2020 年 26 巻 6 号 p. 813-824
The safety of 6-(methylsulfinyl) hexyl isothiocyanate (6-MSITC) and Wasabi sulfinyl (WS), a 6-MSITC-containing supplement from Wasabi rhizome, was evaluated by a toxicity test and human clinical trial. Synthetic 6-MSITC (SS) and WS were used in the Ames test to clarify their mutagenic activity, and then used in acute and sub-acute toxicity tests via oral administration in five-week-old Sprague-Dawley rats. WS was further estimated by a human clinical trial. Eleven healthy subjects, aged 20–64 years, were enrolled in the clinical trial; they ingested five WS capsules daily for four weeks. The incidence and severity of WS ingestion-related adverse events were estimated by physical examination, weight and vital sign measurements (resting systolic and diastolic blood pressures, and heart rate), blood analysis, and urinalysis. In the Ames test, no mutagenicity of SS was observed in the range of 20–1 250 µg/plate. Lethal dose 50 (LD50) values of SS were estimated to be 451.2 mg/kg in male rats and 400.7 mg/kg in female rats. No sub-acute toxicity was observed in Sprague-Dawley rats administrated WS at 2 500 mg/kg/day (containing 2.5 mg 6-MSITC/kg/day). In the human clinical trial, three adverse events occurred in 2 of 11 (18.2%) subjects, which were classified not to be side effects from WS ingestion by clinical physicians. Although some fluctuations were observed in clinical laboratory values and physical measurement values, these fluctuations were within the reference ranges without clinical significance. Thus, ingestion of 500 mg/day of WS was demonstrated for the first time to be safe at the least. Taken together, our data on the safety evaluation of 6-MSITC and WS, a 6-MSITC-containing supplement, provide the first standard references for Wasabi supplement application.
Wasabi originating in Japan is the plant of Brassicaceae Wasabia. “Wasabi” written on wooden strips was found in the ruins of a medicinal herb garden in Asuka-Kyo that existed about 1300 years ago (Hasegawa et al., 2004), and was recorded in “Honzo Wamyo,” the oldest Japanese encyclopedia of medicinal plants, about 1100 years ago. Wasabi was traditionally used for medicine at this time, and then used as a food condiment about 800 years ago (Hasegawa et al., 2004). Now, approximately 2 200 tons of wasabi are produced annually for consumption (Ministry of Agriculture, Forestry and Fisheries, 2017).
Wasabi contains 21 kinds of isothiocyanates (ITCs; Ina, 1982; Etoh et al., 1990). Allyl ITC (AITC), a pungent ingredient, is the most well-known compound. In recent years, 6-(methylsulfinyl) hexyl ITC (6-MSITC) has attracted attention due to various functional reports. Many physiological functions of 6-MSITC, including detoxification (Morimitsu et al., 2002), antioxidation (Hou et al., 2000, 2011; Lee et al., 2010), anti-inflammation (Uto et al., 2007, 2012; Yamada-Kato et al., 2012), blood flow improvement (Morimitsu et al., 2000), anticancer functions (Fuke et al., 2014), and the protection of neuronal cells (Trio et al., 2016), have been reported. Recently, 6-MSITC has been reported to improve outcomes in mouse models of Parkinson's and Alzheimer's diseases (Morroni et al., 2014, 2018). In addition, the data from a human clinical intervention including 37 healthy males and females who ingested Wasabi sulfinyl (WS), a wasabi extract powder containing 0.8% 6-MSITC, for eight weeks revealed that the attention and judgment of the clinical intervention volunteers were improved (Okunishi et al., 2019). Therefore, WS has recently been developed as a health supplement.
Although 6-MSITC and WS have been reported to have benefits to health, there are few data on their safety assessment. Thus, safety confirmation for WS is considered to be crucial and important. No-observed-adverse-effect levels have been reported for AITC, benzyl ITC, phenethyl ITC, and 3-methylthiopropyl ITC at 12, 5, 5, and 30 mg/kg body weight/day, respectively (Valerio and Knaap, 2006). Additionally, sulforaphane (4-methylsulfinylbutyl isothiocyanate (4-MSITC)) found in broccoli has been investigated by several clinical trials for its internal metabolism and functionality (Shapiro et al., 2001; Kensler et al., 2005). No clinical trial on 4-MSITC safety has been performed even though it was used in a perfume (JECFA No. 1892, FEMA No. 4414).
Based on this information, in this study, we estimated the safety of 6-MSITC by the Ames test, acute toxicity test, and sub-acute toxicity test. Finally, a human clinical trial was also carried out for WS containing 0.8% 6-MSITC. According to the previous results that middle-aged and older adults who ingested 100 mg WS for eight weeks daily showed improved cognitive performance (Okunishi et al., 2019), a five-time overdose was designed in the present trial to examine the safety of 500 mg WS intake for four weeks in healthy adults.
