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Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans a
a
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Sari Honda , Yukari Miura , Akiko Masuda & Toshiya Masuda a
Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima, Japan b
Faculty of Human Life Science, Shikoku University, Tokushima, Japan Published online: 15 Aug 2014.
Click for updates To cite this article: Sari Honda, Yukari Miura, Akiko Masuda & Toshiya Masuda (2014) Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans, Bioscience, Biotechnology, and Biochemistry, 78:12, 2110-2116, DOI: 10.1080/09168451.2014.946397 To link to this article: http://dx.doi.org/10.1080/09168451.2014.946397
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 12, 2110–2116
Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans Sari Honda1, Yukari Miura1, Akiko Masuda2 and Toshiya Masuda1,* 1
Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima, Japan; 2Faculty of Human Life Science, Shikoku University, Tokushima, Japan
Received June 5, 2014; accepted July 9, 2014
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http://dx.doi.org/10.1080/09168451.2014.946397
Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee. Key words: cryptochlorogenic lactone; neochlorogenic lactone; chlorogenic acid; roasted coffee beans; xanthine oxidase inhibition
Introduction Coffee is a popular beverage throughout the world. The coffee plant belongs to the Rubiaceae family, and the most economically important species is Coffea arabica (Arabica coffee), the beans of which account for over 60% of global coffee production. The coffee tree is a tropical plant, which grows in the countries between the latitudes of 25°N and 25°S, and over 8000 tons of coffee beans were produced in 2012/ 2013.1) Coffee beverages are made by brewing roasted coffee beans, and most of the flavor and aroma is generated in the roasting process. During roasting, green coffee beans are heated to around 200 °C for several minutes, and chemical reactions that polymerize, colorize, and develop flavors occur. Of late, various healthful effects of coffee beverages have attracted much attention. Numerous investigations of healthful functions of coffee to humans have been carried out.2) Gout was historically referred to as “the king of diseases and the diseases of kings”, but has now been recognized as a lifestyle-related disease.3,4) Rates of gout and related diseases approximately doubled *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
between 1990 and 2010 in USA.5) In Japan, numbers of patients with gout and hyperuricemia have also increased three times during the last 20 years.4) These rises are believed to be because of changes in diet and increase in metabolic diseases.6) In 2007, Choi et al.7) reported remarkable healthful effects of coffee, showing reduced risk of gout in both male and female coffee drinkers.8,9) Gout is an inflammatory disease that is caused by increase in serum crystalline uric acid.10) Uric acid is the final metabolite of purine catabolism in humans and is produced by oxidation of xanthine and hypoxanthine by xanthine oxidase (XO). In their study, Choi and Curhan8) also clarified that caffeine, one of the main constituents of coffee, did not reduce serum uric acid concentrations and suggested that other coffee constituents may have contributed to this effect. Likewise, similar results in epidemiological studies in Japanese middle-age men and women were also reported.11) Green coffee beans contain 56–63% carbohydrate, 11–13% protein, 12–18% lipid, 1% caffeine, and 6–8% polyphenols of their dry weight.13) However, typical XO inhibitors, which probably ameliorate gout, have not yet been discovered. Polyphenol constituents of coffee beans include chlorogenic acid [5-O-caffeoylquinic acid (1)], its isomers [cryptochlorogenic acid (4-O-caffeoylquinic acid, 2) and neochlorogenic acid (3-O-caffeoylquinic acid, 3)],13) and various related phenols such as caffeic acid and ferulic acid (Fig. 1). These polyphenols are not very stable under oxidizing and heating conditions, indicating that the products are produced during the coffee-making process and have altered functions after roasting and brewing. In a recent study, we discovered a very potent XO inhibitor from air-oxidation products of caffeic acid.14) Therefore, we screened for potent XO inhibitors in roasted beans in addition to green and dried (just before roasting process) beans.
Materials and methods Coffee samples, chemicals, and instruments. Green, dried, and roasted coffee beans were purchased from a local coffee trading company (Jet-Coffee Co.
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pre-incubated at 37 °C for 5 min. A XO buffer solution (0.054 unit mL−1, 20 μL) was added to the solution, and after incubation at 37 °C for 10 min, 3% HClO4 aq (25 μL) was added to stop the reaction. Subsequently, 20 μL of the solution was injected into the HPLC system, and uric acid concentrations were determined under the following conditions: column, Daisopak SP120-5-ODS-BP (150 × 4.6 mm i.d., Daiso, Osaka, Japan); solvent, CH3CN–1% phosphoric acid aq = 5:95 (V/V); flow rate, 0.5 mL min−1; temperature, 40 °C; and detection, 290 nm. Percent inhibition was calculated using the following equation: inhibition (%) = [(peak area of uric acid in control experiment) − (peak area of uric acid in sample experiment)] × 100/(peak area of uric acid in control experiment).
Fig. 1. Chemical structures of chlorogenic acids (1–3) and identified lactone derivatives (4–8).
