Chemistry, Biological Activities and In Silico Bioprospection of Sterols and Triterpenes from Mexican Columnar Cactaceae.

The Cactaceae family is an important source of triterpenes and sterols. The wide uses of those plants include food, gathering, medicinal, and live fences. Several studies have led to the isolation and characterization of many bioactive compounds. This review is focused on the chemistry and biological properties of sterols and triterpenes isolated mainly from some species with columnar and arborescent growth forms of Mexican Cactaceae. Regarding the biological properties of those compounds, apart from a few cases, their molecular mechanisms displayed are not still fully understand. To contribute to the above, computational chemistry tools have given a boost to traditional methods used in natural products research, allowing a more comprehensive exploration of chemistry and biological activities of isolated compounds and extracts. From this information an in silico bioprospection was carried out. The results suggest that sterols and triterpenoids present in Cactaceae have interesting substitution patterns that allow them to interact with some bio targets related to inflammation, metabolic diseases, and neurodegenerative processes. Thus, they should be considered as attractive leads for the development of drugs for the management of chronic degenerative diseases.


Introduction
The word Cactaceae derived from the Greek, "káctos", used in antiquity to name a species of thorny thistle, possibly the cardo Cynara cardunculus (Asteraceae), being used as a generic name by Carlos Linneo in 1753, for several plants of diverse sorts of the Cactaceae family [1]. This family, originally from America, groups about 1500 species. It is estimated that about 700 species grouped in 68 genera are present in Mexico, although there is considerable variability in these data [2][3][4].
The most apparent characteristic of cacti is the presence of the areola, considered as buds homologous to the axillary buds of the other dicotyledons. One main characteristic of buds or areolas is its capacity to form reduced leaves, flowers, new stems, thorns, glands, bristles, and hairs, even sometimes adventitious roots [2]. The ethnobotanical importance of cacti has been described extensively in the literature, including their use as food, medicine, among others. Some species of the family Cactaceae have been used as natural remedies for many centuries in Mexican traditional medicine. For example, peyote (Lophophora williamsii) has been used not only as a means of communication of man to the spiritual world but also as an analgesic and antirheumatic. On the other hand, Ariocarpus Table 1. Chemical structures of sterols isolated from Cactaceae.
are not reviewed in this opportunity.

Sterols from Mexican Columnar Cactaceae
There is not enough data to generalize the type of the preferred sterol skeleton, the substitution pattern, and the quantities of sterols present in the different species, genera, clade or subfamily of Cactaceae. With an exception of some Sonoran Desert species (Carnegiea gigantea, Pachycereus pringlei, Machaerocereus gummosus, Lemaireocereus thurberi, among others) whose chemistry has been described exhaustively because of its ecological implications in the Cactus-Microorganism-Drosophila Model System of the Sonoran Desert [39][40][41]. The structures of sterols isolated from Mexican Cactaceae are presented in Table 1.  * Although Opuntia is not in the Pachycereeae tribe (Opuntieae, Opuntioideae, Cactaceae), because of the diversity and special conformation in one of their sterols, we decide to include it in this review. ** O. versicolor is a synonimus of Cylindropuntia versicolor (Engelm. ex J.M.Coulter) F.M.Knuth. 1 AP: aerial parts; R: roots; P: pollen. Table 3. Bioactivities of selected Cactaceae sterols.

Compound Activity Description Reference
Viperidone MM Strong inhibition binding to LXRα with an IC 50 value of 0.10 µM. [57] Peniocerol AI, CT and IN AI = Potent edema inhibition in TPA induced edema assay. CT = inhibition of breast and colon carcinoma MCF-7 and HCT-15 cell lines proliferation through cell cycle arrest and apoptosis in both cell lines. Also, peniocerol causes Mitochondrial permeability transition (MPT) induction. IN = insect growth regulatory activity against Spodoptera frugiperda and Tenebrio molitor. [26,33,74,75] Macdougallin AI, CT and IN AI = moderate edema inhibition in TPA induced edema assay. CT = inhibition of leukemia K-562 cell line proliferation. IN = insect growth regulatory activity against Spodoptera frugiperda and Tenebrio molitor.

