The plant Angelica keiskei contains two main physiologically active flavonoid chalcones, 4-hydroxyderricin and xanthoangelol. Known as ashitaba in Japan, powder from the sap is widely consumed for its medicinal properties in Asia as a dietary supplement. Limited previously reported mammalian studies were without evidence of toxicity. GLP studies reported here, including a bacterial reverse mutation assay, a chromosome aberration assay, and an in vivo micronucleus assay are negative for genotoxicity. A GLPcompliant 90-day repeated oral gavage study of ashitaba yellow sap powder containing 8.45% chalcones in Sprague Dawley rats resulted in expected known physiological effects on coagulation parameters and plasma lipids at 300 and 1000 mg/kg/day. Ashitaba-related pathology included a dose-related male ratspecific alpha 2-urinary globulin nephropathy at 100, 300, and 1000 mg/kg/day and jejunal lymphangiectasia in both sexes at 1000 mg/kg/day. All other study parameters and histopathological changes were incidental or not of toxicological concern. Based on these studies ashitaba chalcone powder is not genotoxic with a NOAEL of 300 mg/kg in male and female rats.
Chalcones, Flavonoids, Lymphangiectasia, Alpha 2-urinary globulin nephropathy, 4-Hydroxyderricin, Xanthoangelol
The plant Angelica keiskei, native to the Japanese Izu Islands and the Izu, Bouso, and Miura peninsulas, is known in Japan as ashitaba, and is widely cultivated in Korea where it is known as sinsuncho or tomorrow leaf. The powder derived from the sap contains several flavonoid chalcones. The most abundant and physiologically active ashitaba chalcones are 4-hydroxyderricin and xanthoangelol. These chalcones have myriad health benefits from inducing apoptosis in cancer cells, to anti-oxidant, anti-inflammatory, anti-angiogenic, and anti-diabetic properties (Dimmock et al., 1999; Dorn et al., 2010). They have also been demonstrated to inhibit platelet aggregation (Lo et al., 2009), have vasorelaxant properties (Ko et al., 2004; Lin et al., 2001; Lo et al., 2009), and suppress differentiation of preadipocytes to adipocytes (Zhang et al., 2013). Ashitaba green tea is widely consumed in China, Japan, and India as a health-promoting drink; is a perennial herb in Korea as a vegetable juice ingredient; and ashitaba leaves have been consumed as food and medicine for many years on the Izu Islands in Japan (Chang et al., 2014; Enoki et al., 2007; Raj et al., 2013).
The safety of ashitaba chalcone in a 28-day dietary administration of 17, 170, and 1700 mg/kg body weight of ashitaba powder to male Wistar rats was without evidence of toxicity (Nagata et al., 2007). Absorption and metabolism of 4-hydroxyderricin and xanthoangelol following gavage administration of 50, 100, 200,and 500 mg/kg body weight of ashitaba extract in male ICR mice (Nakamura et al., 2012) was without adverse effect. A feeding study of xanthohumol, a related prenylated chalcone present in hops, was conducted in female BALB/c mice for three weeks, achieving a daily dose of approximately 1000 mg/kg body weight without toxicity (Dorn et al., 2010). To further document the safety profile of ashitaba, data from a battery of toxicity tests are presented.
2. Materials and methods
2.1. Studies reviewed
A battery of genotoxicity assays, an acute oral toxicity study in rats, and a 13- week rat subchronic oral toxicity study were contracted by Japan Bio Science Laboratory (JBSL), Osaka City, Japan. The test substance for all studies was provided by JBSL and prepared under Good Manufacturing Practices from plants organically cultivated in Indonesia. Ashitaba powder is obtained from the sap of cut stems. The sap is pasteurized, mixed with cyclodextrin, sterilized, freeze-dried, shattered and passed through 100 mesh to obtain 8% ashitaba powder. Analytical data from three non-consecutive batches are presented in Table 1. Data from the following studies are presented in this report by the author at the request of JBSL:
1. Bacterial reverse mutation test conducted at TNO Nutrition and Food Research Laboratory, The Netherlands (TNO Study Number 4405/16; 30 July 2002)
2. Chromosome aberration test conducted at TNO Nutrition and Food Research Laboratory, The Netherlands (TNO Study Number 5002/02; 3 June 2003)
3. In vivo mouse micronucleus test conducted at Nucro-Technics, Ontario, Canada (Study Number 282452; 13 June 2014)
4. Acute oral toxicity in rats conducted at TNO Nutrition and Food Research Laboratory, The Netherlands (TNO Study Number 4410/06; 31 May 2006)
5. 13-Week rat oral toxicity study conducted at Bozo Research Center, Japan (Study No. B-5530; 31 May 2006) with a subsequent independent pathology peer review (Experimental Pathology Laboratories, Inc., Research Triangle Park, NC; EPL Project No. 946-001; 27 March 2012).
