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Effects of Juzen-Taiho-to on Carcinogenesis, Tumor Progression,
and Metastasis In Vivo
The following is a chapter from the text Juzen-taiho-to (Shi Quan Da Bu Tang in Chinese): Scientific Evaluation and Clinical Applications as written by Ikuo Saiki. In June of 2000 it was published in the peer-reviewed journal Pharmaceutical Society of Japan . This book has been reviewed by Oxford University Press (included in the appendix of papers), and published by CRC Press in 2005. There has been very minor editing done to maintain both topic and grammatical continuity.
Modern Application of Juzen-taiho-to:
This formula is used in Japan to balance homeostasis of the human body. It is also currently administered to patients weakened by long illness, fatigue, loss of appetite, night sweats, circulatory problems and anemia. It is specifically used for cancer patients under going chemotherapy and radiation (Karuki & Saiki 2005). Clinically, Juzen-Taiho is known to improve the general systemic condition of cancer patients and to reduce the adverse effects of chemotherapy, radiation therapy, and surgical treatment, which may lead to a weakness in what is defined in both TCM and Japanese Kampo Medicine as a weakness in Vital Energy and Blood. The following list of modern mechanisms have been defined for Juzen-Taiho, as published in Juzen-taiho-to Scientific Evaluation and Clinical Applications (Karuki & Saiki 2005):
· Enhancement of phagocytosis (Maruyama et a., 1988)
· Cytokine induction (Haranaka et al., 1985; Kubota et al., 1992)
· Antibody production (Hamada et al., 1988)
· Mitogenic activity of spleen cells (Kiyohara et al., 1991)
· Antitumor activity with or without other drugs (Haranaka et al., 1988; Sugiyama et al., 1995a)
· Protection from deleterious effects of anti-cancer drugs (Sugiyama et al., 1995b)
· Radiation-induced immuno-suppression and bone marrow toxicity (Kawamura et al., 1989; Ohninshi et al., 1990)
Cancer
The development of cancer, otherwise known as carcinogenesis, is a multi-step process. Each step is governed by multiple factors; some depend on the genetic constitution of the individual and others on their environment and way of life. Most cancers are likely initiated by a change in the cell's DNA sequences, but a single mutation is not enough to cause cancer. Tumor progression is the process by which an initial population of slightly abnormal cells evolves from bad to worse through successive cycles of mutation and natural selection. Metastasis, one of the major causes of mortality in cancer, is a complex cascade of events involving tumor dissemination from the primary site of growth to distant organs. Following tumor development and progression, the pathogenesis of metastases can be subdivided into a variety of sequential steps.
- Release from the primary tumor and invasion of the surrounding tissues
- Entry into the vascular or lymphatic circulation
- Transit in the circulation
- Arrest in the capillary bed of a distant organ
- Extravagation from the circulation
- Growth at apparently selected sites distant from the original tumor site.
Few cells in a primary tumor can complete all the steps necessary to achieve metastasis. Specific tumor interaction with host cells or components are therefore fundamental events in preferential organ colonization whereby metastases occur in specific organs and not randomly.
Despite advances in diagnostic techniques for the early detection of various cancers and significant improvement in surgical procedures, the mortality rate of cancer has been increasing steadily (Eisenberg et al., 1982; Galadivk et al., 1992; Gastrointestinal Tumor Study Group, 1985), and metastasis is a frequent cause of death by cancer. For instance, the liver is the most common target of the hematogenous metastasis in gastrointestinal tract cancer, especially colon cancer; the prognosis for cases with liver metastasis is extremely poor (Gengmark and Hafstrom, 1969; Govowski et all., 1994). If occult micro-metastases that had been established at the time of surgery could be inhibited, the prognosis of patients with colon carcinoma would improve.
The following is a collection of information that describes the effect of Juzen-Taiho (known in Chinese as Shi Quan Da Bu) on carcinogenesis, tumor progression and metastasis in vivo, and it's inhibitory mechanism of action. It was written by Ikuo Saiki and first published in June of 2000 in the peer-reviewed journal Pharmaceutical Society of Japan. Later, it was included in the text Juzen-taiho-to Scientific Evaluation and Clinical Applications compiled by Karuki & Saki and published in 2005 by CRC Press.
