Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4284-4288, April 1996 Biochemistry

Potentiation of the bioavailability of by an extract ofRadix puerariae (antidipsotropic activity/dose-response curve) WING-MING KEUNG, OSCAR LAzo, LISA KUNZE, AND BERT L. VALLEE Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Contributed by Bert L. Vallee, January 2, 1996

ABSTRACT The dose effect of pure daidzin on the sup- these hamsters. Further, two of its constituents were pression of ethanol intake in Syrian golden hamsters was purified and also shown to suppress ethanol intake in these compared with that of crude daidzin contained in a methanol animals. Daidzin and at doses of 150 and 230 mg/kg extract of Radix puerariae (RP). ECso values estimated from per day, respectively, suppress hamster ethanol intake by the graded dose-response curves for pure daidzin and RP '50% (4). Preliminary analyses by HPLC showed that the RP extract daidzin are 23 and 2.3 mg per hamster per day, extract contains only 22 and 2.6 mg/g of daidzin and daidzein, respectively. Apparently the antidipsotropic activity of the RP respectively; comparison of the activity of RP extract to that of extract cannot be accounted for solely by its daidzin content pure daidzin and daidzein indicated that these two known (22 mg/g). In addition to daidzin, six other were active principles alone could not account for the total anti- identified in the RP extract and quantified-namely, dipsotropic activity of the extract. This present study was (160 mg per g of extract), (3.7 mg/g), daidzein (2.6 undertaken to reveal the origin of the additional antidipso- mg/g), daidzein-4',7-diglucoside (1.2 mg/g), (0.2 tropic activity of the methanol extract of RP remaining unac- mg/g), and (0.16 mg/g). None of these, admin- counted for by daidzin and daidzein. istered either alone or combined, contributes in any signifi- cant way to the antidipsotropic activity of the extract. Plasma AND daidzin concentration-time curves determined in hamsters MATERIALS METHODS administered various doses of pure daidzin or RP extract by RP extract was prepared by refluxing 100 g of fibrous RP i.p. injection indicate that the crude extract daidzin has "10 (Tung Ren Tang Traditional Chinese Medicine Extraction times greater bioavailability than the pure compound. Recon- Factory, Beijing) in 700 ml of methanol for 5 h. The methanol struction of the dose-response effects for pure and crude extract was filtered through Whatman no. 1 filter paper. The daidzin using bioavailable daidzin rather than administered residue was refluxed in 700 ml of fresh methanol for another dose gives a single curve. Synthetic daidzin added to the RP 5 h and also filtered. The combined filtrates were evaporated extract acquires the bioavailability of the endogenous daidzin under vacuum to yield a syrup that was suspended in 200 ml that exists naturally in the extract. These results show that (i) of water and dried to a powder by lyophilization. The extract daidzin is the major active principle in methanol extracts of was analyzed for daidzin, daidzein, daidzein-4',7-diglucoside, RP, and (ii) additional constituents in the methanol extract of puerarin, genistin, genistein, and formononetin by HPLC. The RP assist uptake of daidzin in golden hamsters. column (Beckman C18, 5 ,um, 4.6 