24-dehydropollinastanol AD
Agonist of PPAR-α and PPAR-γ, changing the expression of genes involved in fatty acid transport, binding and oxidation in mouse liver. [61,62] 24-methylenecholesterol AI, AN, and CT AI = Low inhibition of key inflammatory enzymes like COX and NF-κB1. AN = Inhibitory effect for Trypanosoma brucei brucei and Mycobacterium marinum. CT = Potent inhibition of aromatase which is a therapeutic target for breast cancer treatment and has a cytotoxic effect on HS27 cell line.
[ [76][77][78] Pollinastanol CT Inhibition of aromatase which is a therapeutic target for breast cancer treatment. [77] Fucosterol AD, AO, AN, CR, AI, CT and HT AD = Inhibition of sorbitol accumulation and diabetic key enzymes like RLAR, HRAR, PTP1B, and α-glucosidase. It also has a downregulation effect of PPAR-γ, C/EBPα, and SREBP1. AO = Regulate transaminase activity (sGOT, sGPT) and enhances the antioxidant activity of SOD and GSH-px. AN = High inhibitory effect against the parasite P. falciparum. CR = Increases serotonin and noradrenaline in the central nervous system, and it also increases central BDNF levels. Also, it showed cholinesterase inhibitory activity and neuroprotective effects.AI = Represses iNOS, TNF-α, and IL-6 binding to NF-κB and inhibits COX-2. CT = Induced HL-60 and HeLa cell line apoptosis through a mitochondrial pathway. HT = Inhibits the synthesis of glucocorticoid receptors involved in the regulation of ACE decreasing its levels.
[ [79][80][81][82][83][84][85] β-sitosterol AI, AD, AN, CT, MM, and IM AI = Inhibits TNF-α, and NF-κB AD = Decreases glycated hemoglobin, serum glucose, and nitric oxide and increases insulin levels slightly. All this is a result of its potent antioxidant activity in the pancreas. AN = Growth inhibitory activity against bacteria P. smartii and the parasite P. falciparum. CT = Strong cytotoxic effect against A549 cell line by inducing apoptosis via ROS-mediated mitochondrial dysregulation. MM = Mediates cholesterol metabolism by increasing sterol excretion and decreasing cholesterol absorption and synthesis. IM = Inhibits T cell proliferation and blocks the secretion of Th2 and cytokines IL-4 and IL-10.

