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Journal of the American College of Nutrition, Vol. 26, No. 3, 209-218 (2007)
Published by the American College of Nutrition

Clinical Safety of Licorice Flavonoid Oil (LFO) and Pharmacokinetics of Glabridin in Healthy Humans

Fumiki Aoki, MSc, Kaku Nakagawa, MSc, Mitsuaki Kitano, PhD, Hideyuki Ikematsu, MD, Kenjirou Nakamura, MD, Shinichi Yokota, PhD, Yuji Tominaga, MSc, Naoki Arai, MSc and Tatsumasa Mae, PhD

Functional Food Ingredients Division, (F.A., S.Y., Y.T., N.A.)
Life Science Research Laboratories (M.K.), Life Science RD Center, Kaneka Corporation, Hyogo
Functional Food Ingredients Division, Kaneka Corporation, Osaka (K.N., T.M.)
Haradoi Hospital (H.I.)
Tenjin Sogo Clinic (K.N.), Fukuoka, JAPAN

Address reprint requests to: Fumiki Aoki, MSc, Functional Food Ingredients Division, Kaneka Corporation, 1–8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676-8688, JAPAN. E-mail: Fumiki.Aoki{at}kn.kaneka.co.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Objective: Licorice flavonoids have various physiological activities such as abdominal fat-lowering, hypoglycemic and antioxidant effects. Licorice flavonoid oil (LFO: Kaneka Glavonoid Rich OilTM) is a new dietary ingredient containing licorice flavonoids dissolved in medium-chain triglycerides (MCT). Glabridin is one of the bioactive flavonoids included specifically in licorice Glycyrrhiza glabra L. and is the most abundant flavonoid in LFO. In this study, we assessed the safety of LFO in healthy humans and determined the plasma concentration profile of glabridin as a marker compound.

Methods: A single-dose and two multiple-dose studies at low (300 mg), moderate (600 mg) and high (1200 mg) daily doses of LFO were carried out using a placebo-controlled single-blind design. In each study the safety of LFO and the pharmacokinetics of glabridin were assessed.

Results: Pharmacokinetic analysis in the single-dose study with healthy male subjects (n = 5) showed that glabridin was absorbed and reached the maximum concentration (Cmax) after approximately 4 h (Tmax), and then eliminated relatively slowly in a single phase with a T1/2 of approximately 10 h at all doses. The Cmax and AUC0–24 h increased almost linearly with dose. The multiple-dose studies with healthy male and female subjects for 1 week and 4 weeks suggested that plasma glabridin reached steady state levels within 2 weeks with a single daily administration of 300 to 1200 mg/day LFO. In these human studies at three dose levels, there were no clinically noteworthy changes in hematological or related biochemical parameters. All clinical events observed were mild and considered to be unrelated to LFO administration even after repeated administration for 4 weeks.

Conclusion: These studies demonstrated that LFO is safe when administered once daily up to 1200 mg/day. This is the first report on the safety of licorice flavonoids in an oil preparation and the first report on the pharmacokinetics of glabridin in human subjects.

Key words: licorice, glabridin, safety, pharmacokinetics, flavonoid, human

Abbreviations: ADME = absorption, distribution, netabolism and excretion • A/G ratio = albumin/globulin ratio • ALP = alkaline phosphatase • ALT = alanine aminotransferase • APTT = activated partial thromboplastin time • AST = aspartate aminotransferase • AUC = area under the curve • BUN = blood urea nitrogen • CL/F = total clearance • Cmax = the maximum concentration in plasma • LDH = lactate dehydrogenase • LFO = licorice flavonoid oil • MCT = medium-chain triglycerides • NOAEL = no-observed-adverse-effect-level • PT = prothrombin time • T1/2 = elimination half-life • Tmax = time required to reach maximum concentration • {gamma}-GTP = {gamma}-glutamyltranspeptidase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Licorice, the root of the leguminous Glycyrrhiza plant species, has been consumed for over 4000 years since ancient Egyptian times and is one of the most frequently employed botanicals in foods and traditional medicines [1]. Licorice and its aqueous extract are also used as flavoring and sweetening agents in food products because they contain glycyrrhizin, which is 100–200 times sweeter than sugar, as a major component. It is also known that licorice contains bioactive polyphenols. Glabridin is a major polyphenolic flavonoid specific for licorice Glycyrrhiza glabra L. and has antioxidant activity [2–7], anti-Helicobacter pylori activity [8], estrogen-like activity [9–11] and antinephritic and radical scavenging activities [12]. In addition, glabridin inhibits serotonin re-uptake [13], melanogenesis [14], inflammation [14] and cytochrome P450 3A4, 2B6, and 2C9 [15].