mm x 25 cm, IP) was eluted at 1 ml/min with a linear acetonitrile gradient (0-40 min, Pueraria lobata (Willd.) Ohwi or Puerariapseudohirsuta Tan et 12-36%) in 0.1% trifluoroacetic acid. Pure daidzin and daid- Wang (leguminosae) is one of the earliest medicinal plants to zein were synthesized according to published procedures and be used in China. Radix puerariae (RP; root of the Pueraria identified by mass and NMR spectroscopy (5, 6). Daidzein- plant) was first described in the Chinese materia medica, Shen 4',7-diglucoside was synthesized by reacting 1 mol of daidzein, Nong Ben Cao Jing (Anonymous, ca. 200 B.C.), as sweet and 2.1 mol of 2,3,4,6-tetra-0-acetyl-a-D-glucopyranosyl bromide, acrid in taste, cool in nature, and something of a medicinal and 2.1 mol of sodium ethoxide at room temperature for 4 h. panacea: it was used as an antipyretic, antidiarrhetic, dia- Reaction products were precipitated with ethanol and acetyl phoretic, and antiemetic agent-a general antimicrobial agent groups were removed with 1 M NaOH. Reaction side products in today's parlance. In the most celebrated medical treatises of were precipitated by acidification and daidzein-4',7- China, Huang Di Nei Jing (Anonymous, ca. 200 B.C.) and diglucoside in the supernatant was further purified on a Shanghan Lun (Zhang Zhongjing, ca. A.D. 200), RP was semipreparatory HPLC column and identified by NMR and recommended for stiffness and pain of the neck, pain in the mass spectroscopy. Genistein, genistin, formononetin, and eye, "consumptive thirst" (increase in appetite and thirst), and puerarin were products of Indofine Chemical (Somerville, for the induction of early measles eruption (for review, see ref. NJ). Daidzin-supplemented RP extract was prepared by son- 1). Hundreds of years later, medications based on RP and Flos icating 5 g of RP extract and 200 mg of synthetic daidzin in 700 puerariae (flower of the Pueraria plant) were found useful in ml of methanol for 10 min. Methanol was then removed by the treatment of alcohol-related problems, first as an amethys- flash evaporation on a 50°C water bath. RP extract and tic (anti-intoxication) agent (Sun Simiao, ca. A.D. 600) and daidzin-supplemented RP extract contain 22 and 62 mg of later as an antidipsotropic (antidrinking) agent (Li, Dongyuan, daidzin per g dry extract, respectively. ca. A.D. 1200) (for review, see ref. 2). Animal Drinking Experiments. The effect of various agents Recently, we have confirmed the antidipsotropic effect of on ethanol intake was assessed with golden hamsters housed RP in ethanol-preferring Syrian golden hamsters (3). A crude in metabolic cages under a two-bottle free-choice paradigm as methanol extract of RP at a dose of 1.5 g/kg per day signifi- described (3, 4). To determine the dose effects of synthetic cantly suppresses (>50%) the free-choice ethanol intake of daidzin, RP extract, and daidzin-supplemented RP extract on hamster ethanol intake, 45 ethanol-preferring hamsters (121- 130 were used. Before all The publication costs of this article were defrayed in part by page charge g; Sasco, Omaha, NE) drug testing, payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: RP, Radix puerariae; AUC, area under curve. 4284 Downloaded by guest on October 1, 2021 Biochemistry: Keung et aL. Proc. Natl. Acad. Sci. USA 93 (1996) 4285

