Principal Bioactive Properties of Oleanolic Acid, Its Derivatives, and Analogues.

Natural products have always played an important role in pharmacotherapy, helping to control pathophysiological processes associated with human disease. Thus, natural products such as oleanolic acid (OA), a pentacyclic triterpene that has demonstrated important activities in several disease models, are in high demand. The relevant properties of this compound have motivated re-searchers to search for new analogues and derivatives using the OA as a scaffold to which new functional groups have been added or modifications have been realized. OA and its derivatives have been shown to be effective in the treatment of inflammatory processes, triggered by chronic diseases or bacterial and viral infections. OA and its derivatives have also been found to be effective in diabetic disorders, a group of common endocrine diseases characterized by hyperglycemia that can affect several organs, including the liver and brain. This group of compounds has been reported to exhibit significant bioactivity against cancer processes in vitro and in vivo. In this review, we summarize the bioactive properties of OA and its derivatives as anti-inflammatory, anti-bacterial, antiviral, anti-diabetic, hepatoprotective, neuroprotective, and anticancer agents.

Modulating anti-inflammatory and anticancer properties by designing a family of metal-complexes based on 5-nitropicolinic acid.

A new family of six complexes based on 5-nitropicolinic acid (5-npic) and transition metals has been obtained: [M(5-npic)2]n (MII = Mn (1) and Cd (2)), [Cu(5-npic)2]n (3), and [M(5-npic)2(H2O)2] (MII = Co (4), Ni (5), and Zn (6)), which display 1D, 2D, and mononuclear structures, respectively, thanks to different coordination modes of 5-npic. After their physicochemical characterization by single-crystal X-ray diffraction (SCXRD), elemental analyses (EA), and spectroscopic techniques, quantum chemical calculations using Time-Dependent Density Functional Theory (TD-DFT) were performed to further study the luminescence properties of compounds 2 and 6. The potential anticancer activity of all complexes was tested against three tumor cell lines, B16-F10, HT29, and HepG2, which are models widely used for studying melanoma, colon cancer, and liver cancer, respectively. The best results were found for compounds 2 and 4 against B16-F10 (IC50 = 26.94 and 45.10 µg mL-1, respectively). In addition, anti-inflammatory studies using RAW 264.7 cells exhibited promising activity for 2, 3, and 6 (IC50 NO = 5.38, 24.10, and 17.63 µg mL-1, respectively). This multidisciplinary study points to complex 2, based on CdII, as a promising anticancer and anti-inflammatory material.

Evaluation of Anticancer and Anti-Inflammatory Activities of Some Synthetic Rearranged Abietanes.

Synthesis of the rearranged abietane diterpenes pygmaeocins C and D, viridoquinone, saprorthoquinone, and 1-deoxyviroxocine has been successfully achieved. The anticancer and anti-inflammatory activities of selected orthoquinonic compounds 5, 7, 13, and 19, as well as pygmaeocin C (17), were evaluated for the first time. The antitumor properties were assessed using three cancer cell lines: HT29 colon cancer cells, Hep G2 hepatocellular carcinoma cells, and B16-F10 murine melanoma cells. Compounds 5 and 13 showed the highest cytotoxicity in HT29 cells (IC50 = 6.69 ± 1.2 µg/mL and IC50 = 2.7 ± 0.8 µg/mL, respectively). Cytometric studies showed that this growth inhibition involved phase S cell cycle arrest and apoptosis induction, possibly through the activation of the intrinsic apoptotic pathway. Morphological apoptotic changes, including nuclear fragmentation and chromatin condensation, were also observed. Furthermore, the anti-inflammatory activity of these compounds was evaluated on the basis of their ability to inhibit nitric oxide production on the lipopolysaccharide activated RAW 264.7 macrophage cell line. Although all compounds showed high anti-inflammatory activity, with percentages between 40 and 100%, the highest anti-inflammatory potential was obtained by pygmaeocin B (5) (IC50NO = 33.0 ± 0.8 ng/mL). Our results suggest that due to their dual roles, this type of compound could represent a new strategy, contributing to the development of novel anticancer agents.

