Abstract
Touchi, a traditional Chinese food used mainly for seasoning is obtained by first steaming soybeans followed by fermentation with Aspergillus oryzae (koji). A series of toxicological studies was conducted to evaluate the mutagenic and genotoxic potential and subchronic toxicity of a water extract of Touchi, a known inhibitor of α-glucosidase activity. Touchi extract (TE) did not induce reverse mutations in Salmonella typhimurium strains TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA at concentrations up to 5000 μg/plate, in either the absence or presence of exogenous metabolic activation. No deaths occurred and no abnormal clinical signs were observed in any animal in any group in an in vivo micronucleus test, and TE was devoid of clastogenic activity when administered orally to mice at doses up to 2000 mg/kg/day. Thus, TE was evaluated as negative in the bacterial reverse mutation and mouse bone marrow micronucleus tests under the conditions of these assays. To evaluate its subchronic toxicity, SPF rats were administered TE at doses of 0,250,1000, and 2500 mg/kg/day via oral gastric intubation. No treatment-related toxic changes were seen in clinical signs, body weight, food consumption, urinalysis, hematology, blood chemistry, necropsy, organ weight, or histopathology. The no observed adverse effect level for TE was thus considered to be more than 2500 mg/kg/day in both males and females. These results are consistent with Touchi’s status as a traditional Chinese food derived from fermented soybeans and its purported long history of use. Specifically, these data are consistent with the expected safety of human consumption of TE up to at least 5 g/day.
Touchi, a traditional Chinese food used mainly for seasoning, is obtained by first steaming soybeans followed by fermentation with Aspergillus oryzae. This organism, also referred to as koji, is a nontoxic, nonpathogenic microbial commonly used in the Japanese fermentation industry, with applications in sake, soy sauce, and miso manufacturing, as well as commercial enzyme production (Gomi 2002).
Enzyme inhibitory assays conducted with Touchi extract (TE) have revealed a 50% inhibitory concentration (IC50) of 0.33 g/L for rat intestinal α-glucosidase inhibition using sucrose as a substrate (Fujita, Yamagami, and Ohshima 2005). Inhibitors of α-glucosidase have been shown to inhibit disaccharide hydrolases that convert disaccharides into monosaccharides, thereby impeding the digestion and absorption of glucose and suppressing the postprandial rise in plasma glucose levels (Clissold and Edwards 1988; Toeller 1994; Fujita, Yamagami, and Ohshima 2005).
At present, the active components of TE have not been fully characterized. It is believed that an unidentified amino sugar may be involved in its mechanism of α-glucosidase inhibition. This action may be indirectly connected to that of insulin, which also plays a central role in postpranidal lipid metabolism. Dietary carbohydrates are involved in this process because they are the key precursors of lipogenesis. TE has also been shown to significantly lower fasting blood glucose and HbA1c (glycated hemoglobin), as well as triglyceride levels in KKAy diabetic mice and diabetic subjects (Fujita, Yamagami, and Ohshima 2001a, 2001b, 2001c, 2003).
Food products containing TE have been approved as Food for Specified Health Uses (FOSHU) since 2001 in Japan. In order to provide additional evidence of the safety of TE, the mutagenicity, genotoxicity, and subchronic oral toxicity of TE were evaluated. Results of these studies are reported here for the first time.
MATERIALS AND METHODS
Study Design
The studies were performed at Kobuchisawa Research Laboratories, Fuji Biomedix (Yamanashi, Japan) in accordance with relevant national or international standards and guidelines.
Although studies were considered non-GLP (good laboratory practice) compliant because analyses of the test article and dosing material were not performed, the studies were otherwise performed in compliance with OECD Principles of Good Laboratory Practice (adopted by the OECD Council on 26th November, 1997) or the Ordinance on Standards for Conduct of Non-clinical Studies on the Safety of Drugs (Ordinance No. 21 of the Ministry of Health and Welfare dated March 26 1997). The bacterial reverse mutation test was conducted in accordance with OECD Guideline for The Testing of Chemicals 471—Bacterial Reverse Mutation Test. The in vivo micronucleus assay followed the Guideline for Genetic Toxicity Studies (Notification No. 1604 of Pharmaceutical Affairs Bureau, November 1, 1999, MHLW) and the 28-day oral toxicity study complied with Guidelines for Toxicity Studies of Drugs (Notification No. 24 of the First Evaluation and Registration Division, Pharmaceutical Affairs Bureau dated September 11, 1989; Notification No. 88 of the New Drug Division, Pharmaceutical Affairs Bureau dated August 10, 1993). The use of animals in the micronucleus test and the 28-day toxicity study was reviewed by the Institutional Animal Care and Use Committee (IACUC) of Fuji Biomedex.