We prepared licorice flavonoid oil (LFO) by extracting licorice ethanolic extract with medium-chain triglycerides (MCT) and previously reported that licorice flavonoids exhibited abdominal fat-lowering and hypoglycemic effects in obese diabetic KK-Ay mice [16]. Based on these finding, licorice flavonoids are expected to lower abdominal fat and blood glucose in diabetic or obese humans. Therefore we are now developing LFO as a dietary ingredient or a dietary supplement, and the product "Kaneka Glavonoid Rich OilTM" which has a standardized glabridin concentration of 1% in oil, will be on the market in the near future. It is important to assess the safety of potential products when developing dietary ingredients or supplements. We have already conducted mutagenic evaluation such as in vitro Ames test and chromosome aberration test, and in vivo micronucleus test. These studies demonstrated that LFO had no mutagenic potential. In addition, we have also conducted multiple-dose study in rats for 90 days. Based on this study, no-observed-adverse-effect-level (NOAEL) of LFO was 1200 mg/kg/day in rats. In the present study, we assessed the safety of LFO product (Kaneka Glavonoid Rich Oil TM) when administered orally to humans.

It is also important to determine the pharmacokinetics of bioactive compounds in assessing their safety and effects. Although LFO contains a variety of hydrophobic polyphenolic flavonoids from licorice G. glabra L., glabridin is the most abundant flavonoid among all licorice flavonoids [7,17–19]. Glabridin can thus be considered an appropriate marker compound for the study on the pharmacokinetics of licorice flavonoids. To date, no studies have been reported on the pharmacokinetics of glabridin in humans. Therefore, we recently developed and reported the validated analytical method for the determination of glabridin in human plasma using solid-phase extraction (SPE) and LC-MS/MS [20]. In this study, we also used plasma glabridin as a marker compound in assessing the safety of LFO in humans.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 REFERENCES
 
Subjects
The subjects were 117 healthy Japanese volunteers (66 men and 51 women) aged 20–52 y. Criteria for subject selection were: age 20–60 y; body weight >50 kg for men and >40 kg for women; no intake of pharmaceutical products or dietary supplements including licorice ingredients; no history of serious disease such as diabetes, hepatic disease, renal disease, or cardiac disease; no allergy to licorice ingredients; no history of alcoholism; and nonpregnancy. In addition, a subject who was judged to be ineligible for the study by the attending physician was excluded. All subjects kept a diary to record symptoms and intake of test samples. The criteria for exclusion of subjects during the study were as follows: those with low intake rate of the test samples; those who did not visit the clinic and thus did not complete the study, for personal reasons unrelated to the study such as an extended business trips, forgetting to visit clinic, contingency, and hospitalizations obviously unrelated to side effects of the test samples; those doing other acts of noncompliance which might compromise the credibility of the study result, such as many blanks in their diary; and those with other reasons considered a reasonable bases for exclusion from the study. The subjects were instructed to maintain a normal lifestyle, including eating and exercise. This study was conducted in accordance with the Helsinki Declaration after approval by the Ethics Committee of Haradoi Hospital and was fully explained to all subjects, who gave written, informed consent.