Synthesis of Tricyclic Pterolobirin H Analogue: Evaluation of Anticancer and Anti-Inflammatory Activities and Molecular Docking Investigations.

Pterolobirin H (3), a cassane diterpene isolated from the roots of Pterolobium macropterum, exhibits important anti-inflammatory and anticancer properties. However, its relatively complex tetracyclic structure makes it difficult to obtain by chemical synthesis, thus limiting the studies of its biological activities. Therefore, we present here a short route to obtain a rational simplification of pterolobirin H (3) and some intermediates. The anti-inflammatory activity of these compounds was assayed in LPS-stimulated RAW 264.7 macrophages. All compounds showed potent inhibition of NO production, with percentages between 54 to 100% at sub-cytotoxic concentrations. The highest anti-inflammatory effect was shown for compounds 15 and 16. The simplified analog 16 revealed potential NO inhibition properties, being 2.34 higher than that of natural cassane pterolobirin H (3). On the other hand, hydroxyphenol 15 was also demonstrated to be the strongest NO inhibitor in RAW 264.7 macrophages (IC50 NO = 0.62 ± 0.21 µg/mL), with an IC50NO value 28.3 times lower than that of pterolobirin H (3). Moreover, the anticancer potential of these compounds was evaluated in three cancer cell lines: HT29 colon cancer cells, Hep-G2 hepatoma cells, and B16-F10 murine melanoma cells. Intermediate 15 was the most active against all the selected tumor cell lines. Compound 15 revealed the highest cytotoxic effect with the lowest IC50 value (IC50 = 2.45 ± 0.29 µg/mL in HT29 cells) and displayed an important apoptotic effect through an extrinsic pathway, as evidenced in the flow cytometry analysis. Furthermore, the Hoechst staining assay showed that analog 15 triggered morphological changes, including nuclear fragmentation and chromatin condensation, in treated HT29 cells. Finally, the in silico studies demonstrated that cassane analogs exhibit promising binding affinities and docking performance with iNOS and caspase 8, which confirms the obtained experimental results.

Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes.

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize, in yields of 72-86%, 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids (Aa) of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, 12 NBD-Aa-triterpene conjugates have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these NBD-Aa-triterpene conjugates enabled their uptake and subcellular distribution to be followed in order to probe in detail their biological properties at the cellular and molecular level.

Semisynthesis and Evaluation of Anti-Inflammatory Activity of the Cassane-Type Diterpenoid Taepeenin F and of Some Synthetic Intermediates.

A new strategy for the semisynthesis of the aromatic cassane-type diterpene taepeenin F (6) is reported. The introduction of the methyl group at C-14, characteristic of the target compound, was achieved via dienone 13, easily prepared from abietic acid (10), the major compound in renewable rosin. Biological assays of selected compounds are reported. The antiproliferative activity against HT29, B16-F10, and HepG2 tumor cell lines has been investigated. Salicylaldehyde 21 was the most active compound (IC50 = 7.72 µM). Products 16 and 21 displayed apoptotic effects in B16-F10 cells, with total apoptosis rates of 46 and 38.4%, respectively. This apoptotic process involves a significant arrest of the B16-F10 cell cycle, blocking the G0/G1 phase. Dienone 16 did not cause any loss of the mitochondrial membrane potential (MMP), while salicylaldehyde 21 caused a partial loss of the MMP. The anti-inflammatory activity of the selected compounds was investigated with the LPS-stimulated RAW 264.7 macrophages. All compounds showed potent NO inhibition, with percentages between 80 and 99% at subcytotoxic concentrations. Dienone 16 inhibited LPS-induced differentiation of RAW 264.7 cells, by increasing the proportion of cells in the S phase. In addition, salicylaldehyde 21 had effects on the cell cycle, recovering the cells from the G0/G1 full arrest produced in response to LPS action.

