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Epidemiological studies support the association of coffee-specific diterpenes, with various beneficial health effects. Although anti-antiangiogenic properties of free cafestol and kahweol have been recently described, available data regarding their esterified form, in particular palmitate esters as the main diterpene esters present in coffee, is still rare. Given that angiogenesis plays an important role in many pathological conditions, including cancer growth and metastasis, this study aimed to assess and compare the potential anti-angiogenic effects of cafestol palmitate (CP) and kahweol palmitate (KP) in an in vitro angiogenesis model. According to our findings, both compounds inhibited angiogenesis steps on human microvascular endothelial cells (HMVECs), although a more significant effect was observed for KP.Compared to control, HMVECs viability decreased in a dose-dependent manner upon incubation either with CP or KP. Concentrations of 75 and 100 µM of each compound were cytotoxic. Cell proliferation was also dramatically reduced by both diterpene esters at 50 µM, although KP had a stronger inhibitory effect. However, CP and KP did not induce apoptosis on HMVECs. Both compounds reduced cell migration, but this effect was only statistically significant after KP incubation. Inhibition of VEGFR2 expression and its downstream effector Akt, but not Erk, was also observed in CP and KP-treated HMVECs. The following results were confirmed using ELISA assay that indicated the inhibitory effects of CP and KP on phosphorylated (active) VEGFR-2. Taken together, these data indicate that both CP and KP can be considered potent compounds against angiogenesis-dependent disorders. Our findings further indicate that KP exerts more potent anti-angiogenic effects than CP, in most of assays.3

The influence of different brewing conditions on the concentration of the main caffeoylquinic acids (3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), and 5-caffeoylquinic acid (5-CQA)) was investigated. For this purpose, twenty-four coffee brews were extracted and analyzed using HPLC-DAD at 325 nm. Our findings demonstrate the great impact of brewing techniques on the caffeoylquinic acids (CQAs) content. The major isomer was 3-CQA, accounting for about 50% of the total CQAs, followed by 5-CQA and 4-CQA, accounting for about 24–36% for each one. The total content of CQAs was in the range of 45.79 to 1662.01 mg/L, found in iced cappuccino and pod espresso, respectively. In conclusion, this study demonstrates that coffee brews, in particular those prepared using pressurized methods, can be considered as the potential sources of antioxidants such as CQAs.

The aim of the present study was to evaluate the effect of preparation conditions of espresso coffee (EC) on the diterpenes profile. ECs were prepared from roasted and ground (R&G) Arabica coffee and analyzed for the content of cafestol and kahweol by liquid-liquid extraction followed by HPLC-DAD, as well as their lipid content. The main variables in the present study were: the water quantity, the amount of coffee, the particle size, the percolation time, the water temperature and pressure.Average cafestol, kahweol and lipid content of R&G Arabica coffee were 467 ± 20 mg/100 g, 638 ± 33 mg/100 g, and 15.1 ± 0.1 g/100 g, respectively. Although all parameters influenced the diterpenes content of ECs (21 samples), the particle size and water quantity were the most significant ones. It was possible to reduce the total diterpenes from 58.8 ± 0.7 mg/L (2.3 mg/40 mL) to 30.7 ± 0.8 mg/L (1.2 mg/40 mL) by varying the brewing conditions. The extraction yield of diterpenes and lipids were in the range of 1.5-2.5% and 7.0-9.0%, respectively. Regarding total cafestol and kahweol, very fine particles seem to be more desirable for the production of highly concentrated brew (2.3 mg/40 mL) with cafestol and kahweol extraction yields of 2.8 and 2.9%, respectively, than other studied ECs. On the other hand, samples brewed at 70 ºC exhibited lower diterpenes content (1.2 mg/40 mL) and diterpenes extraction efficiency (1.4%) with respect to all other considered parameters. This study clearly shows that parameters for coffee brew preparation may be changed to modify the diterpenes content of ECs according to the desired purpose.Keywords Espresso coffee, Brewing, Diterpenes, Roasted coffee beans IntroductionCoffee is a globally consumed beverage and can be prepared in different ways. One of the common coffee brewing techniques is the Italian "Espresso" which are consumed over than 50 million cups per days [1]. Espresso coffee (EC) is an intense beverage with special aroma made for immediate consumption [2]. For the preparation of espresso coffee, a limited amount of hot water (90±5 °C), under pressure (9±2 bar), passes through a compressed finely roasted and ground coffee (R&G, 6.5±1.5 g) in a short period of time (30±5 s) and produces a brew (15-50 mL) with strong taste and flavor topped with crema (dense foam layer) [1,

Several coffee brews, including classical and commercial beverages, were analyzed for their diterpene esters content (cafestol and kahweol linoleate, oleate, palmitate and stearate) by high performance liquid chromatography with diode array detector (HPLC-DAD) combined with spectral deconvolution. Due to the coelution of cafestol and kahweol esters at 225 nm, HPLC-DAD did not give accurate quantification of cafestol esters. Accordingly, spectral deconvolution was used to deconvolve the co-migrating profiles. Total cafestol and kahweol esters content of classical coffee brews ranged from 5-232 to 2-1016 mg/L, respectively. Commercial blends contained 1-54 mg/L of total cafestol esters and 2-403 mg/L of total kahweol esters. Boiled coffee had the highest diterpene esters content, while filtered and instant brews showed the lowest concentrations. However, individual diterpene esters content was not affected by brewing procedure as in terms of kahweol esters, kahweol palmitate was the main compound in all samples, followed by kahweol linoleate, oleate and stearate. Higher amounts of cafestol palmitate and stearate were also observed compared to cafestol linoleate and cafestol oleate. The ratio of diterpene esters esterified with unsaturated fatty acids to total diterpene esters was considered as measure of their unsaturation in analyzed samples which varied from 47 to 52%. Providing new information regarding the diterpene esters content and their distribution in coffee brews will allow a better use of coffee as a functional beverage.

14In this manuscript, the separation of four kahweol esters and four cafestol esters that are present in 15Arabica coffee brews was investigated using HPLC/DAD. Those compounds could not be baseline 16 separated in a single chromatogram using RP-LC but the kahweol esters could be selectively detected by 17 setting the wavelength at 290 nm. In this case the four kahweol esters were baseline separated allowing 18 for their quantification. Such approach was not possible for the cafestol esters and spectral deconvolution 19 was used to obtain deconvolved chromatograms that were specific to cafestol and kahweol esters 20 respectively. In each of those chromatograms, the 4 esters were baseline separated allowing for the 21 quantification of the eight targeted compounds. Because kahweol esters could be quantified either using 22 the chromatogram obtained by setting the wavelength at 290 nm or using the deconvolved chromatogram, 23 those compounds were used to compare the analytical performances. Slightly better LOD were obtained 24 using the deconvolved chromatogram (average LOD of 5.7 mg/L against 6.7 mg/L). Identical 25 concentrations were found in a real sample with both approaches. The peak areas of the different diterpene 26 esters in the deconvolved chromatograms were proven to be repeatable with an average intra-day 27 repeatability of 0.8 % (6 replicates) and an inter-day repeatability of 1.0% (3 successive days, two 28 replicates each day). This work demonstrates the accuracy of spectral deconvolution in conjunction with 29 HPLC-DAD (HPLC-DAD/SD) to mathematically separate co-eluting compounds using the full spectra 30 recorded by the DAD. 31 32

The present paper aimed the optimization of the saponification and extraction of diterpenes from coffee brews using a simple methodology based on HPLC-DAD, as well as to quantify the diterpenes and their palmitate esters content in several coffee brews. Regarding cafestol and kahweol, the best conditions to maximize total extraction