Ltd, Sakai, Japan). The beans had been imported from Brazil as Santos No.2 grade and were processed in the company. Chlorogenic acid (5-O-caffeoylquinic acid) was purchased from Funakoshi (Tokyo, Japan). Cryptochlorogenic acid (4-O-caffeoylquinic acid) and neochlorogenic acid (3-O-caffeoylquinic acid) were synthesized from chlorogenic acid according to the method reported by Nagels et al.15) XO was obtained from Wako Pure Chemicals (Osaka, Japan). A Toyopearl HW-40F gel was purchased from Tosoh (Tokyo, Japan) for size-exclusion chromatography (SEC). Xanthine, uric acid, and Cosmosil 140 C18 OPN for ODS SPE column were obtained from Nacalai Tesque (Kyoto, Japan). HPLC-grade solvents were purchased from Kishida Chemicals (Osaka, Japan). All other solvents and reagents were obtained from Nacalai Tesque. NMR spectra were recorded using an ECS-400 spectrometer (400 MHz for 1H, JEOL, Tokyo, Japan), and ESI-MS spectra were measured using a XEVO-QTOF spectrometer (Waters Japan, Tokyo, Japan). Analytical HPLC was performed using an LC-10 low-pressure gradient system (Shimadzu, Kyoto, Japan) equipped with an SPD-M10 AVP photodiode array detector and a CTO-10ASVP column oven (Shimadzu). Data were analyzed using the Class-M10A software (Shimadzu). Preparative HPLC was performed using an LC-6AD pump (Shimadzu) equipped with a UV-8011 detector (Tosoh). XO inhibitory assay. XO inhibitory assays were performed using the method reported by Masuda et al.16) with slight modifications. In brief, the reaction media comprised 2.2 mmol L−1 xanthine aq (10 μL), test sample in DMSO (10 μL), and 12.5 mmol L−1 phosphate buffer (pH 7.8, 160 μL) and were
Boiling water extraction of green, dried, and roasted coffee beans and HPLC analysis of constituents. Beans were ground using an MJ516 coffee mill (Melita, Tokyo, Japan). Boiling water (5 mL) was added to the powder (1 g), allowed to stand for 1 h, and then filtered. The filtrate was evaporated under reduced pressure to produce coffee bean extracts (150, 177, and 214 mg from roasted beans, dried beans, and green beans, respectively). Constituents of each extract were analyzed using HPLC. Extracts (15 mg) were dissolved in 50% CH3CN–H2O (2 mL) and were passed through a C18 SPE cartridge (Strata C18-E 100 mg, Phenomenex, Torrance, CA, USA) to make 4 mL solution by eluting CH3CN through the cartridge. Aliquots of 10 μL were then injected into the HPLC instrument and analyzed under the following conditions: column, Cosmosil 5C18-AR-II (250 × 4.6 mm i.d.); solvent, A: 1% acetic acid in H2O, B: CH3CN; gradient conditions were linear gradient from 5% solvent B (0 min) to 45% solvent B (40 min) and then to 100% solvent B (50–55 min); flow rate, 1.0 mL min−1; detection, 254, 280, and 320 nm. SEC fractionation of roasted coffee bean extracts. The roasted coffee bean extract (4.5 g) was prepared from 30 g of roasted ground beans and 150 mL of boiling water (150 mL) as described above. Addition of a small amount of CH3OH–H2O (1:1) to the extract (3.0 g) produced a white precipitate. After filtration of the precipitate (426 mg), the filtrate was charged to a HW40F SEC column (150 × 1.6 cm i.d.) and was eluted with CH3OH–H2O (1:1). Eluted solutions of ca. 15 mL were collected and then combined to 7 fractions according to HPLC analysis under the conditions described above. Yields were 149, 240, 919, 1004, 133, 15, and 17 mg in fractions 1, 2, 3, 4, 5, 6, and 7, respectively. LC-MS analysis of the most active fraction of SEC. A 1 mg mL−1 CH3CN–DMSO (1:1) solution containing a sample of fraction 7 was prepared and filtered through Millex-LG (0.2 μm, Waters). Subsequently, 5 μL of sample solution was injected into UPLC (Cosmosil 5C18-ARII (250 × 2.0 mm i.d.)) using Acuity UPLC auto sampler and was eluted with 1%
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acetic acid in H2O–CH3CN (85:15) at a flow rate of 0.5 mL min−1. Photodiode array (PDA) detection of the eluted peaks was performed between 230 and 400 nm. MS analysis was also performed under the following conditions: mode, ESI negative; capillary V, 1.2 kV; cone V, 23 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L h−1; desolvation gas flow, 800 L h−1, MSE low collision energy, 6 V; MSE high collision energy, 20–30 V. Elemental compositions of peak compounds were calculated using high-resolution MS data from deprotonated molecular ions using the MassLynx software (V. 4.1, Waters). Isolation of observed peak compounds from active fraction 7. Roasted coffee beans (1 kg of ground beans) were extracted twice with 5 L and then 2.5 L of boiling water. After cooling, the combined extract solution was charged into a Cosmosil 140 C18-OPN gel column (2 L) and eluted with CH3OH (2 L). The methanol solution was evaporated to give 98 g of brown solid, and 10 g of the solid was purified twice using HW-40F column (1.3 L) eluted with methanol. After evaporation of solvent, the remaining 241 mg of solid contained the compounds that corresponded to the peaks observed in the active fraction 7. Finally, peak compounds B–E were separated using preparative HPLC under the following conditions: column, Cosmosil 5C18-AR-II (250 × 20 mm i.d.); solvent, 1% acetic acid in H2O–CH3CN (85:15); flow rate, 6 mL min−1; and detection, 320 nm. Yields of peak compounds were 17, 8, 21, and 10 mg for peak compounds B–E, respectively. Peak compound B (4), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0766 [M − H]−, 179, 173, 161; 1H-NMR (CD3OD) δ 2.34 (1H, m, H-2a), 2.47 (1H, brd, J = 11.2 Hz, H-2b), 4.71 (1H, brd, J = 4.0 Hz, H-3), 3.94 (1H, br, H-4), 5.11(1H, brd, J = 4.4 Hz, H-5), 2.03(1H, brd, J = 14.4 Hz, H-6ax), 2.38 (1H, dd, J = 14.4 and 4.4 Hz, H-6b), 6.13 (1H, d, J = 16.0 Hz, H-2′), 7.51 (1H, d, J = 16.0 Hz, H-3′), 7.00 (1H, d, J = 2.0 Hz, H-2′′), 6.75 (1H, d, J = 8.0 Hz, H-5′′), 6.91 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′). Peak compound C, HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0757 [M − H]−, 179, 173, 161; major compound C-1 (5): 1H-NMR (CD3OD) δ 2.36 (1H, d, J = 12.6 Hz, H-2a), 2.41 (1H, ddd, J = 12.6, 6.8, and 2.7 Hz, H-2b), 4.80 (1H, overlapped with signal of HOD, H-3), 5.05 (1H, brd, J = 4.4 Hz, H-4), 4.10 (1H, brd, J = 4.4 Hz, H-5), 2.05 (1H, brd, J = 14.0 Hz, H-6a), 2.26 (1H, dd, J = 14.0 and 4.8 Hz, H-6b), 6.27 (1H, d, J = 16.0 Hz, H-2′), 7.58 (1H, d, J = 16.0, H-3′), 7.03 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 8.0 Hz, H-5′′), 6.94 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′); minor compound C-2 (6): readable signals of 1H-NMR (CD3OD) δ 1.72–1.80 (2H, complex, H-2a and H-6a), 2.35–2.36 (1H, complex, H-2b and H-6a), 4.46 (1H, dt, J = 9.9 and 2.2 Hz, H-3), 4.64 (1H, brd, J = 4.5 Hz, H4), 4.48 (1H, dt, J = 10.4 and 4.5 Hz, H-5), 6.24 (1H, d, J = 15.6 Hz, H-2′). Peak compound D (7), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0765 [M − H]−, 179, 161; 1H-NMR (CD3OD) δ 2.06 (1H, t, J = 11.6 Hz, H-2a), 2.16 (1H, ddd, J = 11.6, 6.4, and 2.0 Hz, H-
