J Nutr Sci Vitaminol, 53, 358–365, 2007
Absorption of Dietary Licorice Isoflavan Glabridin to Blood Circulation in Rats
Chinatsu ITO1, Naomi OI1, Takashi HASHIMOTO1, Hideo NAKABAYASHI1, Fumiki AOKI2, Yuji TOMINAGA3, Shinichi YOKOTA3, Kazunori HOSOE4 and Kazuki KANAZAWA1,*
1Laboratory of Food and Nutritional Chemistry, Graduate School of Agriculture, Kobe University, Rokkodai, Nada-ku, Kobe 657–8501, Japan 2Functional Food Ingredients Division, Kaneka Corporation, 3–2–4 Nakanoshima, Kita-ku, Osaka 530–8288, Japan 3Functional Food Ingredients Division, and 4Life Science Research Laboratories, Life Science RD Center Kaneka Corporation, 18 Miyamae-machi, Takasago, Hyogo 676–8688, Japan (Received February 19, 2007)
Summary Bioavailability of glabridin was elucidated to show that this compound is one of the active components in the traditional medicine licorice. Using a model of intestinal absorption, Caco-2 cell monolayer, incorporation of glabridin was examined. Glabridin was easily incorporated into the cells and released to the basolateral side at a permeability coef- ficient of 1.70 0.16 cm/s 105. The released glabridin was the aglycone form and not a conjugated form. Then, 10 mg (30 mol)/kg body weight of standard chemical glabridin and licorice flavonoid oil (LFO) containing 10 mg/kg body weight of glabridin were adminis- tered orally to rats, and the blood concentrations of glabridin was determined. Glabridin showed a maximum concentration 1 h after the dose, of 87 nmol/L for standard glabridin and 145 nmol/L for LFO glabridin, and decreased gradually over 24 h after the dose. The level of incorporation into the liver was about 0.43% of the dosed amount 2 h after the dose. These detected glabridins were in the aglycone form and not conjugated forms. The bioavail- ability was calculated to be AUCinf of 0.825 and 1.30 M·h and elimination T1/2 of 8.2 and 8.5 h for standard glabridin and LFO, respectively. Adipocytokine levels were determined in the rats. The secreted amount of monocyte chemoattractant protein-1 was significantly lower in the glabridin group compared to control vehicle group. Thus, dietary glabridin was at least partly incorporated into the body in an unchanged form, though most dietary fla- vonoids are converted to non-active conjugate forms during intestinal absorption. Key Words dietary glabridin, licorice, intestinal absorption, bioavailability, MCP-1
Licorice (Glycyrrhiza glabra L.) is an authoritative any metabolic conversions. However, the bioavailability medicinal plant and its root has been used for a tradi- of glabridin is still unclear. tional medicine since the Egyptian Era. The root extract Bioavailability of dietary factors is closely associated includes flavonoids, glycyrrhizin, and saponin, among with susceptibility to conjugation during intestinal other compounds. The active compound in determining absorption. Non-nutrients such as polyphenols and fla- uptake efficiency is believed to be a flavonoid, glabridin. vonoids undergo conjugation in intestinal cells while Glabridin has been reported to possess a strong antioxi- nutrients are metabolized in the liver (14, 15). The con- dative potency that can inhibit oxidation of low-density jugations mainly occur on functional groups such as lipoprotein (1–5), nephritis (6), Helicobacter pylori activ- hydroxyls with UDP-glucuronosyltransferase and/or ity (7), and melanogenesis and inflammation (8), and phenol sulfotransferase, and consequently the conju- also to exhibit a modulating function on several pro- gates do not exhibit bioactivities. Thus, highly bioavail- teins such as cytochrome P450 enzymes (9), peroxi- able compounds are those which escape conjugation some proliferators-activated receptor- (10), and regu- and can then exhibit a considerable range of bioactivi- latory proteins of serotonin re-uptake (11), blood ties in the body. We have assumed that prenyl groups glucose level (12), and bone disorders (13). This infor- attached to functional groups occasionally resist the mation on the bio-functions of glabridin attracted us to conjugation activity by obstructing the access of conju- examine the bioavailability of dietary glabridin. The bio- gation enzymes, and found that one prenyl phenol, functions of glabridin can not be supported without bio- artepillin C, was incorporated in the active free form availability data, because it needs to be shown that gla- and exhibited a strong activity (16). An oral dose of bridin is incorporated into the body by itself without artepillin C clearly suppressed the formation of aberrant crypt foci (17) and induced tumor cells to arrest at G0/
*To whom correspondence should be addressed. G1 by stimulating an expression of Cip1/p21 in the cells E-mail: [email protected] (18). Glabridin is a prenyl compound (Fig. 1), and may
358
Absorption of Dietary Glabridin in Rats 359
