Cafestol, a Bioactive Substance in Coffee, Has Antidiabetic Properties

Article pubs.acs.org/jnp

Cafestol, a Bioactive Substance in Coffee, Has Antidiabetic Properties in KKAy Mice Fredrik Brustad Mellbye,*,† Per Bendix Jeppesen,† Pedram Shokouh,‡,§ Christoffer Laustsen,⊥ Kjeld Hermansen,† and Søren Gregersen† †

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-Hansens Gade 2, 8000 Aarhus C, Denmark Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 82, 8200 Aarhus N, Denmark § The Danish Diabetes Academy, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark ⊥ MR Research Centre, Department of Clinical Medicine, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark ‡

ABSTRACT: Daily coffee consumption is inversely associated with risk of type-2 diabetes (T2D). Cafestol, a bioactive substance in coffee, increases glucose-stimulated insulin secretion in vitro and increases glucose uptake in human skeletal muscle cells. We hypothesized that cafestol can postpone development of T2D in KKAy mice. Forty-seven male KKAy mice were randomized to consume chow supplemented daily with either 1.1 (high), 0.4 (low), or 0 (control) mg of cafestol for 10 weeks. We collected blood samples for fasting glucose, glucagon, and insulin as well as liver, muscle, and fat tissues for gene expression analysis. We isolated islets of Langerhans and measured insulin secretory capacity. After 10 weeks of intervention, fasting plasma glucose was 28−30% lower in cafestol groups compared with the control group (p < 0.01). Fasting glucagon was 20% lower and insulin sensitivity improved by 42% in the high-cafestol group (p < 0.05). Cafestol increased insulin secretion from isolated islets by 75−87% compared to the control group (p < 0.001). Our results show that cafestol possesses antidiabetic properties in KKAy mice. Consequently, cafestol may contribute to the reduced risk of developing T2D in coffee consumers and has a potential role as an antidiabetic drug.

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showed that caffeic acid increases insulin secretion10 and that extremely high doses of chlorogenic acid are able to increase insulin secretion and glucose uptake into myocytes.11 Most recently, we demonstrated that the diterpene cafestol, at concentrations ranging from 10−12 to 10−8 M, is capable of increasing glucose uptake in human skeletal muscle cells and stimulating insulin secretion after short- and long-term incubation of INS-1E beta cells.12 Such concentrations of cafestol are likely to be achieved in subjects drinking unfiltered coffee, considering the high bioavailability of cafestol.13 These concentrations may even be achieved in subjects drinking filtered coffee. However, cafestol poorly passes a cellulose coffee filter, and many of the studies focusing on coffee consumption and T2D risk are based on filtered coffee. It has been found that a cup of Scandinavian boiled coffee contains 6.2 mg, Turkish coffee contains 4.2 mg, and French press coffee contains 2.6 mg of cafestol, while a cup of filtered coffee may contain only 0.1 mg of cafestol.14 No studies have elucidated the potential antidiabetic effects of cafestol in a living animal or human model. The KKAy mouse is a genetically modified model that becomes obese

ype-2 diabetes (T2D) is characterized by impaired insulin secretion, enhanced glucagon secretion, and/or reduced insulin sensitivity.1 Globally, T2D represents a great burden to both patients and society. Consequently, it is imperative to exploit preventive means and to identify modifiable risk factors to minimize disease-related complications. Randomized intervention studies have established that lifestyle intervention with diet and exercise can delay or prevent T2D.2 Recently, it was demonstrated that coffee drinkers daily consuming 3−4 cups of coffee have a 25% lower relative risk of developing T2D than subjects drinking 1 or fewer cups per day.3,4 The question arises, what is the mechanism of action behind the beneficial effects of coffee on the development of T2D? Coffee contains a large number of bioactive substances that can be categorized as alkaloids including methylxanthines (mainly caffeine) and trigonelline, phenolic acids (e.g., caffeic, ferulic, and quinic acids and their ester-like chlorogenic acids), diterpenes (e.g., cafestol and kahweol), lignans (secoisolaricresenol), flavonoids (e.g., daidzein and catechins), and Maillard reaction products (in roasted coffee).5−7 The main stimulant in coffee, caffeine, has attracted major interest. However, since decaffeinated coffee displays the same inverse association with T2D development as caffeinated coffee, it is less likely that all the beneficial effects of coffee are merely or mainly attributed to caffeine.4,8,9 Previously, animal studies © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 5, 2017 Published: August 1, 2017 2353

