Assessment of mutagenicity, acute and sub-acute toxicity and human trial safety of wasabi leaf extract powder

Abstract

Wasabi leaf has been reported to show human health benefits without assessment of its safety. This study aims to investigate the mutagenicity, acute and sub-acute toxicity and human trial safety of wasabi leaf extract (WLE). The Ames test was used to assess mutagenicity, while acute and sub-acute toxicity were assessed by oral administration in five-week-old Slc:ICR mice (SPF) and five-week-old Sprague-Dawley (SD) rats, respectively. Human trial safety was further determined in a clinical trial. Twelve healthy subjects, aged 20–64 years and mildly obese (BMI 23.0 to 30.0 kg/m²), were enrolled in the clinical trial, and participants ingested 200 mg WLE daily for 12 weeks. The effect of WLE on fat metabolism was evaluated by visceral fat area (VFA), subcutaneous fat area (SFA), VFA/SFA(V/S) area ratio, body weight, BMI, TG, T-Cho, HDL-C, LDL-C, waist circumference, and body fat percentage. In the Ames test, WLE did not show mutagenicity in the range of 1.2–5000 µg/plate. No acute toxicity was observed in Slc:ICR mice (SPF) administered 5000 mg/kg/day WLE, and no sub-acute toxicity was observed in Crl:CD (SD) rats administered 2500 mg/kg/day WLE. In the human clinical trial, there were no significant differences between the WLE and placebo groups for any outcome measure assessed. Thus, ingestion of 200 mg/day of WLE was demonstrated for the first time to be safe. Taken together, our data on the mutagenicity, acute and sub-acute toxicity and human trial safety of WLE provide the first standard references for wasabi leaf supplement application.

Introduction

Wasabi is a uniquely pungent vegetable that originates from Japan and is an indispensable condiment in modern Japanese cuisine. Currently, approximate 2200 tons of wasabi are produced annually for consumption (Ministry of Agriculture, Forestry and Fisheries, 2017).

In general, the rhizome of the wasabi plant is used as a spice. The majority of studies have focused on the biological functions of wasabi rhizome extract, which contain 21 kinds of isothiocyanates (ITC) (Ina, 1982; Etoh et al., 1990). Part of the wasabi leaf is used for wasabi paste, but most wasabi leaves are discarded because they are less pungent and more bitter than the rhizome. On the other hand, wasabi leaf extract (WLE) is reported to have anti-obesity activity (Yamasaki et al., 2013; Oowatari et al., 2016; Misawa et al., 2018). Animal experiments further demonstrated that the body weight of rats fed a high-fat diet supplemented with wasabi leaf was significantly reduced compared to the control (Yamada-Kato et al., 2016). Regarding the active ingredients, it has been reported that wasabi leaf contains some flavonoid glycosides (Hosoya et al., 2005) and relatively low isothiocyanate content. Of them, isosaponarin is reported to have antioxidant (Hosoya et al., 2008; Sekiguchi et al., 2010) and anti-inflammatory (Yoshida et al., 2015) activities. Moreover, isosaponarin could promote collagen production (Nagai et al., 2010) and showed increased expression of β-3 adrenergic receptors and the enzyme UCP-1, which is related to fat β-oxidation. Isovitexin is another flavonoid that has been isolated from wasabi leaf (Taguchi et al., 2019), and has been reported to have some bioactive functions, e.g., antioxidative (Ramarathnam et al., 1989) and renoprotective effects (Liu et al., 2020). Isovitexin also reduced postprandial blood glucose levels in sucrose-loaded normoglycemic mice (Choo et al., 2012).

Although these studies have suggested that wasabi leaf has health benefits, there are minimal data on its safety. Confirmation of the safety of WLE is necessary for the application of wasabi leaf as a health food supplement. In this study, we thus investigated the mutagenicity of WLE using the Ames test, as well as its acute toxicity in mice and sub-acute toxicity in rats. In addition, to enable future large-scale clinical trials, small-scale human trials were conducted to evaluate the safety of WLE, in which adverse events as well as hematological and biochemical parameters were investigated.

Materials and Methods

Preparation of WLE powder, the Ames test, and acute and sub-acute toxicity tests    Wasabi leaves were harvested from Hokkaido by Kinjirushi Co., Ltd., a manufacturer of wasabi products, and were then grated into a paste. WLE used in this study was extracted from the paste using 50% ethanol, which was shown to be an efficient solvent for the extraction of many kinds of polyphenols in plants in our previous study and is also used in the food industry. The ethanol extracts were concentrated in an evaporator and then lyophilized to a powder. The yield of extract from raw wasabi leaves was 2.8%. The WLE concentration was adjusted with distilled water to prepare samples for each experiment. The flavonoids were analyzed by high performance liquid chromatography, and total polyphenol content was measured by the Folin-Ciocalteau method with gallic acid as a standard (Gabriel et al., 2014). WLE contained three major flavonoids, isovitexin, isosaponarin, and isoorientin, at concentrations of 0.272, 0.012, and 0.030 mg/g, respectively, as well as a total polyphenol content of 2.28 mg GAE/g.

The Ames test and acute/sub-acute toxicity tests were carried out by TTC Co., Ltd. (Tokyo, Japan). Salmonella typhimurium TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA strains were used for the Ames test. The test was performed using the S9 (±) pre-incubation method (37 °C, 20 min) with a culture time of 48 h, a maximum volume of 5 000 µg/plate, a common ratio of 4, and seven volumes of two plates each. The acute toxicity test consisted of single oral gavage administration of WLE (adjusted to 1 250, 2 500, and 5 000 mg/kg in 20 mL of water) to 20 male and female Slc:ICR mice (SPF) (5 weeks old). Administration was started on day 0 in all cases, and the general condition of mice was monitored for 14 days.