bonate membrane 6.5 mm in diameter and 0.4 m in pore size (Corning Costar Co., NY) at a density of 2.5 105 cells/well. The cells on the Transwells were used to test the cellular uptake of glabridin. Alternatively, Caco- 2 was seeded in 96-well plates at a density of 0.6 106 cells/well, and after an overnight culture, was used for measuring cell viability. Incubation of Caco-2 monolayers with glabridin. The Caco-2 cells at passages 55–61 were used for experi- ments 21–22 d postseeding in the Transwells according Fig. 1. Chemical structure of glabridin. to the methods of Murota et al. (20). The transepithelial electrical residence value of the monolayers, which was measured by the method of Hidalgo et al. (21) with a also escape conjugation. We have previously found that Millicell-ERS (Millipore Co.) exceeded 600 ·cm2. The dietary glabridin appeared in human plasma in its free inserts were washed with Hank’s balanced salt solution form (19). The reported obvious bio-functions may cor- (HBSS) pH 7.4, for 30 min, in a CO2 incubator, and then relate with the high bioavailability of glabridin. To used in the cellular uptake experiments. Glabridin or improve our understanding of the incorporation of LFO in DMSO was diluted with HBSS to 10 M. The dietary glabridin it is necessary to show whether glabri- final concentration of solvent was 0.1% for DMSO. The din is absorbed into the body unchanged in its active HBSS containing glabridin (200 L) was added to the form or not by providing pharmacokinetic parameters. apical side of the Caco-2 monolayer at a concentration In the present study, we examined the incorporation of 10 M glabridin (2 nmol/well) and incubated for up of glabridin first with a human colon adenocarcinoma to 24 h. Following incubation at 37˚C, the apical and cell line Caco-2, which has been used as a model of basolateral solutions were collected. Cellular extracts intestinal absorption. Then, glabridin was administered were prepared from whole inserts by shaking slowly for orally to rats, and glabridin itself but not the metabo- 30 min with 100 L methanol for the apical side and lized conjugates were determined in the blood. 700 L for the basolateral side. The recovered solutions were submitted to solid-phase extraction by methanol MATERIALS AND METHODS with Sep-PakR (Vac 1 cc C8, Waters), and analyzed by Chemicals. Glabridin of the highest grade was pur- HPLC (19). chased from Wako Pure Chemical Industries, Ltd. With the transportation data, a permeability (Papp) (Osaka, Japan). This was dissolved in dimethylsulfoxide coefficient of glabridin was calculated according to the (DMSO) for cell line experiments, or dissolved in method described previously (22) using the following medium-chain triglycerides (MCT) with a fatty acid equation: composition of C8 : C10 99 : 1 at a concentration of P V/AC ·dC/dT 1% glabridin (w/w) for animal experiments. We also app 0 prepared a flavonoid extract from licorice as described where, V the volume of the solution in the basolateral previously (12). Briefly, licorice (Glycyrrhiza glabra L.) side, A the membrane surface area, C0 the initial con- root was extracted with ethanol. This was used as the centration in the apical side, and dC/dT the change in licorice-ethanol extract for cell line experiments after glabridin concentration in the basolateral solution over dissolving in DMSO at a concentration of 10% glabri- time. din. The extract was filtered through a paper filter, con- Cell viability. To establish the cytotoxicity of glabri- densed, and mixed with MCT. Removing insoluble din and LFO, Caco-2 cells in 96-well plates were incu- materials by filtration, MCT was further added to adjust bated with these reagents, and the cell viability basi- the concentration of glabridin to 1%. The MCT solution cally was evaluated with a MTS assay using 3-(4,5- was used as licorice flavonoid oil (LFO) for animal dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- experiments. Other chemicals were commercial prod- (4-sulfophenyl)-2H-tetrazolium according to the previ- ucts of the highest grade available. ously described method (23). Briefly, 10 L MTS (2 mg/ Caco-2 cells. Caco-2 cells were kindly provided by mL phosphate-buffered saline) was added to the wells Dr. Junji Terao (The University of Tokushima, School of and incubated at 37˚C for 4 h, and then the difference Medicine, Japan), and cultured in Dulbecco’s modified in absorbance between 492 and 630 nm was measured Eagle’s medium (DMEM) (GIBCO BRL, NY), supple- with a Microplate reader MTP-120 (Corona Electric). mented with 100 mL/L fetal bovine serum (Sigma Animals and animal study. Male Crlj:CD(SD) rats (8 Chemical), 10 mL/L non-essential amino acid solution wk old) were purchased from Charles River Laborato- (GIBCO), 25,000 units/L antibiotics penicillin, and 25 ries Japan Inc. The rats were housed in a temperature- mg/L streptomycin (GIBCO). The cells were cultured as and humidity-controlled room (23 1˚C and 60 5% a monolayer under a humidified atmosphere of 95% air relative humidity) with a 12-h light-and-dark cycle, and 5% CO2 at 37˚C. Subcultured until 80–90% conflu- and were given free access to food (Rodent Lab diet EQ, ent, they were harvested by trypsin treatment and then Shizuoka Laboratory Animal Center) and water. After seeded on the apical side of a Transwell with a polycar- 1-wk acclimation, 36 rats were randomly assigned to
