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Atractylodis Rhizoma extract and its component, atractylon, inhibit tumor promotion in mouse skin two-stage carcinogenesis
Phytomedicine Vol. 1/1994, pp. 55-58 © 1994 by Gustav Fischer Verlag, Stuttgart· Jena . New York
Atractylodis Rhizoma Extract and Its Component, Atractylon, Inhibit Tumor Promotion in Mouse Skin Two-Stage Carcinogenesis S. YU, K. YASUKAWA* and M. TAKIDO College of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274, Japan
Summary The methanol extract of Atractylodis Rhizoma, a traditional Chinese medicine, was found to have antitumor-promoting activity in two-stage carcinogenesis. From the active fraction of the extract, atractylon was isolated. The isolated compound showed inhibitory activity against 12-0tetradecanoylphorbol-13-acetate (TPA)-induced ear inflammation in mice. Furthermore, atractyIon markedly inhibited tumor promotion by TPA following 7,12-dimethylbenz[a]anthracene initiation in mice. Key words: Atractylon; Atractylodis Rhizoma; Antitumor promotion; Two-stage carcinogenesis; Anti-inflammatory activity.
Introduction Atractylodis Rhizoma, the rhizome of Atractylodes [aponica Koidzumi, is used for the treatment of pain, gastritis and enteritis (Namba, 1980). The pharmacological action of Atractylodis Rhizoma includes inhibition of stress ulceration of the stomach (Kubo, et aI., 1983). Atractylon, the major essential oil component of Atractylodis Rhizoma, has been shown to have a significant preventive effect against carbon tetrachloride- and D-galactosamine-induced lesions in primary-cultured rat hepatocytes (Kiso, et aI., 1983; 1985). In the present study, the methanol extract from Atractylodis Rhizoma was found to inhibit TPA-induced tumor promotion during two-stage carcinogenesis in mice. Atractylon (fig. 1) was isolated from the active fraction. Furthermore, atractylon was found to inhibit tumor promotion markedly in two-stage carcinogenesis in mice.
Fig. 1. Chemical structure of atractylon,
Materials and Methods Instruments: Melting points were determined on a Yanagimoto micro-melting point apparatus and are presented uncorrected. NMR spectra were measured with a JEOL JNM-GSX 400 (tH, 400 MHz; BC, 100 MHz) spectrometer, Japan, and chemical shifts are presented as 0 values relative to tetramethylsilane (TMS) as an internal standard. Mass spectra (MS) were measured with a Hitachi M-2000 double-focusing mass spectrometer, Japan, at an ionization voltage of 70 eV. Chemicals: 7,12-Dimethylbenz[a]anthracene (DMBA) was purchased from Sigma Chemical Co., St. Louis, USA. TPA was obtained from Chemicals for Cancer Research Inc., Chicago, USA. Column chromatography (CC) was carried out on silica gel (E. Merck, Germany). Materials: Atractylodis Rhizoma (the rhizome of Atractylodes [aponica Koidzumi) was obtained from the market in Tokyo. This was identified as the rhizome of Atractylodes [aponica by description, chemical analysis and purity test (Namba, 1980; Takitani, 1991). Extraction from Atractylodis Rhizoma: One hundred grams of Atractylodis Rhizoma was extracted by refluxing with methanol for 3 h. The organic solvent was evaporated in vacuo to dryness. The yield of MeOH extract was 18.9 g.
