Active ingredients of traditional Japanese (kampo) medicine, inchinkoto, in murine concanavalin A-induced hepatitis

Journal of Ethnopharmacology 127 (2010) 742–749

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Active ingredients of traditional Japanese (kampo) medicine, inchinkoto, in murine concanavalin A-induced hepatitis Akihito Mase a,d,∗ , Bunsho Makino b , Naoko Tsuchiya a , Masahiro Yamamoto a , Yoshio Kase a , Shuuichi Takeda c , Takaaki Hasegawa d a

Tsumura Research Laboratories, Tsumura & Co., Ami, Ibaraki 300-1192, Japan Botanical Raw Materials Research Dept., Tsumura & Co., Ami, Ibaraki 300-1192, Japan Analytical Technology Center, Tsumura & Co., Ami, Ibaraki 300-1192, Japan d Department of Hospital Pharmacy and Pharmacokinetics, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan b c

a r t i c l e

i n f o

Article history: Received 22 June 2009 Received in revised form 25 November 2009 Accepted 28 November 2009 Available online 3 December 2009 Keywords: Capillarisin Concanavalin A Hepatitis IFN-␥ Inchinkoto Nitric oxide

a b s t r a c t Aim of the study: The traditional Japanese (kampo) medicine inchinkoto (ICKT) is used in Eastern Asia as a choleretic and hepatoprotective agent. Previously, we reported that ICKT ameliorates murine concanavalin A (con A)-induced hepatitis via suppression of interferon (IFN)-␥ and interleukin (IL)-12 production. In the present study, we investigated the active ingredients of ICKT. Materials and methods: ICKT and extracts of its component herbs were fractionated, and their effects on liver injury and cytokine production in vivo (biochemical markers of liver injury and cytokine levels in serum) and in vitro (cytokine and nitrite production in the cultures of splenocytes and peritoneal macrophages). Results: Decoctions of component herbs, Artemisiae Capillari Spica (Artemisia capillaris Thunberg: ‘Inchinko’ in Japanese), Gardeniae Fructus (Gardenia jasminoides Ellis: ‘Sanshishi’) and Rhei Rhizoma (Rheum palmatum Linné: ‘Daio’) were administered orally. Inchinko and Sanshishi decreased serum transaminases and IFN-␥ concentrations. Examination of fractions of component herbs suggested that capillarisin, a component of Inchinko, has potent hepatoprotective activity in vivo. In in vitro studies, capillarisin and genipin, an intestinal metabolite of geniposide that is contained in Sanshishi, were examined. IFN-␥ production was significantly suppressed by capillarisin and genipin in con A-stimulated splenocyte culture. Genipin also suppressed IL-1␤, IL-6, and IL-12p70 synthesis. Capillarisin and genipin decreased nitrite release from IFN-␥-stimulated macrophages. Conclusions: These results suggested that both Inchinko and Sanshishi may contribute to the protective effects of ICKT against con A hepatitis. Capillarisin was found to be potently hepatoprotective, and genipin may also contribute, especially via modulation of cytokine production. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Since certain herbal medicines of pharmaceutical grade were approved as ethical drugs by the Ministry of Health and Welfare of Japan over 30 years ago, they have been used by physicians practicing Western medicine in Japan. Of these traditional Japanese herbal medicines, inchinkoto (abbreviated as ICKT) has been frequently administered to patients with various liver diseases, especially hepatic cirrhosis and jaundice. Many basic, experimental, and clinical studies on the effects of ICKT have been

∗ Corresponding author at: Tsumura Research Laboratories, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan. Tel.: +81 29 889 3857; fax: +81 29 889 2158. E-mail address: mase [email protected] (A. Mase). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.11.029

reported (Onji et al., 1990; Yamamoto et al., 1996, 2000; Itoh et al., 1997; Yamashiki et al., 2000). We previously showed that ICKT ameliorated concanavalin A (con A)-induced murine hepatitis through the suppression of serum IFN-␥ and IL-12 at 8 h after con A treatment (Yamashiki et al., 2000). Con A-induced hepatitis has been frequently used as a model of liver injury in which helper T cells, Kupffer cells, and NKT cells play important roles (Tiegs et al., 1992; Schumann et al., 2000; Takeda et al., 2000). In this experimental hepatitis model, IL-10, IL-12, and IL-2 are produced during the early stage and IFN-␥ is produced during the effector (late) stage after con A injection (Louis et al., 1997), and the Th1 immune response induced by IL-12 plays an important role in the pathogenesis (Kusters et al., 1996). IL-10 antagonizes the action of IL-12 as an anti-inflammatory cytokine. Thus the effect of ICKT on IFN-␥ and IL-12 suggests that ICKT contains active compound(s) whose hepatoprotective effects are