Synthesis and Biological Evaluation of Cassane Diterpene (5α)-Vuacapane-8(14), 9(11)-Diene and of Some Related Compounds.

A set of thirteen cassane-type diterpenes was synthesized and an expedient synthetic route was used to evaluate 14-desmethyl analogs of the most active tested cassane. The anti-inflammatory activities of these 13 compounds were evaluated on a lipopolysaccharide (LPS)-activated RAW 264.7 cell line by inhibition of nitric oxide (NO) production, some of them reaching 100% NO inhibition after 72 h of treatment. The greatest anti-inflammatory effect was observed for compounds 16 and 20 with an IC50 NO of 2.98 ± 0.04 µg/mL and 5.71 ± 0.14 µg/mL, respectively. Flow-cytometry analysis was used to determine the cell cycle distribution and showed that the inhibition in NO release was accompanied by a reversion of the differentiation processes. Moreover, the anti-cancer potential of these 13 compounds were evaluated in three tumor cell lines (B16-F10, HT29, and Hep G2). The strongest cytotoxic effect was achieved by salicylaldehyde 20, and pterolobirin G (6), with IC50 values around 3 µg/mL in HT29 cells, with total apoptosis rates 80% at IC80 concentrations, producing a significant cell-cycle arrest in the G0/G1 phase, and a possible activation of the extrinsic apoptotic pathway. Additionally, initial SAR data analysis showed that the methyl group at the C-14 positions of cassane diterpenoids is not always important for their cytotoxic and anti-inflammatory activities.

Nutraceutical Role of Polyphenols and Triterpenes Present in the Extracts of Fruits and Leaves of Olea europaea as Antioxidants, Anti-Infectives and Anticancer Agents on Healthy Growth.

There is currently a worldwide consensus and recognition of the undoubted health benefits of the so-called Mediterranean diet, with its intake being associated with a lower risk of mortality. The most important characteristics of this type of diet are based on the consumption of significant amounts of fruit, vegetables, legumes, and nuts, which provide, in addition to some active ingredients, fiber and a proportion of vegetable protein, together with extra virgin olive oil (EVOO) as the main sources of vegetable fat. Fish and meat from poultry and other small farm animals are the main sources of protein. One of the main components, as already mentioned, is EVOO, which is rich in monounsaturated fatty acids and to a lesser extent in polyunsaturated fatty acids. The intake of this type of nutrient also provides an important set of phytochemicals whose health potential is widely spread and agreed upon. These phytochemicals include significant amounts of anthocyanins, stilbenes, flavonoids, phenolic acids, and terpenes of varying complexities. Therefore, the inclusion in the diet of this type of molecules, with a proven healthy effect, provides an unquestionable preventive and/or curative activity on an important group of pathologies related to cardiovascular, infectious, and cancerous diseases, as well as those related to the metabolic syndrome. The aim of this review is therefore to shed light on the nutraceutical role of two of the main phytochemicals present in Olea europaea fruit and leaf extracts, polyphenols, and triterpenes, on healthy animal growth. Their immunomodulatory, anti-infective, antioxidant, anti-aging, and anti-carcinogenic capabilities show them to be potential nutraceuticals, providing healthy growth.

Efficient In Vitro and In Vivo Anti-Inflammatory Activity of a Diamine-PEGylated Oleanolic Acid Derivative.

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti-inflammatory effects of a diamine-PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub-cytotoxic concentrations for anti-inflammatory assays of OADP in RAW 264.7 cells. The inflammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 µg/mL of OADP. Cell-cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS-stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF-α, IL-1ß, iNOS, and COX-2; and the blocking of p-IκBα production in LPS-stimulated RAW 264.7 cells. Finally, we have analyzed the anti-inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti-inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes.

Diclofenac N-Derivatives as Therapeutic Agents with Anti-Inflammatory and Anti-Cancer Effect.