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One hundred grams of Atractylodis Rhizoma was extracted by refluxing with water for 3 h. The solvent was freezed to dryness. The yield of water extraxt was 12.1 g. Isolation of active agent from the methanol extract of Atractylodis Rhizoma: Ten grams of the methanol extract of Atractylodis Rhizoma was separated using a n-hexanemethanol-water mixture (19:19:2). Yields were: n-hexanesoluble fraction: 4.49 g; methanol-water-soluble fraction: 4.89 g. The n-hexane-soluble fraction was then subjected to CC on silica gel using n-hexane as a solvent to obtain six fractions. The inhibition ratio of the fractions were showed in table 1 on TPA-induced inlfammatory ear edema. The active agent (I) was isolated from the potent inhibitory fraction (5, yield: I = 718 mg) and was identified as atractylon by standard physical and chemical procedures and comparison with an authentic sample (Hikino, et aI., 1964; Nishikawa, et aI., 1976). Animals: Female ICR mice were obtained from japan SLC, Shizuoka, japan, and housed in an air-conditioned room (22-23 "C) lit from 8:00 to 20:00. Food and water were available ad libitum. Assay ofTPA-induced inflammation in mice: TPA (1 nM) dissolved in acetone (20 Ill) was applied to the right ear of each mouse with a micropipette. A volume of 10 III was delivered to both the inner and outer surfaces of the ear. Twenty microliters of the sample solution, their vehicles, methanol-water (1:1) or chloroform-methanol (1:1) (as a control) was applied topically about 30 min before each TPA treatment. For ear thickness determinations, a pocket thickness gauge (Mitsutoyo Co., Ltd., Tokyo, japan) with a range of 0-9 mm, graduated at O.Ol-mm intervals and modified so that the contact surface area was increased, thus reducing the tension, was applied to the tip of the ear. Ear thickness was determined at before treatment (a). Edema was measured 6 h after TPA treatment (b: TPA alone; b': TPA plus sample). The following values were then calculated: Edema A: edema induced by TPA alone (b-a). Edema B: edema induced by TPA plus sample (b'-a). Edema A - Edema B Inhibition ratio (%) = X 100 Edema A Each value was the mean of individual determinations from 5 mice. The 50 % inhibitory dose (ID so) values were determined by pro bit-graphic interpolation for four dose levels. Two-stage carcinogenesis experiments: Three groups of 15 mice underwent initiation by application of 50llg of DMBA in acetone (100 Ill) to the dorsal skin. Promotion with Lug of TPA in acetone (100 Ill), applied twice weekly, was begun 7 days after the initiation. Atractylon (2 umol), or the methanol extract (2 mg) of Atractylodis Rhizoma and their vehicle, acetone-dimethylsulfoxide-water (18:1:1, 100 Ill) were applied topically 30 min before TPA treatment. These treatments were continued for 20 weeks.
Statistical analysis: Statistical analysis was performed by Student's t-test.
Results
Inhibition by the methanol extract ofAtractylodis Rhizorna on TPA-induced inflammatory ear edema and TPA-induced tumor promotion: The methanol extract of Atractylodis Rhizoma markedly inhibited the inflammation induced by TPA in mice (table 1). Figure 2A illustrates the time course of skin tumor formation in the groups treated with DMBA plus TPA, with or without the methanol extract from Atractylodis Rhizoma. The first tumor appeared at week 6 and all mice had tumors by week 9 in the group treated with DMBA plus TPA. In the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma, the first tumor appeared during week 10. The percentage of tumor-bearing mice treated with DMBA plus TPA was 100 % at week 20, whereas that in the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma was 33 %. Figure 2B shows the average number of tumors per mouse. The group treated with DMBA plus TPA produced 10.4 tumors per mouse at week 20, whilst the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma had 0.6 tumors per mouse. Treatment with the methanol extract of Atractylodis Rhizoma therefore caused a 94 % reduction in the average number of tumors per mouse at week 20. Table1. Inhibitory effect of atractylon and extracts from Atractylodis Rhizoma on TPA-induced inflammation in mice. Name Water extract Methanol extract Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Atractylon Quercetin Indomethacin Hydrocortisone
IDso (mg/ear)
I.R. 43*1 80*1 29*1 30*1 60*1 77*1 82*1
91 0.9 1.6 0.3 0.03
82*1 40*2 96*2 99*2
The samples were applied 30 min before TPA treatment; ear edema was determined at 6 h after TPA treatment. *p < 0.01 by Student's t-test as compared to the control group. I.R.: Inhibition ratio (at 12 mg/ear; 21 mg/ear).
