The Disposition of Diammonium Glycyrrhizinate and Glycyrrhetinic Acid in the Isolated Perfused Rat Intestine and Liver

• Abstract
• Introduction
• Materials and Methods
• Instruments, reagents and animals
• Experimental preparation
• Determination of GZ, GA and phenol red in collected samples by HPLC
• Data analysis
• Results
• Discussion
• References

Abstract

The major component of liquorice root extract, glycyrrhizinate (GZ), has been formulated as an injection for the treatment of hepatitis. If given orally, GZ has poor bioavailability and is catalysed to glycyrrhetinic acid (GA) by intestinal bacteria. GA is subsequently responsible for significant side effects. This study was conducted to clarify the relationship between GZ and GA absorption and bioavailability. GZ and GA absorption were investigated using the in situ single pass isolated perfused intestine (IPI). Hepatic disposition was investigated using isolated perfused liver (IPL) and in vivo biliary excretion models. The apparent permeability and absorption rate constants in the IPI for GZ were 0.36 ± 0.31 cm/min and 0.35 ± 0.33 min⁻¹, while those for GA were 5.73 ± 0.11 cm/min and 1.53 ± 0.05 min⁻¹, respectively. The hepatic extraction ratios of unbound GZ and GA in the IPL were 0.22 ± 0.01 and 0.44 ± 0.15, respectively. Seven hours after intra-portal venous injection in vivo, the cumulative biliary excretion ratio for GZ was 96 %. There was negligible biliary excretion of unchanged GA during the same period. It was apparent in all models used that in the absence of intestinal bacteria GZ was not metabolised to GA, or vice versa. Hence, GZ can be absorbed unchanged from the intestine provided it has sufficient time and is protected from intestinal bacteria. This opens up the possibility that the use of pharmaceutical carrier systems or similar formulation approaches may allow effective oral administration of therapeutic levels of GZ without the side effects associated with GA.

Introduction

Liquorice has a long history of use as a medicinal plant, being commonly used for a range of ailments including cough and gastro-intestinal disorders. Within traditional Chinese medicine, liquorice is considered a very important herb. It is often used as a ‘servant herb’ to help other herbs to exert their pharmacological effects, reduce other herbs’ toxicity and improve the taste of the concoction [1]. In addition, liquorice extract or more commonly glycyrrhizinate (GZ), a triterpene saponin and major component of liquorice root, has been used in treatment of a variety of diseases including acute and chronic hepatitis, especially chronic hepatitis C [2].

When GZ is administered orally, a considerable part of the dose can be catalysed to glycyrrhetinic acid (GA) by intestinal bacteria, which is then absorbed [3]. When a large oral dose of GZ (e. g., > 50 mg/kg in rat) is administered, not all of the GZ is metabolised to GA. Unchanged GZ can be absorbed and detected in plasma. However, the bioavailability of GZ is only 4 % following oral administration (200 mg/kg) in rats. In the past, it was believed that GA is the major pharmacologically active component in the body after administration of GZ [4]. However, it has been reported that GZ has a better therapeutic effect on hepatitis and less systemic adverse reactions if administered intravenously. Following i. v. injection of GZ, GA plasma concentrations are negligible. This suggests that unchanged GZ directly exerts the pharmacological effect against hepatitis. Indeed, it has been reported that GZ has a variety of biological activities including protecting the liver, as well as having antiviral and anti-allergy properties [5], [6], [7].

Both GA and GZ may induce adverse effects on the body through inhibition of the type 2 isozyme of 11β-hydroxysteroid dehydrogenase (11-β-HSD2). 11-β-HSD2 is responsible for the conversion of cortisol to its inactive metabolite cortisone in the kidney and other organs. When 11-β-HSD2 is inhibited, cortisol accumulates and binds to the mineralocorticoid receptors which leads to hypertension and hypokalemia [8]. It has been estimated that GA possesses about a 200-fold stronger inhibitory effect on 11-β-HSD2 than does GZ. Therefore, GA is responsible for the major adverse effects on the body after oral administration of liquorice extract or GZ. Due to its significant therapeutic outcome and reduced side effects, intravenous administration of GZ is preferred to oral administration in clinical practice for the treatment of chronic hepatitis [9].