Preparation of 6-MSITC, acute toxicity and sub-acute toxicity tests Synthetic 6-MSITC (SS) was synthesized at Ogawa & Co., Ltd. (Tokyo, Japan) through the oxidation of 6-(methylthio) hexyl ITC, which is an analog of 6-MSITC (Murata et al., 2004). WS containing 0.1% 6-MSITC was extracted from rhizome.
The Ames tests were carried out at the Koei Techno Service Co., Ltd. (Osaka City, Osaka, Japan) by a reverse mutation assay with two strains of Salmonella typhimurium (TA100 and TA98). The assay was conducted for SS with seven doses from 20 µg to 1 250 µg/plate for each strain, and for WS with seven doses from 78 µg to 5 000 µg/plate for each strain.
The acute toxicity tests were performed at the Japan Bio Science Laboratory Co., Ltd. (Kaizu-cho, Gifu, Japan) by oral administration once to both sexes of Sprague-Dawley (SD) rats [Crj:CD(SD)IGS, SPF]. The dosage levels were designed for seven doses from 125 mg/kg to 759 mg/kg, individually based on the body weight at the first day of administration (Day 0). Body weight was measured at Day 0 and at Days 1, 3, 5, 7, 10 and 14 (day of autopsy). The LD50 value was calculated according to the 14-day cumulative mortality with the probit method.
The sub-acute toxicity tests were done at TTC Co., Ltd. (Tokyo, Japan) by repeated administration to both sexes of SD rats [Crl:CD(SD)] for 28 days. The dosage levels were designed for 500 mg/kg/day and 2 500 mg/kg/day, individually based on the body weight at Day 0. The toxicity was evaluated based on the 28-day observation data, including the general condition, confirmation of life and death, hematology, blood chemistry, urinalysis, and necropsy.
Ethics The toxicity tests adhered to the Good Laboratory Practices (GLP, the Ministry of Health, Labour and Welfare Ordinance No. 21 of 1997) and the Organization for Economic Co-operation and Development Guidelines for the Testing of Chemicals as a non-GLP test.
All of the animal experiments adhered to the Guidelines for the Proper Implementation of Animal Experiments (Science Council of Japan, 2006), Act on Welfare and Management of Animals (Amendment of Act No. 68 of 2005) and Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals (Notice of the Ministry of the Environment No. 88 of 2006). Sub-acute toxicity tests were conducted according to the Ministerial Ordinance on Good Laboratory Practice for Nonclinical Safety Studies of Drugs (Ordinance of the Ministry of Health and Welfare No. 21 of 1997) and Partial Revision of the Guidelines for Repeated Dose Toxicity Studies (PMSB/ELD Notification No. 902).
The human clinical trial adhered to all guidelines set forth in the Declaration of Helsinki (amended in October 2013), the Ethical Guidelines for Medical Research in Humans (observed by the Ministry of Education and Science and the Ministry of Health, Labour and Welfare, the Japanese Government, in December 2014 [amended in February 2017]), and the guidance of the Ethical Guidelines for Medical Research in Humans (enacted by the Ministry of Education and Science and the Ministry of Health, Labour and Welfare, the Japanese Government, in February 2015 [amended in March 2017]). The protocol of the study was approved by the Aisei Hospital Ueno Clinic Research Ethics Committee, Tokyo, Japan (IRB number: 12000071) on April 27 2017 before starting the clinical trial. All volunteers received a full explanation of the aims and procedures. Written informed consent was obtained from all participants before starting the study. All records were classified with an anonymous subject identification number to ensure privacy.
The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR; study ID UMIN000027402).
Participants Of the 26 subjects who participated in the screening, 11 healthy male and female subjects, between the ages of 20 to 64 years, were initially enrolled in this study. Individuals were excluded if: (1) they were allergic to Wasabi or likely to get allergies by participating in this research; (2) they were under treatment for a disease; (3) they had a severe disease, such as diabetes, liver disease, kidney disease, heart disease or cancer, or had a history of a severe disease; (4) their physical measurement, physical examination and clinical laboratory values before the start of ingestion significantly deviated from the reference range; (5) they had lifestyle habits that would affect the research results, such as working day and night shifts, or were scheduled to go on a shift day and night or overseas during the research period; (6) they had participated in another clinical research within one month before obtaining acquisition, or were planning to participate in another clinical study after acquiring consent to participate in this study; (7) they were pregnant or nursing, or planned to get pregnant during the research period; (8) they were unsuitable as subjects from the questionnaire on lifestyle habits; and (9) they were judged unsuitable for this study by the medical investigator.
