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Effect of the culture extract of Lentinus edodes mycelia on splenic sympathetic activity and cancer cell proliferation
Autonomic Neuroscience: Basic and Clinical 145 (2009) 50–54
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Effect of the culture extract of Lentinus edodes mycelia on splenic sympathetic activity and cancer cell proliferation Jiao Shen a,⁎, Mamoru Tanida a, Yoshiyuki Fujisaki a, Yuko Horii a, Kazuko Hashimoto b, Katsuya Nagai a a b
ANBAS Corporation, 4-12-17 Toyosaki, Kita-Ku, Osaka 531-0072, Japan Humalabo Co., Ltd., 1-11-9 Azabudai, Minato-ku, Tokyo 106-0041, Japan
a r t i c l e
i n f o
Article history: Received 25 September 2008 Accepted 9 November 2008 Keywords: Spleen Rat Athymic nude mice Nerve Human cancer In vivo Tumor inhibition
a b s t r a c t The spleen is an important organ for tumor immunity, and the splenic sympathetic nerve has a suppressive effect on splenic natural killer (NK) cytotoxicity. On the basis of this and reports that Lentinus edodes (Shiitake mushroom) has tumor-inhibitory effects, the authors hypothesized that an extract of a mycelial culture of L. edodes grown in a solid medium of sugar-cane bagasse and defatted rice bran–L·E·M–might affect the sympathetic splenic sympathetic nerve activity (Splenic-SNA) and thus inhibit tumor proliferation. Thus, the effect of L·E·M on Splenic-SNA and human cancer cell proliferation was examined. Splenic-SNA was found to be suppressed by an intraduodenal L·E·M injection in urethane-anesthetized rats, which significantly inhibited increases in the tumor volume of human colon and breast cancer cells implanted in athymic nude mice. These findings suggest that L·E·M has an inhibitory effect on tumor proliferation possibly via a reduction in NK cytotoxicity through the suppression of Splenic-SNA. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
The tumor-inhibitory effects of some mushrooms and their extracts have been suggested. It has also been suggested that extracts of Lentinus edodes (Shiitake mushroom, a kind of basidiomycete) show antitumor activity in animals and humans (Hibino et al., 1994; Israilides et al., 2008). The whole extract of the mycelial culture of L. edodes grown in a solid medium of sugar cane bagasse and defatted rice bran is known as L·E·M (Sugano et al., 1982), which was reported to have tumor-inhibitory activity (Sugano et al., 1982; Morinaga et al., 1994). Moreover, the spleen is an important organ involved in immune functions, and the splenic sympathetic nerve was suggested to mediate the central modulation of immunity (Hori et al., 1995) and suppress splenic natural killer (NK) cytotoxicity (Katafuchi et al., 1993). These facts raised the possibility that L·E·M realizes its tumorinhibitory activity by enhancing splenic NK cytotoxicity through the suppression of splenic sympathetic nerve activity (Splenic-SNA). Therefore, we examined whether intraduodenal administration of L·E·M inhibited Splenic-SNA in urethane-anesthetized rats. In this experiment, we found that intraduodenal infusion of L·E·M suppressed Splenic-SNA. Furthermore, we investigated the effects of L·E·M–by providing it as the sole drinking water–on the proliferation of human colon cancer (HCT116) cells and human breast cancer (MCF117) cells implanted in BALB/c athymic nude mice lacking acquired immunity. The details of the results are described here.
2.1. Animals
⁎ Corresponding author. Tel.: +81 6 6486 1080; fax: +81 6 64861081. E-mail address: [email protected] (J. Shen). 1566-0702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.11.004
Male Wistar rats (age, 8 weeks; weight, 250–300 g) and female BALB/ c athymic nude mice (BALB/c Slc-nu/nu; Japan SLC, Inc.) (4 weeks old, weighing 14.5–18.8 g) were used. All the animals were group-housed (3 or 4 animals per cage) in a room maintained at 24 ± 1 °C and illuminated for 12 h (07:00–19:00 h). Food (type MF; Oriental Yeast Co., Tokyo) and water were freely available. The animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of the ANBAS Corporation in accordance with the Science Council of Japan's guidelines for animal experiments issued on June 1, 2006. 2.2. Determination of Splenic-SNA Splenic-SNA was determined using urethane-anesthetized rats, as described previously (Tanida et al., 2005a). Briefly, the rats were fasted for 4–6 h and anesthetized with urethane intraperitoneally (1 g/kg, IP) prior to the surgery. For recording the neural activity of the sympathetic nerves innervating the spleen, a splenic branch of the splanchnic nerve was dissected, ligated, and then attached to a pair of silver wire electrodes. The electrical signals from the electrodes were amplified 5000–10,000 times with a band path of 100 to 1000 kHz, filtered, and monitored on an oscilloscope. The raw nerve activity was converted to standard pulses using a window discriminator. Data were obtained as described previously (Tanida et al., 2005a,b).