Investigation of atractylon from the methanol extract of Atractylodis Rhizoma for inhibitory activity against TPAinduced ear edema: The component active against TPA-induced inflammatory ear edema was separated from the methanol extract of Atractylodis Rhizoma, and was identified as atractylon (Table 1). Inhibitory effect of fractions 3
Atractylodis Rhizoma Extract and Its Component
Fig. 2. Inhibitory effect of methanol extract of Atractylodis Rhizoma on the tumor promotion of skin papillomas by TPAin DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 ug of DMBA, 1 Jlg of TPA was applied twiceweekly. Topicalapplication of methanol extract (2mg) of Atractylodis Rhizoma and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A),and as average numbers of papillomas per mouse (B) . • =+TPAwith vehicle alone; 0 =+TPAwith rnerhanol extract of Atractylodis Rhizoma.
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Fig. 3. Inhibitory effect of atractylon on the tumor promotion of skin papillomas by TPAin DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 Jlg of DMBA, 1 Jlg of TPA was appliedtwiceweekly. Topicalapplication of atractylon (2umol) and vehicle was performed 30 min before each TPAtreatment. Data are expressedas percentage of mice bearing papillomas (A), and as average numbers of papillomasper mouse (B) . • =+TPAwith vehicle alone; 0 =+TPAwith atracrylon.
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Inhibitory effect of atracty/on against TPA-induced inflammation in mice: As table 1 shows, the 50 % inh ibitory dose of atractylon for TPA-induced inflammatory ear edema was 0.9 mg/ear. Application of the sample completely inhibited TPA-induced inflammation and this inhibitory activity was dose-dependent. By comparison with standard drugs, this compound was a less effective inhibitor than hydrocortisone and indomethacin, but was more effective than quercetin, a known inhibitor of tumor promotion. Atractylon inhibited TPA-induced inflammation in mice to a similar extent as cepharanthine, a known inhibitor of tumor promotion and inflammation.
Inhibitory effect of atracty/on on the tumor-promoting activity ofTPA: Figure 3A illustrates the time course of skin tumor formation in the groups treated with DMBA plus TPA, with or without atractylon. The first tumor appeard during week 6 and all mice had tumors by week l O in the group treated with DMBA plus TPA. In the group treated with DMBA plus TPA and Zurnol atractylon, the first tumor appeared during week 8. The percentage of tumor-bearing mice treated with DM BA plus TPA was 100 % at week 20, whereas that in the group trea ted with DMBA plu s TPA and 21lmol atractylon was 33 %. Figure 3B sho ws the aver -
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Weeks of promoti on
age number of tumors per mouse. The group treated with DMBA plus TPA produced 10.4 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and 21lmol atractylon had 0.4 tumors per mouse. Treatment with 2 1lmol atractylon therefore caused a 96 % reduction in the average number of tumors per mouse at week 20.
Discussion Our previous studies have demonstrated that extracts from edible plants and crude drugs can inhibit TPA-induced inflammatory ear edema, and have identified active components separated from hop and stevia (Yasukawa, et al., 1993b). Furthermore, plant constituents, such as flavonol glycosides (Yasukawa, et al., 1990), sterols and triterpenes (Yasukawa, et al., 1991a), lignan (Yasukawa, et aI., 1992), and alkaloids (Yasukawa, et aI., 1988a; Yasukawa, et al., 1989; Yasukawa, et al., 1991b; Yasukawa, et aI., 1993) have been shown to inhibit tumor promotion during two-stage carcinogenesis in mouse skin. The traditional Chinese medicines (Kampo medicines in Japanese) are today frequently used to treat human cancer as complementary drugs in Japan. The major uses of the Kampo medicines Rikkunshi-to and Zyuzen-taiho-to are in the treatment of immunosuppression caused by anti-cancer
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drugs, and poor health after operations. Atractylodis Rhizoma, one of the crude drugs used, is of importance in the formulation of Kampo medicines. Oral administration of Zyuzen-taiho-to slightly suppressed TPA-induced tumor promotion in two-stage carcinogenesis in mouse skin (Haranaka, et aI., 1987).