Study design and procedure The study was an open-label trial. It consisted of a screening test, a four-week WS ingestion period, and a two-week follow-up period without WS ingestion. The trial schedule is shown in Table 1.
After the screening test, each eligible subject received one capsule that contained 100 mg WS (0.8 mg 6-MSITC) adsorbed on α-cyclodextrin and a 110 mg vehicle (consisting of starch, silicon dioxide and calcium stearate). The WS capsules were provided by Kinjirushi Co., Ltd. (Nagoya, Japan). The nutritional components of the test foods (per daily intake; five capsules) are shown in Table 2. During the trial period, a medical investigator instructed all subjects on the following points: (1) avoid excessive exercise or eating too little or too much; (2) take five capsules per day, according to the safety test standards for food for specified health uses (Precautions for Preparing Application Forms Pertaining to Food Applications for Specified Health Uses, Consumer Affairs Agency, Japan, April 2020), and record a daily report; (3) maintain a healthy daily lifestyle; (4) do not ingest other health foods (foods for a specified health use, functional display foods, health supplements, or other supplements); (5) on the day before the examination, go to bed before midnight to ensure enough sleep; (6) on the day before the examination, do not eat or drink anything other than water after 10 pm until the end of the examination.
| Item | Amount |
|---|---|
| Energy (kcal) | 5.04 |
| Protein (mg) | 257.8 |
| Fat (mg) | 78.6 |
| Carbohydrate (mg) | 826.3 |
| Sodium (mg) | 0.57 |
Analysis was carried out in Japan Food Research Laboratories by standard methods.
This study was conducted from May to August 2017 in the Medical Station Clinic (Tokyo, Japan) and the Public Interest Incorporated Foundation Aiseikai Aisei Hospital Ueno Medical Clinic (Tokyo, Japan).
Safety assessment Safety was evaluated by the incidence and severity of WS ingestion-related adverse events in subjects during the research period. An adverse event was defined as an undesirable or unintended injury or symptom, including an abnormality in clinical laboratory test values for the subjects after the preliminary examination, regardless of whether or not there was a causal relationship with the conducted study. A safety evaluation was also conducted through physical examinations, measurements of the subjects' weights and vital signs (resting systolic and diastolic blood pressures, and heart rate), and laboratory assessments, including biochemical, hematological and urinal analysis. The laboratory biochemical indexes included total protein (TP), albumin (Alb), total bilirubin (TB), direct bilirubin (D-B), indirect bilirubin (I-B), aspartate-aminotransferase, alanine-aminotransferase, γ-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), alkaline phosphatase, blood urea nitrogen, creatinine (Cr), uric acid, lipid profile (total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), triglycerides, sodium, potassium, chloride, glucose (GLU), and hemoglobin A1c (screening only). Hematological indexes consisted of the white blood cell count, red blood cell count, hemoglobin (Hb), hematocrit and platelets (PLT). The urinalysis consisted of the occult blood reaction, protein, and GLU in urine.
Data analyses An analysis of adverse events was conducted for all subjects, and the number of adverse event occurrences was counted. The values at each time point before and after ingestion were compared using the paired t-test. A p-value <0.05 was considered statistically significant. No statistical adjustment for multiple testing was performed. Microsoft Excel 2013 was used for these analyses, and the results are expressed as the mean ± standard deviation.
Results of the Ames test, acute test, and sub-acute test The Ames test was conducted to investigate the mutagenicity of SS (purity: 95% or more) and WS in the concentration ranges of 20–1 250 µg/plate and 78–5 000 µg/plate, respectively. In the case of SS, growth inhibition of TA100 (−S9) and TA98 (both −S9 and +S9) was observed at a concentration of 313 µg/plate. In the case of WS, growth inhibition of TA100 (−S9) was observed in the concentration range of 156–5000 µg/plate. On the other hand, no mutagenicity was observed at any concentration of either SS or WS (Table 3).
| SS | WS | |||||||
|---|---|---|---|---|---|---|---|---|
| TA100 (Base pair substitution type) |
TA98 (Frame shift type) |
TA100 (Base pair substitution type) |
TA98 (Frame shift type) |
|||||
| −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | |
| 20 µg/plate | — | — | — | — | / | / | / | / |
| 39 µg/plate | — | — | — | — | / | / | / | / |
| 78 µg/plate | — | — | — | — | — | — | — | — |
| 156 µg/plate | — | — | — | — | — | — | —* | — |
| 313 µg/plate | —* | — | —* | —* | — | — | —* | — |
| 625 µg/plate | N | N | N | N | — | — | —* | — |
| 1250 µg/plate | N | N | N | N | — | — | —* | — |
| 2500 µg/plate | / | / | / | / | — | — | —* | — |
| 5000 µg/plate | / | / | / | / | — | — | —* | — |
| Positive Control | + | + | + | + | + | + | + | + |
*: Growth inhibition was observed, N: No Growth was observed, —: No more than 2 times back mutation, +: More than 2 times back mutations were seen, /: No data
The strain −S9 or +S9 was pre-incubated at 30 °C for 20 minutes, and further cultured for 48 hours after addition of test substance. The solvent used was dehydrated DMSO, and no precipitation of the test substance was observed.
