Coffee-specific Diterpenes Research Articles

Pressurized-fluid extraction of cafestol and kahweol diterpenes from green coffee

Abstract Cafestol and kahweol were extracted from green coffee beans ( C. arabica ) using a pressurized-fluid extraction (PFE) system. The effects of extracting solvent (i.e. water, ethanol and methanol), flow rate (0.5, 1.0, 2.0 and 3.0 mL · min − 1 ), extraction temperature (60, 80 and 100 °C) and time on extraction efficiency of coffee diterpenes were investigated. Quantification of diterpenes extracts was achieved by HPLC-UV method. The solvent flow rate was clearly the most significant process parameter. Diterpenes recovery resulted also influenced by temperature, except for the lowest flow rate, indicating solubility and mass transfer constraints. Kinetic degradation was also examined suggesting a thermolability of the diterpenes at 100 °C. The optimum extraction conditions were 100 °C and 3.0 mL · min − 1 using methanol. Under these conditions, PFE showed a remarkably higher efficiency compared with conventional extraction techniques. Industrial relevance Coffee is the world's most traded food commodity. Coffee-specific diterpenes (i.e. cafestol and kahweol) are high-value bioactive compounds, naturally occurring in the coffee lipid fraction. However, they are not easy to separate and likely to be unstable when isolated. High-pressure extraction, namely pressurized fluid extraction (PFE), appears as an attractive option as it reduces extraction time, thus avoiding degradation of thermolabile compounds, while requiring smaller quantities of solvent than traditional extraction methods (e.g. Soxhlet). PFE offers the possibility to operate with a wide range of liquid solvents well above their boiling point, but very little information can be found on the effect of operational conditions to efficiently recover coffee diterpenes. Such knowledge provides a basis for the rational design of PFE processes and, may be useful to allow industrial scale-up interpretation for coffee diterpenes extraction.

Anti-Angiogenic Properties of Cafestol and Kahweol Palmitate Diterpene Esters.

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, are 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. These findings were confirmed using ELISA assay for 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. J. Cell. Biochem. 117: 2748-2756, 2016. © 2016 Wiley Periodicals, Inc.

Abstract: S1-12 COFFEE AND CAFESTOL

There is an increasing evidence that oxidative stress is implicated in the processes of inflammation and carcinogenesis. It has been shown that kahweol and cafestol, coffee-specific diterpenes, exhibit chemoprotective effects. This study investigated the effects of kahweol and cafestol, coffee-specific diterpenes, on the hydrogen peroxide (H2O2)-induced oxidative stress and DNA damage in NIH3T3 cells. When the cells were treated with kahweol or cafestol, cytotoxicity, lipid peroxidation, and reactive oxygen species production induced by H2O2 were markedly reduced in a dose-dependent manner. Moreover, kahweol and cafestol were shown to be highly protected against H2O2-induced oxidative DNA damage as determined by the Comet (single cell gel electrophoresis) assay and the measurement of 8-oxoguanine content in NIH3T3 cells. Kahweol and cafestol also protected hydroxyl radical-induced 2-deoxy-d-ribose degradation by ferric ion-nitrilotriacetic acid and H2O2. In addition, kahweol and cafestol efficiently removed the superoxide anion generated from the xanthine/xanthine oxidase system. These results suggest that kahweol and cafestol are effective in protecting against H2O2-induced oxidative stress and DNA damage, probably via scavenging free oxygen radicals, and that kahweol and cafestol act as antioxidants.

Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1, UGT1A6 and GCLC were lower in tissues from nrf2 −/− mice than from nrf2 +/+ mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2 +/+ mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C + K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2 −/− mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C + K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C + K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5′-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 μmol/l furan, suggesting that this ring structure within C + K is insufficient for gene induction. Priming of nrf2 +/+ MEFs, but not nrf2 −/− MEFs, with C + K conferred 2-fold resistance towards acrolein.