Triterpenes from Mexican Columnar Cactaceae
One of the best-studied chemical groups of cacti is the triterpenes since they constitute, in many cases, the most abundant compounds found in Cactaceae. The triterpenes display a wide diversity of structural features, with characteristic oxidation patterns, and a particular distribution in the species, reasons why they are used for chemotaxonomic studies [94]. In addition to the above, many of the triterpenes isolated from these plants present biological activities ranging from their ecological effects [40], as well as effects on models of inflammation and anti-nociception [13,26,95], which makes their research attractive. Information of structures with an emphasis on the origin of the skeleton of each compound isolated from Cactaceae is presented in Tables 4-6. In Table 4, pentacyclic triterpenes cycloartenol, 24-methylenecycloartenol and 25(27)-dehydrolanost-8-enol are described, while lupaneand oleanane-type triterpenes are described in Tables 5 and 6 respectively.  (27) * The cyclopropane moiety is absent instead of a C-19 β-methyl group, and the presence of a double bond between 8(9).   (27) * The cyclopropane moiety is absent instead of a C-19 β-methyl group, and the presence of a double bond between 8(9).   Triterpene Name R1 R2 R3 CH3 * Dumortierigenin and machaerogenin possess a lactone moiety at C-15, C-28, and C-21, C-28, respectively.
Although only Mexican columnar cacti were considered to the construction of the present review, a special mention is necessary to describe the triterpene content of Trichocereus pachanoi, a cactus plant from South America, which is an important source of some compounds named pachanols A-D (Table 7). These compounds possess a particular type of triterpene skeleton, called pachanane, characterized by the presence of five six-membered rings, and the position of the 27α-CH3 attached to C-15 [96,97]. * Dumortierigenin and machaerogenin possess a lactone moiety at C-15, C-28, and C-21, C-28, respectively.
Although only Mexican columnar cacti were considered to the construction of the present review, a special mention is necessary to describe the triterpene content of Trichocereus pachanoi, a cactus plant from South America, which is an important source of some compounds named pachanols A-D (Table 7). These compounds possess a particular type of triterpene skeleton, called pachanane, characterized by the presence of five six-membered rings, and the position of the 27α-CH 3 attached to C-15 [96,97].
Additionally, for the construction of the present review, only the aglycones of triterpenes were considered. In the first reports regarding the isolation and elucidation of those compounds, the routine procedure began with the hydrolysis of the organic extracts, isolating only the aglycones. The pattern of glycosylation of the natural compounds, and the number and relative positions of sugar moieties should be discussed in another review. Specific information about the content of triterpenes by species, as well as their biological activities, are described in Tables 8 and 9, respectively. Although only Mexican columnar cacti were considered to the construction of the present review, a special mention is necessary to describe the triterpene content of Trichocereus pachanoi, a cactus plant from South America, which is an important source of some compounds named pachanols A-D (Table 7). These compounds possess a particular type of triterpene skeleton, called pachanane, characterized by the presence of five six-membered rings, and the position of the 27α-CH3 attached to C-15 [96,97]. Pachanol A R1 = -, R2 = H, R3 = CH3; △ 12(13), 14 (15) Pachanol B R1 = H, R2 = -, R3 = αCH3; △ 11(12), 13 (18) Pachanol C Pachanol D Although only Mexican columnar cacti were considered to the construction of the present review, a special mention is necessary to describe the triterpene content of Trichocereus pachanoi, a cactus plant from South America, which is an important source of some compounds named pachanols A-D (Table 7). These compounds possess a particular type of triterpene skeleton, called pachanane, characterized by the presence of five six-membered rings, and the position of the 27α-CH3 attached to C-15 [96,97]. Pachanol A R1 = -, R2 = H, R3 = CH3; △ 12(13), 14 (15) Pachanol B R1 = H, R2 = -, R3 = αCH3; △ 11(12), 13 (18) Pachanol C Pachanol D Although only Mexican columnar cacti were considered to the construction of the present review, a special mention is necessary to describe the triterpene content of Trichocereus pachanoi, a cactus plant from South America, which is an important source of some compounds named pachanols A-D (Table 7). These compounds possess a particular type of triterpene skeleton, called pachanane, characterized by the presence of five six-membered rings, and the position of the 27α-CH3 attached to C-15 [96,97].  (12) Morolic acid 27-desoxiphillirigenin 3β-hydroxy-11α,12αepoxyolean-28,13β-olide Additionally, for the construction of the present review, only the aglycones of triterpenes were considered. In the first reports regarding the isolation and elucidation of those compounds, the routine procedure began with the hydrolysis of the organic extracts, isolating only the aglycones. The pattern of glycosylation of the natural compounds, and the number and relative positions of sugar moieties should be discussed in another review. Specific information about the content of triterpenes by species, as well as their biological activities, are described in Tables 8 and 9, respectively. Morolic acid 27-desoxiphillirigenin 3β-hydroxy-11α,12αepoxyolean-28,13β-olide Additionally, for the construction of the present review, only the aglycones of triterpenes were considered. In the first reports regarding the isolation and elucidation of those compounds, the routine procedure began with the hydrolysis of the organic extracts, isolating only the aglycones. The pattern of glycosylation of the natural compounds, and the number and relative positions of sugar moieties should be discussed in another review. Specific information about the content of triterpenes by species, as well as their biological activities, are described in Tables 8 and 9, respectively. Morolic acid 27-desoxiphillirigenin 3β-hydroxy-11α,12αepoxyolean-28,13β-olide Additionally, for the construction of the present review, only the aglycones of triterpenes were considered. In the first reports regarding the isolation and elucidation of those compounds, the routine procedure began with the hydrolysis of the organic extracts, isolating only the aglycones. The pattern of glycosylation of the natural compounds, and the number and relative positions of sugar moieties should be discussed in another review. Specific information about the content of triterpenes by species, as well as their biological activities, are described in Tables 8 and 9, respectively.    It should be noted that of the following genera listed in Mexico: Acanthocereus, Bergerocactus, Cephalocereus, Echinocereus, and Neobuxbaumia, have not been revised for their sterol nor triterpenoid content, so the chemistry of those species will have to be explored, to generate knowledge about the kind of natural products presents in each genus and their bioactivities.
From this review, it is evident that sterols and triterpenes from Cactaceae exhibit a myriad of bioactivities, mainly anti-inflammatory, metabolic regulatory, and CNS protective activities standing out. The lack of information about the molecular mechanism displayed of sterols and terpenoids prompt us to perform an in silico bioprospection in some molecular targets. Among the targets that have been commonly assayed against each biological process, only COX-1, COX-2, PTP1B, PPAR-α, PPAR-γ, acetylcholinesterase (AChE), LXR-α and LXR-β were selected for the in silico study to explore the theoretical molecular affinity of sterols and triterpenoids against each target. Then, the information presented in the results section, do not represent a validation of the molecular mechanism in the case of good molecular coupling between the ligands and the targets, nor do they represent the verification of the null activity of the ligands in the molecular targets. The data from molecular docking only represents a guide to future research designed to discover the specific molecular mechanisms that each molecule presents in biological models. Table 9. Most representative biological activities of triterpenes isolated from Mexican columnar Cactaceae.   upregulating the phosphorylation of MAPKs. HP = inhibition of liver oxidative stress in the iron/ascorbate system and showed hepatoprotective effects against D-GalN/TNF-α induced cell dead. [13,19,87,127,133,[135][136][137][138]143,147,160,161,163,[167][168][169][170][171] Maniladiol AN, AI AN = growth inhibitory activity against M. tuberculosis and the reverse transcriptase inhibition of HIV-1. AI = decreases the production of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6 and inhibits NO production. coli, and C. albicans and the reverse transcriptase inhibitor of HIV-1.