hamsters received (i.p.) 1 ml of sterile phosphate-buffered 100 saline (PBS) each day for 6 days to establish baseline ethanol consumption. They were then divided into nine groups of five animals each and doses for 6 consecutive given daily days (i.p.) 80 of synthetic daidzin, RP extract, or daidzin-supplemented RP I1 extract suspended in 1 ml of sterile PBS. Groups I, II, and III 80> received 6, 18, and 60 mg of synthetic daidzin, respectively; 0 and VI received and 370 of RP 60 - groups IV, V, 37, 111, mg (A extract containing 0.81, 2.44, and 8.1 mg of daidzin, respec- 0 tively; and groups VII, VIII, and IX were given 11, 33, and 111 co4) mg of daidzin-supplemented RP extract containing 0.69, 2.07, CI 40 1 and 6.9 mg of daidzin, respectively. Water and a 15% aqueous ethanol solution were available continuously. Drugs were administered between 3 and 4 p.m. each day and amounts of ethanol consumed were measured between 9 and 10 a.m. The 20 response of each hamster to daidzin or RP extract treatment is expressed as % ethanol intake suppression (% suppression) I I I I given by (Vo - Vd) x 100/Vo, where Vo and Vd are the average 0 I daily intakes of 15% aqueous ethanol solution during the 1 3 10 30 100 baseline and treatment periods, respectively. Throughout this Dose, mg/day study (12 days), hamster weights stayed relatively constant at 124-140 g. Hence, the dosages given were based on an average FIG. 1. Semi-log plot of graded dose-response of ethanol intake body weight of 130 g and were not corrected for the weight of suppression by daidzin administered either as the compound (groups each individual hamster. I, II, and III; open squares) or as a crude RP extract (groups IV, V, Time Course of Plasma Daidzin Concentration and Area and VI; filled squares). Values are mean ± SE for five different Under Curve (AUC). Thirty ethanol-preferring hamsters (121- hamsters. 130 selected as described were used in this g) (3, 4) study. extract is very similar to that of the pure However, Animals were first anesthetized with urethane 1.2 compound. (Sigma; g/kg crude daidzin is 10 times more than the and from each one a 0-time blood was apparently potent pure i.p.) sample (20 ,ul) This can be one or both of the drawn from the orbital venous into a tube 5 compound. finding explained by plexus containing following hypotheses: (i) in addition to daidzin, the RP extract pul of 9% K3EDTA. Animals were then divided into six groups. contains another that could either the Groups I, II, and III were given (i.p.) 6, 18, and 60 mg of pure agent(s) potentiate effect of, or act or with daidzin, and daidzin, respectively. Groups IV, V, and VI were given 37, 111, additively synergistically and of RP extract and 8.1 of (ii) the extract contains a factor(s) that increases the bioavail- 370 mg containing 0.81, 2.44, mg of daidzin. daidzin, respectively. Blood samples (20 ,ul) were taken 15, 30, ability 60, 120, 240, and 540 min after drug administration. Plasma Content and Antidipsotropic Activity of Daidzin Analogs Found in RP Extract. The methanol extract of RP contains a samples were obtained by centrifugation and the proteins were number of other isoflavones that are of daidzin precipitated with an equal volume of ice-cold methanol/ analogs (8, 9) perchloric acid (7.5:2.5) and removed by centrifugation. Daid- and hence could be antidipsotropic. The methanol extract of RP in our constitutes -19% of the zin concentration in the supernatant was analyzed by HPLC as prepared laboratory dry described above. Plasma daidzin concentration-time curves weight of the fibrous root. HPLC analysis of this extract has were constructed from these data and the AUC between the identified seven isoflavones. The most abundant is puerarin zero and ninth hour was estimated by the trapezoid method (160 mg/g extract), followed by daidzin (22 mg/g), genistin and that after the ninth hour was calculated based on the (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 elimination rate constant estimated from the elimination mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). phase of the curve (7). Both daidzin and daidzein have been shown to suppress free-choice ethanol consumption by golden hamsters (3, 4) whereas puerarin, the most abundant isoflavone, does not (4). RESULTS AND DISCUSSION In this study, we find that daidzin and daidzein do not Dose Effect of Pure Daidzin and RP Extract Daidzin on potentiate each other's activity, but rather they act additively Hamster Ethanol Intake. Daidzin, administered both as a pure (Table 1). However, at a dose corresponding to the amounts compound and as a component of a methanol extract of RP, present in a standard sample of RP extract, neither of them suppresses ethanol intake by golden hamsters in a dose- exhibits any measurable antidipsotropic effect (Table 1, lines dependent manner (Fig. 1). The right-hand curve (open 6 and 8). Further, none of the other isoflavones identified in squares) shows the graded dose-response of pure daidzin on the crude RP extract suppresses hamster ethanol intake, either free-choice ethanol intake. A dose of 6 mg per hamster per day alone or in combination at the same amounts and proportions suppresses ethanol intake by -25% and a dose of 60 mg per as found in an EC50 dose of the extract (Table 1). This lack of hamster per day suppresses it by -76%. The EC50 value (the synergism among these isoflavones makes it unlikely that the dose that produces a 50% suppression) estimated from the small amounts of these compounds, including daidzin and dose-response curve is -23 mg per hamster per day. daidzein, could account for the enhanced antidipsotropic The dose-response curve for the RP extract, plotted in activity of the exact. terms of dry weight, gives an estimated ECso of 111 mg per Relative Bioavailability: Synthetic Daidzin vs. Daidzin in hamster per day (see Fig. 6, filled squares). If plotted on the RP Extract. Herbalists who practice traditional Chinese med- basis of daidzin content, however, as shown in the left-hand icine generally believe that crude drugs are superior to pure curve of Fig. 1 (filled squares), it gives an estimated ECso50 value ones for a number of reasons. Among them, crude drugs are of -2.3 mg daidzin per hamster per day, a tenth of that thought to be less toxic and have higher bioavailability. To obtained for pure daidzin. determine whether or not the RP extract constitutes a more The slopes of both lines in Fig. 1 are typical dose-responses bioavailable form of daidzin than the pure compound, the two (-0.8). That the two lines are virtually parallel suggests that dosage forms were evaluated in golden hamsters in terms of the action of daidzin administered as a component of the the time course for appearance of daidzin in blood (7). Downloaded by guest on October 1, 2021 4286 Bohmsr:Kuge l Proc. Natl. Acad. Sci. USA 93 (1996)

