## 4 Discussion  
This study aimed to examine changes in the chemical composition of elephant dung coffee compared to more common civet coffee and conventionally processed coffee. Research on the chemical profile of elephant coffee is still very limited, with only two published studies addressing the topic [22, 23]. The first study analyzed commercially available elephant coffee (Black Ivory coffee--the same brand used in our study), while the second used elephant coffee obtained through experimental means. However, to the best of our knowledge, neither study included control samples such as conventionally processed coffee or coffee derived from other animals (e.g., civets). Additionally, this is the first report to use HPLC-UV analysis for profiling caffeine and phenolic compounds in elephant coffee, alongside in vitro assessments of antioxidant potential using specific assays such as ORAC and ABTS. It is well known that coffee processing--particularly roasting--can significantly affect its chemical composition [2, 24]. Ideally, green beans from all sample types should be obtained and roasted under identical conditions. Unfortunately, we were unable to acquire green beans from all commercial sources. It is also widely recognized that a coffee's country of origin can significantly influence its chemical profile [25, 26]. To minimize variability, we selected samples that were as similar as possible in key aspects: all were Arabica varieties, roasted to a medium level, and sourced from Southeast Asia. Elephant coffee, along with civet coffee, contained lower caffeine levels and generally exhibited reduced antioxidant activity compared to conventionally processed coffee. This  
Figure 1: Score plot of three sample groups—civet coffee (red), conventional coffee (green), and elephant coffee (blue)—based on a total of nine samples. Ten variables with the greatest contribution to the separation in the coordinate system of PC2 and PC3 are expressed as concentrations [mg/g DW] and were Pareto-scaled. aligns with the idea that some caffeine and other compounds, such as antioxidants, are absorbed or broken down during digestion in the animal's gastrointestinal tract [4; 22; 27]. Given that an elephant's digestive system is much larger than a civet's, this effect is thought to be more pronounced in elephant coffee. Interestingly, the levels of chlorogenic, gallic, cinnamic, and caffe acids were very similar across all samples, with minimal differences. A comparable pattern was observed in other studies where civet coffee showed chlorogenic acid levels equal to or even higher than conventional coffee [28]. Although some research on green, unroasted coffee reported opposite findings [29], the differences we observed were so small that they are likely negligible from a consumer's perspective. The most significant differences between the samples were observed in their volatile compound profiles. Compounds such as 2-methylfuran, 3-methylbutanal, and 2-methylbutanal were absent in conventional coffee but present in both civet and elephant coffees. These compounds are typically found in higher concentrations in Robust coffee than in Arabica [30], which may explain their absence in our conventional Arabica samples. Additionally, some studies suggest that these compounds, along with various pyrazines, may result from bacterial fermentation either before the coffee is consumed by the animal or within its gastrointestinal tract [31; 32]. As such, they could serve as potential markers for animal-derived coffee--especially 2-methylbutanal, which was notably abundant in our elephant coffee sample. Conversely, the elephant coffee sample lacked 2-ethyl-5-methylpyrazine and 3-ethyl-2-hydroxy-2-cyclopent-1-one but was relatively rich in 3-ethyl-2,5-dimethylpyrazine. Derivatives of these compounds are commonly found in coffee and contribute to its characteristic aroma [33].