J. Shen et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 50–54
2.3. Cell cultures, implantations, and tumor proliferation assays HCT116 and MCF7 cells were cultured in the DMEM medium supplemented with 10% fetal bovine serum. They were harvested, and their viabilities were determined by trypan blue staining; cells with more than 95% viability were used. HCT116 (2.5 × 106 cells) or MCF7 cells (5.0 × 106 cells) were re-suspended in 200 μl of serum-free DMEM, and injected into the subcutaneous space in the right lower back of each BALB/c nude mouse using a 26-gauge needle. Because MCF-7 is a human breast cancer cell line, which requires estradiol supplementation for its proliferation in athymic mice (White et al., 1982), each nude mouse including the control was implanted with a 1.25-mg 17β-estradiol pellet (release ability for 60 days; Innovation Research, Sarasota, FL) a week before the injection of MCF7 cells. 2.4. Reagent administrations L·E·M (an extract of cultured L. Edodes Mycelia; Humalabo Co., Tokyo) was dissolved in water. For the intraduodenal injection, a polyethylene tube (PE-60; Clay Adams, Parsippany, NJ) was inserted into the duodenum with its end at a point approximately 5 mm toward the jejunum. For the tumor proliferation assays, the mice were randomly divided into 2 experimental groups of 6 animals each. Group 1 (control) received water and Group 2 received the L·E·M solution (100 mg/ml) as the sole drinking water, respectively, from the time after the implantation of either HCT116 or MCF7 cells. The bottles of drinking water were changed every 2 or 3 days. Tumor volume was measured using a digital caliper (graduated in 0.01 mm), and the tumor volume was calculated using the following formula as previously described (Gonzalez et al., 2006): 0.5 × L × W2 (L, length of the longest axis; W, width of the line meeting the longest axis at right angles). At the end of the experiment, which was day 20 for the HCT116 implantation experiment and day 27 for the MCF7 implantation experiment, the tumors were excised from the mice, which were sacrificed with an overdose of ketamine hydrochloride, and the excised tumor weights were determined. The L·E·M used is a product of Humalabo Co., Tokyo, and it was produced by growing a specific type of mycelia of L. edodes (Shiitake mushroom). It was cultured for up to 7 months in a solid culture medium composed of defatted rice bran and bagasse (sugarcane fibers) mixed in a fixed ratio. The active ingredients of this culture were extracted under strict examination, using a special technique. This extraction technique was based on enzymatic degradation without the use of chemicals.
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L·E·M solution (500 mg/ml) are shown in Fig. 1a. Means ± SEMs of the percentage change from the 0-min values of the Splenic-SNA of 3 rats are shown in Fig. 1b. A slight increase was observed from 25 min after the intraduodenal injection of water (Fig. 1b). In contrast, an intraduodenal injection of 1 ml of the L·E·M solution (500 mg/ml) gradually and markedly lowered Splenic-SNA, with the lowest level being 60.2 ± 14.3% at 60 min after the injection (Fig. 1b). The statistical significance of the difference between the groups with regard to the values of Splenic-SNA 5–60 min after the administration of the L·E·M solution and water was analyzed by ANOVA, and the difference was found to be statistically significant (P b 0.0005, F = 64.8 by ANOVA). The basal (0 min) values of Splenic-SNA were obtained as the mean values of the 5-min period before the intraduodenal administration of water and the L·E·M solution. The absolute values of Splenic-SNA at 0 min were as follows: the water group, 163.1 ± 5.5 spikes/5 s; L·E·M group, 239.9 ± 61.5 spikes/5 s. No statistically significant difference was detected between these 0-min values. 3.2. Effect of L·E·M on the proliferation of human colon cancer cells implanted in BALB/c athymic nude mice Intraduodenal administration of L·E·M reduced Splenic-SNA in the urethane-anesthetized rats (Fig. 1). Therefore, it is possible that L·E·M is also able to suppress Splenic-SNA in mice. If this is the case, and Splenic-SNA is involved in the mechanism of tumor immunity, L·E·M might affect Splenic-SNA, which in turn might affect the proliferation rate of malignant tumors. To examine this possibility, we studied the effect of an L·E·M solution (100 mg/ml) on the proliferation of HCT116 cells implanted into the subcutaneous space in the back of female BALB/c athymic nude mice lacking acquired immunity but not innate
2.5. Data and statistical analyses Splenic-SNA (spikes/5 s) measured during each 5-min period after the intraduodenal injection of either L·E·M solution or water was evaluated by digital signal processing and statistical analyses. Because of the interindividual variability in the preinjection state, the percent change from the 0-min value was calculated for Splenic-SNA. All the data were expressed as means ± SEMs. Statistical significances of differences in the respective basal (0 min or 0 day) values were detected by nonparametric Mann–Whitney U test. Analysis of variance (ANOVA) with repeated measures was applied to compare group responses in terms of Splenic-SNA and tumor volume induced by L·E·M and water. In all cases, P b 0.05 was applied as the level of significance. 3. Results 3.1. Effect of intraduodenal administration of L·E·M on Splenic-SNA in urethane-anesthetized rats Typical recordings of Splenic-SNA in urethane-anesthetized rats following intraduodenal injection of 1 ml of water and 1 ml of the
Fig. 1. Effect of intraduodenal administration of 1 ml of L·E·M solution (500 mg/ml) on splenic sympathetic nerve activity (Splenic-SNA). (a) Representative recordings of Splenic-SNA in rats intraduodenally administered water or the L·E·M solution. Vertical scale bars to the left of the recordings represent the neural discharge rate of 100 spikes/ 5 s. (b) Changes in neural activity expressed as a percentage of the 0-min value. Data (means ± SEMs) are expressed as the percentages of change in the values from the 0-min value. Each of the 3 rats in the water group and L·E·M group were used. Analysis of variance was used to analyze the statistical significance of the difference between Splenic-SNA of the 2 groups.
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J. Shen et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 50–54
immunity. Fig. 2a shows the changes in tumor volume (m3, means ± SEMs) of HCT116 cells in the subcutaneous space of the nude mice during the 20-day test period. The control group of mice were provided with water, and the experimental group of mice, with L·E·M solution (100 mg/ml) as the sole drinking for this 20-day test period. The tumor volume of the control mice given water gradually and markedly increased, and it reached a value of 645.45 ± 114.20 mm3 on day 20 after the implantation (Fig. 2a). The tumor volume of HCT116 cells in the mice given the L·E·M solution also increased; however, this increase was lower than that of the control mice who were given water. Moreover, the tumor volume of the L·E·M group on day 20 was 465.62 ± 57.54 mm3 (72.14% of the tumor volume of the control group). The difference between the control and L·E·M groups with regard to the tumor volumes from day 4 to day 20 was statistically analyzed by ANOVA with repeated measures, and it was found to be statistically significant (P b 0.0005, F = 14.7 by ANOVA). The tumor weights (means ± SEMs) of HCT116 cells excised from the mice 20 days after the implantation are shown in Fig. 2b. The tumor weight of the control mice given water was 0.357 ± 0.051 g, and that of mice given L·E·M was 0.290 ± 0.052 g (81.2% of the tumor weight of the control group). Although there was no statistically significant difference between the tumor weights on day 20 of these 2 groups of mice, a borderline statistical difference (0.05 b P b 0.1) was detected by the Mann–Whitney U test (Fig. 2b).
