Oxonitrogenated Derivatives of Eremophilans and Eudesmans: Antiproliferative and Anti-Trypanosoma cruzi Activity.

Cancer is one of the most important causes of death worldwide. Solid tumors represent the vast majority of cancers (>90%), and the chemotherapeutic agents used for their treatment are still characterized by variable efficacy and toxicity. Sesquiterpenes are a group of natural compounds that have shown a wide range of biological activities, including cytotoxic and antiparasitic activity, among others. The antiproliferative activity of natural sesquiterpenes, tessaric acid, ilicic acid, and ilicic alcohol and their semisynthetic derivatives against HeLa, T-47D, WiDr, A549, HBL-100, and SW1573 cell lines were evaluated. The effect of the compounds on Trypanosoma cruzi epimastigotes was also assessed. The selectivity index was calculated using murine splenocytes. Derivatives 13 and 15 were the most antiproliferative compounds, with GI50 values ranging between 5.3 (±0.32) and 14 (±0.90) µM, in all cell lines tested. The presence of 1,2,3-triazole groups in derivatives 15−19 led to improvements in activity compared to those corresponding to the starting natural product (3), with GI50 values ranging between 12 (±1.5) and 17 (±1.1) µM and 16 being the most active compound. In relation to the anti-T. cruzi activity, derivatives 7 and 16 obtained from tessaric acid and ilicic acid were among the most active and selective compounds with IC50 values of 9.3 and 8.8 µM (SI = 8.0 and 9.4), respectively.

In vivo photoinduced [4 + 2] dimerization of a neo-clerodane diterpene in Baccharis flabellata. ROS and RNS scavenging abilities.

Secondary metabolites play a major role in the adaptation of plants to the environment. Furan neo-clerodane diterpenes are characteristic secondary metabolites in Baccharis flabellata Hook. & Arn. var. flabellata. One of the main compounds is the diene ent-15,16-epoxy-19-hydroxy-1,3,13(16),14-clerodatetraen-18-oic acid (DAC). In this work a new dimeric compound (DACD) has been isolated and identified by NMR and MS techniques. The presence of other minor dimers was also observed in the same plant methanolic extracts. Assuming that they may be the products of [4 + 2] condensation of two monomeric moieties, the formation of adducts by photochemical dimerization was checked by inducing the in vitro [4 + 2] cycloaddition of DAC. Moreover, the DAC and DACD accumulation rates in aerial parts of B. flabellata specimens were analyzed monthly during a complete phenological cycle. The accumulation of monomer depends on the plant phonological stage; meanwhile the dimer proportion arises in detriment of the monomer as the solar UV radiation increases. Since plants exposed to strong UV intensities produce radical species, the scavenger properties of these compounds toward reactive nitrogen species (RNS), and reactive oxygen species (ROS), were analyzed. Albeit DAC and DACD show significant superoxide radical scavenger activities, the monomer proved to be more effective than the dimer toward ROS, while DACD was an excellent RNS scavenger.

Gardenin B-induced cell death in human leukemia cells involves multiple caspases but is independent of the generation of reactive oxygen species.

Flavonoids have attracted great interest due to their possible anticancer activities. Here we investigated the antiproliferative activity of the flavonoids isolated from Baccharis scandens against human leukemia cell lines and found that the methoxyflavonoid gardenin B was the most cytotoxic compound against HL-60 and U-937 cells, showing IC50 values between 1.6 and 3.0 µM, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. These effects on viability were accompanied by the concentration- and time-dependent appearance of apoptosis as evidenced by DNA fragmentation, formation of apoptotic bodies and a sub-G1 ratio increase. Comparative studies with the best-studied bioflavonoid quercetin indicate that gardenin B is a more cytotoxic and more apoptotic inducer than quercetin. Cell death induced by gardenin B was associated with: (i) a significant induction of caspase-2, -3, -8 and -9 activities; (ii) cleavage of the initiator caspases (caspase-2, -8 and -9), of the executioner caspase-3, and of poly(ADP-ribose) polymerase; and (iii) a concentration-dependent reactive oxygen species generation. In conclusion, apoptosis induced by gardenin B is associated with activation of both the extrinsic and the intrinsic apoptotic pathways of cell death and occurs through a mechanism that is independent of the generation of reactive oxygen species.