While i. v. treatment with GZ may be more effective and have fewer side effects than oral treatment, i. v. therapy does not lend itself to the treatment of chronic diseases. Understanding the processes that contribute towards the low oral bioavailability of GZ may provide information that will allow GZ to be developed as an oral therapy. Knowledge of intestinal absorption of GZ and GA to date mainly derives from in vivo studies in animals and humans, intestinal absorption being estimated indirectly from plasma concentrations [4].

Both the intestine and the liver may affect oral bioavailability. Neither GZ nor GA has been tested in the isolated perfused intestine (IPI) to determine the mechanisms affecting their intestinal absorption. Whilst hepatic disposition of GZ has been characterised using an isolated perfused liver technique [10], the disposition of GA in the liver is unknown. Therefore, this study was designed to investigate intestinal absorption and hepatic disposition of diammonium GZ and GA.

Materials and Methods

Instruments, reagents and animals

A high-performance liquid chromatography (HPLC) system (Model LC-2010C) was supplied by Shimadzu and consisted of a 4-component gradient pump, an on-line degasification device, an auto-sampler, a UV detector and a column oven. A high-speed centrifuge (Model 1612 – 1) was supplied by Surgical Instrument Factory, Shanghai Medical Apparatus Company. A thermostat (Model 501 super) was supplied by Shanghai Equipment Factory Co. Ltd. A peristaltic pump (Model Lead-1) was purchased from Baoding Lange Peristaltic Pump Co. Ltd. A microinfusion pump (Model WZS-50) was supplied by Medical Instrument Factory, Zhejiang Medical University.

Phenol red (analytical grade) was supplied by Guoyao Chemicals Co. Ltd. NaCl, KCl, MgCl₂, NaH₂PO₄ and NaHCO₃ were all of analytical grade and purchased from Nanjing No. 1 Chemicals Factory. Glucose (analytical grade) was supplied by Shanghai Huixing Biochemicals Co. Ltd. Methanol (HPLC grade) was purchased from Merck. Ammonium acetate (analytical grade) was supplied by Nanjing Chemicals Factory. Diammonium GZ and GA (purity of both GZ and GA > 97 %) were supplied by Jiangsu Zhengdatianqing Medicine Co. Ltd.

Male Sprague-Dawley rats (200 to 250 g) were supplied by the Laboratory Animal Centre of Southeast University (Jiangsu, China), maintained on 12-h light/dark cycle and acclimatised for at least 5 days prior to the experiment. The studies were approved by the Animal Ethics Committee of China Pharmaceutical University.

Experimental preparation

The in situ single pass perfused intestine: The perfused intestine preparation was based on methods described previously [11], [12] with appropriate modifications. The perfusion medium consisted of Krebs-Henseleit (K-H) solution at pH 7.4 supplemented with phenol red (1 g/L). Rats were fasted with free access to water overnight, and then anesthetised with 25 % urethane at a dose of 5 mL/kg by i. p. injection and placed on a 37 °C heating pad during surgery and perfusion. Upon verification of the absence of a pain reflex, a midline abdominal incision of 3 – 4 cm was made. The intestine was cannulated at the beginning of the duodenum and the end of ileum with plastic tubing and then was rinsed with 37 °C saline (2.5 mL/min) for 20 min to clear the contents. Bacterial culture for the outflow liquid was E. coli positive. K-H perfusion medium was then pumped into the intestine at a constant flow rate of 1 mL/min for 10 min. The perfusion medium was then expelled from the intestine by air. Following this, the intestine was perfused (5 mL/min) with K-H medium containing GZ (12.0 μg/mL) or GA (8.16 μg/mL) for 10 min. Bacterial culture for the outflow perfusate at this stage was E. coli negative. Perfusion flow rate was adjusted to 0.2 mL/min and this point of time was set as zero. Perfusate samples (1 mL each) were collected from the ileum tubing at 0, 30, 45, 60, 75 and 90 min. Samples were frozen immediately and stored at −20 °C until assayed. The concentrations of GZ and GA in the perfusate samples were determined by HPLC. The loss of perfusate volume in the intestine cavity (absorption of water by intestine) during perfusion was estimated by determining the increase of phenol red concentration in the perfusate. At the end of the experiment, the length (l) and interior diameter (r) of the perfused intestine were measured without stretching.

Stability investigation of drugs in blank perfusate: Four replicates of perfusate containing diammonium GZ (12.0 μg/mL) or GA (4.08 μg/mL) were prepared and incubated at 37 °C for 3 h. The drug concentrations in perfusate before and after incubation were determined and compared.