2.2. Animal husbandry and care
The animal care and use committee at each performing laboratory approved study protocols. Animals were housed and maintained according to the AAALAC International Guide for the Care and Use of Laboratory Animals and CCAC Guidelines for Care and Use of Experimental Animals.
2.3. Study details
2.3.1. Bacterial reverse mutation assay
The bacterial reverse mutation assay was carried out in compliance with Good Laboratory Practices and OECD Guideline 471 utilizing Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 with tryptophan-requiring Escherichia coli strain WP2 uvrA. All assays were conducted with and without metabolic activation (S9 mix from liver homogenate from rats treated with Aroclor 1254). Ashitaba chalcone powder was cytotoxic in Salmonella strain TA 100 and negative for other strains tested both with and without metabolic activation. The positive controls (see Table 2) gave the expected response in revertant colonies with and without S9-mix. The highest concentration of test agent that could be dissolved in DMSO was 125 mg/mL. Given the 8% purity of chalcone, the highest concentration of chalcone was 10 mg/mL resulting in a highest concentration of 1000 μg/plate. The assay used 5 different concentrations of test substance from 12.3 to 1000 μg/plate. Assay results were considered positive if the mean number of revertant colonies had a concentrationdependent increase or if there was a reproducible two-fold increase over negative control.
2.3.2. Chromosome aberration
Two chromosome aberration tests were conducted in compliance with Good Laboratory Practice and OECD Guideline 473. Tissue culture media was purchased from Life Technologies (Gibco, The Netherlands) and Chinese hamster ovary cells (CHO K-1 line) were obtained from Prof. Dr. A.T. Natarajan, University of Leiden, The Netherlands. Other reagents included S9 mix (as above), colcemid (Fluka AG, Switzerland), DMSO (Sigma Chemical Company, USA), and methanol, acetic acid, and Giemsa stain (Merck-Darmstadt, FRG). The purity of the ashitaba powder was set at 100% (actual concentration of chalcone was 8%) and final concentrations of test substance in culture media ranged from 10 to 2500 μg/mL. Positive controls consisted of mitomycin C in the absence of S9 and cyclophosphamide in the presence of S9 mix. All cells at the highest concentrations (2500, 1250, 625 and 313 μg/mL) died before the end of a 4-hour treatment and these concentrations were considered too toxic for further study. For non-cytotoxic dose concentrations, a two-sided Fisher’s exact probability test was used to compare treated versus control. Study details are as follows:
Test 1. In the absence of S9 metabolic activation, treatment time was 4 hours (pulse treatment) and 18 hours (continuous treatment) while in the presence of S9 metabolic activation treatment time was 4 hours (pulse treatment). Harvesting of cells was 18 hours after onset of treatment.
Test 2. In the absence of S9 continuous treatment was for 18 hours with harvesting at 18 hours and at 32 hours. In the presence of S9 there was a 4-hour pulse exposure followed by harvesting at 18 and 32 hours. A repeat assay was done when the expected cyclophosphamide positive control did not yield expected results at a 32-hour sampling period.
2.3.3. In vivo MN assay
An in vivo micronucleus assay using ashitaba chalcone powder following OECD guideline 474 and in compliance with Good Laboratory Practices was conducted in Swiss mice (CD-1, Charles River, Canada). The range-finding study used groups of 3 male and female mice at doses of 500, 1000, and 2000 mg ashitaba chalcone power/ kg body weight. The ashitaba yellow powder consisted of 30% sap and 70% branched cyclodextrin and contained analytically confirmed 7.2% chalcone. Based on no sex difference in response in the range-finding study, the main study was done in groups of 14 male mice at the limit dose of ashitaba (2000 mg/kg), with a methylcellulose vehicle control, and 70 mg cyclophosphamide/kg body weight as a positive control. Seven mice in each group were terminated 24 and 36 hours post-dosing, harvested bone marrow was stained with Giemsa, and 2000 polychromatic erythrocytes were blindly scored by high magnification microscopy for the presence of micronuclei. The ratio of normochromatic to polychromatic erythrocytes was determined based on 500 cells and the number of micronuclei in normochromatic erythrocytes was also determined.
Any intergroup difference in mean number of polychromatic erythrocytes with micronuclei was analyzed using ANOVA on ranks (Kruskal–Wallis One Way Analysis of Variance on Ranks) at p < 0.05. To isolate the group(s) that differed from others, All Pair-wise Multiple Comparison Procedures (Dunn’s Method and Duncan’s Method) were used.
