Food Suppl Biomater Health. 2021 Jun;1(2):e22 https://doi.org/10.52361/fsbh.2021.1.e22 pISSN 2765-4362·eISSN 2765-4699

Original Article Effects ofRaphanus sativus var. niger (Black ) Fermented with Lactobacillus plantarum on Alcohol Metabolism in Rats

Kyung-Ku Kang,1,* Min-Soo Seo ,1,* Soo-Eun Sung ,1 Joo-Hee Choi ,1 Sijoon Lee ,1 KilSoo Kim ,1,2 Wookju Jang,3 Hak Sung Lee 4

1Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea 2Department of Veterinary Toxicology, College of Veterinary Medicine, Kyungpook National University, Daegu, Korea Received: Apr 28, 2021 3Efficacy Evaluation Team, Food Science R&D Center, Kolmar BNH CO., LTD, Seoul, Korea Revised: Jun 24, 2021 4Natural Product Research Team, Food Science R&D Center, Kolmar BNH CO., LTD., Seoul, Korea Accepted: Jun 24, 2021

Correspondence: Hak Sung Lee, PhD ABSTRACT Natural Product Research Team, Food Science R&D Center, Kolmar BNH CO., LTD., 61 Excessive drinking causes various side effects including hepatic and neurological diseases Heolleung-ro 8-gil, Seocho-gu, Seoul 06800, that can lead to serious social problems, and thus, efforts to search for a way to help to Korea. E-mail: [email protected] promote alcohol decomposition are increasingly needed. sativus var. niger commonly known as black radish has been reported to have biologically active glycosides such as *These authors contributed equally to this and also known to improve liver functions, suggesting that this vegetable work. may have an action to promote alcohol metabolism. In the present study, this possibility © 2021 Health Supplements Future Forum was tested with an extract prepared from the black radish fermented with Lactobacillus This is an Open Access article distributed plantarum in rats. This fermented black radish extract (75 or 300 mg/kg) or saline (2 mL/ under the terms of the Creative Commons kg) was administered orally. After 30 minutes, ethanol (4 g/kg) was administered orally Attribution Non-Commercial License (https:// using 25% ethanol in water and blood was sampled at 0, 1, 2 and 4 hours after ethanol creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial administration and then euthanasia was performed to get the liver. The sera obtained from use, distribution, and reproduction in any the blood samples were used for the concentration analysis of ethanol and acetaldehyde and medium, provided the original work is properly the examination of blood chemistries. The liver tissues were used for the activity assays for cited. alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). It was observed that in

