Masatoshi NOHMI, Minoru FURUYA, Kuniya KOIZUMI, Koji IIJIMA, Sadao NAKAYAMA and Katsuji OGucHI

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1 Showa Univ. J. Med. Sci. 7(2), 151 `161, December 1995 Original Effects of Kampo Medicines on Hepatic Drug-Metabolizing Enzymes in Rats Masatoshi NOHMI, Minoru FURUYA, Kuniya KOIZUMI, Koji IIJIMA, Sadao NAKAYAMA and Katsuji OGucHI Abstract: We studied the effects of the kampo medicines, sho-saiko-to (SST), oren-gedoku-to (OGT), sanmotsu-ogon-to (SOT), keigai-rengyo-to (KRT), senkyu-chacho-san (SCS), on rat hepatic drug-metabolizing enzymes in vivo. Hot-water extracts of each kampo medicine were given orally at doses of 100 and 1000 mg/kg. After a single administration, SST increased activities of aniline hydroxylase (ANH), aminopyrine N-demethylase (APD), and cytochrome c reductase (cyt. c red.) and levels of cytochrome P-450 (P-450) and cytochrome b5 (b5) in the liver. Single administrations of OGT, SOT, and SCS decreased activities of ANH and APD and P-450 content and increased cyt. c red. activity. A single administration of KRT at 100 mg/kg increased ANH and APD activities, but at a dose of 1000 mg/kg decreased both activities. Repeated administration of test drugs at doses of 100 and 1000 mg/kg/day for 14 days resulted in the following: SST and OGT reduced ANH activity; SCS and KRT reduced APD activity; OGT and KRT lowered P-450 content; cyt, c. red. activity was increased by SST, OGT, and SOT and was reduced by KRT. These results suggest that the examined kampo medicines may modify the effectiveness of simultaneously administered drugs that are metabolized by hepatic enzymes. Key words: kampo medicine, cytochrome P-450, aniline hydroxylase, aminopyrine N-demethylase, cytochrome c reductase Introduction Kampo is a important traditional medicine in Japan. Decoctions of crude drugs, mostly of herbal origin, are used in kampo medicine; they have been proved to be often effective, especially for chronic diseases. Although most kampo medicines are administered orally and absorbed via the alimentary tract to be transported to the liver, few studies have investigated their effects on liver function. Several reports indicate that extracts from some herbs affect hepatic drug-metabolizing enzymes (HDMEs)1-3). We have previously examined the effects of 32 crude herbal drugs from the families Umbellif erae, Compositae, and Labiatae on rat HDMEs in vitro, and reported that several herbs, including byakushi and ogon (Table 1), significantly suppress aniline hydroxylase (ANH) and aminopyrine N-demethylase (APD) activities4-6). Further more, we investigated the effects of byakushi and ogon on rat HDMEs in vivo and showed that byakushi and ogon reduce ANH activity and cytochromoe P-450 (P-450) content and enhanced cytochrome c reductase (cyt. c red.) activity. These reports suggest that kampo medicines Department of Pharamacology, School of Medicine, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142, Japan.

2 152 Masatoshi NOHMI, et al. Table 1. Formulations of kampo medications.