Test Article and Chemicals
TE (lot no. 06.02.25) was supplied by Nippon Supplements. All tests were performed on TE dissolved in water for injection. The S9 fraction in the reverse mutation assay was prepared by mixing commercially available Rat Liver S9 (Oriental Yeast; lot no. 06080405) prepared from the livers of 7-week-old male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone and cofactor (Oriental Yeast; lot nos. 999602 and 999603) in 1:9 ratio at the time of use.
Positive-control substances were as follows: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2; lot no. CKO1402), 2-aminoanthracene (2-AA; lot no. ELJ6826), and sodium azide (SA; lot no. ELG7946) obtained from Wako Pure Chemical Industries. Mitomycin C (MMC; lot no. 075K1921) was obtained from Sigma-Aldrich, and 9-aminoacridine (9-AA; lot no. 6698C) was obtained from ICN Biomedicals. Water served as a negative control.
In Vivo Bone Marrow Micronucleus Test
Because Touchi has been eaten as a fermented food, the toxicity of the test article is assumed to be very low. Therefore, the high-dose level for this study was determined to be 2000 mg/kg/day. The mid- and low-dose levels were selected to be 1000 and 500 mg/kg/day by a common ratio of 2, respectively.
Male SPF rats of Crl:CD(SD) strain were obtained from Charles River Japan at the age of 7 weeks. Animals were quarantined for 3 days and subsequently acclimated for 5 days. Six healthy animals of were randomly assigned to five groups in an attempt to equalize mean group body weights by a computerized randomization procedure. Rats were housed individually in metal cages (W14.5 × D26 × H12 cm) in animal rooms with a set temperature of 22°C (actual temperature ranged from 21.1 °C to 23.4°C) and a relative humidity of 50% (actual range: 42.6% to 62.6%), air ventilation at 15 to 25 times per hour, and artificial lighting (on/off for 12 h per day). The animals were allowed free access to autoclaved sterilized CRF-1 pellets (Oriental Yeast). Ultraviolet (UV)-irradiated tap water was supplied ad libitum using water bottles.
In accordance with the Guideline for Genetic Toxicity Studies, rats were administered TE by oral gavage on 2 successive days. The dosing volume was 10 ml/kg. Control animals received water at the same volume. A single 0.4-mg/ml dose of MMC was administered intraperitoneally (dosing volume 10 ml/kg) as the positive-control substance.
Rats were sacrificed by cervical dislocation approximately 24 h after the last dose, and femurs were removed. Fetal bovine serum (~0.2 ml) was injected into the bone marrow of each femur and the bone marrow cells were washed out into a centrifugation tube. The cell suspension was centrifuged at 1000 rpm for 5 min. The supernatant was discarded and a little serum remaining in the tube was used to suspend the cells uniformly. The cell suspension was smeared on the slide (three slides per animal), air-dried, fixed with methanol for 5 min, and stained at room temperature for about 25 min with 3% Giemsa solution prepared by using neutral-buffered phosphate solution (pH 6.8). The smears were then washed with water and air-dried.
The incidence of micronuclei was examined for 2000 polychromatic erythrocytes (PCEs) per rat, or 1000 per slide of one animal (two slides) using 1000-fold microscopy. Polychromatic erythrocytes were counted similarly for 1000 erythrocytes per animal, or 500 erythrocytes were counted per slide of one animal (two slides). The frequency (%) of polychromatic erythrocytes to the total erythrocytes was calculated for each bone marrow specimen.
The Kastenbaum and Bowman’s method was performed to compare the incidences of micronuclei between the control group and each of the treated groups. The frequency (%) of polychromatic erythrocytes to the total erythrocytes and body weight values were compared between the control group and each of the treated groups by Dunnett’s multiple-comparison test. A significant level will be 5% (two-tailed). A significant result was indicated by either 5% or 1%. A micronucleus induction activity was judged as positive when the incidence of polychromatic erythrocytes with micronuclei in the treated groups increases significantly as compared with the control group and when the incidence of polychromatic erythrocytes with micronuclei increases dose-dependently.
