Researchers discovered three novel coffee diterpene esters, named caffaldehydes A, B and C, in roasted Arabica beans with strong blood sugar–lowering activity. Using advanced NMR and LC-MS/MS, the team also identified three additional novel trace diterpenes, highlighting coffee’s hidden role as a functional food.
Study: Bioactive oriented discovery of diterpenoids in Coffea arabica basing on 1D NMR and LC-MS/MS molecular network. Image credit: amenic181/Shutterstock.com
A recent study published in Beverage Plant Research analyzed coffee diterpenes' composition and potential activity. Researchers identified three novel coffee diterpene esters from roasted Arabica coffee beans, named caffaldehyde A, B, and C, with significant alpha-glucosidase inhibitory activity (IC₅₀ values stronger than the positive control, acarbose).
What are functional foods?
Functional foods are whole, enriched, or enhanced foods that offer additional benefits beyond basic nutrition. These are naturally occurring foods like berries or modified ingredients like probiotics. In addition to essential nutrients, functional foods provide many biologically active ingredients to humans. These bioactive ingredients may possess anti-oxidation, neuroprotection, hypoglycemic, and lipid-lowering properties.
Coffee is one of the most widely consumed beverages worldwide and is considered one of the most economically valuable plants. Arabica (Coffea arabica) and Robusta (Coffea canephora) are popular coffee species that are widely planted and traded globally. Coffee diterpenoids are key characteristic components of coffee; most exist as diterpene esters. Although 24 fatty acids form these esters, linoleic acid and palmitic acid diterpene esters are abundantly found.
The content of coffee diterpenoids varies among different coffee species. To date, over 70 types of coffee diterpenes have been documented. Certain coffee diterpenoids, such as kahweol and cafestol, have been identified to have anticancer and blood sugar-lowering effects. Since coffee roasted beans are an extremely complex mixture system, it is important to explore if any other novel active coffee diterpenes exist.
Analytical methods to detect bioactive compounds
Scientists experience significant difficulties in extracting functional ingredients from complex food extracts. Traditional phytochemical analysis involves multiple steps, including extraction, separation, structure analysis, and activity evaluation, which are tedious, time-consuming, and not always accurate.
In contrast, technological advances, such as Nuclear Magnetic Resonance (NMR), High-Performance Liquid Chromatography-Solid-Phase Extraction-Nuclear Magnetic Resonance spectroscopy (HPLC-SPE-NMR), Liquid Chromatography-multiple-stage Mass Spectrometry (LC-MSn), and High-Performance Liquid Chromatography coupled with multiple Mass Spectrometry (MSn), can promote biological activity detection or metabolomics analysis in a relatively accelerated manner.
NMR and LC-MSn are commonly used to identify bioactive compounds and assess their activities. Scientists also use an LC-MS/MS-based molecular networking approach to track active compounds. Considering the benefits of these analytical methods, coupling NMR with LC-MS/MS could be beneficial in identifying bioactive compounds.
About the study
The current research combined NMR and LC-MS/MS technology to identify new coffee diterpene esters in roasted Arabica coffee beans. The structure of the novel bioactive diterpenes was characterized through spectral analysis. The objective was to minimize chromatographic separation wherever possible to reduce the use of solvents and consumables, making the process more environmentally friendly and efficient.
A three-step scheme was proposed; the first step involved subjecting the sub-fraction samples to 1H-NMR and activity detection. The cluster-heatmap approach was used to group 1H NMR and activity data and screen out the active groups. In the second step, the fractions of interest were screened out for 13C-DEPT NMR (Carbon-13 Distortionless Enhancement by Polarization Transfer Nuclear Magnetic Resonance) detection. Here, fractions most likely to contain active novel compounds were purified by semi-preparative HPLC.
The isolated compounds were further determined by 2D NMR and High-Resolution Electrospray Ionization Mass Spectrometry (HRESIMS), and their functions were validated. The third step involved an LC-MS/MS-based molecular network to examine potential trace novel active compounds.
Study findings
The current study divided the diterpene extract of roasted coffee into 19 fractions using silica gel column chromatography. The fractions' 1H NMR spectra were analyzed to obtain the integral value of the characteristic region. The cluster heatmap generated a matrix with 19 rows and 1,167 columns.
According to the cluster result based on 1H NMR data, fractions were divided into five groups. Since the Fr.1−Fr.3 group did not exhibit any α-glucosidase inhibitory activity, it was dropped from further analysis. Among the four groups that exhibited varying levels of inhibitory activity, Fr.9−Fr.13 exerted maximum α-glucosidase inhibitory activity.
Combining clustering heatmap and 1H NMR spectral data indicated compounds in Fr.9−Fr.13 group exert inhibitory activity. Subsequently, the representative sample Fr.9 from the active group (Fr.9−Fr.13) was assessed using 13C DEPT NMR data, indicating a characteristic carbon signal of aldehyde groups. HPLC analysis of sample Fr.9 resulted in three chromatographic peaks. Semi-preparative HPLC purified these compounds to obtain compounds 1−3.
1H NMR spectrum indicated the presence of two methyl groups, one oxidation methylene group, three olefinic protons, and one aldehyde proton group. 13C-DEPT NMR spectrum highlighted the presence of a total of 36 carbon atoms. 13C NMR analysis revealed compound 1 is a diterpene ester with sixteen carbon saturated fatty acids.
Although 1H NMR and 13C NMR data of compounds 2 and 3 were alike, 13C NMR and HRESIMS data indicated that compound 2 possesses two additional methylenes and compound 3 contains four additional methylenes in the long chain of fatty acids compared to compound 1. Notably, compounds 1-3 of Fr.9 exert α-glucosidase inhibitory activity.
Combining these data with the HRESIMS identified three other novel trace coffee diterpene esters: magaric acid, octadecenoic acid, and nonadecanoic acid, with potential α-glucosidase inhibitory activity.
Conclusions
Three novel diterpene ester compounds, caffaldehyde A, B, and C, with IC₅₀ values stronger than acarbose, were identified from the crude extract of Coffea arabica. In addition, three additional novel coffee diterpene esters with similar activity were determined using an LC-MS/MS-based molecular network. The method proposed in this study could be applied to separate targeted active metabolites of other foods in future research, while reducing solvent use and research time.
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Compounds show significant inhibitory effects on α-glucosidase