Test Sample Preparations
Licorice flavonoid oil (LFO) is a new dietary ingredient in the form of an edible oil with the brand name Kaneka Glavonoid Rich OilTM. LFO was produced as reported Nakagawa et al. [16] with slight modification of glabridin concentration, which was reduced from 1.2% to 1% by diluting with MCT in order to produce standardized products. LFO includes licorice (Glycyrrhiza glabra L.) hydrophobic polyphenols and is produced by further extraction of licorice ethanolic extract with medium-chain triglycerides (MCT; C8:C10 = 99:1) and has a standardized concentration of 1% glabridin in oil. LFO contains MCT at about 90% (w/w), and a solid fraction derived from licorice ethanolic extract at about 10% (w/w) in the total weight. The polyphenol content in LFO is approximately 8% as determined by the Folin-Ciocalteau method using glabridin as a standard compound. A large proportion of the polyphenols in LFO are polyphenolic flavonoids. The contents of licorice flavonoids other than glabridin in LFO were about 0.2% glabrene, 0.2% glabrol, and 0.1% 4'-O-methylglabridin. The glycyrrhizin content in LFO is less than 0.005%. The concentrations of pesticides in LFO were well below the values established by Ministry of Health, Labour and Welfare of Japan. For oral administration to the subjects, active and placebo capsules were prepared using non-transparent brown-colored soft capsules made of gelatin. The active capsules were filled with 300 mg LFO and 33 mg beeswax, and the placebo capsules with 300 mg MCT, instead of LFO. The MCT was the same oil used in producing LFO. The capsules were produced in accordance with GMP (Good Manufacturing Practice) by Cardinal Health Japan 408 KK corp. (Tokyo, Japan). The capsules are stable at 25°C for at least 1 year.

Study Design
A single-dose study and two multiple-dose studies were carried out using a placebo-controlled single-blind design. The studies were conducted step by step with an increase in the dosing period while confirming the safety of each dosing design. The dosing regimen was decided based on the our in-house study (90 day multiple-dose study in rat), exhibiting NOAEL (No Observed Adverse Effect Level) of 1200 mg/kg/day in rat.

In study 1, the single-dose study, the male subjects were randomly assigned to three groups (n = 5) and received single oral administration of 300, 600, or 1200 mg LFO after breakfast. Blood samples for measurement of plasma glabridin were collected at 0 (pre-dose), 2, 4, 6, 8, and 24 h post-dose. Blood and urine samples for safety laboratory tests were collected pre-dose, 24 h post-dose, and 7 days after dosing.

In study 2, the 1-wk multiple-dose study, the male and female subjects were randomly assigned to four groups and received once daily oral administration of 300 (n = 5/sex), 600 (n = 5 /sex), or 1200 mg (n = 5/sex) LFO, or placebo (n = 6/sex) after breakfast for 1 week. Fasting blood samples for measurement of plasma glabridin were collected at pre-dose and post-dose (4 and 24 h) on the first (day 1) and last (day 7) days during the study. Blood and urine samples for laboratory safety tests were collected pre-dose on the first (day 1) day, 24 h post-dose on the last (day 7) day during the study, and a week after the dosing period.

In study 3, the 4-wk multiple-dose study, the male and female subjects were randomly assigned to four groups and received once daily oral administration of 300 (n = 7/sex), 600 (n = 7 /sex), or 1200 mg (n = 7/sex) LFO, or placebo (n = 9/sex) after breakfast for 4 weeks. Fasting blood samples for measurement of plasma glabridin were collected before consuming the capsules pre-dose, 2 and 4 weeks after initiation of treatment. Blood and urine samples for laboratory safety tests were collected at pre-dose, 2 and 4 weeks after initiation of treatment, and 2 weeks after the dosing period.

In all studies, all subjects were administered a combination of four LFO and/or placebo capsules daily according to the study design. On each sampling day of all studies, subjects were checked for any clinical events by a physician and evaluated for body weight, heart rate, blood pressure, electrocardiogram, and body temperature.

Plasma Glabridin Measurement
Plasma glabridin was measured by solid-phase extraction and LC-MS/MS according to Aoki et al [20]. The lower limit of quantitation of glabridin was 0.1 ng/mL in plasma. In study 1, plasma glabridin concentration-time data were analyzed with a non-compartmental method using WINNONLIN software (version 1.1; SCI, Morrisville, NC). The parameters measured were the maximum concentration in plasma (Cmax), time required to reach the maximum concentration (Tmax: the time at observed Cmax), area under the curve (AUC0–24h, AUC0-{infty}), total clearance (Cl/F: where F denotes bioavailability) and elimination half-life (T1/2). The AUC for the entire 24 h blood-sampling period of study 1 (AUC0–24h) was calculated by the trapezoidal method. The T1/2 was calculated as ln 2/z, where z was the terminal elimination rate constant. In addition, to simulate the change of plasma glabridin during the 1-week multiple-dose, we separately conducted a one-compartmental model analysis using the mean plasma glabridin profiles in each group. In studies 2 and 3, plasma glabridin concentration was measured to assess accumulation of glabridin after repeated multiple administrations.