360 ITO C et al. three groups so that there were no significant differ- cal Co. Ltd. (Tokyo, Japan), respectively. ences in the starting body weight among the groups Statistical analysis. Data was expressed as the (318–367 g). One group received a single oral dose of mean SE. The significance of the difference was deter- 10 mg/kg body weight (30 mol/kg) of glabridin in mined by Student’s t-test. Differences were considered MCT by stomach tube, another group was given LFO in significant when p 0.05. MCT containing 10 mg/kg body weight of glabridin, RESULTS and the other group was given MCT alone to serve as a control for the following cytokine analysis. The rats Incorporation of glabridin in Caco-2 cells were fasted for 8 h before the oral doses, and allowed Caco-2 monolayers have been frequently used as a free to access the diet and water after the doses. At reg- model system of intestinal absorption, and were ular intervals, blood was collected from the tail vein, employed here to understand whether glabridin was and submitted to an analysis of glabridin. The rats were able to be incorporated with or without metabolic con- sacrificed 48 h after the oral doses. Mesenteric fat and version. Before the transportation experiments, the kidney leaf fat were removed, and the feces and urine cytotoxicity of glabridin was examined (Fig. 2). Stan- were collected. dard chemical glabridin did not affect cell viability at Another three groups of rats were treated similarly concentrations up to 25 M after 24-h incubation. Lic- with oral doses of 30 mol/kg and 15 mol/kg body orice-ethanol extract at a concentration of 10 M gla- weight of glabridin in MCT and LFO containing 30 bridin slightly decreased the cell viability after 24-h mol/kg body weight of glabridin in MCT. Two hours incubation. As a result, the standard chemical and lico- later the rats were sacrificed, and the liver, kidney, rice-ethanol extract were added at the concentrations of mesenteric fat, and kidney leaf fat were taken for glabri- 10 M (2.0 nmol/well) glabridin to the apical phase of din analysis. the Caco-2 monolayer and the distribution of glabridin All animal treatments in this study conformed to the determined after 12 h (Fig. 3A). In the Caco-2 assay “Guidelines for the Care and Use of Experimental Ani- with standard glabridin, 1.80 nmol/well glabridin was mals, Rokkodai Campus, Kobe University.” recovered, and 47% was in the apical phase, 27% in the Determination of glabridin by HPLC. Blood samples cells, and 26% in the basolateral phase. Similarly, 1.64 were treated with heparin and centrifuged, and the nmol/well was recovered in the cells treated with lico- supernatant was extracted with 9-volumes of ethyl ace- rice-ethanol extract, and 38% was on the apical side, tate 3 times. Liver, kidney, fats, urine, and feces were 36% in the cells, and 25% in basolateral phase. Glabri- homogenized in 0.1 M sodium acetate buffer (pH 5.0) din of both standard chemical and licorice-ethanol with a Teflon homogenizer and then extracted with extract was incorporated into intestinal cells and ethyl acetate. These extracts were submitted to solid- released to the basolateral side mostly without meta- phase extraction by methanol with Sep-PakR, and ana- bolic conversion. The time-dependent pass-through rate lyzed on a HPLC (Model LC-10AD VP, Shimadzu, Japan) was determined in Caco-2 cells after the addition of 2 equipped with an electrochemical detector (Coullochem nmol/well of standard glabridin to the apical side (Fig. III; analysis cell, Model 5010; ESA Inc., USA). The 3B). With increasing incubation time, apical glabridin HPLC conditions were basically similar to those in our decreased and basolateral glabridin increased. On the previous report: column, YMC J’Sphere ODS-H80 JH- basis of the detection data, the Papp coefficient of glabri- 303 4.6 250 mm; mobile phase, phosphate buffer (pH 6.9) : methanol : acetonitrile 35 : 35 : 30 (v/v/v); col- umn temperature, 40˚C; flow rate, 1.0 mL/min; sample injection, 20 L. The detector was set under 850 mV for the guard cell, 350 mV for E1 and 800 mV for E2, the detection limit of glabridin was 1.0 nM, and the quantitative limit was 1 to 2.5 nM. The above extraction conditions gave the following recovery % when an accurate amount of glabridin was incubated for 1 h with plasma, homogenates of liver, kidney, adipose tissue, and urine: 