3 Kampo Medicines and Drug-metabolizing Enzymes 153 affect the activity of HDMEs, causing drug interactions in the liver7). In the present study, we investigated the effects on rat HDMEs of five common kampo formulations, sho-saiko-to (SST), oren-gedoku-to (OGT), sanmotsu-ogon-to (SOT), keigairengyo-to (KRT), and senkyu-chacho-san (SCS), all containing byakushi or ogon. Materials and Methods Animals and crude drugs Seven-week-old male Sprague-Dawley (SD) rats (Sankyo Labo Service Co., Tokyo, Japan) were individually housed in wire mesh cages and kept in a room controlled at a temperature of 24 }1 Ž and a relative humidity of 55 }10% with a 12-hour light and dark cycle. Rats were given a standard diet (MF: Oriental Yeast Co., Tokyo, Japan) and water ad libitum. The ingredients of the five kampo formulations are listed in Table 1. Crude drugs were purchased from Uchida Wakan-Yaku Co., Ltd. (Tokyo, Japan), Tsumura Co., Ltd. (Tokyo, Japan), and Tochimoto-Tenkaido Co. (Osaka, Japan). Preparation and administration of kampo medicines Hot-water extracts (HWEs) were prepared by immersion of crude drugs in a 20-fold (w/w) volume of distilled water and boiled at 100 Ž for 30 min, and then immediately passed through filter paper (Advantec 2: Toyo Roshi Kaisha Co., Tokyo, Japan). The filtrate was evaporated under reduced pressure to half volume, then lyophilized and stored. The yields (w/w) of lyophilized HWEs were 23.3% for SST, 23.6% for OGT, 37.1% for SOT, 22.5% for KRT, and 22.7% for SCS. In single and repeated administration studies, HWEs were dissolved in distilled water and administered orally once a day at doses of 100 or 1000 mg/ 4 ml/kg/day; these doses corresponded to amounts approximately equal to and 10-fold the normal human adult dose, respectively. The activities and contents of HDME were determined 1, 3, 6, 12, and 24 hr after single administrations and 24 hr after the last of repeated daily administration for 14 days. Control rats received the same volume of distilled water Chemicals. Nicotinamide adenine dinucleotide phosphate (oxidized form and reduced form), glucose- 6-phosphate and cytochrome c III were purchased from Oriental Yeast (Tokyo, Japan) and Sigma Chemical Co. (St. Louis, Mo, USA), respectively. All other chemicals used were commercial products of the highest available purity. Preparation o f hepatic enzymes and microsomal fractions Rat were fasted for 18 h before liver removal. The liver was perfused with ice-cold saline, then removed and weighed. The livers were homogenized with 4 volumes of 1.15% KCl-0.01 M Na+/K+ phosphate buffer (ph 7.4) in a Potter Elvehjem homogenizer. The homogenate was centrifuged at g for 20 min at 4 Ž, and the supernatant obtained was further centrifuged at g for 60 min to obtain the microsomal fraction. Microsomal pellets (MsP) were suspended in 20% glycerin-0.1 M Na+/K+ phosphate buffer (ph 7.4). The supernatant centrifuged at g was used for the ANH and APD assays, and the microsomal fraction was used for the P-450, cytochrome b5 (b5), and cyt. c red. assays. Determinations of HDME activity and protein content The ANH and APD activities in the g supernatant were determined spectrophotometrically by the method of Mazel8. The P-450 and b5 contents and cyt. c red. activity in the microsomal fraction were determined by the method of Omura and Sato9) or Omura and Takesue10)

4 154 Masatoshi NOHMI, et al. Statistical analysis Data are expressed as mean }S.D. of five rats. Statistical significance was evaluated using Student's t-test, and a P value less than 0.05 was considered significant. Results Effect of single oral administration of kampo HWEs. SST Single oral administration of SST at 100 mg/kg induced the following: a significant in- Fig. 1. Effect of a single administration of SST on rat HDMEs. Each datum was plotted as percent of the control value with a bar expressing the S.D. (n=5). Control value (C) in the plots of respective dose (100 and 1000 mg/kg) were as follows: for ANH, }0.093 and }0.117 nmol p-aminophenol,/mg protein/20 min; for APD, 17.0 }0.6 and 20.0 }1.7 nmol HCHO/mg protein/30 min; for cytochrome P-450, 0.18 }0.01 and nmol cytochrome P-450/mg protein; for cytochrome b5, }0.009 and }0.010 nmol cytochrome b;/mg protein; for cyt. c red., 388 }24 and 388 }29 nmol NADPH-cytochrome c reductase/ml, protein/min. Significant difference from the control, * P<0.05, ** P<0.01, *** P<0.001.

5 Kampo Medicines and Drug-metabolizing Enzymes 155 Fig. 2. Effect of a single administration of OGT on rat HDMEs. Each datum was plotted as percent of the control value with a bar expressing the S.D. (n=5). Control values (C) in the plots of respective dose (100 and 1000 mg/kg) were as follows: for ANH, }0.108 and }0.052 nmol p-aminophenol/mg protein/20 min; for APD, 22.9 }1.0 and 23.4 }0.8 nmol HCHO/mg protein/30 min; for cytochrome P-450, 0.20 }0.01 and 0.22 }0.01 nmol cytochrome P-450/mg protein; for cytochrome b }0.004 and }0.009 nmol cytochrome b,/mg protein; for cyt. c red., 318 }23 and 266 }16 nmol NADPH-cytochrome c reductase/mg protein/min. Significant difference from the control, * P<0.05, ** P<0.01, *** P< crease in ANH activity from 1 h to 12 h, but a nonsignificant increase at 24 h; a significant increase in APD activity from 3 h to 24 h; a significant increase in the P-450 content from 6 to 24 h; a significant increase in the b, content from 1 to 12 h, except at 6 h; a significant increase in cyt. c red. activity from 1 h to 24 h. At a dose of 1000 mg/kg SST had no effect on ANH and APD activities or P-450 and bs amounts, while cyt. c red. activity was increased at 1 h and 12 h (Fig. 1). OGT A single administration of OGT at 100 mg/kg reduced APD activity at 12 h and enhanced

