METABOLOMIC ANALYSIS AND IN-VITRO ANTIDIABETIC ACTIVITY OF CIVET COFFEE AND STUDY OF SECONDARY METABOLITE ENDOPHYTIC FUNGI FROM COFFEE BEANS

• Main Author: Febrina, Lizma
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64835
• Institution: Institut Teknologi Bandung

Luwak (civet) coffee is originated from coffee cherries fermented in the digestive system of the Asian palm civet (Luwak, Paradoxurus hermaphoditus). The fermentation process leads to the civet coffee having a unique flavor and different from the regular coffee. It indicates a change in the coffee metabolite profile and is considered to affect its antidiabetic activity. To the best of our knowledge, antidiabetic acitivity of civet coffees, had not been reported before. Besides that, the endophytic fungi of coffee had also received the attention of researchers. However, their studies mostly focused on the collection of the endophytic fungi, and not on the chemical investigation yet. Based on these reasons, our study aims to analyze comprehensively the metabolite profile of the civet coffees. The samples used in this work consisted of the wild and the caged civet coffees, and the regular arabica coffee as the control. The in-vitro antidiabetic activity of the coffee samples was also evaluated. Furthermore, the secondary metabolites of Clonostashys rosea, endophytic fungi collected from regular arabica coffee, were successfully isolated. Metabolite profile of the coffee samples were analyzed with 1H NMR-based metabolomics. The metabolite identification was validated by 2D NMR spectra analysis including 1H-1H gCOSY, 1H-1H zTOCSY, and J-Resolved. In total, 18 metabolites in the green coffee beans, and 27 compounds in the roasted coffee samples were successfully identified. The results showed that the civet coffees and regular arabica coffee were qualitatively similar. However, there were differences in the metabolite composition in each coffee sample. Multivariate statistical analysis was applied to reveal the similarity and the differences within the coffee samples. The results showed that the green beans of civet coffees contained high concentrations of alanine, asparagine, citric acid, malic acid, and trigonelline, when compared with the regular coffee. Among those, citric acid and malic acid were the most important distinguishing compounds of civet coffees. Meanwhile, the roasted civet coffees were characterized with higher levels of acetic acid, trigonelline, quinic acid, citric acid, and malic acid, when compared with the control coffee. The most discriminant metabolites of the roasted civet coffees were malic acid and citric acid.Antidiabetic activity of the coffee samples was evaluated by performing in-vitro test related to the type 2 diabetes mellitus, including ?-glucosidase inhibitory and antioxidant assays. The test result showed that the wild civet coffee (the green and the roasted beans) possessed the highest antioxidant activity and ?-glucosidase inhibition. The wild civet coffee inhibited ?-glucosidase with mixed inhibition mechanism. The correlation of the antidiabetic activity with the quantified metabolites was evaluated by the Pearson correlation coefficient method. The statistical data showed that various metabolites contributed to the activity. Malic acid was discovered as the most positively correlated compound with the antidiabetic activity. Furthermore, this result indicated that the fermentation of coffee beans in the civet digestive system which increased the malic acid contain, provided an advantage for the in-vitro antidiabetic activity. Interestingly, it is the first scientific report regarding the in- vitro antidiabetic activity of the civet coffees.C. rosea, an endophytic fungi, was successfully collected from the green beans of regular arabica coffee. The extract of C. rosea was fractionated and purified using various chromatographic techniques, yielding 2 pure secondary metabolites, namely 3,7-dimethyl-6-hydroxy-8-methoxyisochroman and tanzawaic D acid. The molecular structures of the isolated compounds were determined by analysis of 1D (1H, 13C, TOCSY) and 2D (HSQC, HMBC 1H-1H COSY) NMR spectra. These two compounds were discovered for the first time from the C. rosea and have not been isolated from the host plant yet. In this study, NMR-based metabolomics was applied to analyze the metabolite profile of the civet coffees comprehensively without any separation. Identification of the 1H NMR spectra revealed that the metabolites contained in the coffee samples were qualitatively similar but quantitatively different. The fermentation in the civet digestive system positively impacted the antioxidant and the ?-glucosidase inhibitory activities of the coffees. In-vivo validation would allow the application of the civet coffees as the functional food related to type 2 diabetes mellitus. In addition, this study had isolated for the first time 2 polyketide derivative compounds, namely 3,7-dimethyl-6-hydroxy-8-methoxyisochroman and tanzawaic D acid, from C. rosea, the endophytic fungi collected from the green coffee beans.