Protective effects of kahweol and cafestol against hydrogen peroxide-induced oxidative stress and DNA damage

There is an increasing evidence that oxidative stress is implicated in the processes of inflammation and carcinogenesis. It has been shown that kahweol and cafestol, coffee-specific diterpenes, exhibit chemoprotective effects. This study investigated the effects of kahweol and cafestol, coffee-specific diterpenes, on the hydrogen peroxide (H 2O 2)-induced oxidative stress and DNA damage in NIH3T3 cells. When the cells were treated with kahweol or cafestol, cytotoxicity, lipid peroxidation, and reactive oxygen species production induced by H 2O 2 were markedly reduced in a dose-dependent manner. Moreover, kahweol and cafestol were shown to be highly protected against H 2O 2-induced oxidative DNA damage as determined by the Comet (single cell gel electrophoresis) assay and the measurement of 8-oxoguanine content in NIH3T3 cells. Kahweol and cafestol also protected hydroxyl radical-induced 2-deoxy- d-ribose degradation by ferric ion-nitrilotriacetic acid and H 2O 2. In addition, kahweol and cafestol efficiently removed the superoxide anion generated from the xanthine/xanthine oxidase system. These results suggest that kahweol and cafestol are effective in protecting against H 2O 2-induced oxidative stress and DNA damage, probably via scavenging free oxygen radicals, and that kahweol and cafestol act as antioxidants.

Hepatoprotective and antioxidant effects of the coffee diterpenes kahweol and cafestol on carbon tetrachloride-induced liver damage in mice

The hepatoprotective effects of kahweol and cafestol, coffee-specific diterpenes, on the carbon tetrachloride (CCl 4)-induced liver damage as well as the possible mechanisms involved in these protections were investigated. Pretreatment with kahweol and cafestol prior to the administration of CCl 4 significantly prevented the increase in the serum levels of hepatic enzyme markers (alanine aminotransferase and aspartate aminotransferase) and reduced oxidative stress, such as reduced glutathione content and lipid peroxidation, in the liver in a dose-dependent manner. The histopathological evaluation of the livers also revealed that kahweol and cafestol reduced the incidence of liver lesions induced by CCl 4. Treatment of the mice with kahweol and cafestol also resulted in a significant decrease in the cytochrome P450 2E1 (CYP2E1), the major isozyme involved in CCl 4 bioactivation, specific enzyme activities, such as p-nitrophenol and aniline hydroxylation. Kahweol and cafestol exhibited antioxidant effects on FeCl 2-ascorbate induced lipid peroxidation in a mouse liver homogenate, and on superoxide radical scavenging activity. These results suggest that the protective effects of kahweol and cafestol against the CCl 4-induced hepatotoxicity possibly involve mechanisms related to their ability to block the CYP2E1-mediated CCl 4 bioactivation and free radical scavenging effects.

Suppressive effects of the kahweol and cafestol on cyclooxygenase-2 expression in macrophages

Inducible cyclooxygenase-2 (COX-2) has been suggested to play a role in the processes of inflammation and carcinogenesis. Recent studies have shown the chemoprotective effects of kahweol and cafestol, which are coffee-specific diterpenes. This study investigated the effects of kahweol and cafestol on the expression of COX-2 in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Kahweol and cafestol significantly suppressed the LPS-induced production of prostaglandin E2, COX-2 protein and mRNA expression, and COX-2 promoter activity in a dose-dependent manner. Furthermore, kahweol blocked the LPS-induced activation of NF-κB by preventing IκB degradation and inhibiting IκB kinase activity. These results will provide new insights into the anti-inflammatory and anti-carcinogenic properties of kahweol and cafestol.

Coffee diterpenes prevent benzo[ a]pyrene genotoxicity in rat and human culture systems

The coffee-specific diterpenes cafestol and kahweol (C + K) have been identified as two important chemoprotective agents in coffee. In the present study, the potential preventive effects of C + K against the genotoxicity of B[ a]P were investigated in rat primary hepatocytes and in human bronchial Beas-2B cells. Several independent mechanisms were identified and their respective contribution to the overall protective effects was determined. A marked dose-dependent inhibition by C + K of B[ a]P DNA-binding was found in cells of both origins. However, data showed that the significant induction by C + K of the detoxifying enzyme GST-Yp subunit is the key mechanism of protection against B[ a]P DNA-binding in rat liver. In contrast, the phase I-mediated mechanism where C + K produce an inhibition of CYP 1A1 induction by B[ a]P is of key significance for the C + K protection in human Beas-2B cells. Moreover, this effect suggests a novel mechanism of chemoprotection by the coffee diterpenes cafestol and kahweol.