Fig. 3. Tumor volumes and weights of human breast cancer (MCF7) cells implanted into BALB/c athymic nude mice provided with water and L·E·M as the sole drinking water. (a) Changes in the tumor volumes of MCF7 cells implanted into the nude mice. Means ± SEMs of the tumor volume (mm3) in the 2 groups are shown. Analysis of variance with repeated measures was used to analyze the statistical difference in the tumor volumes between the 2 groups. (b) Tumor weights (means ± SEMs) of each group on day 27. The tumor weights of the 2 groups of rats provided with water and the L·E·M solution as the sole drinking water showed no statistically significant difference by the Mann–Whitney U test.
3.3. Effect of L·E·M on proliferation of human breast cancer cells implanted in BALB/c athymic nude mice
Fig. 2. Tumor volumes and weights of human colon cancer (HCT116) cells implanted into BALB/c athymic nude mice provided with water or L·E·M as the sole drinking water. (a) Changes in the tumor volumes of HC116 cells implanted into nude mice. Means ± SEM of the tumor volume (mm3) in these 2 groups are shown. Analysis of variance with repeated measures was used to analyze the statistical significance of the difference in tumor volumes between the 2 groups. (b) Tumor weights (means ± SEMs) of each group on day 20. The tumor weights of the 2 groups administered water or the L·E·M solution showed no statistically significant difference by the Mann–Whitney U test.
Finally, we examined the effect of the L·E·M solution on the proliferation of MCF7 cells implanted into the subcutaneous space in the back of female BALB/c athymic nude mice. Fig. 3a shows the changes in tumor volume (m3) of MCF7 cells in the subcutaneous space of the nude mice during the 27-day test period. Mice in the control group were given water (water-control group), and mice in the experimental group were given the L·E·M solution (100 mg/ml) (L·E·M group) as the sole drinking for the 27-day test period. The tumor volume of the control mice given water gradually and markedly increased, and it reached a value of 326.02 ± 97.14 mm3 on day 27 after the implantation (Fig. 3a). The tumor volume of MCF7 cells in the mice given the L·E·M solution was also elevated gradually (Fig. 3a). However, the increase in the tumor volume in the L·E·M group was much lower than that in the water-control group, and the tumor volume of the L·E·M group was 162.73 ± 75.74 mm3 (49.9% of the tumor volume of the water-control group) on day 27 after the implantation (Fig. 3a). The difference between the tumor volumes of the control and L·E·M groups on days 4 to 27 was statistically analyzed by ANOVA with repeated measures, and it was found to be statistically significant (P b 0.0005, F = 20.5 by ANOVA). The tumor weights of MCF7 cells excised from the mice 27 days after the implantation are shown in Fig. 3b. The tumor weight of the control mice given water was 0.142 ± 0.047 g, and the tumor weight of
J. Shen et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 50–54
mice given the L·E·M solution was 0.070 ± 0.034 g (49.3% of the tumor weight of the water-control group). Although there was no statistically significant difference between the tumor weights on day 27 for these 2 groups of mice (P = 0.16 by the Mann–Whitney U test), the ratio of the tumor weight of the L·E·M group to that of the water-group was 0.493 on day 27, which was similar to the ratio of the tumor volume of the L·E·M group to that of the water-group (0.499) on day 27. 4. Discussions In this study, the following observations were made: (1) SplenicSNA was suppressed by oral administration of 1 ml of L·E·M solution (500 mg/ml) in urethane-anesthetized rats (Fig. 1); (2) the volume of the tumor due to HCT116 cells implanted into the subcutaneous space of athymic nude mice was lowered by the L·E·M solution (100 mg/ml), which was provided as the sole drinking water (Fig. 2); (3) the tumor volume due to MCF7 cells implanted into the subcutaneous space of athymic nude mice was also reduced by the L·E·M solution (100 mg/ml), which was provided as the sole drinking (Fig. 3). The autonomic nervous system in collaboration with the endocrine system is important for maintaining the homeostasis of important physiological phenomena for survival in mammals. Recently, we observed that in rats, olfactory stimulation with the scents of grapefruit oil (GFO) and lavender oil (LVO) affected autonomic nerve activities and thus physiological phenomena. The following observations were made: (1) Olfactory stimulation with the scent of GFO elevated the activities of sympathetic nerves innervating white adipose tissue, brown adipose tissue, the adrenal glands, and the kidney, and thus increased the plasma glycerol level, body temperature, and blood pressure. (2) Olfactory stimulation with the scent of GFO lowered the gastric parasympathetic (vagal) nerve activity and thus reduced food intake. (3) Olfactory stimulation with the scent of LVO suppressed the activities of sympathetic nerves innervating white adipose tissue, brown adipose tissue, the adrenal glands, and the kidney, and thus decreased the plasma glycerol level, body temperature, and blood pressure. (4) Olfactory stimulation with the scent of LVO enhanced gastric parasympathetic (vagal) nerve activity and thus increased food intake (Shen et al., 2005a,b; Tanida et al., 2005b, 2006). Moreover, it was observed that intraduodenal administration of a type of lactobacillus affected autonomic neurotransmissions, blood pressure, and blood glucose (Tanida et al., 2005a,b; Yamano et al., 2006). These findings suggest that changes in the autonomic nerve activity are good indicators of physiological markers such as lipolysis, body temperature, blood pressure, blood glucose, appetite, etc. Additionally, we previously observed that intraduodenal administration of 1 ml of a mixture of L-arginine (50 mM) and L-lysine (50 mM) suppressed Splenic-SNA in urethane-anesthetized rats. We observed that a mixed solution of L-arginine and L-lysine (50 mM each) provided as the sole drinking water inhibited the tumor volumes of HCT116 cells implanted into athymic nude mice, and we detected a significant correlation between the suppression