In vivo antitumor effects of bitter principles from the antlered form of fruiting bodies of ganoderm by Oswald Gekko Kierkegaard

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In vivo antitumor effects of bitter principles from the antlered form of fruiting bodies of Ganoderma lucidum Article in Journal of Natural Medicines · December 2005 Impact Factor: 1.59 · DOI: 10.1007/s11418-005-0003-5

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J Nat Med (2006) 60: 42–48 DOI 10.1007/s11418-005-0003-5

O R I GI N A L P A P E R

Jiang Jing Gao Æ Akiko Hirakawa Æ Byung Sun Min Norio Nakamura Æ Masao Hattori

In vivo antitumor effects of bitter principles from the antlered form of fruiting bodies of Ganoderma lucidum

Received: 9 May 2005 / Accepted: 1 July 2005 / Published online: 23 September 2005 The Japanese Society of Pharmacognosy and Springer-Verlag 2005

Abstract Two triterpene fractions and a single ganoderma alcohol obtained from an antlered form of the fruiting bodies of Ganoderma lucidum were examined for their antitumor effects on the growth of inoculated mouse Lewis lung carcinoma in mice by intraperitoneal administration. The ganoderma alcohol fraction significantly suppressed the tumor growth at doses of 50 and 100 mg/kg in the treatment period, and even after the administration, showing antitumor activity with a T/C value of 70.6% at a dose of 100 mg/kg. On the other hand, no obvious activity was shown at each dose in the ganoderma-acid-fraction-treated groups. Furthermore, ganoderiol F, which exhibited the strongest cytotoxicity against four tumor cell lines among five ganoderma alcohols examined, remarkably inhibited the tumor growth, accounting for 63.7% and 78.7% of control group at a dose of 5 mg/kg, 54.1% and 63.0% at a dose of 10 mg/kg, and 47.7% and 53.9% at a dose of 20 mg/ kg in and after the administration period, respectively, in a dose-dependent manner. These results suggest that the antitumor effects of bitter principles in G. lucidum are mainly due to ganoderma alcohols. Keywords Antitumor effect Æ Ganoderma lucidum Æ Bitter principle Æ Triterpene Æ Ganoderiol F Æ Lewis lung carcinoma Æ Cytotoxicity

J. J. Gao Æ A. Hirakawa Æ B. S. Min Æ N. Nakamura M. Hattori (&) Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan E-mail: saibo421@ms.toyama-mpu.ac.jp Tel.: +81-76-4347630 Fax: +81-76-4345060 B. S. Min Immunomodulator Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea

Introduction Medicinal mushrooms have been used for a long time in traditional medicine. The potential medicinal value and wide acceptability of these edible mushrooms have attracted increasing interests in the search for biologically active substances from them [1, 2]. Ganoderma lucidum Karst (Reishi in Japanese), a species of basidiomycetes that belongs to Ganodermataceae of Aphyllophorales [3], is one of the most popular chemopreventive mushroom in China, Japan, Korean, and other Asian countries and has been under modern pharmacological research during the last 30 years. Nowadays, according to the confirmation and elucidation of its pharmacological activities and clinical effects, G. lucidum and related products are widely used as not only health foods but also clinical drugs for the prevention and treatment of various kinds of diseases, such as hypertension, leucopenia, and cancer [4]. Many biological activities of components from fruiting bodies, mycelia, and spores of G. lucidum have been identified. The antitumor and immune-enhancing properties of polysaccharides have been deeply investigated both in vitro and in vivo [5–8], and the water-soluble extracts of this mushroom have been used in traditional medicine as antitumor and immunomodulating agents [9]. In contrast, for another major group of constituents, triterpenes, which are bitter in taste and characteristic principles in this mushroom, few studies have been conducted about their antitumor activities in vivo although over 140 highly oxygenated and pharmacologically active lanostane-type triterpenes have been isolated from this mushroom [10]. Extraordinarily, because of the difficulty in obtaining sufficient amounts of pure compounds for animal experiments, no in vivo study on the antitumor effect of G. lucidum using a single triterpene component has been reported. Recently, the triterpene-enriched fractions and extracts from the umbrella form of the fruiting bodies and mycelia of G. lucidum were reported to have antitumor