Inhibition of Carcinogenesis:
Tatsuta et al. (1994) investigated the effect of Juzen-taiho on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) in Sprague-Dawley rats. Administered in the diety for 15 weeks, 2.0 or 4.0 % Juzen-taiho significantly reduced the size, volume and/or number of glutathione-S-transferase (GST-P) positive and gamma-glutamyl transpeptidase (GGT) positive hepatic lesions. This treatment also caused a significant increase in the proportion of IL-2 receptor-positive lymphocytes among the lymphocytes infiltrating the tumors, as well as a significant decrease in the labeling index of preneoplastic lesion. This suggests that the inhibition of hepatocarcinogenesis by Juzen-taiho-to is in part due to activation of the immune system. Similarly, Juzen-Taiho significantly inhibited the formation of GAT-P positive foci treated with diethylnitrosameine and two-thirds partial hepatectomy.
The inhibitory effects of Juzen-taiho-to on the development of bladder cancer induced by N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) were also evaluated in C57BL/6 strain mice (Watanabe et al., 1997). The group administered Juzen-taiho-to a lower rate of bladder cancer incidents, compared to a control group. Furthermore, neither normal epithelia nor hyperplasia was observed in the control group at 12 weeks after BBN administration, whereas 70% of the treated group retained normal epithelia or had simple hyperplasia of the bladder tissue. This suggests that Juzen-taiho-to is likely to be effective in preventing the early neoplastic changes that occur in the mouse urinary bladder cancer induced by BBN.
Sakamoto et al. (1994) have reported that Juzen-taiho-to suppressed the activities of theymidylate synthetase and thymidine kinase involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, in spontaneous mammary tumors of SHN mice with a reduced level of serum prolactin. This indicates that this formula may have anti-tumor effects on mammary tumors.
Prevention of Malignant Tumor Progression:
Malignant tumor progression is the process by which tumor cells acquire a more malignant phenotype, such as enhancement of the ability to proliferate, to invade, or to metastasize, and is affected by various factors. However, few studies have been conducted on the mechanisms, facilitating factors, and inhibitors of progression because of the lack of a suitable experimental model. In one tumor progression model (figure 2) spontaneously regressive QR-32 tumor cells, when coimplanted s.c. with a foreign body (a gelatin sponge), irreversibly acquired the ability to grow progressively at inoculated sites - even without a gelatin sponge (Okada e al. 1992). In contrast, QR-32 cells alone gradually grew over 15 days after inoculation and thereafter regressed for up to 25 days.
It has also been shown that such progressive growth of tumors was provoked through the enhancement of PGE2 production in the tumors or by oxygen radicals and inflammatory cytokines produced by host cells reactive with the gelatin sponge (Okada et al., 1992, 1993, 1994). This seems analogous to the fact that malignant progression of tumors followed by metastasis is clinically observed to be elicited by various factors and circumstances, including stresses, anticancer drugs, inflammation, etc. The progressive growth after s.c. coimplantation of QR-32 cells with a gelatin sponge was prevented by endogenous induction of anti-oxidative enzymes or scavengers such as manganese superoxide-dismutase (Mn-SOD) or metallotheonein at the tumor sites by orally administered bismuth subnitrate and lipohilic vitamin C (Okada et al., 1995).
The inhibitory effect of oral administration of Juzen-taiho-to on progressive growth of this mouse fibrosarcoma was investigated in this tumor progression model. Oral administration of Juzen-taiho-to caused significant inhibition of the progressive growth of QR-32 regressor tumors after coimplantation with gelatin sponge (Figure 3) and prolonged the survival of tumor bearing mice. These results indicate that Juzen-taiho-to may be effective for preventing weakly maliganant tumor from growing progressively upon coimplantation with a gelatin sponge (Ohnishi et al., 1996).
Such progressive growth, however, may not necessarily be equivealent to malignant progression because even cells that do not acquire a more malignant phenotype can show transient proliferation depending on the host circumastance and the implantation conditions. Resulant progressive tumor (QRsP) showed gelatin sponge-independent growth after reinoculation (six out of xis mice, Figure 3), as also demonstrated previously (Okada et al., 1992). This phenomenon could be regarded as progression. Actually, tumors obtained from the group treated with Juzen-taiho-to did not grow progressively after reinoculation into syngeneic mice without a gelatin sponge (zero out of six mice, Figure 3).
