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Isolation of coffee diterpenes by means of high-speed countercurrent chromatography
Journal of Food Composition and Analysis 22 (2009) 233–237
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Original Article
Isolation of coffee diterpenes by means of high-speed countercurrent chromatography Heike Scharnhop, Peter Winterhalter * Institute of Food Chemistry, University of Braunschweig, Institute of Technology, Schleinitzstrasse 20, 38106 Braunschweig, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2007 Received in revised form 1 August 2008 Accepted 24 October 2008
Many different physiological activities have been ascribed to coffee diterpenes, such as cholesterol increasing activity for the diterpenes kahweol and cafestol. Another member of this class, namely the diterpene 16-O-methylcafestol, is used as a marker substance for robusta coffee. Up to now, methodologies for the preparative isolation of these key coffee ingredients are still limited. In this study, high-speed countercurrent chromatography (HSCCC) was successfully employed for the isolation and purification of different diterpenes (i.e. kahweol, cafestol, 16-O-methylkahweol, 16-O-methylcafestol, dehydrokahweol, and dehydrocafestol) from Coffea arabica and Coffea canephora var. robusta. The solvent systems consisted of hexane–ethyl acetate–ethanol–water mixtures. Identity and purity of the isolated compounds were confirmed by high-performance liquid chromatography with photo diode array detection (HPLC-PDA) and HPLC–multiple mass spectrometry (HPLC–MSn) as well as NMR measurements. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Coffee 16-O-methylcafestol Cafestol Kahweol Coffea canephora var. robusta Diterpene High-speed countercurrent chromatography Bioactive non-nutrients Food quality Food analysis Food composition
1. Introduction Coffee is one of the most popular beverages in the world, with an annual consumption of 144 L per capita in Germany in the year 2006. Of major importance for the worldwide coffee production are the two coffee species arabica and canephora var. robusta. Many different physiological effects are associated with coffee consumption, and many of them are likely to be related to compounds of the coffee oil, namely the diterpenes. Major diterpenes of coffee are kahweol and cafestol (Fig. 1). In the coffee oil the diterpenes are mostly esterified with various fatty acids and only a small amount of the diterpenes is present in the free form. Various studies have shown high physiological activity for kahweol and cafestol, and both beneficial and harmful effects have been reported. On the one hand, these compounds are considered to increase serum cholesterol level (Bak and Grobbee, 1989; Weusten-Van der Wouw et al., 1994); on the other hand, some beneficial effects, such as enhanced glutathione S-transferase activity (detoxifying enzyme system) (Lam et al., 1987) or protection against benz[a]pyrene- and aflatoxin B1-induced
* Corresponding author. Tel.: +49 531 391 7200; fax: +49 531 391 7230. E-mail address: [email protected] (P. Winterhalter). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.10.018
genotoxicity are known (Cavin et al., 2001, 2003). These effects depend on the amount of kahweol and cafestol present in the coffee brew and consequently, on the way coffee is prepared. The highest content of diterpenes was found in boiled, unfiltered coffee brews. The content of diterpenes in drip-filtered coffee is negligible (Urgert et al., 1995). Apart from the physiological effects, diterpenes are also important marker compounds for coffee processing, e.g. free kahweol in the case of steaming treatment (Speer and Kurt, 2001) as well as for coffee authenticity (16-O-methylcafestol, 16-OMC) (Speer et al., 1992). One unit of arabica costs almost twice as much as the same unit of robusta coffee. Often the high-quality arabicas, described as ‘‘100% arabica’’ or ‘‘Highland coffee’’, are mixed with the less expensive robustas. Therefore it is important to control the authenticity of the products with appropriate methods enabling a differentiation of the two species. Robusta beans are smaller and rounder than arabica beans. These differences persist during the roasting process. Hence a differentiation by comparison of the bean-size before grinding is possible. Also quantities over 15% of robusta in arabica blends can be detected by trained coffee tasters in sensory testing (Speer et al., 1992). The most important method for discrimination, however, is the analysis of the coffee ingredients. Many different methods have been described to discriminate between arabica
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Fig. 1. Structures of coffee diterpenes isolated by HSCCC.
and robusta, e.g. by the presence of different metals (Martı´n et al., 1998), trigonelline and caffeine (Casal et al., 2000), amino acid enantiomers (Casal et al., 2003), biogenic amines (Casal et al., 2004), sterol fraction (Valdenebro et al., 1999) or the ratio between sterols and diterpenes (Frega et al., 1994). However, the most suitable indicator for robusta coffees is 16-O-methylcafestol (1). A validated method by high-performance liquid chromatography enables the detection of up to 2% of robusta in arabica blends (DIN 10779, 1999). By using more selective detectors like LC–MSn in the multiple reaction monitoring mode (MRM), trace amounts of approx. 0.2% robusta can be detected (Bonnla¨nder et al., 2007). For an accurate analysis of coffee authenticity, 16-OMC is required as a standard compound. In the frame of this study a preparative method for the isolation of 16-O-methylcafestol from robusta coffee by high-speed countercurrent chromatography (HSCCC) is reported. HSCCC is an all liquid chromatographic technique that operates under gentle conditions and allows non-destructive isolation even of labile natural compounds. Due to the absence of any solid stationary phase, adsorption losses are minimised and hence a 100% sample recovery is guaranteed (Ito, 2005). Many different substance-classes have been successfully separated by means of HSCCC, e.g. triterpenes like steroids (Lee et al., 1988). Diterpene isolation so far – for example, by means of preparative RP-HPLC – has been very time-consuming (Lam et al., 1982; Pettitt, 1987; Bertholet, 1987). Isolation of diterpenes using HSCCC, on the other hand, takes advantage of the high sample load (1 g per separation), the use of inexpensive solvents and the high purity of the isolated substances.