A. Mase et al. / Journal of Ethnopharmacology 127 (2010) 742–749

mediated via suppression of the production of proinflammatory/Th1 cytokines. ICKT consists of three crude drugs: Artemisiae Capillari Spica (Artemisia capillaris Thunberg: ‘Inchinko’ in Japanese), Gardeniae Fructus (Gardenia jasminoides Ellis: ‘Sanshishi’) and Rhei Rhizoma (Rheum palmatum Linné: ‘Daio’). To identify anti-inflammatory substances with IL-12 and IFN-␥ modulating ability, we examined ICKT component herbs, their fractions, and ingredients for their hepatoprotective and cytokine modulating activities using the in vivo con A-induced hepatitis model and in vitro immune cell culture. The results provide insights into the ingredients responsible, at least partly, for the beneficial effects of ICKT and raise the possibility of developing new pharmacotherapeutic strategies targeting inflammatory liver diseases. 2. Materials and methods 2.1. Mice and reagents Five-week-old male BALB/c mice were purchased from Charles River Japan (Tokyo, Japan). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals proposed by the National Institute of Health. Con A, dexamethasone (dexa) and prednisolone (pred) were purchased from Sigma Chemical Co. (St. Louis, MO). Tween 80 was from Tokyo Kasei Co. (Tokyo, Japan). Con A was dissolved in sterilized phosphate-buffered saline (PBS), and dexa was dissolved in 1% Tween 80. Genipin and dimethyl sulfoxide (DMSO) were purchased from Wako Pure Chemical Industries (Osaka, Japan). The preparation ICKT (TJ-135) was industrially prepared by Tsumura & Co. as a spray-dried powder from a hot-water extract composed of three crude drugs in fixed proportions (expressed in dry weight corresponding to a daily intake of the medicine for an adult): 4.0 g Artemisia Capillari Spica (Inchinko in Japanese), the spike of Artemisia capillaris Thunberg (Shikoku-region and Nagano prefecture, Japan), 3.0 g Gardeniae Fructus (Sanshishi in Japanese), the fruit of Gardenia jasminoides Ellis (Sichuan province, China) and 1.0 g Rhei Rhizoma (Daio in Japanese), the rhizome of Rheum palmatum Linné (Sichuan and Qinghai Prov., China). Spraydried extract powders of the componental crude drugs, Artemisiae Capillari Spica (Lot No. 2001085010), Gardeniae Fructus (Lot No. 251044010), and Rhei Rhizoma (Lot No. 2991028010) were also prepared and provided by Tsumura & Co. In in vivo experiments, serum levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using an automatic analyzer 7070 (Hitachi Ltd., Tokyo, Japan). The levels of nitrate/nitrite and various cytokines in the samples were determined using commercially available assay kits (Cayman Chemical Company, Ann Arbor, MI; Biosource International Inc., Camarillo, CA). In in vitro experiments, multiple cytokine assays were performed using a Bio-Plex cytokine assay kit (Bio-Rad Laboratories, Tokyo, Japan). Cell proliferation tests (viability) were performed using Celltiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corp., Madison, WI). 2.2. Fractionation of the prescription ICKT and its component crude drug extract powder In order to study the active ingredients of ICKT, the fractionations according to the polarity of ingredients were performed using an adsorption chromatography. Prior to the fractionation, the contaminant of the crude drug residue and high molecular weight components were separated by 80% ethanol-precipitation method as follows: the extract powder (1.0 kg) of the prescrip-

