## 4 Discussion
One was reported by [PERSON] and colleagues [31] in 1977 in which the plant part used was the stem. Another one was reported by [PERSON] and colleagues [15] in 2017 in which the plant parts used were the leaf and stem. Our study is the first report on the isolation of lupeol from the root part. Owing to its high abundance in the roots and simple root profile obtained via GC-MS analysis, we developed a simpler and straight-forward isolation method for lupeol from the plant's roots. In our study, we used an initial 80 g of freeze-dried root powder and macerated it with ethyl acetate for five days during the first extraction, followed by three days for the second extraction on the root residues with ethyl acetate, and combined the two extracts and dried them. The vacuum-dried extract was re-dissolved in methanol and underwent recrystallization to yield 1.138 g white amorphous compound, which was identified to be lupeol (yield of extraction = 1.423%). In [31], an initial two kilograms of dried and milled _C. mutans_ stems were extracted by using light petroleum in Soxhlet apparatus, followed by silica gel column chromatography using ether and light petroleum for elution, then further purified by using preparative thin layer chromatography and recrystallization to yield 3.7 g of lupeol (yield = 0.185%). In [15], an initial four kilograms of dried and powdered _C. mutans_ leaves were macerated with methanol and filtered, followed by solvent-solvent partitioned with hexane to yield a crude extract of 153.9 g. The crude hexane extract was later dissolved in methanol and chromatographed over Diaion HP20 SS using ethanol to yield five sub-fractions (H1, H2, H3, H4, and H5). These five sub-fractions were further subjected to column chromatography using different gels to produce four pure compounds (shaftoside, stigmasterol, \(\beta\)-sitosterol, and lupeol). Unfortunately, the final yield for each of the pure compounds was not mentioned in the literature and thus could not compare to our method. However, compared to Dampawan, our method manages to produce a higher extraction yield of lupool. Additionally, our method is simple and straightforward compared to [15; 31] as it does not involve solvent-solvent partition, and also without the hassle to go through the column or preparative thin-layer chromatography. The successful isolation of lupool from C. _nutans_ roots in this study also suggests the importance of GC-MS- or LC-MS-based profiling of every part of the plant first prior to the selection of potential candidate compounds for the isolation process. Figure 5: The biosynthesis of pentacyclic terpenoids and phytosterols. The biosynthesis structure and pathway were produced using the KingDraw v2.0 for Windows software ([[http://www.kingdraw.cn/en/index.html](http://www.kingdraw.cn/en/index.html)]([http://www.kingdraw.cn/en/index.html](http://www.kingdraw.cn/en/index.html)), Qingdao, China. Downloaded and installed on 1 May 2021). In addition, the extensive characterization of the isolated compound using UV-visible spectrophotometry, Fourier transform infrared spectroscopy, GC-MS, and nuclear magnetic resonance have been able to identify the isolated compound as lupeol. Further analysis of the anti-proliferative activity of the isolated lupeol against human breast cancer cells has revealed that the isolated lupeol was able to demonstrate comparable anti-proliferative activity with the standard lupeol and positive control camptothecin. A study reported by [22] has postulated the possible mechanism of lupeol on human breast cancer cells by down-regulating the anti-apoptosis proteins, Bcl-2 and Bcl-XL, which lead to cell apoptosis. Our previous study [7] also shows that the root extract from _C. mutans_ is capable of causing apoptosis of the breast cancer cells via the condensation of chromatin, down-regulation of the BCL2 gene, and the loss of mitochondrial membrane potential of the cancer cells. We therefore highly speculate that the pro-apoptotic capability of _C.