Bacterial Reverse Mutation Test
TE was tested in Salmonella typhimurium strains TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA. Based on the results of a concentration-range finding test (data not shown), TE was dissolved in water and repeatedly tested at five dose levels of 5000,2500, 1250,625, and 313 μg/plate both with and without metabolic activation. The positive-control substances were AF-2, 9-aminoacridine, 2-aminoanthracene, and sodium azide; a vehicle control (water) was also included. Tests were performed by the pre-incubation method, and three plates per dose were used.
After the 48-h incubation period, revertant colony counts were measured manually. A colony analyzer (CA-11D; System Science) was used for the counting of about 100 or more colonies (TA100 strain and positive-control group). The precipitation of the test article on the plate was checked grossly, and the state of growth inhibition (background lawn) was examined under a stereoscope. The revertant colony counts, the mean and standard deviation for test article groups (each dose), negative-control group, and positive-control group were tabulated for each cell strain. Concentration-response curves were drawn for the test article groups. The test was repeated to determine reproducibility of the results.
Statistical analysis of data was not performed. Instead, the test results were judged as positive when (1) the revertant colony counts in the test article group were two times or more than that in the negative control; (2) a dose-response relationship was found in at least one strain with or without the metabolic activation system; and (3) reproducibility was obtained in the main studies.
28-Day Toxicity Study
Animals and Maintenance
Male and female SPF rats of Crl:CD(SD) strain were obtained from Charles River Japan at the age of 4 weeks. Animals were quarantined for 3 days and subsequently acclimated for 5 days (male) or 6 days (female). Ten healthy animals of each sex were randomly assigned to four groups as shown in Table 1 in an attempt to equalize mean group body weights by a computerized randomization procedure.
Rats were housed individually in metal cages (W15 × D30 × H17 cm) in animal rooms with a set temperature of 22°C (actual temperature ranged from 21.3°C to 23.2°C) and a relative humidity of 50% (actual range: 44.5% to 69.5%), air ventilation at 15 to 25 times per hour, and artificial lighting (on/off for 12 h per day). The animals were allowed free access to autoclaved sterilized CRF-1 pellets (Oriental Yeast). UV-irradiated tap water was supplied ad libitum.
Observations and Measurements
Clinical Observations, Body Weight, and Ophthalmoscopy
All animals were observed for clinical signs and mortality more than twice a day (before dosing and during the 2 h after dosing). Body weights were measured at the initiation of dosing and thereafter at weekly intervals during the dosing period. At the scheduled sacrifice, final body weights were determined following overnight fast and were thus excluded from evaluation.
Food Consumption and Water Consumption
Food consumption was measured for one day on a once-weekly basis for each animal.
Urinalysis
Urinalysis was performed before week 4 for half of the males and females of each group. Animals were placed in a metabolism cage during the urine collection, and only drinking water was available freely from a water bottle. The examination was performed using fresh urine (urine within 2 h after excretion). Urinary pH, protein, glucose, kentone bodies, urobilinogen, bilirubin, and occult blood were measured using the test paper method (Multistix; Bayer Medical).
Hematology
All animals were fasted from the evening on the day before necropsy, and blood samples obtained from the abdominal aorta under ether anesthesia at necropsy were collected into a bottle containing EDTA-K2. Red and white blood cell (WBC) counts, hematocrit (Ht) values, hemoglobin (Hb) contents, mean corpuscular hemoglobin (MCH), reticulocyte (ret) counts, platelet (Plt) counts, and differential leukocyte counts were determined using an automatic blood cell counter (XT-2000iV; Sysmex Corporation). For differential leukocyte counts (lymphocyte [Lym], neutrophil [Neut], monocyte [Mono], basophil [Baso], and eosinophil [Eosi]), blood smears were prepared and stained with Wright stain.