ORCID iDs the rats administered the fermented black radish extract, the concentrations of ethanol and Min-Soo Seo acetaldehyde decreased more and simultaneously the activities of ADH and ALDH increased https://orcid.org/0000-0001-7817-2222 more than those in the saline-treated rats. Any significant changes in the blood chemistries Soo-Eun Sung and histology of liver tissues were not observed. These results suggest that the fermented https://orcid.org/0000-0001-9971-2443 black radish extract has a potential that may ameliorate hangover symptoms caused by Joo-Hee Choi excessive alcohol ingestion. https://orcid.org/0000-0003-4319-7950 Sijoon Lee Keywords: Black Radish; Fermentation; Alcohol; Alcohol Dehydrogenase; https://orcid.org/0000-0002-5870-687X KilSoo Kim Aldehyde Dehydrogenase; Hangover https://orcid.org/0000-0001-7370-2685 Hak Sung Lee https://orcid.org/0000-0001-9594-7173 INTRODUCTION Funding This research was supported by Korea Institute Drinking rates, that is, alcohol consumption rates, are sharply increasing as alcohol of Planning and Evaluation for Technology consumption has been linked to the relieving of stress. Overdrinking has resulted in an https://e-fsbh.org 1/8 Fermented Black Radish Ameliorates Hangover Symptoms in Food, Agriculture, Forestry and fisheries increase in various physical and social problems. In the stomach, 20%–30% of alcohol is (IPET) through Agri-Bio industry Technology absorbed within 30 minutes, and 90% or more is absorbed in the small intestine within Development Program, Funded by Ministry of 2 hours on an empty stomach. Then, the alcohol is metabolized mainly in the liver. Agriculture, Food and Rural Affairs (MAFRA) 1 (Grant No. 316006-5-HD030). Unabsorbed alcohol is discharged through the lungs, urine, and sweat. Alcohol is oxidized to acetaldehyde in the liver by alcohol dehydrogenase (ADH) and then further becomes Disclosure acetic acid by aldehyde dehydrogenase (ALDH).2 Acetaldehyde produced during this process The authors have no potential conflicts of is known as the cause of hangover symptoms such as nausea, vomiting, dizziness, thirst, interest to disclose. lethargy, headache, and muscle pain.3 Acetaldehyde is also known to induce alterations in the Author Contributions plasma membrane structures of hepatic cells due to its high affinity with sulfur-containing Conceptualization: Lee HS; Data curation: substances, such as cysteine and glutathione, leading to liver damages, such as fatty liver or Jang W; Investigation: Lee S, Kim K; hepatic necrosis. Methodology: Sung SE, Choi JH; Resources: Lee HS, Jang W; Supervision: Kang KK, Lee HS; Writing - original draft: Kang KK, Seo MS; Raphanus sativus var. niger, which is commonly known as black radish or oriental radish, is Writing - review & editing: Lee HS. a type of radish and is a vegetable of the family. It is characterized by black exterior skin and white interior flesh. Black radish is known to contain -based compounds, such as glucosisymbrin, glucoputrajivin, sinigrin, and glucoraphanin, which are components that have shown physiological activities.4-8 However, upon ingestion, glucosinolate is decomposed by myrosinase to form isothiocyanate, which is not easy to intake because of its spicy taste.9 For this reason, in Korea, black radish, like kimchi, has since long ago been ingested after the flavor has been improved through fermentation.

Black radish has recently been reported to have an effect in improving liver functions,6,10-12 and there is an increasing demand for foods that can quickly improve hangover symptoms. Thus, this study prepared black radish as a fermented product through the lactic acid fermentation process to confirm its function in improving hangover symptoms related to alcohol metabolism.

METHODS

Sample preparation Black radish provided by Seongsan Sunrise Peak NH, Jeju, was washed, cut and then pulverized after primary sterilization for 15 minutes at 95°C. Thereafter, secondary sterilization was performed at 121°C for 30 minutes to prepare a black radish medium. Lactobacillus plantarum obtained from the Korean Culture Center of Microorganisms (Seoul, Korea) was inoculated into the black radish medium at a concentration of 5 × 108 CFU/100 g and then cultured at 37°C for 2 days. After completion of the culture, the sample was subjected to sterilization at 95°C for 15 minutes, freeze-dried and then powdered to prepare the fermented powder.

Treatments of rats with fermented black radish extract Sprague-Dawley rats (male, 8 weeks old) purchased from ORIENT BIO Inc. (Seongnam, Korea) were used as experimental animals. The environment of the breeding room was maintained at an average temperature of 22°C ± 1°C and humidity of 50% ± 10%. The contrast was adjusted at intervals of 12 hours, and feed and water were freely supplied during the one-week acclimatization period. This experiment was conducted in accordance with the Laboratory Animal Act (Act No. 11987, the Ministry of Food and Drug Safety) and the Animal Protection Act (Act No. 13023, the Ministry of Agriculture, Food, and Rural Affairs) and further approved by the Institutional Animal Care and Use Committee of the Daegu- Gyeongbuk Medical Innovation Foundation (DGMIF-20071502-02). Rats were divided https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e22 2/8 Fermented Black Radish Ameliorates Hangover Symptoms

