Effects of Colestimide And/Or Bofu-Tsusho-San on Plasma and Liver Lipids in Mice Fed a High-Fat Diet

in vivo 19: 1029-1034 (2005)

Effects of Colestimide and/or Bofu-tsusho-san on Plasma and Liver Lipids in Mice Fed a High-fat Diet

SHINOBU SAKAMOTO1, SHINGO TAKESHITA1, SHUJI SASSA1, SATOE SUZUKI2, YUKO ISHIKAWA3 and HIDEKI KUDO3

1Medical Research Institute, Tokyo Medical and Dental University, Tokyo; 2Institute of Medical Technology, Bunkyo-gakuin University, Tokyo; 3Laboratory of Clinical Nutrition, Kagawa Nutrition University, Saitama, Japan

Abstract. Hypercholesterolemia is known to enhance the risk sheng-san (in Chinese)], a traditional Japanese herbal of coronary heart disease and fatty liver. Colestimide is an remedy, contains 18 components (Table I). This remedy has anion-exchange resin, which is not absorbed in the small long been used against obesity in China and Japan (5). The intestine, decreases the intestinal reabsorption of bile acids colestimide anion-exchange resin is not absorbed in the synthesized from cholesterol in the liver and consequently small intestine, but decreases the intestinal reabsorption of increases bile acid excretion into the feces. Bofu-tsusho-san, a bile acids synthesized from cholesterol in the liver and traditional Japanese herbal remedy, contains 18 components consequently increases bile acid excretion into the feces, and has long been used as an anti-obesity agent. In the present without any known serious adverse effects. study, we investigated the effects of colestimide and/or Bohu- In the present study, the effects of colestimide and/or tsusho-san in young male mice fed a high-fat diet. The high- Bohu-tsusho-san on body growth and plasma and liver lipids fat diet supplemented with both colestimide and Bofu-tsusho- were investigated in young male mice fed a high-fat diet. san markedly reduced the plasma levels of lipids, the liver weight and number of fatty droplets in the liver cytoplasm, and Materials and Methods the body growth, compared with animals fed a high-fat diet alone. Neither medicine affected the blood biochemistry. Thus, Animals and experimental procedures. Male ICR mice (Charles the hypocholesterolemic action of colestimide, sometimes River Japan, Yokohama, Japan), 8 weeks of age, were used in the bringing light flatulence, which is improved by simultaneous present study. They were housed in plastic cages with wood administration of Bofu-tsusho-san, which activates the shavings under controlled conditions (24±0.5ÆC and 12 h of light thermogenesis of brown adipose tissue, is suggested to reduce from 06.00 to 18.00 h), in accordance with the principles outlined body mass and liver lipids, lowering the plasma levels of lipids. in the Guide for Animal Care and Use of the Committee of Tokyo Medical and Dental University, Japan. All mice had free access to a commercial normal-diet (AIN-76A; Oriental Yeast Co., Ltd., Hypercholesterolemia is known to enhance the risk of Tokyo, Japan), which is a purified diet based on casein as the sole coronary heart disease (1), fatty liver disease (2) and source of protein (6), and tap water ad libitum. The daily food carcinogenesis, which is associated with hydroxyl radical intake was approximately 3.0±0.5 g/mouse. The mice were divided formation (3). Colestimide is a newly developed anion- into four groups of 15 at the age of 10 weeks. The animals in the exchange resin with an imidazolium salt on an epoxide high-fat diet group (HF-group) were each fed 3.0 g of a polymer skeleton (2-methylimidazole-epichlorohydrin commercial high-fat diet (F2HFD2; Oriental Yeast Co., Ltd.), which consisted of 58% lard (wt/wt), 30% fish powder, 10% skim copolymer), that has been used for the treatment of milk and a 2% vitamin and mineral mixture (equivalent to 7.5% hypercholesterolemia (4). Bofu-tsusho-san [Fang-feng-tong- carbohydrate, 24.5% protein and 60% fat) (7), daily for 4 weeks. Since the human doses of colestimide and Bofu-tsusho-san did not affect the plasma levels of lipids in mice in the preliminary experiments (data not shown), the colestimide-treated group (CO- Correspondence to: Shinobu Sakamoto, MD, Ph.D., Medical group) received 3.0 g of the high-fat diet supplemented with Research Institute, Tokyo Medical and Dental University, 1-5-45, 12.6 mg of colestimide (Cholebineì, Mitsubishi Pharma Co., Tokyo, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel: +81-3-5803- Japan), i.e. approximately 10-fold the human dose (3.0 g/day) in 3.0 g 5837, Fax: +81-3-5803-5853, e-mail: [email protected] of the diet (given the ratio of mouse body weight 35.0 g/ human body weight 50.0 kg). The Bofu-tsusho-san-treated group (BO-group) Key Words: Anion-exchange resin colestimide, herbal medicine was given 3.0 g of the high-fat diet supplemented with 31.5 mg of Bofu-tsusho-san, hyperlipidemia, liver lipids, mice. Bofu-tsusho-san (Bofu-tsusho-san extract granules, Tsumura & Co.,

0258-851X/2005 $2.00+.40 1029 in vivo 19: 1029-1034 (2005)

Table I. Components of Bofu-tsusho-san. Table II. Body growth and organ weights in each group.