Safety Assessment
In each study, laboratory safety tests and monitoring of clinical events were performed. The laboratory safety tests included the following hematological and relevant biochemical parameters: white blood cell count, differential white blood count, red blood cell count, hemoglobin, hematocrit, and platelet count, activated partial thromboplastin time (APTT), and prothrombin time (PT) in whole blood; total protein, albumin, albumin/globulin ratio (A/G ratio), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), {gamma}-glutamyltranspeptidase ({gamma}-GTP), total bilirubin, creatinine, blood urea nitrogen (BUN), uric acid, total cholesterol, HDL cholesterol, LDL cholesterol, VLDL cholesterol, ketone body fraction, RLP cholesterol, lipoprotein fractions (HDL, LDL and VLDL), triglycerides, free fatty acid, phospholipids, blood glucose, hemoglobin A1C, insulin, sodium, potassium, and chloride in serum; and pH, protein, glucose, urobilinogen, occult blood, and bilirubin in urine. Qualitative analysis was used for urinary parameters other than pH.

As clinical events, all symptoms during the studies were recorded and assessed for relationship to LFO administration by the study physician using a four-point scale ("Unrelated": Does not follow a reasonable temporal sequence after the investigational food is administrated, Can likely be explained by the subject's underlying clinical state or other factors, There is another obvious cause of the clinical event; "Possible": Does not follow a reasonable temporal sequence after the investigational food is administrated, Can likely be explained by the subject's underlying clinical state or other factors; "Probable": Follows a reasonable temporal sequence after the investigational food is administrated, Can be equally explained by the subject's underlying clinical state or other factors as administration of the investigational food; "Definite": Follows a reasonable temporal sequence after the investigational food is administrated, Can be most likely to be explained by administration of the investigational food than the subject's underlying clinical state or other factors.)

Statistical Analysis
Values are presented as the mean ± the standard error of the mean (SEM). The paired t-test was used to analyze changes before and after dosing in each study. One-way ANOVA was used to compare pre-dose demographic data among study groups. The 2 by 2 c2 (chi-square) test was used for examining changes among study groups in clinical events and urinary occult blood. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
REFERENCES
 
Subjects
Table 1 summarizes the demographic data of the subjects in studies 1–3. In studies 1 and 2, all subjects completed the study. In study 3, all subjects completed the study except two female subjects in the placebo group. One subject did not visit the clinic when required and did not participate in the study 3 for personal reasons unrelated to the study. The other was found to have low hemoglobin before the study 3. In all studies, no significant differences were observed in age, weight, or height among placebo and treatment groups.


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  		Table 1. Demographic Data of the Subjects before Experiment in the Studies 1–3a,b

 
Plasma Glabridin Concentration
Fig. 1 illustrates the plasma concentration-time profiles of glabridin after LFO administration in the single-dose study (study 1). The pharmacokinetic parameters of glabridin after a single administration are summarized in Table 2. Following the dose, glabridin was absorbed and reached Cmax with a mean Tmax of 3.2–6.0 h (4.27 h; overall mean value), and was eliminated linearly with a mean T1/2 of 8.3–13.9 h (10.7 h; overall mean value). The total clearance values were unchanged or slightly decreased with different dose levels. In addition, AUC0–24h and Cmax increased almost linearly with doses (Fig. 2).


Figure 1
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  		Fig. 1. Plasma glabridin (ng/mL) in healthy male subjects (n = 5) after a single oral administration of 300, 600 or 1200 mg licorice flavonoid oil (LFO). Symbols are expressed as mean ± SEM.

 

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  		Table 2. Pharmacokinetic Parameters of Glabridin in Healthy Male Subjects after a Single Oral Administration of Licorice Flavonoid Oil (LFO)a,b

 

Figure 2
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  		Fig. 2. Relationship between dose of LFO and Cmax (upper panel) or AUC0–24h (lower panel) in healthy male subjects after a single administration of 300, 600 or 1200 mg licorice flavonoid oil (LFO). In each figure, linear regression lines and correlation coefficients (r) are described. AUC = area under the curve, Cmax = the maximum concentration in plasma.

