Glycyrrhizae Radix (GR, root of licorice) has several pharmacological activities, including chemopreventive, hypoglycemic, anti-inflammatory, and antioxidative activities (Xie et al. 2014; Ng et al. 2021). It has been used as an adjuvant to increase the therapeutic efficacy of other drugs (Qiao et al. 2014) and as a sweetener in traditional medicines, chewing gums, and chocolates (Ishida et al. 1992; Cantelli-Forti et al. 1994; Kobayashi et al. 1995; Lin et al. 2005). Moreover, orally administered GR extract (0.1-0.5 g/kg) in mice for nine days effectively ameliorated interferon-γ-related autoimmune responses and demonstrated neuroprotective efficacy (Yang et al. 2013; Yang et al. 2019). To understand the relationship between the response elicited by GR extract and its pharmacokinetics, bioanalysis of the predominant or pharmacological components of GR extract in biological samples following supplementation and investigation of their pharmacokinetics are necessary.

Glycyrrhizin (Fig. 1A), a major marker component of GR (Kobayashi et al. 1995; Qiao et al. 2014; Peng et al. 2015; Dong et al. 2016), exerts strong neuroprotective effects on experimental autoimmune encephalomyelitis and glutamate-induced apoptosis in neuronal cells and shows therapeutic effects against arthritis, hepatotoxicity, leukemia, allergies, stomach ulcers, and inflammation (Maggiolini et al. 2002). The major active flavonoids of GR, including isoliquiritin, liquiritin, isoliquiritigenin, and liquiritigenin (Gao et al. 2009) (Fig. 1B-1E), are frequently used as antidepressant, anticancer, cardioprotective, antimicrobial, and neuroprotective agents (Cuendet et al. 2006; Cuendet et al. 2010). The pharmacological activities of the major active flavonoids can overlap; however, the main activities could be differentiated. For instance, liquiritin, isoliquiritin, and glycyrrhizic acid mainly and positively influence the anti-inflammatory, anticancer, cardioprotective, and antioxidant activities with different potencies, whereas their deglycosylated metabolites, liquiritigenin, isoliquiritigenin, and glycyrrhetinic acid, mainly and positively influence the immunoregulatory and neuroprotective activities (Qin et al. 2022; Wu et al. 2022; He et al. 2023). Additionally, chemically interconnected liquiritigenin and isoliquiritigenin showed significant efficacy in Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases and disorders in numerous in vitro and in vivo experimental studies (Ramalingam et al. 2018).

Figure 1. Structure of (A) glycyrrhizin, (B) liquritin, (C) isoliquiritin, (D) liquiritigenin, and (E) isoliquiritigenin.

Based on literature search, glycyrrhizin, liquiritin, isoliquiritigenin, and liquiritigenin were selected as the predominant or pharmacological components of the GR extract. We previously developed an analytical method for glycyrrhizin, liquiritin, isoliquiritigenin, and liquiritigenin analysis using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) system with a relatively higher sensitivity (e.g., lower limit of quantification: glycyrrhizin [2 ng/mL] and isoliquiritigenin, liquiritigenin, and liquiritin [0.2 ng/mL each]) with a small volume (50 μL) of plasma samples (Han et al. 2019). Using these analytical methods, glycyrrhizin, isoliquiritigenin, liquiritin, and liquiritigenin plasma concentrations in rats were determined following a single oral GR extract administration at a dose of 1 g/kg (Han et al. 2019). In this study, glycyrrhizin concentration in plasma showed the highest and most stable profile for 12 h. Isoliquiritigenin and liquiritigenin concentrations in plasma were similar and eliminated rapidly after 4 h; however, they rebounded to the initial plasma concentration for 10 h, which could be explained from the biotransformation of liquiritin to isoliquiritigenin (Han et al. 2019). Similarly, co-administration with Jiegeng changed glycyrrhizin and liquiritigenin metabolism in rat fecal lysate, consequently altering their pharmacokinetic profiles (Mao et al. 2017). Besides this biotransformation, compared with the addition of an equal amount of liquiritin alone, liquiritin permeability was much greater when added as a part of the GR extract (Mao et al. 2017), consistent with our case.

Therefore, this study aimed to pharmacokinetically compare GR extract administration with that of an equal amount of a single component contained in the GR extract, including glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, in rats (Wang et al. 2016). This study also aimed to investigate and compare the intestinal absorption and metabolism of these major pharmacological components in coexisting rats. Isoliquiritin was included in the pharmacokinetic and permeability study to better understand the in vivo biotransformation process.