Furthermore, rats were used for the sub-acute toxicity test because various organs, blood, and urine were required to assess symptoms of toxicity. Eighteen male and 18 female Crl:CD (SD) rats (5 weeks old) were administrated by repeated oral gavage with WLE for 28 days at a dose of 500 mg/kg/day or 1 500 mg/kg/day with water. The general condition of rats was observed before and after administration. During the study period, solid feed CRF-1 (Oriental Yeast Co., Ltd.) was provided, and drinking water was freely available. Each animal was subjected to urinalysis, necropsy, organ weight measurement, hematology, and blood biochemical analysis after 28 days.

Ethics    The toxicity tests adhered to the Good Laboratory Practices (GLP, 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; however, the assays were performed under non-GLP conditions.

All 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 Practices 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 (revised by WMA Fortaleza General Assembly, Brazil, in October 2013) and Ethical Guidelines for Medical Research on Humans (partially revised December 22, 2014/February 28, 2017). The human rights of the subjects were respected and the trial protocol was confirmed with the approval of the Ethics Review Committee. The trial was carried out under the supervision of a doctor. This study was conducted following review and approval by the Medical Association Hakusuikai Suda Clinic Investigational Review Board. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry, study ID UMIN000036597.

Participants    Of 22 subjects initially screened, 12 healthy male and female subjects, aged 20–64 years and mildly obese (BMI 23.0 to 30.0 kg/m²), were enrolled in this study. Individuals were excluded if they were (1) undergoing treatment with any drug at the time of the study, including drugs used to treat obesity, hyperlipidemia, or lipid metabolism; (2) on a diet or exercise regimen under the supervision of a physician; (3) unable to stop taking health foods or supplements during the study; (4) unable to refrain from drinking alcohol on the day before the test; (5) illness or history of serious illness; (6) food allergy or drug dependence; (7) history of alcohol dependence; (8) metal in the body due to surgery; (9) presence of a cardiac pacemaker or another medical device implanted in the body; (10) claustrophobia; (11) shift worker or working night shift; (12) pregnancy, nursing, or wishing to become pregnant during the study period; (13) current participation in another clinical trial; (14) participation in another clinical trial within 1 month of the present study; and (15) judged by the investigator to be inappropriate for participation in the clinical trial on vitals (blood pressure/pulse) were excluded.

Study design and procedure    The study was a randomized, placebo-controlled, double-blind, parallel-design, twelve-week treatment study (Table 1). A flow chart detailing the selection and grouping of study subjects is presented in Fig. 1. The study was designed to assess the efficacy and safety of WLE powder intake for improved body composition. After medical screening, each eligible subject was randomly assigned to one of two groups: WLE powder group, who received two capsules containing 100 mg WLE powder/capsule embedded in dextrin daily, and the placebo group, who received two capsules containing 100 mg dextrin/capsule daily. Tablets were taken with water before going to bed. As a restriction, during the test period, the subjects were required to record specified items, sleep time, activities and physical condition changes, in an electronic diary every day, and to keep a food diary for 3 days before the start of WLE powder intake and for 3 days before visiting the clinic. The ingestion of medicines and all health foods and supplements that potentially affect body fat and lipid metabolism was prohibited (if normally taken, these were discontinued from the date of consent to participate). Subjects were instructed to not make any changes to their lifestyle habits including diet, exercise, smoking, and administration of medicines, before participating in the trial. The Examination Consultation Service was used to notify the subjects to avoid any lifestyle changes. Any event including excessive exercise that greatly deviated from their normal daily range, more or less food intake than usual, temporary illnesses, or the intake of any medication was recorded in the electronic diary.

Table 1. Human clinic trial schedule

* 1: Height is measured only for screening.

* 2: Nutrition survey (recorded 3 d before week 0 (ingestion starting date), 8 weeks after ingestion, 3 d before visiting doctor at 12 weeks of intake)

Fig. 1.

Flow chart for the selection and grouping of subjects in the human clinic trial

On the day before visiting the clinic, subjects were instructed not to consume anything except water, and the intake of food and drink must be completed by 21:00. Additionally, no excessive exercise or drastic change in caloric intake was allowed. On the day of the test, subjects could not smoke from the time they woke up until the end of the test. They also did not eat breakfast or consume WLE powder on that morning.

Evaluation/observation items    Doctor interviews, physical measurements, and blood examinations were performed three times, at the initial screening and at the following 8 and 12 weeks.

Abdominal VFA, abdominal SFA, and total abdominal fat area were calculated using PC software (FAT scan) with image data obtained from the CT scans (Brivo CT385; GE Healthcare). The V/S area ratio was determined from the VFA and SFA. Body weight, BMI, waist circumference, body fat percentage, total cholesterol (T-Cho), HDL cholesterol, and LDL cholesterol were also evaluated.

Safety assessment    Safety was evaluated by the incidence and severity of WLE powder ingestion-related adverse events in subjects during the research period. An adverse event was defined as any undesirable or unintended injury or symptom, including abnormalities in clinical laboratory test values, occurring in subjects after the medical screening.

The incidence of side effects, vital signs (blood pressure and pulse), and other symptoms were monitored. Blood tests were performed to measure white blood cell count, red blood cell count, platelets, hematocrit, hemoglobin, total protein, alkaline phosphatase, aspartate transaminase, alanine transaminase, γ-glutamyltransferase, lactate dehydrogenase, uric acid, urea nitrogen, total bilirubin, albumin, creatinine, creatine phosphokinase, glucose, and hemoglobin A1c.