Crude drugs (18) Contents# (g) Groups HF CO BO CB

1) Scutellariae Radix (ougon) 2.0 Body growth 2) Glycyrrhizae Radix (kanzou) 2.0 3) Platycodi Radix (kikyou) 2.0 Initial BW 34.4±0.6 32.6±0.4 33.3±0.6 33.7±0.5 4) Gypsum Fibrosum (sekkou) 2.0 (g) 5) Atractylodis Rhizoma (byakujyutsu) 2.0 Final BW 39.9±0.4 38.6±0.6 38.8±1.1 38.3±0.5* 6) Rhei Rhizoma (daiou) 1.5 (g) 7) Schizonepetae Spica (keigai) 1.2 % Growth 16.0±0.9 17.6±0.8 15.0±1.4 10.8±1.3** 8) Gardeniae Fructus (sanshishi) 1.2 (%) 9) Paeoniae Radix (shakuyaku) 1.2 10) Cnidii Rhizoma (senkyuu) 1.2 Organ weights 11) Angelicae Radix (touki) 1.2 12) Menthae Herba (hakka) 1.2 Testis (g/BW) 5.21±0.27 5.96±0.47 5.92±0.49 5.41±0.31 13) Ledebouriellae Radix (bouhuu) 1.2 14) Ephedrae Herba (maou) 1.2 Spleen (g/BW) 2.70±0.19 2.55±0.14 2.40±0.13 2.72±0.19 15) Forsythiae Fructus (rengyou) 1.2 16) Zingiberis Rhizoma (shoukyou) 0.3 Kidney (g/BW) 14.3±0.5 14.5±0.6 14.2±0.7 15.4±0.5 17) Kadinum (kasseki) 3.0 18) Natrium Sulfurieum (boushou) 0.7 Liver (g/BW) 48.3±0.9 48.6±1.1 47.4±1.1 42.2±0.9**

#The amount of each crude drug required to prepare 4.5 g HF: high-fat diet group, CO: colestimide-treated group, of a powdered extract in 7.5 g of Bofu-tsusho-san extract granules. See BO: Bofu-tsusho-san-treated group, Materials and Methods for details. CB: group treated with colestimide plus Bofu-tsusho-san. BW: body weight Data are means±SEM. ** and *significantly different from that of the HF-group; p<0.01 and 0.05 Tokyo, Japan), i.e. approximately 10-fold the human dose (7.5 g/day) in 3.0 g of the diet. The group treated with both colestimide and Bofu-tsusho-san (CB-group) was given 3.0 g of the high-fat diet supplemented with 12.6 mg of colestimide and (Table II). Although no differences in the wet weights of the 31.5 mg of Bofu-tsusho-san extract in 3.0 g of the diet. All experimental procedures conformed to the regulations described testis, spleen and kidney were observed among the groups, in the Guide to the Care and Use of Laboratory Animals of the the liver weights in the CB-group were markedly reduced in U. S. National Institutes of Health (NIH). comparison with the other groups (p<0.01) (Table II). Five mice a cage were daily given 15 g of the diet (3.0 g of diet/day/ mouse), and the body growth was checked weekly Histological examination of the liver. Histological throughout the experiment. All mice were killed at 14 weeks of age examination showed that hepatic lipid deposits appeared as by cervical dislocation after cardiac puncture under deep urethane small vacuoles within the cytoplasm of liver cells in mice fed anesthesia (1.5 g urethane/ kg of body weight, Merck, Darmstadt, Germany) to measure the plasma levels of lipids and biochemical the high-fat diet (HF-group) (Figure 1A). However, the substances, while the sera obtained were later commercially diets supplemented with colestimide and/or Bofu-tsusho-san measured (SRL, Inc., Tokyo, Japan). At autopsy, the removed markedly reduced the number of these fatty droplets in testis, spleen, kidney and liver were weighed and recorded. Each mice in the other groups (Figures 1B, 1C and 1D). liver obtained was immediately fixed in a 10% formaldehyde buffer solution (pH 7.2), embedded in paraffin, prepared as 5-Ìm serial Plasma levels of lipids. The plasma levels of total cholesterol sections and stained with Mayer’s hematoxylin and eosin for (TC) in the CO- and CB-groups were reduced to 75.0% histological examination. (p<0.05) and 69.8% (p<0.01) of that in the HF-group, Statistical analysis. All parameters were expressed as the respectively (Figure 2A). Free total cholesterol (FC) in the mean±SEM. Statistical analysis was performed using the unpaired experimental groups (CO-, BO- and CB-groups) was t-test. A p value less than 0.05 was considered statistically significant. decreased to less than 70% of that in the HF-group (p<0.01) (Figure 2B). Phospholipid (PhL) in the CB-group Results was lowered to 72.6% of that in the HF-group (p<0.01) (Figure 2C). The plasma levels of triglyceride (TG) in the Body growth and organ weights. All mice in each cage CO- and CB-groups were reduced to 57.3% (p<0.05) and consumed the provided diet completely. The least body 46.7% (p<0.01) of that in the HF-group, respectively weight increase was observed in the CB-group (p<0.01) (Figure 2D).