Materials

Glycyrrhizin (purity > 95.0%), isoliquiritigenin (purity > 99.0%), isoliquiritin (purity > 90.0%), liquiritin (purity > 98.0%), liquiritigenin (purity > 97.0%), berberine (purity ≥ 98.0%), dimethyl sulfoxide (DMSO), and Hank’s balanced salt solution (HBSS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water, methanol, and other solvents were obtained from J.T. Baker Korea (Seoul, Korea) and TEDIA (Fairfield, OH, USA). All other chemicals were of reagent and analytical grade.

Plant material and extraction

Dried GR (1 kg), obtained from CK Pharm Co. (Seoul, Korea), was extracted with 12 L of 94% ethanol for 3 h and filtered through Whatman Qualitative filter paper grade 1. The filtrate was concentrated using a rotary evaporator (EYELA, Tokyo, Japan) to obtain the GR extract. The voucher specimen (#KNUNPM GR-2015-002) was deposited at the laboratory of Natural Products Medicine at Kyungpook National University (Daegu, Korea).

Animals

Male Sprague–Dawley rats (7 weeks old, 249 ± 6 g) were obtained from Samtako (Osan, Korea) and were acclimated for one week at the animal facility of the College of Pharmacy, Kyungpook National University. Rats were maintained in an environmentally clean room at 21℃-27℃ with 12-h light (09:00-21:00) and relative humidity of 60% ± 5%. Furthermore, they were fasted for 12 h with water ad libitum. The Kyungpook National University Animal Committee approved the protocols and procedures for the animal studies (IACUC No.: KNU-2017-0126, approval date: September 23, 2017).

Pharmacokinetic study

Rats were fasted for 16 h with water ad libitum before the pharmacokinetic experiments. Their femoral arteries were cannulated with a polyethylene tube (PE50, Jungdo, Seoul, Korea) under anesthesia with isoflurane (isoflurane vaporizer set to 2%, with 0.8 L/min oxygen flow).

The GR extract (1 g/kg/2 mL suspended in distilled water containing 10% DMSO) was orally administered to four rats through gavage. The corresponding doses of glycyrrhizin (20 mg/kg/2 mL suspended in distilled water containing 10% DMSO), liquiritin (5.8 mg/kg/2 mL), isoliquiritin (1.5 mg/kg/2 mL), liquiritigenin (0.5 mg/kg/2 mL), or isoliquiritigenin (0.2 mg/kg/2 mL) were orally administered to rats (four rats per component) through gavage. Blood samples (approximately 120 μL) were taken via the femoral artery at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 12 h and centrifuged at 10,000 × g for 1 min at 4℃. Supernatant plasma samples (50 μL each) were stored at −80℃ until analysis.

Intestinal permeability test

Rats were fasted for 16 h. However, they had nonrestricted access to water before the study commenced. They were anesthetized using isoflurane (isoflurane vaporizer set to 2%, with 0.8 L/min oxygen flow), and the proximal part of the ileal segments (approximately 20 cm) were excised, opened along the mesenteric border, and washed in prewarmed HBSS (pH 7.4). Segments were mounted in a tissue holder of a Navicyte Easy Mount Ussing Chamber (Warner Instruments, Holliston, MA, USA) with a 0.76 cm² surface area. They were acclimated in HBSS for 15 min with continuous oxygenation (95% O₂/5% CO₂ gas). Intestinal permeability studies were conducted by adding HBSS to both sides of the intestinal segments, which included 1 mL of prewarmed HBSS containing the GR extract or equivalent single component (e.g., GR extract [100 mg/6 mL], glycyrrhizin [2 mg/6 mL], liquiritin [0.6 mg/6 mL], isoliquiritin [0.15 mg/6 mL], liquiritigenin [0.05 mg/6 mL], and isoliquiritigenin [0.02 mg/6 mL]) on the apical side of the ileal segments and 1 mL of prewarmed fresh HBSS on the basal side. Sample aliquots (400 μL) were withdrawn at 30-min intervals for 2 h from the basal side, and an equal volume of prewarmed fresh HBSS was subsequently replenished. During the experiment, carbogen gas (5% CO₂/95% O₂) was bubbled into the Ussing chambers at 150 drops/min. Samples were stored at −80℃ until analysis.

