Clinical and Pharmacological Studies on Liver Diseases Treated with Kampo Herbal Medicine

Clinical and Pharmacological Studies on Liver Diseases Treated with Kampo Herbal Medicine

Jong-Chol Cyong

Abstracts: Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which is linked to the development of liver cirrhosis and hepatocellular carcinoma. Most physicians who practice Kampo medicine in Japan have observed that Kampo medicine can be as effective as interferon therapy in the treatment of chronic hepatitis C. In the present study, to evaluate the effect of Kampo medicine on chronic hepatitis C, clinical treatment was assessed in short-term and long-term study, and it was shown that Ninjin-yoei-to (Formula ginseng compositae: TJ-108) was very effective. Therefore, to find the most active herbal component of TJ-108 in the treatment of HCV, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, which are specific to TJ-108, were screened using an in vitro HCV infection model. Among the three herbs, Schisandra Fruit was found to be most active. In the next step, Gomisin A, an active component of Schisandra Fruit, was studied using an in vitro model with MOLT-4 cells and an animal model of immunologically induced acute hepatic failures. It is concluded that the therapeutic effect of TJ-108 on chronic hepatitis C is from the inhibitory effect on HCV infection, and also from the protective effect on immunological hepatopathy of Schisandra Fruit and its lignan component, Gomisin A.

More than 170 million people are infected by Hepatitis C virus (HCV) world wide. While interferon is currently the most used single agent therapy, it is reported that interferon is successful in only up to 15% of patients in getting rid of the virus (Gish, 1999). A new effective therapeutic option is expected to be developed for treating HCV. Patients with chronic hepatitis C usually complain of systemic weakness, fatigue and loss of appetite (Luo et al., 1998). Most physicians who practice Kampo medicine have observed that Kampo medicine can easily improve these symptoms (Tajiri et al., 1991). We investigated the clinical effect of Kampo medicine on the course of chronic hepatitis C. In Asian countries, the Schisandra Fruit has traditionally been used for hepatitis and bronchitis. Gomisin A, a lignan component of Schisandra Fruit, has been reported to have hepatoprotective effect (Nomura et al., 1994; Shiota et al., 1996). We have found that Gomisin A has anti-viral-adhesion effect and anti-hepatitis effect, which were screened by using an in vitro model with MOLT-4 (human lymphoblastoma) and an animal model of immunologically induced acute hepatic failures (Arai et al., 1995).

Materials and Methods

Herbal Preparations and Isolation of Active Substances

Ninjin-yoei-to (Formula ginseng compositae: TJ-108), Juzen-taiho-to (Formula decem supplementrum major: TJ-48) and Dai-saiko-to (Formula bupleuri major: TJ-8) (Table 1) were obtained from Tumura Pharmaceutical Company (Tokyo, Japan) as commercial products.

Table 1. Composition of Ninjin-yoei-to, Juzen-taiho-to and Dai-saiko-to

Ninjin-yoie-to TJ- 108

9.0 g (one day dosage) contains 6.0 g of dried extract obtained

from mixed raw herbs in the following ratio:

Rehmannia Root 4.0g

Japanese Angelica Root 4.0g

Atractylodes Rhizome 4.0g

Hoelen 4.0g

Ginseng Root 3.0g

Cinnamon Bark 2.5g

Polygala Root 2.0g

Peony Root 2.0g

Citrus Unshiu Peel 2.0g

Astragalus Root 1.5g

Glycyrrhiza Root 1.0g

Schisandra Fruit 1.0g

Juzen-taiho-to TJ-48

7.5g (one day dosage) contains 5.0g of dried extract obtained from

mixed raw herbs in the following ratio:

Astragalus Root 3.05g

Cinnamon Bark 2.5g

Rehmannia Root 4.0g

Peony Root 2.0g

Cnidium Rhizome 3.0g

Atractylodes Lancea Rhizome 3.0g

Japanese Angelica Root 3.0g

Ginseng Root 3.0g

Hoelen 3.0g

Glycyrrhiza Root 1.5g

Dai-saiko-to TJ-8

7.5g (one day dosage) contains 4.5g of dried extract obtained from

mixed raw herbs in the following ratio:

Bupleurum Root 6.0g

Pinellia Tuber 4.0g

Scutellaria Root 3.0g

Peony Root 3.0g

Jujube Root 3.0g

Immature Orange 2.0g

Ginger Rhizome 1.0g

Rhubarb Rhizome 1.0g

Three herbs, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, specific to TJ-108 were purchased from Uchida Wakanyaku (Tokyo, Japan), and the water extracts of these three herbs were used for in vitro experiments to study the effect on the viral infection in MOLT-4 cells.

Isolated Gomisin A from Schisandra Fruit was obtained from Tumura Pharmaceutical Company (Tokyo, Japan) for in vitro experiments on immunologically-induced acute hepatic failure.