A series of diclofenac N-derivatives (2, 4, 6, 8c, 9c, 10a-c) were synthesized in order to test their anti-cancer and anti-inflammatory effects. The anticarcinogen activity has been assayed against three cancer cell lines: HT29, human colon cancer cells; Hep-G2, human hepatic cells; and B16-F10, murine melanoma cells. First, we determined the cytotoxicity of the different compounds, finding that the most effective compound was compound 8c against all cell lines and both compounds 4 and 6 in human Hep-G2 and HT29 cell lines. Compounds 4 and 8c were selected for the percentage of apoptosis determination, cell cycle distribution, and mitochondrial membrane potential measure because these products presented the lowest IC50 values in two of the three cancer cell lines assayed (B16-F10 and HepG2), and were two of the three products with lowest IC50 in HT29 cell line. Moreover, the percentages of apoptosis induction were determined for compounds 4 and 8c, showing that the highest values were between 30 to 60%. Next, the effects of these two compounds were observed on the cellular cycle, resulting in an increase in the cell population in G2/M cell cycle phase after treatment with product 8c, whereas compound 4 increased the cells in phase G0/G1, by possible differentiation process induction. Finally, to determine the possible apoptosis mechanism triggered by these compounds, mitochondrial potential was evaluated, indicating the possible activation of extrinsic apoptotic mechanism. On the other hand, we studied the anti-inflammatory effects of these diclofenac (DCF) derivatives on lipopolysaccharide (LPS) activated RAW 264.7 macrophages-monocytes murine cells by inhibition of nitric oxide (NO) production. As a first step, we determined the cytotoxicity of the synthesized compounds, as well as DCF, against these cells. Then, sub-cytotoxic concentrations were used to determine NO release at different incubation times. The greatest anti-inflammatory effect was observed for products 2, 4, 8c, 10a, 10b, and 9c at 20 µg·mL-1 concentration after 48 h of treatment, with inhibition of produced NO between 60 to 75%, and a concentration that reduces to the 50% the production of NO (IC50 NO) between 2.5 to 25 times lower than that of DCF. In this work, we synthesized and determined for the first time the anti-cancer and anti-inflammatory potential of eight diclofenac N-derivatives. In agreement with the recent evidences suggesting that inflammation may contribute to all states of tumorigenesis, the development of these new derivatives capable of inducing apoptosis and anti-inflammatory effects at very low concentrations represent new effective therapeutic strategies against these diseases.

Synthesis and Biological Activity of Triterpene-Coumarin Conjugates.

A set of 12 maslinic acid-coumarin conjugates was synthesized, with 9 being maslinic acid-diamine-coumarin conjugates at the C-28 carboxylic acid group of triterpene acid and the other three being maslinic acid-coumarin conjugates at C-2/C-3 and/or C-28 of the triterpene skeleton. The cytotoxic effects of these 12 triterpene conjugates were evaluated in three cancer cell lines (B16-F10, HT29, and Hep G2) and compared, respectively, with three nontumor cell lines from the same or similar tissue (HPF, IEC-18, and WRL68). The most potent cytotoxic results were achieved by a conjugate with two molecules of coumarin-3-carboxylic acid coupled through the C-2 and C-3 hydroxy groups of maslinic acid. This conjugate showed submicromolar IC50 values in two of the three cancer cell lines tested (0.6, 1.1, and 0.9 µM), being between 110- and 30-fold more effective than its corresponding precursor. Furthermore, this conjugate (10) showed percentages of cell viability for the three nontumor lines of 90%. Four maslinic acid-coumarin conjugates displayed apoptotic effects in the treated cells, with total apoptosis rates of between 40 and 85%, relative to the control. Almost all the compounds assayed caused cell-cycle arrest in all cancer cell lines, increasing the number of these cells in the G0/G1 phase.

Effects of Erythrodiol on the Antioxidant Response and Proteome of HepG2 Cells.