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food consumption (4.7 g (±0.2)) than the low- and highcafestol groups (5.4 g (±0.2), p < 0.05, and 5.9 g (±0.1), p < 0.001, respectively). Fasting Blood Glucose. There were no significant differences between the two intervention groups and the control group at study start (Figure 2). During the 10-week

shortly after birth and serves as a model for T2D. The aim of our study was to clarify the potential antidiabetic effects of cafestol by investigating the longer-term effects of cafestol in vivo on blood glucose, insulin, and glucagon levels, weight, and food consumption in KKAy mice. Furthermore, we measured the impact of the dietary supplement with cafestol on insulin secretion from isolated islets in vitro, as well as gene expressions in liver, muscle, and fat tissues and liver fat content. We hypothesized that cafestol can prevent or delay the development of T2D in KKAy mice by improving beta cell function and insulin sensitivity.

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RESULTS AND DISCUSSION Body Weight and Food Consumption. During the study, the mice increased body weight as illustrated in Figure 1A. At

Figure 2. Fasting tail blood glucose of the KKAy mice at intervention week one, three, five, seven, and 10. Data are presented as mean ± SEM. Comparisons are made between the control group and each cafestol group individually. * = p < 0.05. ** = p < 0.01. *** = p < 0.001.

intervention, fasting blood glucose gradually increased in all groups. At week 10, mean fasting blood glucose was 43% higher in the control group (18 mmol/L (±1.2)) than in the lowcafestol group (12.6 mmol/L (±0.7), p < 0.001) and 39% higher in controls than in the high-cafestol group (12.9 mmol/ L (±0.6), p < 0.01). At week 10, nonfasting blood glucose was 51% higher in the control group (26.2 mmol/L (±1.2)) compared to the low-cafestol group (17.3 mmol/L (±1.6), p < 0.0001) and 33% higher than the high-cafestol group (19.7 mmol/L (±1.6), p < 0.01) (Figure 3). Plasma Hormones and Lipids. Plasma insulin, glucagon, total cholesterol, HDL, and LDL concentrations were measured at intervention week 10 (study end) in a blood sample collected

Figure 1. Body weight of the KKAy mice at intervention week two, four, six, and eight (A); 24 h food consumption by the KKAy mice at intervention week two and week 10 (B). Data are presented as mean ± SEM. Comparisons are made between control and cafestol groups within each week. * = p < 0.05. ** = p < 0.01. *** = p < 0.001.

week four, there were no significant weight differences between the groups. At week eight, the high-cafestol group weighed on average 44.9 g (±0.8), almost 3 g more than the control group (42.1 g (±0.65), p < 0.01). The average 24 h food consumption at the study’s start for the three groups was not significantly different. At the study’s end, the control group had a lower 24 h

Figure 3. Nonfasting tail blood glucose at intervention week 10, measured 5 h after administration of chow with 0 (control), 0.4 (low), or 1.1 mg (high) of cafestol. Data are presented as mean ± SEM. Comparisons are made between the control group and each cafestol group individually. * = p < 0.05. ** = p < 0.01. *** = p < 0.001. 2354

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from the retrobulbar plexus of the KKAy mice. The highcafestol control group had a 20% lower plasma glucagon level (54.3 pg/mL (±2.8)) than the control group (68.1 pg/mL (±4.8), p < 0.05) and 28% lower plasma glucagon level than the low-cafestol group (74.7 pg/mL (±2.4), p < 0.05) (Figure 4A). The high-cafestol group tended to have a lower plasma insulin level than the low-cafestol and the control groups (Figure 4B). Insulin resistance (HOMA-IR) was 42% lower in the high-cafestol group compared to the control group (p < 0.05), while there were no significant differences between the low-cafestol group and the control or the high-cafestol group (Figure 4C). Total (Figure 5A), HDL (Figure 5B), and LDL

