. In contrast, the recovery of ginsenoside from the RG powders in each digestion phase was mostly >90%, except for ginsenoside Rk1 (Table 2). Thus, the recovery of ginsenosides Rb1, Rb2, and Rc was significantly decreased, whereas that of Rk1 was remarkably increased from the duodenum to the ileum. It was reported that PPD type ginsenosides such as Rb1, Rb2, and Rc were mainly converted to Rk1 and Rg5 through Rg3 (Figure 2).26 In the above result, the change in the recovery rate was thought to be due to the conversion of Rb1, Rb2, and Rc to Rk1 during simulated digestion. Kim et al.23 reported that stability of the ginsenosides in red ginseng extract was observed when they were placed in a 37 °C water bath for 4 h, and no significant changes in the ginsenoside composition were found. Moreover, the ginsenosides Rb1 and Rg1 added to serum samples in rats were stable as the recovery range was 90.13−105.66%.16 Serventi et al.19
the WG and RG powders were identified and quantitated by UPLC-MS analysis. As shown in Figure S2 (Supporting Information), nine ginsenosides were detected in WG powder. The total ginsenoside content of WG was 6.47 ± 0.33 mg/g dry weight (Table 1). Ginsenoside Re (2.08 ± 0.27 mg/g) was most abundant, followed by Rb1, Rg1, Rb2, Rc, and Rf, and there were traces of Rg2 and Rd. The PPD/PPT ratio was 0.55. These results were similar to the total ginsenoside content (7.4 mg/g) and PPD/PPT ratio (0.46) of WG reported by Choi et al.27 There were no differences in the composition of ginsenosides when compared to the results of Kim et al.28 and Sun et al.,8 but the total quantities of ginsenosides were somewhat different. The PPD/PPT ratio also differed as it ranged from 0.5 to 1.5. This is thought to be influenced by the cultivated areas, growth years, and harvest time of ginseng. Meanwhile, 17 ginsenosides were detected in RG powder (Figure S2 and Figure 1). The total ginsenoside content of RG was 11.82 ± 0.46 mg/g, 1.8-fold higher than that of WG (Table 2). Unlike WG, the main ginsenoside of RG was Rb1 (3.30 ± 0.27 mg/g). The levels of polar PPT type ginsenosides, such as Re and Rg1, decreased, and levels of all of the ginsenosides of the PPD type, such as Rb1, Rb2, Rc, Rd, Rg3 (S,R), Rk1, and Rg5, and the less polar ginsenosides of the PPT type, such as Rg2 (S,R), Rg6, F4, Rk3, and Rh4, increased. The mechanisms involved were deduced to be the demalonylation of malonyl ginsenosides in WG27 and the loss of a glycosyl moiety at C20−OH of dammarane type ginsenosides through thermal hydrolysis by steam processing.3 Thus, the PPD/PPT ratio increased to 2.18. The formation of less polar ginsenosides (Rg2 (20S), Rg2 (20R), Rg3 (20S), Rg3 (20R), Rg5, Rg6, Rh1, Rh4, F4, Rk1, and Rk3) in steamed ginseng has been reported for Korean ginseng.7,29 Recently, many studies had been conducted on the quantitative analysis of ginsenosides in RG. The ginsenoside contents varied by preparation method, such as the steaming temperature, steaming time, and number of D
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Figure 2. Possible enzymatic or chemical bioconversion of protopanaxadiol (A) and protopanaxatriol (B) type ginsenosides.