Erythrodiol (EO) is a pentacyclic triterpenic alcohol found in olive tree leaves and olive oil, and it has important effects on the health properties and quality of olive oil. In this study, we characterized the cytotoxic effects of EO on human hepatocarcinoma (HepG2) cells by studying changes in cell viability, reactive oxygen species (ROS) production, antioxidant defense systems, and the proteome. The results reveal that EO markedly decreased HepG2 cell viability without changing ROS levels. The concentrations of glutathione and NADPH were significantly reduced, with selective changes in the activity of several antioxidant enzymes: glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Proteomic data reveal that EO led to the complete elimination or decreased abundance of 41 and 3 proteins, respectively, and the abundance of 29 proteins increased. The results of functional enrichment analysis show that important metabolic processes and the nuclear transport of mature mRNA were impaired, whereas AMP biosynthesis and cell cycle G2/M phase transition were induced. The transcription factors and miRNAs involved in this response were also identified. These potent antiproliferative effects make EO a good candidate for the further analysis of its hepatic antitumor effects in in vivo studies.

Anti-cancer and anti-inflammatory activities of a new family of coordination compounds based on divalent transition metal ions and indazole-3-carboxylic acid.

A new family of mononuclear coordination compounds has been synthetized and characterized: [M(3-ind)2(H2O)2] (M = Co (1), Ni (2), Zn (3), Fe (4), Mn (5); 3-ind = indazole-3-carboxylate). These materials are mononuclear coordination compounds that possess strong hydrogen bond interactions. The anti-inflammatory effects of these compounds were assayed in lipopolysaccharide activated RAW 264.7 macrophages by inhibition of NO production. Moreover, the cytotoxicity of the complexes and the ligand in RAW 264.7 cells were determined for the first time. The most significant results were obtained for the compounds 4 and 5 reaching values of NO inhibition close to 80% at 48 h, and above to 90% at 72 h of treatment. The highest inhibitory effects on NO production were showed at the range 7-23 µg/mL for compounds 4 and 5. As a consequence, compounds 4 and 5 could be potential drugs due to the interesting anti-inflammatory properties showed. The anti-cancer potential of these compounds has been also tested against different tumor cell lines. The cytotoxicity of the ligand and of compounds 2 and 3 were assayed in three cell lines: HT29, colon cancer cells, Hep-G2, hepatoma cells and B16-F10 melanoma cells. The best results have been achieved with compound 2 in HepG2 and B16-F10 cell lines, being between 1.5 and 2 times more effective that the ligand in HepG2 cells, and B16-F10 cells. All in all, indazole-3-carboxylic acid is a promising ligand for the formation of coordination compounds with biochemical properties.

A Diamine-PEGylated Oleanolic Acid Derivative Induced Efficient Apoptosis through a Death Receptor and Mitochondrial Apoptotic Pathway in HepG2 Human Hepatoma Cells.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Our recent studies have shown that the diamine-(PEG)ylated oleanolic acid (OADP) has strong anti-tumor effects in HCCs. In this study, we evaluated the anti-tumor mechanisms of OADP in the HepG2 HCC cell line. The cytotoxicity results showed that HepG2 cell viability was markedly reduced, with a very low 50% of cell growth inhibitory concentration (IC50, 0.14 µg/mL). We then investigated the anti-tumor mechanisms of OADP in HepG2 cells. The flow-cytometry analysis was used to evaluate cell apoptosis, indicating that 74-95% of cells were apoptotic. OADP caused cell cycle arrest in the G0/G1 phase and the loss of the mitochondrial membrane potential (MMP). Western blot analysis was performed to assess the expression levels of key proteins associated with the underlying molecular mechanism. The results showed the clear upregulation of caspase-8, caspase-9, caspase-3, Bak, p21, and p53, accompanied by the downregulation of Bcl-2. Similar results were obtained by the cotreatment with OADP and the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Agents such as OADP, which are capable of activating extrinsic and intrinsic apoptotic pathways, may represent potential HCC cancer therapies.