cholesterol (Figure 5C) and plasma triglycerides (Figure 5D) showed no significant differences between intervention groups and the control group. Insulin Secretory Capacity. Ten weeks of intervention elicited increased glucose (16.7 mM)-stimulated insulin secretion from isolated islets by 87% (20.9 ng/mL (±1.9), p < 0.001) and 75% (19.6 ng/mL (±1.6), p < 0.01) from lowand high-cafestol groups, respectively, compared to the control group (11.2 ng/mL (±1.4)) (Figure 6). At low (3.3 mM) glucose we observed a reduced insulin secretion (by 67% (1.0 ng/mL (±0.1), p < 0.001, and 44% (2.2 ng/mL (±0.4), p < 0.05)) in low- and high-cafestol groups compared to the control group (5.0 ng/mL (±0.7)). Gene Expression Results. After 10 weeks of intervention, gene expression analysis showed significant group differences only for the glucagon-receptor (GCRG) and insulin-receptor (INSR) genes. In white adipose tissue from the mice in the high-cafestol group, there was an increase of GCRG expression compared to the control group (p < 0.05) (Figure 7A). In muscle tissue, however, the low-cafestol group showed a reduction of GCRG expression compared to the control group (p < 0.05) (Figure 7B). In liver tissue, INSR gene expression was increased in the high-cafestol group compared to the control group (p < 0.05) (Figure 7C). Apart from these, there were no significant differences in expression of genes listed in Table 1 in the analyzed tissues. MR Spectroscopy and Triglyceride Measurement of Liver Tissue. There were no significant differences in water− fat fraction of liver tissue measured by MR-spectroscopy or triglyceride concentration in liver tissue samples between groups. First Demonstration of Antidiabetic Effects of Cafestol in Animals. In the present study, we demonstrated that 10 weeks of oral administration of both low and high doses of cafestol resulted in reduced blood glucose in diabetic KKAy mice and concomitantly exaggerated insulin secretion in vitro along with lower plasma glucagon levels and reduced insulin resistance. The higher weight in the mice group treated with high-dose cafestol is probably in part due to less glucosuria and reduced loss of calories as well as larger food intake than in controls. To our knowledge, this is the first demonstration of an antidiabetic effect of cafestol in animals. Consequently, cafestol could postpone frank hyperglycemia in this T2D animal model of KKAy mice that develops diabetes at 7 to 10 weeks of age.15 Concomitantly, we observed lower plasma insulin levels and improved insulin sensitivity in the cafestol groups. During the course of diabetes development, it is believed that beta cells initially compensate the increasing insulin resistance with higher insulin secretion, while later on the secretory capacity decreases. T2D is also characterized by increased circulating levels of glucagon.16 Interestingly, cafestol supplementation elicited lower glucagon levels. The combined hormonal and metabolic features of cafestol observed are considered beneficial and diabetes-preventive. We did not observe significantly higher plasma LDL levels in the high-cafestol group, although it tended to be higher. An LDL-cholesterol elevating effect of cafestol in humans has previously been shown. Thus, for every 10 mg daily of cafestol there is an LDL increase of 0.13 mmol/L.14,17−20 Cafestol is believed to increase cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activity.20 We anticipate that the oral antidiabetic dose in humans will be relatively low and that the antidiabetic benefits will not be

Figure 4. Fasting plasma glucagon (A) and insulin (B) of KKAy mice at intervention week 10. Insulin resistance (HOMA-IR) of KKAy mice at intervention week 10 (C). Data are presented as mean ± SEM. Comparisons are made between the control group and each cafestol group individually. * = p < 0.05. 2355

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Figure 5. Fasting plasma total cholesterol (A), high-density lipoprotein (HDL) (B), low-density lipoprotein (LDL) (C), and triglycerides (D) of KKAy mice at intervention week 10. Data are presented as mean ± SEM.

Dyslipidemia and obesity are known risk factors and precursors in the development of T2D, and therefore we would expect the measured weight developments to benefit the control group. Our results indicate that positive effects from either increased islet insulin secretory capacity, altered gene expression, or both exceed the potential diabetogenic effects of weight gain. Interestingly, we observed that isolated islets of the cafestol groups had improved beta cell function and secreted more insulin in response to high glucose than the control group, but less insulin in response to low glucose. This corroborates our previous findings in INS-1 cells, where we found that cafestol increased insulin secretion at high glucose and lowered insulin secretion at low glucose.12 Thus, it appears that the effects of cafestol are glucose-dependent and that cafestol apparently does not tend to induce hypoglycemia, a feared feature of some antidiabetic drugs, e.g., insulin and sulfonylureas. The present study adds further knowledge about mechanisms of action of bioactive substances in coffee. It has earlier been shown that, for example, polyphenols contained in coffee can modulate insulin sensitivity by increasing the postprandial glucagon-like peptide 1 response in humans and rodents.21,22 We studied the impact of cafestol on gene expression in adipose and liver tissue and observed an increased expression of glucagon receptor genes in adipose tissue and increased expression of insulin receptor genes in liver tissue. A simultaneous increased expression of glucagon receptors in adipose tissue and reduced expression of glucagon receptors in muscle tissue may theoretically result in increased lipolysis and reduced muscle tissue loss in catabolic states, which may contribute to the antidiabetic effect seen. We also found that