into 3 groups (24 rats in each group); first group was administered 2 mL/kg of saline, second and third group was administered 75 and 300 mg/kg of the fermented black radish extract orally, respectively. After 30 minutes, 25% ethanol (Merck, Darmstadt, Germany) was orally administered in all the rats at a dose of 4 g/kg. At 1, 2, and 4 hours after ethanol administration, animals were anesthetized with isoflurane (Virbac, Carros, France). Then, their whole blood was collected from the abdominal vein by opening the abdominal cavity, and liver tissue was further extract. Subsequently, some were stored at −70°C, and the rest were fixed in 10% neutral formalin. The collected whole blood was centrifuged at 3,000 rpm and 4°C for 15 minutes, and then, the serum was separated and stored at −70°C. All experimental rats were fasted for at least 18 hours before alcohol administration, and water feed intake were restricted after alcohol administration.

Measurement of blood ethanol and acetaldehyde content Ethanol in serum collected by time intervals was measured with a blood biochemical analyzer (TBA-120FR; Toshiba, Tokyo, Japan). After acetaldehyde was subjected to reaction using an acetaldehyde assay kit (EACT-100; Biovision, Milpitas, CA, USA), acetaldehyde was measured at 565 nm using a spectrophotometer (Synergy H4; Biotek, Winooski, VT, USA).

Measurement of ADH and ALDH activities in liver tissue After autopsy, the liver tissues stored at −70°C were each divided into 50 mg portions and further subjected to homogenization and reaction by utilizing the ADH activity colorimetric assay kit (K787-100; Biovision) and the ALDH activity colorimetric assay kit (K787-100; Biovision). Subsequently, the activity of each enzyme at 450 nm was measured by using a spectrophotometer (Synergy H4; Biotek).

Histopathology of liver tissue Liver tissues fixed in 10% neutral formalin were subjected to dehydration, tissue clearing, and paraffin penetration by using a tissue processor (Excelsior ES; Thermo Fisher Scientific, Waltham, MA, USA). Tissue samples were then embedded into paraffin blocks using a tissue embedding system (Histostar; Thermo Fisher Scientific). After paraffin blocks were cut into 4 μm using a microtome, to prepare slides, hematoxylin and eosin (H&E) staining was performed using an autostainer (Coverstainer; Aglient, Santa Clara, CA, USA). H&E-stained slides were image-scanned using a slide scanner (Panoramic SCAN; 3DHistech, Budapest, Hungary).

Serum biochemical analysis Using the serum obtained from animals 4 hours after ethanol administration, 16 items (sodium, potassium, chloride, total protein, albumin, blood urea nitrogen, creatinine, glucose, total bilirubin, calcium, phosphorus, total cholesterol, triglyceride, aspartate transaminase, alanine, aminotransferase, and alkaline phosphatase) were measured with a blood biochemical analyzer (TBA-120FR; Toshiba).

Statistical analysis The measured values in this study are represented as the mean value ± standard deviations. Student's t-test was used to test for statistical significance between the control and experimental groups, and the measurements were determined to be significant when theP value was less than 0.05.

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RESULTS

Changes in the blood concentrations of ethanol and acetaldehyde According to the results of the analysis of the blood ethanol concentration, the highest concentration in the control group was attained at 113.27 mg/dL 1 hour after alcohol administration, which decreased to 59.00 mg/dL after 2 hours and 23.85 mg/dL after 4 hours (Fig. 1A). The concentration in the low-dose group significantly decreased to 50.07 mg/dL 1 hour after alcohol administration compared to the control group and further significantly decreased to 7.37 mg/dL after 2 hours and 11.22 mg/dL after 4 hours. In comparison to the control group, the concentration in the high-dose group significantly decreased to 61.32 mg/dL 1 hour after alcohol administration and further significantly decreased to 59.30 mg/ dL after 2 hours and 5.60 mg/dL after 4 hours. According to the results of the analysis of the blood acetaldehyde concentrations, the control group reached a concentration of 155.05 µM 1 hour after alcohol administration, and the concentration slightly decreased to 169.58 µM after 2 hours and to 131.31 µM after 4 hoursFig. ( 1B). The low-dose group reached the concentration of 155.58 µM 1 hour after alcohol administration, and its concentration further decreased to 135.54 µM after 2 hours and 122.93 µM after 4 hours. The concentration in the high-dose group significantly decreased to 179.65 µM 1 hour after alcohol administration and further significantly decreased to 94.36 µM after 2 hours and 48.27 µM after 4 hours compared to the control group. At this research, the basal blood levels which before 25% ethanol uptake of alcohol and acetaldehyde were 14.45 ± 3.47 mg/dL and 29.57 ± 1.37 µM, respectively (data not shown).