2b), 4.90 (1H, ddd, J = 11.6, 4.9, and 2.0 Hz, H-3), 4.26 (1H, brt, J = 4.9 Hz, H-4), 4.72 (1H, dd, J = 5.8 and 4.9 Hz, H-5), 2.28 (1H, ddd, J = 11.7, 5.8, and 2.0 Hz, H-6a), 2.53 (1H, d, J = 11.7 Hz, H-6b), 6.30 (1H, d, J = 16.0 Hz, H-2′), 7.60 (1H, d, J = 16.0 Hz, H-3′), 7.02 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 8.0 Hz, H-5′′), 6.93 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′). Peak compound E (8), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0777 [M − H]−, 179, 161; 1H-NMR (CD3OD) δ 1.94 (1H, t, J = 11.2 Hz, H-2a), 2.13 (1H, ddd, J = 11.4, 6.5, 1.5 Hz, H-2b), 3.94 (1H, ddd, J = 11.4, 6.5, and 4.5 Hz, H-3), 4.85 (1H, overlapped with signal of HOD, H-4), 5.28 (1H, brt, J = 4.5 Hz, H-5), 2.36 (2H, m, H-6ab), 6.34 (1H, d, J = 16.5 Hz, H-2′), 7.64 (1H, d, J = 16.5 Hz, H-3′), 7.05 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 9.0 Hz, H-5′′), 6.96 (1H, dd, J = 9.0 and 2.0 Hz, H-6′′).
Results and discussion XO inhibitory activities of green, dried, and roasted coffee bean extracts In many countries, coffee beans are imported as green beans and are then roasted for brewing. In the present study, we obtained three kinds of coffee beans in different stages such as green, heat-dried, and roasted beans from the same origin. The beans were then ground using a coffee mill, and boiling water was used to prepare the extracts. Fig. 2 shows the XO inhibitory activity of each extract at 0.3 and 0.6 mg mL−1. Although two extracts from green and dried beans weakly promoted XO activity at 0.6 mg mL−1, extracts from roasted beans exerted significant XO inhibitory activity in a dose-dependent manner. These data indicate that only roasted beans can ameliorate gout by inhibiting XO, whereas green and dried beans had no XO inhibitory activity. This suggests that the constituents of green and dried beans may not play a role for the amelioration of gout. Coffee beans are consumed as beverages and rarely as raw beans. Therefore, our health benefits of coffee drinking are dependent on roasting and brewing. Therefore, production of potent
Fig. 2. XO inhibitory activities of boiling water extracts (0.3 and 0.6 mg mL−1) from green, dried, and roasted coffee beans. Note: Data are expressed at the mean ± SD (n = 3). *Significant against control (t-test, p < 0.05).
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absorbed from coffee beverages in sufficient quantities to inhibit XO.
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Fig. 3. XO inhibitory activities of eluted fractions (0.15 mg mL−1) from size-exclusion chromatography of roasted coffee bean extract. Note: Data are expressed at the mean ± SD (n = 3). *Significant against control (t-test, p < 0.05).
XO inhibitors from raw bean constituents may arise from the roasting process. Fractionation and XO inhibitory activities of SEC from roasted been extracts Boiling water extracts from roasted coffee beans were expected to contain large amounts of polymeric compounds, which were formed during high-temperature treatments. Initially, we employed SEC to separate constituents according to molecular size. In particular, 3 g of roasted bean extract was eluted in seven fractions using a Toyopearl HW-40F column, and XO inhibitory activities of these fractions were measured (Fig. 3). These experiments demonstrated that the compounds that were eluted earlier with higher molecular weights had increasing effects on XO activity. Although fractions of moderate molecular weight compounds were eluted in the middle of the procedure and showed no significant activity, later eluted fractions showed remarkable potent inhibitory activity. Because high molecular weight substances are poorly absorbed through the human digestive tract, only low-weight molecules with high inhibitory activities may be
LC-PDA and LC-MS analyzes of the most active fraction To obtain information for the structures of active compounds in roasted coffee beans, PDA and MS were used to detect HPLC analysis eluted from the most active fraction (No. 7). Five major peaks were observed in the HPLC profile (Fig. 4). UV spectra of all peaks, which were obtained using a PDA detector, were very similar and had λ max at 320 and 290(sh) nm. This absorption pattern is characteristic of caffeoyl structures, indicating that these peaks corresponded with caffeic acid derivatives. Chlorogenic acid is a well-known caffeic acid derivative and is recognized as a major polyphenol of coffee.17) However, neither chlorogenic acid nor caffeic acid itself were detected in this fraction. HR-MS analyzes of these peaks indicated molecular formulas of C17H20O9 for peak A [m/z 367.1017 (M − H)−], C16H16O8 for peaks B–E [m/z 335.0766 (M − H)− for B, m/z 335.0757 (M − H)− for C, m/z 335.0765 (M − H)− for D, m/z 335.0777 (M − H)− for E]. Both UV spectra and molecular data indicate that (1) these are chlorogenic acid derivatives, (2) the compound in peak A may be a methylated chlorogenic acid, and (3) B–E are dehydrated derivatives of chlorogenic acid. Isolation and structure identification of the XO inhibitory active compounds The peak compounds in fraction 7 were isolated after large-scale purification. Solid-phase extraction of boiling water extracts of roasted coffee beans (1 kg) gave 98 g of methanol-soluble substances, including the peak compounds in fraction 7. Compounds of peaks B–E were collected using HW-40F SEC elution. Compound of peak A was not found in the elution and may be an artifact such as a methyl ester of chlorogenic acids in the previous procedure. Finally, preparative HPLC using an ODS column separated each peak compounds for structure analyzes, and all compounds were
Fig. 4. HPLC analytical profile of active fraction 7 from size-exclusion chromatography of roasted coffee bean extracts. Note: HPLC conditions: column, Cosmosil 5C18-ARII (250 × 2.0 mm i.d.); solvent, 1% acetic acid in H2O–CH3CN (85:15); flow rate of 0.5 mL min−1.