of SplenicSNA in rats and the reduction of HCT116 tumor volume in nude mice that caused by the administration of the L-arginine and L-lysine mixture (Shen et al., submitted). It was also suggested that extracts of L. edodes exhibit antitumor activity in animals and humans (Hibino et al., 1994; Israilides et al., 2008). Therefore, we hypothesized that a whole extract of the mycelial culture of L. edodes grown in a solid medium of sugar-cane bagasse and defatted rice bran, i.e., L·E·M, might suppress either Splenic-SNA in urethane-anesthetized rats or tumor growth of HCT116 and MCF7 cells implanted into athymic nude mice lacking acquired immunity but not innate immunity. Consequently, it was observed that an intraduodenal injection of 1 ml of the L·E·M solution (500 mg/ml) significantly reduced Splenic-SNA in urethaneanesthetized rats (Fig. 1) and that providing the L·E·M solution (100 mg/ml) as the sole drinking water inhibited the growth of both HCT116 and MCF7 cells in the nude mice (Figs. 2 and 3). These finding
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suggest that L·E·M has suppressive actions on Splenic-SNA and tumor proliferation. The spleen is an important organ involved in immune functions; Splenic-SNA is suggested to mediate the central modulation of immunity (Hori et al., 1995); and the excitation of Splenic-SNA causes the suppression of splenic NK cytotoxicity (Katafuchi et al., 1993). In other words, the reduction of Splenic-SNA might elicit an increase in NK cytotoxicity. Considering these facts and the present findings, it is possible that L·E·M might increase the spleen NK cytotoxicity through a reduction in Splenic-SNA, and thus function to suppress tumor proliferation due to HCT116 and MCF7 cells. This needs to be examined in the future. L·E·M contains β-glucan (Wasser, 2002), and β-glucan is suggested to inhibit tumor growth through the activation of macrophages (Lee et al., 2002), NK cytotoxicity (Fujimiya et al.,1998), and the complement system (Gelderman et al., 2004) as well as through increases in the number and activity of lymphocytes (Ng and Yap, 2002). Therefore, β-glucan might be an effective substance with regard to the action of L·E·M on Splenic-SNA and tumor proliferation observed in this experiment. This possibility must be examined in the future. In conclusion, L·E·M inhibited Splenic-SNA in urethane-anesthetized rats and suppressed the proliferation of human colon cancer cells and human breast cancer cells implanted in athymic nude mice. These findings suggest that L·E·M has antitumor action. Acknowledgements The authors would like to express their deep gratitude to Professor Masato Okada and Dr. Chitose Oneyama, Research Institute for Microbial Diseases, Osaka University, for their help in the culturing of cancer cells. References Gonzalez, L., Aqullo-Ortuno, M.T., Garcia-Martinez, J.M., Calcaabrini, A., Gamollo, C., Palacios, J., Aranda, A., Martin-Perez, J., 2006. Role of c-Src in human MCF7 breast cancer cell tumorigenesis. J. Biol. Chem. 281, 20851–20854. Fujimiya, Y., Suzuki, Y., Oshima, K., Kobori, H., Moriguchi, K., Nakashima, H., Matumoto, Y., Takahara, S., Ebina, T., Katakura, R., 1998. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis. Cancer Immunol. Immunother. 46, 147–159. Gelderman, K.A., Tomlinson, S., Ross, G.D., Gorter, A., 2004. Complement function in mAb-mediated cancer immunotherapy. Trends Immunol. 25, 158–164. Hibino, Y., Konishi, Y., Koike, J., Tabata, T., Ohashi, Y., Sugano, N., 1994. Productions of interferon-gamma and nitrite are induced in mouse splenic cells by a hateroglycanprotein fraction from culture medium of Lentinus edodes mycelia. Immunopharmacology 28, 77–85. Hori, T., Katafuchi, T., Take, S., Shimizu, N., Niijima, A., 1995. The autonomic nervous system as a communication channel between the brain and the immune system. Neuroimmunomodulation 2, 203–215. Israilides, C., Kletsas, D., Arapoglou, D., Philippossis, A., Pratsinis, H., Ebringerova, A., Hribalova, F., Haarding, S.E., 2008. In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinus edodes. Phytomedecine 15, 512–519. Katafuchi, T., Take, S., Hori, T., 1993. Roles of sympathetic nervous system in the suppression of cytotoxicity of splenic natural killer cells in rat. J. Physiol. 465, 343–357. Lee, D.Y., Ji, I.H., Chang, H.I., Kim, C.W., 2002. High-level TNF-α secretion and macrophage activity with soluble β-glucans from Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 66, 233–238. Morinaga, H., Tazawa, K., Tagoh, H., Muraguchi, A., Fujimaki, M., 1994. An in vivo study of hepatic and splenic interleukin-1 beta mRNA expression following oral PSK or LEM administration. Jpn. J. Cancer Res. 85, 1298–1303. Ng, M.L., Yap, A.T., 2002. Inhibition of human colon carcinoma development by lentinan from shiitake mushrooms (Lentinus edodes). J. Altern. Complement. Med. 8, 581–589. Shen, J., Niijima, A., Tanida, M., Horii, Y., Maeda, K., Nagai, K., 2005a. Olfactory stimulation with scent of grapefruit oil affects autonomic nerves, lipolysis and appetite in rats. Neurosci. Lett. 380, 289–294. Shen, J., Niijima, A., Tanida, M., Horii, Y., Maeda, K., Nagai, K., 2005b. Olfactory stimulation with scent of lavender oil affects autonomic nerves, lipolysis and appetite in rats. Neurosci. Lett. 383, 188–193. Sugano, N., Hibino, Y., Choji, Y., Maeda, H., 1982. Anticarcinogenic actions of watersoluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia. Cancer Lett. 17, 109–114.
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Tanida, M., Niijima, A., Fukuda, Y., Sawai, H., Tsuruoka, N., Shen, J., Yamada, S., Kiso, Y., Nagai, K., 2005a. Dose-dependent effects of L-carnosine on the renal sympathetic nerve and blood pressure in urethane-anesthetized rats. Am. J. Physiol. 288, R447–R455. Tanida, M., Niijima, A., Shen, J., Nakamura, T., Nagai, K., 2005b. Olfactory stimulation with scent of essential oil of grapefruit affects autonomic neurotransmission and blood pressure. Brain Res. 1058, 44–55. Tanida, M., Niijima, A., Shen, J., Nakamura, T., Nagai, K., 2006. Olfactory stimulation with scent of lavender oil affects autonomic neurotransmission and blood pressure in rats. Neurosci. Lett. 398, 155–160.