Data analyses    In this study, the null hypothesis that each measured value would not differ before and after ingestion of WLE or between groups was tested. The significance level was 5% in a two-sided test, and the null hypothesis was rejected if a significant difference was found. A significance level of 5–10% was considered to indicate a tendency. The normality of evaluation items obtained was examined using the Shapiro-Wilk test for continuous data; a paired t-test was performed if normality was shown, and the Wilcoxon signed-rank test was performed if normality was not shown. For unpaired data, if normality was indicated, a test of variance (F-test) was performed, followed by the Student's t-test (for equal variance) or Aspin-Welch's t-test (for unequal variance). The Mann–Whitney U-test was performed for unpaired data when normality was not shown. For ordered data with many stages, corresponding data were subjected to the Wilcoxon signed-rank test, and uncorrelated data were subjected to the Mann-Whitney U-test. For ordinal data and nominal data with few steps, the corresponding data were subjected to McNemar's test, and the uncorrelated data were analyzed by Fisher's direct method or the χ2 test according to the number of data points. When multiple comparisons were required, Bonferroni's, Tukey's or Dunnett's method was used according to the format of the data. Variance and correlation coefficient analysis were also performed as necessary. For safety evaluation items, in principle, only intra-group comparison of values before and after intake was performed, and comparison between groups before and after intake was not performed. Statistical analysis was performed using IBM SPSS Statistics (ver. 25).

Results

Results of the Ames, acute and sub-acute tests    The Ames test was conducted to investigate the mutagenicity of WLE in the concentration range of 1.2–5 000 µg/plate. Growth inhibition was observed in all strains at 1 250–5 000 µg/plate. In the case of +S9, growth inhibition was only observed at 5 000 µg/plate for the TA98 strain. On the other hand, no mutagenicity was observed at any concentration of WLE (Table 2).

Table 2. Results of the Ames test (revertant colony number)

		Base pair substitution type						Frame shift type
		TA100		TA1535		WP2 uvrA		TA98		TA1537
		−S9	+S9	−S9	+S9	−S9	+S9	−S9	+S9	−S9	+S9
Negative control		152	151	12	15	24	30	17	32	8	17
Wasabi leaf extract	1.2 µg/plate	143	178	11	18	29	47	17	33	11	11
	4.9 µg/plate	173	172	16	6	30	35	23	37	7	7
	20 µg/plate	173	181	15	10	36	30	13	34	6	9
	78 µg/plate	171	174	15	13	33	35	21	27	5	9
	313 µg/plate	160	169	12	9	25	33	25	31	5	11
	1250 µg/plate	144	208	13 *	17	30	21	15 *	29	5 *	14
	5000 µg/plate	178 *	224	12 *	14	26 *	33	22 *	36 *	3 *	7
Positive control		628 +	1063 +	550 +	326 +	142 +	1363 +	464 +	331 +	2465 +	154 +
reagent		AF-2	2-AA	NaN3	2-AA	AF-2	2-AA	AF-2	2-AA	ICR-191	2-AA
concentrtion(µg/plate)		0.01	1.0	0.5	2.0	0.01	10	0.1	0.5	1.0	2.0

*  Significant growth inhibition was observed (p < 0.05)

+  More than 2 times mutations than control was observed.

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.

Positive control reagents. AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide, 2-AA; 2-Aminoanthracene, NaN3; Sodium azide, ICR-191; 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride.

The acute toxicity of WLE was tested in male and female Slc:ICR mice (SPF). No mortality was observed at 5 000 mg/kg. Compared to the control group, no significant increase or decrease in body weight after 14-day administration was observed (Fig. 2). At necropsy, liver discoloration was observed in two male rats administered 2 500 mg/kg WLE; however, it was clarified that the effect was not due to the test substance, as no dose-dependency was observed in the administration groups. From these data, the non-toxic equivalent of WLE was estimated as 5 000 mg/kg.

Fig. 2.

Effect of single dose administration of WLE on body weight of male and female rats

WLE was dissolved in 20 ml of water in the mentioned concentration ranges, and was orally forcibly administered using a syringe and a feeding needle. Control group was administered with water in the same manner. Body weight was measured using an electronic balance (PM2000, METTLER TOLEDO Co., Ltd.). Values are expressed as mean ± SD (n = 5).

The sub-acute toxicity of WLE was further evaluated by repeated oral administration to five-week-old Sprague-Dawley rats for 28 days. No mortality was observed in any case throughout the observation period. No significant changes were observed in the general observation states and body weight as compared with the control group, and no significant changes were observed in water intake, food consumption, urinalysis, and hematological indexes (Tables 3–7, Fig. 3).

Table 3. Effect of WLE on feed consumption (g) in rats

Sex	Group	Dose (mg/kg)		Day
				0	4	7	11	14	18	21	25	27
Male	Control	0	Mean	21.7	24.2	24.3	26.2	27.7	26.3	28.2	27.3	26.8
			SD	1.4	1.2	2.3	1.9	1.8	2.1	2.3	2.0	2.1
	WLE	500	Mean	22.2	23.5	25.0	26.5	27.5	28.8	29.2	30.7	28.3
			SD	2.3	1.0	3.2	2.9	2.3	2.6	3.5	3.8	2.0
		1500	Mean	22.5	23.5	24.0	25.8	27.0	28.0	27.5	29.7	27.8
			SD	1.2	2.2	1.7	1.9	2.0	2.9	2.5	4.6	4.6
Female	Control	0	Mean	17.7	17.8	17.7	18.0	19.0	19.0	18.0	20.0	21.0
			SD	1.2	1.0	1.5	2.2	2.1	1.4	2.5	3.7	1.5
	WLE	500	Mean	17.8	18.7	17.7	18.0	18.2	19.0	17.3	17.2	19.3
			SD	1.8	2.0	3.4	2.2	1.5	2.1	3.7	2.1	2.9
		1500	Mean	17.5	17.7	18.5	16.7	16.5	17.2	19.0	18.8	21.2
			SD	2.4	2.1	1.9	3.1	2.1	2.5	4.2	2.7	2.8

Each Value represented mean ± SD (g/day, n = 6). Solid feed CRF-1 (lot numbers 060411B1 and 060511B1, manufactured by Oriental Yeast Co., Ltd.) was freely fed throughout the test period.