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identical with several chlorogenic acid lactones by comparison of their structure analytical data with reported data, in particular those of 1H NMR.18) Peak compound B (4) has the molecular formula C16H16O8 as estimated using negative high-resolution ESI-MS of peak B. Typical UV absorption maxima at 320 and 290 (sh) nm and MS fragment ions at m/z 179 and 161 suggest the presence of a caffeoyl moiety. 1H NMR data and coupling systems observed in COSY show a proton-coupling pattern of CH2–CH(O–)–CH (O–)–CH(O–)–CH2 in addition to the proton signal set on the caffeoyl moiety, which indicated a typical caffeoxylated quinic acid structure. These data indicate that compound B is similar to chlorogenic acid. However, chemical shifts and coupling constants of protons at oxygenated carbons differed from those of chlorogenic acid. In particular, small coupling constants ( 100 μmol L−1.19,20) In the present study, we measured XO inhibitory activity of chlorogenic acids using our assay system. Initially, XO inhibitory activities of the isolated compounds B–E (4–8), chlorogenic acid (1), and isomers (2 and 3) at relatively high concentrations (1000 μmol L−1) were measured (Table 1). Under these conditions, chlorogenic acid (1), cryptochlorogenic acid (2), and neochlorogenic acid (3) showed very weak XO inhibitory activity, whereas lactone compounds D (7) and E (8) showed remarkable XO inhibitory activities with IC50 values of 210 and 360 μmol L−1, respectively. In contrast, compounds B (4) and C (5 and 6) did not demonstrate strong activities, despite their similar lactone structures. In terms of structure activity relationships, all strongly active compounds carry 1,5lactone structure, whereas weakly active compounds have a lactone moiety at the other position. These data indicate that the lactonization between the 1-carboxylic acid and 5-hydroxy groups is important for XO inhibitory activity, whereas the caffeoyl moiety may be benign. Chlorogenic acid lactones are bitter taste constituents of coffee beverages18) and were found in roasted coffee beans.21) Numerous biological activities of chlorogenic acids and related compounds including their lactones, have been reported.13) These chlorogenic lactones were
XO inhibitory activities of chlorogenic acids, isolated compounds, and allopurinol (positive control).
Compound
a
of 3:1. Proton signals and coupling networks observed in COSY data of major C-1 were very similar to those of B, indicating that C-1 is an isomer of B. The clear difference between C-1 and B was detected in chemical shifts of the H-4. The low field-shifted H-4 proton signal (δ 5.05 ppm) strongly indicates that a caffeoyl group exists at the 4-position. Therefore, compound C-1 (5) has the structure 5. The 1H NMR data of C-2 were difficult to be assigned completely because C-2 existed was a minor compound in peak C. Comparison with reported NMR data for chlorogenic acid lactones18) revealed that compound C-2 was 3-O-caffeoylepi-δ-quinide. Therefore, the structure of compound C-2 is depicted as structure 6 in Fig. 1.
Percent inhibition at 1000 μmol L−1(mean ± SD)a
IC50 (μmol L−1)
22.5 ± 4.5d 16.8 ± 0.9d 32.9 ± 1.8d 38.8 ± 2.7d 50.2 ± 2.7d 81.0 ± 2.4d 70.1 ± 2.3d 53.8 ± 2.8b,d
–c – – – – 210 360 –
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Fig. 5. HPLC analytical profiles of extracts from green, dried, and roasted coffee beans. Note: HPLC conditions: column, Cosmosil 5C18-AR-II (250 × 4.6 mm i.d.); solvent, (A): 1% acetic acid in H2O, (B): CH3CN; gradient conditions [percent of solvent B (time)], 5% (0 min), 45% (40 min), 100% (50–55 min); flow rate, 1.0 mL min−1.
produced from chlorogenic acid during the early stage of the roasting process.22) To confirm the contribution of these chlorogenic lactones to the XO inhibitory activity of the extract of roasted coffee beans, we performed constituent analyzes of three extracts using HPLC. As shown in Fig. 5, the HPLC peaks of chlorogenic lactones (B–E) were clearly observed only in the extract of roasted coffee beans. A large number of minor cinnamoylated quinic acids similar to chlorogenic acid have been found in coffee beans,23) and some of these have XO inhibitory activities.24) Although our assay indicated that XO inhibitory activities of chlorogenic lactones were not very high compared with that of the synthetic XO inhibitor and gout medicine such as allopurinol, the lactones, particularly compounds D (cryptochlorogenic lactone) and E (neochlorogenic lactone), should play an important role in the XO inhibitory activity of the roasted coffee beans because of their clear observation in HPLC of the roasted coffee beans. In conclusions, the present data indicate that roasted coffee beans may ameliorate gout and hyperuricemia by significantly inhibiting XO. In contrast, raw coffee beans appear to lack XO inhibitory activities. Lactone derivatives of chlorogenic acids, particularly crypto- and neochlorogenic lactones, are responsible for the XO inhibitory activity of roasted coffee beans. These lactones are typical components
of roasted beans and are produced from the raw beans during the heating process that is necessary for making coffee beverages.
Acknowledgment This research was supported financially by The All Japan Coffee Association (Tokyo, Japan).