6 156 Masatoshi NoHMI, et al. Fig. 3. Effect of a single administration of SOT on rat HDMEs. Each datum was plotted as percent of the control value with a bar expressing the S.D. (n=5). Control values (C) in the plots of respective dose (100 and 1000 mg/kg) were as follows: for ANH, }0.061 and }0.098 nmol p-aminophenol/mg protein/20 min; for APD, 21.5 }1.4 and nmol HCHO/mg protein/30 min; for cytochrome P-450, 0.22 }0.02 and nmol cytochrome P-450/mg protein; for cytochrome b5, }0.004 and }0.005 nmol cytochrome b ;/mg protein; for cyt. c red., 447 }30 and 452±14 nmol NADPH-cytochrome c reductase/mg protein/min Significant difference from the control, * P<0.05, ** P<0.01, *** P< cyt. c red. activity from 1 h to 24 h. A dose of 1000 mg/kg reduced both ANH and APD activities; ANH activity was reduced to 74% of control at 12 h and APD to 79% of control at 12 h. The b5 content was reduced at a dose of 1000 mg/kg after 6 h (Fig. 2). SOT A single administration of SOT at 100 mg/kg reduced ANH and APD activities at 3 h. The P-450 content was also decreased to 81% of control. A dose of 1000 mg/kg enhanced ANH activity at 24 h, and decreased P-450 content at 6 h. The b5 content was decreased at a dose of 1000 mg/kg at 6 h. Activity of cyt. c red. was enhanced at both doses (Fig. 3).

7 Kampo Medicines and Drug-metabolizing Enzymes 157 Fig. 4. Effect of a single administration of KRT on rat HDMEs. Each datum was plotted as percent of the control value with a bar expressing the S.D. (n=5). Control values (C) in the plots of respective dose (100 and 1000mg/kg) were as follows: for ANH, 0.482±0.038 and 0.780±0.118 nmol p-aminophenol/mg protein/20 min; for APD, 12.1 }1.4 and 19.5 }2.7 nmol HCHO/mg protein/30 min; for cytochrome P-450, 0.11 }0.01 and 0.15 }0.01 nmol cytochrome P-450/mg protein; for cytochrome b5, }0.002 and }0.002 nmol cytochrome b;/mg protein; for cyt. c red., 306±20 and 374 }40 nmol NADPH-cytochrome c reductase/mg protein/min. Significant difference from the control, * P<0.05, ** P<0.01, P< KRT A single administration of KRT at 100 mg/kg enhanced both ANH and APD activities at 1 h, whereas administration at 1000 mg/kg induced decreases in both activities at 3 h. The P-450 content was increased at 12 h by a dose of 1000 mg/kg. The b5 content was increased at 1 h by a dose of 100 mg/kg and at 12 h by a dose of 1000 mg/kg. Activity of cyt. c red. was decreased by the administration of KRT at 1000 mg/kg at 6 h (Fig. 4). SCS A single administration of SCS at 1000 mg/kg reduced ANH and APD activities and re-

8 158 Masatoshi NoHML, et al. Fig. 5. Effect of a single administration of SCS on rat HDMEs. Each datum was plotted as percent of the control value with a bar expressing the S.D. (n=5). Control values (C) in the plots of respective dose (100 and 1000 mg/kg) were as follows: for ANH, }0.030 and nmol p-aminophenol/mg protein/20 min; for APD, 11.0 }0.4 and nmol HCHO/mg protein/30 min; for cytochrome P-450, 0.11 }0.01 and 0.25 }0.02 nmol cytochrome P-450/mg protein; for cytochrome b5, }0.001 and }0.006 nmol cytochrome b 5/mg protein; for cyt. c red., 224 }31 and 438 }46 nmol NADPH-cytochrome c reductase/mg protein/min. Significant difference from the control, * P<0.05, ** P<0.01, *** P< duced P-450 content. At a dose of 100 mg/kg SCS increased the b, content at 1 h and decreased b5 content at 3 h. Activity of cyt. c red. was increased at both doses (Fig. 5). Effect of repeated administrations of kampo HWEs. Repeated treatments did not affect body weight or liver weight. However, microsomal protein content (MsP) were significantly increased (P<.05) with SST at doses of 100 mg/kg (26.2 }1.1 mg/g liver weight, mean }S.D.) and 1000 mg/kg (24.5 }1.7 mg/g), OGT at 100 mg/kg (24.8 }1.3 mg/g) and SOT at 1000 mg/kg (24.0 }1.5 mg/g) compared with controls (21.9 }0.5 mg/g),

