Diterpenes in Coffee - ACS Symposium Series (ACS Publications)

Chapter 25

Diterpenes in Coffee K . Speer, A. Hruschka, T. Kurzrock, and I. Kölling-Speer

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 10, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch025

Institute of Food Chemistry, Technical University of Dresden, D-01728 Dresden, Germany In recent years, the diterpenes in the lipid fraction of coffee have become of considerable interest: 16-O-methylcafestol as an indicator for Robusta coffee, the diterpenes cafestol and kahweol as components with different physiological effects. In comparison to green coffee, a large number of additional diterpenes were obtained in roasted coffee samples. Aside from diterpene derivatives formed by the loss of a water molecule, with cafestal and kahweal two further degradation products were elucidated. Many of these compounds have been detected and quantified in differently prepared coffee beverages as well.

The two most important coffee species, Coffea arabica and Coffea canephora, var. robusta, contain between 7 and 17% fat in the coffee beans. The lipid content of Arabica coffee averages some 15%, whilst Robusta coffee contains much less, namely around 10% lipids. The main components in coffee oil are triglycerides, followed closely by diterpenes with a share of 20%. These compounds are important not only because of their amount, but also because of their physiological effects, which is also the reason for the increasing interest in them in recent years. The structural formulae of four diterpenes are assembled in Figure 1. Until now, the physiological activity of only cafestol and kahweol has been researched and in several studies reported that, through the enjoyment of coffee, the serum cholesterol level can increase. Initially, triglycerides were said to be responsible for this effect. Since then, however, it has been established that it was the diterpenes which influence the serum cholesterol level (1,2,3). In addition, a substantial number of scientific publications exist where positive effects of diterpenes are introduced. It was shown that cafestol stimulates the giutathion-S-transferase activity, through which the decomposition of xenobiotica is accelerated (4). Other authors reported that the diterpenes cafestol and kahweol protect against Β1-induced genotoxicity (5,6). Up until today, only a few of these compounds have been examined in detail. To what extent additive, reversible or even synergistic effects show up, can only

© 2000 American Chemical Society

Parliment et al.; Caffeinated Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

241

242


be judged when the entirety of the diterpene compounds found in coffee is included in this study. Hence, the following shall be an overview of the current state of knowledge with regard to diterpene compounds in coffee, where, above all, the results of this research team, gathered from 1985 to the present, w i l l be presented.

i 6-O-MethyicafestoI

16-O-MethyIkahweol

R =H : Free Diterpene R = Fatty A c i d : Diterpene Ester

Figure L Structural formulae of the diterpenes.

Diterpenes in the Lipid Fraction of Green Robusta and Arabica Coffees It has been well-known since the 1930s that coffee contains diterpenes (7). Working groups under Wettstein (8), Chakravorty (9), Djerassi (10), and Haworth ( i i ) worked for several years to identify the structure of two of the coffee diterpenes, namely kahweol and cafestol. Through later work by Kaufmann and Hamsagar (12,13) and also by Folstar (14,15), it could be shown that both diterpenes are predominantly esterified with different fatty acids and exist freely only i n small amounts. In order to analyze the total amount, it was necessary to extract the fat from the coffee bean, to saponify and then to determine the diterpenes i n the unsaponifiable matter by means of G C or faster by H P L C (16,17) (Figure 2). Several green Arabica and Robusta coffees were investigated (Figure 3). In Arabica coffee beans, the diterpenes cafestol and kahweol were found, whereas Robusta coffee beans contain cafestol, small amounts of kahweol and, additionally, another diterpene - the 16-O-methylcafestol (16-OMC) (18,19). The new diterpene was present only i n the Robusta coffee; therefore, it is the



243

Figure 2. HPLC-chromatograms of green Arabica and Robusta coffees. Conditions: column 250x4mm, Nucleosil 120-3 C , eluent: acetonitrile/water (50:50), detection: UV 220 nm. 18

Figure 3. Contents of diterpenes in the unsaponiflable matter of different green Arabica and Robusta coffees.


244


indicator substance used to detect parts of Robusta i n Arabica coffee mixtures. A validated method of determination used i n Germany has just been published as the D I N 10779 of the German Institute for Normalization. This method makes it possible to identify very small parts of Robusta ~ parts under 2 percent - i n mixtures of Arabica coffees. Recently, a further diterpene was found and tentatively introduced as a 16O-methyl derivative of kahweol. It should be present exclusively i n coffea stenophylla, a coffee species which is not being used commercially (20). However, this derivative has now also been found i n many Robusta coffee samples and, using different spectroscopic methods, it was clearly identified as 16-O-methylkahweol (21).