Next, we investigated the permeability of isoliquiritigenin (0.02 mg/6 mL) in the absence or presence of a single component of the GR extract using the same experimental procedures described above. Briefly, its permeability was assessed by adding 1 mL of prewarmed HBSS containing isoliquiritigenin (0.02 mg/6 mL) in the absence or presence of a single component of the GR extract (e.g., glycyrrhizin [2 mg/6 mL], liquiritin [0.6 mg/6 mL], isoliquiritin [0.15 mg/6 mL], and liquiritigenin [0.05 mg/6 mL]) on the apical side of the ileal segments and 1 mL of prewarmed fresh HBSS on the basal side. Sample aliquots (400 μL) were withdrawn at 30-min intervals for 2 h from the basal side, and an equal volume of prewarmed fresh HBSS was subsequently replenished. Samples were stored at −80℃ until analysis.

Biotransformation of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin

Rats were fasted for 16 h with water ad libitum before ileal dissection. They were anesthetized using isoflurane (isoflurane vaporizer set to 2%, with 0.8 L/min oxygen flow), and the proximal part of the ileal segments (approximately 20 cm) were excised. The dissected ileal segments were washed using a 10 mL syringe filled with prewarmed HBSS (pH 7.4), and the eluent was vortexed for 1 min followed by centrifugation at 1,000 × g for 5 min at 4℃. The supernatant of the intestinal eluent was used for incubation with isoliquiritigenin, liquiritigenin, and liquiritin.

The ileal segments were mounted onto a Navicyte Easy Mount Ussing Chamber and acclimatized with HBSS for 30 min. Experiments started by changing HBSS with a prewarmed intestinal eluent (1 mL) containing liquiritin, isoliquiritin, liquiritigenin, or isoliquiritigenin (100 μg/mL each) to the apical side of the ileal segments and incubated for 2 h. During the experiment, carbogen gas (5% CO₂/95% O₂) was bubbled into the Ussing chambers at 150 drops/min. Sample aliquots (50 μL) were mixed with 300 µL methanol containing berberine (0.1 ng/mL), vortexed for 10 min, and centrifuged at 10,000 × g for 10 min at 4℃. An aliquot (10 µL) of the supernatant was injected into the LC-MS/MS system.

LC-MS/MS analysis of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin

An Agilent 6470 triple quadrupole mass spectrometer equipped with an Agilent Infinity 1260 Infinite II HPLC system (Agilent Technologies, Santa Clara, CA, USA) was used to analyze glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin. The method developed by Han et al. (2019) was used with slight modifications. Table 1 summarizes the MS/MS conditions for the detection of these analytes. The mobile phase consisted of methanol:water (65:35, v/v) with 0.1% formic acid and was eluted in the isocratic mode. A Synergi Polar-RP column (4 µm, 150 × 2 mm; Phenomenex, Torrance, CA, USA) equipped with a polar-RP guard column (4 × 2 mm; Phenomenex) was used for chromatographic separation.

Table 1 . MS/MS parameters for the detection of the analytes and IS.

Compounds	MRM transitions (m/z)		Ion mode	TR (min)	CE (eV)
	Precursor ion	Product ion
Liquiritin	417.2	254.9	Negative	2.0	20
Isoliquritin	417.2	254.9	Negative	2.4	20
Liquiritigenin	255.1	135.0	Negative	3.2	15
Isoliquiritigenin	255.1	135.0	Negative	4.2	15
Glycyrrhizin	845.5	669.4	Positive	8.7	35
Berberine (IS)	336.1	320.0	Positive	4.9	30

For the analysis of the glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin contents in the GR extract, 100 mg of the GR extract was diluted 200 times with methanol. The diluted samples (50 μL) were added to 300 µL methanol containing berberine (0.1 ng/mL), vortexed for 10 min, and centrifuged at 10,000 × g for 10 min at 4℃. A supernatant aliquot (10 µL) was injected into the LC-MS/MS system.

For the analysis of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin plasma concentrations, plasma samples (50 µL) were added to 300 µL methanol containing berberine (0.1 ng/mL), vortexed for 10 min, and centrifuged at 10,000 × g for 10 min at 4℃. A supernatant aliquot (10 µL) was injected into the LC-MS/MS system. For the analysis of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin in HBSS, HBSS samples (50 µL) were added to 300 µL methanol containing berberine (0.1 ng/mL), vortexed for 10 min, and centrifuged at 10,000 × g for 10 min at 4℃. A supernatant aliquot (10 µL) was injected into the LC-MS/MS system.