Compound
Inhibitor of tyrosinase and could be used as an insecticidal agent AI = edema inhibition in the TPA-induced inflammation assay. CT = potent anti-proliferative effect inducing apoptosis, cell cycle arrest, and ROS generation. HT = reduce cardiac remodeling by inhibiting angiotensin II-induced proliferation via PPAR-γ. IM = reduce the synthesis of pro-inflammatory mediators, auto-antibody production, suppression of endogenous leptin production, and inhibits migration of leukocytes in the CNS. HP = high protection of human hepatoma cells against CCl4-induced injury with ALT level decreased. [13,153,164,167,[174][175][176][177][178][179] Longispinogenin AN, AI, CT AN = growth inhibitory activity against M. tuberculosis. AI = edema inhibition in the TPA-induced inflammation assay. CT = inhibition of cervical (HeLa) cancer cell line proliferation. [173,180,181] Pachanol D AC AC = strong inhibitory effect on the acetic acid-induced writhing test. [95] Cochalic acid CT CT = potent inhibition of cervical (HeLa) cancer cell line proliferation. [180] Chichipegenin AI, CT AI = edema inhibition in the TPA-induced inflammation assay. CT = inhibition of breast and colon carcinoma MCF-7 and HCT-15 cell lines proliferation. [26] Stellatogenin AC AC = strong inhibitory effect on the acetic acid-induced writhing test. [95] β-amyrin AD, AN, AI, CR, CT, HP AD = moderate inhibition of α-Glucosidase and α-Amylase. AN = growth inhibitory activity against several bacteria as B. subtilis, S.
aureus, and C. albicans, the parasite T. cruzi, and antiviral inhibitory activity against the reverse transcriptase of HIV-1 and IAV. AI = decreases the production of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6 and inhibits NO production CR = interaction with the GABAA receptor and produce sedative and hypnotic, increasing noradrenergic activity. It also showed neuroprotective activity reducing α-synuclein aggregation upregulating LGG-1 expression. CT = inhibition of proliferation in the cervical (HeLa), ovary (SK-OV-3), breast (MCF-7), colon (DLD-1), and other cancer cell lines. HP = hepatoprotective effect against acetaminophen-induced hepatotoxicity.

Compound Activity Description Reference
Oleanolic aldehyde AD, AN AD = dose-dependent enhancement of insulin secretion by INS-1 cells. AN = growth inhibitory activity against S. mutans and P. gingivalis. [187,188] Gummosogenin AC, CT AC = strong inhibitory effect on the acetic acid-induced writhing test. CT = inhibition of cervical (HeLa) cancer cell line proliferation. [95,180] Lupeone AN, AD, AI, RN AN = reverse transcriptase inhibitor of HIV-1. AD = moderate inhibition of α-Glucosidase and selective allosteric inhibition of PTP1B. AI = decreases the production of pro-inflammatory cytokines such as IL-12 and IL-6. RN = strong protective effect of cisplatin-induced nephrotoxicity. [128,133,135,164,183,189] Turberogenin AC, CT AC = strong inhibitory effect on the acetic acid-induced writhing test. CT = low inhibition of cervical (HeLa) cancer cell line proliferation. [95,180] Morolic acid AD, AI AD = induced a significant reduction of blood glucose levels by inhibition of 11β-HSD1. AI = inhibition of the leukocyte dermal infiltration and inhibition of key inflammatory enzymes as PLA2 and 5-LOX. [190,191] Queretaroic acid CT CT = inhibition of cervical (HeLa) cancer cell line proliferation. [180] Calenduladiol CR, CT CR = inhibition of acetylcholinesterase and butyrylcholinesterase in vitro. CT = inhibition of leukemia (NB4 and K562) cancer cell lines proliferation. [192,193] Cycloartenol AD, AN, and CT AD = Decrease glucose intestinal absorption that could be associated with SGLT1 regulation as well as α-glucosidase inhibition. Downregulation of fatty acid synthesis and interferes with the absorption of cholesterol. AN = Inhibitory effect against bacteria E. coli and P. aeruginosa and low inhibition of the parasite P. falciparum. CT = Weak cytotoxic activity against HL60 cell line and p38MAPK-mediated apoptosis in the U87 cell line. [91,[194][195][196][197][198][199][200] Betulinic