ADH and ALDH activities in liver tissue According to the results of measurements in liver tissue, the ADH activity of the control group was determined to be 108.43 mU/mL, while activity in the low- and high-dose groups significantly increased to 123.09 mU/mL and 204.35 mU/mL, respectively, compared to the control group (Fig. 2A). According to the results ALDH activity measurements in liver tissue, the activity of the control group was determined to be 8.55 mU/mL, while activity in the low- and high-dose groups significantly increased to 13.23 mU/mL and 19.42 mU/mL, respectively, compared to the control group (Fig. 2B).

A B 140 Saline 350 Saline 120 Low 300 Low 100 High 250 High 80 200

60 µM 150 mg/dL * 40 * 100 20 50 * 0 * * 0 1 2 4 1 2 4 Time (hr) Time (hr) Fig. 1. Effects of the black radish extract fermented with lactobacillus on the blood concentration of ethanol and acetaldehyde in the ethanol-administered rats. Oral administration of the fermented extract (75 [low] or 300 [high] mg/kg) or saline (2 mL/kg) were done to rats and after 30 minutes, ethanol (4 g/kg) was administered orally. And then, blood was sampled at the times shown in the figures and used for the concentration analysis of ethanol and acetaldehyde. (A) Ethanol. (B) Acetaldehyde. Each point represents the mean ± standard deviation (n = 24). *P < 0.05 compared with saline group. https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e22 4/8 Fermented Black Radish Ameliorates Hangover Symptoms

A B 250 * 25 * 200 20

150 15 mU/mL 100 mU/mL 10

50 5

0 0 Saline Low High Saline Low High Fig. 2. Effects of the black radish extract fermented with lactobacillus on the activities of ADH and ALDH in the livers of ethanol-administrated rats. The experimental conditions were the same as in the Fig. 1, except that the livers were removed at 4 hours after the ethanol administration and used for the enzyme assays. (A) ADH. (B) ALDH. Each bar represents the mean ± standard deviation (n = 24). ADH = alcohol dehydrogenase, ALDH = acetaldehyde dehydrogenase. *P < 0.05 compared with saline group.

Histopathological evaluation and serum biochemical analysis of liver tissue According to the results of histopathological evaluations of liver tissue, neither the low- nor the high-dose groups showed any specific changes compared to the control group Fig.( 3). Serum biochemical analysis of sodium, potassium, chloride, total protein, albumin, blood urea nitrogen, creatinine, glucose, total bilirubin, calcium, phosphorus, total cholesterol, triglyceride, aspartate transaminase, alanine aminotransferase, and alkaline phosphatase showed no significant changes in either the low- nor high-dose groups compared to the control group (Table 1).

A B C

Fig. 3. Effects of the black radish extract fermented with lactobacillus on the histology of the livers of ethanol-administrated rats. The experimental conditions were the same as in the Fig. 2. But the livers were used for histological examination after hematoxylin and eosin staining. The representative photographs (× 400 magnification) are shown. (A) Saline group, (B) 75 mg group and (C) 300 mg group. Bars indicate 20 µm.