Clinical Chemistry
Clinical chemistry assessment was performed for all rats using blood samples collected from the abdominal aorta via a procedure similar to that used for blood collection for hematological assessment. The blood samples stood at room temperature for 1 h and then were centrifuged at 3000 rpm for 15 min at 4°C. The aspartate aminotransferase and lactate dehydrogenase were examined using the plasma containing sodium heparin (Novo Heparin Injection 1000; MochidaPharma). Serum and plasma were used for examination of the following parameters using an automatic clinical chemistry analyzer (Synchron CX-7; Beckman K.K.): aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ-glutamyltranspeptidase (γ-GTP), glucose (Glu.), total cholesterol (T.Cho.), triglyceride (TG), phospholipid (PL), total protein (TP), albumin (Alb.), urea nitrogen (BUN), creatinine (Crea.), total bilirubin (T.Bil.), sodium (Na), potassium (K), chloride (Cl), inorganic phosphorus (P), and calcium (Ca). The albumin/globulin ratio (A/G ratio) was calculated.
Pathology
All animals were fasted from the evening on the day before necropsy after the end of the dosing period and necropsied after blood collection and euthanasia by bleeding from the abdominal aorta under ether anesthesia. Absolute organ weights were measured for the following organs and their relative weights to body weight were calculated: heart, lungs (including bronchus), liver, kidneys, spleen, thymus, and adrenals. The paired organs were weighed all together.
The following organs and tissues in all groups were fixed in neutral buffered 10% formalin solution: brain, pituitary gland, thyroid gland, heart*, thymus*, spleen*, mesenteric lymph nodes, lung* (including bronchus), liver*, stomach*, small intestines (duodenum, jejunum, ileum) and large intestines (cecum, colon, rectum), kidney*, adrenal gland*, testis, epididymis, prostate, ovary, uterus, urinary bladder, sciatic nerve, sternum* (including bone marrow). (An asterisk denotes organs of control and high-dose group animals that were embedded, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically.) Lungs were inflated with neutral buffered 10% formalin solution. Bone tissues were decalcified with 10% formic acid–formalin solution.
Statistical Analysis
Quantitative data were analyzed for homogeneity of variance using Bartlett’s test. The significance of any difference between the control and test article groups was determined using Dunnett’s multiple comparison test for homogeneous data or Steel’s test for heterogeneous data. A significant level was 5% for Bartlett’s test. For other tests, the statistical analysis was two-tailed, with 5% used as level of significance. A significant result was indicated by either 5% or 1%.
RESULTS
In Vivo Micronucleus Test
Results are shown in Table 2. The mean incidence of polychromatic erythrocytes with micronuclei in the TE-treated groups was equivalent or less than in the negative-control group (0.14%, 0.09%, and 0.06% in the 500, 1000, and 2000 mg/kg groups, respectively, and 0.13% in the negative-control group). Thus, no dose-dependent increase was observed in the incidence of polychromatic erythrocytes with micronuclei among TE-treated groups, whereas a significant increase in the incidence of polychromatic erythrocytes with micronuclei (4.92%) was observed in the positive-control group treated with MMC.
The mean frequency of polychromatic erythrocytes to the total erythrocytes was 48.3%, 49.2%, and 47.4% in the 500, 1000, and 2000 mg/kg groups, respectively, and 47.8% in the negative-control group. In contrast, a significant decrease in the frequency of polychromatic erythrocytes was observed in the MMC dosing group (41.8%)
Bacterial Reverse Mutation Test
The mutagenicity test was conducted at five concentrations (313 to 5000 μg/plate), with a common ratio of 2. As shown in Table 3, the number of revertants did not increase to more than twice that of the negative control in any strain used, with or without S9 mix. No microbial contamination was detected in the test solution at the highest concentration, or S9 mix in any of the tests. The mutagenicity of the positive-control substances was confirmed, and the numbers of revertants in positive and negative controls were within the range (mean ± 3SD) of historical control values (data not shown). No precipitation of the test article or growth inhibition was observed in any cell strains. Moreover, TE did not increase the number of colonies on the plates at the maximal dose. The data of the test were judged as acceptable because (1) the negative-control values were appropriate in comparison with the background values; (2) the positive-control value was more than twice the negative-control value and appropriate in comparison with the background values; and (3) there were no abnormalities in the sterility test.
