Microwave-assisted extraction versus Soxhlet extraction to determine triterpene acids in olive skins.

Microwave-assisted extraction is compared with a more classical technique, Soxhlet extraction, to determine the content of triterpene acids in olive skins. The samples used in their original unmilled state and milled were extracted with ethyl acetate or methanol as solvents. The optimized operating conditions (e.g., amount and type of solvent, and time and temperature of extractions) to attain the better extraction yields have been established. For the identification and quantitation of the target compounds, an ultra high performance liquid chromatography with tandem mass spectrometry method was employed. The best results were achieved using the microwave-assisted extraction technique, which was much faster than the Soxhlet extraction method, and showed higher efficiency in the extraction of the triterpenic acids (oleanolic and maslinic).

Synthesis and Antioxidant Activity of Hydroxytyrosol Alkyl-Carbonate Derivatives.

Three procedures have been investigated for the isolation of tyrosol (1) and hydroxytyrosol (2) from a phenolic extract obtained from the solid residue of olive milling. These three methods, which facilitated the recovery of these phenols, were chemical or enzymatic acetylation, benzylation, and carbomethoxylation, and subsequent carbonylation or acetonation reactions. Several new lipophilic alkyl-carbonate derivatives of hydroxytyrosol have been synthesized, coupling the primary hydroxy group of this phenol, through a carbonate linker, using alcohols with different chain lengths. The antioxidant properties of these lipophilic derivatives have been evaluated by different methods and compared with free hydroxytyrosol (2) and also with the well-known antioxidants BHT and α-tocopherol. Three methods were used for the determination of this antioxidant activity: FRAP and ABTS assays, to test the antioxidant power in hydrophilic media, and the Rancimat test, to evaluate the antioxidant capacity in a lipophilic matrix. These new alkyl-carbonate derivatives of hydroxytyrosol enhanced the antioxidant activity of this natural phenol, with their antioxidant properties also being higher than those of the commercial antioxidants BHT and α-tocopherol. There was no clear influence of the side-chain length on the antioxidant properties of the alkyl-carbonate derivatives of 2, although the best results were achieved mainly by the compounds with a longer chain on the primary hydroxy group of this natural phenolic substance.

Semi-synthesis and antiproliferative evaluation of PEGylated pentacyclic triterpenes.

Several PEGylated derivatives of oleanolic and maslinic acids have been semi-synthesized, attaching one acid-PEG reagent to the hydroxyl group/s at C-2 or C-2/C-3 of the A rings of these natural triterpenes, and also to their corresponding C-28 benzyl derivatives. Several monomeric and dimeric PEGylated compounds have also been produced by linking one diamine-PEG reagent to the carboxyl group at C-28 of the same natural triterpenes and also to their corresponding C-2 or C-2/C-3 acetylated derivatives. The cytotoxic effects of 12 triterpenic PEGylated derivatives in three cancer-cell lines (B16F10, HT29, and Hep G2) have been assayed. The best results have been achieved by the PEGylated-amine derivative of oleanolic acid, with IC50 concentrations between 0.22 and 3.78 µM, being between 28- and 963-fold more effective than its natural precursor. The percentages of apoptosis induction have also been determined for the five PEGylated derivatives with the lowest cytotoxicity data. All five compounds showed apoptotic effects on the treated cells, with a total apoptosis rate of 99% in the B16F10 cells, 80% in the Hep G2 cells, and 51% in the HT29 cells. We have also studied the changes in the mitochondrial membrane potential (MMP) to elucidate the possible mechanism involved in the apoptotic responses (intrinsic or extrinsic). Finally, to verify the results found in the cytometry assays, we have used fluorescence microscopy techniques to determine changes in the cell morphology. These PEGylated derivatives of natural triterpenoids, which can induce apoptosis at very low concentrations in different tumour lines, may represent new effective therapeutic drugs against these diseases.

Biotransformation of oleanolic and maslinic methyl esters by Rhizomucor miehei CECT 2749.

