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...