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in roast coffee. Nippon Shokuhin Kogyo Gakkaishi [J. Jpn. Soc. Food Sci. Technol.]. 1975;22:545–548. Frank O, Zehentbauer G, Hofmann T. Bioresponse-guided decomposition of roast coffee beverage and identification of key bitter taste compounds. Eur. Food Res. Technol. 2006;222:492–508. Özyürek M, Bektaşoğlu B, Güçlü K, Apak R. Measurement of xanthine oxidase inhibition activity of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method. Anal. Chim. Acta. 2009;636:42–50. Sarawek S, Feistel B, Pischel I, Butterweck V. Flavonoids of Cynara scolymus possess potent xanthine oxidase inhibitory activity in vitro but are devoid of hyperuricemic effects in rats after oral application. Planta Med. 2008;74:221–227. Schrader K, Kiehne A, Engelhardt UH, Maier HG. Determination of chlorogenic acids with lactones in roasted coffee. J. Sci. Food Agric. 1996;71:392–398. Farah A, Paulis TD, Trugo LC, Martin PR. Effect of roasting on the formation of chlorogenic acid lactones in coffee. J. Agric. Food Chem. 2005;53:1505–1513. Clifford MN, Knight S, Surucu B, Kuhnert N. Characterization by LC-MSn of four new classes of chlorogenic acids in green coffee beans: dimethoxycinnamoylquinic acids, diferuloylquinic acids, caffeoyl-dimetoxycinnamoylquinic acids, and feruloyl-dimethoxycinnamoylquinic acids. J. Agric. Food Chem. 2006;54:1957–1969. Nguyen MTT, Awale S, Tezuka Y, Ueda J, Tran QL, Kadota S. Xanthine oxidase inhibitors from the flowers of Chrysanthemum sinense. Planta Med. 2006;72:46–51.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans a
a
b
a
Sari Honda , Yukari Miura , Akiko Masuda & Toshiya Masuda a
Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima, Japan b
Faculty of Human Life Science, Shikoku University, Tokushima, Japan Published online: 15 Aug 2014.
Click for updates To cite this article: Sari Honda, Yukari Miura, Akiko Masuda & Toshiya Masuda (2014) Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans, Bioscience, Biotechnology, and Biochemistry, 78:12, 2110-2116, DOI: 10.1080/09168451.2014.946397 To link to this article: http://dx.doi.org/10.1080/09168451.2014.946397
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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 12, 2110–2116
Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans Sari Honda1, Yukari Miura1, Akiko Masuda2 and Toshiya Masuda1,* 1
Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima, Japan; 2Faculty of Human Life Science, Shikoku University, Tokushima, Japan
Received June 5, 2014; accepted July 9, 2014
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http://dx.doi.org/10.1080/09168451.2014.946397
Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee. Key words: cryptochlorogenic lactone; neochlorogenic lactone; chlorogenic acid; roasted coffee beans; xanthine oxidase inhibition
Introduction Coffee is a popular beverage throughout the world. The coffee plant belongs to the Rubiaceae family, and the most economically important species is Coffea arabica (Arabica coffee), the beans of which account for over 60% of global coffee production. The coffee tree is a tropical plant, which grows in the countries between the latitudes of 25°N and 25°S, and over 8000 tons of coffee beans were produced in 2012/ 2013.1) Coffee beverages are made by brewing roasted coffee beans, and most of the flavor and aroma is generated in the roasting process. During roasting, green coffee beans are heated to around 200 °C for several minutes, and chemical reactions that polymerize, colorize, and develop flavors occur. Of late, various healthful effects of coffee beverages have attracted much attention. Numerous investigations of healthful functions of coffee to humans have been carried out.2) Gout was historically referred to as “the king of diseases and the diseases of kings”, but has now been recognized as a lifestyle-related disease.3,4) Rates of gout and related diseases approximately doubled *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
between 1990 and 2010 in USA.5) In Japan, numbers of patients with gout and hyperuricemia have also increased three times during the last 20 years.4) These rises are believed to be because of changes in diet and increase in metabolic diseases.6) In 2007, Choi et al.7) reported remarkable healthful effects of coffee, showing reduced risk of gout in both male and female coffee drinkers.8,9) Gout is an inflammatory disease that is caused by increase in serum crystalline uric acid.10) Uric acid is the final metabolite of purine catabolism in humans and is produced by oxidation of xanthine and hypoxanthine by xanthine oxidase (XO). In their study, Choi and Curhan8) also clarified that caffeine, one of the main constituents of coffee, did not reduce serum uric acid concentrations and suggested that other coffee constituents may have contributed to this effect. Likewise, similar results in epidemiological studies in Japanese middle-age men and women were also reported.11) Green coffee beans contain 56–63% carbohydrate, 11–13% protein, 12–18% lipid, 1% caffeine, and 6–8% polyphenols of their dry weight.13) However, typical XO inhibitors, which probably ameliorate gout, have not yet been discovered. Polyphenol constituents of coffee beans include chlorogenic acid [5-O-caffeoylquinic acid (1)], its isomers [cryptochlorogenic acid (4-O-caffeoylquinic acid, 2) and neochlorogenic acid (3-O-caffeoylquinic acid, 3)],13) and various related phenols such as caffeic acid and ferulic acid (Fig. 1). These polyphenols are not very stable under oxidizing and heating conditions, indicating that the products are produced during the coffee-making process and have altered functions after roasting and brewing. In a recent study, we discovered a very potent XO inhibitor from air-oxidation products of caffeic acid.14) Therefore, we screened for potent XO inhibitors in roasted beans in addition to green and dried (just before roasting process) beans.
Materials and methods Coffee samples, chemicals, and instruments. Green, dried, and roasted coffee beans were purchased from a local coffee trading company (Jet-Coffee Co.
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pre-incubated at 37 °C for 5 min. A XO buffer solution (0.054 unit mL−1, 20 μL) was added to the solution, and after incubation at 37 °C for 10 min, 3% HClO4 aq (25 μL) was added to stop the reaction. Subsequently, 20 μL of the solution was injected into the HPLC system, and uric acid concentrations were determined under the following conditions: column, Daisopak SP120-5-ODS-BP (150 × 4.6 mm i.d., Daiso, Osaka, Japan); solvent, CH3CN–1% phosphoric acid aq = 5:95 (V/V); flow rate, 0.5 mL min−1; temperature, 40 °C; and detection, 290 nm. Percent inhibition was calculated using the following equation: inhibition (%) = [(peak area of uric acid in control experiment) − (peak area of uric acid in sample experiment)] × 100/(peak area of uric acid in control experiment).
Fig. 1. Chemical structures of chlorogenic acids (1–3) and identified lactone derivatives (4–8).