Determination of physical adsorption of GZ and GA to intestinal mucus: Approximately 10 cm of intestine were taken between duodenum and ileum from a healthy rat. The mucus surface of the intestine was flipped to face the outside and rinsed with blank perfusate. Then, the intestine was placed into the perfusate containing diammonium GZ (12.0 μg/mL) or GA (4.08 μg/mL) and incubated at 37 °C for 3 h. The drug concentrations in perfusate before and after incubation were determined and compared.

The isolated perfused rat liver (IPL): A Radnoti IPL System (Model 130 003; Radnoti Glass Technology Inc.) was used. The recirculating IPL preparation was based on the methods described previously [13] Briefly, the blank perfusion medium consisted of Krebs-Henseleit solution (pH 7.4) that had been supplemented with glucose (15 mmol/L) and sodium taurocholate (8.4 μmol/L). A part of the blank perfusate was used to prepare GZ perfusate (12.0 μg/mL) and GA perfusate (2.04 μg/mL). Rats were anesthetised and a midline incision of the skin from the pelvis to the ensisternum made. The bile duct was isolated and a cannula (PE-10) inserted. The rat was injected with 0.5 mL of sodium heparin (72 U/mL) through the inferior vena cava and a cannula (inflow) filled with heparinised Krebs-Henseleit buffer introduced into the portal vein and another cannula (outflow) into the inferior vena cava. Approximately 100 mL of preheated oxygenated Krebs-Henseleit buffer (KHB) were infused (15 mL/min) through the liver. The liver was then removed from the body and placed in the organ container in the Radnoti IPL System. The portal vein cannula was connected to a perfusate reservoir containing GZ, GA or no-drug perfusate, which was pumped into the liver at 20 mL/min. The outflow perfusate from the liver was directed into waste for 1 min. Then the outflow cannula was connected (t = 0) to the perfusate reservoir allowing recirculation of perfusate, of which the total volume was adjusted to 220 mL. Perfusate samples were taken from the outflow tubing at 0.5, 20, 30, 45, 60, 80, 100 and 120 min.

Assessment of viability of the isolated perfused liver preparation: Parameters used to assess viability and functionality of the isolated perfused liver preparation included an assessment of liver appearance, and measurement of LDH, AST and ALT perfusate concentrations in the IPL with or without drug using previously described methods [14], [15], [16].

Cumulative biliary excretion rate of GZ and GA in rats in vivo: Eight rats were fasted overnight with free access to water, and then anesthetised with 25 % urethane at a dose of 5 mL/kg by i. p. injection and placed on a 37 °C heating pad. A midline abdominal incision of 2 cm was made and the bile duct was cannulated. The axifugal end of the pyloric vein was ligated to block blood flow from the pyloricum. Diammonium GZ (7.5 mg/kg, n = 4) or GA (5.0 mg/kg, n = 4) solution was injected (at time 0) into the portal vein from the pyloric vein and the latter was then ligated to stop bleeding. Bile was collected from the cannula at 1 h intervals for 7 h. Samples were frozen immediately and stored at -20 °C until assayed.