Table 1. Effect of RP isoflavones, alone or combined, on ethanol 60 intake by golden hamsters Dose, mg per Suppression, Agent hamster per day % (n)* 50 (1) Daidzin 20 57 ± 12 (71)t (2) Daidzein 30 56 ± 7 (12)t (3) Daidzin + daidzeint 10 + 15 61 ± 14 (3) 40 (4) RP Extract 111 50 ± 8 (5) (5) Puerarin 17.8§ 0 U11 (6) Daidzin 2.4§ 0 30 (7) Genistin 0.4§ 0 (8) Daidzein 0.3§ 0 (9) Daidzein-4',7-diglucoside 0.13§ 0 20 (10) Genistein 0.02§ 0 (11) Formononetin 0.02§ 0 (12) Mixture of agents (5-11)t 0 10 *Data are mean ± SD of the mean of n hamsters. Y tTaken from ref. 4. tTo make mixtures, isoflavones were first dissolved in methanol (1 mg/ml) by sonication for 10 min. Appropriate aliquots were com- 0 10 20 30 bined and methanol was then removed by flash evaporation in a 50°C water bath. The dry materials were redissolved or suspended in sterile Daidzin dose, mg/hamster PBS for i.p. administration. §Dose is equal to the amount of the specified isoflavone found in 111 FIG. 3. Relationships between the area under the plasma daidzin mg of RP extract. concentration-time curves (AUC) and the daidzin doses given to hamsters receiving either pure daidzin (open squares) or RP extract Fig. 2 shows the plasma daidzin concentration-time curves (filled squares). Values are means for five hamsters. for pure daidzin (Fig. 2A) and the methanol extract of RP (Fig. 2B). Maximal plasma daidzin in hamsters given 150 mg of more importantly also has a much higher ("10 times) extent crude extract (which contains 3.3 mg of daidzin) occurs at 33 of availability than pure daidzin. Replotting the dose response min (tma,), whereas in hamsters given 6 mg of pure daidzin, it data shown in Fig. 1 in terms of bioavailable daidzin admin- occurs at 63 min. The peak times (tmax) determined for both istered (AUC), based on the relationships established in Fig. crude and pure daidzin are independent of the dosage admin- 3, gives virtually superimposable lines (Fig. 4). Because the istered. The average maximal plasma daidzin concentration therapeutic effect of a drug is generally related to its extent of (Cmax) obtained in hamsters treated with 150 mg of extract is availability rather than dose administered, these results 65 ,uM, more than 10 times the 5.8 ,uM found in hamsters strongly suggest that daidzin is the major, if not the only, active treated with 6 mg of pure daidzin. The AUC estimated for both principle in the methanol extract of RP. pure daidzin- and RP extract-treated hamsters is directly Bioavailability and Antidipsotropic Activity of Daidzin En- proportional to the dose of daidzin administered (Fig. 3). riched Methanol Extract of RP. The apparently higher bio- Within the dose range studied, daidzin administered as the availability of daidzin given as the RP extract can be attributed extract yields AUC values 10 times those obtained with the to one or both of the following possibilities: (i) the crude same dose of pure daidzin. Thus, daidzin administered as the extract assists daidzin and/or and in extract not has a rate of but delivery uptake, (ii) crude only higher availability (tmax) addition to daidzin itself, there is one or more daidzin pre- cursors in the extract that is rapidly absorbed and transformed 6 A into daidzin in vivo. To evaluate these possibilities, synthetic 100