Antiproliferative and Pro-Apoptotic Effect of Uvaol in Human Hepatocarcinoma HepG2 Cells by Affecting G0/G1 Cell Cycle Arrest, ROS Production and AKT/PI3K Signaling Pathway.

Natural products have a significant role in the development of new drugs, being relevant the pentacyclic triterpenes extracted from Olea europaea L. Anticancer effect of uvaol, a natural triterpene, has been scarcely studied. The aim of this study was to understand the anticancer mechanism of uvaol in the HepG2 cell line. Cytotoxicity results showed a selectivity effect of uvaol with higher influence in HepG2 than WRL68 cells used as control. Our results show that uvaol has a clear and selective anticancer activity in HepG2 cells supported by a significant anti-migratory capacity and a significant increase in the expression of HSP-60. Furthermore, the administration of this triterpene induces cell arrest in the G0/G1 phase, as well as an increase in the rate of cell apoptosis. These results are supported by a decrease in the expression of the anti-apoptotic protein Bcl2, an increase in the expression of the pro-apoptotic protein Bax, together with a down-regulation of the AKT/PI3K signaling pathway. A reduction in reactive oxygen species (ROS) levels in HepG2 cells was also observed. Altogether, results showed anti-proliferative and pro-apoptotic effect of uvaol on hepatocellular carcinoma, constituting an interesting challenge in the development of new treatments against this type of cancer.

Antiparasitic, anti-inflammatory and cytotoxic activities of 2D coordination polymers based on 1H-indazole-5-carboxylic acid.

We report on the formation of two novel multifunctional isomorphous (4,4) square-grid 2D coordination polymers based on 1H-indazole-5-carboxylic acid. To the best of our knowledge, these complexes are the first examples of 2D-coordination polymers constructed with this novel ligand. We have analysed in detail the structural, magnetic and anti-parasitic properties of the resulting materials. In addition, the capability of inhibiting nitric oxide production from macrophage cells has been measured and was used as an indirect measure of the anti-inflammatory response. Finally, the photocatalytic activity was measured with a model pollutant, i.e. vanillic acid (phenolic compound), with the aim of further increasing the functionalities and applicability of the compounds.

Oleanolic Acid Derivatives as Potential Inhibitors of HIV-1 Protease.

Pentacyclic triterpenes, such as oleanolic acid (I), are promising scaffolds for diversification through the use of combinatorial methods to obtain derivatives that improve their biological properties, increasing their bioavailability and enhancing their therapeutic efficacy. The purpose of this study was to evaluate the influence that derivatives of oleanolic acid, conjugated with one or two amino acids and an acyl group, might exert on HIV-1 protease inhibition. The in vitro studies conducted suggested that the presence of a carboxyacyl group generally improves the inhibition of HIV-1 protease, especially when a phthaloyl group is present, with IC50 concentration values below 5 µM. The gain in activity of three 3-phthaloyl derivatives, with sub-micromolar IC50 values, was between 60- and 100-fold more active than oleanolic acid. A molecular docking study has also been performed to elucidate the mode of binding to the protease by these oleanolic acid derivatives. In general, the derivatives that exhibited the highest inhibitory activity of HIV-1 protease also showed the highest binding energies in docking simulations. The overall results suggest that the coupling of one or two amino acids and a phthaloyl group to oleanolic acid improves HIV-1 protease inhibition, implying that these triterpene derivatives may be promising antiviral agents against HIV.

Diamine and PEGylated-diamine conjugates of triterpenic acids as potential anticancer agents.

