## 3 Results and Discussion
### Phytochemical Analysis of the Pure C. Operculatus Leaf Powder
#### 3.1.2 Quantification of Total Flavonoids and Terpenoids  
Flavonoids and terpenoids are the main groups of secondary metabolites found in natural products capable of antibacterial properties via multiple mechanisms of action [18,19]. Additionally, several recent reports have confirmed that the major classes of phytochemicals, including flavonoids and terpenoids, have significant antioxidant and free radical scavenging activities [20]. Thus, we determined the total flavonoid and terpenoid levels in the young/mature/old leaves (1-3/4-5/> 6 weeks old and 7-9/12-14/10-12 cm length, respectively) of 3-years-old _C. operculatus_ plants. The powdered and dried _C. operculatus_ was extracted with boiling water (Table 3), and the percentage yield was further calculated on a dried weight basis (after the extracts were kept at 4 \({}^{\circ}\)C in a capped, airtight container until use). The results of spectrophotometric determination for the total flavonoid and terpenoid contents are presented in Table 3. The determination of the total flavonoid content was based on the reaction of the complexation of flavonoids with AlCl\({}_{3}\) and absorbance measurement at 510 nm [2]. The spectrophotometric quantification of the total terpenoid content was carried out using a vanillin-sulphuric acid assay with absorbance at 560 nm [16]. Means with no letter in common indicate significant differences between treatments (\(p\leq 0.05\)). The results of Table 3 demonstrate the percentage yields of the young, mature, and old leaves extracted with boiling water. For flavonoids, quercetin was used as the reference for semi-quantitative determination. The percentage yield extracted from old leaves is 2.7 mg QE/g (milligrams of quercetin equivalent per gram of the dry matter extracted). For terpenoids, menthol was used as the quantitative reference. The terpenoid yield in the old leaves' extracts is 5.4 mg SE/g (milligrams of sesquiterpenes based on menthol used for relative quantification and expressed as equivalent per gram of the dry matter extracted). The flavonoid and terpenoid contents in the old leaves' extracts are higher than those measured for young and mature leaves. Based on [PERSON] et al. [21], we can speculate that the young and mature leaves suffered less damage and thus contained fewer secondary metabolites than the older leaves, which could accumulate more defense compounds during their lifetime. Effectively, many secondary metabolites found in plants are involved in defense against herbivores, pests, and pathogens. Plant responses to herbivory with traits related to both defense and tolerance are thus affected by plant age [21]. Older plants are capable of maintaining stronger chemical defense mechanisms than young plants, possibly because old plants accumulate higher amounts of plant defense compounds over a longer growth period; another possible hypothesis could be that young plants need to primarily allocate their resources to their growth process [2,22,23]. Nevertheless, regardless of the plant's physiological explanation, each kind of leaf contains relatively high levels of flavonoids and terpenoids, but the older leaves did contain significantly more. Nevertheless, it is not possible to imagine a very precise selection of leaves during a harvest at a real industrial scale. Thus, we decided to continue the study only based on a representative mix of leaf ages. The next samples were composed of young and mature leaves. Additionally, they also contained a proportion of old leaves that may fit with what can be really collected in fields by local farmers.