In Silico Bioprospection of Bioactive Compounds
All sterols and triterpenes described in this review were constructed as ligands using Chem Sketch from Advanced Chemistry Development [202] and exported to Spartan '10 as mol files. The geometry of all molecules was optimized using MMFF//HF 6-31 G*, and final structures were included in an SDF file. The docking studies were carried out using Molegro Virtual Docker v.6.0.1 [203] based on the crystal structures of some bio targets that have been proposed for the biological activities of Cactacaeae extracts or purified compounds derived from them: (a) anti-inflammatory activity-cyclooxygenase-1 (COX-1, PDB code: 1Q4G [204]) and cyclooxygenase-2 (COX-2, PDB code: 3NT1 [205]); (b) metabolic activity-PPAR-α (PDB code: 2ZNN [206]), PPAR-γ (PDB code: 5Y2T [82]) and PTP1B (PDB code: 1C83 [207]); (c) neuroprotective activity-LXR-α (PDB code: 3IPU [208]) LXR-β (PDB code: 1P8D [209]) and acetylcholinesterase (PDB code: 4EY6 [210]). All 3D protein structures were retrieved from the Protein Data Bank [211]. Docking studies were carried out using a previously reported methodology [17,18]. Briefly, all the solvent molecules and cocrystallized ligands were removed from the downloaded structures. The active sites of each enzyme or the ligand-binding domain (LBD) were chosen as the searching sites centered on the cocrystallized ligand, except for PTP1B where the docking area was centered on allosteric site B as previously described [212], and delimited with a 15 Å radius sphere. Standard software procedure was used using the MolOptimizer algorithm. The assignments of charges on each protein were based on standard templates; no other charges were necessary to be set. The Root Mean Square Deviation (RMSD) threshold for multiple cluster poses was set to <2.00 Å. The docking algorithm was set to 5000 maximum iterations with a simplex evolution population size of 50 and 25 runs for each ligand. After docking, MolDock Score was calculated as the theoretical binding affinity, lower values of the score are related to better binding. For each ligand, the pose with the lowest score was selected for further analysis. Cocrystallized ligands were also docked to their respective receptors to verify the efficacy of this procedure, the top-ranking score was recorded, and the RMSD of that pose from the PDB original structure was computed. In all the cases, the RMSD values were lower than 2.5 Å. Targets   Tables 10 and 11 show the results obtained for the docking study carried on COX-1 and COX-2. In both tables, the top-10 compounds with higher affinity are displayed, but a complete table of all the results is included as part of Supplementary information. Table 10 shows the data from sterol derivatives, and Table 11 shows the data from triterpene derivatives that had lower MolDock scores. The analysis of the binding mode of the sterols with higher affinity reveals some structural features that improve ligand binding. In the first instance, among sterols, the oxidation pattern at C3, C6, and C7 appears to be relevant for the interaction with residues Leu 384, Tyr 385, Trp 387 and Met 522 in both COX enzymes. Notably, a hydrogen bond interaction to Met 522 is constant in all 6-OH substituted derivatives like peniocerol. Interestingly, a small hydrophobic pocket is located between these residues (in yellow in Figure 1a) and is occupied by the methyl group in position 4 of locereol, slightly increasing theoretical affinity. Other critical structural factors for enzyme binding are the unsaturations in rings B and D. The unsaturation in ring B seems to be necessary for optimal interaction of substituents in position 6 and potentially 7 with Met 522. Whereas, unsaturation in ring D is needed for accommodation of the aliphatic chain in a hydrophobic pocket formed by Val 116, Val 349, and Tyr 355 (for example, thurberol has better theoretical affinity than peniocerol). Additional substituents in the aliphatic chain improve enzyme binding, as compounds with additional methylene or ethylene groups have slightly more affinity than their analogs than do not bear these groups. (i.e., 24-methylenelophenol has more affinity than lophenol). COX-2 is one of the preferred molecular targets of NSAIDs [17]. The literature is full of research papers about the design, synthesis or isolation together with in silico, in vivo and ex vivo studies to the development of new anti-inflammatory drugs. Although the clinical importance of selectivity against COX-2 vs COX-1 was earlier discussed [213], many groups focus their research programs to discover selective COX-2 drugs. The top ten sterols and triterpenes with the highest MolDock scores cannot be considered selective against any COX enzyme if we consider the MolDock Score as a measure of the theoretical affinity of the compound. From the top 10 ligands with better MolDock score, fucosterol, spinasterol, 24-methylenecholesterol, β-sitosterol, and peniocerol in the sterols group and lupeone and lupeol of triterpene group are well known anti-inflammatory molecules, with mechanisms including COX-2 inhibitory activities. It is possible to focus the next survey to isolate and determine the anti-inflammatory activities and their molecular mechanisms of compounds like thurberol, locereol or lophenol, together with thurberogenine, betulinic aldehyde, 16β-and 22βhydroxystellatogenin or machaerogenine which anti-inflammatory activities or their inhibitory activities against COX enzymes are still unknown. On the other hand, the analysis of the interaction of triterpenes shows that they could similarly bind to COX-1 and COX-2 as sterols. Interestingly, the first 9 compounds with better theoretical affinities belong to the lupane skeleton. In general, the ring A of all compounds interacts with Leu 384, Tyr 385, Trp 387, and Met 522 in the same fashion as sterols (in yellow in Figure 1a,b). The 4,4-dimethyl group in ring A occupies the small hydrophobic group ubicated between these residues. However, it seems to be too large to fit on this site, leading to a lower theoretical affinity. The most oxidized rings (usually rings D and E) in 22β-hydroxystellatogenin interact with the same residues that the side chain of sterol does. The interaction is through hydrogen bonding to residues Ser 353, Tyr 349, and Try 355 of COX-1 or Tyr 348 and Try 385 in COX-2 (in green in Figure 1b). Although hydroxylation in rings D and E improves theoretical affinity, the number of hydroxy or keto groups is not correlated with theoretical affinity, suggesting that hydrophobic interaction is more critical of ligand binding.

Antidiabetic and Metabolic Activities
COX-2 is one of the preferred molecular targets of NSAIDs [17]. The literature is full of research papers about the design, synthesis or isolation together with in silico, in vivo and ex vivo studies to the development of new anti-inflammatory drugs. Although the clinical importance of selectivity against COX-2 vs COX-1 was earlier discussed [213], many groups focus their research programs to discover selective COX-2 drugs. The top ten sterols and triterpenes with the highest MolDock scores cannot be considered selective against any COX enzyme if we consider the MolDock Score as a measure of the theoretical affinity of the compound. From the top 10 ligands with better MolDock score, fucosterol, spinasterol, 24-methylenecholesterol, β-sitosterol, and peniocerol in the sterols group and lupeone and lupeol of triterpene group are well known anti-inflammatory molecules, with mechanisms including COX-2 inhibitory activities. It is possible to focus the next survey to isolate and determine the anti-inflammatory activities and their molecular mechanisms of compounds like thurberol, locereol or lophenol, together with thurberogenine, betulinic aldehyde, 16β-and 22β-hydroxystellatogenin or machaerogenine which anti-inflammatory activities or their inhibitory activities against COX enzymes are still unknown.