Subjects for Clinical Study

1) Thirty-four outpatients with chronic hepatitis C were enrolled at the Kampo Medicine Department, Kitasato Institute, Tokyo, for short term observation. The serum amount of HCV-RNA was measured by reverse transcriptase polimerase chain reaction (RT-PCR) before the administration of Kampo medicine. After six months treatment with Kampo medicine (oral administration disage as shown in Table 1), the amount of HCV-RNA was remeasured.

2) For long-term study, another 37 HCV carriers were observed for the change of viral titer every 6 months and for the symptoms and clinical courses.

Cell Cultures and RNA Extraction for RT-PCR

For three days, TJ-108 was administered (p.o. nine gm/day, 3 gm each time) to three healthy volunteers. Two weeks later, TJ-48 was administered to the same volunteers for three days (p.o. 7.5 gm/day, 2.5 gm each time). Finally, two weeks later, TJ-8 was administered to the same volunteers for three days (p.o 7.5 gm/day, 2.5 gm each time). Blood samples were collected after each treatment.

The method of detecting HCV infection was based on that of Shimizu et al. (1992). Firstly, human cell line MOLT-4 cells were incubated for 48 hrs in the culture media containing the 10% serum of previously Kampo-administrated healthy volunteers. Virus-positive serum was added to the cultured cells, and the cells were incubated at 37 [degrees] C for 2 hrs for absorption of the virus. After washing 4 times with PBS, RNA was extracted using an RNA extraction kit according to the manufacturer’s instruction (ISOGEN, Tokyo, Japan) for the following nested RT-PCR. In the other in vitro experiment, MOLT-4 cells were cultured in the medium with water extracts of three herbs, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, respectively, for 48 hrs followed by the same process of RNA extraction and RT-PCR as the previous one. And also, a different dose of Gomisin A, lignan component of Schisandra Fruit, was added to the culture media to confirm the anti-viral absorption effect in MOLT-4 cells. All experiments were done in triplicate.

Flow Cytometry for Anti-HCV

Intracellular viral antigen was stained by indirect fluorescent antibody technique using anti-HCV antibody. Monoclonal anti-HCV antibody that is specific to the peptide translated from the NS region of HCV-RNA was used as the first antibody, and FITC-labeled mouse anti-IgG was used as the second antibody. TJ-108 solution and viral serum were added to MOLT-4 cells, and the virus antigens in the cells were assayed by flow cytometry.

Immunologically-induced Acute Hepatic Failure

Immunologically-induced acute hepatic failure was induced according to the modified method of Ferluga and Allison (Koda et al., 1987; Iijima et al., 1995). Heat-killed propioni-bacterium acnes (P. acnes) (162 [micro]g/mouse) were injected intravenously into male ICR mice (n = 22, 35-40 g, 7 wks, Japan) and after 7 days, acute hepatic failure was induced by intraperitoneal injection of LPS (1 [micro]g/mouse). Each test sample suspended in saline containing 10% Tween 80 was intraperitoneally administered to the mice 90 mins before LPS injection. The survival rate of mice was compared until 24 hrs later with P. acnes-LPS-treated mice (control), which were injected with saline containing 10% Tween 80.

Results

Effects of Kampo Medicine on the Treatment of Chronic Hepatitis C

1) Short-term study

In the short-term study, out of 34 patients treated with different Kampo medicines, 8 showed a decrease of more than 10-2 in the amount of virus during the follow-up period (Table 2). Of these 8 cases, 6 patients had been treated with formula ginseng compositae (Ninjin-yoei-to: TJ-108). The change in the amount of the virus is shown in Figure 1. The age range of effective cases was 44 to 81 years, and the mean age was 61.2.

[Figure 1 ILLUSTRATION OMITTED]

Table 2. Changes of HCV-RNA Titer Due to Kampo Medicine (6 months)

Titer change serum HCV RNA No. of cases

decrease (< 10-2 x) 8

no change (10-2 x ~ 102 x) 24

increase (> 102 x) 2

2) Long-term study

We found that more than one year of administration of Kampo medicine might induce seroconversion. Therefore, we administered Kampo medicine, mainly TJ-108 (p.o., 9.0 gm/day, 3.0 gm each time), to 37 outpatients with chronic hepatitis C for a long-term study. It was found that some subjective symptoms were improved in all cases. Furthermore, 8 patients were confirmed to be viral-seroconverted by cRT-PCR. This is equivalent to 21.6% of the cases (Table 3). The mean age of the patients with viral seroconversion was 51.8 with mean treatment period of 3.1 yrs. In 5 patients, virus positive was first confirmed by PCR after interferon treatment, and it was converted to negative after Kampo medicine treatment (Tables 4, 5).