On the other hand, oral administration of Juzen-taiho-to for 7 days after inoculation of QRsP progressive cells resulted in a significant reduction of the tumor growth and enhancement of the survival rate in tumor-bearing mice compared with the control (Ohnishi et al., 1996). Because Juzen-taiho-to has been reported to possess antitumor effects based on the activation of macrophages (Maruyama et al., 1988), cystyokine induction (Haranaka et al., 1985; Kubota et al., 1992). Augmentation of NK cell activity (Takahashi and Nakazawa, 1995), etc., the inhibitory mechanisms of the progressive growth of QR-32 regressor cells and the growth of the resultant QRsP progressor cells may be in part associated with induction of host-mediated immune surveillance by Juzen-taiho-to.
However, oral administration of bismuth subnitrate, which induces metallothionein as a scavenger of oxygen radicals in the tumor tissue (Satoh et al., 1989), resulted in a significant inhibition of gelatin sponge-elicited progressive growth (Ohnishi et al., 1996). These results suggest that Juzen-taiho-to may act to induce atioxidants and to reduce PGE2 production (Okada et al., 1990) during tumor progression, in addition to augmenting the host mediated immune responses.
Inhibition of Tumor Growth and Metastasis and the Inhibitory Mechanism
The anti-tumor activity of Juzen-taiho-to has been investigated using various tumor models (Table 1). When Juzen-taiho-to (150 or 300 mg/kg) was administered orally twice a day for 10 days after i.p. inoculation of Ehrlich ascites tumor in ICR mice, it significantly suppressed the tumor growth and also prolonged the survival time of tumor-bearing mice (Itoh and Shimura, 1985a). It should be noted that Juzen-taiho-to was not effective or was only slightly effective in inhibiting the growth of P388 leukemia, Liwis lung carcinoma (LLC), and sarcoma-180 ascites tumor (Aburada et al., 1983; Itoh, 1989; Komiyama et al., 1988).
Oral administration of Juzen-taiho-to or Sho-aiko-to resulted in a significant increase in relative organ weight of the spleen, thymus, and liver and also enhcance of reticuloendiotherlial cell function such as the phagocytic index, compared with the control (Itoh & Shimura, 1985b). The lung metastasis of LLC was inhibited by i.v. injection of peritoneal macrophages activated with Juzen-taiho-to (Itoh, 1989). Thus, the mechanism of antitumor activity of Juzen-taiho-to may be party due to the stimulation of the reticuloendthelial system, C3 activation, and dpression of the liver microsomal drug metabolizing enzymatic system.
Oral administration of Juzen-taiho-to significantly suppressed the growth of human U-87MG glioma cells transplanted s.c. into BALB/c nude mice and prolonged the survival time of the mice (Takahashi and Nakazawa, 1995). Endogenous TNF production was augmented by administration of Juzen-taiho-to without a secondary stimulus. Similar results were also obtained by using murine 203-glioma cells in syngeic mice. The anti-tumor mechanism of Juzen-taiho-to appears in part to involve the ability to induce endogenous TNF production.
We also examined the effect of oral administration of Juzen-taiho-to on liver metastasis resulting from intraportal vein injection of colon 26-L5 carcinoma cells in vivo (Ohnishi et al., 1997) and the role of the immune system after the administration. Oral administration of Juzen-taiho-to before tumor inoculation resulted in the dose-dependant inhibition of liver metastasis of colon 26-L5 carcinoma cells and a significant enhancement of survival rate compared to the untreated control (Ohnishi et al., 1998a) (Figure 4 through Figure 6). Cis-Diamminedichloroplatinum II (CDDP) significantly inhibited liver metastasis at 80 µg/mouse (Sugiyama et al., 1995a, b), but it produced severe adverse effects such as decrease of body weight and a 50% death rate of the mice, Juzen-taiho-to did not produce any side effect, nor did it directly affect the tumor cells in vitro . Thus, Juzen-taiho-to may be a biological response modifier that inhibits micrometastasis and differs from chemotherapeutic agents. Similarly, oral administration of Juzen-taiho-to before i.v. inoculation of B16-BL6 melanoma cells resulted in marked inhibition of lung metastasis without causing any loss of body weight (Ohnishi et al., 1998b).
