Arctium lappa was purchased in October 2016 from a traditional market in Naju-si Jeonnam province, Korea. A voucher specimen (DSUOB-AL-01) was deposited at the College of Oriental Medicine, Dongshin University. The root was separated for the study and air-dried. The powdered A. lappa roots (250 g) were extracted twice with hot water (1 L) at room temperature for 3 days. After filtration, the water was evaporated, freeze dried, and stored at −50℃. The crude extract was resuspended in distilled water and filtered through a 0.4-µm membrane. The yield of A. lappa roots extract with hot water was 5%.

In order to evaluate the safety of A. lappa, 32 C57BL/6 mice were purchased from SamTako BioKorea (Osan, Korea) and acclimatized to experimental conditions for 7 days. The mice were divided into four treatment groups and treated for 8-weeks with ad libitum access to the relevant diet and water: (1) control; (2) 60% fat diet; (3) 60%-fat diet and 50 mg/kg/day A. lappa treatment; and (4) 60%-fat diet and 250 mg/kg/day A lappa treatment. The 60% fat-diet was provided by Research Diets, Inc. (Product #D12492, New Brunswick, NJ, USA; Table 2). All experiments were approved by the Institutional Animal Care and Use Committee at Chonnam National University (Approval No. CNU IACUC-YB-R-2016-50).

All animals were observed twice per day for physiological changes, such as movement, fur color/grooming, appetite, aggression, and body weight and dietary consumption was measured twice per week. At 3 h before sacrifice, feeding was restricted and the rats were anesthetized with intraperitoneal injections of 50 mg/kg Zoletil (Virbac, Fort Worth, TX, USA). After sacrifice, the appearance of all organs was observed with the naked eye to identify any toxicological changes. Whole blood was collected from the heart and analyzed with a Hemavet Multispecies Hematology System (Drew Scientific Inc., Waterbury, CT, USA) (n=8 per group). Five organs (heart, lung, kidney, brain, and spleen) were collected for the evaluation of the morphological changes that result from the treatment with A. lappa. All organs were fixed in 10% (v/v) formaldehyde solution, dehydrated in a graded ethanol series (99.9, 90, 80, and 70%), and embedded in paraffin. The paraffin-embedded lung tissue was then sectioned (4 µm) longitudinally and stained with hematoxylin and eosin.

The data are shown as the mean±standard deviation (SD). Group differences were evaluated by one-way analysis of variance followed by Dunnett's multiple comparison test. Statistical significance was set at P<0.05 or P<0.01.

After acclimatization for 7 days, all mice were weighed and the mean body weight in each group was adjusted from 22.1±1.66 g in order to standardize the differences in body weight among each group (Figure 1). In the control group, the mice were fed a normal diet and the body weight increase was approximately 6.7 g; in comparison, the body weight of animals fed a 60%-fat diet had increased by approximately 15.8 g after 8-weeks. Although the body weight gain was the lowest in the control group, among the 60%-fat diet-fed groups, the mean body weight in the 250 mg/kg/day A. lappa-treated group was 33.7±2.96 g.

The comparison of the white blood cell (WBC)/differential cell counts in the control group in indicated a significant increase in the high-fat diet groups (Table 1). The number of WBC in the control group was 16.6±8.32×10² cells/mL and in the 60%-fat diet group it was 52.4±18.36×10² cells/mL. The number of WBC in the control group three times higher than that of the 60%-fat diet group. However, there was no difference between the animals fed the 60-fat diet and those fed the 60%-fat diet and treated with A. lappa. This result indicated that A. lappa treatment under 250 mg/kg/day for 8-weeks did not affect the changes in WBC. The results of the differential cell counts were similar to those of WBC. Although each cell involving WBC was increased by the 60% fat diet there, was no change between the 60%-fat diet and those fed the 60% fat-diet and treated with A. lappa. The results indicated that A. lappa L. treatment did not influence the changes of each cell in WBC.

However, the increased blood glucose level that was observed in the 60%-fat diet group was dose-dependently suppressed by A. lappa treatment. The blood glucose level in the 60%-fat diet group was approximately two times higher (474±41 mg/dL) compared with the control group (241±32 mg/dL), but the levels were 341±41 mg/dL in with 60%-fat diet and 50 mg/kg A. lappa treatment and 230±41 mg/dL with 60% -fat diet and 250 mg/dL A. lappa treatment. Thus, A. lappa treatment significantly suppressed the increase in blood glucose.

Many blood glycemic controllers have been reported as culinary ingredients and/or traditional medicine materials, such as Aloe vera [9], bilberry (Vaccinium myrtillus) [10], Okra (Abelmoschus esculentus) [11], and Cinnamon sp. [12]. However, most of their blood glucose suppressive effects were induced by extracts, not by a single isolated component of the extracts. Recently, biological effects of A. lappa have been reported, including the suppression of renal interstitial fibrosis [5], neuroprotection [6], gastroprotection [7], and anti-metastasis [8]. However, no reports have covered the blood glucose suppression effect and although most of anti-diabetic agents have anti-glucose effect, it is necessary to investigate the mode of action and candidate molecules of glucose suppression.

In order to evaluate the histopathological changes in lung (Figure 2A), heart (Figure 2B), kidney (Figure 2C), brain (Figure 2D), and spleen (Figure 2E), the relevant organs were stained with H&E. No morphological changes related to the A. lappa treatment were found in the organs, except for the liver. All images in the “a” panels in each photograph were considered normal as they were from the animals fed the normal diet. The “b” panels are representative images of the 60%-fat diet group. There was no difference between the control group and the 60%-fat diet group. A. lappa treatment did not induce morphological changes in any of the organs observed.

Many natural products have been used worldwide as a culinary material or medicine for centuries. However, although ingredients may have been previously considered safe, recent toxicological analysis techniques has classified them as toxic. In 2015, Cynanchum auriculatum was distributed as C. wilfordii [13] in Korea, but the U.S. Food & Drug Administration classified C. auriculatum as a poisonous plant based on the research of Han et al. [14]. For hundred years, A. lappa has been used as a culinary ingredient that is boiled down in soy sauce in Korea and Japan and is a frequently ingested food. However, as the toxicity of A. lappa repeated administration had not been previously evaluated, we conducted an assessment of the toxicity of a 8-weeks oral administration of A. lappa. The results indicated that A. lappa was very safe and had therapeutic effects, including the suppression of body weight gain and blood glucose.