28-Day Toxicity Study
No clinical signs or changes in body weight (Table 4) or food consumption related to the administration of TE were observed. No abnormal changes were observed in any urinalysis parameter in treated animals as compared with the control animals. As shown in Table 5, a statistically significant decrease was seen in mean corpuscular hemoglobin and mean corpuscular volume for males in the 1000 mg/kg group, whereas mean corpuscular hemoglobin concentration was statistically increased in these animals. However, these changes were considered to be unrelated to the test substance, because no dose-dependent effects were noted, or changes were within the range of background data. No other significant changes were seen in hematological parameters. Similarly, a statistically lower chloride value for males in the 1000 and 2500 mg/kg groups, and a lower γ-GTP value for females in the 250 mg/kg group were observed (Table 6). These changes were likewise judged to be unrelated to the test substance, because they were unrelated to dose or considered a mild change within the range of background data. No other significant effects on clinical chemistry parameters were noted.
Although a statistically lower value was seen in the relative thymus weight for males in the 1000 mg/kg group (123 ± 24 versus 156 ± 33 mg%; p < .05 by Dunnett’s test), this change was not considered to be related to the administration of TE because it was a mild change unrelated to dose and was within the range of background data.
Unilateral pelvic dilation was observed in the right kidney of one male at the 2500 mg/kg dose group at necropsy. Upon histopathological examination, slight interstitial mononuclear cell infiltration and unilateral dilatation of pelvis in the kidney were observed. These changes were observed in only one animal and thus considered to be of spontaneous origin. No other changes were seen upon pathological or histopathological examination.
DISCUSSION
It has been known for many years that certain plant foods contain a substance that inhibits the activity of salivary and pancreatic amylase. More recently, these components have been purified and marketed for use in weight control under the generic name “starch blockers” (Bo-Linn 1982). For example, an extract derived from the common white kidney bean (Phaseolus vulgaris) has been shown to have alpha-amylase-inhibiting activity (Chokshi 2007).
Similarly, inhibitors of α-glucosidase, such as TE, have been shown to improve the time relationship between plasma insulin and glucose increases after a meal through inhibition of disaccharide hydrolases that convert disaccharides into monosaccharides (Clissold and Edwards 1988; Toeller 1994; Raptis and Dimitriadis 2001; Fujita, Yamagami, and Ohshima 2005). These substances may have use as dietary supplements to support healthy glucose metabolism and carbohydrate digestion.
The safety of TE has previously been established to support its approval as a FOSHU (Food for Specified Health Uses) by the Japanese Ministry of Health, Labour and Welfare. The administration of 133 or 655 mg/kg/day TE to KKAy mice for 60 days had no adverse effects on blood biochemistry or organ pathology. A single oral dose administration test has been performed on healthy subjects at a dose of 10 g/day, a level 10 times higher than the recommended daily intake (RDI) of 1 g/day. Moreover, we also studied borderline diabetic subjects were administered TE at a level of 5 g/day, fivefold higher than the RDI, for 5 weeks. No subjective symptoms or adverse effects on hematology, clinical chemistry, or urinalysis parameters were reported in either study (data not shown). Two products containing TE have been marketed for almost 10 years in Japan. No reports of side effects associated with these products have been reported by consumers.
Under the conditions of the studies reported herein, TE exhibited no potential to induce micronuclei and caused no suppression of the hematopoietic cell proliferation in the bone marrow of mice. Likewise, TE was not found to induce reverse mutation in bacteria. Often, when the extracts of food materials are tested, there could be stimulation of bacterial growth due to the presence of protein, or specifically, the amino acid histidine. However, TE was fermented by Aspergillus oryzae; therefore stimulation materials for the growth of bacteria such as proteins or carbohydrates were degraded during the fermentation period. As shown in Table 3, TE did not stimulate any bacterial growth at the maximum concentration.
Because no toxic changes were observed at 2500 mg/kg in both sexes, the no observed adverse effect level for TE was considered to be more than 2500 mg/kg in males and females. These results are consistent with Touchi’s status as a traditional Chinese food derived from fermented soybeans and its purported long history of use.
Specifically, these data are consistent with the expected safety of human consumption of TE up to at least 5 g/day in individuals with a normal body weight of 60 kg, determined as follows:
(*A 30-fold safety factor was applied rather than the more conservative 100-fold safety factor and is considered appropriate due to considerable human experience with Touchi.)
Footnotes
Tables
Conflict of Interest Statement. The authors are presently employed by Nippon Supplement, Inc., the manufacturer of the TE product used in the study and is the sponsor and sole source of funding for the present study.