Antidiabetic and Metabolic Activities
Several studies have positioned both sterols and triterpenes as hits for the development of drugs for the treatment of metabolic diseases, including diabetes mellitus type 2 [214,215]. Table 12 shows the top 10 sterol ligands, which exhibited the highest theoretical affinity for PTP1B, PPAR-α, and PPAR-γ. Ligands showed in Table 12 exhibited the highest affinity for all the three bio targets and could be considered as potential multitarget molecules for the management of complex metabolic diseases like metabolic syndrome. However, other sterols not shown in this Table exhibited a good affinity for two of the targets like lophenol, locereol, and 5a-cholesta-8,14-dien-3β-ol, which had good theoretical affinity against PPAR-α and PPAR-γ, some synthetic ligands have been developed as PPARα/γ dual agonists to achieve a broad spectrum of metabolic effects with actions against dyslipidemia and hyperglycemia. Deoxyviperidone, 5β-deoxyviperidone, and lathosterol had good scores against PTP1B and PPAR-γ, which could be a good combination of bio targets for the treatment of diabetes mellitus type 2. Well known antidiabetic sterols like fucosterol, lophenol and β-sitosterol have demonstrated in several studies their potential to regulate at different levels both PTP1B and/or PPAR proteins [61,216,217]. Although lophenol does not belong to the top 10 molecules with higher theoretical affinity, the Moldock score values for both PPAR-α and PPAR-γ proteins were -130.9 and -129.7, suggesting that the methodology used correctly predicts the biological activity. Special attention should be taken with sterols like schottenol, spinasterol, thurberol cyclostenol 24-methylenecholesterol, peniocerol, among other sterols that were considered as good candidates to future antidiabetic research. In the case of triterpenes (Table 13), the results strongly suggest that the hydroxylation pattern in rings D and E seems important for enzyme binding. Notably, it is remarkable that many of the triterpene derivatives bear a 17-carboxylic group. Analysis of the predicted complexes show that despite the searching area was centered on the close allosteric site B, which is comprised by residues Arg 24, Arg 254, Glu 262, Tyr 46, Asp 48, Val 49, Ile 219, and Met 258 [218], both sterols and triterpenes can interact with this site but also with the catalytic site, particularly with two of the most critical residues Cys 215 and Arg 221. This finding could explain the non-competitive and mixed-inhibition properties of some compounds, which has been experimentally demonstrated [212,219]. For example, fucosterol and β-sitosterol mainly occupy allosteric site B, while 6-hydroxy substituted sterols bind to both the catalytic site (via the 3β-OH group, the 6α-OH interacts through a hydrogen bond to Tyr 46 and Lys 120) and the allosteric site (Figure 2a). In the case of triterpenes, some of the derivatives with better affinity have hydroxy or carboxylic groups in rings D and E which can interact with amino acids located in site B like Arg 47 (for 17-COOH substituted derivatives) and Asp 48 (for lactone derivatives as shown in Figure 2b).  (Table 13), the results strongly suggest that the hydroxylation pattern in rings D and E seems important for enzyme binding. Notably, it is remarkable that many of the triterpene derivatives bear a 17-carboxylic group.  [218], both sterols and triterpenes can interact with this site but also with the catalytic site, particularly with two of the most critical residues Cys 215 and Arg 221. This finding could explain the non-competitive and mixed-inhibition properties of some compounds, which has been experimentally demonstrated [212,219]. For example, fucosterol and β-sitosterol mainly occupy allosteric site B, while 6-hydroxy substituted sterols bind to both the catalytic site (via the 3β-OH group, the 6α-OH interacts through a hydrogen bond to Tyr 46 and Lys 120) and the allosteric site (Figure 2a). In the case of triterpenes, some of the derivatives with better affinity have hydroxy or carboxylic groups in rings D and E which can interact with amino acids located in site B like Arg 47 (for 17-COOH substituted derivatives) and Asp 48 (for lactone derivatives as shown in Figure 2b).  Analysis of the predicted poses of the testes compounds to PPAR-α revealed three potential binding modes (Figure 3): (a) sterols with no hydroxyl groups in ring B interact in a cavity distinct to the one occupied by known synthetic partial agonists; (b) 6-hydroxy substituted sterols (like thurberol and peniocerol) and lactone bearing triterpenes (like stellatogenin) bind to the same site of synthetic agonists; and (c) acidic triterpenes occupy the same site as known partial agonists, with the carboxyl group approximating to the same residues that interact with the carboxyl group present in the majority of PPAR partial agonists. This mode of interaction could explain the demonstrated activity of oleanolic acid as a PPAR-α activator [220]. For the PPAR-γ docking study, two different modes of binding were found. The first one was common for most triterpenes, these compounds bounded in a different site than that of known synthetic agonists, away from the thiazolidinedione (TZD) binding site formed by residues Ser 289, His 323, His 449 and Tyr 473. The second one was common for sterols; the tetracyclic skeleton occupied an alternative pocket, and the lateral chain could locate either inside or outside of the TZD binding site (Figure 3b). It would be expected that these alternative modes of binding in both nuclear receptors lead to none or different levels of activation. Thus, additional studies should be carried out. and peniocerol) and lactone bearing triterpenes (like stellatogenin) bind to the same site of synthetic agonists; and c) acidic triterpenes occupy the same site as known partial agonists, with the carboxyl group approximating to the same residues that interact with the carboxyl group present in the majority of PPAR partial agonists. This mode of interaction could explain the demonstrated activity of oleanolic acid as a PPAR-α activator [220]. For the PPAR-γ docking study, two different modes of binding were found. The first one was common for most triterpenes, these compounds bounded in a different site than that of known synthetic agonists, away from the thiazolidinedione (TZD) binding site formed by residues Ser 289, His 323, His 449 and Tyr 473. The second one was common for sterols; the tetracyclic skeleton occupied an alternative pocket, and the lateral chain could locate either inside or outside of the TZD binding site (Figure 3b). It would be expected that these alternative modes of binding in both nuclear receptors lead to none or different levels of activation. Thus, additional studies should be carried out. Some triterpenes from Cactaceae like β-amyrin, oleanolic aid, oleanolic aldehyde, lupeol, betulin, betulinic acid, lupeone and cycloartenol which are well known to regulate some molecular targets of diabetes pathology, with a good prediction in this review. We need to explore the antidiabetic activities, particularly the activity against molecular targets involved in metabolic diseases, of some triterpenes like 16β-hydroxystellatogenin, thurberogenin, stellatogenin, myrtillogenic acid, alamosogenin, 22β-hydroxystellatogenin, machaeric acid, and machaerinic acid, all of them characteristic items of the Cactaceae chemistry, which display some specific oxidation patterns as was discussed above.