In addition to 16-O-methylcafestol, the diterpenes 16-Omethylkahweol 2 (robusta, green), cafestol 3 and kahweol 4 (arabica and robusta, green and roasted), and the roasting products dehydrocafestol 5 and dehydrokahweol 6 (arabica and robusta, roasted) were also accessible by HSCCC separations. 2. Materials and methods For the isolation of diterpenes Coffea arabica (Mexico) and Coffea canephora var. robusta (India) were used. From both coffee samples green as well as roasted beans were examined. 2.1. Extraction and saponification of coffee samples A 500 g amount of green or roasted coffee beans was ground and extracted with methyl tert-butyl ether (MTBE) at 80 8C in a Soxhlet apparatus over 5 h. After evaporating the solvent in vacuo, the remaining coffee oil was saponified with 1 L of an ethanolic potassium hydroxide solution (10%) at 90 8C for 4 h. After removing the alcohol, the residue was solubilised in water (1 L, 70 8C) and 250 mL of sodium chloride solution (10%) and extracted with MTBE (3 400 mL). The organic phase was washed with 1 L of an aqueous sodium chloride solution (2%) and dried over sodium sulfate. The solvent was evaporated in vacuo and the residues were used for the separation by high-speed countercurrent chromatography. 2.2. High-speed countercurrent chromatography (HSCCC) A high-speed countercurrent chromatograph manufactured by Pharma-Tech Research (Baltimore, MD, USA) was equipped
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with three coils (2.6 mm internal diameter of the tubing) to give a final volume of the liquid phase of 850 mL. The separations were run at a revolution speed of 800 rpm. The mobile phase was delivered by a Biotronik HPLC pump BT 3020 (Jasco, GroßUmstadt, Germany). The sample (unsaponifiable matter of different coffee samples) was dissolved in 20 mL of a 1:1 mixture of the upper and lower phase of the solvent system. The amount of sample injected was around 1 g. Elution was monitored with a UV–vis detector (Knauer, Berlin, Germany) at l = 220 nm. The solvent system used for the separation of the unsaponifiable matter consisted of hexane–ethyl acetate–ethanol–water (5:2:5:2. v/v/v/v; the less dense layer acting as the stationary phase, flow rate: 4.0 mL/min, head-to-tail mode; all chemicals were of analytical grade). Sixteen milliliter fractions were collected with an automatic collector. 2.3. Thin-layer chromatography (TLC) First control of identity and purity of the obtained HSCCCfractions was made by TLC. The stationary phase was silica gel 60 F254 from Merck (Darmstadt, Germany), the mobile phase diethyl ether–chloroform (1:1). After development, the TLC plate was dried and sprayed with a solution of 1 g vanillin in 250 g ethanol acidified with 20 mL of sulphuric acid (conc.). Red and blue spots were visible. 2.4. HPLC-PDA analysis HPLC-PDA analyses were performed on an MD-910 multiwavelength detector (220–650 nm), equipped with a DG-980-50 three-line degasser, a LG-980-02 ternary gradient unit, a PU-980 Intelligent HPLC pump, and Borwin PDA chromatography software (Jasco, Germany). Diterpenes were detected at 220 and 280 nm. The samples were injected via a Rheodyne 7125 injection valve (Techlab, Germany) equipped with a 20 mL loop, and separations were carried out on a 250 mm 4.6 mm i.d., 5 mm, Synergi MaxRP 80A column (Phenomenex, Germany) at a flow rate of 0.8 mL/min. A two solvent system was used. Solvent A: acetonitrile, solvent B: water. The linear gradient was as follows: 0 min, 40% B; 20 min, 30% B; 40 min, 0% B; 45 min, 0% B; 46 min, 40% B. 2.5. HPLC–ESI-MSn HPLC–MSn analyses of fractions and purified diterpenes were performed on a Bruker Esquire LC–MS system (Bruker Daltonik, Germany). The HPLC system consisted of a System 1100 binary pump G1312A (Agilent, Germany) and a Lichrograph L-4000 UV–vis detector (Merck Hitachi, Japan). UV chromatograms were recorded with a Chromatopac C-R6A integrator (Shimadzu, Japan). The LC part of the system was controlled by ChemStation version A.06.01, and MS data were processed by Esquire NT 4.0 software (Bruker Daltonik). MS parameters: positive ion mode; capillary, 2500 V; capillary exit offset, 70 V; end plate offset, 500 V; skimmer 1, 25 V; skimmer 2, 10 V; dry gas, N2, 11 L/ min; dry temperature, 355 8C; nebulizer, 60 psi; and scan range, 50–2200 m/z; chromatographic conditions were as described above.
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3. Results and discussion According to their provenance, Coffea canephora var. robusta beans contain between 0.6 and 1.8 g 16-O-methylcafestol (16OMC) 1 per kg. Due to the high stability of 16-OMC, the content in crude and roasted coffee is almost the same. Therefore this diterpene is an ideal marker for robusta coffee (Speer et al., 1992). In coffee diterpenes are hardly present in the free form. They are mostly esterified with fatty acids like palmitic and linolic acid (Kurzrock and Speer, 2001). For an enrichment of diterpenes, the coffee oil has to be saponified with ethanolic potassium hydroxide solution (10%) and afterwards extracted with organic solvent according to the validated method for the quantification of 16OMC by HPLC (DIN 10779, 1999). For the isolation of 16-OMC, the unsaponifiable matter of the coffee oil was fractionated by means of HSCCC. In order to obtain suitable solvent systems for HSCCC, the partition coefficient K of the sample was monitored by TLC. Suitable partition coefficients for HSCCC are around K = 1, i.e. the sample is soluble in the lighter phase as well as in the more dense phase of the two phase solvent system (Degenhardt, 2002). All cafestol derivates exhibit under the used TLC conditions red spots and can be distinguished from kahweol derivates which appear in blue colour. TLC of the unsaponifiable matter of arabica and robusta shows red spots at Rf 0.15 indicating cafestol 3. Furthermore, the arabica sample has a blue spot at Rf 0.14 indicating kahweol 4 which is not detectable by TLC in robusta coffee due to its low content. Robusta coffee shows a red spot at Rf 0.45 which is absent in arabica coffee. This marks 16-O-methylcafestol 1. TLC of roasted arabica and robusta samples show more spots at Rf 0.60. In arabica red and blue bands could be recognised, whereas robusta coffee only shows the red one. These signals indicate the roasting products dehydrocafestol 5 and dehydrokahweol 6. 3.1. Isolation and purification of 16-O-methylcafestol (1) and 16-Omethylkahweol (2) by HSCCC In order to find a suitable solvent system some nonpolar two phase solvent mixtures containing hexane, ethyl acetate, methanol or ethanol and water in different ratios were tested and the partition coefficient K was determined by TLC (Ito, 2005). Mixtures of these solvents were used, e.g. for the separation of nonpolar natural compounds like steroids (Lee et al., 1988). As suitable solvent system (K 1) for the isolation of 16-O-methylcafestol 1 hexane–ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v) was elaborated. Fig. 2 shows the preparative HSCCC separation of 1 g of the unsaponifiable matter from green robusta coffee. First characterisation of isolated fractions was performed by means of TLC. 16-Omethylcafestol 1 could be detected by a red spot at Rf 0.45 in fractions eluting approximately after 3 h. Some previous fractions show a blue spot at similar Rf. In these fractions 16-O-methylkahweol 2 could be enriched. The identity of all isolated diterpenes was besides the TLC monitoring confirmed by means of UV–vis, LC–MS/MS (positive mode), and 1D/2D-NMR experiments (1H, 13C, DEPT, HMBC, HMQC). In one HSCCC run 120 mg of 16-O-methylcafestol 1 could be obtained. The purity was around 95% (HPLC). A final purification (>99%) was achieved by preparative HPLC.
2.6. Nuclear magnetic resonance (NMR) spectroscopy 3.2. Isolation and purification of cafestol (3) and kahweol (4) 1
H, 13C, DEPT and 1H–1H-COSY spectra were measured on a Bruker AMX 300 spectrometer (Bruker Biospin, Germany) at 300.1 and 75.4 MHz, respectively. HMQC and HMBC experiments were performed on a Bruker AM 600 instrument. Solvent was chloroform-d1 (Merck, Darmstadt, Germany).
The content of kahweol in robusta coffee is very low. Hence, for the isolation of this diterpene green arabica coffee beans were separated by means of HSCCC using the solvent system hexane– ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v). Under the condi-
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Fig. 2. HSCCC separation of the unsaponifiable matter of green robusta coffee for the isolation of 16-O-methylcafestol. Solvent system: hexane–ethyl acetate–ethanol– water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm, retention of stationary phase: 29.3%.