Table 4. Effect of WLE on water consumption (mL) in rats

Sex	Group	Dose (mg/kg)		Day
				0	4	7	11	14	18	21	25	27
Male	Control	0	Mean	26.7	25.2	28.3	28.8	30.5	31.3	32.0	29.8	30.2
			SD	1.5	1.7	2.4	3.3	3.8	3.6	3.3	3.7	3.4
	WLE	500	Mean	25.8	25.2	27.5	27.7	31.0	29.7	28.5	28.2	26.0
			SD	1.9	1.6	3.7	3.3	3.3	5.0	5.6	3.6	2.8
		1500	Mean	27.5	28.5	30.2	32.8	32.7	32.5	32.0	32.3	32.7
			SD	2.0	2.8	2.7	4.6	4.4	4.8	4.9	5.2	6.5
Female	Control	0	Mean	22.5	20.3	20.8	20.0	22.2	19.5	20.5	20.3	21.8
			SD	1.4	2.2	3.4	3.1	5.6	3.6	4.0	3.1	2.5
	WLE	500	Mean	22.7	22.7	24.3	21.8	24.5	22.7	22.7	23.3	22.7
			SD	1.9	2.9	3.4	4.4	2.9	2.5	4.0	5.1	4.2
		1500	Mean	22.8	20.5	23.3	20.5	20.7	19.8	22.8	21.2	22.3
			SD	1.5	2.3	2.4	3.6	3.4	4.0	4.5	4.8	3.9

Each value represented mean ± SD (g/day, n = 6). Tap water was freely ingested by a water absorbing bottle.

Table 5. Urinalysis in rats treated orally with WLE for 28 days (3hr stock urine)

Sex			Control	WLE (mg/kg B.W.)
				500 mg/kg	1500 mg/kg
Male	pH	7.5	1	0	0
		8.0	5	3	5
		8.5	0	3	1
	Protein	−	6	6	5
		+	0	0	1
	Glucose	−	6	6	6
	Ketones	−	6	6	6
	Occult blood	−	6	6	6
	Urobilinogen	0.1	6	6	6
	Bilirubin	−	6	6	6
Female	pH	7.5	0	1	1
		8.0	4	3	3
		8.5	2	2	2
	Protein	−	6	6	6
	Glucose	−	6	6	6
	Ketones	−	6	6	6
	Occult blood	−	6	6	6
	Urobilinogen	0.1	6	6	6
	Bilirubin	−	6	6	6

All rats in each group were housed in cages for 4 weeks administration. After the end of administration on the day, the urine was collected and measured for up to 3 hours under fasting and water intake. Number of rats (N = 6), − Negative, +(Protein); 30mg/dL, Urobilinogen; Ehrlich unit/dL

Table 6. Urinalysis in rats treated orally with WLE for 28 days (21 hr stock urine)

Sex			Control	WLE (mg/kg B.W.)
				500 mg/kg	1500 mg/kg
Male	Volume	(mL/21hr)	10.8 ± 2.5	12 ± 2.8	13.9 ± 4.2
	Specific gravity		1.054 ± 0.016	1.055 ± 0.013	1.048 ± 0.014
	Na	(mEq/21hr)	1.565 ± 0.619	1.787 ± 0.230	1.587 ± 0.229
	K	(mEq/21hr)	2.739 ± 0.616	3.143 ± 0.205	3.298 ± 0.475
	Cl	(mEq/21hr)	1.860 ± 0.553	2.004 ± 0.173	1.877 ± 0.204
Female	Volume	(mL/21hr)	9.2 ± 4.9	7.2 ± 2.8	10.3 ± 4.4
	Specific gravity		1.043 ± 0.014	1.055 ± 0.015	1.034 ± 0.010
	Na	(mEq/21hr)	0.832 ± 0.572	0.851 ± 0.307	0.781 ± 0.444
	K	(mEq/21hr)	1.451 ± 0.796	1.510 ± 0.445	1.411 ± 0.824
	Cl	(mEq/21hr)	0.957 ± 0.614	0.927 ± 0.294	0.812 ± 0.469

All rats in each group were housed in cages for 4 weeks administration. After the end of administration on the day, the urine was collected and measured for up to 21 hours under fasting and water intake.