The results of the acute toxicity test for SS performed in male and female SD rats are shown in Table 4. No death was observed in rats administrated WS at the dose of 2 000 mg/kg, but death was observed with SS at the dose of 308 mg/kg or more. The LD50 value was estimated to be 451.2 mg/kg in males (95% confidence limit: 357.5–573.8 mg/kg) and 400.7 mg/kg in females (95% confidence limit: 322.1–507.6 mg/kg) for SS. Necropsy of dead rats at 1–3 days after administration revealed signs of erosion due to irritant substances, ulceration and/or reddening of the forestomach and glandular mucosa. No changes in the other organs were observed. Autopsies were performed for all surviving rats. Secondary symptoms, such as an increase in forestomach thickening, were observed in the rats fed 169 mg/kg or more.
| Acute | Sub-acute | ||
|---|---|---|---|
| LD50 | NOAEL | ||
| SS | WS | WS | |
| SD-rats(male) | 451.2 mg/kg | — | |
| 2500 mg/kg | |||
| SD-rats(female) | 400.7 mg/kg | — | |
⋇ —: No deaths were observed at 2000 mg/kg, NOAEL: No Observed Adverse Effect Level
The SD rats were bred in a stainless steel wire hanger cage in a barrier breeding room with a temperature of 20 to 26 °C., a relative humidity of 40 to 70%, a ventilation rate of 10 to 17 times / hour under controlled light (12-h light/day), where they had free access to water and feed.
The sub-acute toxicity test for WS (containing 0.1% 6-MSITC) was further evaluated by repeated oral administrations to five-week-old male and female SD rats for 28 days. No animal died even when 2 500 mg/kg WS was administered, and no change was observed in the general observations, including body weight, food consumption, water rehydration, urinalysis, hematological examination, and pathological anatomical examination.
Characteristics of the subjects in the human clinical trial Of the 26 subjects who participated in the screening test, 11 met the selection criteria and did not fall under the exclusion criteria. The subjects' backgrounds are shown in Table 5. Some subjects whose values were outside of the reference range in the clinical examination were also included in the study after confirming no problems for participating in the study by a medical investigator. All 11 subjects completed this study with a 100.0% ingestion rate of the test food.
| Item | All examples | Male | Female |
|---|---|---|---|
| Numbers | 11 | 6 | 5 |
| Age (years) | 45.2 ± 13.0 | 45.0 ± 13.0 | 45.4 ± 14.5 |
| Height (cm) | 169.11 ± 7.85 | 173.95 ± 6.86 | 163.30 ± 4.21 |
| Body weight (kg) | 59.52 ± 8.06 | 65.47 ± 5.41 | 52.38 ± 2.98 |
| BMI (kg/m2) | 20.72 ± 1.29 | 21.60 ± 0.78 | 19.66 ±0.92 |
| Systolic blood pressure (mmHg) | 108.9 ± 9.4 | 108.7 ± 7.8 | 109.2 ± 12.1 |
| Diastolic blood pressure (mmHg) | 63.6 ± 8.5 | 61.5 ± 6.7 | 66.2 ± 10.5 |
| Pulse rate (beat/minute) | 67.6 ± 10.4 | 69.5 ± 13.4 | 65.4 ± 5.7 |
Values are expressed as the mean ± standard deviation (other than the number of people). Abbreviations: body mass index (BMI).
Men and women from age 20s to 60s were incorporated in human clinical trial in order to assess the safety of food in a wide range of ages and genders. All of subjects were required to have medical check to confirm their health background.
Evaluation of safety The results on the adverse events are summarized in Table 6. Three adverse events occurred in 2 of 11 subjects (18.2%), including nausea, stomachache, heavy stomach, and anorexia. As causes of the adverse events, there were self-reports of excessive drinking of cold beverages and poor physical condition due to high temperatures and high humidity levels. Therefore, the adverse events were not caused by the test food. The medical investigator concluded that the test food had no clinical problem.
| Items | Number |
|---|---|
| Subjects to safety analysis | 11 |
| Persons expressing secondary effects | 0 |
| Incidence rate of side effects (%) | 0 |
| Occurrences of secondary effects | 0 |
| Adverse events | 2 |
| Incidence of adverse events (%) | 18.2 |
| Occurrences of adverse events | 3 |
| Adverse events by symptom: | |
| Nausea, stomachache | 1 |
| Heavy Stomach | 1 |
| Anorexia | 1 |
If the same adverse event occurred multiple times in the same subject, it was accounted as one event. Subject's subjective symptoms and adverse events were judged by the responsible physician with objective findings.