Figure 6. Insulin secretion from isolated islets of Langerhans from KKAy mice harvested from the control, low-cafestol, and high-cafestol groups: insulin secretion was measured after 60 min of incubation at low (3.3 mmol/L) or high (16.7 mmol/L) glucose. Comparisons are made between the low or high glucose incubated islets in the control group and the low or high glucose incubated islets from each cafestol group individually. Data are presented as mean ± SEM.* = p < 0.05. ** = p < 0.01. *** = p < 0.001.

outweighed by a small increase in LDL cholesterol. During the 10-week intervention, we found the food consumption and weight of the high-cafestol group to be higher than the lowcafestol and control group. The control group’s relatively lower body weight may to some extent be secondary to glucosuria. 2356

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Figure 7. Glucagon receptor gene expression in adipose (A) and muscle (B) tissue and insulin receptor gene expression in liver tissue (C). Data are presented as mean ± SEM. Comparisons are made between the control group and each cafestol group individually. * = p < 0.05.

HOMA-IR does not seem to be mediated via changes in the central transcription factors PPARalpha, PPARgamma, and Sirt1 since they were unchanged by cafestol supplementation in both liver and muscle tissues. It should, however, be underlined that the interpretation of the gene changes observed is open for discussion. We chose to supplement with two doses of cafestol. The dosages were empirically set to 0.4 and 1.1 mg cafestol daily, translating to 41 and 113 mg cafestol/m2 mouse body surface area. There is about 6.2 mg of cafestol in a 150 mL cup of boiled coffee.14 If extrapolating our intervention mouse data to humans, this is comparable to a human, with a 1.71 m2 body surface area, receiving 11 and 31 cups of boiled coffee every day, respectively.23 These amounts were chosen due to the well-known higher rodent mass-specific metabolic rate in mice. Thus, we anticipate that the cafestol dosage chosen may be comparable to realistic human coffee consumption patterns. Our study lasted for 10 weeks, during which the control group developed severe hyperglycemia (T2D). It would have been of interest to see if the beneficial effects of cafestol are lasting, e.g., during longer-term supplementation with cafestol and after discontinuation. Furthermore, it would have been of interest to include an additional group receiving a conventional antidiabetic drug as a positive control. Our results indicate that daily intake of cafestol can delay the onset of T2D in KKAy mice, a T2D animal model. Our in vivo results support the antidiabetic effects previously demonstrated in vitro.12 More studies are needed to confirm and clarify whether cafestol can be used for the treatment or prevention of T2D in humans.

Table 1. List of Genes Investigated for Expression in Liver, Muscle, and Adipose Tissue from KKay Mice, with TaqMan, ID, and Name TaqMan

ID

Hs99999901_s1 Rn00710172_m1 Rn00573474_m1 Rn01483784_m1 Rn00690901_m1

18s Abca1 Acaca Adipor1 Akt2

Rn00576935_m1

AMPKα2

Rn00580728_m1 Rn00670361_m1 Rn01463550_m1 Rn00566576_m1 Rn00597158_m1 Rn00563565_m1 Rn00565296_m1 Rn01527840_m1

CD36 FABP4 FAS G6PD Gcgr GLUT2 GYS2 Hprt1

Rn01637243_m1 Rn01482270_s1 Rn01473307_m1 Rn00561474_m1 Rn00581185_m1 Rn00571541_m1 Rn00564547_m1 Rn00440891_m1 Rn00580241_m1