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tion ICKT was suspended in water (4 L) and ethanol (16 L) was then added into the suspension and stirred for 1 h. The precipitate was filtered through a filter paper and washed with 80% ethanol. The residue was further precipitated by 80% ethanol in a same manner followed by filtration and lyophilization (P fraction). The combined solutions were concentrated under reduced pressure and then subjected to an adsorption chromatography with a porous polymer gel (DIAION HP20, Mitsubishi Chemical Co., Japan, 4.0 kg), and eluted with 20 L of water (W-fraction, containing polar components such as sugars and amino acids), 50% aqueous methanol (50 M-fraction, containing mid-polar components such as glycosides), and methanol (100 M-fraction, containing many lipophilic components), successively (Fig. 3). These eluates were concentrated under reduced pressure followed by lyophilization (W- and 50 M-fractions) or drying under reduced pressure (100 M-fraction) to afford the fraction samples for the pharmacological study. The yields of the fractions, P, W, 50 M, and 100 M, were 29.1, 37.4, 21.2, and 10.6% (w/w), respectively, vs. dry weight of ICKT. The fractionation procedure was also applied for the extract powders of the componental crude drugs, Artemisiae Capillari Spica (Lot No. 2001085010) and Gardeniae Fructus (Lot No. 251044010), in a half scale (500 g). The yields (in % (w/w) vs. dry weight of extract powder) of each fraction of Artemisiae Capillari Spica and Gardeniae Fructus were as follows: P (33.2 and 36.9), W (40.1 and 27.4), 50 M (12.5 and 23.5), and 100 M (14.4 and 9.3), respectively. 2.3. Protocols of in vivo experiments ICKT, crude drugs, separated fractions, and ingredients were suspended in 1% Tween 80 and orally administered to the mice except for the dose-dependency examination of capillarisin. Administration schedules for the respective drugs are given in the figure legends. The tested doses of ICKT have been selected from the previous studies which have shown various beneficial effects of ICKT in rodent models (Yamashiki et al., 2000). The doses of component herbs and fractions have been calculated from the effective dose of ICKT according to the ratio of the mixed herbs. After 20 mg/kg bodyweight of con A was injected via the BALB/c tail vein, and blood samples were directly collected from the heart at various time points under ether anesthesia. Sera were separated by centrifuging blood samples at 900 × g for 10 min at 4 ◦ C and stored at −80 ◦ C until analysis. 2.4. In vitro assessments using splenocytes and peritoneal macrophages Splenocytes and peritoneal macrophages were isolated using conventional procedures (Nemoto et al., 1999; Byun et al., 2006). Cells were cultured in RPMI medium 1640 (Gibco Laboratories, Grand Island, NY) containing 2 mmol/L glutamine, 1% penicillin–streptomycin solution (Sigma Chemical Co.), 50 ␮mol/L 2-mercaptoethanol (Sigma Chemical Co.), and 10% heat-inactivated fetal bovine serum (Gibco Laboratories). Herbal ingredients were dissolved in DMSO and diluted with the RPMI medium to the desired concentrations, and then sterilized by filtering through 0.22 ␮m filters (Millipore, Bedford, MA). Splenocytes were adjusted to a concentration of 5 × 106 cells/ml and plated onto 96-well microplates (Beckton Dickinson Labware, Lincoln Park, NJ). At the same time, con A (final concentration: 5 ␮g/ml) or culture medium, 20 ␮L of 0.5% DMSO (final concentration: 0.05%) or 20 ␮L of the test sample was added. After 24 h culture, the culture supernatant was collected and stored at −80 ◦ C until analysis. BALB/c mice were injected i.p. with 2.5 ml of 4.05% thioglycolate medium I (Eiken Chemicals Co., Tokyo, Japan). After 4 days, peri-

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12 h, and ICKT showed potent hepatoprotective activity in a dosedependent manner (Fig. 1). 3.2. Effects of component herbs of ICKT on con A-induced hepatitis ICKT consists of three component herbs: Artemisiae Capillari Spica, Gardeniae Fructus, and Rhei Rhizoma (ratio 4:3:1). The appropriate doses at this ratio of decoctions were orally administered to mice at 42, 18, and 1 h before con A injection, and serum levels of AST, ALT, and IFN-␥ were measured after 12 h. Among these three crude drugs, Artemisia Capillaris Spica (Inchinko) potently and Gadenia Fructus (Sanshishi) slightly suppressed transaminases. Inchinko and Sanshishi decreased blood levels of IFN-␥ (Fig. 2) significantly. Rhei Rhizoma (daio) showed neither effect, therefore, we have excluded this herb from the target of subsequent investigation. 3.3. Effects of W, 50 M, and 100 M fractions of Inchinko and Sanshishi on con A-induced hepatitis In a preliminary study, the effects of four fractions (P, W, 50 M, and 100 M) of ICKT on con A-induced hepatitis were examined. In

Fig. 1. Effect of ICKT on con A-induced hepatitis in mice. For 7 days before the intravenous injection of PBS or con A, 1% Tween 80 was administered orally every day to the normal and control groups and inchinkoto (ICKT) or dexamethasone (dexa) treatment groups. Mice were sacrificed 12 h after con A or PBS treatment. Serum parameters were analyzed as described in Section 2. LDH, lactose dehydrogenase activity (IU = international unit); AST, aspartate aminotransferase (IU); ALT, alanine aminotransferase (IU). Each group included 8–12 mice. Statistical significance from control at *p < 0.05 and **p < 0.01, respectively. Each value represents mean ± S.E.M. The effect of dexamethasone as a positive control is also shown.