Data and statistical analysis

Using noncompartmental analysis (WinNonlin 6.3; Pharsight Corp., Mountain View, CA, USA), the following pharmacokinetic parameters of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were calculated: terminal half-life (t_1/2), area under the plasma concentration–time curve during the observation period (AUC_12h), AUC to infinite time (AUC_inf), and mean residence time (MRT). The maximum concentration (C_max) and the time to reach C_max (T_max) were obtained from the experimental data.

To calculate permeability, the transport rate of the analytes (i.e., glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin) was calculated from the slope of the regression line from the mean permeated amounts versus the incubation time plot. The apparent permeability (P_app) was calculated using the following equation (Jeon et al. 2021; Lim et al. 2022):

$${\text{P}}_{\text{app}}\text{(cm/s)=}\frac{\text{transport rate (nmol/s)}}{{\text{Initial concentration of analyte (μM)×surface area of membrane (cm}}^{\text{2}}\text{)}}$$

The concentrations of the analytes (i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin) were quantitated, and their formation rates (pmol/mg protein/min) were calculated by dividing the amounts of metabolites formed from the incubation with enterocytes in the rats’ ileal segments by the incubation time.

Student’s t-test (IBM SPSS Statistics, version 26; Armonk, NY, USA) was used to determine the statistical significance of the pharmacokinetic parameters and treatment groups. p-values of < 0.05 were considered statistically significant.

Content of the GR extract

Table 2 summarizes the glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin concentrations in the GR extract. Glycyrrhizin had the highest concentration (2.0%), consistent with that of previous studies (Kobayashi et al. 1995; Lin et al. 2005; Wang et al. 2009; Xie et al. 2014). Liquritin, isoliquiritin, liquiritigenin, and isoliquiritigenin had lower concentrations (0.6%, 0.1%, 0.05%, and 0.03%, respectively).

Table 2 . The contents of glycyrrhizin and 4 flavonoids in the GR extract.

Compounds	Content (mg/g extract)	Content (%)
Glycyrrhizin	20.2 ± 3.2	2.0 ± 0.3
Liquiritin	5.8 ± 1.1	0.6 ± 0.1
Isoliquiritin	1.5 ± 0.3	0.1 ± 0.0
Liquiritigenin	0.49 ± 0.06	0.05 ± 0.01
Isoliquiritigenin	0.29 ± 0.06	0.03 ± 0.01

For the pharmacokinetic comparison, we administered 1 g of GR extract/kg to the rats, and the corresponding doses of saponin and flavonoids, 20 mg/kg of glycyrrhizin, 5.8 mg/kg of liquiritin, 1.5 mg/kg of isoliquiritin, 0.5 mg/kg of liquiritigenin, and 0.2 mg/kg of isoliquiritigenin were determined based on the content in the GR extract (Table 2). Additionally, to mimic the intestinal situation that occurred when the GR extract was orally administered to the rats, isoliquiritigenin, liquiritigenin, and liquiritin concentrations were determined based on the oral dose of the GR extract, their content in the GR extract, and the fluid volume of the small intestine. For example, the GR extract was administered at a dose of 1 g/kg/2 mL to rats with stomach and intestinal fluid volumes of 3.38-6.63 and 11.1-16.5 mL, respectively (McConnell et al. 2008), resulting in a 15-30-fold dilution. Therefore, for the intestinal permeability study, the GR extract (100 mg/6 mL), glycyrrhizin (2 mg/6 mL), liquiritin (0.6 mg/6 mL), isoliquiritin (0.15 mg/6 mL), liquiritigenin (0.05 mg/6 mL), and isoliquiritigenin (0.02 mg/6 mL) were used (30-fold dilution).

Pharmacokinetic study of the GR extract

Next, we investigated the glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin plasma concentrations following a single oral administration of the GR extract at a 1 g/kg dose. Considering the glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin contents in the GR extract, equal amounts of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were also orally administered at a single dose of 20, 5.8, 1.5, 0.5, and 0.2 mg/kg, respectively, and their plasma concentrations were compared with the corresponding profile obtained from the oral administration of the GR extract.

Glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin plasma concentrations over time and their pharmacokinetic parameters are shown in Fig. 2 and Table 3, respectively. Of the major components in the rats’ plasma, glycyrrhizin was maintained at the highest concentration for 12 h, and the glycyrrhizin plasma concentrations from the GR extract administration were similar for 2 h but lower than that following orally administered glycyrrhizin (20 mg/kg) during the 4-12 h period. Liquiritin and isoliquiritin plasma concentrations gradually decreased at elimination half-lives (T_1/2) of 2.13 ± 0.83 h and 4.68 ± 3.09 h, respectively. Moreover, liquiritin and isoliquiritin plasma concentrations from the GR extract administration (1 g/kg) were similar to those following the oral administration of liquiritin and isoliquiritin at the corresponding doses (5.8 and 1.5 mg/kg, respectively). In contrast, liquiritigenin and isoliquiritigenin plasma concentrations from the GR extract administration were similar, which increased for 10 h and maintained. However, liquiritigenin and isoliquiritigenin plasma concentration profiles following a single administration of liquiritigenin (0.5 mg/kg) or isoliquiritigenin (0.2 mg/kg), respectively, differed from the corresponding liquiritigenin and isoliquiritigenin concentrations of the GR extract. Therefore, C_max, AUC, T_max, and MRT values of liquiritigenin and isoliquiritigenin from the GR extract differed significantly from those of liquiritigenin and isoliquiritigenin following the administration of a single component (Table 3). These results suggested that isoliquiritigenin and liquiritigenin levels increased during the late intestinal absorption.

Figure 2. Plasma concentration–time profiles of (A) glycyrrhizin, (B) liquiritin, (C) isoliquiritin, (D) liquiritigenin, and (E) isoliquiritigenin following the oral administration of the GR extract (1 g/kg) or an equivalent single component to rats. The following are oral doses of individual component: (A) glycyrrhizin 20 mg/kg, (B) liquiritin 5.8 mg/kg, (C) isoliquiritin 1.5 mg/kg, (D) liquiritigenin 0.5 mg/kg, and (E) isoliquiritigenin 0.2 mg/kg. Data are expressed as mean ± SD (n = 4).

Table 3 . Pharmacokinetic parameters of glycyrrhizin and 4 flavonoids in rats.

Component	Parameters	Single component	GR extract
Gglycyrrhizin	Cmax (ng/mL)	260.4 ± 117.3	311.5 ± 154.3
	Tmax (h)	2.44 ± 1.81	0.50 ± 0.35
	AUC12h (ng·h/mL)	1653 ± 368.1	1191 ± 808.8
	T1/2 (h)	4.95 ± 1.94	8.63 ± 8.95
	MRT (h)	9.08 ± 2.79	12.8 ± 11.3
Liquiritin	Cmax (ng/mL)	18.47 ± 2.19	26.82 ± 8.50
	Tmax (h)	0.31 ± 0.13	0.38 ± 0.14
	AUC12h (ng·h/mL)	26.71 ± 9.52	35.01 ± 5.05
	T1/2 (h)	1.67 ± 0.65	2.13 ± 0.83
	MRT (h)	1.85 ± 0.54	2.22 ± 0.39
Isoliquiritin	Cmax (ng/mL)	23.00 ± 11.9	12.29 ± 4.72
	Tmax (h)	0.31 ± 0.13	0.31 ± 0.13
	AUC12h (ng·h/mL)	26.99 ± 10.9	20.85 ± 7.66
	T1/2 (h)	4.26 ± 3.94	4.68 ± 3.09
	MRT (h)	2.89 ± 0.73	3.07 ± 0.61
Liquiritigenin	Cmax (ng/mL)	3.07 ± 0.48	10.1 ± 3.83*
	Tmax (h)	0.25 ± 0.0	11.0 ± 1.15***
	AUC12h (ng·h/mL)	8.66 ± 0.63	40.4 ± 11.7**
	T1/2 (h)	7.29 ± 3.68	NC
	MRT (h)	4.74 ± 0.80	8.64 ± 0.53***
Isoliquiritigenin	Cmax (ng/mL)	7.84 ± 5.01	16.52 ± 2.66*
	Tmax (h)	0.31 ± 0.13	8.13 ± 5.17*
	AUC12h (ng·h/mL)	8.09 ± 3.13	68.70 ± 12.49***
	T1/2 (h)	2.02 ± 0.26	NC
	MRT (h)	2.07 ± 0.51	7.91 ± 1.12 ***

C_max, maximum plasma concentration; T_max, time to reach C_max; AUC_12h, area under plasma concentration-time curve from zero to 12 h; T_1/2, elimination half-life; MRT, mean residence time; NC, not calculated. *p < 0.05, **p < 0.01, ***p < 0.001; statistically significant compared with equivalent single component. Data were expressed as mean ± SD (n = 4)..

