## 4 Discussion
### Altitude Variation in Secondary Metabolite Profiling  
Gas chromatography coupled with mass spectrometry (GC-MS) provides more precise information for qualitative and quantitative analysis of secondary metabolites [30]. During the present study, GC-MS analysis of chicory leaves collected at different altitudes and localities revealed the presence of different secondary metabolite compounds in different concentrations. The highest number of metabolites, i.e., 78, were identified at an elevation of 2172 m (17%) and the lowest number of metabolites, i.e., 61 (12%), were identified at the lowest elevation of 1554 m. Thus, altitudinal variation strongly influences the landscape topography, geology, amount of rainfall and, consequently, soil texture and moisture, groundwater depth, hydrology, evaporation, soil type, and the vegetation itself [58]. There are usually noticeable variations in secondary metabolite production between different taxa compared to intra-specific variations of a particular taxon at different elevations [59]. The present study also revealed the presence of eight common major metabolites from chicory samples at all altitudes and localities, among which methyl commate B had the highest concentration in terms of percent area (10.14) with an average value of 8.79% followed by \(\gamma\)-sitosterol and 9, 12, 15-octadecartionic acid, with potent known biological activities. Methyl commate B is a triterpene glycoside, which is a natural, polar compound with low volatility. Triterpene glycosides are well known for their cytotoxic, antibacterial, antimicrobial, antiviral, insecticide, nematicide, anticoagulant, hemolytic, antiparasitic, wound healing, and antitumor activities [60]. Another major metabolite that was observed is \(\gamma\)-sitosterol (9.3%), which is a steroid that possesses antibacterial potential [61] and anti-inflammatory activity after topical application [62]. Furthermore, the fatty acid 9,12,15, octadecatrienoic acid, a key metabolite identified in the GC-MS analysis in the present study, has anti-inflammatory, anticancer, hypocholesterolememia, insectifuge, and antiandrogenic activities [63]. This study also revealed the presence of three terpenoid compounds, including betulin, kolavelool, and betulin aldehyde, at high altitudes of 1790, 1901, and 2172 m that were not present at low altitudes, among which betulin had the highest concentration with an average value of 21.02% followed by kolavelool with 7.37% and betulinaldehyde with 6.98%. These compounds possess significant biological activities, for instance, betulin possesses anti-inflammatory [64], hepatoprotective activity [65], antiplasmodal effects and some of its derivatives were reported to exhibit antibacterial [66; 67], anticancer [68] and antileishman activity [69]. Furthermore, kolavelool exhibited cytotoxic activity against human leukemia [70]. However, the plants' ecological and physiological relations aresignificantly affected by different environmental factors such as sunlight, temperature, relative humidity, wind velocity, availability of water, etc. Altitudinal differences results in significant changes in these parameters, which in turn affects secondary metabolite production in plants [27]. Variation in the same taxon at different altitudes may be the result of differences in their genetic makeup. Thus, the presence and absence of a particular secondary metabolite in the chicory leaf at different altitudes might be due to the genetic variability, different geographical conditions, physiological development, and growth of the plant as revealed by the multivariate metabolomics data analysis.