activities in vitro or in vivo [11, 12]. Indeed, we have found previously that the triterpenes isolated from G. lucidum exhibited cytotoxicities against mouse sarcoma Meth-A (lucidumols A and B, ganodermanondiol, ganodermanontriol, and ganoderiol F) and S-180 (ganodermanonol and ganodermanondiol), mouse LLC (lucidumol A, ganoderiol F, and ganodermanontriol), and human breast carcinoma T-47D (ganodermanonol and ganodermanondiol) cells [13, 14]. These results led us to evaluate ganoderma triterpenes as antitumor drugs. In this connection, we investigated the in vivo antitumor eﬀects of ganoderma alcohol and acid fractions obtained from an antlered form of the fruiting bodies of G. luidum using LLC-bearing mice. Furthermore, on the basis of the cytotoxicity assay, ganderiol F was selected to examine its antitumor eﬀect in LLC-inoculated mice as a representative ganoderma alcohol from this mushroom.

Materials and methods Plant materials An antlered form of the fruiting bodies of G. lucidum was provided by TDK Co. Ltd (Akita, Japan), and their specimens are deposited at Museum of Material Medica, Toyama Med. Pharm. University, Toyama, Japan.

following two fractions, corresponding to ganoderma alcohols (688.5 mg) and acids (2.15 g), respectively, were evaporated in vacuo and used in this experiment as alcohol and acid fractions. Isolation of ganoderma alcohols Eighteen grams of the CHCl3 extract obtained from 380 g of pulverized fruiting bodies of G. lucidum (antlered form) mentioned as above was applied to a column of Florisil (40 g). The elution was started with hexane– acetone (9:1, 8:2, and 7:3), then CHCl3–MeOH (1:1) to yield six fractions (fractions 1–6, 264 mg, 540 mg, 960 mg, 1.34 g, 8.14 g, and 444 mg, respectively). Florisil chromatography of fraction 2 (hexane–acetone, 8:2) gave ganodermadiol ( 6.0 mg) [16]. Repeated column chromatography of fraction 3 on silica gel (hexane– acetone, 8:2) followed by preparative HPLC using a RPODS-80TS column, and a linear gradient of CH3CN (75% fi 95%) in 2% AcOH; 5 ml/min, afforded lucidumol B ( 6.2 mg) and ganoderiol F ( 84.8 mg) [17]. Separation of fraction 4 by preparative HPLC (CH3CN in 2% AcOH, 70% fi 90%) after chromatography on Florisil and silica gel columns furnished ganodermanontriol ( 48.2 mg) and ganodermatriol ( 28.0 mg) [18, 19]. The structures of these compounds were determined by spectroscopic methods including 1D- and 2D-NMR, UV, IR, and EIMS (Fig. 1).

Chemicals and media Column chromatography was carried out on Florisil (100–200 mesh, Nacalai Tesque, Kyoto, Japan) and silica gel (Kieselgel 60, 70–230 mesh, Merck Co., Darmstadt, Germany). Thin-layer chromatography (TLC) was done on pre-coated silica gel 60 F254 plates (0.25 mm, Merck), and spots were detected under a UV light and by spraying with 10% H2SO4 followed by heating. Roswell Park Memorial Institute (RPMI) 1640 was purchased from ICN Biomedicals Inc. (Ohio, USA) and fetal bovine serum from JRH Biosciences Co. (Lenexa, USA). Streptomycin and penicillin G potassium obtained from Wako Pure Chemical Co. (Osaka, Japan), sulforhodamine B (SRB) and adriamycin from Sigma Chemical Co. (St. Louis, USA).

Tumor cells Meth A (mouse sarcoma) and Lewis lung carcinoma (LLC, mouse) cells were purchased from RIKEN Cell Line Bank (Tsukuba, Japan). Sarcoma 180 (mouse sarcoma) and T-47D (human carcinoma) were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan). The cells were maintained as monolayer cultures in RPMI 1640 medium supplemented with 5% FBS, sodium bicarbonate (2 g), penicillin G (100,000 units), and streptomycin (100 mg) in a humidiﬁed 5% CO2 atmosphere at 37 C. Animals