Intestinal permeability of the GR extract

The apparent permeability (P_app) of glycyrrhizin, liquiritin, and isoliquiritin differed insignificantly when added as a part of the GR extract or an equivalent amount of a single component. However, the P_app of liquiritigenin and isoliquiritigenin was significantly higher when added as part of the GR extract than when added individually. The fold increase in the P_app of liquiritigenin and isoliquiritigenin from the GR extract was 2.2- and 4.8-fold, respectively, compared with that of a single component (Fig. 3).

Figure 3. Apparent permeability (P_app) of glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin from the GR extract (100 mg/6 mL) or an equivalent single component in the rat ileum. The following are the initial concentration of individual component: glycyrrhizin 2 mg/6 mL, liquiritin 0.6 mg/6 mL, isoliquiritin 0.15 mg/6 mL, liquiritigenin 0.05 mg/6 mL, and isoliquiritigenin 0.02 mg/6 mL. *p < 0.05, **p < 0.01, and ***p < 0.001; statistically significant compared with an equivalent single component. Data are expressed as mean ± SD (n = 3).

To investigate the mechanisms underlying the increased P_app of liquiritigenin and isoliquiritigenin from the GR extract, the P_app of isoliquiritigenin in the presence of a major GR extract component was measured. Isoliquiritigenin was selected given that it showed the highest increase in P_app among the tested flavonoids in the GR extract. Fig. 4A shows that adding glycyrrhizin did not change the P_app of isoliquiritigenin. Coexistence of liquiritin, isoliquiritin, and liquiritigenin significantly increased it with fold-changes of 2.2, 5.4, and 2.3, respectively (Fig. 4A), but not vice versa (Fig. 4B). This suggested that liquiritin, isoliquiritin, and liquiritigenin coexistence increased the P_app of isoliquiritigenin, thereby enhancing its absorption and plasma exposure.

Figure 4. (A) Apparent permeability (P_app) of isoliquiritigenin in the absence or presence of glycyrrhizin, liquiritin, isoliquiritin, and liquiritigenin. (B) P_app of liquiritin, isoliquiritin, and liquiritigenin in the absence or presence of isoliquiritigenin in the rat ileum. The following are the initial concentrations of individual component: liquiritin 0.6 mg/6 mL, isoliquiritin 0.15 mg/6 mL, liquiritigenin 0.05 mg/6 mL, and isoliquiritigenin 0.02 mg/6 mL. *p < 0.05, **p < 0.01, and ***p < 0.001; statistically significant compared with an equivalent single component. Data are expressed as mean ± SD (n = 3).

Biotransformation of the GR extract in the rat intestines

Next, we investigated the biotransformation of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin based on their structural features and previous studies (Ramalingam et al. 2018; Han et al. 2019). After a 2 h incubation of a single component of the GR extract, including liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, with enterocytes from the rats’ ileal segments, the components’ formation rates were calculated (Fig. 5). Liquiritin transformed into isoliquiritigenin and liquiritigenin, and the liquiritigenin formation rate was 4-fold greater than that of isoliquiritigenin. Isoliquiritin incubation resulted in isoliquiritigenin and liquiritigenin formation with isoliquiritigenin having a 12.2-fold higher formation rate than that of liquiritigenin (Fig. 5A and 5B). Moreover, liquiritigenin or isoliquiritigenin incubation with rat enterocytes resulted in isoliquiritigenin or liquiritigenin formation with a much lower formation rate than that of liquiritin or isoliquiritin (Fig. 5C and 5D). These results suggested that liquiritigenin and isoliquiritigenin were interchangeable with lower formation rates; however, they did not transform into liquiritin or isoliquiritin (Fig. 5E). The major biotransformation processes were from liquiritin to liquiritigenin and from isoliquiritin to isoliquiritigenin. They had similar formation rates. However, the biotransformation process from liquiritin to isoliquiritigenin and from isoliquiritin to liquiritigenin also occurred with lower formation rates (Fig. 5E).