A new iridoid, verbascoside and derivatives with inhibitory activity against Taq DNA polymerase.

DNA polymerases are enzymes that play a crucial role in DNA metabolism such as replication, repair, transcription, recombination, and chromosome segregation during mitosis. Herein we report the isolation of a new iridoid (6-epi-catalpol, 2) and per-O-acetyl-verbascoside (11) from aerial part of Buddleja cordobensis Grisebach (Buddlejaceae). From compound 2, we have obtained eight compounds by chemical transformation. This group of compounds at a concentration of 500µM was assayed against Taq DNA polymerase. Compound 11 (per-O-acetyl-verbascoside) was the most active with an IC50 of 1.21±0.18µM; compounds 9, 2 and 8 were strong inhibitors with IC50 values of 5.57±0.70, 21.62±0.22 and 78.13±0.93µM, respectively. Compounds 11 and 9 could be a leader structures to development new anticancer chemotherapy medicines and a useful tool to investigate DNA polymerase activity.

Mass spectrometry studies of lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

RATIONALE: Sauroxine and N-demethylsauroxine are lycodine-type Lycopodium alkaloids. In recent years, Lycopodium alkaloids have gained significant interest due to their unique skeletal characteristics as well as due to their acetylcholinesterase activity. It is known that drugs that inhibit acetylcholinesterase can be used to treat the early stages of Alzheimer's disease. METHODS: Sauroxine and N-demethylsauroxine were isolated from the aerial parts of Huperzia saururus (Lam.) Trevis. Electron ionization mass spectrometry (EI-MS) (low resolution) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation was conducted using an ion trap, GCQ Plus mass spectrometer with MS/MS. Electron ionization high-resolution mass spectrometry (EI-HRMS) was performed in a magnetic sector mass spectrometer (Micromass VG). RESULTS: Using GC/EI-CID-MS/MS we obtained different fragmentation routes that connect all the ionic populations. In addition, the use of EI-HRMS allowed us to measure the exact masses of all the fragment ions, and, with all this information gathered, we tried to establish a fragmentation scheme concordant with the ascendant and descendant species. CONCLUSIONS: The mass spectrometry studies presented in this work complete our mass studies of Lycopodium alkaloids. The mass spectrometry work presented has been very useful to confirm the structures as well as to support the biogenetic relationships between the lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

Chemistry and biological activity of coumarins at molecular level.

Synthetic coumarins were prepared in high yields using ionic liquids as an environmental friendly alternative. 3,4-Dimethyl-7-hydroxycoumarin (3ab) and 3-isopropyl-4-methyl-5,7-dihydroxycoumarin (3bc) showed interesting activity against Taq DNA polymerase with IC50 values of 115.7 microM and 82.2 microM, respectively. Also, 4-methyl-7-hydroxycoumarin (3aa) and 4-methyl-5,7-dihydroxycoumarin (3ba) exhibited inhibitory activity against MMLV-RT with IC50 values of 23.5 microM and 18.3 microM, respectively. These inhibitors could have importance as antiretroviral chemotherapeutic agents and also for the development of antitumor drugs.

A new semisynthetic derivative of sauroine induces LTP in hippocampal slices and improves learning performance in the Morris Water Maze.