1030 Sakamoto et al: Colestimide and Herbal Medicine on Mouse Liver Lipids

Figure 1. Histological structure of liver in each group (HE staining, original magnification x 400). A: HF (high-fat diet)-group; arrowheads indicate fatty droplets, B: CO (colestimide-treated)-group, C: BO (Bofu-tsusho-san-treated)-group, D: CB (treated with colestimide plus Bofu-tsusho-san)-group.

Plasma parameters. There were no differences in the plasma is characterized by a wide spectrum of liver damage, i.e. levels of total protein (TP), albumin (Alb), glucose (Glc), simple steatosis may progress to advanced fibrosis and to blood urea nitrogen (BUN), creatinine (Crt), calcium (Ca) cryptogenic cirrhosis via steatohepatitis, and ultimately to or inorganic phosphorus (iP) and activities of glutamic- hepatocellular carcinoma (2). It is suggested that chronic oxaloacetic transaminase (GOT), L-lactate dehydrogenase hepatocellular injury, necroinflammation, stellate cell (LDH), alkaline phosphatase (ALP) or choline esterase I activation, progressive fibrosis and, ultimately, cirrhosis are (ChE) among the groups (Table III). initiated by the peroxidation of hepatic lipids and injury- related release of cytokines. Obesity is the single most Discussion significant risk factor for the development of fatty liver, i.e. obesity is also predictive of the presence of fibrosis, The relationship between elevated plasma levels of low- potentially progressing to advanced liver diseases. From a density lipoprotein (LDL)-cholesterol and increased risk for histopathological point of view, insulin resistance plays a coronary heart disease (8) has been demonstrated by large- central role in the accumulation of triglycerides within the scale cohort studies. Fatty liver disease is a new hepatocytes and in the initiation of the inflammatory clinicopathological entity of emerging importance, now cascade (2). Fatty diets, composed of much meat and little recognized as the most common cause of abnormal liver. It fiber, have been known to increase the risk of

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Figure 2. Plasma levels of lipids in each group. A: total cholesterol (TC), B: free total cholesterol (FC), C: phospholipid (PhL), D: triglyceride (TG). ** and *significantly different from that of the HF-group; p<0.01 and 0.05.

carcinogenesis, associated with hypercholesterolemia and stimulated with ephedrine contained in Ephedrae Herba) hydroxyl radical formation (3, 9). and to inhibit phosphodiesterase activity (that was inhibited Colestimide is a newly developed anion-exchange resin, with licoricidin contained in Glycyrrhizae Radix, that has demonstrated more potent hypolipidemic activity d-pinorestinol contained in Forsythiae Fructus and than that of cholestylamine (10). Colestimide reduces Schizonepetae Spica), resulting in weight loss in mice (5). plasma levels of large, light LDL, a precursor of small, Sennosides contained in Rhei Rhizoma and Natrium heavy LDL, and is used widely for the treatment of Sulfurieum are known to improve flatulence and hypercholesterolemia (4). Colestimide is not absorbed in the constipation. In general, Chinese herbal medicines are small intestine, but decreases the intestinal reabsorption of believed to cause few adverse effect in a clinical dose, if not bile acids synthesized from cholesterol in the liver and administered for excessive time-periods. consequently increases bile acid excretion into the feces. No In the present study, the high-fat diet supplemented with serious adverse effects have been linked with the use of both colestimide and Bofu-tsusho-san markedly reduced the colestimide, although it is known to bring light flatulence plasma levels of lipids, i.e. in addition to cholesterol, with swelling of the abdomen as a minor side-effect. Bofu- triglyceride was also lowered by supplementation with tsusho-san contains 18 components, and a mixture colestimide, with or without Bofu-tsusho-san. Further, the consisting of each chopped ingredient is extracted with a 12- liver weight, the number of fatty droplets in the liver fold volume of hot water, filtered and spray-dried to give a cytoplasm and the body growth, were all reduced in powdered extract. This Chinese medicine has long been comparison to animals fed the high-fat diet alone. Neither used as an anti-obesity agent in China and Japan. It has substance affected the blood biochemistry, indicating been demonstrated to activate thermogenesis of brown correct function of the liver, kidneys, and a balance of adipose tissue (that was activated by norepinephrine release electrolytes.