Free Diterpenes In their free form, the diterpenes cafestol, kahweol and 16-OMC occur only as minor components i n coffee oil (22,23). Quantifying them requires an effective separation from the major compounds which interfere with the analysis. B y means of gel permeation chromatography, it is possible to simultaneously analyze and quantify the small amounts of these compounds by subsequent HPLC. Several coffees were analyzed. In the Arabica coffees, both free cafestol and free kahweol were determined (Figure 4). In addition to cafestol and 16-OMC, traces of kahweol were detected i n some of the Robusta coffees. Only traces of free kahweol, i f at all, had been expected, because the contents of total kahweol achieved after saponification were very small, with amounts between 10 mg/kg to 60 mg/kg.

Figure 4. Contents of free diterpenes in green Arabica and Robusta coffees.


245


When the content of the free diterpene was contrasted with the total content of the diterpene determined after saponification, a similar result for each examined diterpene was obtained. In Arabicas, the proportions ranged from 0.7 to 2.5%, i n Robustas the proportions of the free diterpenes were slightly higher with 1.1 to 3.5%. Diterpene Fatty Acid Esters Until now only a few diterpene esters have been reported. In order to identify the individual diterpene esters, it was necessary to separate the other compounds of coffee oil such as sterol esters, sterols, phosphatides, free fatty acids, free diterpenes and mainly the triglycerides. Using the same gel chromatographic system, the diterpene esters were separated with success. However, they were still present i n the same fraction together with sterol esters, which were removed by using solid phase extraction on silica cartridges. Finally for Arabicas, one fraction was achieved containing the cafestol and kahweol esters; for Robustas a second fraction was achieved which included the 16-OMC esters. The fractions were analyzed by H P L C , and as exemplarily shown i n Figure 5 for a Robusta coffee sample, it was possible to determine the individual cafestol esters.

Figure 5. HPLC-chromatogram of cafestol fatty acid esters. Conditions: column 250x4mm, Nucleosil 120-3 C , eluent: acetonitrile/iso-propanol (60:40), detection: UV220 nm. 18

Esters with fatty acids such as C H , Cie, Q g , C i s i , Cni, C1&3, C o, C > C 4 were identified, as well as esters with the fatty acid C o i and some oddnumbered fatty acids such as Cn,Ci9» C i and C23. The individual diterpene esters were irregularly present i n the coffee oil. The odd-numbered fatty acid esters were minor components, whereas the diterpenes esterified with palmitic, linoleic, oleic, stearic, arachidic and behenic 2

22

2

2


2

246


acid existed i n larger amounts. The focus was therefore placed on these six diterpene esters, which make up a sum of nearly 98% of the respective diterpenes (24,25,26). The total content of these six quantitatively significant cafestol esters i n different Arabica coffees was analyzed. The established content fell between 9.4 and 21.2 g/kg dry weight, corresponding to 5.2 to 11.8 g/kg cafestol. In different Robusta coffees, it was determined to be between 2.2 and 7.6 g/kg dry weight, corresponding to 1.2 to 4.2 g/kg cafestol, thus, notably less than i n the Arabica coffees.

Diterpenes in the Lipid Fraction of Roasted Robusta and Arabica Coffees During the roasting process, temperatures i n the middle of the coffee bean reach up to 230 °C. Through this process, a large number of compounds are changed. Therefore, the question presents itself, to what extent the diterpenes are also affected. Free Diterpenes In order to investigate the free diterpenes, one type of green Arabica and one of green Robusta coffee were roasted at different temperatures for three minutes each, and then analyzed. The results are shown in Figure 6. In this figure, the amounts of the free diterpenes are referred to i n kilogram of lipid, i n contrast to the previous data, where they were referred to i n kilogram of dry matter. This modification makes sense because the dry matter of coffee highly increases during roasting, whereas the total lipid content remains stable. The results illustrate that increasing roasting temperature diminishes the contents of the free diterpenes kahweol, cafestol, and 16-OMC. Up to 80% of the initial amounts, which were analyzed in the green coffees, were lost. During roasting, two additional peaks become clear i n the H P L C chromatogram of Arabica coffee. It is a question of decomposition products from cafestol and kahweol which could be identified as dehydrocafestol and dehydrokahweol (Figure 7). Both compounds increased with raising roasting temperatures (27,28,29). Diterpene Fatty A c i d Esters The stable behaviour of diterpene fatty acid esters during roasting is quite different. Examinations of the 16-OMC esters have shown that these are clearly stable during roasting and i n spite of different roasting temperatures, the proportional distribution for the diterpene esters is almost the same (30). O n the contrary, the contents of the diterpene esters i n cafestol and kahweol decrease depending on the roasting temperature. A n increase i n roasting temperature leads to a decrease i n the total cafestol ester content, but the distribution of the esters remains nearly the same. Subsequently, all of the esters decompose i n a similar manner.