Because metastasizing tumor cells interact with host cells such as lympthocytes, NK cells, and monocytes, which as important in the destruction of tumor cells (Fidler, 1986; Hanna, 1982), we investigated whether Juzen-taiho-to could stimulate immune cells to inhibit tumor metastasis. Antiasialo GM 1 serum can selectively eliminate NK cells (Habu et al., 1981; Saiki et al., 1989) and 2 chloroadenosine can eliminate macrophages (Saiki et al., 1989; Saito & Yamaguchi, 1981). Figure 7 shows that liver metastasis was enhanced in mice pretreated with antiasialo GM1 serum or 2-chloroadenosine, and in T-cell-deficient nude mice, compared to untreated normal mice. This indicates that NK cells, macrophages, and T-cells play important roles in the prevention of the metastatic spread of tumor cells. Juzen-taiho-to significantly inhibited the experimental liver metastasis of colon 26-L5 cells in mice pretreated with antiasialo GM1 serum as well as untreated normal mice, but it did not inhibit the metastasis in 2-chloroadenosine pretreated mice (Figure 7) and T-cell deficient nude mice.
Juzen-taiho-to was inactive when the contributions of macrophages and T-cells were eliminated from our system, so its inhibitory mechanism is likely to be related to the activation of these cells. We also found that the oral administration of Juzen-taiho-to caused peritoneal exudates macrophages (PEMs) to become cytostatic against tumor cells in vitro . Although the exact mechanism responsible for the inhibition of liver metastasis by Juzen-taiho-to is not fully understood, this inbitory effect is partly associated with the activation of macrophages (Ohnishi et al., 1998a). Further investigation will be needed to determine the detailed mechanisms responsible for the inhibition of tumor metastasis by Juzen-taiho-to. However, it may be difficult to evaluate the results of the in vitro study using this formulation, the so-called "Furikake" (in Japanese) study, because the expression of in vivo efficacy by the formulation is sometimes mediated by the active (intestinal bacterial) metabolites after the oral administration.
In conclusion, oral of Juzen-taiho-to Inhibited liver metastasis of colon 26-L5 carcinoma cells and enhanced the survival rate, possibly through the activation of macrophages and T-cells (Ohnishi et al., 1998a). Thus, Juzen-taiho-to may be therapeutically effective for the prevention of cancer metastasis.
Combination with Other Treatment Modalities (Chemotherapy, Hyperthermia, Radiation, ETC.)
In addition to identifying new types of anticancer agents, it is also important to develop methods or agents that can auument the therapeutic efficacy and/or reduce the side effects of other anticancer agents. Combination therapy with Juzen-taiho-to and other treatment modalities including anticancer drugs has also been investigated using various tumor models to examine whether Juzen-taiho-to could potentiate the efficacy. The results are summarized in Table 1.
Daily administration of Juzen-taiho-to alone after subcutaneous inoculation of Meth A fibrosarcoma, sarcoma-180, or B16 melanoma cells resulted in almost no inhibition of tumor growth (Komiyama et al., 1988). In contract, combination treatment with Juzen-taiho-to (p.o.) and mitomycin C (i.p.) resulted in more effective inhibition of the growth of Meth A fibrosarcoma or B16 melanoma cells than treatment with mitocycin C or Juzen-taiho-to alone (Komiyama et al.,1988). Treatment with the combination of Juzen-taiho-to mitocycin C also resulted in a significant enhancement of the survival rate of mice inoculated with P388 leukemia cells and markedly reduced the side effects caused by mitomycin C (Aburada, et al., 1983).
Juzen-taiho-to was given orally (days 1 to 50) to ICR mice inoculated s.c. with sarcoma-180. Mice were administered mitomycin C on various days and treated with hyperthermia (43 degrees celcius, 30 min). Juzen-taiho-to markedly potentieated the antitumor effect of the combination of mitomycin C and hyperthermia (Komiyama et al., 1989). Similar results using the treatment modality were obtained after the inoculation of B16 melanoma cells into BDF1 mice. In addition to the augmentation of the combined effect of mitomycin C and the subsequent marked growth of the tumors were reduced by the administration of Juzen-taiho-to (Komiyama et al., 1989).
In the MBT-2 bladder tumor-C3H/He mouse model, treatment with a combination of Juzen-taiho-to and cis-diamine dichloroplatinum (CDDP) inhibited tumor growth and prolonged the survival rate of mice more effectively than CDDP alone (Ebinsuno et al., 1989). In addition, oral administration of Juzen-taiho-to for 2 weeks significantly ameliorated the adverse effects caused by treatment with a high dose of CDDP, including lethal toxicity, renal and hepatic toxicity, and myelosuppression (Ebinsuno et al., 1989)
Administration of Juzen-taiho-to alone exerted no inhibitory effect on the growth of intradermally inoculated Meth A tumor cells. Howver, Juzen-taiho-to displayed a marked antitumor effect when combined with surgical excision (Maruyama et al., 1993). When oral adminstration of Juzen-taiho-to (0.5g/kg/day) was begun on postexcision day 1, the growth of a secondary tumor inoculated on postexcision day 7 was great inhibited. Because the antitumor immunity of spleen cells from mice treated with Juzen-taiho-to was abolished by treatment of the spleen cells with anti-L3T4 monoclonal antibody + complement, the effect is mediated by L3T4-positive helper T-cells.