Preparation of ganoderma alcohol and acid fractions Ganoderma alcohol and acid fractions were separated according to our previous report [15]. One hundred and ninety grams of pulverized fruiting bodies of G. lucidum (antlered form) was extracted three times with 20 volumes of CHCl3 by reﬂuxing in a boiling water bath for 3 h, and the combined extracts were evaporated to dryness in vacuo. Half of the CHCl3 extract (4.45 g) was chromatographed on a Florisil column (25.0 g) eluted with hexane–acetone (9:1 and 7:3), and CHCl3–MeOH (1:2) to give three fractions, monitoring by TLC. The

Speciﬁc pathogen-free female BDF-1 mice, purchased from Japan SLC Inc. (Shizuoka, Japan), were used at 4 weeks of age, weighing 14–16 g. The animals were fed with a commercial pellet chow (Clea Japan Inc., Tokyo, Japan) in a temperature-controlled room at 25 ± 2 C and water ad libitum. Cytotoxicity assay The in vitro tumor cell assay was carried out by SRB method as described previously [14, 20]. The 50%

44 Fig. 1 Structures of ganoderma alcohols isolated from G. lucidum

R2 25 20

R R5 3

Compound

Ganodermadiol

(1)

Lucidumol B

(2)

OH OH

Ganodermanontriol (4)

Ganodermatriol

inhibitory concentration (IC50) was calculated by the Probit method [21]. Animal experiment For the animal experiments, alcohol and acid fractions and ganoderiol F were completely suspended in Tween 20 followed by dilution with 0.9% NaCl solution. The ﬁnal concentration of Tween 20 in sample solution was 2%. Adriamycin was dissolved in 0.9% NaCl solution. For the subcutaneous tumor assay [22], a suspension of 5 · 105 LLC cells in 0.1 ml of 0.9% NaCl solution was carefully inoculated intradermally into the left axilla of mice. Then, the tumor-bearing mice were randomly assigned to treatment experimental groups (ﬁve or six mice in each group). Twenty-four hours after the tumor inoculation, mice were intraperitoneally given ganoderma alcohol and acid fractions (both of 50 and 100 mg/ kg), ganoderiol F (5, 10, and 20 mg/kg), and adriamycin (2 mg/kg, as a positive control) once daily for 14 consecutive days. Control animals were given 0.1 ml of 0.9% NaCl solution. The tumors were measured on each alternate day using a vernier caliper from the initiation of treatment to the time when grosses ulceration of the tumor was developed in control mice. The tumor size was calculated from the following equation [22]:

(5)

∆24(25)

(3)

Ganoderiol F

∆24(25) OH

∆24(25)

CH2OH

CH3

CH2OH

CH3

CH2OH

tumor vol: ðmm3 Þ ¼ 0:5 a b2 where a is the longest diameter and b is the shortest diameter. The eﬀects by treatments were represented as follows: T=Cð%Þ ¼ðmean value of a treated group=mean value of a control groupÞ 100

Statistical analysis The significance of differences between the experimental groups and control group was calculated by Dunnett’s t test. P < 0.05 was considered significant.

Results Eﬀects of triterpene fractions from the antlered form of the fruiting bodies of G. lucidum on subcutaneously (s.c.) inoculated LLC The fractions of ganoderma alcohols and acids separated from the antlered form of the fruiting bodies were intraperitoneally administrated to LLC-bearing mice with doses of 50 and 100 mg/kg per day for 14

consecutive days starting from the day after the intradermal inoculation of tumor cells. As shown in Fig. 2, the tumor sizes of ganoderma-alcohol-fraction-treated groups were 1,714±245 and 1,451±201 mm3 at doses of 50 and 100 mg/kg per day, respectively, on day 13 in the administration period while 2,542±336 mm3 for the saline control group. Administration of the ganoderma alcohol fraction resulted in significant suppression of tumor growth at both doses with T/C values of 67.5 and 57.1%, compared with the control (data not shown). Moreover, after the administration period, the tumor size of the 100 mg/kg group was 2,226±374 to 5,005±1,158 mm3 (on day 16–20), accounting for 67.5– 72.9% of the control level (3,297±390 to 6,865±390 mm3 on days 16–20). As for the acid fraction, no appreciable activity was observed at each dose. Cytotoxicity of ganoderma alcohols isolated from G. lucidum against four tumor cell lines Five ganoderma alcohols, including ganodermadiol, lucidumol B, ganoderiol F, ganodermanontriol, and ganodermatriol, isolated from G. lucidum were tested for their cytotoxic activities against LLC, T47D, S-180, and Meth-A cells in vitro (Table 1). Among these compounds, ganoderiol F showed the most appreciable toxicity, with IC50 values of 2.0, 3.0, 7.1, and 10.1 lg/ml against LLC, T47D, S-180, and Meth-A cell lines, respectively. Effect of ganoderiol F on subcutaneously (s.c.) implanted LLC To evaluate the antitumor effect of a single ganoderma alcohol, ganoderiol F, which exhibited the strongest