Two semisynthetic acetyl derivatives of the alkaloid sauroine from Huperzia saururus, monoacetyl sauroine, and diacetyl sauroine (DAS) were obtained and their chemical structures were analyzed by NMR. While monoacetyl sauroine is the typical product of acetylation, DAS is an unexpected derivative related to the keto-enol formation of sauroine. Recordings of field excitatory post-synaptic potentials from the CA1 region of rat hippocampal slices showed that only DAS acutely applied induced chemical long-term potentiation (LTP) in a dose-dependent manner with an EC50 of 1.15 ± 0.09 µM. This effect was blocked by 10 µM D(-)-2-amino-5-phosphonopentanoic acid (AP5), suggesting dependence on the NMDA receptor. DAS significantly increased NMDA receptor-dependent excitatory post-synaptic currents without affecting α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-dependent currents. Repetitive administration of DAS improved visuo-spatial learning in the Morris Water Maze. In slices from rats tested in the Morris Water Maze, LTP resulting from electrical synaptic stimulation was 2.5 times larger than in controls. Concentration of DAS measured in the brain after repetitive administration was 29.5 µM. We conclude that slices perfused with DAS display a robust NMDA receptor-dependent chemical LTP. During chronic treatment, DAS enhances learning abilities through a metaplastic mechanism as revealed by the augmentation of LTP in slices. DAS, therefore, may be a promising compound as a nootropic therapeutic drug. A semisynthetic derivative of sauroine, diacetyl sauroine (DAS), induces chemical long-term potentiation in rat hippocampal slices increasing the NMDA receptor-dependent current. 2 mg/kg prior to each session in a Morris Water Maze (MWM) improves behavior performance. In slices prepared from the tested rats the electrical stimulation-dependent long-term potentiation (LTP) was greatly enhanced. Therefore, DAS may have potency as a nootropic drug against the memory decline.

Inhibition of reverse transcriptase and Taq DNA polymerase by compounds possessing the coumarin framework.

Coumarin derivatives were prepared using natural products isolated from plants belonging in the Pterocaulon genus (Asteraceae) and commercial drugs. Some molecules have displayed interesting activity against myeloid murine leukemia virus-reverse transcriptase (MMLV-RT) (compounds 20 and 28 produced inhibition with IC50 values of 38.62 and 50.98 µM, respectively) and Taq DNA polymerase (analogues 13 and 14 produced inhibition with IC50 values of 48.08 and 57.88 µM, respectively). Such inhibitors may have importance as antiretroviral chemotherapeutic agents and also in the development of anticancer drugs.

Derivatives of grindelic acid: from a non-active natural diterpene to synthetic antitumor derivatives.

Using several reactions that include homologations and asymmetric epoxidations as well as Ugi and Huisgen couplings, we generated a small focused library of new derivatives from the labdane-type diterpene grindelic acid. These compounds were evaluated as cytotoxic agents against a panel of five human solid tumor cell lines (HBL-100, HeLa, SW1573, T-47D, and WiDr). The presence of the diamide functionalizations enhanced the cytotoxic effect. N-Benzyl-N-(1-(benzylamino)-2-methyl-1-oxopropan-2-yl)grindelicamide, proved to be the most active product in all cell lines tested, with values of 0.95 (±0.38) µM against HBL-100 cells.

Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana.

Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host's cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc), dehydroleucodine (DhL), psilostachyin (Psi), and, at lesser extent, psilostachyin C (Psi C) is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.

Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae).

INTRODUCTION: The aim of the present study was to analyze the larvicidal activity of different crude extracts of Larrea cuneifolia and its most abundant lignan, nordihydroguaiaretic acid (NDGA), against Culex quinquefasciatus. METHODS: Chloroform, methanol, and aqueous extracts from L. cuneifolia and NDGA were tested against larvae of Cx. quinquefasciatus under laboratory conditions. RESULTS: The chloroform extract showed the highest larvicidal effect, with an estimated LC50 of 0.062 mg/ml. NDGA also demonstrated significant larvicidal activity with an estimated LC50 of 0.092 mg/ml. CONCLUSIONS: These results indicate that the chloroform extract of L. cuneifolia and NDGA are promising insecticides of botanical origin that could be useful for controlling Cx. quinquefasciatus.

Larvicidal activity of crude extracts from Larrea cuneifolia (Zygophyllaceae) and of its metabolite nordihydroguaiaretic acid against the vector Culex quinquefasciatus (Diptera: Culicidae)

INTRODUCTION: The aim of the present study was to analyze the larvicidal activity of different crude extracts of Larrea cuneifolia and its most abundant lignan, nordihydroguaiaretic acid (NDGA), against Culex quinquefasciatus. METHODS: Chloroform, methanol, and aqueous extracts from L. cuneifolia and NDGA were tested against larvae of Cx. quinquefasciatus under laboratory conditions. RESULTS: The chloroform extract showed the highest larvicidal effect, with an estimated LC50 of 0.062 mg/ml. NDGA also demonstrated significant larvicidal activity with an estimated LC50 of 0.092 mg/ml. CONCLUSIONS: These results indicate that the chloroform extract of L. cuneifolia and NDGA are promising insecticides of botanical origin that could be useful for controlling Cx. quinquefasciatus.