80.9 3.8, 90.8 2.3, 86.2 1.5, 68.2 2.5, and 73.5 1.2 (mean SE, n 5), respectively. Determination of adipocytokines in rat circulation. The blood samples were also submitted for the determina- Fig. 2. Effect of glabridin on viability of Caco-2 cells. tion for 5 kinds of cytokines; monocyte chemoattrac- Caco-2 cells were seeded at a density of 0.5 106 cells/ tant protein-1 (MCP-1), leptin, tumor necrosis factor- mL and cultured overnight, and then incubated with (TNF- ), insulin, and adiponectin. These cytokines the indicated amounts of standard glabridin (open cir- were determined using antibody kits obtained from cles) or licorice-ethanol extract (closed circles) in DMSO Amersham Biosciences (Buckinghamshire, UK), Mori- for 24 h. The cell viability was evaluated by an MTS assay and expressed as % of cells treated without glabri- naga & Co. Ltd. (Yokohama, Japan), Amersham Bio- din. Values are means SE, n 5. sciences, Morinaga & Co. Ltd., and Otsuka Phamaceuti- Absorption of Dietary Glabridin in Rats 361
Fig. 3. Distribution of glabridin in Caco-2 monolayer. A, standard glabridin (2 nmol/well) and licorice-ethanol extract (2.0 nmol/well of glabridin) were added to the apical phase of Caco-2 cells and incubated for 12 h. The apical (closed bar), intracellular (open), and basolateral (hatching) glabridin levels were determined as described in “Material and Methods.” The figures are % of total-recovered glabridin, 1.80 nmol/well for the standard glabridin and 1.64 nmol/well for LFO glabri- Fig. 4. HPLC detection of glabridin in blood collected din. B, standard glabridin (2.0 nmol/well) was incu- from tail vein of rats that had received oral doses of gla- bated and the apical (closed bar), intracellular (open), bridin. A, plasma of untreated rat; B, untreated-rat and basolateral (hatching) glabridin were determined plasma spiked with an authentic glabridin; C, plasma of at the time indicated. Values are mean SE, n 4. Aster- rat 0.5 h after an oral dose of 30 mol/kg body weight isks show a significant difference from the value at the of glabridin; D, plasma of rat 0.5 h after an oral dose of respective previous time. LFO containing 30 mol/kg body weight of glabridin. din from apical to basolateral sides in Caco-2 cells was calculated to be 1.70 0.16 cm/s 105. In these cells treated with standard and licorice-ethanol extract gla- bridin, the recovery of glabridin was 90 and 82%, respectively, and was the glabridin itself but not conju- gates. These results clearly indicate that glabridin was incorporated into Caco-2 cells and released to the baso- lateral side mostly without metabolic conversion. Standard and LFO glabridin were administered intra- gastrically to rats, and 30 min later blood was collected and analyzed by HPLC (Fig. 4). In rats dosed with both standard glabridin and LFO, a single peak was detected at around 16 min retention time (Fig. 4C and D) and Fig. 5. Incorporation of dietary glabridin in blood. Stan- coincided with the peak of authentic glabridin (Fig. 4B). dard chemical glabridin (open circle) or LFO glabridin This means that dietary glabridin at least partly appears (closed circle) were administered orally to rats at the in the blood circulation without metabolic conversion. amounts of 30 mol/kg body weight of glabridin, and Incorporation of dietary glabridin in rats the concentration in blood determined at the indicated The standard glabridin 30 mol/kg body weight and times after the doses. Values are mean SE, n 12. LFO containing 30 mol/kg body weight of glabridin were doses to rats intragastrically and blood from tail vein was collected at regular intervals (Fig. 5). The oral 24 h after the doses. dose of 30 mol/kg of standard glabridin showed a peak The incorporation peaks of glabridin were observed concentration of 87 nmol/L glabridin at 1 h after the at 1 h after the doses. Then, 15 mol/kg and 30 mol/ dose, and decreased gradually thereafter. LFO glabridin kg of the standard glabridin and LFO containing 30 was more easily incorporated into the blood and mol/kg of glabridin were administered to another showed a peak of 145 nmol/L at 1 h after the dose and three groups and the accumulations were determined a shoulder peak of 56 nmol/L at 8 h after the dose. Both in liver, kidney, mesenteric fat, and kidney leaf fat 2 h glabridin forms almost disappeared from the blood by after the doses (Fig. 6). A considerable amount of glabri- 362 ITO C et al. din was detected in the liver, 23.0 2.3 nmol/liver in ney, 1.14 0.13 nmol/kidney in the 15 mol/kg group, the 15 mol/kg standard glabridin group, 53.6 5.2 2.12 0.20 nmol/kidney in the 30 mol/kg group, and nmol/liver in the 30 mol/kg standard glabridin group, 2.24 0.29 nmol/kidney in the LFO group, and were and 52.0 11.2 nmol/liver in the LFO group, represent- about 0.02, 0.02 and 0.02% of the doses, respectively ing about 0.37, 0.43 and 0.41% of the