2.3.4. Acute toxicity
An acute gavage toxicity study of ashitaba chalcone powder (8% as chalcone) following OECD Guideline 423 and in compliance with Good Laboratory Practice was conducted in three male and three female Wistar outbred rats (Charles River, Germany) at a dose level of 2000 mg/kg following an overnight fast. Clinical signs and body weight were monitored during a 14-day observation period and rats were examined for macroscopic changes at study termination.
2.3.5. 90-day repeated dose oral toxicity study
A 90-day repeated dose oral gavage study of ashitaba chalcone was conducted in Sprague Dawley rats (Atsugi Breeding Center, Charles River, Japan) at 0, 100, 300 and 1000 mg/kg/day using 12 rats per dose per sex. Dose levels were selected based on result from a preliminary 14-day oral toxicity study. The ashitaba yellow sap powder (30.8% solids and 69.2% branched cyclodextrin) contained 8.45% chalcone and was suspended in olive oil for daily (7 days per week) gavage delivery using a dose volume of 5 mL/kg body weight. The study was done in compliance with Good Laboratory Practice and followed repeated dose toxicity study guidelines (No. 655, Ministry of Health and Welfare, Japan, April 5, 1999). Study parameters included daily clinical observations, frequent body weight measurements, food and water consumption, ophthalmological examinations, urinalysis, hematology, and clinical chemistry analyte measurements. At necropsy abnormal macroscopic findings were recorded and organ weights were obtained for brain, pituitary, thyroids (including parathyroids), adrenals, thymus, spleen, heart, lung, submandibular and sublingual salivary glands, liver, kidneys, testes, prostate, seminal vesicles, ovaries, and uterus. Histopathology slides for gross abnormalities and normal appearing tissues were stained with hematoxylin and eosin. Histopathology slides were peer reviewed by an experienced independent toxicologic pathologist (Seely, 2012).
Microscopic examination was done on gross lesions and all tissues from the high doses and controls. Potential test-article-related tissues examined microscopically from the middle- and low-dose groups of both sexes included adrenal, stomach, jejunum, and liver plus heart, cecum, and kidneys in males. Tissues examined microscopically from all high-dose and control rats included cerebrum, cerebellum, spinal cord (thoracic region), sciatic nerve, eyeballs, optic nerves, Harderian glands, pituitary, thyroids, parathyroids, adrenals, thymus, spleen, mandibular lymph node, mesenteric lymph node, heart, thoracic aorta, trachea, lung (with bronchus), tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, submandibular and sublingual glands, liver, pancreas, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovary, uterus, vagina, oviduct, mammary gland, sternum (with bone marrow), femur (with bone marrow), femoral skeletal muscle, skin (inguinal region), nasal cavity, Zymbal’s glands and gross pathological abnormalities.
For statistical analyses, mean and standard deviation of body weight, food consumption, water consumption, quantitative measurements in urinalysis, hematology and clinical chemistry analytes and organ weights were calculated and analyzed for homogeneity of variance by Bartlett’s test (level of significance = 1%, two-tailed). For homogeneous data, group mean differences were analyzed by Dunnett’s test (level of significance: 5% and 1%, two-tailed). For heterogeneous data, the mean rank differences between groups were analyzed by a Dunnett-type mean rank test (levels of significance: 5% and 1%, two-tailed).
3.1. Bacterial reverse mutation assay
Ashitaba chalcone powder was toxic to Salmonella TA100 both with and without metabolic activation as indicated by a decrease of revertant colonies versus vehicle control. All other bacterial strains were negative under conditions of the assay with vehicle and positive control values within acceptable ranges. Data are presented in Table 2.
3.2. Chromosome aberration
Test 1. Non-cytotoxic doses at and below 156 μg/mL were tested for chromosome aberrations. The 156 μg/mL dose reduced the mitotic index to 66% while lower doses were without effect on mitoses compared to the vehicle control. Pulse treatment with or without metabolic activation and 18 hours of continuous treatment without metabolic activation did not cause a statistically significant increase in aberrant cells at any of the concentrations tested (Table 3).
Test 2. Continuous treatment of cells for 18 and 32 hours without metabolic activation did not induce a statistically significant increase in aberrant cells at non-cytotoxic concentrations of ashitaba chalcone powder (Table 3). Short term treatment (4-hour pulsedose) with metabolic activation (S9 mix) did not induce statistically significant increases in aberrant cells at harvest times of 18 or 32 hours with one exception. A significant increase in aberrant cells was present at 32 hours for the 300 μg/mL dose. However, this dose exhibited transient cytotoxicity at 4 hours and was considered non-relevant by the laboratory conducting the assay.