InsR IRS2 JNK1 Lipc LXRα Mtor Pi3K PKCδ PPARGC1α

Rn00566193_m1

PPARα

Rn00440945_m1

PPARγ

Rn01428094_m1 Rn00566938_m1 Rn00690587_g1

Sirt1 SOD1 SOD2

name 18s rRNA ATP-binding cassette transporter acetyl-CoA carboxylase 1 adiponectin receptor 1 V-akt murine thymoma viral oncogene homologue 2 5′-AMP-activated protein kinase catalytic subunit alpha-2 fatty acid translocase adipocyte protein 2 Fas cell surface death receptor glucose-6-phosphate dehydrogenase glucagon receptor glucose transporter 2 glycogen synthase 2 hypoxanthine phosphoribosyl transferase 1 insulin receptor insulin receptor substrate 2 mitogen-activated protein kinase 8 hepatic lipase liver x receptor alpha mechanistic target of rapamycin phosphoinositide 3-kinase protein kinase c delta peroxisome proliferator-activated receptor gamma coactivator 1-alpha peroxisome proliferator-activated receptor alpha peroxisome proliferator-activated receptor gamma sirtuin 1 superoxide dismutase 1 superoxide dismutase 2

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EXPERIMENTAL SECTION

Chemicals. Cafestol acetate was from Sigma (Steinheim, Germany) and was ≥98% pure (tandem mass spectrometry). Study Design. Forty-seven male KKAy mice were randomized to consume 1.1 mg (high), 0.4 mg (low), or 0 mg (control) of cafestol daily added to normal chow for 10 weeks. The mice were 4 weeks old at study start and were provided by Taconic Biosciences (Hudson, NY, USA). At study start, a tail blood sample was collected and bodyweight was measured. Every second week, fasting tail blood glucose was evaluated and body weight was measured using a Precision Xceed strip glucose measurement system (Abbot Laboratories, North Chicago, IL, USA). Food consumption of the individually caged animals was measured over 24 h at study start and after 8 weeks. At study end, the mice were anesthetized, and blood samples were collected from the retrobulbar plexus. Chilled tubes containing a 3:l mixture of aprotinin/

high cafestol had significantly higher expression of insulin receptor genes in liver tissue compared to the control group. Probably, the increased expression of insulin receptor genes in liver tissue can contribute to the antidiabetic effects and improved insulin sensitivity found in the high-cafestol group. Interestingly, the improved insulin sensitivity demonstrated by 2357

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and 40 cycles of 95 °C, 15 s, and 60 °C, 1 min. The relative gene expression was calculated using the (1 + efficiencies) − ΔCT method, and the fold change (FC) was used to compare the expression levels. ΔCT is the difference in cycle threshold (CT) value between each target gene based on the average of the triplicates and the geometric mean CT values of reference genes. MR Scan and Triglyceride Measurement of Liver Tissue. Liver tissue, collected as stated above, was also used for a water−fat fraction NMR and triglyceride determination, respectively. NMR water−fat-fraction determination was performed on a 9.4 T preclinical MR system (Agilent, UK) equipped with a 1H volume mouse coil (RAPID Biomedical, Würzburg, Germany), using a single pulse and acquired NMR sequence with a 20 kHz spectral width and 2048 complex points. The water−fat fraction was estimated using the iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) analysis25 in MATLAB (Mathworks, Natick, MA, USA). Triglyceride concentration was measured by a COBAS c111 analyzer. Statistical Analysis. All statistical analysis was performed with GraphPad Prism software and IBM SPSS Statistics 22. One-way analysis of variance (ANOVA) and Tukey’s post hoc test was used to compare groups in all results except for HOMA-IR and gene expression analysis results, where an unpaired Student’s t test was used. Data are presented as mean ± SEM; p-values < 0.05 were considered significant.