80

I: 0 60 C 80 0. 60 40 40 20 20

- In*' I _ 1 10 2 4 6 8 10 0 0 10 20 Time, hour 50 100 Bioavailable daidzin, AUC (/Lg.h/ml) FIG. 2. Plasma daidzin concentration-time curves determined in hamsters given (i.p.) 6 mg of pure daidzin suspended in 1 ml of PBS FIG. 4. Replots of the graded dose-response curves shown in Fig. (A) and 150 mg of crude RP extract containing 3.3 mg of daidzin and 1 using bioavailable daidzin (AUC) instead of daidzin doses given. O, suspended in 1 ml of PBS (B). Values are mean ± SE for five hamsters. Pure daidzin; *, RP extract. Downloaded by guest on October 1, 2021 _1 Biochemistry: Keung et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4287

140 plasma daidzin tma values and terminal-elimination half-lives 10 testablished in these two groups of hamsters are also similar the of 120 r >100 !1 (Fig. 5, Inset). Hence, pharmacokinetics synthetic ¢ ^ daidzin into the RP extract are !30 ' = 1.4h incorporated indistinguishable S\f^ N^ Dbs /2 from those of the endogenous daidzin. 100 \ |4 10 - \\ The pharmacological effects of the two different dosage forms of daidzin are also similar. Fig. 6 shows the graded \ | 3 N\ dose-response curve of the daidzin-supplemented RP extract 80 &- 1\ \. ^one the suppression ofethanol intake (open squares) versus that of the natural RP extract (filled squares). The supplemented 60 ) 0.3 . ,extract contains 2.8 times more daidzin than the original 0 2 4 6 8 extract and its dose curve is shifted left with an ECso50 value of \ Time, hour 40 mg per hamster per day, compared with 110 mg per 40 hamster per day for the unsupplemented extract. Clearly, pure daidzin incorporated into the extract and daidzin originally present in the extract are bioequivalent. This not only estab- 20 lishes that the RP extract indeed increases the bioavailability of daidzin, both from endogenous and exogenous sources, and I |Is^- v hence boosts its but also rules out the 0 ^^^^L , antidipsotropic potency, 0 2 4 6 8 10 possibility that different forms of daidzin, e.g., daidzin pre- cursor(s), are present in the methanol extract. Time, hour Solubility of Pure and Crude Daidzin in PBS. The obser- .... that daidzin administered as an RP extract is more FIG. 5. PlasmaPlasma daidzin concentration-time .curvecurves determvationdetermined in hamsters aidministered (i.p.) 150 mg of RP extract (filled squares) or readily absorbed than the pure compound may be related to 150 mg off daidzin-supplemented RP extract (open squares). The the specific physicochemical properties of daidzin. The prin- former coritained 3.3 mg of daidzin and the latter 9.3 mg of daidzin. ciples governing the absorption of daidzin, a small non-ionized Values are mean ± SE for five hamsters. (Inset) Semilogarithmic plot. molecule, from the peritoneum are likely the same as those for the passage of low molecular weight drugs through biological daidzin wras added to an RP extract and its bioavailability as membranes elsewhere, i.e., high lipid/water partition coeffi- well as anitidipsotropic activity were examined. cient and water soluble substances with small molecular radius Added daidzin exhibits the same bioavailability as that favor rapid absorption. To determine whether or not the water naturally ]present in the extract. Fig. 5 shows the plasma daidzin solubility of the two dosage forms of daidzin are indeed concentraition-time curves determined in hamsters that have different, the solubility of pure daidzin and that incorporated been given 150 mg of RP extract that contains 3.3 mg of into an RP extract were determined. Fig. 7 shows the effect of naturally existing daidzin (filled squares) and in those admin- RP extract on the solubility of daidzin in PBS. In the absence istered 15i0 mg of daidzin-supplemented RP extract that con- of RP extract, the solubility of daidzin in PBS is -83 ,g/ml. tains 9.3 mg (3.3 mg naturally existing plus 6 mg synthetic) Incorporation of even a small amount (0.6 mg) of extract daidzin ((open squares). The AUC values determined in the markedly increases the solubility of daidzin fivefold to =416 hamsters treated with these two different dosages are 43 Ag/ml. Daidzin solubility continues to increase as the amount tuh/ml anId 127 ug.h/ml, respectively, which are directly pro- of extract increases but at a diminishing pace. It reaches -830 portional to the amounts of daidzin received by the two groups ,g/ml at 10 mg of extract, and in the presence of 20 mg of of hamsters (3.3 and 9.3 mg, respectively). Further, the ratio of extract, all added daidzin (2 mg) was recovered in the super- the Cmax vvalues determined in these two groups of hamsters is natant. These results clearly indicate that by a mechanism(s) 2.67, also similar to that of the daidzin doses administered. The 100

80 1 1.5 (6.9) (8.1)