Fig. 4. HSCCC separation of the unsaponifiable matter of roasted arabica coffee for the isolation of dehydrokahweol and dehydrocafestol. Solvent system: hexane– ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm; retention of stationary phase: 26.1%.
tions employed, partition coefficient K for kahweol was 0.5 and for cafestol 0.55. The HSCCC chromatogram is shown in Fig. 3. Analysis of separated HSCCC fractions was made by TLC. The chromatogram recorded at 220 nm shows a main peak eluting after 1.5 h. This peak contains kahweol as well as cafestol. Blue spots at Rf 0.14 for kahweol 4 could be detected by TLC in the first peak fractions. Some later fractions showed blue and red spots. In these fractions a mixture of kahweol 4 and cafestol 3 was eluted. At a retention time of 2 h the fractions of this first peak contained pure cafestol 3. Cafestol and kahweol partly co-eluted and approximately 35% of total cafestol/kahweol amount was obtained as a mixture. Hence the yield of separated kahweol and cafestol was around 65%. Purity of isolated kahweol was 88%, and of cafestol >92% (HPLC) and was raised by preparative HPLC up to 99%.
Under the same HSCCC conditions as described before, the K value for dehydrokahweol was 1.1 and for dehydrocafestol 1.3. Fig. 4 shows the obtained chromatogram of this HSCCC separation. TLC analysis gave blue spots at Rf 0.6 in fractions after a retention time of approximately 3.5 h and red spots at Rf 0.6 in fractions collected after 4 h. Isolated fractions were analysed by LC–MS and the presence of kahweol and cafestol was confirmed. Purity of dehydrokahweol 6 after HSCCC separation was around 87% (HPLC). Dehydrocafestol 5 could be isolated in a purity of 92% (HPLC). Both compounds were further cleaned up by preparative HPLC. In summary, HSCCC allowed the isolation of six diterpenes from the unsaponifiable matter of green and roasted coffee beans. Over 100 mg of 16-O-methylcafestol (purity 95%), a marker substance for robusta coffee, could be obtained by the HSCCC fractionation of 1 g of unsaponifiable matter of robusta coffee.
3.3. Isolation and purification of dehydrocafestol (5) and dehydrokahweol (6)
3.4. Analytical data of isolated compounds
Dehydrokahweol and dehydrocafestol are roasting products of kahweol and cafestol (cf. Fig. 1). Formation of these dehydration products is due to the degradation of kahweol and cafestol and depends on the length and temperature of the roasting process (Ko¨lling-Speer et al., 1997). Hence, for the isolation of dehydrocafestol 5 and dehydrokahweol 6, the unsaponifiable matter of roasted arabica coffee was used.
Fig. 3. HSCCC separation of the unsaponifiable matter of green arabica coffee for the isolation of kahweol and cafestol. Solvent system: hexane–ethyl acetate–ethanol– water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm; retention of stationary phase: 25.6%.
For coffee diterpenes only a few 1H NMR and 13C NMR data have been reported in the literature so far (Lam et al., 1982; Pettitt, 1987; Corey and Xiang, 1987; Tewis et al., 1993). With this preparative method, it was possible to complete the existing data of 16-O-methylcafestol 1, cafestol 3, and kahweol 4 and present new ones for 16-O-methylkahweol 2, dehydrocafestol 5, and dehydrokahweol 6. 16-O-Methylcafestol (1) (numbering of the atoms refers to the structure given in Fig. 1), C21H30O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 223 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 331, 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.23 (1H, d, J = 1.5 Hz, H19), 6.20 (1H, d, J = 1.6 Hz, H18), 3.77 (2H, s, H17), 3.17 (3H, s, H21), 2.62 (2H, dt, J = 2.8, 5.8 Hz, H2), 2.30 (1H, dd, J = 2.3, 4.9 Hz, H9), 2.25 (1H, dd, J = 2.3, 4.8 Hz, H13), 2.07 (2H, m, H1), 2.00 (1H, d, J = 13.4 Hz, H14a), 1.82 (1H, q, J = 2.9 Hz, H6a), 1.78 (1H, q, J = 3.1 Hz, H6b), 1.75–1.47 (8H, m, H7, H11, H12, H14b, H15a), 1.43 (1H, dd, J = 1.9, 14.6 Hz, H15b), 1.30–1.18 (1H, m, H5), 0.83 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3d1, ppm): d = 148.8 (C, C3), 140.5 (CH, C19), 120.2 (C, C4), 108.2 (CH, C18), 87.0 (C, C16), 60.5 (CH2, C17), 52.2 (CH, C5), 49.2 (CH2, C15), 48.9 (CH3, C21), 44.4 (C, C8), 44.3 (CH, C9), 41.5 (CH, C13), 41.0 (CH2, C7), 38.6 (C, C10), 37.8 (CH2, C14), 35.8 (CH2, C1), 25.7 (CH2, C12), 23.1 (CH2, C6), 20.6 (CH2, C2), 19.1 (CH2, C11), 13.3 (CH3, C20). 16-O-Methylkahweol (2), C21H28O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 329, 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.27 (1H, sbroad, H19), 6.30 (1H, sbroad, H18),
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6.25 (1H, d, J = 9.9 Hz, H2), 5.90 (1H, d, J = 9.9 Hz, H1), 3.78 (2H, s, H17), 3.18 (3H, s, H21), 2.61 (1H, dd, J = 3.1, 11.5 Hz, H5), 2.23 (2H, dd, J = 0.9, 2.2 Hz, H9, H13) ppm 1.94–1.80 (3H, m), 1.73 (2H, dd, J = 5.5, 9.9 Hz, H15), 1.69–1.18 (7H, m), 0.98 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 141.1 (CH, C19), 138.6 (CH, C1), 121.8 (CH, C4), 115.4 (CH, C2), 108.7 (CH, C18), 87.0 (C, C16), 60.6 (CH2, C17), 49.0 (CH, C5), 48.9 (CH3, C21), 48.4 (CH2, C15), 44.8 (C, C8), 44.5 (CH, C9), 42.0 (C, C10), 41.4 (CH, C13), 40.5 (CH2, C14), 37.8 (CH2, C7), 25.6 (CH2, C12), 22.2 (CH2, C6), 19.2 (CH2, C11), 15.6 (CH3, C20). Cafestol (3), C20H28O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 223 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 317, 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.26 (1H, sbroad, H19), 6.21 (1H, d, J = 1.1 Hz, H18), 3.86 (1H, d, J = 10.9 Hz, H17a), 3.72 (1H, d, J = 10.9 Hz, H17b), 2.64 (2H, dd, J = 2.7, 5.8 Hz, H2), 2.31 (1H, d, J = 2.1 Hz, H9), 2.27 (1H, d, J = 2.1 Hz, H13), 2.14–2.02 (3H, m, H1, H14a), 1.84 (2H, dd, J = 3.1, 12.5 Hz, H6), 1.78–1.60 (8H, m, H7, H11, H12, H14b, H15a), 1.31–1.13 (2H, m, H5), 0.84 (3H, s, H20); 13 C NMR (75.5 MHz, CDCl3-d1, ppm): d = 148.7 (C, C3), 140.6 (CH, C19), 120.2 (C, C4), 108.3 (CH, C18), 81.9 (C, C16), 66.4 (CH2, C17), 53.5 (CH2, C15), 52.2 (C, C5), 45.5 (C, C9), 44.7 (C, C8), 44.3 (CH, C13), 40.9 (CH2, C7), 38.7 (C, C10), 38.2 (CH2, C14), 35.8 (CH2, C1), 26.1 (CH2, C12), 23.1 (CH2, C6), 20.6 (CH2, C2), 19.1 (CH2, C11), 13.3 (CH3, C20). Kahweol (4), C20H26O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 315, 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm): d 7.27 (1H, d, J = 1.6 Hz, H19), 6.30 (1H, d, J = 1.6 Hz, H18), 6.24 (1H, d, J = 10.0 Hz, H2), 5.89 (1H, d, J = 10.0 Hz, H1), 3.83 (1H, d, J = 10.9 Hz, H17a), 3.70 (1H, d, J = 11.0 Hz, H17b), 2.60 (1H, dd, J = 3.4, 12.1 Hz, H5), 2.14– 1.84 (3H, m), 1.79 (2H, dd, J = 1.8, 7.4 Hz, H15), 1.76–1.48 (8H, m), 0.98 