Wasser, S.P., 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60, 258–274. White, A.C., Levy, J.A., McGrath, C.M., 1982. Site-selective growth of a hormoneresponsive human breast carcinoma in athymic mice. Cancer Res. 42, 906-012. Yamano, T., Tanida, M., Maeda, K., Okumura, N., Fukushima, Y., Nagai, K., 2006. Effects of the probiotic strains Lactobacillus johnsonii strain in La1 on autonomic nerves and blood glucose in rats. Life Sci. 79, 1963–1967.
Contents lists available at ScienceDirect
Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Effect of the culture extract of Lentinus edodes mycelia on splenic sympathetic activity and cancer cell proliferation Jiao Shen a,⁎, Mamoru Tanida a, Yoshiyuki Fujisaki a, Yuko Horii a, Kazuko Hashimoto b, Katsuya Nagai a a b
ANBAS Corporation, 4-12-17 Toyosaki, Kita-Ku, Osaka 531-0072, Japan Humalabo Co., Ltd., 1-11-9 Azabudai, Minato-ku, Tokyo 106-0041, Japan
a r t i c l e
i n f o
Article history: Received 25 September 2008 Accepted 9 November 2008 Keywords: Spleen Rat Athymic nude mice Nerve Human cancer In vivo Tumor inhibition
a b s t r a c t The spleen is an important organ for tumor immunity, and the splenic sympathetic nerve has a suppressive effect on splenic natural killer (NK) cytotoxicity. On the basis of this and reports that Lentinus edodes (Shiitake mushroom) has tumor-inhibitory effects, the authors hypothesized that an extract of a mycelial culture of L. edodes grown in a solid medium of sugar-cane bagasse and defatted rice bran–L·E·M–might affect the sympathetic splenic sympathetic nerve activity (Splenic-SNA) and thus inhibit tumor proliferation. Thus, the effect of L·E·M on Splenic-SNA and human cancer cell proliferation was examined. Splenic-SNA was found to be suppressed by an intraduodenal L·E·M injection in urethane-anesthetized rats, which significantly inhibited increases in the tumor volume of human colon and breast cancer cells implanted in athymic nude mice. These findings suggest that L·E·M has an inhibitory effect on tumor proliferation possibly via a reduction in NK cytotoxicity through the suppression of Splenic-SNA. © 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
The tumor-inhibitory effects of some mushrooms and their extracts have been suggested. It has also been suggested that extracts of Lentinus edodes (Shiitake mushroom, a kind of basidiomycete) show antitumor activity in animals and humans (Hibino et al., 1994; Israilides et al., 2008). The whole extract of the mycelial culture of L. edodes grown in a solid medium of sugar cane bagasse and defatted rice bran is known as L·E·M (Sugano et al., 1982), which was reported to have tumor-inhibitory activity (Sugano et al., 1982; Morinaga et al., 1994). Moreover, the spleen is an important organ involved in immune functions, and the splenic sympathetic nerve was suggested to mediate the central modulation of immunity (Hori et al., 1995) and suppress splenic natural killer (NK) cytotoxicity (Katafuchi et al., 1993). These facts raised the possibility that L·E·M realizes its tumorinhibitory activity by enhancing splenic NK cytotoxicity through the suppression of splenic sympathetic nerve activity (Splenic-SNA). Therefore, we examined whether intraduodenal administration of L·E·M inhibited Splenic-SNA in urethane-anesthetized rats. In this experiment, we found that intraduodenal infusion of L·E·M suppressed Splenic-SNA. Furthermore, we investigated the effects of L·E·M–by providing it as the sole drinking water–on the proliferation of human colon cancer (HCT116) cells and human breast cancer (MCF117) cells implanted in BALB/c athymic nude mice lacking acquired immunity. The details of the results are described here.
2.1. Animals
⁎ Corresponding author. Tel.: +81 6 6486 1080; fax: +81 6 64861081. E-mail address: [email protected] (J. Shen). 1566-0702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.11.004
Male Wistar rats (age, 8 weeks; weight, 250–300 g) and female BALB/ c athymic nude mice (BALB/c Slc-nu/nu; Japan SLC, Inc.) (4 weeks old, weighing 14.5–18.8 g) were used. All the animals were group-housed (3 or 4 animals per cage) in a room maintained at 24 ± 1 °C and illuminated for 12 h (07:00–19:00 h). Food (type MF; Oriental Yeast Co., Tokyo) and water were freely available. The animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of the ANBAS Corporation in accordance with the Science Council of Japan's guidelines for animal experiments issued on June 1, 2006. 2.2. Determination of Splenic-SNA Splenic-SNA was determined using urethane-anesthetized rats, as described previously (Tanida et al., 2005a). Briefly, the rats were fasted for 4–6 h and anesthetized with urethane intraperitoneally (1 g/kg, IP) prior to the surgery. For recording the neural activity of the sympathetic nerves innervating the spleen, a splenic branch of the splanchnic nerve was dissected, ligated, and then attached to a pair of silver wire electrodes. The electrical signals from the electrodes were amplified 5000–10,000 times with a band path of 100 to 1000 kHz, filtered, and monitored on an oscilloscope. The raw nerve activity was converted to standard pulses using a window discriminator. Data were obtained as described previously (Tanida et al., 2005a,b).