Neuroprotective Activity
As life expectancy has increased, neurodegenerative diseases have become a growing concern, and the need for efficient treatments has become an urgency. Acetylcholinesterase inhibitors have been used for the management of Alzheimer's disease, and some have suggested their neuroprotective potential. Also, agonists of LXR-α and LXR-β have been studied for their potential use as neuroprotectors. Then, the development of agonists of these receptors that could also act as AChE inhibitors could lead to interesting treatments. Tables 14 and 15, respectively, show the sterols and triterpenes ligands with a higher affinity to the previously mentioned targets. Three different modes of potential binding to PPAR-α were suggested in the docking study, as exemplified with peniocerol (in cyan), oleanolic acid (in blue), and schottenol (in magenta); (b) A similar situation was found for PPAR-γ, with two potential binding modes. The LBD site is shown in yellow and red for the thiazolidinedione binding site. Peniocerol is shown in cyan, oleanolic acid in blue, and known agonist lobeglitazone in magenta is included for comparison purposes. Some triterpenes from Cactaceae like β-amyrin, oleanolic aid, oleanolic aldehyde, lupeol, betulin, betulinic acid, lupeone and cycloartenol which are well known to regulate some molecular targets of diabetes pathology, with a good prediction in this review. We need to explore the antidiabetic activities, particularly the activity against molecular targets involved in metabolic diseases, of some triterpenes like 16β-hydroxystellatogenin, thurberogenin, stellatogenin, myrtillogenic acid, alamosogenin, 22β-hydroxystellatogenin, machaeric acid, and machaerinic acid, all of them characteristic items of the Cactaceae chemistry, which display some specific oxidation patterns as was discussed above.