1032 Sakamoto et al: Colestimide and Herbal Medicine on Mouse Liver Lipids

Table III. Plasma parameters in each group. References

Groups HF CO BO CB 1 Anderson JW and Hanna TJ: Impact of nondigestible carbohydrates on serum lipoproteins and risk for cardiovascular TP (g/dl) 3.88±0.18 3.57±0.12 3.44±0.13 3.40±0.13 disease. J Nutr 129(7 Suppl): 1457S-1466S, 1999. Alb (g/dl) 2.06±0.14 1.90±0.12 1.93±0.06 1.89±0.14 2 Festi D, Colecchia A, Sacco T, Bondi M, Roda E and Marchesini G: Hepatic steatosis in obese patients: clinical Gluc (mg/dl) 165±8 171±8 172±7 173±14 aspects and prognostic significance. Obes Rev 5(1): 27-42, 2004. 3 Tseng TH, Hsu JD, Chu CY and Wang CJ: Promotion of colon GOT (IU/l) 40.0±4.4 49.8±4.1 44.3±5.0 50.8±6.5 carcinogenesis through increasing lipid peroxidation induced in rats by a high cholesterol diet. Cancer Lett 100(1-2): 81-87, 1996. LDH (IU/l) 168±35 148±31 94±28 134±47 4 Homma Y, Kobayashi T, Yamaguchi H, Ozawa H, Sakane H and Nakamura H: Specific reduction of plasma large, light low- ALP (IU/l) 94.3±7.3 112.2±7.6 88.8±9.1 103.3±13.3 density lipoprotein by a bile acid sequestering resin, cholebine (MCI-196) in type II hypercholesterolemia. Atherosclerosis 129: ChE (IU/l) 11.8±1.3 13.5±1.4 10.0±1.1 10.9±1.8 241-248, 1997. 5 Yoshida T, Sakane N, Wakabayashi Y, Umekawa T and Kondo BUN (mg/dl) 20.4±1.3 18.0±0.6 20.3±1.0 19.9±1.8 M: Thermogenic, anti-obesity effects of bofu-tsusho-san in MSG-obese mice. Int J Obes 19: 717-722, 1995. Creat (mg/l) 0.79±0.14 1.00±0.06 1.11±0.08 1.11±0.11 6 Kim H, Hall P, Smith M, Kirk M, Prasain JK, Barnes S and Grubbs C: Chemoprevention by grape seed extract and Ca (mg/dl) 7.41±0.22 7.19±0.16 7.11±0.12 6.84±0.20 genistein in carcinogen-induced mammary cancer in rats is diet dependent. J Nutr 134: 3445S-3452S, 2004. iP (mg/dl) 5.03±0.44 5.64±0.24 6.15±0.35 4.99±0.35 7 Anai M, Funaki M, Ogihara T, Kanda A, Onishi Y, Sakoda H, Inukai K, Nawano M, Fukushima Y, Yazaki Y, Kikuchi M, Oka HF: high-fat diet group, CO: colestimide-treated group, BO: Bofu-tsusho-san-treated group, Y and Asano T: Enhanced insulin-stimulated activation of CB: group treated with colestimide plus Bofu-tsusho-san. phosphatidylinositol 3- kinase in the liver of high-fat-fed rats. Data are means±SEM. Diabetes 48: 158-169, 1999. 8 Fernandez ML: Soluble fiber and nondigestible carbohydrate effects on plasma lipids and cardiovascular risk. Curr Opin Lipidol 12(1): 35-40, 2001. 9 Bobek P, Galbavy S and Mariassyova M: The effect of red beet In conclusion, the hypocholsterolemic action of (Beta vulgaris var. rubra) fiber on alimentary hypercholesterolemia and chemically induced colon carcinogenesis in rats. Nahrung colestimide, enhanced by the simultaneous administration 44(3): 184-187, 2000. of Bofu-tsusho-san, seems effective in reducing body mass, 10 Shimada H, Mitsuka M and Umezu K: The interaction between liver lipids and lowering the plasma lipid level. MCI-196 and bile acids or other biological substances. Jpn Pharmacol Ther 24: 127-132, 1996. Acknowledgements

We gratefully acknowledge Emeritus Professor Dr. Hiroshi Nagasawa, of Meiji University, Japan and Dr. Bonjin Izawa for Received June 29 their consultancy. Accepted August 31, 2005

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