The results of the blood biochemical examination, hematological examination and urinalysis are shown in Tables 7, 8 and 9, respectively. Significant fluctuations after ingestion were observed in the Hb (female), TP and Alb values at the second week, Hb (female), PLT, γ-GT (female), Cr (female) and HDL-C (male) values at the fourth week, and TB, I-B, γ-GT (male), LDH, Cr (female), and CL after two weeks of observation. These fluctuations were within the reference ranges, and the medical investigator concluded that there was no clinical significance. The physical examination values are shown in Table 10. When compared to the screening results significant fluctuations after ingestion were observed in the body weight at the second week, and in the body weight (female) and body mass index (female) at the fourth week. These fluctuations were minor, and the medical investigator concluded that there was no clinical significance.
| Item | Reference values | Sex | n | Screening | 2nd week | 4th week | After a 2-week observation |
|---|---|---|---|---|---|---|---|
| TP (g/dL) | 6.7–8.3 | 11 | 7.22 ± 0.49 | 7.45 ± 0.43* | 7.29 ± 0.46 | 7.23 ± 0.43 | |
| Alb (g/dL) | 3.8–5.2 | 11 | 4.34 ± 0.27 | 4.48 ± 0.26* | 4.43 ± 0.24 | 4.39 ± 0.28 | |
| TB (mg/dL) | 0.2–1.2 | 11 | 0.62 ± 0.15 | 0.74 ± 0.20 | 0.71 ± 0.19 | 0.75 ± 0.21* | |
| D-B (mg/dL) | 0.0–0.2 | 11 | 0.10 ± 0.04 | 0.13 ± 0.05 | 0.13 ± 0.05 | 0.13 ± 0.05 | |
| I-B (mg/dL) | 0.2–1.0 | 11 | 0.52 ± 0.13 | 0.61 ± 0.20 | 0.58 ± 0.15 | 0.62 ± 0.19* | |
| AST (GOT) (U/L) |
10–40 | 11 | 20.3 ± 6.8 | 20.8 ± 6.5 | 18.6 ± 6.4 | 21.4 ± 6.3 | |
| ALT (GPT) (U/L) |
5–45 | 11 | 18.0 ± 10.0 | 18.2 ± 8.5 | 16.2 ± 9.5 | 18.7 ± 9.8 | |
| γ-GT (U/L) | M : 0–80 | male | 6 | 24.2 ± 6.6 | 23.5 ± 5.4 | 23.2 ± 6.4 | 22.3 ± 6.4* |
| F : 0–30 | female | 5 | 14.8 ± 5.0 | 16.0 ± 6.0 | 16.0 ± 4.5* | 16.2 ± 8.1 | |
| LDH (U/L) | 120–240 | 11 | 156.5 ± 20.0 | 166.2 ± 28.7 | 162.6 ± 17.6 | 168.3 ± 19.0* | |
| ALP (U/L) | 100–325 | 11 | 193.2 ± 48.8 | 196.5 ± 60.6 | 188.9 ± 60.3 | 189.8 ± 58.9 | |
| UN (mg/dL) | 8.0–20.0 | 11 | 12.83 ± 3.51 | 12.63 ± 3.01 | 13.58 ± 3.17 | 12.82 ± 3.80 | |
| Cr (mg/dL) | M : 0.61–1.04 | male | 6 | 0.785 ± 0.079 | 0.830 ± 0.072 | 0.798 ± 0.081 | 0.807 ± 0.085 |
| F : 0.47–0.79 | female | 5 | 0.602 ± 0.036 | 0.666 ± 0.052 | 0.672 ± 0.067* | 0.652 ± 0.047** | |
| UA (mg/dL) | M : 3.8–7.0 | male | 6 | 5.50 ± 0.47 | 5.37 ± 0.37 | 5.12 ± 0.35 | 5.50 ± 0.59 |
| F : 2.5–7.0 | female | 5 | 4.28 ± 0.63 | 4.56 ± 1.10 | 4.52 ± 0.87 | 4.32 ± 1.11 | |
| TC (mg/dL) | 120–219 | 11 | 191.8 ± 29.7 | 197.0 ± 31.8 | 192.5 ± 36.6 | 194.5 ± 35.6 | |
| HDL-C (mg/dL) | M : 40–85 | male | 6 | 60.7 ± 5.2 | 58.0 ± 8.9 | 53.8 ± 9.7* | 56.2 ± 13.5 |