247

Figure 6. Contents of free diterpenes in roasted Arabica and Robusta coffees.

AU

Dehydrocafestol Dehydrokahweol

Cafestol

AJ Time (min)"" Figure 7. HPLC-chromatogram

u

60*"

of free diterpenes in roasted Arabica coffee.


248


Formation of Dehydrocafestol and Dehydrokahweol Fatty A c i d Esters As reported earlier, the free diterpenes cafestol and kahweol were dehydrated to their dehydro compounds during the roasting process. Up to the present, it has been unknown whether only the free diterpenes or the diterpene fatty acid esters, too, could be decomposed to the respective dehydro derivatives. In model experiments with cafestol palmitate and cafestol linoleate it has been proved that cafestol fatty acid esters can be dehydrated as well. Dehydrocafestol palmitate and dehydrocafestol linoleate were identified i n roasted coffee, too. From this point, it is possible to assume that further fatty acid esters from both dehydrocafestol and dehydrokahweol may be identified (31) (Figure 8).

Dehydrocafestol

Dehydrokahweol

R = H: Free Diterpene R = Fatty A c i d : Diterpene Ester

Figure 8. Structural formulae of decomposed diterpenes.

Cafestal - A Degradation Product of Cafestol Aside from the described compounds, a further component has been discovered in the unsaponifiable matter of commercial roasted coffee (32). The structure elucidation was carried out by means of N M R , FTIR, and mass spectrometry and checked by chemical conversion. Following the designation of cafestol, cafestal is proposed as the common name. In order to study the roasting behaviour of cafestal, one Arabica and one Robusta coffee sample were roasted at two different temperatures (Figure 9). As expected, Cafestal was not found i n the green coffees. In the roasted coffees, the contents of cafestal were determined from 0.25 to 0.95 mg/g coffee oil. B y raising the roasting degree, the content of cafestal increased as well. In addition to cafestal, one corresponding compound of kahweol has, i n the meantime, also been identified.

Diterpenes in Coffee Beverages In 1993, using the example of 16-OMC ester, it could already be shown that when preparing a coffee drink, lipophile diterpene esters flow into the coffee and are even detectable i n instant coffee granules (33).



249

Figure 9. Contents of cafestal in green and roasted coffees.

The amount of diterpenes i n the drink is decisively dependent on the method of preparation and is directly connected with the amount of lipids i n the drink. With filtered coffee prepared i n a common household coffee machine, the amount of lipids is less than 0.2%. On the contrary, when one prepares an espresso, then between 1-2% of the lipids and thereby diterpenes as well, flow from the finely ground espresso coffee into the drink. When coffee is prepared Scandinavian style, it can even contain up to 22% of the coffee fat. The proportional distribution of diterpenes i n the coffee drink is nearly identical to the distribution i n the roasted coffee which was used. In an espresso prepared from Arabica coffee, a total of 1.3 mg cafestol fatty acid esters and 0.5 mg kahweol esters per 50 ml cup were determined, corresponding with approximately 1.5% of cafestol esters and approximately 1.0% of kahweol esters i n the roasted ground coffee. These results confirm the findings for the 16-OMC (34). In addition, the decomposition by-products dehydrokahweol, dehydrocafestol and cafestal as well as some esters from dehydrocafestol were identified in the coffee beverage.

References 1. 2.

M.

P.

Bak, Α. Α. Α.; Grobbee, D . E . New England J. Med. 1989, 23, 1432-1437. Weusten-Van der Wouw, M. P. M. E; Katan, M. B.; Viani, R.; Huggett, A . C.; Liardon, R.; Meyboom, S. J. Lipid Res. 1994, 35, 721-733. 3. Mensink, R. P.; Lebbink, W . J.; Lobbezoo, I. E . ; Weusten V a n der Wouv, M. E.; Zock, P. L . ; Katan, M. B. J. Int. Med. 1995, 237, 543-550.