Discussion The methanol extract of Atractylodis Rhizoma markedly suppressed tumor promotion by TPA in DMBA-initiated mice. The active compound, atractylon (one of sesquiterpenes), was isolated from the extract and was found to have an inhibitory effect against TPA-induced inflammation in mice. By comparison with standard drugs, this compound was a less effective inhibitor than hydrocortisone and indomethacin, but was more effective than quercetin, a known inhibitor of tumor promotion. In addition, atractyIon markedly inhibited TPA-induced tumor promotion in mice. By comparison with known inhibitors on tumor promotion, atractylon was similar in activity to cepharanthine (Yasukawa, et aI., 1993a), azaphilones (Yasukawa, et aI., 1994), and more effective than quercetin (Yasukawa, et aI., 1988b). Other sesquiterpenes, warburganal and polygodial from the fresh sprouts of Polygonum hydropiper L., were previously shown to inhibit tumor promotion on two-stage carcinogenesis in mouse skin (Matsumoto and Tokuda, 1990). Atractylodis Rhizoma is used in combined Kampo prescriptions, it is of interest from the viewpoints of both Chinese traditional medicine and pharmacognostical science that such sesquiterpenes have inhibitory activity against tumor promotion of carcinogenesis. Acknowledgements This study was supported in a part by the research funds from "Traditional Oriental Medical Science Program" of the Public Health Bureau of Tokyo Metropolitan Gavament. We acknowledge the support in a part by Interdisciplinary General Joint Research Grant for Nihon University.
References Haranaka, R., Kosoto, H., Hirama, N., Hanawa, T., Hasegawa, R., Hyun, S., Nakagawa, S., Haranaka, K., Satomi, N., Sakurai, A., Yasukawa, K., Takido, M.: Antitumor activities of Zyuzen-taiho-to and Cinnamoi Cortex. [. Med. Pharmaceut. Soc. Wakan- Yaku 4: 49-58, 1987. Hikino, H., Hikino, Y., Yoshioka, I.: Studies on the constituents of Atractylodes. IX. Structure and autoxidation of atractylon, Chem. Pharm. Bull. (Tokyo) 12: 755-760, 1964. Kiso, Y, Tohkin, M., Hikino, H.: Antihepatotoxic principles of Atractylodes rhizomes. J. Natl. Prod. 46: 651-654, 1983. Kiso, Y., Tohkin, M., Hikino, H.: Validity of the oriental medicines. 72. Liver-protective drugs. 17. Mechanism of antihepatoxic activity of atractylon I: effect on free radical generation and lipid peroxidation. Planta Med. 51: 97-100, 1985.
Kubo, M., Nogami, M., Nishimura, M., Moriura, T., Arichi, S.: Studies of crude drugs about its origin, process and quality. I. The preventive effects of chinese crude drug "Zhu" on experimental stomach ulcer and its pharmacological evaluation. (1). Yakugaku Zasshi 103: 442-448, 1983. Matsumoto, T., Tokuda, H.: Antitumor-promoting activity of sesquiterpene isolated from an herbal spice. In "Antimutagenesis and Anticarcinogenesis Mechanisms II," Kuroda, Y., Shankel, D.M., Waters, M.D.: (eds) Plenum Press, New York, pp. 423-427,1990. Namba, T.: "Colored Illustrations of Wakan-Yaku," Vol. 1, Hoikusha Publ. Co., Osaka, p. 48, 1980. Nishikawa, Y., Watanabe, Y., Seto, T., Yasuda, I.: Studies on the components of Atractylodes. I. New sesquiterpenoids in the rhizome of Atractylodes lancea. De Candolle. Yakugaku Zasshi 96: 1089-1093, 1976. Sakurai, T., Yamada, H., Saito, Y., Kano, Y.: Enzyme inhibitory activities of acetylene and sesquiterpene compounds in Atractylodes Rhizoma. Bioi. Pbarm. Bull. (Tokyo) 16: 142-145, 1993. Takitani, S.: (ed.) "The Pharmacopoeia of Japan," Twelfth edition, Yakuji Nippo, Ltd., Tokyo, p. 596, 1991. Yasukawa, K., Akasu, M., Takeuchi, M., Takido, M.: Bisbenzylisoquinoline alkaloids inhibit tumor promotion by 12-0-tetradecanoylphorbol13-acetate in two-stage carcinogenesis in mouse skin. Oncology 50: 137-140, 1993a. Yasukawa, K., Ikeya, Y, Mitsuhashi, H., Iwasaki, M., Aburada, M., Nakagawa, S., Takeuchi, M., Takido, M.: Gomisin A inhibits tumor promotion by 12-0-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Oncology 49: 68-71, 1992. Yasukawa, K., Takahashi, M., Natori, S., Kawai, K., Yamazaki, M., Takeuchi, M., Takido, M.: Azaphilones inhibit tumor promotion by 12O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mice. Oncology 51: 108-112, 1994. Yasukawa, K., Takido, M., Matsumoto, T., Takeuchi, M., Nakagawa, S.: Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis. Oncology 48: 72-76, 1991a. Yasukawa, M., Takido, M., Takeuchi, M., Akasu, M., Nakagawa, S.: Inhibitory effect of cepharanthine and its related compounds on 12-0tetradecanoylphorbol-13-acetate-induced inflammation and inhibition of tumor promotion by cepharanthine in two-stage carcinogenesis in mice. Nihon Univ.]. Med. 31: 229-234, 1989. Yasukawa, K., Takido, M., Takeuchi, M., Akasu, M., Nakagawa, S.: Cepharanthine inhibits two-stage tumor promotion by 12-0-tetradecanoylphorbol-13-acetate and mezerein on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene. [, Cancer Res. Clin. Oncol.117:421-424,1991b. Yasukawa, K., Takido, M., Takeuchi, M., Nakagawa, S.: Inhibitory effect of glycyrrhizin and caffeine on two-stage carcinogenesis in mice. Yakugaku Zasshi 108: 794-796, 1988a Yasukawa, K., Takido, M., Takeuchi, M., Nitra, K.: Effect of flavonoids on tumor promoter's activity. In "Plant flavonoids in biology and medicine II: Biochemical, cellular, and medicinal properties," by Cody, V., Middleton, E., Jr., Harborne, J.B., Beretz, A., (eds) Alan R. Liss, New York,pp. 247-250, 1988b. Yasukawa, K., Takido, M., Takeuchi, M., Sato, Y., Nitta, K., Nakagawa, S.: Inhibitory effect of flavonol glycosides on 12-0-tetradecanoylphorbol-13-acetate-induced tumor promotion. Chem. Pharm. Bull. (Tokyo) 38: 774-776,1990. Yasukawa, K., Yamaguchi, A., Arita, J., Sakurai,S., Ikeda, A., Takido, M.: Inhibitory effect of edible plant extracts on 12-0-tetradecanoylphorbol-13-acetate-induced ear oedema in mice. Phytother. Res. 7: 185-189,1993b.
Address Ken Yasukawa, Ph. Do, College of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274, Japan.
Atractylodis Rhizoma Extract and Its Component, Atractylon, Inhibit Tumor Promotion in Mouse Skin Two-Stage Carcinogenesis S. YU, K. YASUKAWA* and M. TAKIDO College of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274, Japan
Summary The methanol extract of Atractylodis Rhizoma, a traditional Chinese medicine, was found to have antitumor-promoting activity in two-stage carcinogenesis. From the active fraction of the extract, atractylon was isolated. The isolated compound showed inhibitory activity against 12-0tetradecanoylphorbol-13-acetate (TPA)-induced ear inflammation in mice. Furthermore, atractyIon markedly inhibited tumor promotion by TPA following 7,12-dimethylbenz[a]anthracene initiation in mice. Key words: Atractylon; Atractylodis Rhizoma; Antitumor promotion; Two-stage carcinogenesis; Anti-inflammatory activity.
Introduction Atractylodis Rhizoma, the rhizome of Atractylodes [aponica Koidzumi, is used for the treatment of pain, gastritis and enteritis (Namba, 1980). The pharmacological action of Atractylodis Rhizoma includes inhibition of stress ulceration of the stomach (Kubo, et aI., 1983). Atractylon, the major essential oil component of Atractylodis Rhizoma, has been shown to have a significant preventive effect against carbon tetrachloride- and D-galactosamine-induced lesions in primary-cultured rat hepatocytes (Kiso, et aI., 1983; 1985). In the present study, the methanol extract from Atractylodis Rhizoma was found to inhibit TPA-induced tumor promotion during two-stage carcinogenesis in mice. Atractylon (fig. 1) was isolated from the active fraction. Furthermore, atractylon was found to inhibit tumor promotion markedly in two-stage carcinogenesis in mice.