Figure 5. Formation rates of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin for a 2 h incubation of (A) liquiritin (0.2 mg/mL), (B) isoliquiritin (0.2 mg/mL), (C) liquiritigenin (0.2 mg/mL), and (D) isoliquiritigenin (0.2 mg/mL) with enterocytes from ileal segments. The arrow indicates the transformation process, and the thickness represents the formation rate. Data are expressed as mean ± SD (n = 3).

GR is a widely used herbal medicine for relieving indigestion and promoting detoxification (Gao et al. 2009; Xie et al. 2014). Glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin have been reported as major pharmacological components of GR (Kobayashi et al. 1995; Xie et al. 2014). Liquiritigenin and isoliquiritigenin are particularly known for their chemopreventive, anti-inflammatory, and antioxidative effects in various carcinomas and disease models (Ramalingam et al. 2018).

Previously, Kim et al. (2020) reported that 2 g/kg of GR extract was orally administered to mice without an acute toxicity profile. In the subchronic study, the GR extract (0.05-1 g/kg) was orally administered to mice for four months with insignificant differences in blood pressure, hematological, biochemical, and histological parameters (Kim et al. 2020). For the pharmacological effect, the GR extract (0.1-0.5 g/kg) was orally administered to mice for nine days to investigate the neuroprotective efficacy (Yang et al. 2019). Repeated oral doses of the GR extract (0.075-0.3 g/kg) for seven days showed dose-dependent antioxidative effects in triptolide-induced liver injury in mice (Cao et al. 2017). As a single-dose therapy, the oral administration of the GR extract (1-10 g/kg) attenuates neuronal apoptosis by inhibiting oxidative stress in D-galactose-induced aging rats (Zhou et al. 2017). Based on the above safety and efficacy profile of the orally administered GR extract, we chose 1 g/kg of single oral administration of the GR extract for the pharmacokinetic study.

GR or its extract is considered generally safe for use in food and approved for use in some over-the-counter drugs by the United States Food and Drug Administration (US FDA) (Isbrucker and Burdock 2006). Traditional Chinese Medicine recommends a decoction of 8-15 g of GR for health protection and up to 100 g for diseases. Intake of the GR extract in the range of 150-300 mg/day appear to be commonly used with supplementation, and 1.8 g/day for four weeks was not associated with any toxicity in humans (Omar et al. 2012). Because of the glycyrrhizin-related mineralocorticoid effect, the daily consumption of glycyrrhizin for therapeutic effects should not exceed 500 mg (Omar et al. 2012). The estimated consumption of glycyrrhizin in the GR extract or powder as a food ingredient is 1.62-216 mg/day in the US, which can be used without concern for safety issues (Isbrucker and Burdock 2006). In our case, the dose of 1 g/kg of the GR extract in rats can be converted to a human dose of 164 mg/kg (9.83 g in 60 kg human) according to the simple practice guide for dose conversion from animal to human (Nair and Jacob 2016). The glycyrrhizin content in 9.83 g of the GR extract was calculated to be 196 mg, which was in the acceptable range for human use.

From the pharmacokinetic comparison of pharmacologically active components from the GR extract and individual components, glycyrrhizin, liquiritin, and isoliquiritin had similar pharmacokinetic profiles regardless of the administration formulation. Glycyrrhizin plasma concentrations after single-component administration were higher than those after the GR extract administration at 4-12 h (Fig. 2A). AUC of glycyrrhizin after single-component administration (1653 ± 368.1 ng·h/mL) decreased to 1191 ± 808.8 ng·h/mL after the GR extract administration but was statistically insignificant. This phenomenon was consistent with a previous report (Cantelli-Forti et al. 1994). AUC and renal excretion of glycyrrhizin in rats after oral administration (480 mg/kg) were significantly smaller than those after the GR extract administration (6.3 g/kg), which could be attributed to the interaction between glycyrrhizin and unknown components in the RG extract during intestinal absorption. A similar interaction was also evidenced in human studies (Cantelli-Forti et al. 1994). High-dose administration of the GR extract (6.3 g/kg in rats and 21 g in humans) may produce a higher interaction than that in our case (1 g/kg in rats).

