3-Hydroxy-6-Methylpyridine with Preventive Activity on Carbon Tetrachloride- Induced Liver Injury Is Produced During Roasting Of

Food Sci. Technol. Res., 17 (1), 39–44, 2011

3-Hydroxy-6-Methylpyridine with Preventive Activity on Carbon Tetrachloride-

Induced Liver Injury Is Produced During Roasting of Coffee Beans

1* 1 2 Kiharu Igarashi , Chihiro Kawai and Shizue Kurakane

1 Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka-shi, Yamagata 997- 8555, Japan 2 Course of the Science of Bioresource, The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan

Received January 10, 2010; Accepted September 24, 2010

Compounds which can be formed over the course of roasting coffee beans were investigated and isolated. One of these compounds was identified as 3-hydroxy-6-methylpyridine (3,6-Py). 3,6-Py and the coffee prepared from light-roasted coffee beans which contain this compound tended to suppress increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities induced by carbon tetrachloride in mice, indicating that 3,6-Py formed during the roasting of coffee beans may be able to mitigate liver injury. The amounts of 3,6-Py were higher in French-roasted than light-roasted coffee beans of the Brazil Santos and Colombia Excelso varieties, suggesting that this compound might be produced under a more severe roasting condition. However, suppressive activity against liver injury was stronger in coffee from light-roasted than French-roasted beans. As the amounts of chlorogenic acid, which is known to have hepatoprotective activity, were higher in coffee from the light-roasted than French-roasted beans, stronger hepatoprotective activity in the coffee from light-roasted beans might be mainly concerned with its higher content of chlorogenic acid.

Keywords: roasted coffee beans, 3-hydroxy-6-methylpyridine, liver injury, roasting of coffee beans Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; DPPH, 1,1-diphenyl-2-pycrylhydrazyl; TE, trolox equivalency.

Introduction and coffee diterpenes, kahweol and cafestrol, and chloro- In ancient times, coffee was drunk for medicinal pur- genic acid are reported to prevent acute liver injury induced poses. Recently, however, coffee has become commonplace by carbon tetrachloride (CCl4) (Ozercan et al., 2006; Lee et in Japan so much so that Japan has become the fourth largest al., 2007; Kapil et al., 1995; Zhou et al., 1993). Moreover, coffee consumer in the world. As a result of its newfound coffee has been reported to be effective in the prevention of popularity, many researchers have studied coffee components liver injury and the mitigation of type 2 diabetes (Dam and and their physiological functions. In particular, many physi- Feskens, 2002), although the validity of these findings is still ological and pharmaceutical functions of caffeine, a major under debate (Ruhl and Everhart, 2005; Haner et al., 2008). component in coffee beans, were studied in relation to the Reports pointed out that it is necessary to determine the prin- benefits of coffee consumption. Caffeine, although suspected ciples by which these effects are demonstrated and to verify to be a cause of cancer, has been shown to have no effect on such effects using such principles. There are many unidenti- cancer. On the other hand, coffee has been found to suppress fied compounds in coffee beans. From roasted beans, we the occurrence of stomach and liver cancers (Bauer et al., previously isolated and identified one of these compound as 1977; Mohr et al., 1984; Inoue et al., 2005). Instant coffee, 3-pyridinol (3-Py) (Kurakane and Igarashi, 2006), which was found to be effective in the prevention of oxidative stress. *To whom correspondence should be addressed. Furthermore, its related compounds are also produced during E-mail: [email protected] the process of roasting coffee beans (Kurakane and Igarashi, 40 K. IGARASHI et al.

2006). beans (without decaffeination) were used. Each bean (10 g) In this report, we identified and isolated another com- was extracted with 150 ml hot water using a coffee maker. pound in the roasted beans and subsequently determined its The filtrate of the extracts (drip coffee) was subjected to contents and preventive effect against CCl4-induced liver HPLC using a Develosil ANIDIUS column (4.6 i.d. × 250 damage in mice. mm, Nomura Chemical Co., Aichi, Japan) for the measurements of 3,6-Py and 3-Py. Elution of each compound from Materials and Methods the column was performed using 95% acetonitrile in water, Isolation of 3-hydroxy-6-methylpyridine and its related and detection occurred at 290 nm. Commercial 3,6-Py and compounds from roasted coffee beans Light- and French- 3-Py were used as the standard for the calculation of each roasted coffee beans were prepared by roasting decaffein- compound after dissolution in water. ated beans of the Colombia Excelso variety at 200℃ for Chlorogenic acid in the filtrate was measured using a De- 14 min, and 220℃ for 17 min, respectively. To isolate the velosil C-30-UG-5 column (4.6 i.d. × 250 mm) and a solvent coffee constituents of the roasted coffee beans, chilled hot system composed of 5% acetonitrile in 1% acetic acid (sol- water extracts from decaffeinated and light-roasted coffee vent A) and 40% acetonitrile in water (solvent B). A linear beans were placed in a Diaion column (20 i.d. × 200 mm, gradient of 0-100% solvent B in solvent A over the course of Mitsubishi Chemical Co., Tokyo, Japan) and then eluted with 180 min at a flow rate of 0.8 mL/min was used. Chlorogenic 50% MeOH after washing the column with distilled water. acid was detected at 330 nm.