toneal exudate cells (PEC) were adjusted to the concentration of 2 × 106 cells/ml and plated on 96-well microplates. Murine IFN-␥ (Toyobo Co. Ltd., Osaka, Japan) was added to the final concentration of 1500 U/ml. Test samples were added immediately as described above. After 24 h culture, the culture supernatant was collected and stored at −80 ◦ C. 2.5. Statistical analysis All values were expressed as the mean ± S.E.M. Data were assessed by one-way ANOVA, and the post hoc test was performed by comparing the results of the treatment groups with those of the control using Dunnett test. p < 0.05 was considered significant. 3. Results 3.1. Effects of ICKT on con A-induced hepatitis ICKT (500, 1000, or 2000 mg/10 ml/kg) and dexa (1 mg/10 ml/kg) were administered orally to mice once a day for 7 days before con A injection. Serum levels of LDH, AST, and ALT were examined at

Fig. 2. Effect of ICKT component herbs on con A-induced hepatitis in mice. Fortytwo, eighteen and one hour before injection of PBS or con A, 1% Tween 80 was administered orally to the normal and control groups, each crude drug, and dexamethasone (dexa) was administered to respective treatment groups. Administration doses were related to the composition ratio of ICKT. Dexa was given p.o. in a dose of 0.5 mg/kg body weight. Mice were sacrificed 12 h after con A or PBS treatment. Serum parameters were analyzed as described in Section 2. Inchinko, Artemisia cappilari spica, Sanshishi, Gardenia Fructus; Daio, Rhei Rhizoma; AST, aspartate aminotransferase (IU); ALT, alanine aminotransferase (IU). Each group included 8–12 mice. Statistical significance from control at *p < 0.05 and **p < 0.01, respectively. Each value represents mean ± S.E.M. The effect of dexamethasone as a positive control is also shown.

Forty-two, eighteen, and one hour before intravenous injection of PBS or con A, 1% Tween 80 was administered orally to the normal and control groups, and each fraction of Inchinko and Sanshishi was administered to respective treatment groups. Administration doses were related to fractionation yield ratios of Inchinko and Sanshishi in ICKT. Prednisolone was given p.o. in a dose of 5 mg/kg body weight. Mice were sacrificed 8 h after con A or PBS treatment. Serum parameters (AST and ALT), cytokines and NO (nitrate/nitrite) levels were analyzed as described in Section 2. The effect of pred as a positive control is also shown. Each group included 7–12 mice. * Statistical significance from control at p < 0.05. Each value represents mean ± S.E.M. ** Statistical significance from control at p < 0.01. Each value represents mean ± S.E.M.

± ± ± ± ± 487.3 314.5 197.1 36922.0 168.4 572.2 404.2 54.0 26232.0 120.6 79.4 120.2 55.4 1831.2 10.9 ± ± ± ± ± 505.5 328.5 308.8 33648.4 133.0 ± ± ± ± ± 634.9 697.2 201.5 41876.8 189.3 ± ± ± ± ±

14.8** 8.5 6.9* 0.0** 5.1**

781.2 727.7 262.7 39636.0 181.1

± ± ± ± ±

208.0 260.8 62.6 2578.1 14.6

539.2 333.7 257.5 35411.1 164.1

± ± ± ± ±

69.0 101.8 57.7 2132.8 14.1

50 M fr. 125 mg/kg

82.8 271.7 51.1 3020.8 25.5

378.5 ± 43.0* 198.0 ± 69.5 265.6 ± 63.6 36431.9 ± 2465.1 155.7 ± 30.6

Sanshishi

W fr. 206 mg/kg 100 M fr. 145 mg/kg

Inchinko

W fr. 400 mg/kg

1% Tween 80 (control) 1% Tween 80 (normal)

206.1 86.9 6.9 0.0 70.0 AST (IU/L) ALT (IU/L) Serum IL-12 (pg/ml) Serum IFN-␥ (pg/ml) Serum NO (␮M)

Fig. 4. Structural formulas of three major ingredients/metabolite of Inchinko (Artemisia Capillari Spica) 100 M and Sanshishi (Gardenia Fructus) 50 M fractions.

Concanavalin A

Next, we examined the effects of three components, capillarisin and 6,7-dimethylesculetin (DMEC) derived from Inchinko 100 M and geniposide derived from Sanshishi 50 M, on con A-induced hepatitis. The structural formulas of the three ingredients and genipin, a metabolic form of geniposide produced by intestinal bacteria, are shown in Fig. 4. The ingredients were administered orally to mice at 42, 18, and 1 h before con A injection. Among these three ingredients, capillarisin seemed to have the

PBS

3.4. Effects of components of Inchinko 100 M and Sanshishi 50 M on con A-induced hepatitis

Table 1 Effect of component herb fractionations for ICKT on con A-induced hepatitis and serum cytokines and NO levels in mice.