Determination of GZ, GA and phenol red in collected samples by HPLC

Determination of GZ and GA in biological samples by HPLC has been reported by several authors [17], [18]. In the current study, phenol red in intestinal perfusate was determined simultaneously with GZ and/or GA. Therefore, a modified and improved HPLC method with both isocratic and gradient elution procedure was used. The perfusate samples were vortex-mixed for 30 seconds, and followed by centrifugation at 6300 × g for 10 min. Aliquots (100 μL) of bile were vortex-mixed with 300 μL of methanol for 3 min, followed by centrifugation at 715 × g for 10 min. The supernatants of the bile samples were transferred into another conical glass tube and further centrifuged at 6300 × g for 10 min. Aliquots (20 μL) of each of the resulting supernatants were injected onto the HPLC. A Lichrospher C₈ column (250 mm × 4.6 mm, 5 μm) was used for HPLC assays at a temperature of 30 °C with the flow rate set to 1 mL/min and detection wavelength at 254 nm. The assay utilised 2 mobile phases mixed in different proportions to 100 %. Mobile phase (MP) A was methanol for perfusate samples or acetonitrile for bile samples, whereas MP B was ammonium acetate (0.2 M, pH 6.8) for both matrices. Simultaneous determination of phenol red and diammonium GZ was done using isocratic elution with 60 % of MP A. Determination of diammonium GZ in bile was by isocratic elution with 21 % of MP A, and determination of GA in bile was by isocratic elution with 70 % of MP A. For simultaneous determination of phenol red and GA the following gradient elution was used: 0 – 5 min, A 60 %; 5 – 5.5 min, A 60 – 80 %; 5.5 – 19 min, A 80 %; 19 – 19.5 min, A 80 – 60 %; 19.5 – 24 min, A 60 %. For simultaneous determination of diammonium GZ and GA the following gradient elution was used: 0 – 6.5 min, A 65 %; 6.5 – 7 min, A 65 – 80 %; 7 – 20 min, A 80 %; 20 – 20.5 min, A 80 – 65 %; 25 min, A 65 %. For simultaneous determination of diammonium GZ, GA and phenol red the following gradient elution was used: 0 – 10 min, MP A 60 %; 10 – 10.5 min, A 60 – 80 %; 10.5 – 24 min, A 80 %; 24 – 24.5 min, A 80 – 60 %; 24.5 – 26 min, A 60 %. The HPLC method was validated using standard reference and quality control samples prior to analysis of experimental samples. The linear ranges of standard curves for GZ and GA were 0.5 – 20 μg/mL and 0.1 – 10 μg/mL, with the lower limits of quantitation being 0.5 μg/mL and 0.1 μg/mL, respectively. The intra- and inter- day variation (RSD) of the assay was < 15 %.

Data analysis

Given that water absorption and secretion during the intestinal perfusion may change the volume of perfusate and drug concentrations, the volume of perfusate in the intestine cavity was corrected using the concentration of phenol red in perfusate. The concentrations of drugs in perfusate were calculated after correction of the volume. Apparent permeability (P_app) values and constant of absorption (K_a) were calculated using equations (1) and (2):

Where C_in and C_out are drug concentrations (μg/mL) in the inflow and outflow perfusate, respectively; C_{PR in} and C_{PR out} are concentrations (μg/mL) of phenol red in the inflow and outflow perfusate, respectively; Q is perfusate flow rate (mL/min); r is radius (cm) of the intestine and l is the length (cm) of the perfused intestinal segment.

The hepatic clearance (CL_H) and the elimination half life (t_1/2) of GZ and GA were calculated from the perfusate GZ and GA concentrations using model-independent analysis in WinNolin V4.1 (Pharsight Corporation) by following equations.

where AUCINF is the area under the perfusate concentration versus time curve extrapolated to infinity.

where λ_Z is the first-order rate constant associated with the terminal (log-linear) portion of the perfusate concentration versus time curve.

The hepatic extraction ratio (E_H) for GZ or GA was calculated by equation (5):

Cumulative biliary excretion ratio (CER) was calculated using equation (6):

The differences of activities of ALT, AST and LDH in perfusate between livers perfused with or without drug were analysed using one-way analysis of variance (ANOVA) and compared with post hoc Dunnet t-test.

Results

The percentages of the drug concentration in the perfusate after 3 h incubation at 37 °C to the original drug concentration (before incubation) were 99.3 % ± 3.0 % and 100.3 % ± 0.8 % (mean ± SD, n = 4) for diammonium GZ and GA, respectively. This indicates that diammonium GZ and GA are stable in the perfusate at 37 °C at least for 3 hours.

The percentages of the drug concentrations in the perfusate after incubation with intestine to the original drug concentration were 99.0 % ± 0.5 % and 98.8 % ± 0.2 % (mean ± SD, n = 4) for diammonium GZ and GA, respectively. Therefore, the physical adsorption of both compounds to intestinal mucus is negligible.

The outflow intestinal perfusate concentrations of GZ or GA decreased during perfusion with reduction of the concentration by 26.0 % and 95.0 % after 90 min perfusion, respectively. P_app and K_a of GA were about 16 and 5 times higher than those for GZ, respectively ([Table 1]).