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 141.1 (CH, C19), 138.5 (CH, C1), 121.8 (C, C4), 115.4 (CH, C2), 108.7 (CH, C18), 81.9 (C, C16), 66.4 (CH2, C17), 53.2 (CH2, C15), 48.3 (CH, C5), 45.4 (CH, C9), 45.1 (CH, C8), 44.5 (CH, C13), 41.9 (C, C10), 40.3 (CH2, C7), 38.1 (CH2, C14), 25.9 (CH2, C12), 22.1 (CH2, C6), 19.0 (CH2, C11),15.6 (CH3, C20). Dehydrocafestol (5), C20H26O2, UV–vis (water–acetonitrile, 40:60, v/v): lmax 203 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm): d = 7.23 (1H, sbroad, H19), 6.21 (1H, d, J = 1.8 Hz, H18), 5.44 (1H, d, J = 1.1 Hz, H15), 4.22 (2H, d, J = 1.1 Hz, H17), 2.61 (2H, dd, J = 2.2, 9.6 Hz, H2), 2.48 (1H, d, J = 2.3 Hz, H9), 2.24 (1H, d, J = 2.5 Hz, H13), 2.21 (1H, d, J = 10.4 Hz, H14a), 2.10 (1H, t, J = 4.1 Hz, H1a), 2.06 (1H, t, J = 4.2 Hz, H1b), 1.84–1.40 (9H, m, H6, H7, H11, H12, H14b), 1.31–1.16 (1H, m, H5), 0.86 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3d1, ppm): d = 148.8 (C, C3), 146.3 (C, C16), 140.5 (CH, C19), 135.9 (CH, C15), 120.2 (C, C4), 108.3 (CH, C18), 61.3 (CH2, C17), 48.9 (C, C8), 44.9 (CH2, C7), 44.1 (CH, C5), 44.0 (CH, C9), 41.2 (CH, C13), 38.6 (C, C10), 38.2 (CH2, C14), 35.8 (CH2, C1), 25.4 (CH2, C12), 22.0 (CH2, C6), 20.7 (CH2, C2), 19.5 (CH2, C11), 13.1 (CH3, C20). Dehydrokahweol (6), C20H24O2, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm): d = 7.27 (1H, d, J = 1.7 Hz, H19), 6.31 (1H, d, J = 1.4 Hz, H18), 6.25 (1H, d, J = 10.0 Hz, H2), 5.92 (1H, d, J = 10.0 Hz, H1), 5.45 (1H, d, J = 0.8 Hz, H15), 4.23 (2H, d, J = 1.5 Hz, H17), 2.60 (1H, dd, J = 2.9, 7.6 Hz, H5), 2.31–2.15 (2H, m, H9, H13), 2.12–1.94 (4H, m), 1.89– 1.04 (6H, m), 1.01 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 146.4 (C, C16), 141.0 (CH, C19), 138.7 (CH, C1), 135.5 (CH, C15), 121.7 (CH, C4), 115.3 (CH, C2), 108.7 (CH, C18), 61.3 (CH2, C17), 49.2 (C, C8), 44.9 (CH2, C7), 44.3 (CH, C5), 44.2 (CH, C9),
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41.8 (C, C10), 41.2 (CH, C13), 37.7 (CH2, C14), 25.4 (CH2, C12), 21.1 (CH2, C6), 19.5 (CH2, C11), 15.4 (CH3, C20). References Bak, A., Grobbee, D., 1989. The effect on serum cholesterol levels of coffee brewed by filtering or boiling. New England Journal of Medicine 321, 1432–1437. Bertholet, R., 1987. Preparation of Cafestol. United States Patentnr 4,692,534. Bonnla¨nder, B., Wu¨nnecke, H., Winterhalter, P., 2007. Rapid and sensitive detection of robusta traces in blends of raw and roasted coffee. Lebensmittelchemie 61, 79–80. Casal, S., Alves, M.R., Mendes, E., Oliveira, M.B.P.P., Ferreira, M.A., 2003. Discrimination between arabica and robusta coffee species on the basis of their amino acid enantiomers. Journal of Agricultural and Food Chemistry 51, 6495–6501. Casal, S., Mendes, E., Alves, M.R., Alves, R.C., Beatriz, M., Oliveira, P.P., Ferreira, M.A., 2004. Free and conjugated biogenic amines in green and roasted coffee beans. Journal of Agricultural and Food Chemistry 52, 6188–6192. Casal, S., Oliveira, M., Alves, M.R., Ferreira, M.A., 2000. Discriminate analysis of roasted coffee varieties for trigonelline, nicotinic acid, and caffeine content. Journal of Agricultural and Food Chemistry 48, 3420–3424. Cavin, C., Bezencon, C., Guignard, G., Schilter, B., 2003. Coffee diterpenes prevent benzo[a]pyrene genotoxicity in rat and human culture systems. Biochemical and Biophysical Research Communications 306, 488–495. Cavin, C., Mace, K., Offord, E.A., Schilter, B., 2001. Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells. Food and Chemical Toxicology 39, 549–556. Corey, E.J., Xiang, Y.B., 1987. A method for the off-ring functionalization of carbonsubstituted furans. Synthesis of kahweol from cafestol. Tetrahedron Letters 28, 5403–5406. Degenhardt, A., 2002. Entwicklung und Anwendung verteilungschromatographischer Trennverfahren in der Lebensmittelanalytik. Thesis. Technische Universita¨t Braunschweig. DIN 10779, 1999. Bestimmung des Gehaltes an 16-O-Methylcafestol in Ro¨stkaffee HPLC-Verfahren, German Institute for Standardization. Frega, N., Bocci, F., Lercker, G., 1994. High resolution gas chromatographic method for determination of robusta coffee in commercial blends. Journal of High Resolution Chromatography 17, 303–307. Ito, Y., 2005. Golden rules and pitfalls in selecting optimum conditions for highspeed counter-current chromatography. Journal of Chromatography A 1065, 145–168. Ko¨lling-Speer, I., Kurt, A., Nguyen Thu, Speer, K., 1997. Cafestol and dehydrocafestol in roasted coffee. In: Proceedings 17th International Scientific Colloquium on Coffee, ASIC/Paris, pp. 201–204. Kurzrock, T., Speer, K., 2001. Diterpenes und diterpene esters in coffee. Food Reviews International 17, 433–450. Lam, L.K.T., Sparnins, V.L., Wattenberg, L.W., 1982. Isolation and identification of kahweol palmitate and cafestol palmitate as active constituents of green coffee beans that enhance glutathione S-transferase activity in the mouse. Cancer Research 42, 1193–1198. Lam, L.K.T., Sparnins, V.L., Wattenberg, L.W., 1987. Effects of derivatives of kahweol and cafestol on the activity of glutathione S-transferase in mice. Journal of Medicinal Chemistry 30, 1399–1403. Lee, Y.-W., Cook, C.E., Ito, Y., 1988. Dual Countercurrent Chromatography. Journal of Liquid Chromatography & Related Technologies 11, 37–53. Martı´n, M.J., Pablos, F., Gonza´les, A.G., 1998. Characterization of green coffee varieties according to their metal content. Analytica Chimica Acta 358, 177– 183. Pettitt, B.C., 1987. Identification of the diterpene esters in arabica and canephora coffees. Journal of Agricultural and Food Chemistry 35, 549–551. Speer, K., Kurt, A., 2001. Effects of steam treatment on diterpenes. In: Proceedings 19th International Scientific Colloquium on Coffee. ASIC/Paris (CD-Rom). Speer, K., Tewis, R., Montag, A., 1992. 16-O-methylcafestol: a quality indicator for coffee. In: Proceedings 14th International Scientific Colloquium on Coffee, ASIC/ Paris, pp. 237–244. Tewis, R., Montag, A., Speer, K., 1993. Dehydrocafestol and dehydrokahweol: two new roasting components in coffee. In: Proceedings 15th International Scientific Colloquium on Coffee, ASIC/Paris, pp. 880–883. Urgert, R., van der Weg, G., Kosmeijer-Schuil, T.G., van de Bovenkamp, P., Hovenier, R., Katan, M.B., 1995. Levels of the cholesterol-elevating diterpenes cafestol and kahweol in various coffee brews. Journal of Agricultural and Food Chemistry 43, 2167–2172. Valdenebro, M.S., Leo´n-Camacho, M., Pablos, F., Gonza´lez, A.-G., Martı´n, M.J., 1999. Determination of the arabica/robusta composition of roasted coffee according to their sterolic content. Analyst 124, 999–1002. Weusten-Van der Wouw, M.P.M.E., Katan, M.B., Viani, R., Huggett, A.C., Liardon, R., Lund-Larsen, P.G., Thelle, D.S., Ahola, I., Aro, A., Meyboom, S., Beynen, A.C., 1994. Identity of the cholesterol-raising factor from boiled coffee and its effects on liver function enzymes. Journal of Lipid Research 35, 721–733.
