# Metabolic Profiling of _Conyza sumatrensis_ ([PERSON].) [PERSON] from Lugazi, Uganda
## 3 Results and Discussion
### GC-MS Allows for Comprehensive Annotation of Volatile Compounds in Dried Leaf Samples  
Headspace GC-MS analysis provided detailed information on volatile thermally stable compounds of the raw leaf material to identify individual plant metabolites. The headspace was thermally extracted directly from the dried leaves without prior liquid extraction. This convenient approach, avoiding lengthy procedures such as prior distillation or liquid extraction, is particularly suitable for analyzing volatile compounds, which are often lost or degraded during liquid extraction; the latter is also often challenged by the selection of a suitable solvent matching subsequent analysis. The high sensitivity and selectivity of GC-MS ensures the generation of accurate and reliable data to enable compound annotation [32]. A first assessment of our data and search against the mass spectral database (Figure 2B) showed that most of the annotated peaks are terpen (44%), followed by other hydrocarbons (36%). Of the terpenes, 65% are predicted as sesquiterpenes, 30% as monoterenes, and only 5% as dieterpenes. The lowest abundant volatile compounds are fatty acid esters, lactones, and carboxylic acids. A detailed analysis assigned hexanal (6.78 min), \(\alpha\)-pinene (10.09 min), D-limonene (13.16 min), \(\alpha\)-copaene (23.40 min), norpinane (23.74 min), aristolene (24.56 min), bergamotene (25.31 min), \(\alpha\)-humulene (25.45 min), \(\beta\)-caryophyllene (25.56 min), and neophytadiene (30.23 min) to the ten most prominent signals (Figure 2A). This corresponds well with previous GC-MS-based studies, which also identified limonene [33], \(\alpha\)-bergamotene, and caryophyllene in \(n\)-hexane and ethyl acetate extracts of \(C\). _sumatrensis_[1]. In accordance, \(\alpha\)-pinene, \(\alpha\)-copaene, \(\alpha\)-humulene, and D-limonene were reported from the essential oil of this plant, contributing to its bioactive properties [10]. The three largest peaks by area in  
Figure 1: HPLC chromatogram of the aqueous and ethanolic extract of \(C\). _sumatrensis_, normalized to relative abundance. Peaks with similar retention times are marked in gray. (*) Peak is only prominent in the ethanolic extract. Detection at 250 nm. (a): ethanolic extract, (b): aqueous extract. our analysis were assigned to terpenes (aristolene, neophytadiene, and D-limonene). In total, 47 volatile compounds were detected in the powdered leaf samples of _C. sumatrenis_ and annotated with a spectral library (Table 2). Of those, 41 compounds could be newly assigned to this plant, including major constituents, e.g., aristolene, norpinane, and neophytadiene. The relative abundance of the metabolites was determined by calculating the share of total peak area [%] as a semiquantitative indicator. For a better and more detailed overview and semiquantitative evaluation based on the peak area of the compounds, please refer to Table 2.  
Aristolene (peak area: 13.03%, retention time: 24.56 min) and cryophyllene (peak area: 5.54%, retention time: 25.56 min) were identified as the most abundant sesquiterpenes. The presence of aristolene has been previously noted in the analysis of other essential oils [34], but to the best of our knowledge, the therapeutic relevance of aristolene has not yet been investigated. Caryophyllene, however, is a widely studied phytochemical and has diverse therapeutic properties, e.g., analgesic, anti-inflammatory, antimicrobial, antifungal, and antioxidant [35]. There is also evidence suggesting potential antidepressant and anxiolytic effects associated with caryophyllene [36]. The most prominent monoterpene was suggested as limonene (peak area: 7.59%, 13.16 min). It is well-investigated, e.g., for its anti-cancer, anti-inflammatory, and antimicrobial activities [37]. Neophytadiene (peak area: 12.94%, retention time: 30.23 min) is the only suggested dipterpene and the second-highest signal overall. It has shown anti-inflammatory activity and neuropharmacological effects and has been suggested as a potential anxiolytic and antidepressant agent [38].