The gross appearance of the perfused livers was normal with a light pink colour. There were no obvious patches or spots on the livers during perfusion. The activities of ALT, AST and LDH in the isolated liver perfusate by the end of perfusion are shown in [Table 2] and are in the normal range [14], [15], [16]. There was no significant difference of the enzyme activities in perfusate between livers perfused with or without drug (p > 0.35). These indicate no adverse effects with the perfusate of GZ and GA on isolated liver and that the viability of each IPL preparation was acceptable.

When recirculating perfusate containing GZ or GA was perfused into the liver through the portal vein, GZ or GA concentration in perfusate reduced continuously during the perfusion ([Table 3]). However, GA was not detected in the perfusate containing GZ and vice versa. The calculated pharmacokinetic parameters are shown in [Table 4].

The average cumulative biliary excretion ratios in rat in vivo for GZ were 85 % and 96 % at 1 and 7 hours after intra-portal vein injection; and the corresponding values for GA were only 0.02 % and 0.11 % ([Fig. 1]).

Discussion

The oral bioavailability of GZ is low in vivo and when GZ is administered in small or medium doses, only GA is detectable in plasma [4]. The results presented above indicate that the main reason for this is that GZ demonstrates low trans-membrane permeability compared to GA. In the IPI, P_app and K_a of GA are about 16 and 5 times higher than those for GZ, respectively. Hence, while GZ can cross the intestinal membrane to a small extent, GA is much more readily absorbed.

However, GZ is detectable in plasma after large dose oral administration [4]. In small and medium oral doses, almost all GZ is catalysed to GA and then absorbed. In large doses, GZ intestinal concentrations exceed the metabolic capacity of intestinal bacterial enzymes, allowing some of the GZ to be absorbed. Our IPI data indicate that in the absence of intestinal bacteria GZ can be absorbed from the intestine as unchanged drug.

The IPL study suggests that both GZ and GA undergo hepatic clearance. However, the hepatic extraction ratio of the unbound drug is relatively low at 0.22 ± 0.01 and 0.44 ± 0.15, respectively. GZ has previously been studied in the IPL where it was found that E_H was in the order of 0.03 to 0.05, depending on the dose administered [10]. Within that report the perfusate flow rate was double what was used in the current study and the perfusate was supplemented with BSA which has a high binding ratio with GZ. Both of these factors would be expected to reduce E_H according to the equation:

E_H = fu·Clint/Q + fu·Clint

Hence, the results from both sets of work are consistent once these factors are taken into account. In either case, it is clear that first pass extraction is not a major factor affecting the bioavailability of GZ. This, alongside the finding that GZ is not metabolised to GA in the IPL, supports the hypothesis that intestinal bacteria are responsible for the metabolism of GZ and hence its low bioavailability.

Study of the hepatic disposition of GZ in the IPL is complicated by the fact that GZ is cholestatic [10]. This impacts the feasibility of collecting sufficient bile to obtain meaningful data in the limited time that the perfused liver remains viable. To overcome this, the in vivo study was conducted where bile was collected for 7 hours. The amount of GA excreted unchanged in the bile over 7 hours was negligible which is consistent with previous reports that GA is mainly metabolised in the liver through conjugation, hydroxylation and oxidation, with metabolites including sulfa-conjugates, mono-glucuronic acid and di-glucuronic acid conjugates, which are excreted into the bile [19]. In contrast, GZ is almost completely excreted unchanged in the bile. This supports the IPL data and previous studies that demonstrate GA could not be detected in systemic venous plasma after GZ was injected into the portal vein [4].

The results presented clarify the fate of GZ and its derivative GA following oral administration. It is apparent that once GZ is absorbed it will not be converted to GA which has a higher capacity to cause side effects. It is also apparent that GZ can be absorbed from the intestine provided it has sufficient time and is protected from intestinal bacteria. This opens up the possibility that the use of pharmaceutical carrier systems or similar formulation approaches may allow effective oral administration of therapeutic levels of GZ without the side effects associated with GA.