Table 7. Effect of WLE on hematological parameters in rats

Sex	Organ		Control		WLE (mg/kg B.W.)
				500 mg/kg	1500 mg/kg
Male	Erythrocytes	(×104/gL)	750 ± 37	749 ± 31	742 ± 41
	Reticulocytes	(‰)	26 ± 3	31 ± 3	29 ± 3
	Hemoglobin	(g/DL)	16.0 ± 0.8	16.0 ± 0.5	15.9 ± 0.7
	Hematocrit	(%)	44.6 ± 2.1	44.8 ± 1.6	44.2 ± 2.8
	MCV	(fL)	59.4 ± 0.5	59.8 ± 1.8	59.6 ± 2.1
	MCH	(pg)	21.3 ± 0.6	21.4 ± 0.8	21.4 ± 0.8
	MCHC	(%)	35.9 ± 1.0	35.8 ± 0.8	35.9 ± 0.7
	Leukocytes	(×102/±L)	97 ± 35	123 ± 29	106 ± 23
	Differential count of leukocytes (%)
	Eosinophils		0.5 ± 1.2	0.8 ± 0.8	0.3 ± 0.8
	Neutrophils	Stab	N.D.	N.D.	N.D.
		Segment	11.8 ± 9.0	10.0 ± 4.6	8.3 ± 2.7
	Basophils		N.D.	N.D.	N.D.
	Monocytes		N.D.	N.D.	0.2 ± 0.4
	Lymphocytes		87.7 ± 8.9	89.2 ± 5.0	91.2 ± 3.3
	Plateles	(×104/gL)	113.9 ± 13.0	111.9 ± 8.6	99.4 ± 11.5
Female	Erythrocytes	(×104/gL)	764 ± 43	751 ± 46	770 ± 43
	Reticulocytes	(‰)	23 ± 3	21 ± 3	22 ± 2
	Hemoglobin	(g/DL)	16.1 ± 0.7	16.3 ± 0.8	16.3 ± 0.4
	Hematocrit	(%)	43.8 ± 2.4	44.1 ± 2.3	44.9 ± 1.3
	MCV	(fL)	57.3 ± 0.9	58.8 ± 1.6	58.4 ± 1.8
	MCH	(pg)	21.1 ± 0.5	21.7 ± 0.7	21.2 ± 0.7
	MCHC	(%)	36.8 ± 0.7	36.9 ± 0.5	36.4 ± 0.4
	Leukocytes	(×102/gL)	73 ± 37	74 ± 10	81 ± 11
	Differential count of leukocytes (%)
	Eosinophils		0.8 ± 1.2	1.2 ± 0.8	0.5 ± 0.8
	Neutrophils	Stab	N.D.	N.D.	N.D.
		Segment	12.8 ± 5.3	12.3 ± 4.3	8.5 ± 2.7
	Basophils		N.D.	0.2 ± 0.4	N.D.
	Monocytes		N.D.	N.D.	0.2 ± 0.4
	Lymphocytes		86.3 ± 6.4	86.3 ± 4.4	90.8 ± 2.4
	Plateles	(×104/gL)	111.7 ± 16.4	116.4 ± 6.0	113.4 ± 6.0

Blood was collected before pathological autopsy after fasting for 18 hours under water intake from the evening of the last administration day. Each value represents mean ± SD (N = 6). N.D.; Not detected (at least less than 5.0 / µL cell count).

Fig. 3.

Effect of 28-day treatment with WLE on body weight of male and female rats

WLE was orally forcibly administered in the mentioned concentration ranges to five-week-old males and females Sprague-Dawley rats for 28 days using a syringe and a feeding needle. All males and females in each group were weighted using an electronic balance (EW-300G, A&D Company, Limited) twice a week during the administration period. Values are expressed as mean ± SD (n = 6).

In blood biochemical indexes, GOT values were 64 ± 4 IU/L in the control group, 76 ± 10 IU/L in the 500 mg/kg/day group, and 75 ± 6 IU/L in the 1 500 mg/kg/day group of male rats. A significant increase in GOT was observed in the 500 and 1500 mg/kg WLE groups, and a low A/G ratio was observed in the 500 mg/kg WLE group of female rats (Table 8).

Table 8. Effect of WLE on serum biochemical parameters in rats

Sex	Organ		Control	WLE (mg/kg B.W.)
				500 mg/kg	1500 mg/kg
Male	AST(GOT)	IU/L	64 ± 4	76 ± 10 **	75 ± 6 *
	ALT(GPT)	IU/L	24 ± 2	27 ± 4	25 ± 1
	ALP	IU/L	704 ± 184	662 ± 71	658 ± 149
	γ-gtp	IU/L	0.2 ± 0.4	0.0 ± 0.0	0.0 ± 0.0
	Total ptotein	g/dL	5.9 ± 0.1	5.9 ± 0.1	5.9 ± 0.2
	Albumin	g/dL	2.5 ± 0.1	2.6 ± 0.1	2.6 ± 0.1
	Globulin	g/dL	3.4 ± 0.2	3.3 ± 0.2	3.3 ± 0.3
	A/G ratio		0.75 ± 0.07	0.79 ± 0.06	0.77 ± 0.07
	Glucose	mg/dL	149 ± 22	155 ± 29	135 ± 6
	Triglyceride	mg/dL	59 ± 19	60 ± 33	40 ± 10
	Total cholesterol	mg/dL	49 ± 5	60 ± 8	49 ± 6
	BUN	mg/dL	11.9 ± 1.0	12.6 ± 2.1	11.3 ± 1.4
	Creatinine	mg/dL	0.21 ± 0.01	0.27 ± 0.06	0.24 ± 0.04
	Total bilirubin	mg/dL	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01
	Na	mEq/L	143.0 ± 1.0	142.9 ± 1.3	143.3 ± 0.9
	K	mEq/L	4.62 ± 0.24	4.54 ± 0.13	4.48 ± 0.28
	Cl	mEq/L	104.7 ± 1.6	103.9 ± 1.6	103.9 ± 1.8
	Inorganic P	mg/dL	8.6 ± 0.4	9.0 ± 0.5	8.9 ± 0.3
	Ca	mg/dL	10.6 ± 0.3	10.5 ± 0.2	10.5 ± 0.2
Female	AST(GOT)	IU/L	71 ± 11	82 ± 14	87 ± 10
	ALT(GPT)	IU/L	21 ± 3	22 ± 3	23 ± 2
	ALP	IU/L	411 ± 62	418 ± 84	408 ± 65
	γ-gtp	IU/L	1.2 ± 0.4	1.0 ± 0.6	1.0 ± 0.0
	Total ptotein	g/dL	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.2
	Albumin	g/dL	2.8 ± 0.1	2.6 ± 0.1	2.9 ± 0.2
	Globulin	g/dL	3.2 ± 0.2	3.4 ± 0.2	3.1 ± 0.2
	A/G ratio		0.89 ± 0.04	0.79 ± 0.06 *	0.92 ± 0.12
	Glucose	mg/dL	134 ± 22	117 ± 19	121 ± 12
	Triglyceride	mg/dL	25 ± 9	23 ± 11	26 ± 20
	Total cholesterol	mg/dL	64 ± 19	66 ± 7	66 ± 15
	BUN	mg/dL	15.4 ± 1.0	13.4 ± 1.1	14.9 ± 1.7
	Creatinine	mg/dL	0.33 ± 0.05	0.31 ± 0.05	0.27 ± 0.05
	Total bilirubin	mg/dL	0.05 ± 0.01	0.06 ± 0.01	0.06 ± 0.01
	Na	mEq/L	142.1 ± 1.4	142.4 ± 0.7	141.3 ± 1.6
	K	mEq/L	4.61 ± 0.08	4.37 ± 0.26	4.72 ± 0.07
	Cl	mEq/L	105.7 ± 1.9	106.3 ± 1.4	105.4 ± 2.5
	Inorganic P	mg/dL	7.8 ± 0.7	7.7 ± 0.5	7.5 ± 0.7
	Ca	mg/dL	10.3 ± 0.3	10.3 ± 0.2	10.3 ± 0.3