 
In the 1-wk multiple-dose study (study 2), plasma glabridin was measured pre-dose, 4 h post-dose which was almost equivalent to the allover mean value of the Tmax observed in the study 1, and 24 h post-dose which was a trough time due to 24 h interval of daily administration. The mean plasma glabridin level at each time point was increased almost linearly (r > 0.96; Table 3) with the doses of LFO, and was higher on day 7 than on day 1. Each plasma glabridin level in the trough time was 1/7–1/4 of the concentration at 4 h on both day 1 and day 7. The accumulation ratios of glabridin, calculated from the mean plasma glabridin levels at 4 h and trough time in day 1 and day 7, were 1.04–1.67 (1.35; overall mean value; Table 3), indicating the slight elevation of plasma glabridin due to the multiple dose. In the 4-wk multiple-dose study (study 3), plasma glabridin level in the trough time increased almost linearly (r > 0.99; Table 3) with the doses of LFO, and the values observed at 2 and 4 weeks were the similar level at each dose of LFO, suggesting that steady-state was maintained (Table 3). There was no sex-related difference in plasma glabridin observed in the studies 2 and 3 (data not shown).


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  		Table 3. Plasma Glabridin (ng/mL) in Healthy Subjects after Multiple-Dose Oral Administration of Licorice Flavonoid Oil (LFO) for 1 Week (Study 2) and 4 Weeks (Study 3)a,b,c

 
Safety Assessment
Throughout the single-dose and two multiple-dose studies, no clinically noteworthy abnormalities were observed. In this report, therefore, we describe the results on the safety of LFO in the 4-week multiple-dose study (study 3), since the study 3 had the longest administration period of these three studies.

In the 4-wk multiple-dose study (study 3), no significant changes were observed in heart rate, blood pressure or the electrocardiogram. Compared with pre-dose value (61.6 ± 3.2 Kg), body weight was slightly but significantly (P < 0.05) higher at 2 weeks (62.1 ± 3.2 Kg) and 4 weeks (62.2 ± 3.2 Kg) after the initiation of treatment, and at 2 weeks after the dosing period (62.2 ± 3.2 Kg) in the 600 mg/day group. Body temperature was significantly (P < 0.05) lower than the pre-dose value (36.30 ± 0.07°C) at 4 weeks after the initiation of treatment (36.04 ± 0.10°C) and at 2 weeks after the dosing period (35.96 ± 0.18 °C) in the placebo group. However, these significant changes were small, and not considered dependent on the dose of LFO, indicating that there were no toxicological implications.

All of the hematological and blood biochemistry parameters remained within normal range in the study 3. Tables 4 and 5 present the hematological and blood biochemistry parameters related to safety assessment at pre-dose and 4 weeks after the initiation of treatment. Blood glucose (P < 0.01) and sodium (P < 0.05) in the placebo group were significantly decreased from the pre-dose values, and chloride in the 300 mg/day group was significantly (P < 0.05) decreased from the pre-dose value. However, the minor concentration changes observed were well within normal range and therefore were not considered clinically significant.


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  		Table 4. Hematological Parameters in Healthy Subjects before and after Multiple-Dose Oral Administration of Licorice Flavonoid Oil (LFO) for 4 Weeksa

 

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  		Table 5. Blood Biochemistry Parameters in Healthy Subjects before and after Multiple-Dose Oral Administration of Licorice Flavonoid Oil (LFO) for 4 weeksa

 
Some parameters of the hematological and blood biochemistry were significantly (P < 0.05) changed from the pre-dose values at 2 weeks after the initiation of treatment and at 2 weeks after the dosing period in any groups in the study 3. At 2 weeks after the initiation of treatment, the values of platelet count in the 1200 mg/day group, AST in the placebo and 1200 mg/day groups, and ALT in the 1200 mg/day group were significantly (P < 0.05) changed from their pre-dose values, and A/G ratio in the 600 and 1200 mg/day groups was significantly (P < 0.05) increased from the pre-dose value. However, their statistically significance were not considered clinically significant since these changes were minor and remained within normal ranges. In addition as shown in Tables 4 and 5, these changes were not observed at 4 weeks after the initiation of treatment, indicating there was no associated time-dependency. Other significantly changed parameters did not exhibit dose-dependent changes (data not shown).