Combination therapy with Kampo [Japanese TCM] preparations (Sho-saiko-to, Juzen-taiho-to, or Cinnamomum cortex) and Streptococcus pyogenes products (OK432) strongly inhibited the growth of Ehlich ascites or Meth A tumor cells through the increased production of endogenous tumor necrosis factor (TNF) (Haranaka et al., 1985, 1988). A significant negative correlation was observed between the TNF activity and tumor weight, and a positive correlation was found between the TNF activity and spleen weight in the Ehrlich ascites tumor-bearing ddY mice receiving the Kampo preparations for OK432.
These results suggest that the anti-tumor activities and capacity to induce TNF production of the preparations are probably due in part to stimulation of the reticuloendothelial system, including macrophages, and induction of host-mediated antitumor substance(s) like TNF as immunopotentiator(s). Also, anti-tumor activity of Juzen-taiho-to and OK432 was observed in one patient with hepatocellular carcinoma (Haranaka et al., 1995).
Protection against Deleterious Effects of Anticancer Drugs and Radiation Induced Immunosupression:
Consumption of a diet containing 1 or 0.5% Juzen-taiho-to for 2 weeks resulted in significant protection against the adverse effects caused by treatment with a high dose of CDDP, including lethal toxicity, renal and hepatic toxicity, and myelosuppression (Ebisuno et al., 1989). Oral administration of Juzen-taiho-to increased the lowered immune response to the normal level in MBT-2 tumor bearing mice (Ebisuno et al., 1990). Juzen-taiho-to also protected aged mice (13 - 15 months old) from CDDP-induced damage to the immune function and restored the lowered cytoxic activity in tumor-bearing mice treated with CDDP.
Sugiyama et al. (1995a) showed that oral administration of Juzen-taiho-to for 12 days prevented increases in blood urea nitrogen, serum creatinine, serum glutamic-oxaloacetic transaminase, serum flutamic-pyruvic transaminases, and relative stomach weight, as well as decreases in white blood cell count, platelet count, relative spleen and thymus weights, food intake, and body weight caused by CDDP to nearly the control levels without reducing the antitumor activity of CDDP against S-180. Also, preventive effects of Juzen-taiho-to were observed against carboplatin-induced myelosuppression and hepatic toxicity (Sugiyama et al., 1995b).
Iijima et al. (1988) reported that treatment with Juzen-taiho-to for 7 days delayed deaths due to lethal doses of MMC or CDDP and markedly improved the survival curves. Also, Juzen-taiho-to reduced the atrophy of the testis, thymus and spleen caused by MMC. It also had protective effects aginst leucopenia, anemia, and body weight loss caused by MMC and against the increases of BUN and creatinine caused by CDDP. These results inducate that combination with Juzen-taiho-to may be a new way to minimize the toxicity of MMC or CDDP.
Kawamura et al. (1989) reported that intrperitoneal injection of MMC resulted in a marked reduction of the numbers of colony-forming units in the spleen (CFU-S) and granulocyte-macrophage colony-forming units (CFU-GM). Administration of Juzen-taiho-to before MMC injection did not protect the mice from damage to hematopoietic function caused by MMC, but remarkably accelerated the recover of CFU-S and DFU-GM. Juzen-taiho-to was effective when its administration was begun after MMC injection. These results suggest that Juzen-taiho-to has the ability to accelerate hematopoietic recovery from bone marrow injury by MMC.
Continuous oral administration of Juzen-taiho-to 2 to 3 weeks before a dose of x-irradiation causing bone marrow death increased the 30 day survival ratios of x-irradiated mice (Hosokawa, 1993(. The administration enhanced the recovery of blood cell counts, especially those of thrombocytes as well as blood forming stem cells (FUs) in bone marrow. Oral administration of Juzen-taiho-to 7 days after x irradiation (29 gy) to the i.m. inoculated Ehrlich tumor significantly prolonged the survival rate of x-irradiated mice. Adminstration of Juzen-taiho-to for 7 days after irradiation showed radioprotective effects by increasing the number and size of day-14 spleen colony forming units (CFU-S) (Ohnishi et al., 1990).