Blood was collected before pathological autopsy after fasting for 18 hours under water intake from the evening of the last administration day. Each value represents mean ± SD (N = 6)

Necropsy results showed an enlarged thymus gland and increased lung weight in females fed 500 mg/kg WLE compared to the control group (Table 9). On the other hand, there were no significant differences in other organs and in absolute weight. The changes in females fed 500 mg/kg WLE did not show dose-dependency and may have been a random change, as these changes were not observed in the 1500 mg/kg WLE group.

Table 9. Relative organ weights of rats treated with WLE for 28 days

Sex	Organ	Weight (/100g B.W.)	Control	WLE (mg/kg B.W.)
				500 mg/kg	1500 mg/kg
Male	Brain	g	0.55 ± 0.02	0.53 ± 0.05	0.54 ± 0.02
	Thymus	g	0.18 ± 0.03	0.19 ± 0.04	0.16 ± 0.02
	Heart	g	0.34 ± 0.01	0.36 ± 0.03	0.36 ± 0.02
	Lung	g	0.33 ± 0.02	0.37 ± 0.03	0.34 ± 0.01
	Liver	g	2.9 ± 0.1	3.1 ± 0.2	2.9 ± 0.1
	Spleen	g	0.20 ± 0.03	0.19 ± 0.04	0.21 ± 0.01
	Kidneys	g	0.77 ± 0.06	0.76 ± 0.07	0.78 ± 0.05
	Adrenals	mg	16 ± 3	15 ± 3	15 ± 2
	Testes	g	0.89 ± 0.07	0.86 ± 0.10	0.89 ± 0.12
Female	Brain	g	0.83 ± 0.09	0.86 ± 0.04	0.84 ± 0.08
	Thymus	g	0.21 ± 0.03	0.24 ± 0.05	0.20 ± 0.03
	Heart	g	0.38 ± 0.07	0.39 ± 0.03	0.37 ± 0.03
	Lung	g	0.43 ± 0.05	0.44 ± 0.04	0.44 ± 0.03
	Liver	g	2.9 ± 0.2	2.8 ± 0.1	2.8 ± 0.1
	Spleen	g	0.20 ± 0.03	0.21 ± 0.02	0.22 ± 0.03
	Kidneys	g	0.79 ± 0.09	0.78 ± 0.03	0.81 ± 0.05
	Adrenals	mg	30 ± 4	32 ± 4	26 ± 4
	Ovaries	mg	35 ± 8	40 ± 6	35 ± 4
	Uterus	g	0.21 ± 0.05	0.2 ± 0.05	0.23 ± 0.08

After the organs were removed, the weight of each organ was measured, and the relative weight (g/100g B.W.) was calculated from the body weight at the time of autopsy. Each value represents mean ± SD (N = 6).

Characteristics of clinical trial subjects    Of the 22 subjects who participated in the screening test, 12 met the selection criteria. The subjects' backgrounds are shown in Table 10. Visceral fat obesity with a VFA and SFA area ratio (V/S) was estimated. As a result, the V/S ratio was 0.57 in the placebo group (mean 0.57 ± 0.23) and 0.56 in the WLE group (mean 0.56 ± 0.27) (p = 0.91). The final basic data of the 12 subjects are presented in Table 11. Initial BMI was significantly higher in the WLE powder group; however, there were no significant differences in VFA, SFA, body weight, body fat percentage, or waist diameter between the groups. Although there was a significant difference in BMI, there was no significant difference in visceral fat, subcutaneous fat, etc. Therefore, the difference in BMI was proposed to be a result of differences in other aspects such as muscle mass, etc.