Table 1. Results of the analysis of serum biochemistry Variables Saline Low High Sodium (mmol/L) 145.65 ± 1.63 144.42 ± 1.12 145.35 ± 0.97 Potassium (mmol/L) 7.12 ± 0.53 6.17 ± 1.31 6.72 ± 0.61 Chloride (mmol/L) 102.92 ± 1.02 102.57 ± 0.80 101.22 ± 1.38 Total protein (g/dL) 5.70 ± 0.25 5.62 ± 0.25 5.60 ± 0.20 Albumin (g/dL) 4.17 ± 0.05 4.14 ± 0.20 4.11 ± 0.18 Blood urea nitrogen (mg/dL) 13.07 ± 0.66 15.1 ± 2.59 14.07 ± 1.51 Creatinine (mg/dL) 0.27 ± 0.05 0.30 ± 0.00 0.30 ± 0.00 Glucose (mg/dL) 46.75 ± 7.63 88.50 ± 13.42 95.25 ± 41.76 Total bilirubin (mg/dL) 0 ± 0 0 ± 0 0 ± 0 Calcium (mg/dL) 11.17 ± 0.17 10.97 ± 0.55 11.67 ± 0.37 Phosphorus (mg/dL) 12.15 ± 1.07 11.12 ± 1.21 11.60 ± 0.45 Total cholesterol (mg/dL) 86.75 ± 8.80 74.75 ± 14.56 77.75 ± 16.45 Triglyceride (mg/dL) 98.00 ± 29.29 67.25 ± 11.35 92.00 ± 18.29 Aspartate transaminase (U/L) 80.75 ± 7.63 78.75 ± 5.50 90.75 ± 10.40 Alanine aminotransferase (U/L) 26.00 ± 2.16 25.5 ± 4.20 32.75 ± 5.18 Alkaline phosphatase (U/L) 956.75 ± 144.66 813.75 ± 291.31 803.50 ± 101.98 The numbers in the table are mean ± standard deviation (n = 24). https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e22 5/8 Fermented Black Radish Ameliorates Hangover Symptoms

DISCUSSION

After ingestion, alcohol is absorbed through the gastrointestinal tract and transported mainly to the liver through blood.1 In the endoplasmic reticulum of the liver, alcohol is oxidized by the microsomal ethanol oxidizing system (MEOS) and then converted to acetate through acetaldehyde. After oxidation, alcohol is converted to carbon dioxide and water while producing energy or to fatty acids or other metabolites.13 Long-term alcohol intake affects hyperlipidemia and the fatty liver. NADH/NAD+ in liver and cells increases due to alcohol metabolism by ADH and MEOS catalysis, and this change is known to increase the activity of the tricarboxylic acid cycle of oxidation and fatty acid in liver cells, leading to the etiology of fatty liver.14 Furthermore, excessive NADH induces hepatocyte necrosis, abnormal hepatocyte metabolism, and inhibited mitochondrial function, and as a result, peroxidation and metabolites induce damage to hepatocytes, thereby accumulating lactic acids, which are fatigue substances, and further reducing the activity of ALDH.3,15