The eluate was evaporated to remove MeOH in a vacuo and Inducement of liver damage by CCl4 Preventive effects then extracted with diethyl ether. The diethyl ether layer was of compounds a and b, in addition to light- and French-roast- evaporated after being dried with Na2SO4 and subsequently ed coffee, on liver injury were determined as follows. Eight- dissolved in a small amount of MeOH for a preparative TLC week old male ddY mice (Japan SLC) were acclimated for 5 with silica gel as an adsorbent and ethyl acetate:CHCl3:88% days in an environment with a temperature of 22 ± 2℃, 40-

HCOOH:H2O (19:1:1:1, v/v) as a developing solvent. Each 60% humidity and light cycle of 6:00-18:00. After feeding on region corresponding to the compounds (compounds a and a basal diet for 3 days, the mice were divided into six groups: b) and exhibiting orange and pink colors on the TLC plate the control group (5 mice), the liver injury group by CCl4 after a visualization of diazotized sulphanilic acid spray- (CCl4 group, 8 mice), the CCl4+3-Py group (3-Py group, 7 ing reagent (each, Rf: 0.32 and 0.15) were scraped from the mice), the CCl4+3,6-Py group (3,6-Py group, 8 mice), the

TLC plate. Each compound was eluted from the scraped CCl4+light coffee (freeze-dried coffee prepared from drip silica gel with a mixture of MeOH and ethyl acetate (1:1, v/ coffee from decaffeinated and light-roasted Colombia Ex- v) and then purified further using Sephadex LH-20 (15 i.d. celso beans) group (DCL group, 7 mice) and CCl4+French × 900 mm, GE Healthcare BioSciences AB, Japan) column coffee (freeze-dried coffee prepared from drip coffee from chromatography with MeOH as the developing solvent. Each decaffeinated and French-roasted Colombia Excelso cof- fraction containing compounds a and b was further purified fee beans) group (DCF group, 7 mice). After fasting for 8 by a preparative HPLC with a Develosil C-30-UG-5 column h, mice were administered one of the following solutions:

(20 i.d. × 250 mm). Elution from the column was performed to the control and CCl4 groups, 0.2 mL of 0.5% sodium car- using 5% MeOH in water from 0 to 75 min, 50% MeOH in boxymethyl cellulose (CMC) solution; to the 3-Py group, 0.2 water from 75 to 230 min, and 100% MeOH after 230 min. mL 3-Py suspension prepared by suspending 100 mg 3-Py The fractions containing compounds a and b were combined in 1 mL of 0.5% CMC solution; to the 3,6-Py group, 0.2 mL and then evaporated. The chemical structures of the obtained 3,6-Py suspension prepared by suspending 115 mg 3,6-Py compounds were determined by NMR and MS spectra analy- in 1 mL of 0.5% CMC solution (in equal moles with 3-Py); ses. The NMR spectra were recorded on a JNM-EX 400 FT- and to the DCL and DCF groups, 0.2 mL of 0.5% CMC solu-

NMR spectrometer (JEOL, Tokyo, Japan) using CD3OD as tion containing 100 mg/ml freeze-dried coffee prepared from the solvent. The FAB-MS spectrum was recorded on a JMX- decaffeinated and light- or French-roasted Columbia Excelso D 300 spectrometer (JEOL, Tokyo, Japan) with glycerol as beans as described above. The amounts of 3-Py and 3,6-Py the matrix. contained in the administered solution were 0.08 and 0.01 Measurement of 3-hydroxy-6-methylpyridine (3,6-Py), mg in the light-roasted beans, and 0.07 and 0.02 mg in the 3-pyridinol (3-Py), and chlorogenic acid contents in coffee French-roasted beans, respectively. beans For the measurement of 3,6-Py and 3-Py contents The control group was injected intraperitoneally with 30 in coffee beans, decaffeinated light- and French-roasted Co- µL olive oil 30 min after the administration of 0.5% CMC lombia Excelso coffee beans along with Brazil Santos coffee solution. The other groups were injected with a mixture of 30 Prevention of Liver Injury by a Coffee Constituent 41