Table 10. Baseline characteristics of subjects at the beginning of the human clinic trial

	Placebo (n=6)	WLE powder (n=6)	P-value
Age (years)	50.3 ± 5.3	50.8 ± 7.2	0.89
Male/female (number)	2/4	3/3	1.00
BMI (kg/m2)	25.5 ± 1.0	27.7 ± 1.4	0.01
Blood pressure (mmHg):
Systolic	119.2 ± 5.9	125.2 ± 8.1	0.18
Diastolic	85.8 ± 6.8	82.5 ± 8.0	0.70
Pulse rate (beats/min)	70.0 ± 4.9	74.7 ± 12.3	0.82
Body fat percentage(%)	33.3 ± 5.5	32.2 ± 6.8	0.76
Waist diameter(cm)	94.7 ± 3.9	98.2 ± 3.2	0.11
Abdominal visceral fat area(VFA)	137.5 ± 49.3	132.0 ± 47.8	0.85
Abdo minal subcutaneous fat area(SFA)	245.5 ± 33.9	248.0 ± 47.3	0.92
VFA/SFA raio(V/S)	0.57 ± 0.23	0.56 ± 0.27	0.91

Values are expressed as the mean ± standard deviation (other than the number of people). Abbreviations: body mass index (BMI).

Table 11. Baseline characteristics of subjects at the end of the trial

	Placebo (n=5)	WLE powder (n=5)	P-value
Age (years)	51.6 ± 4.8	50.6 ± 8.0	0.82
Male/female (number)	2/3	3/2	1.00
BMI (kg/m2)	25.5 ± 1.1	27.7 ± 1.6	0.06
Blood pressure (mmHg) :
Systolic	120.2 ± 5.9	126.6 ± 8.1	0.19
Diastolic	86.6 ± 7.3	84.0 ± 7.9	0.60
Pulse rate (beats/min)	68.4 ± 3.2	71.8 ± 11.2	0.53
Body fat percentage(%)	32.3 ± 5.6	31.1 ± 7.0	1.00
Waist diameter(cm)	94.9 ± 3.7	98.2 ± 3.8	0.15
Abdominal visceral fat area(VFA)	145.5 ± 50.5	141.9 ± 46.1	1.00
Abdominal subcutaneous fat area(SFA)	246.7 ± 37.7	253.3 ± 50.8	0.69
VFA/SFA raio(V/S)	0.61 ± 0.24	0.59 ± 0.28	1.00

Values are expressed as the mean ± standard deviation (other tha n the number of people). Abbreviations: body mass index (BMI).

Evaluation of safety    Results of adverse events are summarized in Table 12. A total of 16 events occurred in five of the twelve subjects. All were mild, except for bruises and fractures, and were randomly distributed in both the placebo and WLE groups. No significant change was observed in blood biochemical indexes. The medical investigator concluded that WLE as the test food showed no clinical issues according to the judgment standards.

Table 12. Adverse events reported by one or more subjects in the trial

	Placebo (n=6)	WLE powder (n=6)
Total subjects reporting one or more adverse event(s)	3	2
Types of adverse events reported:
Gastrointestinal complaints	5
Headache	2	4
Toothache/Gingivitis		1
Rhinitis	2
Bruise		1
Fracture	1
Total of adverse events reported	10	6

Although there are physical condition changes (cold, runny nose/nasal congestion, headache, abdominal pain, constipation, abdominal bloating, toothache/gingivitis, menstrual cramps, etc.), unless otherwise specified, it will not be treated as an adverse event.

Abdominal bloating, abdominal pain, constipation: Those who had symptoms for 3 consecutive days or more were treated as adverse events. Patients who had symptoms for 1–2 days without taking the test foods were not treated as adverse events. However, when the test food was taken, it was treated as an adverse event even if the symptom was only for one day.

Evaluation of body composition    As shown in Tables 13 and 14, no significant differences were observed in the initial endpoint of VFA and in the secondary endpoints of SFA, V/S area ratio, body weight, BMI, TG, T-Cho, HDL-C, LDL-C, waist circumference, or body fat percentage. However, in the placebo group, the mean visceral fat at the start was 145.5 cm², and increased to 165.0 cm² (by 19.6 cm², 13.4%) at the end of the study. On the other hand, in the WLE group, the mean visceral fat at the start was 141.9 cm², and increased to 145.3 cm² (by 3.4 cm², 2.4%) at the end of the test. No statistically significant difference was observed, probably due to the small numbers, i.e., only five subjects per group.

Table 13. Measurement results of body fat-related indexes

Values are expressed as the mean ± standard deviation (SD). p < 0.01 **, p < 0.05 *

Comparison Innergroup: Wilcoxon signed-rank test (Bonferroni correction). Comparison Intergroups: Mann-Whitney's U test.

Although twelve subjects completed the study, the efficacy was evaluated in ten subjects because two subjects who had the abnormal values in the blood test during the study period and a fracture during the study period were excluded.

Table 14. Results of blood lipid measurement

Values are expressed as the mean ± standard deviatio. (SD). p < 0.01 **, p < 0.05 *

Comparison Innergroup: Wilcoxon signed-rank test (Bonferroni correction). Comparison Intergroups: Mann-Whitney's U test.

Moreover, in the placebo group, VFA was increased in all subjects, whereas in the WLE group, VFA was decreased in three of five subjects (Fig. 4) and increased in one subject (by 43.6 cm²). Although the cause of this large increase is unknown, it may be due to external factors such as stress. Blood lipid-related indexes in the WLE group tended to decrease compared with the placebo group. The values of triglycerides, total cholesterol, LDL cholesterol also decreased significantly in the WLE group compared with the placebo group.

Fig. 4.