References

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Dr Jiping Wang

Sansom Institute

School of Pharmacy and Medical Sciences

City East UniSA

Adelaide

SA 5000

Australia

Phone: +61-8-8302-1874

Fax: +61-8-8302-1087

Email: Jiping.Wang@unisa.edu.au

References

• 1 Chen H. The progress of research on the pharmacology of licorice root.  J Jilin AgricSci Technol College. 2006;  15 15-8
  PubMedSearch in Google Scholar
• 2 Liu J, Bai Y, Tian G. Progress of application of composed glycyrrhizin in treating hepatic diseases.  China Pharm. 2006;  17 1823-30
  PubMedSearch in Google Scholar
• 3 Hattori M, Sakamoto T, Kobashi K, Namba T. Metabolism of glycyrrhizin by human intestinal flora.  Planta Med. 1983;  48 38-42
  PubMedSearch in Google Scholar
• 4 Ploeger B, Mensinga T, Sips A, Seinen W, Meulenbelt J, DeJongh J. The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling.  Drug Metab Rev. 2001;  33 125-47
  CrossrefPubMedSearch in Google Scholar
• 5 Li D, Li D, Li Y. Development in the study of chemical components and pharmacological reaction in Glycyrrhiza uralensis Fisch.  Inform Tradit Chin Med. 1995;  5 31-5
  PubMedSearch in Google Scholar
• 6 Pompei R, Flore O, Marccialis M A, Pani A, Loddo B. Glycyrrhizic acid inhibits virus growth and inactivates virus particles.  Nature. 1979;  281 689-90
  CrossrefPubMedSearch in Google Scholar
• 7 Sun X, Huang L, Liu X. Analysis of the chemical components in Glycyrrhiza uralensis Fisch.  Acta Chin Med Pharmacol. 1994;  5 40-2
  PubMedSearch in Google Scholar
• 8 White P C. 11beta-hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess.  Am J Med Sci. 2001;  322 308-15
  CrossrefPubMedSearch in Google Scholar
• 9 Qin G, Shi G, Song Y, Chen M. Meta-analysis of document on diammonium glycyrrhizinate in treatment of patients with chronic hepatitis B.  Chin J Infect Dis. 2005;  23 333-7
  PubMedSearch in Google Scholar
• 10 Ishida S, Sakiya Y, Taira Z. Disposition of glycyrrhizin in the perfused liver of rats.  Biol Pharm Bull. 1994;  17 960-9
  PubMedSearch in Google Scholar
• 11 Barthe L, Woodley J, Houin G. Gastrointestinal absorption of drugs: methods and studies.  Fundam Clin Pharmacol. 1999;  13 154-68
  PubMedSearch in Google Scholar
• 12 Song N -N, Li Q -S, Liu C -X. Intestinal permeability of metformin using single-pass intestinal perfusion in rats.  World J Gastroenterol. 2006;  12 4064-70
  PubMedSearch in Google Scholar
• 13 Wang J, Nation R L, Evans A M, Cox S, Shackleford D. Metabolism and disposition of the antiviral nucleoside analogue AM365 in the isolated perfused rat liver.  Curr Drug Metab. 2005;  6 487-93
  CrossrefPubMedSearch in Google Scholar
• 14 Bais R, Philcox M. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for lactate dehydrogenase.  Eur J Clin Chem Clin Biochem. 1994;  32 639-55
  PubMedSearch in Google Scholar
• 15 Bergmeyer H U, Horder M. IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase.  J Clin Chem Clin Biochem. 1980;  18 521-34
  PubMedSearch in Google Scholar
• 16 Moss D W. Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 3. Revised IFCC method for aspartate aminotransferase.  Eur J Clin Chem Clin Biochem. 1977;  15 719-20
  PubMedSearch in Google Scholar
• 17 Krahenbuhl S, Hasler F, Krapf R. Analysis and pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in humans and experimental animals.  Steroids. 1994;  59 121-6
  CrossrefPubMedSearch in Google Scholar
• 18 Okamura N, Miyauchi H, Choshi T, Ishizu T, Yagi A. Simultaneous determination of glycyrrhizin metabolites formed by the incubation of glycyrrhizin with rat feces by semi-micro high-performance liquid chromatography.  Biol Pharm Bull. 2003;  26 658-61
  CrossrefPubMedSearch in Google Scholar
• 19 Ishida S, Sakiya Y, Ichikawa T, Awazu S. Pharmacokinetics of glycyrrhetic acid, a major metabolite of glycyrrhizin, in rats.  Chem Pharm Bull. 1989;  37 2509-13
  PubMedSearch in Google Scholar

Dr Jiping Wang

Sansom Institute

School of Pharmacy and Medical Sciences

City East UniSA

Adelaide

SA 5000

Australia

Phone: +61-8-8302-1874

Fax: +61-8-8302-1087

Email: Jiping.Wang@unisa.edu.au