In addition, the values remained within their normal range for differential white blood count, free fatty acid, phospholipids, HDL cholesterol, LDL cholesterol, VLDL cholesterol, ketone body fractions, RLP cholesterol, lipoprotein fractions (HDL, LDL and VLDL), hemoglobin A1C, insulin, and urine pH in the study 3 (data not shown). Moreover, glucose, protein, and bilirubin were not detected in urine of any subjects in the study 3. Urobilinogen was not detected for any subjects except one subject in the 1200 mg/day group, for which urobilinogen was detected at the pre-dose, 4 weeks after the initiation of treatment, and 2 weeks after the dosing period. Occult blood in urine was detected for some subjects in all groups throughout the study. However, no relationship between LFO administration and occult blood was observed by the evaluation with the 2 by 2 c2 test between the placebo and each group administrated LFO (data not shown).

All clinical events observed in the study 3 are summarized in Table 6. All clinical events were mild and were not categorized as "Probable" nor "Definite". The total number of the observed events was 13 in the placebo group, 10 in the 300 mg/day group, 9 in the 600 mg/day group, and 17 in the 1200 mg/day group. The number of events categorized as "Possible" was 6 in the placebo group, 6 in the 300 mg/day group, 3 in the 600 mg/day group, and 12 in the 1200 mg/day group. No significant difference was observed between the placebo and each group administered LFO when each of the clinical events and total clinical events were analyzed using the 2 by 2 c2 test.


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  		Table 6. Clinical Events of Repeated Oral Administration of Licorice Flavonoid Oil (LFO) for 4 Weeksa

 

   DISCUSSION
 
Licorice flavonoid oil (LFO: Kaneka Glavonoid Rich OilTM) is the hydrophobic polyphenol-rich fraction of licorice ethanol extract, which is dissolved in MCT. The solid fraction derived from licorice ethanolic extract in LFO accounts for approximately 10% (w/w), of which a large portion is polyphenol (approximately 8% (w/w) in LFO). Polyphenolic flavonoids are considered the major polyphenols in LFO. Glabridin is the most abundant flavonoid among all licorice flavonoids [7,17–19] and is the most typical compound in LFO. Glabridin is standardized at 1% (w/w) in LFO, corresponding to approximately 12.5% polyphenol in LFO. Glabridin is thus a major marker compound among such flavonoids. Glycyrrhizin is a common constituent in licorice root and exhibits adverse effects such as hypertension, arrhythmia, hypermineralocorticoidism, and pseudoaldosteronism via inhibition of 11-hydroxy-steroid dehydrogenase [21]. However, glycyrrhizin is a minor component in LFO, at less than 0.005% in LFO, and the dose of glycyrrhizin in this study was consequently less than 0.06 mg/day, which was approximately 2000 times less than the safety limit (2 mg/kg/day) reported by Van Gelderen et al. [22]. The adverse effect of glycyrrhizin in LFO is thus considered negligible.

In our previous study, we have showed that LFO exhibits abdominal fat-lowering and hypoglycemic effects in obese diabetic KK-Ay mice [16]. The objective of this study was to evaluate the safety of LFO (Kaneka Glavonoid Rich OilTM).

There are several reports that the administration of powdered licorice ethanolic extract in humans exhibits beneficial effects such as inhibition of LDL oxidation [6,23]. In those studies, no adverse effect is reported at the dose of 100 mg licorice ethanol extract, which would be equivalent to 1000 mg LFO. However LFO does not have exactly the same composition as the licorice ethanolic extract, and the absorbed amount of licorice flavonoids could be expected to be higher when dissolved in MCT than that in powdered ethanol extract, since it seems that the absorption of hydrophobic compounds is generally improved when they are administered as solutions in oil. Hence, it is important to evaluate the safety as well as efficacy of LFO as a new dietary ingredient for dietary supplements.

On the basis of the hematological and relevant biochemical results in the 4-wk multiple-dose study, LFO at up to 1200 mg/day appeared to be safe and well tolerated by the study subjects for at least 4 weeks. No clinically noteworthy changes were observed. Although some statistically significant changes were observed, most of those were neither time- nor dose-related. All clinical events observed in all groups including the placebo group were mild and not considered related to the administration of the placebo or LFO. In addition, the number of observed clinical events did not differ significantly between the placebo and LFO-treated groups. Stratified analysis by sex revealed no noteworthy sex-related differences (data not shown). We therefore conclude that administration of LFO is safe when administered up to 1200 mg/day for 4 weeks.