Neuroprotective Activity
As life expectancy has increased, neurodegenerative diseases have become a growing concern, and the need for efficient treatments has become an urgency. Acetylcholinesterase inhibitors have been used for the management of Alzheimer's disease, and some have suggested their neuroprotective potential. Also, agonists of LXR-α and LXR-β have been studied for their potential use as neuroprotectors. Then, the development of agonists of these receptors that could also act as AChE inhibitors could lead to interesting treatments. Tables 14 and 15, respectively, show the sterols and triterpenes ligands with a higher affinity to the previously mentioned targets. For LXR-α, it was found that both sterols and triterpenes bind to the same site ( Figure 4a) but with different interaction patterns. Sterols bind through hydrogen bonding via the 3β-OH group to Glu 267 and Asn 225 while the side alkyl chain interacts with residues Phe 257, Leu 260, Thr 302, and Phe 315. In general, oxidation of ring B improves ligand binding, and slight differences in docking score can be attributed to the nature of the alkyl chain; theoretical affinity increased with the incorporation of additional methylene groups, probably through new hydrophobic interactions. Triterpene derivatives interact with residues Thr 302 and Arg 305 via hydrogen bonding to the lactone or the 17-COOH group of rings D and E. The absence of hydroxy groups in these rings slightly diminishes the theoretical affinity.  According to the results, for the interaction of triterpenes with the enzyme acetylcholinesterase, the substitution pattern in ring A is essential, since that ring A is the one that can approximate the catalytic site. On the other hand, in the B ring, the presence of a hydroxyl group in the C6 helps due to the interaction of the oxygen in the hydroxyl moiety with the Trp 86 located in the protein gorge. In the same set of interactions shown by these compounds, the side chain of cholesterol is stuck with the outer part (mouth) of the enzyme. In triterpenes, the E ring is the one that interacts with the catalytic site through interactions of C17-COOH or lactone moieties, with some residues in the active site. The absence of these features diminishes slightly the affinity. On the other hand, the H atom of 3β-OH of the triterpenes interacts with Tyr 341. In this review is evident the potential that some sterols and triterpenoids from Cactaceae, to the development of new neuroprotectors, via the inhibition of acetylcholinesterase and LXR-α and LXR-β. The docking score calculated for the predicted complexes of the analyzed sterols and triterpenes is close to the score for the known LXR-β ligand 24(S),25-epoxycholesterol (MolDock score = 151.3). The interaction of the 3β-OH group to Asn 239, Glu 281, and Arg 319 are constant in all derivatives ( Figure 4c). As it was the case of LXR-α docking study, oxidation in position 6 improves theoretical affinity through interaction to Phe 243, and Phe 329 and additional differences in docking score among sterols can be accredited to the nature of the alkyl chain side. Whereas triterpenes interact through hydrogen bonding to residue Thr 316 via oxygen atoms of lactone ring or 17-COOH, this interaction pattern was common in most of the derivatives analyzed. Interestingly, ring A lays close to residues His 435 and Trp 457, which interact with the epoxy group of 24(S),25-epoxycholesterol, and could interact with hydroxyl groups of 22-hydroxycholesterol and 24-hydroxycholesterol, known endogenous ligands of LXRs. It has been reported that oxidation at both C-22 and C-24 increases LXR activation [221].

Conclusions
According to the results, for the interaction of triterpenes with the enzyme acetylcholinesterase, the substitution pattern in ring A is essential, since that ring A is the one that can approximate the catalytic site. On the other hand, in the B ring, the presence of a hydroxyl group in the C6 helps due to the interaction of the oxygen in the hydroxyl moiety with the Trp 86 located in the protein gorge. In the same set of interactions shown by these compounds, the side chain of cholesterol is stuck with the outer part (mouth) of the enzyme. In triterpenes, the E ring is the one that interacts with the catalytic site through interactions of C17-COOH or lactone moieties, with some residues in the active site. The absence of these features diminishes slightly the affinity. On the other hand, the H atom of 3β-OH of the triterpenes interacts with Tyr 341. In this review is evident the potential that some sterols and triterpenoids from Cactaceae, to the development of new neuroprotectors, via the inhibition of acetylcholinesterase and LXR-α and LXR-β.

Conclusions
The oxidation pattern in sterols and triterpenes from Cactaceae is an extraordinary feature. The oxidation at C6 to form a 6α-hydroxyl group in cholesterol moiety in sterols like peniocerol, macdougallin, cyclostenol, stenocereol, and thurberol, as well as the C6 ketone in viperidinone, viperidone, deoxyviperidone, and 5β-deoxyviperidone, appears to be essential to explaining the possible molecular mechanisms behind the biological activities of those kinds of compounds, as suggested by the available experimental bioactivities and the results of the present in silico study. On the other hand, the oxidation pattern in E ring in oleanane and lupane triterpenes appears as a pivotal factor in controlling the affinity of compounds. At least in the in vitro assays, the presence of hydroxyl, ketone, carboxylic acid, or lactone groups (thurberogenin and 16β-hydroxystelltogenin) in ring E appears to be important in the selectivity of the sterols and triterpenes with their preferred target.
The chemistry of sterols and triterpenoids of some species, from the subfamily Cactoideae, tribe Phyllocacteae, mainly from the subtribe Echinocereinae, was reviewed, together with their biological activities. The information generated about the sterols reveals the presence of the typical 5 sterols like sitosterol, among others, but interestingly the presence of a very unusual family of 8 sterols with a pattern of oxidation in a sterol moiety of 27 carbons. Two sterols are of biosynthetic importance because it appears as unusual intermediaries in a truncated demethylation process. On the other hand, triterpenes appear as common compounds with unique features, including the oxidation pattern in rings C/D. The compounds found in Cactaceae have been extensively studied because of their biological properties. In this paper, we focused only on three biological activities. At least at an in silico level, it is possible to correlate the biological activities with the theoretical affinities showed between the compounds and some of the specific receptors involved as molecular targets of chronic diseases like inflammation, type 2 diabetes or neurodegenerative disorders. Regarding the in silico bioprospection, this study reveals that the oxidation pattern in ring B of sterol skeleton and rings D and E of triterpenes, together with the presence of lactones, contribute to the biological activities of Cactaceae triterpenes. The above prompt us to continue with this methodological approach to find highly bioactive hits from the sterols and triterpene of Cactaceae, useful for the development of semisynthetic drugs for the management of chronic degenerative diseases with a multitarget approach.