# State-of-the-Art and Opportunities for Bioactive Pentacyclic Triterpenes from Native Mexican Plants
## 2 Pentacyclic Triterpenes from Mexican Plants
### Extraction and Purification Methods  
According to the reviewed literature, the initial step for PCT purification is material preparation. PCTs can be found in the aerial parts, stems, and resin of the identified plants, which must be dried and milled prior to the extraction procedures. Usually, for extraction, organic solvents are used for the extract preparation as binary mixtures, dichloromethane (CH\({}_{2}\)Cl\({}_{2}\)), hexane (C\({}_{6}\)H\({}_{14}\)), chloroform (CHCl\({}_{3}\)), and methyl alcohol (CH\({}_{3}\)OH) are the most adopted solvents [31; 35; 36; 37; 38; 41; 50]. The main extraction techniques used for PCTs from Mexican plants are maceration and Soxhlet extraction [39; 30; 37; 39]. Maceration generally occurs during a period ranging from 24 h to 9 days. It has also been reported that the temperature of the system must be below 40 \({}^{\circ}\)C [29; 35; 40]. Once the maceration is completed, the mixture can be filtered and then the solvents must be completely or partially removed under reduced pressure conditions. In some cases, the maceration-extraction procedure can be carried out in multiple steps, as reported by [PERSON] et al. [37].  
Once the organic extract is prepared, the compounds are fractionated through chromatographic columns (CCs) using silica gel as the stationary phase [29; 31; 32; 33; 34; 35], and hexane, ethyl acetate, acetone, and di- and tri- chloromethane are the most common solvents used for the mobile phase [34; 37; 39; 40]. First, the extract is processed through the CC and when it is required, these fractions are pooled. In the analysis of the solvent systems used in the elution of PCTs through silica gel chromatography, it is notable that the solvent mixtures present estimated dielectric constant values [51] ranging from 4.0 to 20.0 (Table S1), showing that PCTs have a non-polar to slightly polar character, since solvents with a dielectric constant lower than 15 are generally considered non-polar [51]. This nature is highly dependent on the substituents in the PCT. For example, for non-polar compounds such as galphimidins **(38; 43; 44)**, mixtures contain n-hexane with dichloromethane, acetone, and ethyl acetate [45] while for more polar compounds such as oleanolic **(1)**, ursolic **(14)**, and their derivates **(2, 3)**, systems involving hexane-methanol and dichloromethane-methanol are used [29; 33]. Some fractions can be processed again in a second CC to increase the compound purity [29; 36; 37].  
In most cases, during CC, the fractions are analyzed by thin-layer chromatography (TLC) [29; 30; 32; 33; 34; 35], where spraying of a solution of 1% (NH\({}_{4}\))\({}_{4}\)Ce(SO\({}_{4}\))\(\cdot\)2H\({}_{2}\)O reveals the tritterpene [40; 42]. Additionally, in TLC visualization, solutions of olean reagent [30; 32], sulfuric acid 10% or anisaldehyde 10% plus glacial acetic acid 5% plus 5% H\({}_{2}\)SO\({}_{4}\)[29], H\({}_{2}\)SO\({}_{4}\) 10% [33], charring solution [34], Liebermann-Burchard's reagent [35], ceric sulfate in sulfuric acid [36], cerium sulfate (0.1% CeSO\({}_{4}\)/2 N H\({}_{2}\)SO\({}_{4}\)) or vanillin (1% vanillin/5% H\({}_{2}\)SO\({}_{4}\)) [43], or 1% solution of (NH\({}_{4}\))\({}_{4}\)Ce(SO\({}_{4}\))\({}_{4}\) in 2N H\({}_{2}\)SO\({}_{4}\)[42] have been applied too.  
For analytical purposes, HPLC [34; 43] or UHPLC [36] coupled to different detection systems (mass spectrometry, UV, diode-array, evaporative light scattering detector (ELSD)) and gas chromatography [35] have been reported. Nuclear magnetic resonance (NMR) [29; 39; 40; 41; 43; 44; 45], infrared (IR) [29; 36; 37; 38; 41; 42; 39; 43; 44; 36; 38; 41; 45], UV [30; 32; 34; 37; 42; 43; 44] and Fourier transform infrared spectroscopy (FTIR) [36], mass spectrometry [30; 31; 32; 34; 37; 38; 41; 42; 43; 44; 45], optical rotation measurement [30; 31; 32; 34; 37; 41; 42; 43; 44], and X-ray diffraction [42] were used for compound identification.