However, when administered in the form of GR extract, liquiritigenin and isoliquiritigenin plasma concentrations increased significantly compared with those of a single component (Fig. 2D and 2E). These results suggested the beneficial interaction of liquiritigenin and isoliquiritigenin with other components by the orally administered GR extract. Because increased liquiritigenin and isoliquiritigenin plasma concentrations distinctively occurred for 6-12 h, we hypothesized that the interaction could occur in the late phase of the intestinal absorption process. Therefore, we investigated glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin permeability from the GR extract, and the equivalent amount of a single component was measured using the rats’ ileum segments. Consistent with the pharmacokinetic results, the P_app of liquiritigenin and isoliquiritigenin from the GR extract increased significantly compared with that of liquiritigenin and isoliquiritigenin individually (Fig. 3). The increased P_app of isoliquiritigenin was exhibited by the coexistence of liquiritin, isoliquiritin, and liquiritigenin, but not vice versa (Fig. 4).

Literature on the biotransformation of isoliquiritigenin and liquiritigenin have been reported previously. Briefly, β-glucosidase hydrolyzes liquiritin in the intestinal flora, and liquiritigenin is absorbed from the jejunum to the colon and conjugated with UDPGA and sulfate or methylated to produce liquiritigenin-glucuronide and sulfoconjugates or methylconjugates in the gut wall enzymes and in the liver (Németh et al. 2003; Zhang et al. 2016; Ma et al. 2018). Additionally, isoliquiritigenin is biosynthetically and structurally interrelated to liquiritigenin (Simmler et al. 2013; Choi et al. 2015). Chalcone isomerase stereochemically converts isoliquiritigenin to liquiritigenin (Jez and Noel 2002). Liquiritigenin and isoliquiritigenin are interchangeable based on pH and temperature, i.e., isoliquiritigenin undergoes rapid cyclization into liquiritigenin at neutral and lower acidic pH, whereas liquiritigenin isomers undergo a reversible reaction to isoliquiritigenin at basic pH (Jez and Noel 2002). These findings suggest that there is a higher presence of isoliquiritigenin at the lower part compared with liquiritigenin with the help of β-glucosidase and chalcone isomerase, and consequently, intestinal absorption of isoliquiritigenin could be increased at the lower part of intestine. Therefore, in this study, the biotransformation process among liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin was investigated using the Ussing chamber system-mounted rat ileum segments based on the increased plasma exposure at 6 h following oral administration (Fig. 2) and the gastrointestinal transit time (Davies and Morris 1993).

The results showed that isoliquiritigenin was formed from isoliquiritin at the highest rate, converted from liquiritigenin at the lower rate, and from liquiritin at the lowest rate (Fig. 5). Therefore, isoliquiritin, liquiritin, and liquiritigenin could be biosources for isoliquiritigenin, consistent with the increase in the P_app of isoliquiritigenin following the coexistence of isoliquiritin, liquiritin, and liquiritigenin. Similarly, isoliquiritin, liquiritin, and isoliquiritigenin could be biosources for liquiritigenin. Furthermore, this process could occur in intestinal enterocytes, and the converted metabolites are absorbed into the plasma. This could be the underlying mechanism for the increased absorption and pharmacokinetic features of liquiritigenin and isoliquiritigenin after the oral administration of the GR extract.

GR contains at least 400 different chemical constituents, including triterpenoid saponins, flavanones, coumarins, and their glycosides (Fujii et al. 2014; Kao et al. 2014). Furthermore, over 25% of the GR components have been identified as active constituents through oral bioavailability, virtual screening, and drug-likeness (Liu et al. 2013). Therefore, the GR extract has been widely investigated for its pharmacological efficacy in human and animal disease models. Additionally, the beneficial interaction of liquiritigenin and isoliquiritigenin from the oral administration of the GR extract via metabolic conversion in intestinal enterocytes and facilitated absorption could provide a rationale for treatment with GR extract rather than with an individual component. However, the beneficial pharmacological efficacy of these interactions needs further investigation since the pharmacological efficacy of the major flavonoids significantly overlapped in their anti-inflammatory, anticancer, cardioprotective, antioxidant, immunoregulatory, and neuroprotective activities (Ramalingam et al. 2018; Qin et al. 2022; Wu et al. 2022; He et al. 2023).

The authors declare that they have no conflict of interest.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2020R1I1A3074384 and NRF-2020R1A5A2017323) and by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No 316017-3), Republic of Korea.