| F : 40–95 | female | 5 | 66.4 ± 15.4 | 71.0 ± 21.9 | 70.6 ± 24.3 | 70.8 ± 27.3 | |
| LDL-C (mg/dL) | 65–139 | 11 | 112.9 ± 25.1 | 114.5 ± 27.1 | 111.0 ± 27.4 | 111.1 ± 27.2 | |
| TG (mg/dL) | 30–149 | 11 | 72.0 ± 26.7 | 78.1 ± 26.4 | 73.0 ± 34.5 | 86.1 ± 26.2 | |
| Na (mEq/L) | 137–147 | 11 | 142.3 ± 1.9 | 142.6 ± 1.0 | 142.0 ± 1.9 | 141.5 ± 1.8 | |
| K (mEq/L) | 3.5–5.0 | 11 | 4.44 ± 0.32 | 4.35 ± 0.27 | 4.36 ± 0.33 | 4.30 ± 0.23 | |
| CL (mEq/L) | 98–108 | 11 | 105.6 ±2.1 | 105.6 ±2.2 | 105.3 ± 1.7 | 104.4 ± 1.9* | |
| GLU (mg/dL) | 70–109 | 11 | 86.5 ± 7.0 | 85.5 ±8.4 | 87.9 ± 6.9 | 85.5 ± 9.4 | |
| HbAlc (%) | 4.6–6.2 | 11 | 5.20 ± 0.36 |
Values are expressed as the mean ± standard deviation, p < 0.01 **, p < 0.05 *
Abbreviations: Total protein (TP), albumin (Alb), total bilirubin (TB), direct bilirubin (D-B), indirect bilirubin (I-B), aspartate-aminotransferase (AST [GOT]), aknine-aminotransferase (ALT [GPT]), γ-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), blood urea nitrogen (UN), creatinine (Cr), uric acid (UA), lipid profile (total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), triglycerides (TG), sodium (Na), potassium (K), chloride (CL), glucose (GLU), and hemoglobin Ale (HbAlc).
Blood biochemical examinations were carried out in LSI Medience Corporation by standard methods. The adverse events were judged by responsible physician according to the criteria for abnormal changes (antimicrobial safety evaluation criteria) established by the Japanese Society of Chemotherapy, Common Terminology Criteria for Adverse Events v4.0, and the Japan Ningen Dock Society criteria (revised April 2017) (Table7–9).
| Item | Reference values | Sex | n | Screening | 2nd week | 4th week | After a 2-week observation |
|---|---|---|---|---|---|---|---|
| WBC (/µl.) | 3300–9000 | 11 | 5290.9 ± 1164.0 | 5409.1 ± 1432.8 | 5290.9 ± 1116.7 | 5418.2 ± 1050.5 | |
| RBC (×104/µL) | M : 430–570 | male | 6 | 475.0 ± 40.2 | 481.3 ± 42.7 | 466.2 ± 37.0 | 479.3 ± 43.8 |
| F : 380–500 | female | 5 | 440.0 ± 29.3 | 451.0 ± 26.4 | 447.4 ± 25.2 | 444.0 ± 11.6 | |
| Hb (g/dL) | M : 13.5–17.5 | male | 6 | 14.08 ± 1.06 | 14.53 ± 0.80 | 14.18 ± 0.73 | 14.52 ± 0.78 |
| F : 11.5–15.0 | female | 5 | 13.12 ± 0.86 | 13.50 ± 0.60* | 13.60 ± 0.71* | 13.46 ± 0.40 | |
| Ht (%) | M : 39.7–52.4 | male | 6 | 45.13 ± 2.44 | 46.00 ± 2.33 | 44.10 ± 1.98 | 45.95 ± 2.28 |
| F : 34.8–45.0 | female | 5 | 42.14 ± 2.35 | 43.26 ± 1.44 | 42.88 ± 1.71 | 42.88 ± 0.82 | |
| PLT (×104/µL) | 14.0–34.0 | 11 | 26.04 ± 5.30 | 26.47 ± 6.00 | 23.90 ± 5.28* | 26.86 ± 6.37 |
Values are expressed as the mean ± standard deviation, p < 0.01 **, p < 0.05 *
Abbreviations: White blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), and hematocrit (Ht), and platelets (PLT).
