Effect of Burdock Root and the Fermented Product on Alloxan-Induced Mouse Hyperglycemia

Backgrounds: We reported that feeding 5% Asperagillus awamori-fermented burdock root diet was effective in preventing mouse hyperglycemia caused by alloxan. Methods: Diets containing 5% burdock roots were prepared from raw and Asperagillus awamori-fermented burdock root powders. Acatalasemic mice, having a quite low catalase activity in blood, and normal mice were fed these diets for 14 weeks, separately. Then, alloxan (200 mg/kg of body weight) or PBS was intraperitoneally administrated to each mouse. After 5 day from the administration, blood glucose assay and glucose tolerance test were carried out, and then insulin, C-peptide and lipid peroxide in plasma were examined. Results: Incidences of hyperglycemia in normal mice fed control, raw and fermented burdock root diets were 25, 20 and 11%, respectively, and these in acatalasemic mice were 73, 80 and 27%. Insulin and C-peptide in plasma of mice fed raw burdock root diet or control diet were low compared to mice fed the fermented diet. Conclusions: Intake of raw burdock root does not suppress the alloxan-induced hyperglycemia but the fermented burdock root does. It is suggested that Asperagillus awamori plays an important role for the prevention.


Introduction
Diabetes mellitus is a syndrome characterized by hyperglycemia, more than a desirable level of glucose in blood (Taylor, 1995). The morbidity causes blindness, renal failure and amputation, and diabetes is a worldwide disease and one of the major causes of death. Alloxan is a diabetogenic drug for animals, and alloxan with reducing agents in the body generated reactive oxygen species. These species cause oxidative stress and selectively injure β-cells in the pancreas so as to cause hyperglycemia as like diabetes Type 1 (Szkudelski, 2001;Lenzen, 2008). In order prevent this hyperglycemia, many researchers challenged to find (or pursue) anti-hyperglycemic compounds or products (Oloyede, Bello, Ajiboye, & Salawu, 2015;Perumal, Anaswara, Muthurama, & Krishan, 2014). We noticed that Japanese burdock root, Artium lappa, is edible in Korea and Japan and contains a considerable amount of polyphenols such as chlorogenic acid, caffeoylquinic acid, hydroxycinnamoylquinic acids and related compounds (Maruta, Kawabata, & Niki, 1995;Lin & Harnly, 2008), and investigated the effect of Aspergillus awamori-fermented burdock root on hyperglycemia. When mice were fed the Aspergillus awamori-fermented diet, hyperglycemia induced by alloxan was ameliorated (Takemoto, Doi, Zukeran, Inoue, Ishihara, & Masuoka, 2014) like as the intake of antioxidants. However, it is unclear whether raw burdock root is able to modulate alloxan-induced mouse hyperglycemia. In this study, we examined antioxidant activity of raw and the fermented burdock root powders and the effect of raw burdock root diet on alloxan-induced mouse hyperglycemia. Feinstein, Braun, and Howard (1967) were used. Animal experimental procedure was approved by the Ethics Review Committees for Animal Experimentation of Okayama University of Science. Mice were bred and maintained on a laboratory diet (CE-2 diet, Clea Japan, Tokyo) and water ad libitum until the start of the experiments. Catalase activity in the mouse erythrocytes was measured according to previous method (Masuoka, Wakimoto, Ubuka, & Nakano, 1996) and calculated as the difference between the hydrogen peroxide removal rate by hemolysate and the rate (0.73 μmol/s/g of hemoglobin) by hemoglobin (Takemoto, Tanaka, Iwata, Nishihara, Ishihara, Wang, Ogino, Taniuchi, & Masuoka 2009). Raw and fermented burdock root powders were prepared by Ahjikan Co. Ltd (Hiroshima, Japan) from burdock roots (harvested in Japan). Burdock roots were cut into small pieces and then dried using air drying oven at 50 ºC for 4 hrs (raw burdock root powders). The dried pieces were mixed with an equal amount of water and 0.1% Aspergillus awamori spores (final concentration, w/w) and the mixture was fermented at 35 ºC for 40 hrs (Aspergillus awamori-fermented burdock root powders) (Okazaki, Sitanggang, Sato, Ohnishi, Inoue, Iguchi, Watanabe, Tomotake, Harada, & Kato, 2013). Control diet was AIN-93M (Reeves, Nielson, & Fahey, 1993) and each burdock root diet contained 5% burdock root powders was prepared according to the composition, which was indicated in Table 1. Pellets of these diets (1.3 cm) were prepared and stored at -20 ºC until use.  Control diet is AIN-93M diet. Raw burdock root diet is 5% raw burdock root diet. Fermented burdock root diet is 5% Aspergillus awamori-fermented burdock root diet.

Extraction of Raw and Fermented Burdock Root Powders
Five gram of each burdock root powders was extracted five-times with 25 mL of aqueous 50% methanol for 1 h. The extracts were gathered and evaporated at 40 ºC. The residue was dissolved with 10 mL of 50% methanol in water, and the solution was used as sample.