µL olive oil and 30 µL CCl4 30 min after the administration spectra data also confirmed the chemical structure of com- of each sample solution. All mice were anesthetized with pound b to be 3,6-Py. This is the first report to identify this Nembutal (Dainippon Pharmaceutical Co., Osaka, Japan) 22 compound in raw and roasted coffee beans. h after the injection of either olive oil or olive oil/CCl4, fol- The content of 3-Py and 3,6-Py in coffee HPLC chro- lowed by the collection of blood from the heart. The serum matograms of coffee prepared from raw, light-, and French- was obtained by centrifugation of the blood at 3,000 × g for roasted Columbia Excelso beans (decaffeinated) in a coffee 15 min at 15℃ and used for measurement of alanine amino- maker are shown in Fig. 2. Peaks for 3-Py and 3,6-Py were transferase (ALT) and aspartate aminotransferase (AST) activities (Matsumoto, 1955). The basal diet was composed of Authentic 20% casein, 65.5% α-corn starch:sucrose (2:1), 5% cellulose, 3-Hydroxy-6-methylpyridine 3-Pyridinol 3.5% mineral mixture (AIN-93G-MX, Oriental Yeast Co., Columbia Excelso Tokyo, Japan), 1% vitamin mixture (AIN-93-VX, obtained Raw beans from Oriental Co.), and 5% corn oil. The animals were cared for at all times according to the institutional guidelines at Yamagata University. Statistical analyses Each value is given as the mean ± standard error. The significance of the differences between two groups was determined with an unpaired t-test (one- tailed) and Welch’s t-test. One-sided p values less than 0.05 were considered significant.

Results and Discussion Columbia Excelso Brazil Santos Chemical structure of the compounds related to 3-Py (decaffeinated) Light Light Since the Rf value of compound a (0.32) and its color upon being sprayed with diazotized sulphanilic acid on the TLC plate was the same as those of authentic 3-Py, which was previously isolated from roasted coffee beans (Kurakane and Igarashi, 2006) , Compound a was identified as 3-Py.

Compound b (Rf 0.15) exhibited a pink color when sprayed with diazotized sulphanilic acid, suggesting the pres- ence of more than a phenolic hydroxyl group in its chemical structure. The FAB-MS spectrum of compound b showed a pseudo-molecular ion peak at m/z 110 (M+H)+ ; indicat- Columbia Excelso Brazil Santos 13 (decaffeinated) French ing a molecular weight of 109. The C-NMR spectrum of French compound b measured in CD3OD showed the same spectrum with that of authentic 3-hydroxy-6-methylpyridine (3,6-Py)

[100 MHz, δc (CD3OD): 22.5 (CH3), 125.3 (C-4 or C-5), 125.4 (C-5 or C-4), 136.9 (C-2), 149.5 (C-6), 153.6 (C-3) ]. The 1H-, 1H-1H COSY, 1H-13C COSY, and HMBC NMR

Retention time (min) Retention time (min)

Fig. 2. HPLC chromatogram of hot water extracts from raw, light- roasted and French-roasted coffee beans, and of authentic 3-hydroxy-6-methylpyridine and 3-pyridinol. Compound b: Compound a: Analytical conditions: Develosil ANIDIUS column (4.6 i.d. × 250 3-Hydroxy-6-methylpyridine mm, Nomura Chemical Co., Aichi, Japan); flow rate, 0.8 ml/min; solvents, acetonitrile/water (95/5); detection, 290 nm. Arrows with Fig. 1. Chemical structures of 3-pyridinol (3-Py) and 3-hydroxy-6- straight and dotted lines show 3-hydroxy-6-methylpyridine and methylpyridine (3,6-Py). 3-pyridinol, respectively. 42 K. IGARASHI et al. observed in all of the coffee samples except those prepared to be too small to demonstrate the preventive effects against from raw beans. The relationship between the contents liver injury. Therefore, the other compounds such as chloro- of these compounds and roasting conditions are shown in genic acid, kahweol and cafestrol contained in coffee beans,