Cytotoxic bioactivity of some phenylpropanoic acid derivatives.

In this study, we synthesized a series of phenylpropanoic acid derivatives based on modifications at four selected points of the molecular scaffold. The in vitro antiproliferative activities of the compounds were examined in representative human solid tumor cell lines. A SAR was established pointing out the relevance of the substituents. The best activity profiles were obtained for the derivatives bearing more lipophilic esters (GI50 3.1-21 microM).

Sesquiterpene lactones and the diterpene 5-epi-icetexone affect the intracellular and extracellular stages of Trypanosoma cruzi.

Chagas disease is a major health problem in Latin America and is caused by the parasitic protozoan Trypanosoma cruzi. Although many drugs have been used to alleviate the disease, these have been ineffective in the chronic phase and have also presented numerous side effects on patients. In this study we tested the effect of three sesquiterpene lactones (dehydroleucodine, helenalin and mexicanin) and a diterpene (5-epi-icetexone) on parasites (Y-strain) grown in host cells. At 48 h of treatment, the number of amastigotes inside the cells was lower than in the controls. This effect was observable at concentrations of 1.5-3.8 µM, which are of low cytotoxicity to host cells. In addition, the compounds caused a decrease in the percentage of infected cells. The treatments also reduced the presence of trypomastigotes in the extracellular medium. In all cases, helenalin was the most potent. The number of parasites per cell at 24h indicates the occurrence of multiple infection, which would also be affected by the compounds. However, we should not discard an effect on the proliferation and survival of parasites within the host cells. On the other hand, an additional effect on the differentiation of parasites and/or the survival of extracellular trypomastigotes might be possible. We conclude that these compounds are very effective against T. cruzi possibly by multiple mechanisms.

Activation of human leukemic mast cell line LAD2 is modulated by dehydroleucodine and xanthatin.

The aim of the present study was to determine whether dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit the activation of human leukemic LAD2 mast cells induced by compound 48/80 or the calcium ionophore A23187. LAD2 cells were preincubated in the presence of test drugs and then challenged with the secretagogues. This study provides the first evidence in favor of the view that dehydroleucodine and xanthatin inhibit the degranulation of LAD2 cells, thus acting as human mast cell stabilizers. These molecules could be effective in the treatment of human diseases associated with inappropriate mast cell activation.

Antiviral effect and mode of action of methanolic extract of Verbascum thapsus L. on pseudorabies virus (strain RC/79).

The methanolic extract of Verbascum thapsus was evaluated for its antiviral activity against the pseudorabies virus strain RC/79 (PrV), and also for its cytotoxic activity on Vero cells. The extract showed CC50 values of 1100 µg mL⁻¹ and 1426 µg mL⁻¹ by NRU and MTT assays, respectively. The 50% inhibitory concentration of the extract for PrV plaque formation was determined at 35 µg mL⁻¹, and selectivity indices were 31.4 (NRU) and 40.7 (MTT). When cells were pre-treated with the extract prior to virus infection, the inhibition in plaque formation was 70%. PrV was highly inhibited when it was incubated with plant extract or when the extract was added during the adsorption phase (99%). However, no inhibitory effect was observed when the extract was added to the cells after the adsorption period. Thus, these results suggest that the methanolic extract of Verbascum thapsus may contain bioactive compound(s) that affect PrV mostly in the adsorption phase.

Evaluation of antiviral activity of aqueous extracts from Achyrocline satureioides against Western equine encephalitis virus.