A set of 18 amide derivatives of oleanolic or maslinic acid has been semi-synthesised. Twelve were diamine conjugates at C-28 of these triterpenic acids and the other six were PEGylated-diamine derivatives. The cytotoxic effects of these 18 triterpenic derivatives in three cancer-cell lines (B16-F10, HT29, and Hep G2) have been assayed, and have been compared to three non-tumour cell lines of the same or a similar tissue (HPF, IEC-18, and WRL68). The cell viability percentages for the non-tumour HPF line for almost all diamine conjugates of the tested triterpenic acids ranged from 81% to 94%. The best cytotoxic results were achieved with the diamine conjugates of oleanolic or maslinic acid with the shortest and the longest diamine chain (IC50 values from 0.76 µM to 1.76 µM), on the B16-F10 cell line, being between 140- and 20-fold more effective than their corresponding precursors. Four diamine conjugates of these triterpenic acids showed apoptotic effects on treated cells of the B16-F10 line, with total apoptosis rates, relative to control, of between 73% and 90%. The DNA-histogram analysis revealed that all compounds tested produced cell-cycle arrest in B16-F10 cells, increasing the number of these cells in the S phase. All the compounds analysed, except one, did not cause changes in mitochondrial-membrane potential during apoptosis of the B16-F10 cancer cells, suggesting an activation of the extrinsic apoptotic pathway for these compounds.

Synthesis and in vitro antiproliferative evaluation of PEGylated triterpene acids.

A set of PEGylated derivatives of oleanolic and maslinic acids has been semi-synthesised, attaching ethylene glycol, diethylene glycol, triethylene glycol or tetraethylene glycol to the C-28 carboxyl group of these natural triterpenes and some derivatives. Another set of PEGylated derivatives has been semi-synthesised by connecting the same four ethylene glycols to the hydroxyl groups of the A ring of these triterpenic acids, through a carbonate linker, by reaction with trichloromethyl chloroformate. The aqueous solubility of some of these PEGylated derivatives has been compared with that of maslinic acid. The cytotoxic effects of 28 triterpenic PEGylated derivatives in three cancer-cell lines (B16-F10, HT29, and Hep G2) have been assayed. The best results have been achieved with the HT29 cell line, and specifically with the oleanolic acid derivatives having ethylene glycol or tetraethylene glycol attached to the C-28 carboxyl group, which are approximately 27-fold more effective than their natural precursor. Eight PEGylated derivatives have been selected to compare the cytotoxicity results in the HT29 cancer-cell line with those of a non-tumour cell line of the same tissue (IEC-18), four of which were less cytotoxic in the non-tumour cell line. These compounds showed apoptotic effects on treated cells, with percentages of total apoptosis between 20% and 53%, relative to control, at 72h and IC50 concentration, and between 29% to 62%, relative to control, for the same time and IC80 concentration. We have also found that with the treatment of these compounds in HT29 cancer cells, cell-cycle arrest occurred in the G0/G1 phase. Finally, we have also studied changes in mitochondrial membrane potential during apoptosis of HT29 cancer cells, and the results suggest an activation of the extrinsic apoptotic pathway for these compounds.

Target molecules in 3T3-L1 adipocytes differentiation are regulated by maslinic acid, a natural triterpene from Olea europaea.

BACKGROUND: Metabolic syndrome is a set of pathologies among which stand out the obesity, which is related to the lipid droplet accumulation and changes to cellular morphology regulated by several molecules and transcription factors. Maslinic acid (MA) is a natural product with demonstrated pharmacological functions including anti-inflammation, anti-tumor and anti-oxidation, among others. PURPOSE: Here we report the effects of MA on the adipogenesis process in 3T3-L1 cells. METHODS: Cell viability, glucose uptake, cytoplasmic triglyceride droplets, triglycerides quantification, gene transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid-binding protein (aP2) and intracellular Ca2+ levels were determined in pre-adipocytes and adipocytes of 3T3-L1 cells. RESULTS: MA increased glucose uptake. MA also decreased lipid droplets and triglyceride levels, which is in concordance with the down-regulation of PPARγ and aP2. Finally, MA increased the intracellular Ca2+ concentration, which could also be involved in the demonstrated antiadipogenic effect of this triterpene. CONCLUSION: MA has been demonstrated as potential antiadipogenic compound in 3T3-L1 cells.