Fig. 2 In vivo antitumor eﬀects of alcohol and acid fractions separated from the antlered form of fruiting bodies of Ganoderma lucidum on intradermally inoculated Lewis lung carcinoma (LLC) cells in mice by intraperitoneally administration. Data are expressed as mean ± S.E. (n=5–6). Statistical signiﬁcance: *P<0.05, **P<0.01, ***P<0.001 versus control

Table 1 Cytotoxicity of ganoderma alcohols isolated from Ganoderma lucidum on tumor cell growth. IC50 50% inhibitory concentration, Meth-A mouse carcinoma, LLC Lewis lung carcinoma, T-47D human breast carcinoma Compound

Ganodermadiol Lucidumol B Ganoderiol F Ganodermanontriol Ganodermatriol Adriamycina a

IC50 (lg/ml) Meth-A

S-180

LLC

T-47D

>20 >20 10.1 >20 >20 0.13

>20 >20 7.1 >20 >20 0.11

>20 >20 2.0 >20 15.0 0.06

>20 6.5 3.0 >20 >20 0.02

Positive control

activity in a cytotoxicity assay, was administrated to LLC-bearing mice at three doses of 5, 10, and 20 mg/kg/ day. When given intraperitoneally once daily for 2 weeks, the compound signiﬁcantly reduced the tumor size to 1,502±197, 1,275±232, and 1,124±180 mm3 compared with the control group (2,357±188 mm3) at three doses, respectively, and their T/C values were 63.7%, 54.1%, and 47.7% on day 14 in the administration period (Fig. 3). Moreover, on day 20 after the administration period, the tumor sizes of ganoderiol-Ftreated group were 4,086±543, 3,272±576, and 2,802±265 mm3 at doses of 5, 10, and 20 mg/kg, respectively, accounting for 78.7%, 63.0%, and 53.9% of the control group (5,194±573 mm3). Ganoderiol F showed remarkable inhibitory eﬀect on the tumor growth in a dose-dependent manner both in and after the administration period (Fig. 3). The T/C values of the positive control treated with adriamycin were of 38.0% and 44.5% in and after administration period, respectively, at a dose of 2 mg/kg.

46 Fig. 3 In vivo antitumor eﬀects of ganoderiol F isolated from Ganoderma lucidum on intradermally inoculated Lewis lung carcinoma (LLC) cells in mice by intraperitoneal administration. Data are expressed as mean ± S.E. (n=5–6). Statistical signiﬁcance: *P<0.05, **P<0.01, ***P<0.001 versus control

Fig. 4 Body-weight changes of mice treated with alcohol and acid fractions by intraperitoneal administration (n=5–6)

Fig. 5 Body-weight changes of mice treated with ganoderiol F by intraperitoneal. administration (n=5–6)

Toxic and side eﬀects of intraperitoneally administrated ganoderma alcohol and acid fractions and ganoderiol F No evident toxic or side eﬀects were observed, such as the weight reduction observed in the adriamycin-treated-

group at the end of the treatment. The mean body weights of the mice treated with alcohol and acid fractions decreased down to 5 days but then gradually increased, becoming almost the same as control body weights (Fig. 4). There was no signiﬁcant diﬀerence in body weights of mice administrated with ganoderiol F

(Fig. 5). The skin and hair texture, and behavioral pattern did not reﬂect any toxic reaction at experimental doses.