Radical Scavenging Activity on DPPH
Sample (0.03 mL) was added to the mixture consisted of 1.00 mL of 100 mM acetate buffer (pH 5.5), 1.87 mL of ethanol and 0.10 mL of ethanolic solution of 3 mM DPPH at 25 C. The absorbance at 517 nm (DPPH,  =8.32 × 10 3 ) was recorded for 20 min (Blois, 1958). From decrease of the absorbance, scavenging activity was calculated and expressed as scavenged DPPH molecules per 1.0 g of powders.
Sample (0.2 mL) was added to the mixture of 0.2 mL of Folin-Ciocalteau's phenol regent and 4 mL of water, and the mixture was reacted 8 min. Then, 0.6 mL of 7% sodium carbonate aqueous solution was added to the mixture, and the mixture was reacted at 40 C for 30 min. Absorbance at 570 nm was recorded. Gallic acid was used as standard compound, and polyphenol content was expressed as amounts of gallic acid.

Animal Experiments
Acatalasemic mice (n=48) and normal mice (n=48) were used at the age of 14 to 15 weeks old (body weight was between 25 and 36 g) and were housed in a group of four. Acatalasemic and normal mice were divided into three kinds of diet groups, respectively. Control diet, raw and fermented burdock root diets were fed ad libitum for 14 weeks. Then, each diet group further divided two groups. Alloxan (200 mg/kg of body weight) was intraperitoneally administrated using 0.106 M alloxan in phosphate buffered saline (PBS) to each mouse in a group (Kamimura et al., 2013), and the same volume of PBS was injected to each mouse in another group as control. Mice in each group were maintained on the same diet for one more week. After five days from alloxan (or PBS) administration, mice were fasted for 20 hrs, and blood glucose assay and glucose tolerance test (GTT) were carried out. After 2 days from the assay and GTT, mice were fasted for 20 hrs and were killed. Under diethyl ether anesthesia, each mouse blood was collected in test tube containing heparin as the anticoagulant from heart. Then, pancreas in each mouse was isolated, and the sections were prepared for microscopic studies. Blood was centrifuged and the plasma was isolated. Oxidative stress marker, as well as the insulin and C-peptide levels in plasma, was examined.

Assay of Blood Glucose
After fasting for 20 hrs, glucose content in the blood obtained from the tail was determined. As the blood volume for the determination of blood glucose was quite small (approximately 2 μL), the glucose contents in blood were measured with a "Glucose-Test-Ace R" apparatus (Sanwa Kagaku Kenkyusho Co., Nagoya, Japan) applying a glucose oxidase method.

Glucose Tolerance Test (GTT)
After fasting for 20 hrs, a forty percent aqueous glucose solution (5 mL/kg of body weight) was intraperitoneally administered to each mouse (Gao et al., 2007). At 0 and 30 min before and 15, 30, 60, 90 and 120 min after the administration, glucose contents in the blood were measured.

Measurement of Lipid Peroxide in Plasma
Lipid peroxidation in plasma was determined using a Bioxytech LPO-586 KIT (OXIS Health Products Inc, CA, USA). Malondialdehyde and 4-hydroxyalkenals as products of lipid peroxidation were reacted with N-methyl-2-phenylindole at 45 °C. The absorbance at 586 nm was recorded. Values of lipid peroxide in blood were calculated as malondialdehyde.

Determination of the Insulin and C-peptide Levels in Blood
The insulin and C-peptide plasma levels were determined using Mouse Insulin and C-peptide ELISA KITs (U-type) (Shibayagi Co. Ltd., Gunma, Japan). Each determination was carried out according to the manufacturer's instructions. Biotin-conjugated anti-insulin antibody (45 μL) was added to each well in an antibody-coated 96-well plate. To the well, 5 μL of the sample or standard solution was added and reacted for 2 hrs. Then 50 μL of peroxidase-conjugated avidin solution was added and reacted for 30 min. Chromogenic substrate solution (50 μL) was added and reacted for 30 min. The reaction was stopped and the absorbance at 450 nm (sub-wave length, 620 nm) was recorded.

Microscopic Studies of Pancreatic Tissues in the Mice Treated With Alloxan
Isolated pancreatic tissues were fixed in Bouin's fluid and embedded in paraffin. Serial sections (6 μm) were cut from each paraffin-embedded tissue block, and several sections were stained with hematoxylin-eosin and mouse anti-insulin antibody (Santa Cruz Biotechnology) using the Vectastain Elite ABC Rabbit IgG Kit for visualization by light microscopy. The islets and other cells were recorded with a FX380 CCD Camera and a microscope (Olympus, Tokyo, Japan).

Statistical Analysis
Student's t-test (unpaired) was used to evaluate the statistical significance of difference. The difference was considered significant when p < 0.05. www.ccsen

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Blood Administra
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Conclusion
Japanese burdock root is taken as a vegetable in Japan. Raw burdock root does not indicate anti-hyperglycemia caused by oxidative stress but the product fermented with Asperagillus awamori indicates anti-hyperglycemia activity. It is deduced that intake of the fermented burdock root product is useful in preventing hyperglycemia caused by oxidative stress.