The pentacyclic triterpenoids methyl oleanolate, methyl maslinate, methyl 3ß-hydroxyolean-9(11),12-dien-28-oate, and methyl 2α,3ß-dihydroxy-12ß,13ß-epoxyolean-28-oate were biotransformed by Rhizomucor miehei CECT 2749. Microbial transformation of methyl oleanolate produced only a 7ß,30-dihydroxylated metabolite with a conjugated 9(11),12-diene system in the C ring. Biotransformation of the substrate with this 9(11),12-diene system gave the same 7ß,30-dihydroxylated compound together with a 7ß,15α,30-trihydroxyl derivative. The action of this fungus (R. miehei) on methyl maslinate was more varied, isolating metabolites with a 30-hydroxyl group, a 9(11),12-diene system, an 11-oxo group, or an 12-oxo group. Microbial transformation of the substrate with a 12ß,13ß-epoxy function resulted in the isolation of two metabolites with 12-oxo and 28,13ß-olide groups, hydroxylated or not at C-7ß, together with a 30-hydroxy-12-oxo derivative. The structures of these derivatives were deduced by extensive and rigorous spectroscopic studies.

Solid-phase library synthesis of bi-functional derivatives of oleanolic and maslinic acids and their cytotoxicity on three cancer cell lines.

A wide set of 264 compounds has been semisynthesized with high yields and purities. These compounds have been obtained through easy synthetic processes based on a solid-phase combinatorial methodology. All the members of this library have one central core of a natural pentacyclic triterpene (oleanolic or maslinic acid) and differ by 6 amino acids, coupled with the carboxyl group at C-28 of the triterpenoid skeleton, and by 10 different acyl groups attached to the hydroxyl groups of the A-ring of these molecules. According to the literature on the outstanding and promising pharmacological activities of other similar terpene derivatives, some of these compounds have been tested for their cytotoxic effects on the proliferation of three cancer cell lines: B16-F10, HT29, and Hep G2. In general, we have found that around 70% of the compounds tested show cytotoxicity in all three of the cell lines selected; around 60% of the cytotoxic compounds are more effective than their corresponding precursors, that is, oleanolic (OA) or maslinic (MA) acids; and nearly 50% of the cytotoxic derivatives have IC50 values between 2- to 320-fold lower than their corresponding precursor (OA or MA).

Semi-synthesis of acylated triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV agents.

A broad set of potential bioactive conjugate compounds has been semi-synthesized through solution- and solid-phase organic procedures, coupling two natural pentacyclic triterpene acids, oleanolic (OA) and maslinic acids (MA), at the hydroxyl groups of the A-ring of the triterpene skeleton, with 10 different acyl groups. These acyl OA and MA derivatives have been tested for their anti-proliferative (against the b16f10 murine melanoma cancer cells) and antiviral (as inhibitors of the HIV-1-protease) effects. Several derivatives have shown high levels of early and total apoptosis (up to 90%). Most of the compounds that exhibited anti-proliferative effects also generated ROS, probably involving the activation of an intrinsic apoptotic route. The only four compounds that did not cause the release of ROS could be related to the participation of a probable extrinsic activation of the apoptosis mechanism. A great number of these acyl OA and MA derivatives have proved to be potent inhibitors of the HIV-1-protease, the most active inhibitors having IC50 values between 0.31 and 15.6 µM, these values being between 4 and 186 times lower than their non-acylated precursors. The potent activities exhibited in the apoptosis-activation processes and in the inhibition of the HIV-1-protease by some OA and MA acylated derivatives imply that these compounds could be used as new, safe, and effective anticancer and/or antiviral drugs.

Liquid chromatography-mass spectrometry determination in plasma of maslinic acid, a bioactive compound from Olea europaea L.

Maslinic acid, a pentacyclic triterpene from Olea europaea L., exerts hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Here, an LC-APCI-MS method to determine this compound in plasma is presented as a first step in pharmacokinetic studies. Maslinic acid was extracted from plasma with ethyl acetate and separated on a C18 column using a gradient elution of water and acetonitrile. The target ions were m/z 471.3 for maslinic acid and m/z 455.3 for I.S. The method was validated by the analysis of spiked plasma samples obtaining a linear correlation, a recovery of 99.0±0.9% and a quantification limit of 5 nM. The precision and accuracy were ≤8.38% and ≤4.82%, respectively. Finally, the method was verified by measuring the rat plasmatic concentrations 24h after the single oral administration of 10, 25 and 50 mg/kg of maslinic acid, thereby ensuring that the procedure is appropriate for its use in bioavailability studies.

Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei.

Microbial transformation of oleanolic acid by Rhizomucor miehei produced three metabolites. A known compound, a 30-hydroxyl derivative (queretaroic acid), and two 7ß,30- and 1ß,30-dihydroxylated metabolites, respectively. The action of the same fungus (R. miehei) on maslinic acid produced an olean-11-en-28,13ß-olide derivative, a metabolite hydroxylated at C-30, an 11-oxo derivative, and two metabolites with an 11α,12α-epoxy group, hydroxylated or not at C-30. Their structures were elucidated by extensive analyses of their spectroscopic data, and also by chemical correlations.

Antiallodynic and analgesic effects of maslinic acid, a pentacyclic triterpenoid from Olea europaea.

The effects of maslinic acid (1), a pentacyclic triterpenoid obtained from Olea europaea, were studied in several tests for nociception in mice. Systemic administration of 1 reduced acetic acid-induced writhing, the inflammatory phase of formalin-induced pain, and capsaicin-induced mechanical allodynia. However, it did not induce motor incoordination in the rotarod test. The topical administration of 1 also reduced the inflammatory phase of the formalin test, indicating that at least some of its effects are mediated peripherally. The present results demonstrate for the first time that maslinic acid induces antinociceptive and antiallodynic effects.

Assessment of the safety of maslinic acid, a bioactive compound from Olea europaea L.

SCOPE: Maslinic acid, the main pentacyclic triterpene of the cuticle of Olea europaea L. fruit, has multiple beneficial effects on health, most notably antitumor and hypoglycemic properties. Notwithstanding the biological activities, there is a lack of knowledge about its safety. Therefore, the purpose of this study was to evaluate whether high doses of maslinic acid have harmful effects on Swiss CD-1 male mice. METHODS AND RESULTS: The single oral administration of the pentacyclic triterpene at 1000 mg/kg to mice did not produce any signs of morbidity or mortality. The repeated daily oral administration of 50 mg/kg of maslinic acid for 28 days did not induce any sign of toxicity during the experimental period. Body weight did not differ between mice that received the triterpene and the control group. Similarly, hematological and biochemical variables were not affected by the treatment. Histopathologic examination of the organs revealed that there were no differences between the control and the treated mice. CONCLUSION: Taken together the results obtained from the acute and the repeated intake of maslinic acid indicate that the compound does not exert any adverse effects on the variables tested in mice, thus suggesting a sufficient margin of safety for its putative use as a nutraceutical.

Determination of maslinic acid, a pentacyclic triterpene from olives, in rat plasma by high-performance liquid chromatography.

Maslinic acid, a pentacyclic triterpene from olives, has been reported to exert beneficial effects on health, including anticarcinogenic activity. Despite its importance, little is known about its bioavailability in both humans and animals. A fundamental step for this evaluation consisted of measuring this compound in blood. Therefore, a simple high-performance liquid chromatography (HPLC) method with diode array detection has been developed. Maslinic acid contained in plasma was extracted twice using ethyl acetate. After centrifugation, the organic fraction was evaporated to dryness and the residue was reconstituted with methanol/water (75:25, v/v) and analyzed by HPLC. The method was validated by obtaining a linear correlation (r(2) = 0.999) and an average recovery of 99%. Precision expressed as the coefficient of variation ranged from 1.23 to 9.06%. The oral administration of maslinic acid (50 mg/kg) to rats and its subsequent detection in plasma showed that the method is suitable for absorption, distribution, and metabolism studies.

Maslinic acid derivatives induce significant apoptosis in b16f10 murine melanoma cells.