form.30 The aqueous or bioaccessible fractions from undigested material were separated through centrifugation from digestates in each phase generated during simulated digestion. The bioaccessibility of ginsenosides in the aqueous fraction from the food to the small intestinal phase of the simulated digestion of WG (Table 3) and RG (Table 4) generally increased. Among the nine ginsenosides in WG, the bioaccessibility of ginsenoside Re in the food phase (prior to the early stage of enzyme addition in the mouth) had the highest level, at 90% of the food matrix. The ginsenosides Rg1, Rg2 (20S), Rf, and Rg2 (20R)
observed that the recovery of total saponins in soy bread was 83% during in vitro digestion, although recovery of saponins differed considerably by saponin type. In addition, the recovery of carotenoids in meals was >70% during in vitro digestion.17 Therefore, these results indicate that most of the ginsenosides in RG and WG were relatively stable during in vitro digestion. Bioaccessibility of Ginsenosides during Simulated Digestion of WG and RG. Bioaccessibility refers to the amount of a constituent in a food or supplement that is released from the matrix during digestion to a potentially absorbable E
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Table 3. Bioaccessibility of PPD and PPT Ginsenosides from Each Digestate Generated during Simulated Digestion of WGa bioaccessibility in digestion phase (%) ginsenoside T-PPD Rb1 Rb2 Rc Rd T-PPT Re Rf Rg1 Rg2 (20S) Rg2 (20R) T-G
food 60 60 59 60 71 85 90 77 82 80 75 76
± 2.9c ± 2.7b ± 2.8c ± 1.2c ± 4.7b ± 3.9 ± 6.3 ± 2.5b ± 4.3a ± 5.3 ± 0.9ab ± 1.2b
mouth 63 57 67 70 78 86 90 83 84 84 76 78
± 4.3c ± 5.0b ± 3.9b ± 3.7b ± 4.5b ± 3.8 ± 3.8 ± 3.4ab ± 5.4a ± 4.8 ± 1.5ab ± 2.5ab
stomach 73 74 70 75 72 85 89 82 83 86 69 81
± 2.6b ± 1.9a ± 4.0b ± 1.9b ± 2.4b ± 5.8 ± 5.0 ± 0.5ab ± 7.5a ± 10.8 ± 5.4ab ± 4.4ab
duodenum 71 71 67 71 78 84 89 89 76 82 70 79
± 2.7b ± 5.7a ± 2.0b ± 0.3b ± 2.9b ± 3.2 ± 2.3 ± 2.0a ± 4.6ab ± 5.8 ± 1.5b ± 3.0ab
jejunum 78 71 85 83 89 89 91 90 85 88 79 85
± 2.7a ± 2.9a ± 5.0a ± 1.4a ± 1.3a ± 3.9 ± 2.4 ± 5.2a ± 2.7a ± 6.4 ± 2.6a ± 3.3a
ileum 80 74 87 86 88 91 92 91 89 89 81 87
± 2.6a ± 2.6a ± 0.1a ± 4.4a ± 3.1a ± 3.0 ± 5.0 ± 3.2a ± 1.9a ± 3.3 ± 1.4a ± 2.8a
a
Bioaccessibility (%) was calculated from percentage of the ginsenoside concentrations in digestates of each digestion phase on white ginseng. Values represent mean ± SD from four independent experiments. Significant differences at digestion phase of individual ginsenosides as determined by Tukey’s test (p < 0.05) are indicated by different letters.
Table 4. Bioaccessibility of PPD and PPT Ginsenosides from Each Digestate Generated during Simulated Digestion of RGa bioaccessibility in digestion phase (%) ginsenoside T-PPD Rb1 Rb2 Rc Rd Rg3 (20S) Rg3 (20R) Rk1 Rg5 T-PPT Re Rf Rg1 Rg2 (20S) Rg2 (20R) Rg6 F4 Rk3 Rh4 T-G
food 54 60 60 62 65 37 34 32 36 64 85 63 77 62 61 36 30 39 38 57
± 2.8d ± 2.7b ± 2.2b ± 4.7b ± 1.2b ± 2.3d ± 2.5e ± 3.3d ± 3.5d ± 2.4c ± 2.8a ± 3.4b ± 2.1 ± 3.2c ± 1.1b ± 0.8c ± 2.5c ± 1.6c ± 3.8d ± 2.6d
mouth 62 69 70 71 73 44 43 39 43 69 85 75 81 65 66 43 37 45 44 64
± 4.3c ± 5.0a ± 3.9a ± 3.7a ± 4.5a ± 3.8c ± 3.4d ± 5.4c ± 4.8cd ± 2.9c ± 1.5a ± 7.3a ± 5.2 ± 1.9c ± 4.0b ± 1.2b ± 0.8b ± 2.0b ± 2.2c ± 3.6c