Table 1. Although the amounts of 3-Py in Colombia Excelso which are known to be effective in mitigating CCl4-induced was almost the same between the light- and French-roasted hepatotoxicity (Shi et al., 2009; Lee et al., 2007), might beans, the amounts of 3,6-Py was higher in the French-roast- have contributed more strongly to the preventive effects of ed beans. As the amounts of 3,6-Py in Brazil Santos was also coffee extracts. Stronger suppressive effects found in light- higher in the French- than light-roasted, 3,6-Py was thought roasted beans, compared to those in French-roasted beans to be produced when beans were roasted under severe roast- with higher 3,6-Py content, may support the idea that coffee ing conditions. The higher amounts of 3-Py, compared to contains other compounds useful for the prevention of liver those of 3,6-Py, in both the light- and French-roasted coffee, injury. Chlorogenic acid content in coffee, prepared from 100 suggest that this compound has been produced in a relatively g light- and French-roasted beans was 6.6 and 1.3 g, respec- mild roasting condition compared to the conditions for pro- tively. These amounts were much higher than those of 3,6-Py ducing 3,6-Py. in the same coffee samples. Therefore, the stronger suppres- The formation pathway of 3,6-Py is very interesting be- sive effects of coffee against liver injury from the light-roast- cause of its similar chemical structure with 3-Py which was ed beans, compared to that from the French-roasted beans, previously isolated from roasted coffee beans (Kurakane and might be mainly due to the higher content of chlorogenic Igarashi, 2006). The methylation of 3-Py on its 6-position acid in the light-roasted coffee, as chlorogenic acid is known under severe roasting conditions may be a cause of the for- to be available for the prevention of liver injury (Kapil et al., mation of 3,6-Py because of the fact that its content increased 1995; Zhou et al., 1993). in the French-roasted coffee. However, the precise formation The DPPH radical scavenging activity of chlorogenic mechanism still remains to be clarified. acid (1.14 µmol TE/µmol sample) was stronger than that of The effects of 3,6-Py, and coffee prepared from beans 3-Py (9.09 × 10-3) and 3,6-Py (9.07 × 10-3 µmol TE/µmol with different roasting conditions on CCl4-induced liver sample) when measured using the DPPH method (Takahata injury The plasma ALT and AST activities of mice admin- et al., 2001). This result may also support that the stronger istered each coffee sample are shown in Fig. 3. Both 3,6- suppressive effects of coffee from the light-roasted beans, Py and coffee from light-roasted coffee beans suppressed an compared with that from the French-roasted beans, is mainly increase in the AST activities induced by CCl4. ALT activity due to the higher content of chlorogenic acid in coffee from tended to be suppressed by administrating 3,6-Py (0.05 < the light-roasted beans, although the suppression of liver in- p < 0.1). Suppression by 3-Py was much weaker than that jury by the other constituents cannot be excluded. However, of 3,6-Py. Although 3,6-Py showed a suppressive effect for the preventive effects of 3,6-Py, contained only in small increasing the ALT and AST activities caused by CCl4, the amounts, may be important from the standpoint that the content in 20 mg freeze-dried coffee extract which was given roasting process produced hepatoprotective compounds. to each mouse was 0.01-0.02 mg. This amount is considered In this paper, it was reported for the first time that 3,6-

Table 1. Relation between the roasting conditions and contents of 3-hydroxy-6-methylpyridine and 3-pyridinol.

Colombia Excelso Decaffeinated Colombia Excelso Brazil Santos

Raw beans Light French Light French

3-hydroxy-6-methylpyridine nd 5.4 ± 3.5 14.5 ± 1.7 1.4 ± 0.1 3.7 ± 0.3 (mg/100 g beans)

3-pyridinol nd 45 ± 9 46 ± 11 17 ± 1 31 ± 3 (mg/100 g beans)

Light-roasted and French-roasted beans were prepared by heating beans at 200℃ for 14 min, and at 220℃ for 17 min, respectively. Cof- fee was prepared by extracting 10 g decaffeinated roasted beans (Colombia Excelso), roasted beans (Brazil Santos), or raw beans (Colombia Excelso) with 150 mL boiling water using a coffee maker. nd: not detected. Values for decaffeinated Colombia Excelso and Brazil Santos are means of 3 and 5 measurements, respectively. Data are means ± S.E. Prevention of Liver Injury by a Coffee Constituent 43

ALT

AST

Fig. 3. Effects of oral administration of coffee and its constituents on plasma ALT and

AST activities in mice induced with liver injury by CCl4.