J. Shen et al. / Autonomic Neuroscience: Basic and Clinical 145 (2009) 50–54
2.3. Cell cultures, implantations, and tumor proliferation assays HCT116 and MCF7 cells were cultured in the DMEM medium supplemented with 10% fetal bovine serum. They were harvested, and their viabilities were determined by trypan blue staining; cells with more than 95% viability were used. HCT116 (2.5 × 106 cells) or MCF7 cells (5.0 × 106 cells) were re-suspended in 200 μl of serum-free DMEM, and injected into the subcutaneous space in the right lower back of each BALB/c nude mouse using a 26-gauge needle. Because MCF-7 is a human breast cancer cell line, which requires estradiol supplementation for its proliferation in athymic mice (White et al., 1982), each nude mouse including the control was implanted with a 1.25-mg 17β-estradiol pellet (release ability for 60 days; Innovation Research, Sarasota, FL) a week before the injection of MCF7 cells. 2.4. Reagent administrations L·E·M (an extract of cultured L. Edodes Mycelia; Humalabo Co., Tokyo) was dissolved in water. For the intraduodenal injection, a polyethylene tube (PE-60; Clay Adams, Parsippany, NJ) was inserted into the duodenum with its end at a point approximately 5 mm toward the jejunum. For the tumor proliferation assays, the mice were randomly divided into 2 experimental groups of 6 animals each. Group 1 (control) received water and Group 2 received the L·E·M solution (100 mg/ml) as the sole drinking water, respectively, from the time after the implantation of either HCT116 or MCF7 cells. The bottles of drinking water were changed every 2 or 3 days. Tumor volume was measured using a digital caliper (graduated in 0.01 mm), and the tumor volume was calculated using the following formula as previously described (Gonzalez et al., 2006): 0.5 × L × W2 (L, length of the longest axis; W, width of the line meeting the longest axis at right angles). At the end of the experiment, which was day 20 for the HCT116 implantation experiment and day 27 for the MCF7 implantation experiment, the tumors were excised from the mice, which were sacrificed with an overdose of ketamine hydrochloride, and the excised tumor weights were determined. The L·E·M used is a product of Humalabo Co., Tokyo, and it was produced by growing a specific type of mycelia of L. edodes (Shiitake mushroom). It was cultured for up to 7 months in a solid culture medium composed of defatted rice bran and bagasse (sugarcane fibers) mixed in a fixed ratio. The active ingredients of this culture were extracted under strict examination, using a special technique. This extraction technique was based on enzymatic degradation without the use of chemicals.
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L·E·M solution (500 mg/ml) are shown in Fig. 1a. Means ± SEMs of the percentage change from the 0-min values of the Splenic-SNA of 3 rats are shown in Fig. 1b. A slight increase was observed from 25 min after the intraduodenal injection of water (Fig. 1b). In contrast, an intraduodenal injection of 1 ml of the L·E·M solution (500 mg/ml) gradually and markedly lowered Splenic-SNA, with the lowest level being 60.2 ± 14.3% at 60 min after the injection (Fig. 1b). The statistical significance of the difference between the groups with regard to the values of Splenic-SNA 5–60 min after the administration of the L·E·M solution and water was analyzed by ANOVA, and the difference was found to be statistically significant (P b 0.0005, F = 64.8 by ANOVA). The basal (0 min) values of Splenic-SNA were obtained as the mean values of the 5-min period before the intraduodenal administration of water and the L·E·M solution. The absolute values of Splenic-SNA at 0 min were as follows: the water group, 163.1 ± 5.5 spikes/5 s; L·E·M group, 239.9 ± 61.5 spikes/5 s. No statistically significant difference was detected between these 0-min values. 3.2. Effect of L·E·M on the proliferation of human colon cancer cells implanted in BALB/c athymic nude mice Intraduodenal administration of L·E·M reduced Splenic-SNA in the urethane-anesthetized rats (Fig. 1). Therefore, it is possible that L·E·M is also able to suppress Splenic-SNA in mice. If this is the case, and Splenic-SNA is involved in the mechanism of tumor immunity, L·E·M might affect Splenic-SNA, which in turn might affect the proliferation rate of malignant tumors. To examine this possibility, we studied the effect of an L·E·M solution (100 mg/ml) on the proliferation of HCT116 cells implanted into the subcutaneous space in the back of female BALB/c athymic nude mice lacking acquired immunity but not innate
2.5. Data and statistical analyses Splenic-SNA (spikes/5 s) measured during each 5-min period after the intraduodenal injection of either L·E·M solution or water was evaluated by digital signal processing and statistical analyses. Because of the interindividual variability in the preinjection state, the percent change from the 0-min value was calculated for Splenic-SNA. All the data were expressed as means ± SEMs. Statistical significances of differences in the respective basal (0 min or 0 day) values were detected by nonparametric Mann–Whitney U test. Analysis of variance (ANOVA) with repeated measures was applied to compare group responses in terms of Splenic-SNA and tumor volume induced by L·E·M and water. In all cases, P b 0.05 was applied as the level of significance. 3. Results 3.1. Effect of intraduodenal administration of L·E·M on Splenic-SNA in urethane-anesthetized rats Typical recordings of Splenic-SNA in urethane-anesthetized rats following intraduodenal injection of 1 ml of water and 1 ml of the
Fig. 1. Effect of intraduodenal administration of 1 ml of L·E·M solution (500 mg/ml) on splenic sympathetic nerve activity (Splenic-SNA). (a) Representative recordings of Splenic-SNA in rats intraduodenally administered water or the L·E·M solution. Vertical scale bars to the left of the recordings represent the neural discharge rate of 100 spikes/ 5 s. (b) Changes in neural activity expressed as a percentage of the 0-min value. Data (means ± SEMs) are expressed as the percentages of change in the values from the 0-min value. Each of the 3 rats in the water group and L·E·M group were used. Analysis of variance was used to analyze the statistical significance of the difference between Splenic-SNA of the 2 groups.
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immunity. Fig. 2a shows the changes in tumor volume (m3, means ± SEMs) of HCT116 cells in the subcutaneous space of the nude mice during the 20-day test period. The control group of mice were provided with water, and the experimental group of mice, with L·E·M solution (100 mg/ml) as the sole drinking for this 20-day test period. The tumor volume of the control mice given water gradually and markedly increased, and it reached a value of 645.45 ± 114.20 mm3 on day 20 after the implantation (Fig. 2a). The tumor volume of HCT116 cells in the mice given the L·E·M solution also increased; however, this increase was lower than that of the control mice who were given water. Moreover, the tumor volume of the L·E·M group on day 20 was 465.62 ± 57.54 mm3 (72.14% of the tumor volume of the control group). The difference between the control and L·E·M groups with regard to the tumor volumes from day 4 to day 20 was statistically analyzed by ANOVA with repeated measures, and it was found to be statistically significant (P b 0.0005, F = 14.7 by ANOVA). The tumor weights (means ± SEMs) of HCT116 cells excised from the mice 20 days after the implantation are shown in Fig. 2b. The tumor weight of the control mice given water was 0.357 ± 0.051 g, and that of mice given L·E·M was 0.290 ± 0.052 g (81.2% of the tumor weight of the control group). Although there was no statistically significant difference between the tumor weights on day 20 of these 2 groups of mice, a borderline statistical difference (0.05 b P b 0.1) was detected by the Mann–Whitney U test (Fig. 2b).