Table 3. Summary of HCV-RNA Negative Cases (Long-Term Study of TJ-108)

Among 37 HCV carriers 8 cases viral seroconverted (21.6%)

Treatment Period (7 mon ~ 7 yrs)

(mean) 3.8 years

Age (38 ~ 65)

(mean) 51.8

F/M 5/3

Table 4. List of HCV Negative Seroconverted Cases

age/sex route of years of interferon

infection infection

38 M transfusion 26 [Alpha]; 2 term,

[Beta]; 1 term

65 M unknown ? [Alpha]; 1 term

60 M transfusion 5 [Alpha]; 1 term

60 F transfusion 5 [Alpha]; I term

64 F unknown ? [Alpha]; 2 term

60 F transfusion 4 —

59 F transfusion 30 —

62 F unknown ? —

age/sex transaminase years of

s medication

38 M normal 5.0

65 M 100< 5.3

60 M normal 5.4

60 F normal 4.4

64 F 100< 0.6

60 F normal 0.7

59 F normal 1.4

62 F 100< 7.6

Table 5. History of Interferon Treatment and Effect of Kampo Medicine

History of No IFN Total

IFN treatment treatment

HCV-RNA become negative 5 (45%) 3 (13%) 8 (21%)

HCV-RNA persistent 6 23 29

Effects of Kampo Medicine and Gomisin A on the HCV Infection in MOLT-4 Cells

1) Effect of serum containing Kampo medicine

The inoculum used in the present study could absorb the MOLT-4 cells in a 0.001% of serum concentration. The absorption at this level is completely inhibited by interferon [Alpha] (5 x [10.sup.4]) (data not shown).

A band in the neighborhood of 300 bp disappeared at 0.001% inoculum of virus when it was cultured with human serum containing TJ-108. The density of the band markedly decreased even at 0.01% inoculum, while the sera containing TJ-48 and TJ-8 could only slightly inhibit the absorption of the virus (Figure 2).

[Figure 2 ILLUSTRATION OMITTED]

2) Effect of TJ-108

When the TJ-108 solution was directly added to MOLT-4 cells followed by HCV infection, the inhibition of virus infection was shown in a dose-dependent manner (Figure 3). It was also confirmed that the decrease of the intracellular viral antigen was due to the treatment of MOLT-4 cells with TJ-108 solution, in comparison with the controls by flow cytometry (Figure 4).

[Figures 3-4 ILLUSTRATION OMITTED]

3) Effect of Citrus Unshiu Peel, Schisandra Fruit and Polygala Root

The extent of viral absorption to MOLT-4 cells was found to be inhibited by the water extracts of Citrus Unshiu Peel and Schisandra Fruit but not Polygala Root (Figure 5), which are specific to TJ-108.

[Figure 5 ILLUSTRATION OMITTED]

4) Effect of Gomisin-A

Gomisin A isolated from Schisandra Fruit was found to inhibit HCV infection in MOLT-4 cells in a dose-dependent manner (Figure 6).

[Figure 6 ILLUSTRATION OMITTED]

5) Effect of Gomisin A on immunologically-induced acute hepatic failure

The survival curves and mortalities of the mice with acute hepatic failure are shown in Figure 7. When P. acnes and LPS-treated mice were administered only with 10% Tween 80 saline (solvent control) 60 mins prior to LPS treatment, all the mice died within 24 hrs after LPS treatment. However, when Gomisin A (40 [micro]g/kg, 80 [micro]g/kg) isolated from Schisandra Fruit was administered to the mice intraperitoneally, mortality decreased to 20% at 24 hrs after LPS injection. Furthermore, until 8 hrs after LPS injection, survival rate was preserved in a dose-dependent manner (Figure 7).

[Figure 7 ILLUSTRATION OMITTED]

Discussion

In Japan, hepatitis C may often be found in elderly people who have a history of surgery or blood transfusion, and interferon is sometimes not available for such patients. Like chronic hepatitis C, if treatment takes a long time, mild treatment is preferable for maintaining their quality of life. We investigated the effects of Ninjin-yoei-to, TJ-108 on 11 elderly patients (one male, ten females, mean age 72) with chronic hepatitis C for six months. After six months, it was found that the level of transaminase in one patient had become normal. Adverse effects did not appear in any case. The subjective symptoms, such as loss of appetite, fatigue, weakness, and itching were improved in 8 cases. Among these 11, 4 patients were followed up for another six months, which is equivalent to one year. Among these 4 cases, hepatitis C virus antibody titer dropped in one case, and antigen was seroconverted in another.

The clinical data that are shown in the present report suggest that Kampo medicine is very effective in the treatment of chronic hepatitis C. It was not concluded that Kampo medicine is more effective than interferon treatment, but it may be said that it is more effective when the patient has received pretreatment with interferon for decreasing the amount of virus before Kampo treatment. Viral RNA seroconversion is considered to prove that persistent infection in patients may be interrupted, or the virus genome titer remarkably decreased. For these reasons, Kampo medicine, especially TJ-108, is believed to have an effect on the therapeutic course of chronic hepatitis C.

