## 3 Results and Discussion
### GC-MS Analysis of _P. amboinicus_ essential oil Extract
Overall, there was no major qualitative variation in the chemical components related to the collection time except for the additional aliphatic alkane hydrocarbons present in the extract harvested at 8 p.m. (Table 1). Two isomeric phenols (carvacrol and thymol) are chemotypes that are frequently found in _P. ambionicus_[24,25]. Thus, based on these chemical constituents, the _P. ambionicus_ in this study was classified as a carvacrol chemotype. Carvacrol is the signature chemical largely responsible for the sharp, pungent oregano-flavor of oregano, marjoram, and plants from other genera that are also considered as oreganos, such as _Lippia gravecens_ (Mexican oregano) and _P. ambionicus_ (Cuban oregano) [26]. This result was in accordance with the known volatile constituents of most _P. ambionicus_, as previously reviewed by [PERSON] et al. [1]. The variations observed among various reports could be attributed to the different geographical features, climate, seasonal variations, genetic aspects, and extraction methods [1,27]. Figure 6: Chromatographic profiles of _Plectrantthus ambionicus_ essential oil extracted from leaves harvested at different time points. (**A**) 8 a.m.; (**B**) 2 p.m., and (**C**) 8 p.m. The numbers (as indicated by the black arrow) correspond to the terpenoids detected as described in Table 1.  
This result was consistent with our previous _P. amboinicus_ leaf volatile profiles determined using HS-SPME, although the relative amounts of the individual components varied with \(\alpha\)-bergamotene, carvacrol, carvophyllene, p-cymene, and \(\gamma\)-terpinene as major constituents [12]. [PERSON] et al. [28] revealed that the aqueous fraction of _P. amboinicus_ leaves from Uganda extracted using SPME showed linalool (50.30%) as the main compound in this plant. It was also evident that the _P. amboinicus_ growing in the same locality could also exhibit chemical variations. Essential oil of _P. amboinicus_ leaves from UPM Serdang analyzed by [PERSON] et al. [29] showed 3-carene (20.78%), carvacrol (19.29%), camphor (17.96%), \(\gamma\)-terpinene, (8.94%), \(\alpha\)-terpinene (6.04%), and \(\alpha\)-cymene (5.06%) as the main components. In comparison, the wild _P. amboinicus_ collected near the UPM Serdang forest area contained carvacrol (37.73%), tetracontane (16.67%), squalene (15.64%), tetrapentacontane (13.77%), and phytol (12.95%) [30]. It is noteworthy that, in this study, we were able to detect thymoquinone (TQ) among other terpenoids from _P. amboinicus_. TQ was first identified from _Nigella sativa_ black seed essential oil, and later was isolated from other plants such as _O. vulgaris_ L. [31], _O. syriacum_[32; 33; 34], _Satureja montana_ essential oil [35], aerial flowering parts of _Thymus vulgaris_[36], and _Phtyophthora ramorum_[37]. TQ is known for its anticancer activity by deregulating both the mitogen-activated protein kinase (MAPK) and protein kinase B signaling pathways in squamous cell carcinoma [38] and human prostate cancer cell lines [39]. It also inhibits telomerase activity that causes unlimited replication of cancer cells by inducing telomere shortening and apoptosis in glioblastoma cell lines [40]. TQ has been identified as an adjuvant for combination anticancer therapy by enhancing the antitumor activity of chemotherapeutic drugs and/or decreasing their toxicity level similar to normal cells [41]. The presence of TQ in _P. amboinicus_ was also documented by [PERSON] et al. [42] in their water-hexane extract. This shows that TQ is a promising anticancer compound with significant potential for clinical translation. Thus, the presence of this compound in the local herb of _P. amboinicus_ extract is a notable observation, revealing the nutritional value of this plant as a natural source of TQ and the plant's potential to be explored for anticancer drugs. The TQ biosynthetic pathway in _N. sativa_ was proposed by [PERSON] et al. [43] based on the better-studied pathway in the Lamiaceae [44].