9 Kampo Medicines and Drug-metabolizing Enzymes 159 Table 2. Effect of repeated oral administration of HWE on rat hepatic drug metabolizing enzymes. HWE, Hot-water extract. Each value represents the mean }S.D. of data expressed as a percent of the control value (n=5). * P<0.05, ** P<0.01, *** P<0.001: Significant differences from the control value. The effects on the activities of ANH, APD and cyt. c red. and on the contents of P-450 and b5 are shown in Table 2. Repeated administration of SST decreased ANH activity and b5 content, while OGT decreased ANH activity and P-450 content. Decreases of APD and cyt. c red. activity and P-450 content were observed after repeated administration of KRT. Activity of cyt. c red. was increased after administration of SST, OGT, or SOT. Discussion The individual effects of several component crude drugs of the tested kampo medicines on rat HDMEs have previously been investigated. We reported that in vitro most of the crude drugs decreased the activities of ANH and APD, while ogon enhanced the activity of ANH and reduced that of APD6). Byakushi, which significantly reduced activities of ANH and APD in vitro4), also reduced their activities and decreased the P-450 content and increased the b5 content in vivo. In contrast, ogon in vivo decreased the ANH activity and P-450 content and increased the APD activity and b5 content7). In the present study, we observed that each kampo medicine affected HDMEs in different ways; for example, SST increased the activities of ANH and APD and increased P-450 and b5 contents while other medicines decreased them. We also observed that their effects differed with dose. With OGT, SOT, and SCS, all of which generally enhanced activities of HDMEs, the effect appeared to be the sum of the effects of their component crude drugs which could be observed in vitro or in vivo. However, the effects of one kampo formulation cannot be explained by the simple sum of each component herb's effects. For example, SST, which enhanced HDMEs activity, could be regarded as one such formulation. Although some crude component drugs are known to enhance HDME activity, other herbs in SST may also contribute to the enhancement, but their effects on HDMEs are not yet known.

10 160 Masatoshi NoHMI, et al. A single administration of kampo HWE affected HDME activity 1 to 12 h after treatment. The rise in the P-450 content produced by other drugs, such as phenobarbital, is maximal 24 to 48 hours after administration1l). These observations suggest that the effect of HWEs on P-450 content was a direct effect on the enzyme itself rather than an induction of synthesis of heme proteins. After repeated administration, SST reduced ANH activity and b ; content. These results differed from those seen after a single administration. However, repeated administrations of other kampo medicines produced effects similar to those observed after a single administration. Activity of cyt. c red. was enhanced by SST, OGT, SOT, and SCS; the effect observed after single administrations appeared to contribute to these results. It is said that the cyt. c red. content does not always correlate with tht content of P-450. The biological effect of the increase in cyt. c red. activity observed with these kampo administrations is not known. After administration of OGT or KRT, P-450 content was decreased; however, the amount of MsP was increased by repeated treatment. This finding suggests that both OGT and KRT may directly reduce the activity of P-450, even after repeated administration and that the increased MsP may not result from an increase in heme proteins. The pharmacologic effects of kampo medicines when administered with Western drugs have been discussed; several crude drugs, including glycyrrhizae radix, ephedrae herba and rhei rhizoma, might have adverse effects in particular combinations12). Lin and co-workers found no drug interaction between theophylline and tin chun tang or hsaio ching long tang in SD rats of different ages13). On the other hand, Kanamoto et al. found that a 10-day continuous administration of saiko-keishi-to (SKT) significantly increased the plasma clearance of phenytoin and aminopyrine in rabbits14). This report speculated that treatment with SKT might have induced oxidative metabolic enzymes in the rabbit liver. The contents of SKT are almost identical to those of SST; SKT has two herbs, keihi (bark of Cinnamomum cassia Bl.) and syakuyaku (see Table 1), in addition to those in SST. The increase in P-450 observed after SST administration suggests the possible induction of HDMEs as observed after SKT administration. Administration of OGT, SOT, and SCS reduced the activities of ANH and APD and decreased the contents of P-450 and b;. These three kampo medicines may reduce the activities of HDMEs. In summary, SST increased the activities of HDMEs, while OGT, SOT, and SCS reduced them. These effects of HWEs on HDMEs were observed in the early phase after administrations at the normal human dose. These findings suggest that these kampo medicines would affect the pharmacodynamics or pharmacokinetics of other agents when co-administered. References 1) Han YB, Shin KH and Woo WS: Effect of spices on hepatic microsomal enzyme function in mice. Arch P{zarm Res, 7: (1984) 2) Minura Y, Kuki H and Fukushima B: Effect of kampo extracts on rat microsomal fatty acid hydroxylations. Proceedings of Japanese Conference on the Biochemistry of Lipids, 31: (1989) (in Japanese) 3) Tanaka S, Takahashi A, Onoda K, Kawashima K, Nakaura S, Nagao S, Endo T, Ohno Y, Kawanishi T, Takanaka A, Kasuya Y, Yoshihira K, Fukuoka M, Sekita S, Suzuki H, Harada M, Natori S, N akaji Y, Kobayashi K, Suzuki S, Naito K, Uchida O, Yasuhara K, Saito M, Ishii Y, Tobe M and Shoji J: Toxicological Studies Biological Effects of the Herbal Drug Extracts in Rats and Mi ce: Peony Root, Peach Kernel, Japanese Angelica Root and Cnidium Rhizome. Yakugaku Zasshi, 103: (1983) (in Japanese)