Fig. 3. Tumor volumes and weights of human breast cancer (MCF7) cells implanted into BALB/c athymic nude mice provided with water and L·E·M as the sole drinking water. (a) Changes in the tumor volumes of MCF7 cells implanted into the nude mice. Means ± SEMs of the tumor volume (mm3) in the 2 groups are shown. Analysis of variance with repeated measures was used to analyze the statistical difference in the tumor volumes between the 2 groups. (b) Tumor weights (means ± SEMs) of each group on day 27. The tumor weights of the 2 groups of rats provided with water and the L·E·M solution as the sole drinking water showed no statistically significant difference by the Mann–Whitney U test.
3.3. Effect of L·E·M on proliferation of human breast cancer cells implanted in BALB/c athymic nude mice
Fig. 2. Tumor volumes and weights of human colon cancer (HCT116) cells implanted into BALB/c athymic nude mice provided with water or L·E·M as the sole drinking water. (a) Changes in the tumor volumes of HC116 cells implanted into nude mice. Means ± SEM of the tumor volume (mm3) in these 2 groups are shown. Analysis of variance with repeated measures was used to analyze the statistical significance of the difference in tumor volumes between the 2 groups. (b) Tumor weights (means ± SEMs) of each group on day 20. The tumor weights of the 2 groups administered water or the L·E·M solution showed no statistically significant difference by the Mann–Whitney U test.
Finally, we examined the effect of the L·E·M solution on the proliferation of MCF7 cells implanted into the subcutaneous space in the back of female BALB/c athymic nude mice. Fig. 3a shows the changes in tumor volume (m3) of MCF7 cells in the subcutaneous space of the nude mice during the 27-day test period. Mice in the control group were given water (water-control group), and mice in the experimental group were given the L·E·M solution (100 mg/ml) (L·E·M group) as the sole drinking for the 27-day test period. The tumor volume of the control mice given water gradually and markedly increased, and it reached a value of 326.02 ± 97.14 mm3 on day 27 after the implantation (Fig. 3a). The tumor volume of MCF7 cells in the mice given the L·E·M solution was also elevated gradually (Fig. 3a). However, the increase in the tumor volume in the L·E·M group was much lower than that in the water-control group, and the tumor volume of the L·E·M group was 162.73 ± 75.74 mm3 (49.9% of the tumor volume of the water-control group) on day 27 after the implantation (Fig. 3a). The difference between the tumor volumes of the control and L·E·M groups on days 4 to 27 was statistically analyzed by ANOVA with repeated measures, and it was found to be statistically significant (P b 0.0005, F = 20.5 by ANOVA). The tumor weights of MCF7 cells excised from the mice 27 days after the implantation are shown in Fig. 3b. The tumor weight of the control mice given water was 0.142 ± 0.047 g, and the tumor weight of
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mice given the L·E·M solution was 0.070 ± 0.034 g (49.3% of the tumor weight of the water-control group). Although there was no statistically significant difference between the tumor weights on day 27 for these 2 groups of mice (P = 0.16 by the Mann–Whitney U test), the ratio of the tumor weight of the L·E·M group to that of the water-group was 0.493 on day 27, which was similar to the ratio of the tumor volume of the L·E·M group to that of the water-group (0.499) on day 27. 4. Discussions In this study, the following observations were made: (1) SplenicSNA was suppressed by oral administration of 1 ml of L·E·M solution (500 mg/ml) in urethane-anesthetized rats (Fig. 1); (2) the volume of the tumor due to HCT116 cells implanted into the subcutaneous space of athymic nude mice was lowered by the L·E·M solution (100 mg/ml), which was provided as the sole drinking water (Fig. 2); (3) the tumor volume due to MCF7 cells implanted into the subcutaneous space of athymic nude mice was also reduced by the L·E·M solution (100 mg/ml), which was provided as the sole drinking (Fig. 3). The autonomic nervous system in collaboration with the endocrine system is important for maintaining the homeostasis of important physiological phenomena for survival in mammals. Recently, we observed that in rats, olfactory stimulation with the scents of grapefruit oil (GFO) and lavender oil (LVO) affected autonomic nerve activities and thus physiological phenomena. The following observations were made: (1) Olfactory stimulation with the scent of GFO elevated the activities of sympathetic nerves innervating white adipose tissue, brown adipose tissue, the adrenal glands, and the kidney, and thus increased the plasma glycerol level, body temperature, and blood pressure. (2) Olfactory stimulation with the scent of GFO lowered the gastric parasympathetic (vagal) nerve activity and thus reduced food intake. (3) Olfactory stimulation with the scent of LVO suppressed the activities of sympathetic nerves innervating white adipose tissue, brown adipose tissue, the adrenal glands, and the kidney, and thus decreased the plasma glycerol level, body temperature, and blood pressure. (4) Olfactory stimulation with the scent of LVO enhanced gastric parasympathetic (vagal) nerve activity and thus increased food intake (Shen et al., 2005a,b; Tanida et al., 2005b, 2006). Moreover, it was observed that intraduodenal administration of a type of lactobacillus affected autonomic neurotransmissions, blood pressure, and blood glucose (Tanida et al., 2005a,b; Yamano et al., 2006). These findings suggest that changes in the autonomic nerve activity are good indicators of physiological markers such as lipolysis, body temperature, blood pressure, blood glucose, appetite, etc. Additionally, we previously observed that intraduodenal administration of 1 ml of a mixture of L-arginine (50 mM) and L-lysine (50 mM) suppressed Splenic-SNA in urethane-anesthetized rats. We observed that a mixed solution of L-arginine and L-lysine (50 mM each) provided as the sole drinking water inhibited the tumor volumes of HCT116 cells implanted into athymic nude mice, and we detected a significant correlation between the suppression of SplenicSNA in rats and the reduction of HCT116 tumor volume in nude mice that caused by the administration of the L-arginine and L-lysine mixture (Shen et al., submitted). It was also suggested that extracts of L. edodes exhibit antitumor activity in animals and humans (Hibino et al., 1994; Israilides et al., 2008). Therefore, we hypothesized that a whole extract of the mycelial culture of L. edodes grown in a solid medium of sugar-cane bagasse and defatted rice bran, i.e., L·E·M, might suppress either Splenic-SNA in urethane-anesthetized rats or tumor growth of HCT116 and MCF7 cells implanted into athymic nude mice lacking acquired immunity but not innate immunity. Consequently, it was observed that an intraduodenal injection of 1 ml of the L·E·M solution (500 mg/ml) significantly reduced Splenic-SNA in urethaneanesthetized rats (Fig. 1) and that providing the L·E·M solution (100 mg/ml) as the sole drinking water inhibited the growth of both HCT116 and MCF7 cells in the nude mice (Figs. 2 and 3). These finding
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suggest that L·E·M has suppressive actions on Splenic-SNA and tumor proliferation. The spleen is an important organ involved in immune functions; Splenic-SNA is suggested to mediate the central modulation of immunity (Hori et al., 1995); and the excitation of Splenic-SNA causes the suppression of splenic NK cytotoxicity (Katafuchi et al., 1993). In other words, the reduction of Splenic-SNA might elicit an increase in NK cytotoxicity. Considering these facts and the present findings, it is possible that L·E·M might increase the spleen NK cytotoxicity through a reduction in Splenic-SNA, and thus function to suppress tumor proliferation due to HCT116 and MCF7 cells. This needs to be examined in the future. L·E·M contains β-glucan (Wasser, 2002), and β-glucan is suggested to inhibit tumor growth through the activation of macrophages (Lee et al., 2002), NK cytotoxicity (Fujimiya et al.,1998), and the complement system (Gelderman et al., 2004) as well as through increases in the number and activity of lymphocytes (Ng and Yap, 2002). Therefore, β-glucan might be an effective substance with regard to the action of L·E·M on Splenic-SNA and tumor proliferation observed in this experiment. This possibility must be examined in the future. In conclusion, L·E·M inhibited Splenic-SNA in urethane-anesthetized rats and suppressed the proliferation of human colon cancer cells and human breast cancer cells implanted in athymic nude mice. These findings suggest that L·E·M has antitumor action. Acknowledgements The authors would like to express their deep gratitude to Professor Masato Okada and Dr. Chitose Oneyama, Research Institute for Microbial Diseases, Osaka University, for their help in the culturing of cancer cells. References Gonzalez, L., Aqullo-Ortuno, M.T., Garcia-Martinez, J.M., Calcaabrini, A., Gamollo, C., Palacios, J., Aranda, A., Martin-Perez, J., 2006. Role of c-Src in human MCF7 breast cancer cell tumorigenesis. J. Biol. Chem. 281, 20851–20854. Fujimiya, Y., Suzuki, Y., Oshima, K., Kobori, H., Moriguchi, K., Nakashima, H., Matumoto, Y., Takahara, S., Ebina, T., Katakura, R., 1998. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis. Cancer Immunol. Immunother. 46, 147–159. Gelderman, K.A., Tomlinson, S., Ross, G.D., Gorter, A., 2004. Complement function in mAb-mediated cancer immunotherapy. Trends Immunol. 25, 158–164. Hibino, Y., Konishi, Y., Koike, J., Tabata, T., Ohashi, Y., Sugano, N., 1994. Productions of interferon-gamma and nitrite are induced in mouse splenic cells by a hateroglycanprotein fraction from culture medium of Lentinus edodes mycelia. Immunopharmacology 28, 77–85. Hori, T., Katafuchi, T., Take, S., Shimizu, N., Niijima, A., 1995. The autonomic nervous system as a communication channel between the brain and the immune system. Neuroimmunomodulation 2, 203–215. Israilides, C., Kletsas, D., Arapoglou, D., Philippossis, A., Pratsinis, H., Ebringerova, A., Hribalova, F., Haarding, S.E., 2008. In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinus edodes. Phytomedecine 15, 512–519. Katafuchi, T., Take, S., Hori, T., 1993. Roles of sympathetic nervous system in the suppression of cytotoxicity of splenic natural killer cells in rat. J. Physiol. 465, 343–357. Lee, D.Y., Ji, I.H., Chang, H.I., Kim, C.W., 2002. High-level TNF-α secretion and macrophage activity with soluble β-glucans from Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 66, 233–238. Morinaga, H., Tazawa, K., Tagoh, H., Muraguchi, A., Fujimaki, M., 1994. An in vivo study of hepatic and splenic interleukin-1 beta mRNA expression following oral PSK or LEM administration. Jpn. J. Cancer Res. 85, 1298–1303. Ng, M.L., Yap, A.T., 2002. Inhibition of human colon carcinoma development by lentinan from shiitake mushrooms (Lentinus edodes). J. Altern. Complement. Med. 8, 581–589. Shen, J., Niijima, A., Tanida, M., Horii, Y., Maeda, K., Nagai, K., 2005a. Olfactory stimulation with scent of grapefruit oil affects autonomic nerves, lipolysis and appetite in rats. Neurosci. Lett. 380, 289–294. Shen, J., Niijima, A., Tanida, M., Horii, Y., Maeda, K., Nagai, K., 2005b. Olfactory stimulation with scent of lavender oil affects autonomic nerves, lipolysis and appetite in rats. Neurosci. Lett. 383, 188–193. Sugano, N., Hibino, Y., Choji, Y., Maeda, H., 1982. Anticarcinogenic actions of watersoluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia. Cancer Lett. 17, 109–114.
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