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Isolation of coffee diterpenes by means of high-speed countercurrent chromatography Heike Scharnhop, Peter Winterhalter * Institute of Food Chemistry, University of Braunschweig, Institute of Technology, Schleinitzstrasse 20, 38106 Braunschweig, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2007 Received in revised form 1 August 2008 Accepted 24 October 2008
Many different physiological activities have been ascribed to coffee diterpenes, such as cholesterol increasing activity for the diterpenes kahweol and cafestol. Another member of this class, namely the diterpene 16-O-methylcafestol, is used as a marker substance for robusta coffee. Up to now, methodologies for the preparative isolation of these key coffee ingredients are still limited. In this study, high-speed countercurrent chromatography (HSCCC) was successfully employed for the isolation and purification of different diterpenes (i.e. kahweol, cafestol, 16-O-methylkahweol, 16-O-methylcafestol, dehydrokahweol, and dehydrocafestol) from Coffea arabica and Coffea canephora var. robusta. The solvent systems consisted of hexane–ethyl acetate–ethanol–water mixtures. Identity and purity of the isolated compounds were confirmed by high-performance liquid chromatography with photo diode array detection (HPLC-PDA) and HPLC–multiple mass spectrometry (HPLC–MSn) as well as NMR measurements. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Coffee 16-O-methylcafestol Cafestol Kahweol Coffea canephora var. robusta Diterpene High-speed countercurrent chromatography Bioactive non-nutrients Food quality Food analysis Food composition
1. Introduction Coffee is one of the most popular beverages in the world, with an annual consumption of 144 L per capita in Germany in the year 2006. Of major importance for the worldwide coffee production are the two coffee species arabica and canephora var. robusta. Many different physiological effects are associated with coffee consumption, and many of them are likely to be related to compounds of the coffee oil, namely the diterpenes. Major diterpenes of coffee are kahweol and cafestol (Fig. 1). In the coffee oil the diterpenes are mostly esterified with various fatty acids and only a small amount of the diterpenes is present in the free form. Various studies have shown high physiological activity for kahweol and cafestol, and both beneficial and harmful effects have been reported. On the one hand, these compounds are considered to increase serum cholesterol level (Bak and Grobbee, 1989; Weusten-Van der Wouw et al., 1994); on the other hand, some beneficial effects, such as enhanced glutathione S-transferase activity (detoxifying enzyme system) (Lam et al., 1987) or protection against benz[a]pyrene- and aflatoxin B1-induced
* Corresponding author. Tel.: +49 531 391 7200; fax: +49 531 391 7230. E-mail address: [email protected] (P. Winterhalter). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.10.018
genotoxicity are known (Cavin et al., 2001, 2003). These effects depend on the amount of kahweol and cafestol present in the coffee brew and consequently, on the way coffee is prepared. The highest content of diterpenes was found in boiled, unfiltered coffee brews. The content of diterpenes in drip-filtered coffee is negligible (Urgert et al., 1995). Apart from the physiological effects, diterpenes are also important marker compounds for coffee processing, e.g. free kahweol in the case of steaming treatment (Speer and Kurt, 2001) as well as for coffee authenticity (16-O-methylcafestol, 16-OMC) (Speer et al., 1992). One unit of arabica costs almost twice as much as the same unit of robusta coffee. Often the high-quality arabicas, described as ‘‘100% arabica’’ or ‘‘Highland coffee’’, are mixed with the less expensive robustas. Therefore it is important to control the authenticity of the products with appropriate methods enabling a differentiation of the two species. Robusta beans are smaller and rounder than arabica beans. These differences persist during the roasting process. Hence a differentiation by comparison of the bean-size before grinding is possible. Also quantities over 15% of robusta in arabica blends can be detected by trained coffee tasters in sensory testing (Speer et al., 1992). The most important method for discrimination, however, is the analysis of the coffee ingredients. Many different methods have been described to discriminate between arabica
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Fig. 1. Structures of coffee diterpenes isolated by HSCCC.
and robusta, e.g. by the presence of different metals (Martı´n et al., 1998), trigonelline and caffeine (Casal et al., 2000), amino acid enantiomers (Casal et al., 2003), biogenic amines (Casal et al., 2004), sterol fraction (Valdenebro et al., 1999) or the ratio between sterols and diterpenes (Frega et al., 1994). However, the most suitable indicator for robusta coffees is 16-O-methylcafestol (1). A validated method by high-performance liquid chromatography enables the detection of up to 2% of robusta in arabica blends (DIN 10779, 1999). By using more selective detectors like LC–MSn in the multiple reaction monitoring mode (MRM), trace amounts of approx. 0.2% robusta can be detected (Bonnla¨nder et al., 2007). For an accurate analysis of coffee authenticity, 16-OMC is required as a standard compound. In the frame of this study a preparative method for the isolation of 16-O-methylcafestol from robusta coffee by high-speed countercurrent chromatography (HSCCC) is reported. HSCCC is an all liquid chromatographic technique that operates under gentle conditions and allows non-destructive isolation even of labile natural compounds. Due to the absence of any solid stationary phase, adsorption losses are minimised and hence a 100% sample recovery is guaranteed (Ito, 2005). Many different substance-classes have been successfully separated by means of HSCCC, e.g. triterpenes like steroids (Lee et al., 1988). Diterpene isolation so far – for example, by means of preparative RP-HPLC – has been very time-consuming (Lam et al., 1982; Pettitt, 1987; Bertholet, 1987). Isolation of diterpenes using HSCCC, on the other hand, takes advantage of the high sample load (1 g per separation), the use of inexpensive solvents and the high purity of the isolated substances.