stomach 56 60 53 46 64 57 65 38 53 64 84 71 70 63 56 25 34 35 38 58
± 3.1d ± 1.9b ± 4.0b ± 2.8c ± 2.4b ± 5.0b ± 0.5b ± 3.2c ± 5.0c ± 5.0c ± 5.4a ± 2.2b ± 10.5 ± 3.6c ± 2.7c ± 6.4d ± 4.8bc ± 5.5c ± 4.0d ± 4.1d
duodenum 56 58 53 55 63 58 52 43 50 56 68 51 77 54 56 27 29 35 30 56
± 3.9d ± 5.7b ± 4.5b ± 3.0b ± 2.9b ± 2.3b ± 2.0c ± 4.6c ± 5.8c ± 2.8d ± 1.5b ± 4.0c ± 0.5 ± 2.6c ± 3.3c ± 2.6d ± 4.1c ± 3.7c ± 2.5e ± 3.3d
jejunum 75 74 72 71 76 79 71 91 79 75 73 70 73 80 76 78 68 71 66 75
± 3.4b ± 2.9a ± 5.0a ± 1.4a ± 1.3a ± 2.4a ± 5.2b ± 2.7b ± 6.4b ± 3.4b ± 2.6b ± 3.0b ± 2.4 ± 1.8b ± 4.5a ± 2.4a ± 5.6a ± 3.7a ± 4.3b ± 3.4b
ileum 85 82 80 79 85 86 95 131 91 84 84 85 78 88 81 83 75 79 77 85
± 3.0a ± 2.6a ± 0.1a ± 4.4a ± 3.1a ± 5.0a ± 3.2a ± 1.9a ± 3.3a ± 2.9a ± 1.4a ± 3.6a ± 2.8 ± 4.0a ± 1.1a ± 2.4a ± 5.5a ± 3.3a ± 1.74a ± 2.9a
a
Bioaccessibility (%) was calculated from percentage of the ginsenoside concentrations in digestates of each digestion phase on red ginseng. Values represent mean ± SD from four independent experiments. Significant differences at digestion phase of individual ginsenosides as determined by Tukey’s test (p < 0.05) are indicated by different letters.
ginsenosides are classified as belonging to the two major groups according to their structures. The PPD type had sugar moieties attached to the C-3 and/or C-20, and the PPT type has sugar moieties at C-6 and/or C-20 (Figure 1).3 The polarity of PPT, having two hydrophilic tails (sugar moieties) and an additional hydroxyl group, is greater than that of PPD. Thus, the results implied that PPD ginsenosides were more slowly released during the digestion process than the PPT type. Meanwhile, the release of ginsenosides in the aqueous fraction during simulated digestion of the RG increased, especially in the jejunum and ileum (Table 4). Among the 17 ginsenosides in RG, ginsenoside Re (85%) showed the highest bioacessibility in the food phase as with the WG, followed by ginsenoside Rg1 (77%) > Rb1, Rb2, Rc, Rd, Rf, Rg2 (20S), and Rg2 (20R) (60%) > Rg3 (S,R), Rk1, Rg5, Rg6, F4, Rk3, and
ranged from 75 to 82%, and ginsenoside Rd constituted 71% of the matrix, whereas Rb1, Rc, and Rb2 had the lowest level at about 60%. The release percentages of total PPD (T-PPD) and PPT (T-PPT) were 60 and 85%, respectively. In the early phase, the bioaccessibility of ginseng saponins from the food matrix was higher for the PPT type, including Re, Rf, Rg1, and Rg2 (S,R) than for the PPD type, including Rb1, Rb2, Rc, and Rd. Moreover, the percentages of most of the ginsenosides transferred to the bioaccessible fraction after termination of the small intestinal phase (ileum = final stage) of simulated digestion increased to >85%, except for Rb1 (74%) and Rg2 (20R) (81%). The release percentages for T-PPD and T-PPT were 80 and 90%, respectively. The gap between the T-PPD and T-PPT types in the ileum phase (10%) was lower than that in the food phase (25%) during in vitro digestion. The F
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Rh4 (30−40%), in decreasing order. Namely, the bioaccessibility of ginsenoside components present in WG itself was >60%, whereas the less polar RG ginsenosides exhibited low bioaccessibility at