Ltd, Sakai, Japan). The beans had been imported from Brazil as Santos No.2 grade and were processed in the company. Chlorogenic acid (5-O-caffeoylquinic acid) was purchased from Funakoshi (Tokyo, Japan). Cryptochlorogenic acid (4-O-caffeoylquinic acid) and neochlorogenic acid (3-O-caffeoylquinic acid) were synthesized from chlorogenic acid according to the method reported by Nagels et al.15) XO was obtained from Wako Pure Chemicals (Osaka, Japan). A Toyopearl HW-40F gel was purchased from Tosoh (Tokyo, Japan) for size-exclusion chromatography (SEC). Xanthine, uric acid, and Cosmosil 140 C18 OPN for ODS SPE column were obtained from Nacalai Tesque (Kyoto, Japan). HPLC-grade solvents were purchased from Kishida Chemicals (Osaka, Japan). All other solvents and reagents were obtained from Nacalai Tesque. NMR spectra were recorded using an ECS-400 spectrometer (400 MHz for 1H, JEOL, Tokyo, Japan), and ESI-MS spectra were measured using a XEVO-QTOF spectrometer (Waters Japan, Tokyo, Japan). Analytical HPLC was performed using an LC-10 low-pressure gradient system (Shimadzu, Kyoto, Japan) equipped with an SPD-M10 AVP photodiode array detector and a CTO-10ASVP column oven (Shimadzu). Data were analyzed using the Class-M10A software (Shimadzu). Preparative HPLC was performed using an LC-6AD pump (Shimadzu) equipped with a UV-8011 detector (Tosoh). XO inhibitory assay. XO inhibitory assays were performed using the method reported by Masuda et al.16) with slight modifications. In brief, the reaction media comprised 2.2 mmol L−1 xanthine aq (10 μL), test sample in DMSO (10 μL), and 12.5 mmol L−1 phosphate buffer (pH 7.8, 160 μL) and were
Boiling water extraction of green, dried, and roasted coffee beans and HPLC analysis of constituents. Beans were ground using an MJ516 coffee mill (Melita, Tokyo, Japan). Boiling water (5 mL) was added to the powder (1 g), allowed to stand for 1 h, and then filtered. The filtrate was evaporated under reduced pressure to produce coffee bean extracts (150, 177, and 214 mg from roasted beans, dried beans, and green beans, respectively). Constituents of each extract were analyzed using HPLC. Extracts (15 mg) were dissolved in 50% CH3CN–H2O (2 mL) and were passed through a C18 SPE cartridge (Strata C18-E 100 mg, Phenomenex, Torrance, CA, USA) to make 4 mL solution by eluting CH3CN through the cartridge. Aliquots of 10 μL were then injected into the HPLC instrument and analyzed under the following conditions: column, Cosmosil 5C18-AR-II (250 × 4.6 mm i.d.); solvent, A: 1% acetic acid in H2O, B: CH3CN; gradient conditions were linear gradient from 5% solvent B (0 min) to 45% solvent B (40 min) and then to 100% solvent B (50–55 min); flow rate, 1.0 mL min−1; detection, 254, 280, and 320 nm. SEC fractionation of roasted coffee bean extracts. The roasted coffee bean extract (4.5 g) was prepared from 30 g of roasted ground beans and 150 mL of boiling water (150 mL) as described above. Addition of a small amount of CH3OH–H2O (1:1) to the extract (3.0 g) produced a white precipitate. After filtration of the precipitate (426 mg), the filtrate was charged to a HW40F SEC column (150 × 1.6 cm i.d.) and was eluted with CH3OH–H2O (1:1). Eluted solutions of ca. 15 mL were collected and then combined to 7 fractions according to HPLC analysis under the conditions described above. Yields were 149, 240, 919, 1004, 133, 15, and 17 mg in fractions 1, 2, 3, 4, 5, 6, and 7, respectively. LC-MS analysis of the most active fraction of SEC. A 1 mg mL−1 CH3CN–DMSO (1:1) solution containing a sample of fraction 7 was prepared and filtered through Millex-LG (0.2 μm, Waters). Subsequently, 5 μL of sample solution was injected into UPLC (Cosmosil 5C18-ARII (250 × 2.0 mm i.d.)) using Acuity UPLC auto sampler and was eluted with 1%
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acetic acid in H2O–CH3CN (85:15) at a flow rate of 0.5 mL min−1. Photodiode array (PDA) detection of the eluted peaks was performed between 230 and 400 nm. MS analysis was also performed under the following conditions: mode, ESI negative; capillary V, 1.2 kV; cone V, 23 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L h−1; desolvation gas flow, 800 L h−1, MSE low collision energy, 6 V; MSE high collision energy, 20–30 V. Elemental compositions of peak compounds were calculated using high-resolution MS data from deprotonated molecular ions using the MassLynx software (V. 4.1, Waters). Isolation of observed peak compounds from active fraction 7. Roasted coffee beans (1 kg of ground beans) were extracted twice with 5 L and then 2.5 L of boiling water. After cooling, the combined extract solution was charged into a Cosmosil 140 C18-OPN gel column (2 L) and eluted with CH3OH (2 L). The methanol solution was evaporated to give 98 g of brown solid, and 10 g of the solid was purified twice using HW-40F column (1.3 L) eluted with methanol. After evaporation of solvent, the remaining 241 mg of solid contained the compounds that corresponded to the peaks observed in the active fraction 7. Finally, peak compounds B–E were separated using preparative HPLC under the following conditions: column, Cosmosil 5C18-AR-II (250 × 20 mm i.d.); solvent, 1% acetic acid in H2O–CH3CN (85:15); flow rate, 6 mL min−1; and detection, 320 nm. Yields of peak compounds were 17, 8, 21, and 10 mg for peak compounds B–E, respectively. Peak compound B (4), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0766 [M − H]−, 179, 173, 161; 1H-NMR (CD3OD) δ 2.34 (1H, m, H-2a), 2.47 (1H, brd, J = 11.2 Hz, H-2b), 4.71 (1H, brd, J = 4.0 Hz, H-3), 3.94 (1H, br, H-4), 5.11(1H, brd, J = 4.4 Hz, H-5), 2.03(1H, brd, J = 14.4 Hz, H-6ax), 2.38 (1H, dd, J = 14.4 and 4.4 Hz, H-6b), 6.13 (1H, d, J = 16.0 Hz, H-2′), 7.51 (1H, d, J = 16.0 Hz, H-3′), 7.00 (1H, d, J = 2.0 Hz, H-2′′), 6.75 (1H, d, J = 8.0 Hz, H-5′′), 6.91 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′). Peak compound C, HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0757 [M − H]−, 179, 173, 161; major compound C-1 (5): 1H-NMR (CD3OD) δ 2.36 (1H, d, J = 12.6 Hz, H-2a), 2.41 (1H, ddd, J = 12.6, 6.8, and 2.7 Hz, H-2b), 4.80 (1H, overlapped with signal of HOD, H-3), 5.05 (1H, brd, J = 4.4 Hz, H-4), 4.10 (1H, brd, J = 4.4 Hz, H-5), 2.05 (1H, brd, J = 14.0 Hz, H-6a), 2.26 (1H, dd, J = 14.0 and 4.8 Hz, H-6b), 6.27 (1H, d, J = 16.0 Hz, H-2′), 7.58 (1H, d, J = 16.0, H-3′), 7.03 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 8.0 Hz, H-5′′), 6.94 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′); minor compound C-2 (6): readable signals of 1H-NMR (CD3OD) δ 1.72–1.80 (2H, complex, H-2a and H-6a), 2.35–2.36 (1H, complex, H-2b and H-6a), 4.46 (1H, dt, J = 9.9 and 2.2 Hz, H-3), 4.64 (1H, brd, J = 4.5 Hz, H4), 4.48 (1H, dt, J = 10.4 and 4.5 Hz, H-5), 6.24 (1H, d, J = 15.6 Hz, H-2′). Peak compound D (7), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0765 [M − H]−, 179, 161; 1H-NMR (CD3OD) δ 2.06 (1H, t, J = 11.6 Hz, H-2a), 2.16 (1H, ddd, J = 11.6, 6.4, and 2.0 Hz, H-