Discussion Basidiomycetes, such as Agaricus blazei, Lentinan edodes, Polyporus umbellatus, and G. lucidum, have been used by about 1,000,000–2,000,000 people in Japan for the prevention of cancer and/or as an adjuvant with cancer chemotherapy drugs after the removal of malignant tumors [12]. As regards G. lucidum, a large number of research groups have proved the antitumor effects of the polysaccharides and water extracts containing a- and b-glucans, either in vitro or in vivo [6, 7, 23]. But the efficacy on carcinogenesis of triterpenes is not yet well proven. In our recent studies on the comparison of biological activity between two fruiting bodies of germinated and mature antlered forms of G. lucidum, we found that the aqueous extracts of the mature one, which have a much higher content of total triterpenes, tended to be more potent in antitumor activity than another in vivo although the total polysaccharide contents of the two extracts were quite similar [24]. Hence, we speculate that not only the polysaccharide content but also the triterpene content will influence the antitumor activities of G. lucidum. Consequently, we investigated the in vivo antitumor effects of triterpene fractions from G. lucidum in the present study. The intraperitoneal administration for 14 consecutive days of ganoderma alcohol and acid fractions separated from the antlered form of the fruiting bodies of G. lucidum to LLC-bearing mice resulted in significant suppression of tumor growth both in and after the administration period while no effect was observed for the acid fraction. This finding is very interesting when related to the cytotoxic activities of triterpenes isolated from G. lucidum against several tumor cell lines, which have been performed in our previous research [13, 14]. Of the 24 triterpenes tested, all seven alcohols (lucidumol A, lucidumol B, ganodermanondiol, ganoderiol F, ganodermanontriol, ganodermanonol, and ganodermadiol) exhibited cytotoxicities against several cell lines. Ganodermanonol and ganodermanondiol exhibited the most potential cytotoxicity against Meth-A cells, with ED50 values of 2.8 and 3.4 lg/ml, respectively; and lucidumol A, ganoderiol F, and ganodermanontriol had inhibitory activity on LLC cells with ED50 values of 2.3, 6.0, and 9.6 lg/ml, respectively. As for the 17 ganoderma acids, except for ganoderic acids h and G, none showed activities. Subsequently, we isolated five ganoderma alcohols including ganodermadiol, ganodermanontriol, ganodermatriol, lucidumol B, and ganoderiol F from the same strain of G. lucidum in which ganoderiol F exhibited the strongest cytotoxicity against LLC, T47D, S-180, and Meth-A cells. We next examined the in vivo antitumor effect of ganoderiol F. As shown in Fig. 3, when

administrated by the same method, ganoderiol F showed remarkable inhibitory effect on the growth of inoculated LLC both in and after the administration period in a dose-dependent manner over the range tested. The mechanism of the antitumor effects of G. lucidum is not yet well understood. It has been found previously that polysaccharides from G. lucidum exert their in vitro and in vivo antitumor effects via immunomodulatory properties, including the enhancement of lymphocyte proliferation and antibody production [25], and produced both antigenotoxic and antitumor-promoting activities [23, 26]. Triterpene components were reported to exhibit the biological activities of hepatoprotection [27], antihypertension [28], and inhibitory effects on the cholesterol synthesis [29], mediated by inhibiting the activities of enzymes such as b-galactosidase, angiotension-converting enzyme, and cholesterol synthase. A recent report pointed out that a triterpenoid fraction from the fruiting bodies of G. lucidum had antitumor and antimetastatic effects on liver through the inhibition of tumor-induced angiogenesis, but the molecular mechanism was not investigated [12]. More recently, Lin et al. reported a triterpene-enriched extract from G. lucidum inhibited the growth of hepatoma cells by suppressing the protein kinase C, activating the c-Jun Nterminal kinase/stress-activated protein kinase (JNK/ SAPK) and p38 mitogen-activated protein (MAP) kinase, as well as prolonging the G2 cell-cycle phase in Huh-7 cells [11]. It is noteworthy that this is the first report about the in vivo study on antitumor activity of G. lucidum using a single triterpene component. It is said that the polysaccharides of large molecular weights are not absorbed within intestines, and the medicinal actions of mushrooms such as G. lucidum may be mediated by a combination of the polysaccharides and other components [30]. The present study emphasizes that in antitumor activity, the other components may be ganoderma alcohols, and suggests that they are potential antitumor agents. Further studies, including the inhibitory mechanism of antitumor action, are necessary to understand the antitumor effects of ganoderma alcohols in G. lucidum. Acknowledgements This paper is a part of the 21st COE program sponsored by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors are grateful to TDK Service Corporation (Tokyo, Japan) and Reishi Sougou Kenkyu Jo (Tokyo, Japan) for providing ganoderma samples and financial support.

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