## 4 Discussion  
The isolation of anti-proliferative compounds from C. _nutans_ is not easy. This may be due to the complexities of the plant's nature and also various fractionation/chromatographic steps which need to be optimized. We came up with a strategy to pre-screen (profile) the volatile compounds present in every part of the plant; firstly, using a GC-MS method, we have optimized for the detection of volatile compounds [7], then only select the suitable plant part and target compound. In our present study, we found that the aerial part of the plant, i.e., the leaf, was predominated by vitamin E (\(\alpha\)-tocopherol), squalene, and phytos  
Figure 4: Anti-proliferative activities of the isolated lupeol from C. _nutans_ roots compared to the authentic standard lupeol and camptothecin (Supplemental Figure S3) against MCF-7 cells after 72 h with the indicated concentrations. The IC\({}_{50}\) values were expressed as mean \(\pm\) SD of the three independent experiments (\(n\) = 3). Level of significance (\(\star\)\(p\) < 0.01 and \(\star\)\(\star\)\(p\) < 0.001) for each concentration of authentic or isolated lupeol was compared to the control cells, and was determined using one-way ANOVA followed by a post hoc Dunnett test. terols such as stigmasterol, \(\gamma/\beta\)-sitosterol, and campesterol. Interestingly, the intensities of these compounds decreased when moving to the lower part of the plant, i.e., the stem. In the stem, lupeol was found to start to accumulate abundantly and the amount was much higher than squalene and other phytosterols. Lupeol is one of the pentacyclic triterpenes with a 30-carbon skeleton comprising four six-membered rings and one five-membered ring [17]. More intriguingly, when it came to the underground part of the plant, i.e., the root, it was found that this part contained enormous lupeol levels and some betulin. All of the detected phytosterols and pentacyclic terpenoids (lupeol and betulin) in this study have been reported to possess anti-proliferative and apoptotic activities by other researchers [18; 19; 20; 21; 22; 23; 24; 25; 26; 27]. Figure 5 illustrates the biosynthesis of phytosterols and some pentacyclic triterpenoids such as lupeol and betulin. The figure was made by referring to [28; 29; 30]. The biosynthesis of the compounds begins from the reactions of two precursors called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are derived from acetyl-CoA via the mevalonic acid (MVA) pathway [28; 29; 30]. The reaction is catalyzed by the enzyme isopentenyl pyrophosphate isomerase to yield the geranylpyrophosph (GPP) and farnesyl pyrophosphate (FPP) via the catalysis of preyltransferase. Then, two molecules of FPPs are catalyzed by the squalene synthase to yield squalene, which is the important precursor for various phytosterols and pentacyclic triterpenoids. Via the squalene epoxidase, squalene is converted to 2,3-oxidosqualene, which is the branch point for the subsequent biosynthesis of phytosterols or pentacyclic triterpenoids [28; 29; 30]. For phytosterols, multiple enzymatic steps are involved in the plants to convert 2,3-oxidosqualene to cycloartenol and end products such as stigmasterol, sitosterol and campesterol [30]. For pentacyclic triterpenoids such as lupeol and betulin, the first step is the conversion of 2,3-oxidosqualene to lupeol by lupeol synthase, and then followed by three sequential oxidations catalyzed by cytochrome P450 monooxygenases (CyP450), leading to the generation of betulin, betulinic aldehyde, and betulinic acid [28; 29]. Based on the results observed in our study, it can be postulated that squalene biosynthesis may actively take part in the aerial part (leaf). This squalene then becomes the precursor for the biosynthesis of lupeol in the stem part. Finally, the root part may either become the synthesis site or main storage site for lupeol. To date, two studies related to the isolation of lupeol from _C. mutans_ plant have been reported.