Fig. 1. Chemical structure of atractylon,
Materials and Methods Instruments: Melting points were determined on a Yanagimoto micro-melting point apparatus and are presented uncorrected. NMR spectra were measured with a JEOL JNM-GSX 400 (tH, 400 MHz; BC, 100 MHz) spectrometer, Japan, and chemical shifts are presented as 0 values relative to tetramethylsilane (TMS) as an internal standard. Mass spectra (MS) were measured with a Hitachi M-2000 double-focusing mass spectrometer, Japan, at an ionization voltage of 70 eV. Chemicals: 7,12-Dimethylbenz[a]anthracene (DMBA) was purchased from Sigma Chemical Co., St. Louis, USA. TPA was obtained from Chemicals for Cancer Research Inc., Chicago, USA. Column chromatography (CC) was carried out on silica gel (E. Merck, Germany). Materials: Atractylodis Rhizoma (the rhizome of Atractylodes [aponica Koidzumi) was obtained from the market in Tokyo. This was identified as the rhizome of Atractylodes [aponica by description, chemical analysis and purity test (Namba, 1980; Takitani, 1991). Extraction from Atractylodis Rhizoma: One hundred grams of Atractylodis Rhizoma was extracted by refluxing with methanol for 3 h. The organic solvent was evaporated in vacuo to dryness. The yield of MeOH extract was 18.9 g.
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One hundred grams of Atractylodis Rhizoma was extracted by refluxing with water for 3 h. The solvent was freezed to dryness. The yield of water extraxt was 12.1 g. Isolation of active agent from the methanol extract of Atractylodis Rhizoma: Ten grams of the methanol extract of Atractylodis Rhizoma was separated using a n-hexanemethanol-water mixture (19:19:2). Yields were: n-hexanesoluble fraction: 4.49 g; methanol-water-soluble fraction: 4.89 g. The n-hexane-soluble fraction was then subjected to CC on silica gel using n-hexane as a solvent to obtain six fractions. The inhibition ratio of the fractions were showed in table 1 on TPA-induced inlfammatory ear edema. The active agent (I) was isolated from the potent inhibitory fraction (5, yield: I = 718 mg) and was identified as atractylon by standard physical and chemical procedures and comparison with an authentic sample (Hikino, et aI., 1964; Nishikawa, et aI., 1976). Animals: Female ICR mice were obtained from japan SLC, Shizuoka, japan, and housed in an air-conditioned room (22-23 "C) lit from 8:00 to 20:00. Food and water were available ad libitum. Assay ofTPA-induced inflammation in mice: TPA (1 nM) dissolved in acetone (20 Ill) was applied to the right ear of each mouse with a micropipette. A volume of 10 III was delivered to both the inner and outer surfaces of the ear. Twenty microliters of the sample solution, their vehicles, methanol-water (1:1) or chloroform-methanol (1:1) (as a control) was applied topically about 30 min before each TPA treatment. For ear thickness determinations, a pocket thickness gauge (Mitsutoyo Co., Ltd., Tokyo, japan) with a range of 0-9 mm, graduated at O.Ol-mm intervals and modified so that the contact surface area was increased, thus reducing the tension, was applied to the tip of the ear. Ear thickness was determined at before treatment (a). Edema was measured 6 h after TPA treatment (b: TPA alone; b': TPA plus sample). The following values were then calculated: Edema A: edema induced by TPA alone (b-a). Edema B: edema induced by TPA plus sample (b'-a). Edema A - Edema B Inhibition ratio (%) = X 100 Edema A Each value was the mean of individual determinations from 5 mice. The 50 % inhibitory dose (ID so) values were determined by pro bit-graphic interpolation for four dose levels. Two-stage carcinogenesis experiments: Three groups of 15 mice underwent initiation by application of 50llg of DMBA in acetone (100 Ill) to the dorsal skin. Promotion with Lug of TPA in acetone (100 Ill), applied twice weekly, was begun 7 days after the initiation. Atractylon (2 umol), or the methanol extract (2 mg) of Atractylodis Rhizoma and their vehicle, acetone-dimethylsulfoxide-water (18:1:1, 100 Ill) were applied topically 30 min before TPA treatment. These treatments were continued for 20 weeks.