11 Kampo Medicines and Drug-metabolizing Enzymes 161 4) Mayanagi M, Nakayama S and Oguchi K: Effects of Sino-Japanese herbs in the family Umbelliferae on the hepatic drug metabolizing enzymes and lipid peroxidation in rats. Folia Pharmacol Japon, 99: (1992) (in Japanese) 5) Mayanagi M, Nakayama S and Oguchi K: Effects of Sino-Japanese herbs in the family Compositae on the hepatic drug metabolizing enzymes and lipid peroxidation in rats. Folia Pharmacol Japon, 100: (1992) (in Japanese) 6) Nakayama S, Koizumi K, Iijima K, Mayanagi M and Oguchi K: Effects of Sino-Japanese herbs in the family Laviatae on the hepatic drug metabolizing enzymes and lipid peroxidation in rats. Folia Pharmacol Japon, 101: (1993) (in Japanese) 7) Koizumi K, Iijima K, Nohmi M, Nakayama S and Oguchi K: Effects of Byakushi and Ogon on the hepatic drug metabolizing enzymes in rats. Folia Pharmacol Japon, 104: (1994) ( in Japanese) 8) Mazel P: Experiments illustrating drug metabolism in vitro. In: Fundamentals of Drug Metabolism and Drug Disposition, La Du BN, Mandel HG and Way EL (Eds), The Williams and Wilkins Co., Baltimore, pp (1972) 9) Omura T and Sato R: The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem, 239: (1964) 10) Omura T and Takesue S: A new method for simultaneous purification of cytochrome b g and NADPH-cytochrome c reductase from rat liver microsomes. J Biochem, 67: (1970) 11) Remmer H and Merker HJ: Effect of drugs on the formation of smooth endplasmic reticulum and drug-metabolizing enzymes. Ann NY Acad Sci, 123: (1965) 12) Mizuno M, Iinuma M, Yasuda K and Kawada I : Combined effect of Chinese medicine and Western drugs. Ann Proc Gi f u Pharm Univ, 37: (1988) 13) Lin SY, Hou SJ, Wu WH, Chen SH and Young TK: Effect of traditional Chinese herbal medicines on the pharmacokinetics of Western drugs in SD rats of different ages (I) : Aminophylline-Tin Chuan Tang and Aminophylline-Hsiao Ching Long Tang. J Pharmacobio-Dyn, 14: (1991) 14) Kanamoto I, Kodachi M, Horikoshi I and Koizumi T: Effect of saikokeishi-to on the pharmacokinetics of phenytoin in rabbit. Yakugaku Zasshi, 108: (1988) (in Japanese) [Received May 10, 1995: Accepted June 9, 1995]