Sairenji et al. (1992) investigated the effect of myelosuppression on the survival of cachectic mice inoculated with colon 26 adenocarcinoma cells. Juzen-taiho-to prevented a decrease in body weight of tumor-bearing mice. Consequently, it prolonged the survival rate of mice bearing colon 26, suggesting that Juzen-taiho-to has the ability to ameliorate the cachexia induced by transplantable colon 26 carcinoma.
Attempts to Obtain Juzen-taiho-to Preparations with Constant Efficacy:
Herbal prescriptions including Kampo medicines have been recognized by the scientific medical system and have become increasingly popular. Because Kampo formulations are generally prepared from the combination of many crude drugs, they may have effects that differ from the sum of the effects of the individual constituent crude drugs. They must have an acceptable efficacy and quality when used as therapeutic medicines. Formulations prepared from crude drugs with different qualities would have different biological activities and efficacies.
Therefore, it is necessary to control the quality of the formulations and their component crude drugs and their processing procedures to obtain reproducibility of the formulation and efficacy because their quality varies with the origins of the crude drugs and the time and place of harvest, ect. In Japan, the quality of crude drugs is controlled by Japanese Pharmacopeia XIII , which regulates the botanical origin, crude drug test of foreign matter, loss by drying, total ash, acid-insoluble ash, extract content, essential oil content, and microscopic examination. However, to our knowledge, these matters have no been studied in detail in the case of Juzen-taiho-to, although a lot of information about Kampo formulations and their constituents is available in Japanese and Chinese archaic writings.
HPLC Profiles of Juzen-taiho-to and Its Constituent Crude Drugs: (Please see the paper "HPLC Analysis of Juzen-taiho-to and Its Variant Formulations and Their Antimetastatic Efficacies" in our appendix.)
For the purpose of obtaining proper formulations with constant quality and efficacy, we have performed HPLC pattern analysis of Juzen-taiho-to by standard references (Saiki et. al., 1999). Figure 8 shows the HPLC profiles of Juzen-taiho-to by single monitor (220 nm) and contour plot (190 to 400 nm) using a photodiode array system as a detector. The contour plot of the UV absorbance intensity of the compounds shows all of the compounds that have detectable UV absorbance in the extract from the formulation. The origin of each peak of Juzen-taiho-to was identified by comparison with the retention time and UV spectrum of each peak of Juzen-taiho-to was identified by comparison with the retention time and UV spectrum of each extract of crude drug or the chemically defined standard compounds (A-L in figure 8). For example, the peaks of paeoniflorin in Paeoniae Radix and glycyrrhizin from Glycyrrhizae Radix were detected at the D and L positions of the contour plot in Figure 8.
Thus, in addition to testing whether the standard compounds have the pharmacological activity of inhibiting tumor metastasis or not, this HPLC pattern - the so-called fingerprint method - could provide a useful means of identifying the crude drugs and preparing batches of constant formulation. Although the many compounds that have no UV absorbance cannot be detected by this method, a reproducible fingerprint pattern of the formulation may be primarily useful for the assessment of homogeneity of the formulation, which should lead to constant efficacy.
To evaluate the efficacy of thus prepared Juzen-taiho-to formulation (batches #1 and #2), which were independently prepared using the same ten crude drugs by the same procedure. The fingerprint analysis of the two batches of Juzen-taiho-to showed HPLC profiles similar to that shown in Figure 8. Oral administration of the two Juzen-taiho-to preparations (batches #1 and #2) at the effective dose of 40 mg/day (Ohnishi et al., 1998 a, b) significantly reduced the number of colon 26-L5 tumor colonies in the liver. Similar results were observed with both batches of Juzen-taiho-to formulations (Saiki et al., 1999) (Figure 9 upper panel).
Variant Formulations and Their Anti-metastatic Efficacies:
In the usage of preparation of Kampo formulations, some component crude drugs in the formulation are in some cases replace with related crude drugs. To examine the effect on antimetastatic action when original Juzen-taiho-to constituents were replaced with different crude drugs, we prepared variant formulations of Juzen-taiho-to in which one crude drug was substituted with a related crude drug from different sources or places of origin (Table 2). Juzen (Naimo-ogi à Kibaana-ogi) as well as Juzen-taiho-to (#1) significantly inhibited liver metastasis compared with the untreated control (66.7 +/- 28.6 vs. 121.8 +/- 55.1 of untreated control, respectively, in lower panel of Figure 9 lower panel). Also the HPLC pattern of the extract of the root of Astragalus menbranaceus (Kibana-ogi) was very similar to that from Astragalus mongholicus (Naimo-ogi).