Con, control group not administered coffee or its components and CCl4; CCl4, group administered only CCl4; 3-Py, group

administered both 20 mg 3-pyridinol (3-Py) and CCl4; 3,6-Py, group administered both 23 mg 3-hydroxy-6-methylpyri-

dine (3,6-Py) (in equal moles with 3-Py) and CCl4; DCF, group administered both coffee from French-roasted beans (20

mg freeze-dried coffee extracts containing 0.02 mg 3,6-Py and 0.07 mg 3-Py) and CCl4; DCL, group administered both

light-roasted coffee (20 mg freeze-dried coffee extracts containing 0.01 mg 3,6-Py and 0.08 mg 3-Py) and CCl4. Coffee

and its constituents were administered orally 30 min before an intraperitoneal injection of CCl4. Data are means ± S.E. *Significantly different between two groups p( < 0.05). **Significantly different from the other groups p( < 0.05).

Py, which was produced in the course of roasting of coffee 63-70. beans, was effective for mitigating liver injury, although its Dam, R.M. and Feskens, E.J.M. (2002). Coffee consumption and content in the roasted beans is very low. risk of type 2 diabetes mellitus. Lancet, 360, 1477-1478. Haner, M., Witte, D.R., Mosdel, A., Marmot, M.G. and Brunner, E.J. Acknowledgement This study was supported by a grant from the (2008). Prospective study of coffee and tea consumption in rela- All Japan Coffee Association. tion to risk of type 2 diabetes mellitus among men and women ; The Whitehall II study. Br. J. Nutr., 100, 1046-1053. References Inoue, M., Yoshimi, I., Sobue, T. and Tsugane, S. (2005). For the Bauer, A.R., Rank, R.K. and Kerr, R. (1977). The effects of pro- JPHC Study Group: Influence of coffee drinking on subsequent longed coffee intake on genetically identical mice. Life Sci., 21, risk of hepatocellular carcinoma: A prospective study in Japan. 44 K. IGARASHI et al.

Journal of the National Cancer Institute, 97, 293-300. Ozercan, I.H., Dagli, A.F., Utsundag, B., Ozercan, M.R., Bahcecio- Kapil, A., Koul, I.B. and Suri, O.P. (1995). Antihepatotoxic effects glu, I.H., Celik, H., Yalniz M., Poyrazoglu, O.K. and Ataseven, H. of chlorogenic acid from Anthocephalus cadamba. Phytother. (2006). Does instant coffee prevent acute liver injury induced by

Res., 9, 189-93. carbon tetrachloride (CCl4). Hepatol. Res., 35, 163-168. Kurakane, S. and Igarashi, K. (2006). A radical scavenging com- Ruhl, C.E. and Everhart, J.E. (2005). Coffee and tea consumption pound, 3-pyridinol in instant coffee and its heapatoprotective are associated with a lower incidence of chronic liver diseases in activity. Food Sci. Technol. Res., 12, 148-151. the United States. Gastroenterology, 129, 1928-1936. Lee, K.J., Choi, J.H. and Jeong, H.G. (2007). Hepatoprotective and Shi, H., Dong, L., Bai, Y., Zhao, J., Zhang, Y. and Zhang, L. (2009). antioxidant effects of the coffee diterpenes kahweol and cafes- Chlorogeic acid against carbon tetrachloride-induced liver fibro- trol on carbon tetrachloride-induced liver damage in mice. Food sis in rats. Eur. J. Pharmacol., 623, 119-124. Chem. Toxicol., 45, 2118-2125. Takahata, Y., Ohnishi-Kameyama, M., Furuta, S., Takahashi, M. Matsumoto, K. (1955). Transaminase. In “Protein. Nucleic Acid and Suda, I. (2001). Highly polymerized procyanidins in bron and Enzyme”, An extra number No. 24 ’81-11, ed by Ogata, K., soybeans seed coat with a high radical-scavenging activity. J. Ag- Inoue, T., Matsumoto, K. and Uemura, S. Kyouritsu Shuppan, ric. Food Chem., 49, 5843-5847. Tokyo, pp. 94-96 (in Japanese). Zhou, J., Ashoori, F., Suzuki, S., Nishigaki, I. and Yagi, K. (1993). Mohr, U., Althoff, J., Ketkar, M.B., Conradt, P. and Morgareidge, K. Protective effect of chlorogenic acid on lipid peroxidation in- (1984). The influence of caffeine on tumor incipience in Sprague- duced in the liver of rats by carbon tetrachloride or cobalt-60 ir- Dawley rats. Food Chem. Toxicol., 22, 377-382. radiation. J. Clin. Biochem. Nutr., 15, 119-25.