We measured glabridin as a marker compound for LFO in this study, since it is the most abundant flavonoid among all licorice flavonoids [7,17–19] and is the major representative flavonoid in LFO. Our findings comprise the first report on the pharmacokinetics of glabridin. First, we conducted a single-dose study in male subjects with 300, 600 and 1200 mg LFO, which contained 3, 6 and 12 mg of glabridin, respectively. Glabridin was absorbed and reached to a Cmax with a Tmax of approximately 4 h and eliminated relatively slowly in a single phase with a T1/2 of approximately 10 h in all dose groups. The values of AUC0–24h and Cmax increased almost linearly, indicating that glabridin exhibited linear pharmacokinetics over the dose range of 300 to 1200 mg/man of LFO.

Next, we separately analyzed the plasma glabridin profiles by the one-compartmental model, since these were well fitted with the pharmacokinetic model. Based on the pharmacokinetic parameters calculated by the one-compartmental model analysis and the single daily dose interval, plasma glabridin was estimated to reach a steady-state level within 1 week and the accumulation ratio was 1.1 to 1.2, indicating minimal accumulation of glabridin (Fig. 3). We then conducted the 1-wk multiple-dose study of LFO in male and female subjects and observed accumulation ratios ranging from 1.04 to 1.67 (allover mean 1.35), which were calculated from the plasma glabridin levels 4 and 24 h post-dose (trough time). Taking into account the variation in plasma glabridin levels among individuals, this findings was considered roughly consistent with the predicted values (1.1–1.2) from the single-dose study. The finding suggested that the change of ADME (Absorption, Distribution, Metabolism and Excretion) of glabridin might be minor during the 1-wk multiple-dose of 300–1200 mg/day LFO.


Figure 3
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  		Fig. 3. Simulation of plasma glabridin concentrations after once daily oral administration of 300, 600 or 1200 mg licorice flavonoid oil (LFO) for 7 days. The simulation was based on the one-compartmental model analysis of plasma glabridin in the single-dose study of 300, 600 and 1200 mg LFO.

 
Finally, we conducted the 4-wk multiple-dose study of LFO in male and female subjects, and observed accumulation ratios of 0.96 to 1.06, calculated from the trough plasma glabridin levels at 2 and 4 weeks, indicating that plasma glabridin levels had reached the steady-state levels within 2 weeks with single daily doses of 300, 600, and 1200 mg of LFO.

Plasma glabridin levels in the 1-wk and 4-wk multiple-dose studies of LFO also increased almost linearly with the dose levels, being consistent with the single-dose study, in which AUC0–24h and Cmax increased almost linearly with the dose levels. These results also suggested that glabridin exhibited linear pharmacokinetics over the dose range of 300 to 1200 mg/man of LFO. However, it should be noted that linearity was evaluated based on the studies using only three dose levels of LFO with three different groups of subjects. In addition, because the glabridin dose not necessarily exhibit linear pharmacokinetics when LFO is administered at >1200 mg to humans, more studies may be needed on the pharmacokinetics of glabridin, especially using higher doses of LFO in order to determine the effects and safety of such doses of LFO.

Based on the one-compartmental model analysis of the single-dose study data, the simulated trough concentrations of plasma glabridin at the steady-state after administration of 300, 600 and 1200 mg/day LFO were 0.14, 0.25 and 0.57 ng/mL, respectively. And the values of %RSD in 300, 600, 1200 mg LFO groups, which were the mean values of all time points in single-dose study, were 68%, 43%, and 50%. On the other hand, the observed trough concentrations of plasma glabridin after single daily administration of LFO for 1 week in the 1-wk multiple-dose study (Table 3) with 300, 600 and 1200 mg/day LFO were 0.23 ± 0.05 (%RSD, 68%), 0.60 ± 0.16 (%RSD, 86%) and 0.87 ± 0.13 (%RSD, 48%) ng/mL, respectively. And the observed trough concentrations after single daily administration of LFO for 4 weeks in the 4-wk multiple-dose study (Table 3), which were considered the steady levels for each dosing design of 300, 600 and 1200 mg/day LFO, were 0.30 ± 0.04 (%RSD, 51%), 0.61 ± 0.11 (%RSD, 65%) and 1.75 ± 0.54 (%RSD, 116%) ng/mL, respectively. Taking account of the %RSD values of the plasma glabridin in each study, the elevations of plasma glabridin in 1-wk and 4-wk multiple-dose studies were considered within the ranges of variation of the simulated values calculated by using the data of the single-dose study. In addition, the data from these three studies were not necessarily comparable, since the values of plasma glabridin were obtained in each separate study with different subjects. On the other hand, our finding that the accumulation rate between 2 and 4 weeks was approximately 1 in the 4-wk multiple-dose study suggested that the ADME of glabridin had reached the steady-state within 2 weeks in our dosing design. We therefore conclude that accumulation of glabridin dose not continue after 2 weeks of repeated administration in our daily dosing design. However, further studies on the ADME of glabridin might be needed.