In contrast, Juzen (Sojutsu à Byakujutsu/koera), Juzen (Sojutsu à Byakujutsu/Obana-okera), Jezen (Yamato-toki à Hokkai-toki), Juzen (Naimo-ogi à Shingi) had less antimetastatic effect than the original Juzen-taiho-to (#1). Among these four variant formulations with variant formulations with reduced effects, some differences in the HPLC fingerprint patterns were discernible between the original and substituted crude grugs, except in the case of Atractylodis Rhizoma [Byakuutus/Obana-okera], i.e., the rhizome of Atractylodes ovata [Obana-korea]. For example, some additional peaks were seen by arrowheads on the chromatograms compared with those of the original standards (Figure 10.). This suggest that the reduced effects of the variant formulations may be associated with the marked differences indicated in the fingerprint patterns of the substituted crude drugs.
However, Juzen (Sojutsu à Byakujutsu/Obana-okera) was less effective in inhibiting metastasis than the original Juzen-taiho-to (84.1 +/- 27.1 and 63.3 +/- 28.6, respectively in the lower panel of Figure 9), despite the similarity of the HPLC patterns of Sojutsu and Byakujutsu/Obana-okera (Figure 10). Therefore, other components in Byakujutsu/Obana-okera that cannot be detected by HPLC analysis such as polysaccharides or peptides, may be responsible for the reduced efficacy of Juzen (Sojutsu à Byakujutsu/Obana-okera).
In conclusion, we demonstrated differential effects of variant formulations of Juzen-taiho-to on tumor metastasis. The reduced antimetastatic effect of the variant formulations used in this study may be related to the differences in the fingerprint patterns between the original and substituted crude drugs. Thus, HPLC pattern analysis of Kampo medicines may provide a useful method for obtaining their optimal efficacy as well as constant quality of the formulation (Saiki et al., 1999), although such analysis has problems and limitations.
Antitumor Effects of Constituent Crude Drugs in Juzen-taiho-to:
Because Kampo formulations are generally prepared from the combination of many crude drugs, they may have effects that differ from the sum of the effects of the individual constituent crude drugs. However, some studies using individual constituent crude drugs have been carried out in order to investigate the anti-tumor mechanism of Juzen-taiho-to in vivo and to discover active components, although it is doubtful that the results using crude drugs can completely reflect the efficacy of the whole formulation.
Rhemannia Radix, Atractylodis Lanceae Rhizoma, Angelicae Radix and Astragali Radix
Administration of Juzen-taiho-to in combination with cyclophosphamide after s.c. inoculation of EL-4 lymphoma cells resulted in a significant decrease of tumor size and the prolongation of the life span in mice (Yamaguchi et al., 1991). Combination of cyclophosphamide with the extracts of the individual composed crude drugs in Juzen-taiho-to also inhibited liver metastasis of EL-4 lymphoma cells and enhanced the survival rate of mice. Thus, some of the crude drugs in Juzen-taiho-to are effective at inhibiting metastasis indirectly through the augmentation of host immune responses without affecting direct cytotoxcity against tumor cells.
Cinnamomi Cortex
Haranaka et al. (1998) have shown that the combination of Juzen-taiho-to with citomycin C (MMP) or OK432 inhibited thegrowth of Ehrlich ascites tumor in ddY mice or of Meth A fibrosarcoma in Balb/C mice, and enhanced production of endogenous TNF. Similar effects by combination therapy were observed using an extract of Cinnamomi Cortex in place of Juzen-taiho-to formulation.
Ginseng Radix
Ginseng (the root of Panax Ginseng C.A. Meyer, Araliacae), a constituent crude drugs of Juzen-taiho-to, has been used for traditional medicine in China, Korea, Japan , and other Asian countries for the treatment of various diseases, including psychiatric and neurologic diseases as well as diabetes mellitus. So far, ginseng saponins (ginsenosides) have been regarded as the principal components responsible for the pharmacological activities of ginseng. Ginsenosides are glycosides containing an aglycone (protopanxadiol or protopanaxatriol) with a dammarane skeleton and have been shown to possess various biological activities, including the enhancement of cholesterol biosynthesis, stimulation of serum protein synthesis, immunomodulatory effect, and anti-inflammatory activity (Sakakibara et al., 1975; Shibata et al., 1976; Toda et al., 1990; Scglione et al., 1990; Wu et al., 1992).