3.3. In vivo MN
There was no effect on body weight or frequency of micronuclei in either polychromatic or normochromatic erythrocytes at 24 or 36 hours post-dosing at 2000 mg/kg (Tables 4a and 4b). While the ratio of polychromatic to normochromatic erythrocytes was normal for the ashitaba group, the decreased ratio at 36 hours versus 24 hours for the positive control is indicative of bone marrow suppression.
3.4. Acute toxicity
During a 14-day observation period following oral gavage of 2000 mg/kg ashitaba chalcone powder in aqueous solution, no abnormal clinical signs were observed. By the end of the 14-day observation period, all rats gained weight and were without treatment-related macroscopic alterations at study termination and necropsy. Based on these findings, ashitaba chalcone powder was considered not harmful if swallowed.
3.5. 90-day repeated dose toxicity study
3.5.1. Clinical observations
The study was well conducted with two early deaths consisting of one male control dead at week 12 with disseminated erythroblastic leukemia and one 300 mg/kg male dead at week 10 due to an intubation error. All remaining animals were without abnormal clinical signs throughout the study. Food consumption and body weight gain were comparable among all groups without any statistically significant differences. No ophthalmological abnormalities were present in any of the rats and urinalysis, including microscopic examination of sediment, revealed no statistically significant treatment-related effects.
3.5.2. Macroscopic findings and organ weights
Aside from the splenic enlargement and organ discoloration in a male control decedent and thoracic ascites in a 300 mg/kg group male decedent, the only macroscopic changes noted at terminal sacrifice were minor: red foci in lung, stomach, and liver involving single rats. The only organ weight change of possible relevance was increased relative kidney weight (combined kidneys) in high-dose (1000 mg/kg/day) males. This weight change was minor but may be a reflection of male rat-specific alpha 2-urinary globulin nephropathy seen in this group of rats.
Most hemogram measurements were without treatmentassociated statistical effects and were within normal ranges for rats in typical 13-week studies. A statistically significant reduced hemoglobin value was flagged in high-dose males (Table 5a). Decreased platelet counts were present in high-dose male and female rats along with increased prothrombin time (PT) in male and female rats in the 300 mg/kg and 1000 mg/kg groups and increased activated partial thromboplastin time (APTT) in high-dose (1000 mg/kg/day) males (Tables 5a and 5b).
3.5.4. Clinical chemistry
A broad spectrum of clinical chemistry analytes were measured with statistically significant increases in serum alkaline phosphatase, total cholesterol, and phospholipid in high-dose (1000 mg/kg/day) male and female rats and elevated serum triglyceride in high-dose males (Tables 6a and 6b). Very minor changes in blood urea nitrogen, serum sodium, and serum potassium in highdose males were statistically significant. Elevated serum total cholesterol, triglyceride and phospholipid were present as an expected finding.
The histopathology findings from the 90-day study were peer reviewed by an independent pathologist (Seely, 2012) and subsequently also peer reviewed by R. Maronpot. Principal lesion incidences for potential treatment-related findings are presented in Table 7. Potential treatment-related effects that were con- firmed during peer review included minimal adrenal zona fasciculata cytoplasmic vacuolation in 2 males and 3 females in the 1000 mg/ kg groups, jejunal lacteal dilation (Fig. 1) in the 1000 mg/kg groups for both sexes and intracytoplasmic eosinophilic droplets and associated changes in renal proximal tubules consistent with a dose–response for alpha 2-urinary globulin nephropathy in treated male rats. Minimal thickening of the forestomach limiting ridge and minimal cellular infiltrates in the cecal lamina propria were diagnosed by the study pathologist (Table 7) but were considered within normal variation and not a test-article effect during the independent histopathology peer review (Seely, 2012).
Two types of cytoplasmic vacuolation were reported in the liver with higher incidences in males than in females. Fine periportal hepatocyte cytoplasmic vacuoles (Fig. 2) were present in control and treated rats with a reduced incidence in Ashitaba-chalconeexposed males. Larger vacuoles consistent with intracytoplasmic fat in hepatocytes (Figs. 3 & 4) were present in treated males and females. The lobular localization of cytoplasmic vacuoles in treated rats was described as diffuse by the study pathologist and centrilobular by the peer review pathologist (Seely, 2012), who considered the hepatocyte vacuolation toxicologically insignificant. In my review of all the liver slides from the Bozo Research Center study, I noted that the vacuolation was minimal and variable in lobular distribution and chose to tabulate the lobular localization as diffuse. The hepatocellular vacuolation was minimal to mild and uniform across dose groups without a dose-response in severity. This hepatocyte cytoplasmic vacuolation affected a minority of the hepatic lobules and there was no associated hepatocellular cytotoxicity accompanying the vacuolation.
Occasionally other lesions common in rats of this age were present without any relationship to treatment (data not shown).