Achyrocline satureioides (Asteraceae) is a medicinal plant traditionally used in Argentina for the treatment of intestinal infections and various digestive disorders. Its infusion is widely utilised for respiratory problems and viral infections. The objective of this study was to investigate cytotoxicity, virucidal and antiviral properties of the cold aqueous extract (CAE) and hot aqueous extract (HAE) of this plant against Western equine encephalitis virus (WEEV). Cytotoxicity in Vero cells was evaluated by maximum non-cytotoxic concentration (MNCC), neutral red (NR) uptake and MTT reduction methods. To study the antiviral activity of aqueous extracts, plaque reduction assay was performed after pre-treatment of host cells, adsorption, penetration and post-penetration of the virus. Extracellular virus inactivation was also analysed by the same method. Extracts showed strong inhibitory activity after virus penetration with selective index values of 32 (NR) and 63.3 (MTT) for the CAE, and 16.2 (NR) and 24.3 (MTT) for the HAE. Both extracts exhibited virucidal action with lower efficacy than their antiviral properties. The present results demonstrate that aqueous extracts of A. satureioides are active against WEEV. Further studies are needed in order to identify which compounds could be responsible for this effect, and how they exert antiviral action.

Antibacterial Efficacy of Dihydroxylated Chalcones in Binary and Ternary Combinations with Nalidixic Acid and Nalidix Acid-Rutin Against Escherichia coli ATCC 25 922.

In order to determine the existence of synergism, the bacteriostatic action of flavonoids against Escherichia coli ATCC 25 922 between dihydroxylated chalcones and a clinically interesting conventional antibiotic, binary combinations of 2',3-dihydroxychalcone, 2',4-dihydroxychalcone and 2',4'-dihydroxychalcone with nalidixic acid and its ternary combinations with rutin (inactive flavonoid) were assayed against this Gram negative bacterium. Using a kinetic-turbidimetric method, growth kinetics were monitored in broths containing variable amounts of dihydroxychalcone alone, combinations of dihydroxychalcone variable concentration-nalidixic acid constant concentration and dihydroxychalcone variable concentration-nalidixic acid constant concentration-rutin constant concentration, respectively. The minimum inhibitory concentrations of dihydroxychalcones alone and its binary and ternary combinations were evaluated. All chalcones, and their binary and ternary combinations showed antibacterial activity, being rutin an excellent synergizing for the dihydroxychalcone-nalidixic acid binary combination against E. coli ATCC 25 922. Thus, this synergistic effect is an important way that could lead to the development of new combination antibiotics against infections caused by E. coli.

The effect of the diterpene 5-epi-icetexone on the cell cycle of Trypanosoma cruzi.

Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide. We observed that the diterpene does not affect the growth of the parasites when added within 10 h after removal of hydroxyurea, but the compound was effective on growth when added to the cultures after 12 h. Thymidine incorporation was somewhat inhibited when the diterpene was added at 12 h after removal of hydroxyurea, possibly on the transition S/G2. Using transmission electron microscopy we observed that the diterpene induced a delay in the progression of cell division. We conclude that the compound, at cytostatic dose, affects the cell cycle of T. cruzi, possibly in the transition S/G2 phase and cell division. Further studies will focus to identify the molecular targets for the action of 5-epi-icetexone.

The effect of dehydroleucodine in adipocyte differentiation.

Dehydroleucodine (DhL) is a sesquiterpene lactone of the guaianolide group with gastric cytoprotective activity. Recent studies have also demonstrated that DhL inhibits the proliferation of vascular smooth muscle cells. In this study we examined the effect of DhL in the differentiation of 3T3-L1 preadipocytes. The addition of DhL significantly inhibited the differentiation of 3T3-L1 preadipocytes along with a significant decrease in the accumulation of lipid content by a dramatic downregulation of the expression of adipogenic-specific transcriptional factors PPARγ and C-EBPα. However, phosphorylation of AMPKα, Erk1/2 and Akt1 was not inhibited by DhL treatment. Interestingly, we also found that 11,13-dihydrodehydroleucodine, a derivative of DhL with inactivated α-methylene-γ-lactone function, also inhibited the differentiation of 3T3-L1 preadipocytes. Taken together, these data suggest that DhL has an important inhibitory effect in cellular pathways regulating adipocyte differentiation by modulating the PPARγ expression, which is known to play a pivotal role during adipogenesis.