Several studies using ginsenosides have also reported antitumor effects, particularly the inhibition of tumor-induced angiogenesis (Sato et al., 1994), tumor invasion and metastasis (Mochizuki et al., 1995; Shinkai e al., 1996), and the control of phenotypic expression and differentiation of tumor cells (Odashima et al., 1985; Ota et al., 1987). Previously, it was reported that protopanxadiol-type and propanaxatriol-type ginsenosides are metabolized by intestinal bacteria after oral administration to their final derivative 20-O-Beta-D-glucopyranosul-20(S)-protopanaxdiol (referred to as M1) [Hasegawa et al., 1996] or compound K [Kanaoka et al., 1994; Karikum et al., 1991]or 20(S)-protopanaxatriol [referred to as M4 (Hasegawa et al., 1996)] (Figure 16). This made it unclear whether or not the expression of antimetastatic effect by oral administration of ginsenosides can be induced by their metabolites.
We have recently reported that protopanxadiol- or protopanxadiol-type ginsenosides and their major metabolites M1 and M4 markedly inhibited lung metastasis of B16-BL6 melanoma cells when they were administered five times orally (Wakabayashi et al., 1997 a, b). In contrast three consecutive i.v. administrations of metabolite M4 after tumor inoculation resulted in a significant inhibition of lung metastasis, whereas ginsenosides Re and Rg1 did not show any inhibitory effect. These findings suggest that the expression of the in vivo antimetastatic effect by oral administration of both ginsenosides was primarily based on their metabolites M1 and M4.
These results may be also supported by the finding that metabolites were detected in the serum from mice orally given ginsenosides, but ginsenosides were not detected by HPLC analysis. This pharamacokinetic study is in good agreement with previous reports on the low absorption rate of Tb1 from the intestines (Odani et al., 1983; Tanizawa et al., 1993) and high metabolic rate of Rb1 to M1 Tanizawa et al., 1993) in rat and human by using HPLC and enzyme-immunoassay methods (Hasegawa et al., 1996; Kanaoka et al., 1994). Moreover, it has also been noted that ginsenosides are hardly decomposed by gastric juice with the exception of slight oxygenation (Karikuma et al., 1991). Therefore, our findings support the notion that ginsenosides may act as a natural pro-drug that can be transformed to M1 by intestinal anaerobe(s) after oral administration and consequently induce in vivo anti-metastatic effect.
To investigate the incidence of intestinal bacteria processing ginsenoside Rb1-hydrolyzing potential, the hydrolyzing potential of intestinal bacteria, expressed as the transformation rate of Rb1 to M1 between mother mice and their litters, particularly statistically significant between the groups of litters born from mothers with different rates of hydrolyzing potential (Figure 17). This suggests that the intestinal microflora of a litter is primarily infected from the mother. On the other hand, consecultive administration of ginseng extract to the mice with transformation rate, compared with untreated group (Figure 18). However, induction of Rb1-hydrolysing potential by the administration of ginseng extract was hardly effective for the mice with hydrolyzing potential of less than 10%. For such mice, inoculation of fecal microflora from mice with high hydrolyzing potential was also in effective. Therefore, the location of the bacteria capable of hydrolyzing Rb1 on intestinal epithelium cells may be associated with the genetic factors of the hosts.
To examine the influence of RB1-hydrolyzing potential on antimetastatic efficacy of Rb1, Rb1 was orally administered to two sets of mice with low and high hydrolyzing potential after s.c. inoculation of LLC tumour. Figure 19 shows a significant difference between active and inactive groups and also the tendency of a positive relationship between hydrolyzing potential and inhibition of lung metastasis. These findings indicate that the transformation rate of Rb1 to its active metabolite M1 was dependent on Rb1-hydrolyzing potential of intestinal bacteria, which consequently affected the expression of antimetastatic efficacy of orally administered Rb1. Thus, hydrolyzing potential of intestinal bacteria for a crude drug or a formulation may be an important factor influencing the holistic pattern of symptoms and individual pathogenic alterations, so-called "sho" (Zheng in Chinese), by which the diagnosis of a disease state and the ways of treatment in Kampo are determined.
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