In addition to 16-O-methylcafestol, the diterpenes 16-Omethylkahweol 2 (robusta, green), cafestol 3 and kahweol 4 (arabica and robusta, green and roasted), and the roasting products dehydrocafestol 5 and dehydrokahweol 6 (arabica and robusta, roasted) were also accessible by HSCCC separations. 2. Materials and methods For the isolation of diterpenes Coffea arabica (Mexico) and Coffea canephora var. robusta (India) were used. From both coffee samples green as well as roasted beans were examined. 2.1. Extraction and saponification of coffee samples A 500 g amount of green or roasted coffee beans was ground and extracted with methyl tert-butyl ether (MTBE) at 80 8C in a Soxhlet apparatus over 5 h. After evaporating the solvent in vacuo, the remaining coffee oil was saponified with 1 L of an ethanolic potassium hydroxide solution (10%) at 90 8C for 4 h. After removing the alcohol, the residue was solubilised in water (1 L, 70 8C) and 250 mL of sodium chloride solution (10%) and extracted with MTBE (3 400 mL). The organic phase was washed with 1 L of an aqueous sodium chloride solution (2%) and dried over sodium sulfate. The solvent was evaporated in vacuo and the residues were used for the separation by high-speed countercurrent chromatography. 2.2. High-speed countercurrent chromatography (HSCCC) A high-speed countercurrent chromatograph manufactured by Pharma-Tech Research (Baltimore, MD, USA) was equipped
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with three coils (2.6 mm internal diameter of the tubing) to give a final volume of the liquid phase of 850 mL. The separations were run at a revolution speed of 800 rpm. The mobile phase was delivered by a Biotronik HPLC pump BT 3020 (Jasco, GroßUmstadt, Germany). The sample (unsaponifiable matter of different coffee samples) was dissolved in 20 mL of a 1:1 mixture of the upper and lower phase of the solvent system. The amount of sample injected was around 1 g. Elution was monitored with a UV–vis detector (Knauer, Berlin, Germany) at l = 220 nm. The solvent system used for the separation of the unsaponifiable matter consisted of hexane–ethyl acetate–ethanol–water (5:2:5:2. v/v/v/v; the less dense layer acting as the stationary phase, flow rate: 4.0 mL/min, head-to-tail mode; all chemicals were of analytical grade). Sixteen milliliter fractions were collected with an automatic collector. 2.3. Thin-layer chromatography (TLC) First control of identity and purity of the obtained HSCCCfractions was made by TLC. The stationary phase was silica gel 60 F254 from Merck (Darmstadt, Germany), the mobile phase diethyl ether–chloroform (1:1). After development, the TLC plate was dried and sprayed with a solution of 1 g vanillin in 250 g ethanol acidified with 20 mL of sulphuric acid (conc.). Red and blue spots were visible. 2.4. HPLC-PDA analysis HPLC-PDA analyses were performed on an MD-910 multiwavelength detector (220–650 nm), equipped with a DG-980-50 three-line degasser, a LG-980-02 ternary gradient unit, a PU-980 Intelligent HPLC pump, and Borwin PDA chromatography software (Jasco, Germany). Diterpenes were detected at 220 and 280 nm. The samples were injected via a Rheodyne 7125 injection valve (Techlab, Germany) equipped with a 20 mL loop, and separations were carried out on a 250 mm 4.6 mm i.d., 5 mm, Synergi MaxRP 80A column (Phenomenex, Germany) at a flow rate of 0.8 mL/min. A two solvent system was used. Solvent A: acetonitrile, solvent B: water. The linear gradient was as follows: 0 min, 40% B; 20 min, 30% B; 40 min, 0% B; 45 min, 0% B; 46 min, 40% B. 2.5. HPLC–ESI-MSn HPLC–MSn analyses of fractions and purified diterpenes were performed on a Bruker Esquire LC–MS system (Bruker Daltonik, Germany). The HPLC system consisted of a System 1100 binary pump G1312A (Agilent, Germany) and a Lichrograph L-4000 UV–vis detector (Merck Hitachi, Japan). UV chromatograms were recorded with a Chromatopac C-R6A integrator (Shimadzu, Japan). The LC part of the system was controlled by ChemStation version A.06.01, and MS data were processed by Esquire NT 4.0 software (Bruker Daltonik). MS parameters: positive ion mode; capillary, 2500 V; capillary exit offset, 70 V; end plate offset, 500 V; skimmer 1, 25 V; skimmer 2, 10 V; dry gas, N2, 11 L/ min; dry temperature, 355 8C; nebulizer, 60 psi; and scan range, 50–2200 m/z; chromatographic conditions were as described above.
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3. Results and discussion According to their provenance, Coffea canephora var. robusta beans contain between 0.6 and 1.8 g 16-O-methylcafestol (16OMC) 1 per kg. Due to the high stability of 16-OMC, the content in crude and roasted coffee is almost the same. Therefore this diterpene is an ideal marker for robusta coffee (Speer et al., 1992). In coffee diterpenes are hardly present in the free form. They are mostly esterified with fatty acids like palmitic and linolic acid (Kurzrock and Speer, 2001). For an enrichment of diterpenes, the coffee oil has to be saponified with ethanolic potassium hydroxide solution (10%) and afterwards extracted with organic solvent according to the validated method for the quantification of 16OMC by HPLC (DIN 10779, 1999). For the isolation of 16-OMC, the unsaponifiable matter of the coffee oil was fractionated by means of HSCCC. In order to obtain suitable solvent systems for HSCCC, the partition coefficient K of the sample was monitored by TLC. Suitable partition coefficients for HSCCC are around K = 1, i.e. the sample is soluble in the lighter phase as well as in the more dense phase of the two phase solvent system (Degenhardt, 2002). All cafestol derivates exhibit under the used TLC conditions red spots and can be distinguished from kahweol derivates which appear in blue colour. TLC of the unsaponifiable matter of arabica and robusta shows red spots at Rf 0.15 indicating cafestol 3. Furthermore, the arabica sample has a blue spot at Rf 0.14 indicating kahweol 4 which is not detectable by TLC in robusta coffee due to its low content. Robusta coffee shows a red spot at Rf 0.45 which is absent in arabica coffee. This marks 16-O-methylcafestol 1. TLC of roasted arabica and robusta samples show more spots at Rf 0.60. In arabica red and blue bands could be recognised, whereas robusta coffee only shows the red one. These signals indicate the roasting products dehydrocafestol 5 and dehydrokahweol 6. 3.1. Isolation and purification of 16-O-methylcafestol (1) and 16-Omethylkahweol (2) by HSCCC In order to find a suitable solvent system some nonpolar two phase solvent mixtures containing hexane, ethyl acetate, methanol or ethanol and water in different ratios were tested and the partition coefficient K was determined by TLC (Ito, 2005). Mixtures of these solvents were used, e.g. for the separation of nonpolar natural compounds like steroids (Lee et al., 1988). As suitable solvent system (K 1) for the isolation of 16-O-methylcafestol 1 hexane–ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v) was elaborated. Fig. 2 shows the preparative HSCCC separation of 1 g of the unsaponifiable matter from green robusta coffee. First characterisation of isolated fractions was performed by means of TLC. 16-Omethylcafestol 1 could be detected by a red spot at Rf 0.45 in fractions eluting approximately after 3 h. Some previous fractions show a blue spot at similar Rf. In these fractions 16-O-methylkahweol 2 could be enriched. The identity of all isolated diterpenes was besides the TLC monitoring confirmed by means of UV–vis, LC–MS/MS (positive mode), and 1D/2D-NMR experiments (1H, 13C, DEPT, HMBC, HMQC). In one HSCCC run 120 mg of 16-O-methylcafestol 1 could be obtained. The purity was around 95% (HPLC). A final purification (>99%) was achieved by preparative HPLC.