Hematological examinations were carried out in LSI Medience Corporation by standard methods.
| Item | n | Screening | 2nd week | 4th week | After a 2-week observation | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| (−) | (±) | (+) | (−) | (±) | (+) | (−) | (±) | (+) | (−) | (±) | (+) | |||
| Protein | total | 11 | 10 | 1 | 0 | 10 | 1 | 0 | 10 | 0 | 1 | 11 | 0 | 0 |
| male | 6 | 6 | 0 | 0 | 6 | 0 | 0 | 5 | 0 | 1 | 6 | 0 | 0 | |
| female | 5 | 4 | 1 | 0 | 4 | 1 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | |
| Glucose | total | 11 | 11 | 0 | 0 | 11 | 0 | 0 | 11 | 0 | 0 | 11 | 0 | 0 |
| male | 6 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | |
| female | 5 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | |
| Occult blood | total | 11 | 11 | 0 | 0 | 11 | 0 | 0 | 11 | 0 | 0 | 11 | 0 | 0 |
| male | 6 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | 6 | 0 | 0 | |
| female | 5 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | |
The numerical data is a summary of the measurement results.
Urinalyses including urine protein, glucose and occult blood were carried out in LSI Medience Corporation by standard methods.
| Item | Sex | n | Screening | 2nd week | 4th week | After a 2 week observation |
|---|---|---|---|---|---|---|
| Body weight (kg) | total | 11 | 59.52 ± 8.06 | 59.03 ± 8.36* | 59.06 ± 8.49 | 59.05 ± 8.36 |
| male | 6 | 65.47 ± 5.41 | 65.28 ± 5.35 | 65.37 ± 5.53 | 64.98 ± 6.11 | |
| female | 5 | 52.38 ± 2.98 | 51.52 ± 3.13 | 51.50 ± 3.31* | 51.94 ± 3.44 | |
| BMI (kg/m2) | total | 11 | 20.72 ± 1.29 | 20.55 ± 1.48 | 20.55 ± 1.45 | 20.56 ± 1.42 |
| male | 6 | 21.60 ± 0.78 | 21.57 ± 0.77 | 21.58 ± 0.86 | 21.47 ± 0.98 | |
| female | 5 | 19.66 ± 0.92 | 19.32 ± 1.13 | 19.32 ± 0.94* | 19.48 ± 1.06 | |
| Systolic blood pressure (mmHg) | total | 11 | 108.9 ± 9.4 | 105.1 ± 5.9 | 107.0 ± 9.3 | 104.5 ± 11.0 |
| male | 6 | 108.7 ± 7.8 | 106.3 ± 5.0 | 107.7 ± 10.8 | 104.0 ± 11.1 | |
| female | 5 | 109.2 ± 12.1 | 103.6 ± 7.2 | 106.2 ± 8.3 | 105.2 ± 12.0 | |
| Diastolic blood pressure (mmHg) | total | 11 | 63.6 ± 8.5 | 62.9 ± 6.3 | 62.6 ± 7.4 | 60.5 ± 7.4 |
| male | 6 | 61.5 ± 6.7 | 61.3 ± 6.9 | 60.3 ± 6.4 | 58.0 ± 6.2 | |
| female | 5 | 66.2 ± 10.5 | 64.8 ± 5.5 | 65.4 ± 8.3 | 63.6 ± 8.4 | |
| Pulse rate (beat/minute) | total | 11 | 67.6 ± 10.4 | 69.0 ± 11.8 | 68.1 ± 12.0 | 69.3 ± 9.8 |
| male | 6 | 69.5 ± 13.4 | 70.5 ± 14.9 | 70.3 ± 15.2 | 71.3 ± 10.9 | |
| female | 5 | 65.4 ± 5.7 | 67.2 ± 8.1 | 65.4 ± 7.6 | 66.8 ± 8.6 |
Values are expressed as the mean ± standard deviation, p < 0.01 **, p < 0.05 * Abbreviations: BMI, body mass index.
Physical examinations were carried out in Medical Station Clinic by standard methods.
Wasabi is widely consumed in many areas of the world as a spice, and it is indispensable for Japanese food. Its ingredient, 6-MSITC, has been reported to have many functions. However, there are few reports concerning its safety or toxicity. Therefore, in this study, we estimated the safety of pure SS and an extract from Wasabi rhizome, WS, containing 0.8% 6-MSITC, by examining its mutagenic activity in bacteria, acute and sub-acute toxicities in SD rats, and side effects in a human clinical trial.