Our studies showed that mean trough concentrations of plasma glabridin at the steady-state was 0.30 to 1.75 ng/mL with single daily administration of 300, 600, and 1200 mg LFO, corresponding to 3, 6, and 12 mg glabridin, respectively. Given the observed mean Cmax in the single-dose study (1.12 to 2.65 ng/mL), the Cmax of plasma glabridin at the steady-state was 1.42 to 4.4 ng/mL in our dosing design, which corresponded to approximately 4 to 13 nM.

An in vitro study indicated that glabridin inactivated human cytochrome P450s 3A4, 2B6, and 2C9, and that the concentrations required to obtain 50% inhibition were reported 7 µM for 3A4, 12 µM for 2B6, and about 100 µM for 2C9 [15]. These concentrations were at least 700 times higher than our observed steady-state level of plasma glabridin. Therefore, inactivation of cytochrome P450s by glabridin should be minimal when daily doses of at least 300 to 1200 mg of LFO would be administered for 4 weeks. However, LFO contains not only glabridin but other flavonoids as well, leaving open the possibility that these other flavonoids may additively or synergistically inactivate the activity of cytochrome P450s. Therefore, further studies on the effects of all flavonoids of LFO on cytochrome P450s may be required. It is also reported that glabridin binds to human estrogen receptor with about the same affinity as genistein, the best known phytoestorogen, 104 times lower than estradiol [9]. The in vitro estrogen-like activity of glabridin is reported to be in the range of 3 to 300 nM in vascular smooth muscle cells, 30 to 3000 nM in endothelial cells [11], 0.1 to 10 µM in breast cancer cells [9] and 3 µM in bone cells [10]. Our observed steady-state level of plasma glabridin suggested less estrogen-like activity in vascular smooth muscle cells. Consequently, we conclude that the effects of estrogenic activity of glabridin were minimal in our study design. Moreover, several in vitro studies suggest various effects of glabridin, such as inhibition of serotonin reuptake [13], antinephritic and radical scavenging activities [12], antioxidative activity [2–5,7] and inhibition of melanogenesis and inflammation [14]. However, our observed steady-state level of glabridin was lower than the effective concentrations reported in those in vitro studies.

In conclusion, we demonstrated the safety of licorice hydrophobic flavonoids (LFO; Kaneka Glavonoid Rich OilTM) and the nearly linear pharmacokinetics of glabridin, when 300 to 1200 mg LFO was administered once daily to healthy subjects for 4 weeks. We previously reported the abdominal fat-lowering and hypoglycemic effects of licorice hydrophobic flavonoids in obese diabetic KK-Ay mice [16], though their effects in the human body and effective doses have not been elucidated. Further studies would be needed to determine these parameters. Our findings will facilitate studies not only of the abdominal fat-lowering and hypoglycemic effects but various biological and pharmacodynamic effects of licorice hydrophilic flavonoids (LFO) and glabridin in human subjects.

We would like to express our gratitude for laboratory support to Ms. Kazue Matsuzaki and Mr. Akiyoshi Tanaka, Sumika Chemical Analysis Service Ltd., for clinical support to Tetsuro Yamamoto Ph.D. and Mr. Muneaki Iizuka, Total Technological Consultant Co., Ltd., and for manuscript review to Robert J. Barry Ph.D., Kaneka Nutrients L.P.

Received January 22, 2006. Accepted August 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 

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