When we compared the herbal components of TJ-108, which showed to be more effective than the other two Kampo medicines, with those of the other two Kampo medicines, we found that the herbal components almost overlapped except for three herbs. These three herbs, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, are included only in TJ-108. We therefore examined the effects of these three herbs on HCV infection, since their activities may play an important role in the treatment of HCV infection.

To elucidate the mechanism of treatment of Kampo medicine, we made an experimental study with HCV infection in vitro. The inoculum that we were using in the study could absorb the MOLT-4 cells in a 0.001% serum concentration of virus. It is hypothesized that the decrease of the activity of HCV-RNA and of intracellular antigen in MOLT-4 is from the inhibition of the expression of the membrane protein to which HCV may bind.

An extremely small amount of hepatitis C virus can be the cause of chronic hepatitis, which may often develop into cirrhosis and finally to liver cancer. In the present study, we show that Kampo medicine can improve the symptoms of chronic hepatitis C, reduce serum transaminase level, and finally lead to viral seroconversion. It is somehow difficult to imagine that Ninjin-yoei-to can affect chronic hepatitis C, since it is far from its standard use so far. However, we believe that it could be a new application of Kampo medicine. It is concluded that the therapeutic effect of TJ-108 on chronic hepatitis C may originate from the inhibitory effect on HCV reinfection, and also from the protective effect on immunological hepatopathy of Schisandra Fruit and its lignan component Gomisin A.

Acknowledgment

This work was supported in part by a grant in aid for Scientific Research of Kampo Medicine from Tsumura.

References

[1.] Arai, T., K. Hiromatsu, N. Kobayashi, M. Takano, H. Ishida, Y. Nimura, Y. Yoshikai. IL-10 is involved in the protective effect of dibutyryl cyclic adenosine monophosphate on endotoxin-induced inflammatory liver injury. J. Immunol. 155: 5743-5749, 1995.

[2.] Gish, R.G. Future directions in the treatment of patients with chronic hepatitis C virus infection. Can. J. Gastroenterol. 13: 57-62, 1999.

[3.] Luo, J.C., S.J. Hwang, C.R. Lai, C.L. Lu, C.P. Li, S.H. Tsay, J.C. Wu, F.Y. Chang, S.D. Lee. Relationships between serum aminotransferase levels, liver histologies and virological status in patients with chronic hepatitis C in Taiwan. J. Gastroenterol. Hepatol. 13: 685-690, 1998.

[4.] Koda, A., Y. Ono, T. Nishiyori, H. Nagai, N. Matsuura, A. Mase and T. Matsuyama. Immunopharmalogical studies of wen-qing-yin, a Chinese blended medicine: effects on type IV allergic reactions and humoral antibody production. Int. J. Immunopharmac. 9: 289-295, 1987.

[5.] Iijima, K., H. Kiyohara, M. Tanaka, T. Matsumoto, J.C. Cyong and H. Yamada. Preventive effect of taraxasteryl acetate form Inula britannica subsp. japonica on experimental hepatitis in vivo. Planta Med. 61: 50-53, 1995.

[6.] Nomura, M., Y. Ohtaki, T. Hida, T. Aizawa, H. Wakita and K. Miyamoto. Inhibition of early 3-methyl-4-dimethylaminoazobenzene-induced hepatocarcinogenesis by gomisin A in rats. Anticancer Res. 14(5A): 1967-1971, 1994.

[7.] Shiota, G., S. Yamada and H. Kawasaki. Rapid induction of hepatocyte growth factor mRNA after administration of gomisin A, a lignan component of shizandra fruits. Res. Commun. Mol. Pathol. Pharmacol. 94:141-146, 1996.

[8.] Tajiri, H., K. Kozaiwa, Y. Ozaki, K. Miki, K. Shimuzu and S. Okada. Effect of Sho-saiko-to on HBeAg clearance in children with chronic hepatitis B virus infection and with sustained liver disease. Am. J. Chin. Med. 19: 121-129, 1991.

[9.] Shimizu, Y.K., A. Iwamoto, M. Hijikata, R.H. Purcell and H. Yoshikura. Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc. Nat. Acad. Sci. 89: 5477-5481, 1992.

Jong-Chol Cyong(1), Sun-Min Kim(1), Koji Iijima(2), Takao Kobayashi(2) and Minoru Furuya(1)

(1)Department of Bioregulatory Function, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

(2)Department of Immunology, Mayo Clinic and Foundation

(Accepted for publication January 4, 2000)

COPYRIGHT 2000 Institute for Advanced Research in Asian Science and Medicine

COPYRIGHT 2001 Gale Group