that study, the P fraction had no effect on con A-induced hepatitis (data not shown). Therefore, this study did not further examine the effect of the P fraction in con A-induced hepatitis. To investigate the hepatoprotective ingredients contained in ICKT, Artemisiae Capillari Spica (Inchinko) and Gardeniae Fructus (Sanshishi) were further fractionated according to the scheme demonstrated in Fig. 3. The W, 50 M, and 100 M fractions of Inchinko and Sanshishi were administered orally to mice at 42, 18, and 1 h before con A injection. The administration dosage was determined according to the fractionation yield. Serum levels of AST, ALT, IFN-␥, IL-12, and nitrate/nitrite were assessed at 8 h after con A injection. Among the fractions tested, a significant decrease in deaminase activities was observed only in AST in Inchinko 100 M-treated mice. However, blood levels of IFN-␥ and IL-12p70 were significantly decreased by Sanshishi 50 M (Table 1). Inchinko, Sanshishi, and the fractions of both herbs had no effect on blood nitrite/nitrate levels.

50 M fr. 176 mg/kg

Fig. 3. Fractionation scheme for ICKT and decoctions of its component herbs. Extracts of ICKT, Inchinko (Artemisia Capillari Spica) and Sanshishi (Gardenia Fructus) were fractionated, respectively, according to the same procedure indicated in the figure. The fractionation yields have been described in the text (Section 2.2).

± ± ± ± ±

54.3 80.5 6.19* 1415.0* 8.4

l00 M fr. 70 mg/kg

68.0 123.6 36.7 1223.3 16.9

282.0 62.8 42.0 14855.0 63.9

± ± ± ± ±

38.2** 12.2* 3.89* 2436.3** 11.6**

745

5 mg/kg

Prednisolone

A. Mase et al. / Journal of Ethnopharmacology 127 (2010) 742–749

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Table 2 Effects of capillarisin, genipin, and prednisolone on con A-induced cytokine production by cultured splenocytes (A) and IFN-␥-induced cytokines/NO (nitrite) production by cultured peritoneal macrophages (B). A

Splenocytes Concanavalin A Control

Capillarisin

Genipin

10−6 M IL-1␤ (pg/ml) IL-6 (pg/ml) TNF-␣ (pg/ml) IL-4 (pg/ml) IL-12p70 (pg/ml) IFN-␥ (pg/ml) B

2.73 26.36 9.62 0.44 2.41 17.34

± ± ± ± ± ±

2.20** 21.66** 5.38** 0.15** 2.22** 15.77**

38.03 210.08 116.03 138.65 47.72 1739.44

± ± ± ± ± ±

5.40 35.73 32.61 33.49 9.10 239.08

10−5 M

35.67 217.81 114.85 127.47 38.10 1468.94

± ± ± ± ± ±

35.71 195.26 111.64 129.41 39.09 1341.99

± ± ± ± ± ±

4.88 30.73 31.38 29.39 6.71 148.64

33.88 191.66 98.31 111.12 34.59 993.46

Prednisolone

10−6 M ± ± ± ± ± ±

4.03 39.73 29.42 22.67 6.61 132.67*

36.65 208.07 107.42 147.36 42.49 1644.58

± ± ± ± ± ±

4.59 35.43 29.49 34.19 7.94 248.15

10−5 M

10−4 M

10−6 M

31.89 ± 4.01 168.47 ± 29.23 98.34 ± 28.87 123.31 ± 25.53 37.40 ± 7.25 1369.82 ± 224.19

14.92 ± 2.63** 35.59 ± 6.59** 59.12 ± 15.17 73.67 ± 17.92 9.98 ± 2.60** 146.44 ± 38.23**

7.65 30.96 13.49 13.49 3.88 23.00

± ± ± ± ± ±

1.88** 4.96** 5.89* 2.35** 1.64** 8.08**

Macrophages Spon.