Changes in visceral fat area by individual

Equipment: Brivo CT385 (GE Healthcare)

The abdominal visceral fat area (VFA), abdominal subcutaneous fat area (SFA), and abdominal total fat area (TFA) are calculated from the image data obtained by the CT scan using PC software (FAT scan). The V/S area ratio was calculated from the VFA and SFA.

The results of blood hematological and biochemical analyses showed no significant differences between the two groups (Tables 15 and 16).

Table 15. Results of hematology examinations

The hematology tests were evaluated by all subjects to judge the safety.

Values are expressed as the mean ± standard deviation (SD). p < 0.01 **, p < 0.05 *

Comparison Intergroup: Wilcoxon signed-rank test (Bonferroni correction). Comparison Intergroups: Mann-Whitney's U test.

Table 16. Results of blood biochemical indexes examinations

Values are expressed as the mean ± standard deviation (SD). p < 0.01 **, p < 0.05 *

Abbreviations: total bilirubin (TP), aspartate aminotransferase (AST [GOT]), alanine aminotransferase (ALT [GPT]), lactate dehydrogenase (LDH), and hemoglobin A1c (HbA1c).

Although no statistically significant differences were found in this study, possibly due to the small number of subjects, WLE may have an effect in the reduction of blood cholesterol and VFA.

Discussion

In this study, the mutagenicity, acute and sub-acute toxicity and human trial safety of WLE were investigated. In the acute and sub-acute toxicity tests of WLE, a significant increase in blood GOT value was observed in the 500 (76 ± 10 IU/L) and 1500 mg/kg (75 ± 6 IU/L) WLE groups of male rats, and a low A/G ratio was observed in the 500 mg/kg WLE group of female rats. Since the population mean ± acceptable range of blood GOT value of Sprague-Dawley rats is reported as 86 ± 18 IU/L (n = 135), the change in GOT values in this study could be considered to be toxicologically insignificant. Regarding the change in A/G value, no significant change was observed in albumin and globulin values. Moreover, this change did not show dose-dependency, since it was not observed in the high-dose group (1500 mg/kg of WLE), indicating that this may be a random observation. Based on these data, the non-toxic equivalent for administration of WLE for 28 days is estimated to be 1500 mg/kg/day for both males and females.

In the human clinical trial, dietary intake of 200 mg WLE was determined to be a safe concentration. Although a total of 16 adverse events occurred in five of the twelve subjects, all adverse events were randomly distributed in both the placebo and WLE groups. No significant change was observed in blood biochemical indexes, and the medical investigator concluded that WLE (as the test food) showed no clinical issues within the designed concentration and intake period according to the judgment standards.

Interestedly, the WLE group showed lower VFA (p = 0.15) at the end of the 12 week treatment period. The V/S ratio was increased 0.13 in the placebo group (mean 0.13 ± 0.13) and increased 0.04 in the WLE group (mean 0.04 ± 0.12) (p = 0.15) (Table 13). Although the number of subjects in the present clinical trial was small and no statistically significant differences were observed, we obtained interesting information regarding the effects of WLE on regulation of lipid metabolism. In the placebo group, the mean visceral fat was increased from 145.5 to 165.0 cm² (13.4%) at the end of the trial. Meanwhile, in the WLE group, the mean visceral fat was increased from 141.9 to 145.3 cm² (2.4%). These data suggest that WLE powder may have effects in the reduction of visceral fat. Our data are consistent with the results from other animal experiments. For example, WLE could attenuate liver lipid accumulation and white adipose tissue weight induced by a high-fat diet in C57J/BL mice (Yamasaki et al., 2013). Additionally, WLE was reported to reduce fat hypertrophy of adipose tissue by suppressing PPARγ expression in rats (Oowatari et al., 2016).

Regarding the bioactive compounds in WLE responsible for the visceral fat reduction, we analyzed the bioactive components in WLE, and detected at least three major flavonoids, isovitexin, isosaponarin, and isoorientin, at concentrations of 0.272, 0.012, 0.030 mg/g, respectively. The total polyphenol content in WLE was estimated as 2.28 mg/g. Isosaponarin in wasabi leaf has been reported to have antioxidant (Hosoya et al., 2008; Sekiguchi at al., 2010) and anti-obesity (Yamasaki et al., 2013; Oowatari et al., 2016; Misawa et al., 2018) activities. Specifically, animal experiments demonstrated that the body weight of rats fed a high-fat diet supplemented with WLE was significantly reduced compared to the control (Yamada-Kato et al., 2016). The increase in β3 adrenergic receptors in adipocytes and enhancement in the activity of UCP-1 by WLE could stimulate fat beta-oxidation (Yamada-Kato et al., 2016). Moreover, some acid esters and polyphenols derived from wasabi leaf have also been reported to regulate lipid metabolism. For example, 5-hydroxyferulic acid ester could modulate PPARγ and AMPK to suppress 3T3-L1 cell differentiation to adipocytes (Misawa et al., 2018). Caffeic acid 2-phenylethyl ester and luteolin inhibit fat accumulation in 3T3-L1 cells (Juman et al., 2010; Nishina et al., 2015). Thus, isosaponarin, other flavonoids as well as acid esters present in WLE may contribute to the regulation of lipid metabolism. Confirmation of this will be required in future studies using a larger population, following detailed identification of the active ingredient(s).

Acknowledgements    We are grateful to TTC Co., Ltd. for carrying out the Ames, acute and sub-acute toxicity tests, and ImeQ RD Inc. and Higashikoganei Sakura Clinic for coordinating the clinical trial.

Conflicts of Interest    Isao Okunishi and Yousuke Miura are employees of Kinjirushi Co., Ltd. and received a research grant from their company. Yo Nakamura, Yoshitada Hira and De-Xing Hou declare that they have no conflicts of interest.

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