Simultaneous Determination of Glycyrrhizin, Glycyrrhetic Acid and Glycyrrhetic Acid Mono-Glucuronide in Shakuyaku-kanzo-to Incubated with Rat Feces by Semi-micro High-Performance Liquid Chromatography.

Abstract

Shakuyaku-kanzo-to, a traditional Chinese medicine (Kampo medicine), has been used for its antispasmodic and muscle relaxation effects in treatment of colic, lumbago, ischialgia, cramps, tension of cervical muscles and distortion. 1) This prescription consists of licorice root (Glycyrrhizae radix) and peony root (Paeoniae radix). The combined effects of glycyrrhizin (GL) in licorice root and paeoniflorin in peony root act synergistically to block neuromuscular junctions in skeletal muscle. 2) Licorice root is one of the most frequently used crude drugs in Kampo medicine. 3) Its active ingredient is GL, which has been reported to show various pharmacological effects such as steroid-like, 4) anti-inflammatory, 5) and anti-viral 6) actions. On the other hand, GL is known to be hydrolyzed to glycyrrhetic acid (GA), the aglycone of GL, by human intestinal bacteria prior to absorption, 7) although GL is not hydrolyzed to GA, but to glycyrrhetic acid mono-glucuronide (GAMG), by human liver b-D-glucuronidase. 8) GAMG is an intermediate in the hydrolysis of GL to GA. Moreover, when GL is orally administered to rats, it is detected in the plasma, but its bioavailability is lower than that of GA. 9) These results suggest that GL is hydrolyzed by intestinal bacteria and that most of the GA formed is absorbed from the gastrointestinal tract. Therefore, a method for determination of GL and its metabolites is necessary to investigate the metabolism of GL in Kampo medicine. However, there have been no reports regarding simultaneous analysis of GL, GA and GAMG. Furthermore, the limit of detection is important to determine the metabolites present at low levels.The study presented here describes a simple, sensitive and reproducible semi-micro HPLC method for simultaneous determination of these compounds in rat fecal suspension with Shakuyaku-kanzo-to after anaerobic incubation in vitro. MATERIALS AND METHODS MaterialsThe crude drugs were purchased from Tochimototenkaido (Osaka, Japan). GL and GAMG and GA were purchased from Wako Pure Chemical Industries (Wako: Osaka, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. HPLC grade acetonitrile and analytical reagent grade phosphoric acid (85%) were purchased from Wako. Ultrapure distilled water with a resistivity greater than 18 MW was prepared with deionized-distilled water. The organic solvents and other chemical reagents were of analytical reagent grade, and were supplied by Wako.Preparation of Decoction Daily dosages of Shakuyakukanzo-to (licorice root 6 g, peony root 6 g) 3) were prepared as follows: the daily dosage of crude drugs was boiled with 500 ml of water on an electric heater for more than 40 min, halving the original volume. The decoction was filtered through a colander while hot, and after cooling the volume was adjusted to 1000 ml with water. An aliquot of the adjusted decoction was diluted to 10-fold dilution with water, and the diluted decoction was filtered through a Sartorius membrane (Minisart RC 15, pore size: 0.45 mm, Japan Sartorius, Tokyo, Japan) and an...

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products

“…From a consideration of the above, there is clearly a need for a simple analytical method by which to determine 1 and 2 in plant roots, pharmaceutical formulations, confectionery products and plasma samples. Several papers report analytical methods for the determination of 1 and 2 in different matrices including commercial products and biological fluids (Hurst et al, 1983;Ichikawa et al, 1984;Collinge et al, 1985;Hermesse et al, 1986;De Groot et al, 1988;Spinks and Fenwick, 1990;Yamamura et al, 1991;Raggi et al, 1994a;Okamura et al, 2001). Such procedures are mostly based on reversed phase HPLC with UV (Hurst et al, 1983;De Groot et al, 1988;Raggi et al, 1994b;Collinge et al, 1985;Okamura et al, 2001) or fluorometric detection (Yamamura et al, 1991).…”

High-Performance Liquid Chromatographic Determination and Metabolic Study of Sennoside A in Daiokanzoto by Mouse Intestinal Bacteria

“…[15][16][17] To elucidate the role of glycyrrhiza in DKT, the influence of glycyrrhiza and glycyrrhiza constituents on the activity of sennoside A metabolism was investigated using mouse feces. HPLC methods for the determination of sennoside A in rhubarb have been developed.…”

Lipid‐soluble fraction of Shakuyaku‐kanzo‐to inhibits myometrial contraction in pregnant women

“…Glycyrrhizin, a major component of kanzo , is also glycoside that consists of one molecule of glycyrrhetic acid – a pentacyclic triterpenoid derivative – and two molecules of glucuronic acid. Glycyrrhetic acid is a lipid‐soluble compound that is produced by the hydrolysis of glycyrrhizin in intestinal flora . If a lipid‐soluble substance, such as glycyrrhetic acid, is a candidate of active ingredients of shakuyaku‐kanzo‐to , it must be produced rapidly from a water‐soluble precursor, such as glycyrrhizin; otherwise, it cannot elicit its effect within a few minutes because Shakuyaku‐kanzo‐to is known to present its inhibitory effect on skeletal muscle cramps and smooth muscle contractions within a few minutes of oral administration.…”