Murine IFN-␥ Control

Capillarisin −6

10 IL-12p70 (pg/ml) TNF-␣ (pg/ml) Nitrite (␮M)

5.95 48.15 32.86 34.29 7.53 271.72

10−4 M

0.25 ± 0.13 0.00 ± 0.00 0.00 ± 0.00**

3.46 ± 0.36 2.60 ± 1.78 2.40 ± 0.28

M

3.02 ± 0.37 0.54 ± 0.33 2.05 ± 0.31

Genipin −5

10

M

4.02 ± 1.26 3.40 ± 1.53 0.28 ± 0.18**

−4

10

M

1.82 ± 0.27 2.07 ± 1.26 0.00 ± 0.00**

−6

10

M

5.37 ± 1.12 2.35 ± 0.81 1.66 ± 0.49

Prednisolone −5

10

M

5.45 ± 0.78 4.49 ± 1.11 1.06 ± 0.44**

−4

10

M

2.20 ± 0.31 3.90 ± 1.79 0.00 ± 0.00**

10−6 M 0.76 ± 0.25 0.81 ± 0.42 0.00 ± 0.00**

A: Splenocytes were cultured in the absence (spontaneous) or presence (other groups) of con A. B: Peritoneal exudate cells (PEC) were isolated and cultured in the absence (Spon.) or presence (other groups) of IFN-␥. Ingredients (final concentrations 10−6 , 10−5 , 10−4 mol/L) or prednisolone (final concentration 10−6 mol/L) was added and the cells were cultured for 24 h. Cytokine and nitrite levels in the culture supernatant were measured as described in Section 2. * Statistical significance from control at p < 0.05. Each value represents mean ± S.E.M. ** Statistical significance from control at p < 0.01. Each value represents mean ± S.E.M.

A. Mase et al. / Journal of Ethnopharmacology 127 (2010) 742–749

Spon.

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Fig. 5. Effect of ingredients on con A-induced hepatitis in mice. Forty-two, eighteen, and one hour before intravenous injection of PBS or con A, 1% Tween 80 was administered orally to the control group and capillarisin, 6,7-dimethylesculetin (DMEC), geniposide (10 mg/kg body weight), or dexamethasone (dexa; 0.5 mg/kg body weight) to the respective treatment group. Mice were sacrificed 12 h after con A or PBS treatment. Serum parameters were analyzed as described in Section 2. AST, aspartate aminotransferase (IU); ALT, alanine aminotransferase (IU). Each group included 9–11 mice. Statistical significance from control at **p < 0.01. Each value represents mean ± S.E.M. The effect of dexamethasone as a positive control is also shown.

strongest suppressive activity on con A-induced hepatitis, though its suppressive activity was not significant at the dose of 10 mg/kg (Fig. 5). To clarify whether capillarisin has a hepatoprotective activity, the effects of different dosages of capillarisin administered i.p. at 42, 18, and 1 h before con A injection were examined. Capillarisin markedly decreased serum AST and ALT levels in a dose-dependent manner (Fig. 6). 3.5. Effects of herbal components on cytokine and nitrite production by splenocytes and peritoneal macrophages To obtain information about the mechanisms of action of capillarisin and genipin, their effects on various cytokine productions in in vitro splenocyte cultures after stimulation with con A were assessed (Table 2A). Both capillarisin and genipin significantly decreased IFN-␥ production. Moreover genipin significantly decreased IL-1␤, IL-6, and IL-12p70 production. Finally, using an in vitro culture of peritoneal exudate cells (more than 90% macrophages), we evaluated the effects of the ingredients on cytokine and nitrite productions after stimulation with murine IFN-␥. Peritoneal macrophages are one of the major cell populations that produce nitrite, TNF-␣, and IL-12. IFN-␥ stimulation gradually increased the levels of IL-12p70, TNF␣, IL-6, IL-1␤, and nitrite up to 24 h. Though capillarisin and genipin had no effect on the production of IL-12p70 and TNF-alpha, both capillarisin and genipin significantly and dose-dependently suppressed nitrite production at 24 h (Table 2B). The viability of peritoneal macrophages cultured with these ingredients was determined to be more than 90% by an MTS assay (data not shown).

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Fig. 6. Dose-dependent effect of capillarisin on con A-induced hepatitis in mice. PBS or con A was injected intravenously into mice in the control and treatment groups, respectively. Forty-two, eighteen, and one hour before intravenous injection of PBS or con A, 1% Tween 80 in saline was administered i.p. to the control group and each dose of capillarisin or prednisolone (pred; 5 mg/kg body weight) to the respective treatment group. Mice were sacrificed 12 h after con A or PBS treatment. Serum parameters were analyzed as described in Section 2. AST, aspartate aminotransferase (IU); ALT, alanine aminotransferase (IU). Each group included 8–13 mice. Statistical significance from control at *p < 0.05 and **p < 0.01, respectively. Each value represents mean ± S.E.M. The effect of prednisolone as a positive control is also shown.