In the Ames test, no mutagenicity was observed for SS and WS. However, growth inhibition was observed at 313 µg/plate or more for SS, and 156 µg/plate or more in the TA98 strain (−S9) for WS. No growth inhibition was observed up to 500 µg/plate in the TA98 strain (+S9) for WS. Therefore, the components metabolized by the TA98 strain (−S9) might be involved in the growth inhibition of the bacterium. It has been reported that ITCs, including allyl ITC, ethyl ITC, methyl ITC, sulforaphane and benzyl ITC, accumulate in bacteria as GSH, small thiol-like BSH or thioredoxin-dithiocarbamate conjugates, and they attack the active center of enzymes by binding to thiol or amine groups to affect enzymatic activities such as respiration, metabolism and the transcription of genes. The reactivity of ITC sulfhydryl groups was demonstrated to be a major player for these conjugates, and it disturbs the biochemical processes of bacteria to lead to growth inhibition (Dufour et al., 2915). Thus, 6-MSITC might also inhibit the growth of TA98 strain (−S9) through similar kinds of conjugates, because 6-MSITC is a representative ITC, although direct data are needed for this to be proved in future studies.
In the acute toxicity test, the LD50 value of SS in rats was estimated to be 451.2 mg/kg in males and 400.7 mg/kg in females. It has been reported that the LD50 values of AITC were 339–490 mg/kg body weight, and that the LD50 values of 3-methylthiopropyl ITC were 490 mg/kg body weight in males and 540 mg/kg body weight in females (Jenner et al., 1964). Although the molecular weights of these ITCs differ by more than two-fold, their toxicity doses are close. It is considered that the NCS group of ITCs has higher reactivity, which principally contributes to the toxicity. Wasabi generally contains approximately 0.2% AITC and about 0.04% 6-MSITC (Sultana et al., 2003; Murata et al., 2004). Based on these data and our evaluation results for toxicity, even if 6-MSITC is concentrated five-fold, the dose is within the safe range, because the AITC content is five times higher than 6-MSITC in the natural state.
In the acute toxicity test and sub-acute toxicity test of WS, there were no deaths at doses of 2 000 mg/kg and 2 500 mg/kg/day, respectively, and also, no other obvious changes were observed. In the overdose test of WS, the intake was set to be 500 mg WS containing 0.8% 6-MSITC/day for 4 weeks, which was five times higher than the effective intake in the previous clinical studies on brain function (Okunishi et al., 2019). Although three adverse events, including nausea, stomach pain, stomach slack and anorexia occurred in 2 of 11 (18.2%) subjects, the medical investigator confirmed that all adverse events had a clear cause and were not related to WS intake.
Although the body weight and body mass index of the female group at the forth week were significantly reduced, no subject lost a large amount of weight, and the difference was about 0.02 kg on average, which was a slight difference when compared to the second week. Considering that 2 of 5 women were constipated, it is possible that there was a significant difference depending on the timing of defecation. Therefore, this weight loss was not evaluated as toxicity.
Moreover, significant changes in some of the laboratory test values, anthropometric measurements, and physical test values were also observed; however, these changes were mild and were judged to be not clinically relevant to WS intake. Thus, it is considered that there is no safety problem with the consumption of WS within 500 mg/day.
Additionally, a human clinical trial of sulforaphane found in broccoli was previously performed (Shapiro et al., 2006). Twenty-five µmol (4.43 mg) was consumed three times a day for 7 days, and a total of 525 µmol (93 mg) was consumed by three subjects. As a result, increases in alanine-aminotransferase and thyroid-stimulating hormone were observed, but it was confirmed that these changes had no clinical significance for safety problems. It is clear that sulforaphane has the same backbone chemical structure as 6-MSITC (Hou et al., 2000). There is only a small structural difference in the length of the methyl chain: sulforaphane has four methyl groups and 6-MSITC has six methyl groups. In our previous studies, we demonstrated that 6-MSITC and sulforaphane have the same molecular mechanisms for their antioxidant (Hou et al., 2000) and anti-inflammatory activities (Uto et al., 2007, 2012), although 6-MSITC showed lightly stronger activities than sulforaphane. Therefore, our 6-MSITC data from the human clinical trial are consistent with the results for sulforaphane.
Wasabi contains 21 kinds of ITCs, and most of these ITCs have been reported to have a wide range of effects, such as antioxidant effects (Morimitsu et al., 2002; Lee et al., 2014; Uto et al., 2012; Yamada-Kato et al., 2012; Morimitsu et al., 2000; Fuke et al., 2014; Trio et al., 2016; Morroni et al., 2014, 2018; Okunishi et al., 2019). To establish precision safety, it is necessary to further examine differences in susceptibility between races and differences in sensitivity between different age groups.
Acknowledgments We are grateful to TTC Co., Ltd. for coordinating the clinical trial and the toxicity test. We also thank LSI Medience Corporation, Koei Techno Service Co., Ltd. and the Japan Bio Science Laboratory Co., Ltd. for carrying out tests.
Isao Okunishi and Tomoe Yamada-Kato are employees of Kinjirushi Co., Ltd and received research grant from their company. Jiro Saito and De-Xing Hou declare that they have no conflict of interest.