2.6. Nuclear magnetic resonance (NMR) spectroscopy 3.2. Isolation and purification of cafestol (3) and kahweol (4) 1
H, 13C, DEPT and 1H–1H-COSY spectra were measured on a Bruker AMX 300 spectrometer (Bruker Biospin, Germany) at 300.1 and 75.4 MHz, respectively. HMQC and HMBC experiments were performed on a Bruker AM 600 instrument. Solvent was chloroform-d1 (Merck, Darmstadt, Germany).
The content of kahweol in robusta coffee is very low. Hence, for the isolation of this diterpene green arabica coffee beans were separated by means of HSCCC using the solvent system hexane– ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v). Under the condi-
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Fig. 2. HSCCC separation of the unsaponifiable matter of green robusta coffee for the isolation of 16-O-methylcafestol. Solvent system: hexane–ethyl acetate–ethanol– water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm, retention of stationary phase: 29.3%.
Fig. 4. HSCCC separation of the unsaponifiable matter of roasted arabica coffee for the isolation of dehydrokahweol and dehydrocafestol. Solvent system: hexane– ethyl acetate–ethanol–water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm; retention of stationary phase: 26.1%.
tions employed, partition coefficient K for kahweol was 0.5 and for cafestol 0.55. The HSCCC chromatogram is shown in Fig. 3. Analysis of separated HSCCC fractions was made by TLC. The chromatogram recorded at 220 nm shows a main peak eluting after 1.5 h. This peak contains kahweol as well as cafestol. Blue spots at Rf 0.14 for kahweol 4 could be detected by TLC in the first peak fractions. Some later fractions showed blue and red spots. In these fractions a mixture of kahweol 4 and cafestol 3 was eluted. At a retention time of 2 h the fractions of this first peak contained pure cafestol 3. Cafestol and kahweol partly co-eluted and approximately 35% of total cafestol/kahweol amount was obtained as a mixture. Hence the yield of separated kahweol and cafestol was around 65%. Purity of isolated kahweol was 88%, and of cafestol >92% (HPLC) and was raised by preparative HPLC up to 99%.
Under the same HSCCC conditions as described before, the K value for dehydrokahweol was 1.1 and for dehydrocafestol 1.3. Fig. 4 shows the obtained chromatogram of this HSCCC separation. TLC analysis gave blue spots at Rf 0.6 in fractions after a retention time of approximately 3.5 h and red spots at Rf 0.6 in fractions collected after 4 h. Isolated fractions were analysed by LC–MS and the presence of kahweol and cafestol was confirmed. Purity of dehydrokahweol 6 after HSCCC separation was around 87% (HPLC). Dehydrocafestol 5 could be isolated in a purity of 92% (HPLC). Both compounds were further cleaned up by preparative HPLC. In summary, HSCCC allowed the isolation of six diterpenes from the unsaponifiable matter of green and roasted coffee beans. Over 100 mg of 16-O-methylcafestol (purity 95%), a marker substance for robusta coffee, could be obtained by the HSCCC fractionation of 1 g of unsaponifiable matter of robusta coffee.
3.3. Isolation and purification of dehydrocafestol (5) and dehydrokahweol (6)
3.4. Analytical data of isolated compounds
Dehydrokahweol and dehydrocafestol are roasting products of kahweol and cafestol (cf. Fig. 1). Formation of these dehydration products is due to the degradation of kahweol and cafestol and depends on the length and temperature of the roasting process (Ko¨lling-Speer et al., 1997). Hence, for the isolation of dehydrocafestol 5 and dehydrokahweol 6, the unsaponifiable matter of roasted arabica coffee was used.
Fig. 3. HSCCC separation of the unsaponifiable matter of green arabica coffee for the isolation of kahweol and cafestol. Solvent system: hexane–ethyl acetate–ethanol– water (5:2:5:2, v/v/v/v), flow rate: 4.0 mL/min; detection at l = 220 nm; retention of stationary phase: 25.6%.