Cu 60 -

el0. (2.1) C) 40 I- :z (0.69) 0.5 (0.81) 20 0 0 5 10 15 20 0 0I 1 Ia 10 20 50 100 200 500 RP Extract, mg/ml RP Extract, mg/day FIG. 7. Effect of crude RP extract on the solubility of daidzin in PBS. Daidzin (2 mg) was incorporated into 0, 0.125, 0.25, 0.5, 1, 2, 5, FIG. 6. Graded dose-response curves of ethanol intake suppres- 10, and 20 mg of crude RP extract according to the procedure sion by RP extract (groups IV, V, and VI; filled squares) and described in Materials and Methods. The dry samples were suspended daidzin-supplemented RP extract (groups VII, VIII, and IX; open in 1 ml of PBS, sonicated for 5 min, and centrifuged at 12,000 x g for squares). Values are mean ± SE for five hamsters. Numbers in 15 min. Daidzin content in the supernatant was determined by HPLC parentheses are daidzin content in each dose of crude extract. and is defined as PBS soluble daidzin. Downloaded by guest on October 1, 2021 4288 Bohmsr:Kuge l Proc. Natl. Acad. Sci. USA 93 (1996) yet to be discerned the extract of RP is capable of enhancing experiments; Barton Holmquist, Werner Dafeldecker, and Shaohan the solubility of daidzin in PBS. This could account for at least Shen for the synthesis of daidzin, daidzein, and daidzein-4',7- part of its potentiating effect on daidzin bioavailability. diglucoside; and J. F. Riordan and T. C. French for valuable discus- Although the nature of the factor(s) in the RP extract and sions. This work was supported by the Endowment for Research in the exact mechanism by which it enhances daidzin bioavail- Human Biology, Inc. ability have not been identified so far, it is well known that as 1. Fang, Q. (1980) J. Ethnopharmacol. 2, 57-63. pharmacokinetic processes such absorption, distribution, 2. J. A. & E. S. Medicinal Plants China and biotransformation of one can be altered Duke, Ayensu, (1985) of elimination, drug (Reference, Algonac, MI), vol. 1, pp. 339-340. when administered concurrently with another (10). Indeed, 3. Keung, W.-M. & Vallee, B. L. (1993) Proc. Natl. Acad. Sci. USA knowledge of the mechanism by which a given drug interaction 90, 10008-10012. occurs is often clinically beneficial whereas in many cases, the 4. Keung, W.-M. & Vallee, B. L. (1994) Exper. Suppl. (Basel) 71, lack of it can be harmful. In recent years, the use of herbal 372-281. medicines has increased rapidly in the West as supplements to 5. Iyer, R. N., Shah, K. H. & Venkataraman, K. (1951) Proc. Indian or substitutes for conventional pharmaceutical agents. Yet Acad. Sci. 33A, 116-126. very little is known about herbal-conventional drug interac- 6. Farka, L. & Varady, J. (1959) Ber. Dtsch. Ges. Chem. 92,819-821. tions (11, 12). Although the present study shows that the crude 7. Chow, S.-C. & Liu, J.-P. (1992) Design and Analysis ofBioavail- extract enhances the effect of its own active principle, there is ability and Bioequivalence Studies (Dekker, New York). no reason to believe that the pharmacokinetics and pharma- 8. Zhao, S.-P. & Zhang, Y.-Z. (1985) Acta Pharmaceut. Sinica 20, of conventional could not be altered 203-208. codynamics any drug by 9. Fang, Q., Lin, M., Sun, C.-M., Liu, H.-M. & Lang, H.-Y. (1974) concurrent administration of an herbal medicine. The risk of Chin. Med. J. 5, 271-274. such traditional-conventional drug interactions, especially 10. Cadwaller, D. E. (1983) Biopharmaceutics and Drug Interaction among elderly patients and patients whose drug metabolizing (Raven, New York). systems are compromised (13), should be considered before 11. Tani, T. (1989) Med. Drug J. (Japan) 25, 1987-1993. comedication with an herbal medicine and a conventional drug 12. Hasegawa, T., Yamaki, K., Nadai, M., Muraoka, I., Wang, L., is prescribed. Tagagi, K. & Nabeshima, T. (1994) Int. J. Clin. Pharmacol. Ther. 32, 57-61. We thank Drs. Yan Ye Xia for assistance in the hamster drinking 13. Schmucker, D. L. (1985) J. Pharmacol. Exp. Ther. 37, 133-148. Downloaded by guest on October 1, 2021