4. Discussion and conclusions Con A-induced hepatitis is a model of liver injury developed by Tiegs et al. (1992), in which CD4+ T cells and/or NK T cells and Kupffer cells play important roles (Tiegs et al., 1992; Schumann et al., 2000; Takeda et al., 2000). In this model, large amounts of cytokines are released into the blood before or during the development of injury. Using ICR mice, we observed the kinetics of cytokines in the serum 2, 5, 8, and 12 h after the injection of con A (data not shown). TNF-␣ levels reached their peak at 2 h and decreased to an undetectably low level at 8 h. IFN-␥ gradually increased up to 12 h. Levels of IL-2, IL-4, and IL-12 reached their peaks between 2 and 5 h after con A injection but gradually decreased thereafter. IL-10 levels were maintained at high levels between 2 and 12 h. These findings were similar to those reported by Louis et al. (1997) using BALB/c mice. Many studies reported that these cytokines are involved in the pathogenesis of con A-induced hepatitis. In vivo analyses using recombinant cytokines, transgenes, neutralizing antibodies, or gene knockout revealed that the following cytokines were involved in the development of con A-induced hepatitis: TNF-␣ (Gantner et al., 1995; Trautwein et al., 1998), IFN␥ (Kusters et al., 1996; Nicoletti et al., 2000), IL-12 (Zhu et al., 2007), IL-18 (Faggioni et al., 2000), IL-27 (Siebler et al., 2008), IL-5 (Louis et al., 2002) and IL-4 (Toyabe et al., 1997) as hepatopathy-enhancing cytokines, IL-10 (Louis et al., 1997) IL-15 (Li et al., 2006), and IL11 as hepatopathy-inhibiting cytokines, and IL-6 (Mizuhara et al., 1996) as both. These findings suggested that the development of hepatitis could be inhibited by regulating the production of these cytokines. Previously, we reported that ICKT possibly ameriolated con Ainduced hepatitis through the suppression of IFN-␥ and IL-12p70

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(Yamashiki et al., 2000). In this study, to determine the active ingredients, the component herbs, their fractions, and ingredients were examined for their suppressive activity on liver injury and cytokine production in con A-induced hepatitis. Suppression of liver injury as estimated by transaminase activity was predominantly found in mice treated with the Inchinko 100 M fraction. The major ingredient of this fraction, capillarisin, had the most potent hepatoprotective activity, although the suppression of cytokine production was relatively modest both in vivo and in vitro. The Sanshishi 50 M fraction and genipin, the active metabolite of the major ingredient in this fraction, showed consistent and dose-dependent suppression of cytokine production, though a significant reduction of transaminase activity in vivo was not observed. Thus, the suppressions of liver injury and cytokine production apparently did not correspond to each other, and another mechanism of action must be involved in the protective effect of ICKT ingredients on con A-induced hepatitis. Here we hypothesized that the anti-apoptotic effect of capillarisin may play an important role. In a previous study, we screened several hundred herbal extracts for their possible inhibitory activity on transforming growth factor (TGF)␤1-induced liver cell apoptosis, and Inchinko was found to have most potent anti-apoptotic activity (Yamamoto et al., 1996). Of the components of Inchinko, capillarisin inhibited apoptosis. The cytoprotective effects of capillarisin in glycochenodeoxycholic acid-induced apoptosis (Lee et al., 2009) and tert-butyl hydroperoxide-induced oxidative damage (Chu et al., 1999) in rat primary hepatocytes have also been reported. In another our study, we demonstrated that Sanshishi and genipin inhibited Fas-induced murine lethal liver apoptosis in vivo (Yamamoto et al., 2000). In that model, Inchinko did not have protective effect. The discrepancy of these results, however, may be explained by a difference of the modes of apoptosis. Fas-mediated apoptosis also appears to be involved in con A-induced hepatitis; however, a blockade of Fas-mediated apoptotic pathways did not prevent con A-induced liver injury (Nakashima et al., 2008). TNF␣mediated signaling and/or perforin-mediated cell killing has been suggested to play predominant roles in con A-induced hepatitis (Ksontini et al., 1998; Tiegs et al., 1998). The difference in efficacy between capillarisin and genipin therefore suggests that these compounds exert anti-apoptotic effects via distinct mechanisms of action. Another possible mechanism of hepatoprotection by capillarisin may be the activating/inhibiting effects on transcription factors related to protection against oxidative stress. We previously showed that capillarisin and genipin induce cellular anti-oxidative enzymes such as ␥-glutamyl cysteine synthase, the major limiting factor of glutathione (GSH) synthesis and heme oxygenase-1 suppression (Lee et al., 2009). The effects of capillarisin and genipin were absent in embryonic fibroblasts prepared from nuclear factor E2 (Nrf2) knockout mice (Okada et al., 2006). This suggests that the compounds may exert their cytoprotective activities via activation of Nrf2, which binds antioxidant responsive element. Accordingly, Chu C-Y (Chu et al., 1999) reported that capillarisin stabilizes the GSH system and quenches free radicals. Further, capillarisin has been noted to suppress phorbol myristate acetate (PMA)-induced tumor invasion by inhibition of NF-␬B-dependent MMP-9 expression (Lee et al., 2008). In the present study, both capillarisin and genipin potently inhibited NO production in macrophages in a dose-dependent manner. NF-␬B is critically involved in the induction of NO synthetase in inflammatory situations, and NO released from Kupffer cells is suggested to exacerbate con A-induced hepatitis (Sass et al., 2001). Therefore, modulation of anti-oxidative and/or inflammatory transcription factors may play a role in the beneficial effects of capillarisin and genipin. These transcription factors have also been known to profoundly affect cytokine production, suggesting that the effect of ICKT ingredients on cytokine