Statistical analysis: Statistical analysis was performed by Student's t-test.
Results
Inhibition by the methanol extract ofAtractylodis Rhizorna on TPA-induced inflammatory ear edema and TPA-induced tumor promotion: The methanol extract of Atractylodis Rhizoma markedly inhibited the inflammation induced by TPA in mice (table 1). Figure 2A illustrates the time course of skin tumor formation in the groups treated with DMBA plus TPA, with or without the methanol extract from Atractylodis Rhizoma. The first tumor appeared at week 6 and all mice had tumors by week 9 in the group treated with DMBA plus TPA. In the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma, the first tumor appeared during week 10. The percentage of tumor-bearing mice treated with DMBA plus TPA was 100 % at week 20, whereas that in the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma was 33 %. Figure 2B shows the average number of tumors per mouse. The group treated with DMBA plus TPA produced 10.4 tumors per mouse at week 20, whilst the group treated with DMBA plus TPA and the methanol extract of Atractylodis Rhizoma had 0.6 tumors per mouse. Treatment with the methanol extract of Atractylodis Rhizoma therefore caused a 94 % reduction in the average number of tumors per mouse at week 20. Table1. Inhibitory effect of atractylon and extracts from Atractylodis Rhizoma on TPA-induced inflammation in mice. Name Water extract Methanol extract Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Atractylon Quercetin Indomethacin Hydrocortisone
IDso (mg/ear)
I.R. 43*1 80*1 29*1 30*1 60*1 77*1 82*1
91 0.9 1.6 0.3 0.03
82*1 40*2 96*2 99*2
The samples were applied 30 min before TPA treatment; ear edema was determined at 6 h after TPA treatment. *p < 0.01 by Student's t-test as compared to the control group. I.R.: Inhibition ratio (at 12 mg/ear; 21 mg/ear).
Investigation of atractylon from the methanol extract of Atractylodis Rhizoma for inhibitory activity against TPAinduced ear edema: The component active against TPA-induced inflammatory ear edema was separated from the methanol extract of Atractylodis Rhizoma, and was identified as atractylon (Table 1). Inhibitory effect of fractions 3
Atractylodis Rhizoma Extract and Its Component
Fig. 2. Inhibitory effect of methanol extract of Atractylodis Rhizoma on the tumor promotion of skin papillomas by TPAin DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 ug of DMBA, 1 Jlg of TPA was applied twiceweekly. Topicalapplication of methanol extract (2mg) of Atractylodis Rhizoma and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A),and as average numbers of papillomas per mouse (B) . • =+TPAwith vehicle alone; 0 =+TPAwith rnerhanol extract of Atractylodis Rhizoma.
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Fig. 3. Inhibitory effect of atractylon on the tumor promotion of skin papillomas by TPAin DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 Jlg of DMBA, 1 Jlg of TPA was appliedtwiceweekly. Topicalapplication of atractylon (2umol) and vehicle was performed 30 min before each TPAtreatment. Data are expressedas percentage of mice bearing papillomas (A), and as average numbers of papillomasper mouse (B) . • =+TPAwith vehicle alone; 0 =+TPAwith atracrylon.
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Weeks of promoti on
and 4 was due to atractylon in fraction . Full details of the isolation and identification, and the spectral data, are available on request from the author for correspondence.