250 4. 5.

6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

Lam; L . T. K . ; Sparnis, V . L.; Wattenberg, L . W. Cancer Res. 1982, 42, 1193-1198. Miller, E . G . ; Gonzales-Sanders, A . P.; Couvillion, A . M.; Binnie, W. H.; Sunahara, G. L ; Bertholet, R. 15th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1993; pp 420-425. Cavin, C.; Holzhauser, D . ; Constable, Α.; Huggett, A . C.; Schilter, Β. Carcinogenesis 1998, 19, 1369-1375. Bengis, R.O.; Anderson, R. J. J. Biol. Chem. 1932, 97, 99-113. Wettstein, Α.; Spillmann, M.; Miescher, K . Helv. chim. Acta 1945, 28, 1004-1013. Chakravorty, P. N.; Wesner, M. M.; Levin R. H . J. Amer. Chem. Soc. 1943, 65, 929-932. Djerassi, C.; Finnegan R. A . J. Amer. Chem. Soc. 1960, 82, 4342-4344. Haworth, R. D . ; Johnstone, R. A . W. J. Chem. Soc. 1957, 1492-1496. Kaufmann, H . P.; Hamsagar, R. S. Fette Seifen Anstrichmittel 1962, 64, 206-213. Kaufmann, H . P.; Hamsagar, R. S. Fette Seifen Anstrichmittel 1962, 64, 734-738. Folstar, P.; Pilnik, W.; de Heus, J. G.; van der Plas, H . C. Lebensmwiss. u. Technol. 1975, 8, 286-288. Folstar, P. In Coffee, Chemistry; Clarke R. J.; Macrae, R., Elsevier Applied Science: London, New York, 1985; V o l . 1, pp Vol. 203-222. Speer, K.; Mischnick-Lübbecke, P. Lebensmittelchemie 1989, 43, 43. Speer, K.; Montag, A . Dtsch. Lebensm.-Rundsch. 1989, 85, 381-384. Speer, K.; Mischnick, P. Z. Lebensm. Unters. Forsch. 1989, 189, 219-222. Speer, Κ. Z. Lebensm. Unters. Forsch. 1989, 189, 326-330. De Roos, B.; van der Weg, G . ; Urgert, R.; van de Bovenkamp, P.; Charrier, Α.; Katan, Μ. Β. J. Agric. Food Chem. 1997, 45, 3065-3069. Kölling-Speer, I.; Speer, K . unpublished. Speer, K . ; Tewis, R.; Montag, Α. Z. Lebensm. Unters. Forsch. 1991, 192, 451-454. Kölling-Speer, I.; Strohschneider, S.; Speer, K . J. High Resol. Chromatogr. 1999, 22, 43-46. Speer, K. Proc. Euro Food Chem. V I , Hamburg, 1991, V o l . 1, pp 338-342. Speer, K . 16th Colloq. Sci. Int. Cafe; Association Scientifique International du Café, Paris, 1995, pp 224-231. Kurzrock, T.; Speer, K . 17th Colloq. Sci. Int. Cafe; Nairobi 1997, Association Scientifique International du Café: Paris, 1997, pp 133-140. Speer, K.; Tewis, R.; Montag, A . 14th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1991, pp 615-621. Tewis, R.; Montag, Α.; Speer, K . 15th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1993, pp 880-883. Kölling-Speer, I.; Kurt, Α.; Nguyen, Thu; Speer, K . 17th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1997, pp 201205. Speer, K . ; Sehat, N.; Montag, A . 15th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1993, pp 583-592.


251


31. Kurzrock, T.; Speer, Κ. Proc. 20th Int. Symposium Capillary Chromatogr., Riva del Garda, 1998. 32. Hruschka, Α.; Speer, K . Proc. Euro Food Chem. IX, Interlaken, 1997, pp 655-658. 33. Sehat, N.; Montag, Α.; Speer, K . 15th Colloq. Sci. Int. Cafe; Association Scientifique International du Café: Paris, 1993, pp 869-872. 34. Kurzrock, T. Dissertation, Technical University of Dresden, Dresden, Germany, 1998.


Diterpenes in Coffee - ACS Symposium Series (ACS Publications)

Recommend Documents