administered (Fig. 6B). Lower amounts of glabridin occurred in glabridin, respectively. The dose of 30 mol/kg showed mesenteric and kidney leaf fats (Fig. 6C and D). The twice the amount of incorporation into the liver com- mesenteric concentrations of around 1 nmol/rat were pared to the dose of 15 mol/kg, but the incorporation similar in the three groups regardless of the doses. In rate was similar. Glabridin was also detected in the kid- kidney fat, 0.13 0.05 nmol/rat in the 15 mol/kg group, 0.60 0.16 nmol/rat in the 30 mol/kg group, and 1.80 0.59 nmol/rat in the LFO group were detected and were around 0.002, 0.005 and 0.014% of the administered amounts, respectively. These results showed that dietary glabridin was partly incorporated into the blood and liver in an unchanged form. After a short time in the circulation glabridin was excreted through the kidney and barely accumulated in adipose tissues. Changes in cytokine secretions following with the dose of glabridin In our previous paper (24), we showed that dietary glabridin suppresses body weight gain and fat accumu- lation in obese C57BL/6J mice. Therefore, in the present study, adiposis related cytokines, insulin, leptin, adi- ponectin, MCP-1, and TNF- were determined in the blood collected from the rats in Fig. 5. The cytokine lev- els were compared to those in an MCT-dosed group as a control (Fig. 7). Insulin secretion increased in all three Fig. 6. Accumulation of dietary glabridin in liver (A), groups, which is considered to be due to resumption of kidney (B), mesenteric fat (C), and kidney leaf fat (D) 2 feeding after 8-h fasting for the oral doses. Adiponectin h after the doses of glabridin or LFO. Values are mean SE of glabridin concentration in organ and tis- was almost unchanged in the three groups and TNF- sues of each rat, n 12. showed similar changes in all three groups. Leptin
Fig. 7. Changes in secretions of insulin, adiponectin, leptin, TNF- , and MCP-1 after oral doses of standard glabridin (open circle), LFO glabridin (closed circle), or vehicle MCT (triangle) as a control. Asterisks show a significant difference from the value of MCT group. Absorption of Dietary Glabridin in Rats 363
Table 1. Pharmacokinetic parameters of glabridin in rats.*
AUC C T T Cl/F 30 mol/kg body weight of: inf max 1/2 max (nM·h) (nM) (h) (h) (L/h)
Standard glabridin 825 87 8.2 1 37.4 LFO glabridin 1,301 145 8.5 1 23.7
* Mean values of plasma glabridin profiles in each group were analyzed using non-compartmental model. secretion was a little lower in the LFO group than in the doses. In feces of both groups glabridin was detected at MCT control and the standard glabridin groups, but a similar ratio, around 37% of the administered was not significant. MCP-1 secretion in standard glabri- amounts. Urine glabridin was trace in both groups, at din group was significantly lower at 8–24 h after the less than 2 nmol/rat. This suggests that glabridin did dose than in the MCT group. not show entero-hepatic circulation. The pharmacoki- netic parameters of dietary glabridin, the maximum DISCUSSION concentration in plasma (Cmax), time required to reach Licorice flavonoid glabridin has been expected to pos- the maximum concentration (Tmax), area under the sess a high bioavailability, because glabridin is a prenyl curve (AUCinf), total clearance (Cl/F: where F denotes compound and refractory from undergoing conjuga- bioavailability), and elimination half-life (T1/2), were tions, although most flavonoids are inactivated by con- calculated using WINNONLIN software (version 1.1; jugation during intestinal absorption. In the present SCI, Morrisville, NC) from the results in Fig. 5 (Table 1). study, we have shown that dietary glabridin was at least Standard glabridin gave AUCinf of 0.825 M·h, T1/2 of partly incorporated into the body in the aglycone form 8.2 h, and Cl/F of 37.4 L/h. LFO glabridin had values of using Caco-2 monolayer and rat studies. 1.30 M·h, 8.5 h, and 23.7 L/h, respectively. We also In the Caco-2 monolayer study, glabridin was easily determined the T1/2 of plasma glabridin in humans incorporated into the cells and released to the basolat- using LFO glabridin, and found it to be about 10 h (25). eral side, and furthermore the released glabridin was in This indicates that dietary glabridin shows a similar the free aglycone form not in conjugated forms (Fig. 3). elimination profile in humans and rats. Comparing the Recovery of glabridin from the Caco-2 cells was around parameters between standard and LFO glabridin, AUCinf 90%. This means that most glabridin added to the api- and Cmax values were greater in LFO than for standard cal side passed to the basolateral side without conver- glabridin, while Tmax and T1/2 values were similar. This sion. The Papp coefficient of glabridin from the apical to indicates that LFO glabridin is more easily incorporated basolateral side