Inhibitory effect of atracty/on against TPA-induced inflammation in mice: As table 1 shows, the 50 % inh ibitory dose of atractylon for TPA-induced inflammatory ear edema was 0.9 mg/ear. Application of the sample completely inhibited TPA-induced inflammation and this inhibitory activity was dose-dependent. By comparison with standard drugs, this compound was a less effective inhibitor than hydrocortisone and indomethacin, but was more effective than quercetin, a known inhibitor of tumor promotion. Atractylon inhibited TPA-induced inflammation in mice to a similar extent as cepharanthine, a known inhibitor of tumor promotion and inflammation.
Inhibitory effect of atracty/on on the tumor-promoting activity ofTPA: Figure 3A illustrates the time course of skin tumor formation in the groups treated with DMBA plus TPA, with or without atractylon. The first tumor appeard during week 6 and all mice had tumors by week l O in the group treated with DMBA plus TPA. In the group treated with DMBA plus TPA and Zurnol atractylon, the first tumor appeared during week 8. The percentage of tumor-bearing mice treated with DM BA plus TPA was 100 % at week 20, whereas that in the group trea ted with DMBA plu s TPA and 21lmol atractylon was 33 %. Figure 3B sho ws the aver -
20
5
10
15
20
Weeks of promoti on
age number of tumors per mouse. The group treated with DMBA plus TPA produced 10.4 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and 21lmol atractylon had 0.4 tumors per mouse. Treatment with 2 1lmol atractylon therefore caused a 96 % reduction in the average number of tumors per mouse at week 20.
Discussion Our previous studies have demonstrated that extracts from edible plants and crude drugs can inhibit TPA-induced inflammatory ear edema, and have identified active components separated from hop and stevia (Yasukawa, et al., 1993b). Furthermore, plant constituents, such as flavonol glycosides (Yasukawa, et al., 1990), sterols and triterpenes (Yasukawa, et al., 1991a), lignan (Yasukawa, et aI., 1992), and alkaloids (Yasukawa, et aI., 1988a; Yasukawa, et al., 1989; Yasukawa, et al., 1991b; Yasukawa, et aI., 1993) have been shown to inhibit tumor promotion during two-stage carcinogenesis in mouse skin. The traditional Chinese medicines (Kampo medicines in Japanese) are today frequently used to treat human cancer as complementary drugs in Japan. The major uses of the Kampo medicines Rikkunshi-to and Zyuzen-taiho-to are in the treatment of immunosuppression caused by anti-cancer
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drugs, and poor health after operations. Atractylodis Rhizoma, one of the crude drugs used, is of importance in the formulation of Kampo medicines. Oral administration of Zyuzen-taiho-to slightly suppressed TPA-induced tumor promotion in two-stage carcinogenesis in mouse skin (Haranaka, et aI., 1987).
Discussion The methanol extract of Atractylodis Rhizoma markedly suppressed tumor promotion by TPA in DMBA-initiated mice. The active compound, atractylon (one of sesquiterpenes), was isolated from the extract and was found to have an inhibitory effect against TPA-induced inflammation in mice. By comparison with standard drugs, this compound was a less effective inhibitor than hydrocortisone and indomethacin, but was more effective than quercetin, a known inhibitor of tumor promotion. In addition, atractyIon markedly inhibited TPA-induced tumor promotion in mice. By comparison with known inhibitors on tumor promotion, atractylon was similar in activity to cepharanthine (Yasukawa, et aI., 1993a), azaphilones (Yasukawa, et aI., 1994), and more effective than quercetin (Yasukawa, et aI., 1988b). Other sesquiterpenes, warburganal and polygodial from the fresh sprouts of Polygonum hydropiper L., were previously shown to inhibit tumor promotion on two-stage carcinogenesis in mouse skin (Matsumoto and Tokuda, 1990). Atractylodis Rhizoma is used in combined Kampo prescriptions, it is of interest from the viewpoints of both Chinese traditional medicine and pharmacognostical science that such sesquiterpenes have inhibitory activity against tumor promotion of carcinogenesis. Acknowledgements This study was supported in a part by the research funds from "Traditional Oriental Medical Science Program" of the Public Health Bureau of Tokyo Metropolitan Gavament. We acknowledge the support in a part by Interdisciplinary General Joint Research Grant for Nihon University.
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Address Ken Yasukawa, Ph. Do, College of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274, Japan.