2b), 4.90 (1H, ddd, J = 11.6, 4.9, and 2.0 Hz, H-3), 4.26 (1H, brt, J = 4.9 Hz, H-4), 4.72 (1H, dd, J = 5.8 and 4.9 Hz, H-5), 2.28 (1H, ddd, J = 11.7, 5.8, and 2.0 Hz, H-6a), 2.53 (1H, d, J = 11.7 Hz, H-6b), 6.30 (1H, d, J = 16.0 Hz, H-2′), 7.60 (1H, d, J = 16.0 Hz, H-3′), 7.02 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 8.0 Hz, H-5′′), 6.93 (1H, dd, J = 8.0 and 2.0 Hz, H-6′′). Peak compound E (8), HR-ESI-MS calcd for C16H15O8, 335.0767, found m/z 335.0777 [M − H]−, 179, 161; 1H-NMR (CD3OD) δ 1.94 (1H, t, J = 11.2 Hz, H-2a), 2.13 (1H, ddd, J = 11.4, 6.5, 1.5 Hz, H-2b), 3.94 (1H, ddd, J = 11.4, 6.5, and 4.5 Hz, H-3), 4.85 (1H, overlapped with signal of HOD, H-4), 5.28 (1H, brt, J = 4.5 Hz, H-5), 2.36 (2H, m, H-6ab), 6.34 (1H, d, J = 16.5 Hz, H-2′), 7.64 (1H, d, J = 16.5 Hz, H-3′), 7.05 (1H, d, J = 2.0 Hz, H-2′′), 6.76 (1H, d, J = 9.0 Hz, H-5′′), 6.96 (1H, dd, J = 9.0 and 2.0 Hz, H-6′′).
Results and discussion XO inhibitory activities of green, dried, and roasted coffee bean extracts In many countries, coffee beans are imported as green beans and are then roasted for brewing. In the present study, we obtained three kinds of coffee beans in different stages such as green, heat-dried, and roasted beans from the same origin. The beans were then ground using a coffee mill, and boiling water was used to prepare the extracts. Fig. 2 shows the XO inhibitory activity of each extract at 0.3 and 0.6 mg mL−1. Although two extracts from green and dried beans weakly promoted XO activity at 0.6 mg mL−1, extracts from roasted beans exerted significant XO inhibitory activity in a dose-dependent manner. These data indicate that only roasted beans can ameliorate gout by inhibiting XO, whereas green and dried beans had no XO inhibitory activity. This suggests that the constituents of green and dried beans may not play a role for the amelioration of gout. Coffee beans are consumed as beverages and rarely as raw beans. Therefore, our health benefits of coffee drinking are dependent on roasting and brewing. Therefore, production of potent
Fig. 2. XO inhibitory activities of boiling water extracts (0.3 and 0.6 mg mL−1) from green, dried, and roasted coffee beans. Note: Data are expressed at the mean ± SD (n = 3). *Significant against control (t-test, p < 0.05).
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absorbed from coffee beverages in sufficient quantities to inhibit XO.
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Fig. 3. XO inhibitory activities of eluted fractions (0.15 mg mL−1) from size-exclusion chromatography of roasted coffee bean extract. Note: Data are expressed at the mean ± SD (n = 3). *Significant against control (t-test, p < 0.05).
XO inhibitors from raw bean constituents may arise from the roasting process. Fractionation and XO inhibitory activities of SEC from roasted been extracts Boiling water extracts from roasted coffee beans were expected to contain large amounts of polymeric compounds, which were formed during high-temperature treatments. Initially, we employed SEC to separate constituents according to molecular size. In particular, 3 g of roasted bean extract was eluted in seven fractions using a Toyopearl HW-40F column, and XO inhibitory activities of these fractions were measured (Fig. 3). These experiments demonstrated that the compounds that were eluted earlier with higher molecular weights had increasing effects on XO activity. Although fractions of moderate molecular weight compounds were eluted in the middle of the procedure and showed no significant activity, later eluted fractions showed remarkable potent inhibitory activity. Because high molecular weight substances are poorly absorbed through the human digestive tract, only low-weight molecules with high inhibitory activities may be
LC-PDA and LC-MS analyzes of the most active fraction To obtain information for the structures of active compounds in roasted coffee beans, PDA and MS were used to detect HPLC analysis eluted from the most active fraction (No. 7). Five major peaks were observed in the HPLC profile (Fig. 4). UV spectra of all peaks, which were obtained using a PDA detector, were very similar and had λ max at 320 and 290(sh) nm. This absorption pattern is characteristic of caffeoyl structures, indicating that these peaks corresponded with caffeic acid derivatives. Chlorogenic acid is a well-known caffeic acid derivative and is recognized as a major polyphenol of coffee.17) However, neither chlorogenic acid nor caffeic acid itself were detected in this fraction. HR-MS analyzes of these peaks indicated molecular formulas of C17H20O9 for peak A [m/z 367.1017 (M − H)−], C16H16O8 for peaks B–E [m/z 335.0766 (M − H)− for B, m/z 335.0757 (M − H)− for C, m/z 335.0765 (M − H)− for D, m/z 335.0777 (M − H)− for E]. Both UV spectra and molecular data indicate that (1) these are chlorogenic acid derivatives, (2) the compound in peak A may be a methylated chlorogenic acid, and (3) B–E are dehydrated derivatives of chlorogenic acid. Isolation and structure identification of the XO inhibitory active compounds The peak compounds in fraction 7 were isolated after large-scale purification. Solid-phase extraction of boiling water extracts of roasted coffee beans (1 kg) gave 98 g of methanol-soluble substances, including the peak compounds in fraction 7. Compounds of peaks B–E were collected using HW-40F SEC elution. Compound of peak A was not found in the elution and may be an artifact such as a methyl ester of chlorogenic acids in the previous procedure. Finally, preparative HPLC using an ODS column separated each peak compounds for structure analyzes, and all compounds were
Fig. 4. HPLC analytical profile of active fraction 7 from size-exclusion chromatography of roasted coffee bean extracts. Note: HPLC conditions: column, Cosmosil 5C18-ARII (250 × 2.0 mm i.d.); solvent, 1% acetic acid in H2O–CH3CN (85:15); flow rate of 0.5 mL min−1.