To identify the effects of fermented black radish extract on blood alcohol metabolism, this study orally administered fermented black radish extract and ethanol in Sprague-Dawley rats. Subsequently, serum was obtained at the intervals of 1, 2, and 4 hours to measure the concentrations of ethanol and acetaldehyde in serum. The ethanol concentration decreased over time after reaching the highest value 1 hour after ethanol administration in all groups. In the low-dose group, the ethanol concentration significantly decreased 2 hours after ethanol administration compared to the control group, and in the high-dose group, the ethanol concentration significantly decreased compared to the control group 4 hours after ethanol administration. These results suggest that the administration of fermented black radish extract has an effect on the decrease of ethanol. The results of measuring the acetaldehyde concentrations showed that there was little difference across all groups 1 hour after ethanol administration, while the acetaldehyde concentration in the high-dose group showed a tendency to decrease over time, and the concentration significantly decreased compared to the control group 4 hours after ethanol administration. This result suggests that the administration of fermented black radish extract has an effect on the reduction of the acetaldehyde concentration. However, although the ethanol concentrations of the low- and high-concentration groups were lower than that of the control group 1 hour after ethanol administration, the acetaldehyde concentration did not show a significant difference from the control group. This contrast suggests that the fermented black radish extract that was administrated prior to ethanol administration had an effect on the absorption of ethanol in the stomach. In the human body, about 20%–50% of ethanol is absorbed into the stomach, and 90% or more is absorbed into the small intestine within 2 hours on an empty stomach.1 Because this experiment was conducted with animals on an empty stomach prior to administration, there would be a difference in ethanol absorption in the low- and high- dose groups compared to the control group. Moreover, for the same reason, the ethanol concentration 2 hours after ethanol administration significantly decreased compared to that 1 hour after ethanol administration in the low-dose group, whereas the ethanol concentration in the high-dose group did not show a significant decrease compared to that 1 hour after ethanol administration. This result suggests that there is a difference in the absorption of ethanol in the stomach depending on the concentration of fermented black radish extract ingested.

Ethanol is oxidized to acetaldehyde in the liver through the catalytic reaction of ADH, and acetaldehyde becomes acetic acid through the catalytic reaction of ALDH.2 Acetaldehyde is known to be a major cause of hangover due to alcohol consumption, and it has been https://e-fsbh.org https://doi.org/10.52361/fsbh.2021.1.e22 6/8 Fermented Black Radish Ameliorates Hangover Symptoms

reported that alcohol increases the production of nitric oxide in brain cells, which is toxic to the central nervous system and thereby damages brain cells.16,17 In this experiment, the ADH activity in the liver significantly increased in the high-dose group compared to the control group, and the ALDH activity also showed an increasing tendency in the high-dose group compared to the control group. These results suggest that the administration of fermented black radish extract has an effect on the increase in ADH and ALDH activities and that in relation to the decrease in the concentrations of ethanol and acetaldehyde in the group administered the fermented black radish extract compared to the control group, fermented black radish extract increases ADH and ALDH activity, thereby affecting the decomposition of ethanol and acetaldehyde after ethanol absorption.

Liver damage, which occurs due to foreign substances, could result in hepatocyte degeneration, lipoidosis, fibrosis, and even necrosis progress, which can be histopathologically observed.18 In addition, because enzymes within cells leak into the blood due to hepatocellular necrosis, the concentrations of aspartate transaminase and alanine aminotransferase enzymes in the blood become indicators of liver damage.19 In this experiment, there were no specific findings observed in the liver histopathology of the low- and high-dose groups compared to the control group, and serum biochemical analysis did not show any significant changes in liver-related aspartate transaminase, alanine aminotransferase, and other indicators. These results suggest that fermented black radish extract does not cause acute toxicological changes.

Black radish extract is known to contribute to liver health by strongly inducing enzymes, such as CYP1A1, CYP1A2, glutathione S-transferase, quinone reductase, and microsomeal epoxide hydrolase, in liver cells to activate phases I and II, which are involved in liver detoxification. Furthermore, black radish extract in rats induced with liver damage through a high-fat diet has been found to contribute to the physiological activity of the liver by inhibiting lipid peroxidation in liver tissues.6,7 In this study, the improvability of hangover symptoms by consuming fermented black radish extract was determined by checking whether the fermented black radish extract increased the activity of ADH and ALDH in an ethanol- administered animal model, thereby affecting the reduction of ethanol and acetaldehyde concentrations. Furthermore, this study determined whether fermented black radish extract resulted in acute toxicity by assessing the histopathological and serum biochemical levels in the liver. However, additional studies will be necessary to separate the physiologically active substances from fermented black radish extract and to determine the mechanism that affects ethanol metabolism in relation to substance separation and to compare sex difference between male and female.

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