For coffee diterpenes only a few 1H NMR and 13C NMR data have been reported in the literature so far (Lam et al., 1982; Pettitt, 1987; Corey and Xiang, 1987; Tewis et al., 1993). With this preparative method, it was possible to complete the existing data of 16-O-methylcafestol 1, cafestol 3, and kahweol 4 and present new ones for 16-O-methylkahweol 2, dehydrocafestol 5, and dehydrokahweol 6. 16-O-Methylcafestol (1) (numbering of the atoms refers to the structure given in Fig. 1), C21H30O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 223 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 331, 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.23 (1H, d, J = 1.5 Hz, H19), 6.20 (1H, d, J = 1.6 Hz, H18), 3.77 (2H, s, H17), 3.17 (3H, s, H21), 2.62 (2H, dt, J = 2.8, 5.8 Hz, H2), 2.30 (1H, dd, J = 2.3, 4.9 Hz, H9), 2.25 (1H, dd, J = 2.3, 4.8 Hz, H13), 2.07 (2H, m, H1), 2.00 (1H, d, J = 13.4 Hz, H14a), 1.82 (1H, q, J = 2.9 Hz, H6a), 1.78 (1H, q, J = 3.1 Hz, H6b), 1.75–1.47 (8H, m, H7, H11, H12, H14b, H15a), 1.43 (1H, dd, J = 1.9, 14.6 Hz, H15b), 1.30–1.18 (1H, m, H5), 0.83 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3d1, ppm): d = 148.8 (C, C3), 140.5 (CH, C19), 120.2 (C, C4), 108.2 (CH, C18), 87.0 (C, C16), 60.5 (CH2, C17), 52.2 (CH, C5), 49.2 (CH2, C15), 48.9 (CH3, C21), 44.4 (C, C8), 44.3 (CH, C9), 41.5 (CH, C13), 41.0 (CH2, C7), 38.6 (C, C10), 37.8 (CH2, C14), 35.8 (CH2, C1), 25.7 (CH2, C12), 23.1 (CH2, C6), 20.6 (CH2, C2), 19.1 (CH2, C11), 13.3 (CH3, C20). 16-O-Methylkahweol (2), C21H28O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 329, 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.27 (1H, sbroad, H19), 6.30 (1H, sbroad, H18),
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6.25 (1H, d, J = 9.9 Hz, H2), 5.90 (1H, d, J = 9.9 Hz, H1), 3.78 (2H, s, H17), 3.18 (3H, s, H21), 2.61 (1H, dd, J = 3.1, 11.5 Hz, H5), 2.23 (2H, dd, J = 0.9, 2.2 Hz, H9, H13) ppm 1.94–1.80 (3H, m), 1.73 (2H, dd, J = 5.5, 9.9 Hz, H15), 1.69–1.18 (7H, m), 0.98 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 141.1 (CH, C19), 138.6 (CH, C1), 121.8 (CH, C4), 115.4 (CH, C2), 108.7 (CH, C18), 87.0 (C, C16), 60.6 (CH2, C17), 49.0 (CH, C5), 48.9 (CH3, C21), 48.4 (CH2, C15), 44.8 (C, C8), 44.5 (CH, C9), 42.0 (C, C10), 41.4 (CH, C13), 40.5 (CH2, C14), 37.8 (CH2, C7), 25.6 (CH2, C12), 22.2 (CH2, C6), 19.2 (CH2, C11), 15.6 (CH3, C20). Cafestol (3), C20H28O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 223 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 317, 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm) d = 7.26 (1H, sbroad, H19), 6.21 (1H, d, J = 1.1 Hz, H18), 3.86 (1H, d, J = 10.9 Hz, H17a), 3.72 (1H, d, J = 10.9 Hz, H17b), 2.64 (2H, dd, J = 2.7, 5.8 Hz, H2), 2.31 (1H, d, J = 2.1 Hz, H9), 2.27 (1H, d, J = 2.1 Hz, H13), 2.14–2.02 (3H, m, H1, H14a), 1.84 (2H, dd, J = 3.1, 12.5 Hz, H6), 1.78–1.60 (8H, m, H7, H11, H12, H14b, H15a), 1.31–1.13 (2H, m, H5), 0.84 (3H, s, H20); 13 C NMR (75.5 MHz, CDCl3-d1, ppm): d = 148.7 (C, C3), 140.6 (CH, C19), 120.2 (C, C4), 108.3 (CH, C18), 81.9 (C, C16), 66.4 (CH2, C17), 53.5 (CH2, C15), 52.2 (C, C5), 45.5 (C, C9), 44.7 (C, C8), 44.3 (CH, C13), 40.9 (CH2, C7), 38.7 (C, C10), 38.2 (CH2, C14), 35.8 (CH2, C1), 26.1 (CH2, C12), 23.1 (CH2, C6), 20.6 (CH2, C2), 19.1 (CH2, C11), 13.3 (CH3, C20). Kahweol (4), C20H26O3, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 315, 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm): d 7.27 (1H, d, J = 1.6 Hz, H19), 6.30 (1H, d, J = 1.6 Hz, H18), 6.24 (1H, d, J = 10.0 Hz, H2), 5.89 (1H, d, J = 10.0 Hz, H1), 3.83 (1H, d, J = 10.9 Hz, H17a), 3.70 (1H, d, J = 11.0 Hz, H17b), 2.60 (1H, dd, J = 3.4, 12.1 Hz, H5), 2.14– 1.84 (3H, m), 1.79 (2H, dd, J = 1.8, 7.4 Hz, H15), 1.76–1.48 (8H, m), 0.98 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 141.1 (CH, C19), 138.5 (CH, C1), 121.8 (C, C4), 115.4 (CH, C2), 108.7 (CH, C18), 81.9 (C, C16), 66.4 (CH2, C17), 53.2 (CH2, C15), 48.3 (CH, C5), 45.4 (CH, C9), 45.1 (CH, C8), 44.5 (CH, C13), 41.9 (C, C10), 40.3 (CH2, C7), 38.1 (CH2, C14), 25.9 (CH2, C12), 22.1 (CH2, C6), 19.0 (CH2, C11),15.6 (CH3, C20). Dehydrocafestol (5), C20H26O2, UV–vis (water–acetonitrile, 40:60, v/v): lmax 203 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 299, 281, 147, 133, 1H NMR (300 MHz, CDCl3-d1, ppm): d = 7.23 (1H, sbroad, H19), 6.21 (1H, d, J = 1.8 Hz, H18), 5.44 (1H, d, J = 1.1 Hz, H15), 4.22 (2H, d, J = 1.1 Hz, H17), 2.61 (2H, dd, J = 2.2, 9.6 Hz, H2), 2.48 (1H, d, J = 2.3 Hz, H9), 2.24 (1H, d, J = 2.5 Hz, H13), 2.21 (1H, d, J = 10.4 Hz, H14a), 2.10 (1H, t, J = 4.1 Hz, H1a), 2.06 (1H, t, J = 4.2 Hz, H1b), 1.84–1.40 (9H, m, H6, H7, H11, H12, H14b), 1.31–1.16 (1H, m, H5), 0.86 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3d1, ppm): d = 148.8 (C, C3), 146.3 (C, C16), 140.5 (CH, C19), 135.9 (CH, C15), 120.2 (C, C4), 108.3 (CH, C18), 61.3 (CH2, C17), 48.9 (C, C8), 44.9 (CH2, C7), 44.1 (CH, C5), 44.0 (CH, C9), 41.2 (CH, C13), 38.6 (C, C10), 38.2 (CH2, C14), 35.8 (CH2, C1), 25.4 (CH2, C12), 22.0 (CH2, C6), 20.7 (CH2, C2), 19.5 (CH2, C11), 13.1 (CH3, C20). Dehydrokahweol (6), C20H24O2, UV–vis (water–acetonitrile, 40:60, v/v): lmax 287 nm, ESI-MSn (positive ion mode): [M + H]+ = m/z 297, 279, 145, 131, 1H NMR (300 MHz, CDCl3-d1, ppm): d = 7.27 (1H, d, J = 1.7 Hz, H19), 6.31 (1H, d, J = 1.4 Hz, H18), 6.25 (1H, d, J = 10.0 Hz, H2), 5.92 (1H, d, J = 10.0 Hz, H1), 5.45 (1H, d, J = 0.8 Hz, H15), 4.23 (2H, d, J = 1.5 Hz, H17), 2.60 (1H, dd, J = 2.9, 7.6 Hz, H5), 2.31–2.15 (2H, m, H9, H13), 2.12–1.94 (4H, m), 1.89– 1.04 (6H, m), 1.01 (3H, s, H20); 13C NMR (75.5 MHz, CDCl3-d1, ppm): d = 150.3 (C, C3), 146.4 (C, C16), 141.0 (CH, C19), 138.7 (CH, C1), 135.5 (CH, C15), 121.7 (CH, C4), 115.3 (CH, C2), 108.7 (CH, C18), 61.3 (CH2, C17), 49.2 (C, C8), 44.9 (CH2, C7), 44.3 (CH, C5), 44.2 (CH, C9),
237
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