production may be related to the modulation of these transcription factors. The effect of capillarisin on cytokine production is rather modest, and that of genipin is relatively potent but does not correspond to the hepatoprotection, as mentioned above. However, it must be noted that both compounds significantly suppress IFN-␥ production. It is well known that IFN-␥ is a critically involved in the pathogenesis of con A-induced hepatitis. IFN-␥ is produced by con A stimulation of the liver and various other cells, and subsequently, it directly induces hepatocyte apoptosis and Kupffer cell activation in the liver. Activated Kupffer cells produce various oxidative stress components (nitric oxide, superoxide anion, and hydrogen peroxide) that exacerbate liver injury (Nakashima et al., 2008). Capillarisin and genipin suppressed con A-induced IFN-␥ production from splenocytes and IFN-␥-induced nitrite formation from peritoneal macrophages, suggesting that the compounds may have a pleiotropic suppressive action on the pathogenic route mediated by IFN-␥. The effects may be insufficient to prevent liver injury by themselves, but they might play some adjunct role in the hepatoprotective effect of ICKT. The present study showed that a dose of 30 mg/kg of capillarisin is required to obtain significant hepatoprotection by systemic administration. The content of capillarisin in ICKT (approximately 0.1–0.5% in Inchinko) is therefore insufficient to explain the effect of ICKT in con A-induced hepatitis. However, our previous report showed that Inchinko contains various other ingredients that suppress TGF-␤1 apoptosis (Yamamoto et al., 1996) in addition to capillarisin. Further, although genipin by itself did not exert hepatoprotective activity in this model, the suppression of cytokine production by genipin may give adjunctive support for the hepatoprotective effect of Inchinko, as described previously. In conclusion, the present study revealed that Inchinko and Sanshishi may be responsible for the suppressive effect of ICKT on con A-induced hepatitis. The capillarisin contained in Inchinko and genipin derived from Sanshishi has been found to have suppressive effects on transaminase and cytokine productions in vivo, respectively. Capillarisin and genipin had modulating effects on in vitro cytokine/NO (nitrite) production. Clarifying the mechanisms of action of these two compounds may be not only useful for the clarification of the mechanism of ICKT in the amelioration of hepatitis but also contribute to the development of novel hepatitis therapeutic medicines, because they appear to show different hepatoprotective effects depending on the type of hepatitis model. Acknowledgements The authors are grateful to Dr. Masato Fukutake, Dr. Yoshiki Ikeda, and Dr. Masaru Sakaguchi of Tsumura & Co. for valuable comments and technical assistance. References Byun, J.A., Ryu, M.H., Lee, J.K., 2006. The immunomodulatory effects of 3monochloro-1,2-propanediol on murine splenocyte and peritoneal macrophage function in vitro. Toxicology In Vitro 20, 272–278. Chu, C.-Y., Tseng, T.-H., Hwang, J.-M., Chou, F.-P., Wang, C.-J., 1999. Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes. Archives of Toxicology 73, 263–268. Faggioni, R., Jones-Carson, J., Reed, D.A., Dinarello, C.A., Feingold, K.R., Grunfeld, C., Fantuzzi, G., 2000. Leptin-deficient (ob/ob) mice are protected from T cellmediated hepatotoxicity: role of tumor necrosis factor ␣ and IL-18. Proceedings of the National Academic Science United States of America 97, 2367–2372. Gantner, F., Leist, M., Lohse, A.W., Germann, P.G., Tiegs, G., 1995. Concanavalin Ainduced T-cell mediated hepatic injury in mice: the role of tumor necrosis factor. Hepatology 21, 190–198. Itoh, T., Shibahara, N., Mantani, N., Tahara, E., Shimada, Y., Terasawa, K., 1997. Effect of Kampo treatment on chronic viral hepatitis on the basis of traditional diagnosis. Journal of Traditional Medicines 14, 204–210. Ksontini, R., Colagiovanni, D.B., Josephs, M.D., Edwards III, C.K., Tannahill, C.L., Solorzano, C.C., Norman, J., Denham, W., Clare-Salzler, M., Mackay, S.L.D.,

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