in Caco-2 cells was 1.70 cm/s 105. than standard glabridin. Both forms of glabridin were Though the value is lower compared to the Papp of 2.7 dissolved in the same solvent, MCT, and LFO is a mix- cm/s 105 of another prenyl phenol artepillin C (14), it ture of various licorice flavonoids as mentioned in is clear that glabridin gradually passes-through intesti- “Material and Methods.” Probably the coexistent com- nal cells and glabridin itself is incorporated into the ponents facilitate the incorporation of glabridin in LFO. blood stream. In addition, glabridin was detected in the Pharmacokinetic data has been reported for various blood unchanged in form when orally dosed to rats (Fig. flavonoids. The most abundant flavonoid in our food is 4). These results show that dietary glabridin also quercetin (26). Hollman et al. (27, 28) determined that appears by itself in animal blood. the AUC of quercetin was 18.8 M·h and its elimination The fate of dietary glabridin was determined in rats T1/2 was 16–17 h when about 200 mol of quercetin dosed with 30 mol/kg body weight of standard chemi- glucoside was ingested in humans. Conquer et al. (29) cal glabridin and LFO glabridin (Fig. 5). Standard glabri- showed that the blood concentration of quercetin was din gave a peak of 87 nmol/L for the maximum concen- less than 1.5 M when a 1 g quercetin capsule was sup- tration in blood 1 h after the dose. LFO glabridin showed plied daily. These findings indicate that quercetin enters a peak concentration of 145 nmol/L at 1 h after the the blood more easily than glabridin. However, the dose accompanied by a shoulder of 56 nmol/L at 8 h chemical form of endogenous quercetin is mostly conju- after the dose. Both forms of glabridin decreased gradu- gates of glucuronide or sulfate (30, 31). The conjugates ally and almost disappeared 24 h after the doses. At the fail to produce bioactivity because they are the excre- end of experiments 48 h after the doses, the rats were tion form and can not enter body cells (14, 32, 33). In sacrificed and glabridin accumulation determined. In the present study, both standard chemical glabridin and liver, kidney, mesenteric fat, and kidney leaf fats, glabri- LFO glabridin are one-order of magnitude lower in AUC din was under the detection limit (data not shown). value than quercetin, but glabridin existed in the blood This means that dietary glabridin is partly incorporated as the aglycone form. In a recent study (34), the into the blood circulation and easily excreted by 24 h authors reported that glabridin was mainly metabolized after the doses without accumulation. by glucuronidation and the metabolic capacity of intes- Glabridin was also determined in feces and urine col- tine microsomes was 1/15 to 1/20 of that in liver lected throughout the experiments until 48 h after the microsomes. The information also means that dietary 364 ITO C et al. glabridin circulates in the blood stream mainly as the Free Radic Biol Med 23: 302–313. aglycone form. These strongly indicate that glabridin 2) Fuhrman B, Buch S, Vaya J, Blinky PA, Coleman R, possesses a higher bioavailability than the other fla- Hayek T, Aviram M. 1997. Licorice extract and its major vonoid such as quercetin. polyphenol glabridin protect low-density lipoprotein Using another three groups of rats, the incorporation against lipid peroxidation, in vitro and studies in humans and in atherosclerotic apolipoprotein E-defi- of glabridin in organs and tissues was measured 2 h cient mice. Am J Clin Nutr 66: 267–275. after the dose (Fig. 6), since the maximum concentra- 3) Belinky PA, Aviram M, Fuhrman B, Rosenblat M, Vaya J. tion in blood appeared at 1 h after the dose in Fig. 5. 1998. The antioxidative effects of the isoflavan glabridin Glabridin was incorporated into the liver, kidney, and on endogenous constituents of LDL during its oxidation. kidney leaf fat, and was double in amount in the groups Atherosclerosis 137: 49–61. with 30 mol/kg of standard or LFO glabridin com- 4)Rosenblat M, Belinky P, Vaya J, Levy R, Hayek T, Cole- pared to the group of 15 mol of standard glabridin. man R, Merchav S, Aviram M. 1999. Macrophage Thus, glabridin seems to be incorporated dose-depen- enrichment with the isoflavan glabridin inhibits dently, and the incorporated amount in liver was calcu- NADPH oxidase-induced cell-mediated oxidation of low lated to be around 0.37–0.43% of the dose. Previously, density lipoprotein. A possible role for protein kinase C. we showed that dietary glabridin suppressed the gene J Biol Chem 274: 13790–13799. 