Maslinic acid (2α,3ß-dihydroxyolean-12-en-28-oic acid), a natural dihydroxylated pentacyclic triterpene acid isolated from olive-pressing residues, has been investigated together with some of its derivatives regarding the induction of apoptosis in B16F10 melanoma cells. Some of the compounds tested are described in this work, but others come from previous studies. Ten of these derivatives induce over 80% of apoptosis, clearly promoting cell death in B16F10 melanoma. By contrast, the induction cell death through necrosis was very slightly significant with these compounds. These results indicate that maslinic acid derivatives are promising chemopreventive and chemotherapeutic agents.

Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium falciparum intraerythrocytic stages.

BACKGROUND: Natural products have played an important role as leads for the development of new drugs against malaria. Recent studies have shown that maslinic acid (MA), a natural triterpene obtained from olive pomace, which displays multiple biological and antimicrobial activities, also exerts inhibitory effects on the development of some Apicomplexan, including Eimeria, Toxoplasma and Neospora. To ascertain if MA displays anti-malarial activity, the main objective of this study was to asses the effect of MA on Plasmodium falciparum-infected erythrocytes in vitro. METHODS: Synchronized P. falciparum-infected erythrocyte cultures were incubated under different conditions with MA, and compared to chloroquine and atovaquone treated cultures. The effects on parasite growth were determined by monitoring the parasitaemia and the accumulation of the different infective stages visualized in thin blood smears. RESULTS: MA inhibits the growth of P. falciparum Dd2 and 3D7 strains in infected erythrocytes in, dose-dependent manner, leading to the accumulation of immature forms at IC50 concentrations, while higher doses produced non-viable parasite cells. MA-treated infected-erythrocyte cultures were compared to those treated with chloroquine or atovaquone, showing significant differences in the pattern of accumulation of parasitic stages. Transient MA treatment at different parasite stages showed that the compound targeted intra-erythrocytic processes from early-ring to schizont stage. These results indicate that MA has a parasitostatic effect, which does not inactivate permanently P. falciparum, as the removal of the compound allowed the infection to continue CONCLUSIONS: MA displays anti-malarial activity at multiple intraerythrocytic stages of the parasite and, depending on the dose and incubation time, behaves as a plasmodial parasitostatic compound. This novel parasitostatic effect appears to be unrelated to previous mechanisms proposed for current anti-malarial drugs, and may be relevant to uncover new prospective plasmodial targets and opens novel possibilities of therapies associated to host immune response.

Anticoccidial activity of maslinic acid against infection with Eimeria tenella in chickens.

We propose maslinic acid (2-alpha, 3-beta-dihydroxiolean-12-en-28-oic acid), found in the leaves and fruit of the olive tree (Olea europaea L.), as a new natural coccidiostatic product against Eimeria tenella. Its action in infected animals has been compared with animals treated with sodium salinomycin. The lesion index (LS), the oocyst index (OI) and the anticoccidial index (ACI) were studied with regard to the weight of the chicks. The ACI for maslinic acid was 210.27 and for sodium salinomycin 173.09. Similarly, both LS and OI decreased in the groups treated with maslinic acid. A considerable increase in weight was found in the chicks treated with maslinic acid compared with those in the control group. Histopathological studies of the caecum at 120 h post-infection showed that the infection rate decreased significantly in chicks treated with maslinic acid.

Action of a pentacyclic triterpenoid, maslinic acid, against Toxoplasma gondii.

The action of maslinic acid (2alpha,3beta-dihydroxyolean-12-en-28-oic acid) (1), a pentacyclic derivative present in the pressed fruits of the olive (Olea europaea), has been studied against the tachyzoites of Toxoplasma gondii. The capability of tachyzoites to infect Vero cells treated with 1 was affected. The LD(50) values were 58.2 muM for the isolated tachyzoites and 236 muM for the noninfected Vero cells. Zymograms of the T. gondii proteases incubated with 1 showed a dosage-dependent inhibition of some of the proteases. The parasites treated with 1 showed gliding motility and ultrastructural alterations. The present findings suggest that protease activity of the parasite required for cell invasion is the action target for maslinic acid (1).

Bioactive compounds with added value prepared from terpenes contained in solid wastes from the olive oil industry.