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identical with several chlorogenic acid lactones by comparison of their structure analytical data with reported data, in particular those of 1H NMR.18) Peak compound B (4) has the molecular formula C16H16O8 as estimated using negative high-resolution ESI-MS of peak B. Typical UV absorption maxima at 320 and 290 (sh) nm and MS fragment ions at m/z 179 and 161 suggest the presence of a caffeoyl moiety. 1H NMR data and coupling systems observed in COSY show a proton-coupling pattern of CH2–CH(O–)–CH (O–)–CH(O–)–CH2 in addition to the proton signal set on the caffeoyl moiety, which indicated a typical caffeoxylated quinic acid structure. These data indicate that compound B is similar to chlorogenic acid. However, chemical shifts and coupling constants of protons at oxygenated carbons differed from those of chlorogenic acid. In particular, small coupling constants ( 100 μmol L−1.19,20) In the present study, we measured XO inhibitory activity of chlorogenic acids using our assay system. Initially, XO inhibitory activities of the isolated compounds B–E (4–8), chlorogenic acid (1), and isomers (2 and 3) at relatively high concentrations (1000 μmol L−1) were measured (Table 1). Under these conditions, chlorogenic acid (1), cryptochlorogenic acid (2), and neochlorogenic acid (3) showed very weak XO inhibitory activity, whereas lactone compounds D (7) and E (8) showed remarkable XO inhibitory activities with IC50 values of 210 and 360 μmol L−1, respectively. In contrast, compounds B (4) and C (5 and 6) did not demonstrate strong activities, despite their similar lactone structures. In terms of structure activity relationships, all strongly active compounds carry 1,5lactone structure, whereas weakly active compounds have a lactone moiety at the other position. These data indicate that the lactonization between the 1-carboxylic acid and 5-hydroxy groups is important for XO inhibitory activity, whereas the caffeoyl moiety may be benign. Chlorogenic acid lactones are bitter taste constituents of coffee beverages18) and were found in roasted coffee beans.21) Numerous biological activities of chlorogenic acids and related compounds including their lactones, have been reported.13) These chlorogenic lactones were
XO inhibitory activities of chlorogenic acids, isolated compounds, and allopurinol (positive control).
Compound
a
of 3:1. Proton signals and coupling networks observed in COSY data of major C-1 were very similar to those of B, indicating that C-1 is an isomer of B. The clear difference between C-1 and B was detected in chemical shifts of the H-4. The low field-shifted H-4 proton signal (δ 5.05 ppm) strongly indicates that a caffeoyl group exists at the 4-position. Therefore, compound C-1 (5) has the structure 5. The 1H NMR data of C-2 were difficult to be assigned completely because C-2 existed was a minor compound in peak C. Comparison with reported NMR data for chlorogenic acid lactones18) revealed that compound C-2 was 3-O-caffeoylepi-δ-quinide. Therefore, the structure of compound C-2 is depicted as structure 6 in Fig. 1.
Percent inhibition at 1000 μmol L−1(mean ± SD)a
IC50 (μmol L−1)
22.5 ± 4.5d 16.8 ± 0.9d 32.9 ± 1.8d 38.8 ± 2.7d 50.2 ± 2.7d 81.0 ± 2.4d 70.1 ± 2.3d 53.8 ± 2.8b,d
–c – – – – 210 360 –
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Fig. 5. HPLC analytical profiles of extracts from green, dried, and roasted coffee beans. Note: HPLC conditions: column, Cosmosil 5C18-AR-II (250 × 4.6 mm i.d.); solvent, (A): 1% acetic acid in H2O, (B): CH3CN; gradient conditions [percent of solvent B (time)], 5% (0 min), 45% (40 min), 100% (50–55 min); flow rate, 1.0 mL min−1.
produced from chlorogenic acid during the early stage of the roasting process.22) To confirm the contribution of these chlorogenic lactones to the XO inhibitory activity of the extract of roasted coffee beans, we performed constituent analyzes of three extracts using HPLC. As shown in Fig. 5, the HPLC peaks of chlorogenic lactones (B–E) were clearly observed only in the extract of roasted coffee beans. A large number of minor cinnamoylated quinic acids similar to chlorogenic acid have been found in coffee beans,23) and some of these have XO inhibitory activities.24) Although our assay indicated that XO inhibitory activities of chlorogenic lactones were not very high compared with that of the synthetic XO inhibitor and gout medicine such as allopurinol, the lactones, particularly compounds D (cryptochlorogenic lactone) and E (neochlorogenic lactone), should play an important role in the XO inhibitory activity of the roasted coffee beans because of their clear observation in HPLC of the roasted coffee beans. In conclusions, the present data indicate that roasted coffee beans may ameliorate gout and hyperuricemia by significantly inhibiting XO. In contrast, raw coffee beans appear to lack XO inhibitory activities. Lactone derivatives of chlorogenic acids, particularly crypto- and neochlorogenic lactones, are responsible for the XO inhibitory activity of roasted coffee beans. These lactones are typical components
of roasted beans and are produced from the raw beans during the heating process that is necessary for making coffee beverages.
Acknowledgment This research was supported financially by The All Japan Coffee Association (Tokyo, Japan).
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