heparin were used. Tubes were centrifuged at 1702 RCF for 10 min at 4 °C, and plasma was collected and frozen for later analysis of glucose, insulin, glucagon, triglycerides, and cholesterol. Islets of Langerhans were isolated using the collagenase digestion technique.24 The mice were euthanized, and liver, muscle (soleus), and visceral fat tissues were collected for gene expression analysis. All groups had unlimited access to normal chow (Altromin 1324, Brogaarden, Lynge, Denmark) and water at all times, except every morning, when normal chow was removed and a single chow pellet with (intervention groups) or without (control group) cafestol was administered. This bolus was produced by dissolving cafestol in absolute ethanol and vaporizing it into chow pellets. This vaporizing procedure was repeated every other week to compensate for possible cafestol loss over time. The control group received chow pellets with only vaporized ethanol. When the pellet was completely consumed, usually after 2−3 h, normal chow was given to the animals again. By the end of week 10, nonfasting blood glucose was measured 5 h after administration of a single pellet with (intervention groups) or without (control group) cafestol. Insulin Secretion Studies. After collagenase treatment and isolation of the islets of Langerhans, these were incubated in RPMI 1640 (Gibco, Life Technologies, Naerum, Denmark) for 24 h at 37 °C, 5.0% CO2. Subsequently, RPMI was replaced by Hank’s balanced salt solution (Sigma, Steinheim, Germany). Hereafter, islets were transferred to well plates, one islet in each well, and incubated in a modified Krebs ringer buffer (M-KRB, Sigma), with either 3.3 or 16.7 mM glucose in a 37 °C water bath for 1 h. Hereafter, 0.1 mL of the incubation medium was transferred from each well to plastic tubes and frozen for later insulin measurement, which was carried out using guinea pig anti-porcine insulin antibody, mono-125I-(Tyr A14)labeled human insulin as tracer, and rat insulin as standard (Novo Nordisk, Bagsvaerd, Denmark). Plasma Insulin, Glucagon, Triglycerides, and Cholesterol Analysis. Insulin and glucagon were measured using RIA kits (Linco Research Inc., St. Charles, MO, USA). Plasma total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol were analyzed using enzymatic colorimetric methods on a COBAS c111 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). HOMA-IR is a measure of insulin resistance and was calculated from the following formula: (plasma glucose × plasma insulin)/22.5. Gene Expression Analysis. Liver, muscle, and fat tissues were collected as stated above, immediately frozen in liquid nitrogen, and later transferred to a −80 °C freezer. Total RNA was extracted from frozen liver, adipose, and muscle tissue using the RNeasy minikit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA purity and concentrations were evaluated by measuring absorbance at 260−280 nm (NanoDrop ND-8000 UV− vis spectrophotometer, NanoDrop Technologies, Wilmington, DE, USA). The 18S and 28S ribosomal bands were examined on a 0.7% nondenaturing agarose gel stained with SYBR green to evaluate the integrity of the RNA. Quantitative real-time PCR was performed using the Fluidigm BioMark System (AROS, Applied Biotechnology AS, Denmark). The samples were analyzed for expression of 28 gene transcripts using mice-specific TaqMan assays. Two specific assays were used as endogenous control: 18S (ABI, Hs99999901_s1) and Hprt1 (ABI, Rn01527840_m1). A list of the TaqMan assays used is given in Table 1. Samples were analyzed using Fluidigm 96.96 Dynamic (Fluidigm catalog no. BMK-M-96.96) arrays with assay triplicates in accordance with the manufacturer’s protocol. One hundred nanograms of RNA was used as input in 20 μL reverse transcript reaction using the High Capacity cDNA reverse transcription kit (ABI, PN4368813) in accordance with the manufacturer’s protocol. After reverse transcription, the cDNA samples were amplified according to the instructions given in the Fluidigm Specific Target Amplification Quick Reference Manual. The cDNA was amplified using a targetspecific assay (diluted 1:100) and TaqMan PreAmp master mix (2×) (ABI, PN 4391128) in a 14-cycle thermal cycler reaction: 95 °C 10 min and 14 cycles of 95 °C 15 s and 60 °C 4 min. Amplification was performed using the standard conditions: 50 °C 2 min, 95 °C 10 min,

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45 26 84 44 25. E-mail: [email protected]. ORCID

Fredrik Brustad Mellbye: 0000-0002-1477-1178 Notes

The authors declare the following competing financial interest(s): F. B. Mellbye, P. B. Jeppesen, K. Hermansen, and S. Gregersen in cooperation with Aarhus University and Central Denmark Region have a patent pending on the usage of cafestol as an antidiabetic drug. The authors declare no other conflicts of interest.

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ACKNOWLEDGMENTS L. Trudsø and E. Mølgård Jensen are thanked for skilled technical assistance. The project was awarded a POC grant of DKK 250.000 by Aarhus University.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00395 J. Nat. Prod. 2017, 80, 2353−2359