5) Haraguchi H, Yoshida N, Ishikawa H, Tamura Y, expression of lipogenesis-related enzymes and stimu- Mizutani K, Kinoshita T. 2000. Protection of mitochon- lated gene expression of -oxidation enzymes in the drial functions against oxidative stress by isoflavans liver of C57BL/6J mice (24). Adiposis-related cytokines from Glycyrrhiza glabra. J Pharm Pharmacol 52: 219– were measured in the blood pooled from the glabridin- 232. treated rats in Fig. 5 (Fig. 7). Adipocyte growth is con- 6) Fukai T, Satoh K, Nomura T, Sakagami H. 2003. Anti- sidered to correlate with the secretions of insulin, lep- nephritis and radical scavenging activity of prenylfla- tin, adiponectin, TNF- , and other cytokines (35), and vonoids. Fitoterapia 74: 720–724. described to accompany inflammation and increasing 7) Fukai T, Marumo A, Kaitou K, Kanda T, Terada S, secretion of TNF- (36). MCP-1 which contributes to Nomura T. 2002. Anti-Helicobacter pylori flavonoids macrophage infiltration has been reported to increase from licorice extract. Life Sci 71: 1449–1463. in obese diabetic mice (37). In the present study, the 8)Yokota T, Nishio H, Kubota Y, Mizoguchi M. 1998. The inhibitory effect of glabridin from licorice extracts on oral dose of glabridin did not accompany clear changes melanogenesis and inflammation. Pigment Cell Res 11: of insulin, adiponectin, or TNF- levels. Leptin secretion 355–361. showed a tendency to be lowered in the LFO group, and 9)Kent UM, Aviram M, Rosenblat M, Hollenberg PF. 2002. MCP-1 was significant lower in the standard glabridin The licorice root derived isoflavan glabridin inhibits the group. These indicate that dietary glabridin can miti- activities of human cytochrome P450S 3A4, 2B6, and gate inflammation through suppressing MCP-1 secre- 2C9. Drug Metab Dispos 30: 709–715. tion. However, the suppressing effect was not observed 10) Mae T, Kishida H, Nishiyama T, Tsukagawa M, Konishi in the LFO group. LFO includes various licorice fla- E, Kuroda M, Mimaki Y, Sashida Y, Takahashi K, vonoids other than glabridin, such as glabrene and Kawada T, Nakagawa K, Kitahara M. 2003. A licorice isoliquiritigenin (1, 13). Glabrene has a strong estro- ethanolic extract with peroxisome proliferator-activated gen-like activity (38, 39), and isoliquiritigenin exhibits receptor- ligand-binding activity affects diabete in KK- Ay mice, abdominal obesity in diet-induced obese C57BL a reducing effect of prostaglandin E and nitric oxide 2 mice and hypertension in spontaneously hypertensive (40, 41). These flavonoids probably offset the effect of rats. J Nutr 133: 3369–3377. glabridin in the present experiments. 11) Ofir R, Tamir S, Khatib S, Vaya J. 2003. Inhibition of The bioactivity of LFO should be examined more serotonin re-uptake by licorice constituents. J Mol Neu- thoroughly, but one of the ingredients, glabridin, clearly rosci 20: 135–140. possessed a high bioavailability in the present study. 12) Nakagawa K, Kishida H, Arai N, Nishiyama T, Mae T. Prenyl flavanone glabridin escaped conjugation during 2004. Licorice flavonoids suppress abdominal fat accu- the intestinal absorption process (Figs. 3 and 4) and mulation and increase in blood glucose level in obese existed in the free aglycone form in the blood and liver diabetic KK-Ay mice. Biol Pharm Bull 27: 1775–1778. (Figs. 5 and 6). Free aglycones have been well recog- 13) Somjen D, Katzburg S, Vaya J, Kaye AM, Hendel D, Pos- nized to exhibit strong bioactivity, but the conjugate ner GH, Tamir S. 2004. Estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts forms have low bioactivity. Prenyl flavonoids have been and prepuberal rat skeletal tissues. J Steroid Biochem Mol found to possess a higher bioactivity, which is probably Biol 91: 241–246. attributable to the higher affinity of hydrophobic prenyl 14) Murota K, Terao J. 2003. Antioxidative flavonoid quer- groups to biomembranes (42). Glabridin should be one cetin: implication of its intestinal absorption and metab- of candidate factors in the diet for preventing degenera- olism. Arch Biochem Biophys 417: 12–17. tive diseases. 15) Németh K, Plumb GW, Berrin J-G, Juge N, Jacob R, Naim HY, Williamson G, Swallow DM, Kroon PA. 2003. De- REFERENCES glycosylation by small intestinal epithelial cell -glucosi- 1)Vaya J, Belinky PA, Aviram M. 1997. Antioxidant con- dases is a critical step in the absorption and metabolism stituents from licorice roots, isolation, structure elucida- of dietary flavonoid glycosides in humans. Eur J Nutr tion and antioxidative capacity toward LDL oxidation. 42: 29–42. Absorption of Dietary Glabridin in Rats 365