Starting from solid wastes from two-phase olive-oil extraction, the pentacyclic triterpenes oleanolic acid and maslinic acid were isolated. These natural compounds were transformed into methyl olean-12-en-28-oate (5), which then was transformed into several seco-C-ring triterpene compounds by chemical and photolytic modifications. The triene seco-products were fragmented through several oxidative procedures to produce, simultaneously, cis- and trans-decalin derivatives, both potential synthons for bioactive compounds. The chemical behavior of the isolated fragments was investigated, and a suitable approach to several low-molecular-weight terpenes was performed. These are interesting processes for the value-addition to solid waste from the olive-oil industry.

Different pathways for the deoxygenation of the A-ring of natural triterpene compounds.

Some deoxygenation pathways were tested to remove the hydroxyl groups of the natural triterpenes oleanolic acid and maslinic acid to obtain a practical starting material for the semisynthesis of other interesting organic synthons. Different deoxygenation processes were carried out starting from these triterpenic acids or from several derivatives such as methyl esters and epoxy derivatives. The hydroxyl groups were transformed into some intermediate compounds including xanthyl, thiocarbonyl or tosyl derivatives. The opening of the oxirane ring between C-2 and C-3 was also achieved through different methods using deoxygenating reagents such as Me3SiCl/NaI, WCl4/n-BuLi and Cp2TiCl.

Solution- and solid-phase synthesis and anti-HIV activity of maslinic acid derivatives containing amino acids and peptides.

Maslinic acid (1) has been coupled at C-28 with several alpha- and omega-amino acids by using solution- and solid-phase synthetic procedures. Twelve derivatives (2-13) with a single amino acid residue were prepared in solution phase, whereas a dipeptide (14), a tripeptide (15), and a series of conjugate dipeptides (16-24) were synthesized in solid phase. The anti-HIV activity of these compounds was assessed on MT-2 cells infected with viral clones carrying the luciferase gene as a reporter. While in maslinic acid (1) were present both cytotoxic and antiviral activities, only the derivatives 13 and 24 showed anti-HIV-1 activity and therefore represent a novel class of anti-HIV-1 compounds.

Apolipoprotein E determines the hepatic transcriptional profile of dietary maslinic acid in mice.

The hypothesis that the maslinic acid (MA) of olive oil (OO) dramatically influences hepatic gene expression was tested in mice. Two OOs only differing in the presence of MA were prepared. Using DNA microarrays, we analyzed hepatic gene expression in apolipoprotein E (apoE)-deficient mice with a C57BL/6J genetic background that were fed with isocaloric, isonitrogenous diets containing either 10% (w/w) OO or 10% MA-enriched OO. As an initial screening of potential candidate genes involved in a differential response, this study further considered only genes with remarkably modified expression (signal log(2) ratio higher than1.5 or lower than -1.5). The nine genes fulfilling these prerequisites were confirmed by quantitative reverse transcriptase polymerase chain reaction and analyzed in C57BL/6J wild-type mice. Only Cyp2b9, Cyp2b13 and Dbp expressions appeared significantly increased, and Marco was significantly decreased in apoE-deficient mice receiving the MA-enriched diet. Dbp was up-regulated to an extent depending on the genetic background of the mice and negatively associated with the expression of Marco, a gene strongly up-regulated by the absence of apoE. These expression changes could be used as markers of the intake of the MA-enriched OO and are influenced by genetic background generated by the absence or the presence of apoE. Overall, these results (a) indicate that MA in virgin OO is highly active in controlling hepatic gene expression and (b) highlight the important interaction between the response to MA and the presence of apoE. They also confirm that virgin OO cannot be simplistically classified as monounsaturated fatty-enriched oil without paying attention to its active minor components.

Remote hydroxylation of methyl groups by regioselective cyclopalladation. Partial synthesis of hyptatic acid-A.

Hyptatic acid-A (32), a 2alpha,3beta,24-trihydroxyolean-12-en-28-oic acid, previously isolated from Hyptis capitata, was obtained from maslinic acid (2). The regioselective cyclopalladation of the axial methyl group on C-4 of maslinic acid afforded the C-24 hydroxymethylene group due to the presence of a C-2-OR substituent. Nevertheless, hederagenin (7) (23-hydroxy derivative) was formed when this oxygenated group was not present.