Cafestol and kahweol, two coffee specific diterpenes with anticarcinogenic activity

Epidemiological studies have found an inverse association between coffee consumption and the risk of certain types of cancers such as colorectal cancers. Animal data support such a chemopreventive effect of coffee. Substantial research has been devoted to the identification of coffee components that may be responsible for these beneficial effects. In animal models and cell culture systems, the coffee diterpenes cafestol and kahweol (C+K) were shown to produce a broad range of biochemical effects resulting in a reduction of the genotoxicity of several carcinogens including 7,12-dimethylbenz[ a]anthracene (DMBA), aflatoxin B 1 (AFB 1), benzo[ a]pyrene (B[ a]P) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Different mechanisms appear to be involved in these chemoprotective effects: an induction of conjugating enzymes (e.g. glutathione S-transferases, glucuronosyl S-transferases), an increased expression of proteins involved in cellular antioxidant defense (e.g. γ-glutamyl cysteine synthetase and heme oxygenase-1) and an inhibition of the expression and/or activity of cytochromes P450 involved in carcinogen activation (e.g. CYP2C11, CYP3A2). In animal models, the C+K-mediated induction of conjugating and antioxidant enzymes has been observed in hepatic, intestinal and kidney tissues. In the small intestine, these inductions were shown to be mediated by Nrf2-dependent transcriptional activation. In vitro investigations obtained in cell cultures of human origin indicate that the effects and mechanisms observed in animal test systems with C+K are likely to be of relevance for humans. In human liver epithelial cell lines transfected to express AFB 1-activating P450s, C+K treatment resulted in a reduction of AFB 1–DNA binding. This protection was correlated with an induction of GST-mu, an enzyme known to be involved in AFB 1 detoxification. In addition, C+K was found to inhibit P450 2B6, one of the human enzymes responsible for AFB 1 activation. Altogether, the data on the biological effects of C+K provide a plausible hypothesis to explain some of the anticarcinogenic effects of coffee observed in human epidemiological studies and in animal experiments.

Protective effects of coffee diterpenes against aflatoxin B 1-induced genotoxicity: mechanisms in rat and human cells

The coffee-specific diterpenes cafestol and kahweol (C+K) have been reported to be anticarcinogenic in several animal models. Proposed mechanisms involve a co-ordinated modulation of several enzymes responsible for carcinogen detoxification, thus preventing reactive agents interacting with critical target sites. To address the human relevance of the chemoprotective effects of C+K against aflatoxin B 1 (AFB 1) genotoxicity observed in rat liver, and to compare the mechanisms of protection involved in both species, animal and human hepatic in vitro test systems were applied. In rat primary hepatocytes, C+K reduced the expression of cytochrome P450 CYP 2C11 and CYP 3A2, the key enzymes responsible for AFB 1 activation to the genotoxic metabolite aflatoxin B 1-8,9 epoxide (AFBO). In addition, these diterpenes induced significantly GST Yc2, the most efficient rat GST subunit involved in AFBO detoxification. These effects of C+K resulted in a marked dose-dependent inhibition of AFB 1–DNA binding in this rat in vitro culture system. Their relevance in humans was addressed using liver epithelial cell lines (THLE) stably transfected to express AFB 1 metabolising cytochrome P450s. In these cells, C+K also produced a significant inhibition of AFB 1–DNA adducts formation linked with an induction of the human glutathione S-transferase GST-mu. Altogether, these results suggest that C+K may have chemoprotective activity against AFB 1 genotoxicity in both rats and humans.

The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism.

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB1) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-S-transferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB1. These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.