16) Shimizu K, Ashida H, Matsuura Y, Kanazawa K. 2004. 29) Conquer JA, Maiani G, Azzini E, Raguzzini A, Holub BJ. Antioxidative bioavailability of artepillin C in Brazilian 1998. Supplementation with quercetin markedly propolis. Arch Biochem Biophys 424: 181–188. increases plasma quercetin concentration without 17) Shimizu K, Das SK, Baba M, Matsuura Y, Kanazawa K. effect on selected risk factors for heart disease in healthy 2006. Dietary artepillin C suppresses the formation of subjects. J Nutr 128: 593–597. aberrant crypt foci induced by azoxymethane in mouse 30) Sesink ALA, O’Leary KA, Hollman PCH. 2001. Querce- colon. Cancer Lett 240: 135–142. tin glucuronides but not glucosides are present in 18) Shimizu K, Das SK, Hashimoto T, Sowa Y, Yoshida T, human plasma after consumption of quercetin-3-gluco- Sakai T, Matsuura Y, Kanazawa K. 2005. Artepillin C in side or quercetin-4′-glucoside. J Nutr 131: 1938–1941. Brazilian propolis induces G0/G1 arrest via stimulation 31)Day AJ, Mellom F, Barron D, Sarranzin G, Morgan MRA, of Cip1/p21 expression in human colon cancer cells. Williamson G. 2001. Human metabolism of dietary fla- Mol Carcinog 44: 293–299. vonoids: Identification of plasma metabolites of querce- 19) Aoki F, Nakagawa K, Tanaka A, Matsuzaki K, Arai N, tin. Free Radic Res 35: 941–952. Mae T. 2005. Determination of glabridin in human 32)Day AJ, Bao Y, Morgan MR, Williamson G. 2000. Conju- plasma by solid-phase extraction and LC-MS/MS. J Chro- gation position of quercetin glucuronides and effect on matogr 828: 70–74. biological activity. Free Radic Biol Med 29: 1234–1243. 20) Murota K, Shimizu S, Chujo H, Moon J-H, Terao J. 2000. 33) Piskula MK, Terao J. 1998. Quercetin’s solubility affects Efficiency of absorption and metabolic conversion of its accumulation in rat plasma after oral administra- quercetin and its glucosides in human intestinal cell tion. J Agric Food Chem 46: 4313–4317. line Caco-2. Arch Biochem Biophys 384: 391–397. 34) Cao J, Chen X, Liang J, Yu X-Q, Xu A-L, Chan E, Wei D, 21) Hidalgo IJ, Raub TJ, Borchardt RT. 1989. Characteriza- Huang M, Wen J-Y, Yu X-Y, Li X-T, Sheu F-S, Zhou S-F. tion of the human colon carcinoma cell line (Caco-2) as 2007. Role of P-glycoprotein in the intestinal absorption a model system for intestinal epithelial permeability. of glabridin, an active flavonoid from the root of Glycyr- Gastroenterology 96: 736–749. rhiza glabra. Drug Metab Dispos 35: 539–553. 22)Walgren RA, Walle UK, Walle T. 1998. Transport of 35)Rosen ED, Spiegelman BM. 2006. Adipocytes as regula- quercetin and its glucosides across human intestinal tors of energy balance and glucose homeostasis. Nature epithelial Caco-2 cells. Biochem Pharmacol 55: 1721– 444: 847–853. 1727. 36) Tilg H, Moschen AR. 2006. Adipocytokines: mediators 23) Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwin- linking adipose tissue, inflammation and immunity. ski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Nature Rev Immunol 6: 772–783. Boyd MR. 1988. Feasibility of drug screening with pan- 37)Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, els of human tumor cell lines using a microculture tet- Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira razolium assay. Cancer Res 48: 589–601. K, Kasuga M. 2006. MCP-1 contributes to macrophage 24) Aoki F, Honda S, Kishida H, Kitano M, Arai N, Tanaka infiltration into adipose tissue, insulin resistance, and H, Yokota S, Nakagawa K, Asakura T, Nakai Y, Mae T. hepatic steatosis in obesity. J Clin Invest 116: 1494– 2007. Suppression by licorice flavonoids of abdominal 1505. fat accumulation and body weight gain in high-fat diet- 38)Tamir S, Eizenberg M, Somjen D, Izrael S, Vaya J. 2001. induced obese C57BL/6J mice. Biosci Biotechnol Biochem Estrogen-like activity of glabrene and other constituents 71: 206–214. isolated from licorice root. J Steroid Biochem Mol Biol 78: 25) Aoki F, Nakagawa K, Kitano M, Ikematsu H, Nakamura 291–298. K, Yokota S, Tominaga Y, Arai N, Mae T. 2007. Clinical 39) Nerya O, Vaya J, Musa R, Izrael S, Ben-Arie R, Tamir S. safety of licorice flavonoids oil (LFO) and pharmacoki- 2003. Glabrene and isoriquiritigenin as tyrosinase netics of glabridin in healthy humans. J Am Coll Nutr inhibitors from licorice roots. J Agric Food Chem 51: 26: 209–218. 1201–1207. 26) Sakakibara H, Honda Y, Nakagawa S, Ashida H, 40)Takahashi T, Takasuka N, Iigo M, Baba M, Nishino H, Kanazawa K. 2003. Simultaneous determination of all Tsuda H, Okuyama T. 2004. Isoliquiritigenin, a fla- polyphenols in vegetables, fruits, and teas. J Agric Food vonoids from licorice, reduces prostaglandin E2 and Chem 51: 571–581. nitric oxide, causes apoptosis, and suppresses abberant 27) Hollman PCH, VD Gaag M, Mengelers MJB, van Trup crypt foci development. Cancer Sci 95: 448–453. MP, de Vries JHM, Katan MB. 1996. Absorption and dis- 41)Takahashi T, Baba M, Nishino H, Okuyama T. 2006. position kinetics of the dietary antioxidant quercetin in Cyclooxygenase-2 plays a suppressive role for induction man. Free Radic Biol Med 21: 703–707. of apoptosis in isoliquiritigenin-treated mouse colon 28) Hollman PCH, Bijsman MNCP, van Gameren Y, Cnossen cancer cells. Cancer Lett 231: 319–325. EPJ, de Vries JHM, Katan MB. 1999. The sugar moiety is 42)Kuzuyama T, Noel JP, Richard SB. 2005. Structural a major determinant of the absorption of dietary fla- basis for the promiscuous biosynthetic prenylation of vonoids glycosides in man. Free Radic Res 31: 569–573. aromatic natural products. Nature 435: 983–987.