Antiprotozoal compounds from Mikania periplocifolia (Asteraceae).

Chagas disease, African trypanosomiasis and Leishmaniasis are neglected parasitic diseases which affect millions of people worldwide. In a previous work, we report the antiprotozoal activity of the dichloromethane extract of Mikania periplocifolia Hook. & Arn. (Asteraceae). The aim of this work was to isolate and identify the bioactive compounds present in the extract. The fractionation of the dichloromethane extract has led to the isolation of the sesquiterpene lactone miscandenin and the flavonoid onopordin, together with the sesquiterpene lactones mikanolide, dihydromikanolide and deoxymikanolide, which have previously shown antiprotozoal activity. Miscandenin and onopordin were assayed in vitro against Trypanosoma cruzi, T. brucei and Leishmania braziliensis. Miscandenin was active against T. cruzi trypomastigotes and amastigotes with IC50 values of 9.1 and 7.7 µg/ml, respectively. This sesquiterpene lactone and the flavonoid onopordin showed activity against T. brucei trypomastigotes (IC50 = 0.16 and 0.37 µg/ml) and L. braziliensis promastigotes (IC50 = 0.6 and 1.2 µg/ml), respectively. The CC50 values on mammalian cells were 37.9 and 53.4 µg/ml for miscandenin and onopordin, respectively. Besides, the pharmacokinetic and physicochemical properties of miscandenin were assessed in silico, showing a good drug-likeness profile. Our results highlight this compound as a promising candidate for further preclinical studies in the search of new drugs for the treatment of trypanosomiasis and leishmaniasis.

Germacrane Sesquiterpene Dilactones from Mikania micrantha and Their Antibacterial and Cytotoxic Activity.

Four new germacrane sesquiterpene dilactones, 2ß-hydroxyl-11ß,13-dihydrodeoxymikanolide (1), 3ß-hydroxyl-11ß,13-dihydrodeoxymikanolide (2), 1α,3ß-dihydroxy-4,9-germacradiene-12,8:15,6-diolide (3), and (11ß,13-dihydrodeoxymikanolide-13-yl)-adenine (4), together with five known ones (5-9) were isolated from the aerial parts of Mikania micrantha. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compound 4 is featured with an adenine moiety in the molecule, which is the first nitrogen-containing sesquiterpenoid so far isolated from this plant species. These compounds were evaluated for their in vitro antibacterial activity against four Gram-(+) bacteria of Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC) and Curtobacterium. flaccumfaciens (CF), and three Gram-(-) bacteria of Escherichia coli (EC), Salmonella. typhimurium (SA), and Pseudomonas Solanacearum (PS). Compounds 4 and 7-9 were found to show strong in vitro antibacterial activity toward all the tested bacteria with the MIC values ranging from 1.56 to 12.5 µg/mL. Notably, compounds 4 and 9 showed significant antibacterial activity against the drug-resistant bacterium of MRSA with MIC value 6.25 µg/mL, which was close to reference compound vancomycin (MIC 3.125 µg/mL). Compounds 4 and 7-9 were further revealed to show in vitro cytotoxic activity toward human tumor A549, HepG2, MCF-7, and HeLa cell lines, with IC50 values ranging from 8.97 to 27.39 µM. No antibacterial and cytotoxic activity were displayed for the other compounds. The present research provided new data to support that M. micrantha is rich in structurally diverse bioactive compounds worthy of further development for pharmaceutical applications and for crop protection in agricultural fields.

Ent-kaurenoic acid-enriched Mikania glomerata leaves-complexed ß-cyclodextrin: Pharmaceutical development and in vivo antitumor activity in a sarcoma 180 mouse model.

The extract obtained from Mikania glomerata leaves rich in ent-kaurenoic acid (ERKA) shows cytotoxic activity in vitro, but its hydrophobic nature and thermosensitivity are issues to be solved prior to in vivo antitumor studies. The purpose of this study was to investigate the antitumor activity of inclusion complexes formed between ERKA and ß-cyclodextrin (ERKA:ß-CD) in rodents. ERKA:ß-CD complexes obtained by malaxation (MX) and co-evaporation (CE) methods were firstly characterized regarding their physical properties, encapsulation efficiency, and cytotoxicity againts L929 cells. The antitumor activity study was then performed in mice with sarcoma 180 treated with saline, 5-fluouracil (5FU) and ERKA:ß-CD at 30, 100 and 300 µg/kg. The weight, volume, percentage of inhibition growth, gross and pathological features and positivity for TUNEL, ki67, NFκB and NRF2 in the tumors were assessed. Serum lactate-dehydrogenase activity (LDH), white blood cells count (WBC) and both gross and pathological features of the liver, kidneys and spleen were also evaluated. The formation of the inclusion complexes was confirmed by thermal analysis and FTIR, and they were non-toxic for L929 cells. The MX provided a better complexation efficiency. ERKA:ß-CD300 promoted significant tumor growth inhibition, and attenuated the tumor mitotic activity and necrosis content, comparable to 5-fluorouracil. ERKA:ß-CD300 also increased TUNEL-detected cell death, reduced Ki67 and NF-kB immunoexpression, and partially inhibited the serum LDH activity. No side effect was observed in ERKA:ß-CD300-treated animals. The ERKA:ß-CD inclusion complexes at 300 µg/kg displays antitumour activity in mice with low systemic toxicity, likely due to inhibition on the NF-kB signaling pathway and LDH activity.

Mikania micrantha Extract Inhibits HMG-CoA Reductase and ACAT2 and Ameliorates Hypercholesterolemia and Lipid Peroxidation in High Cholesterol-Fed Rats.

The present study aimed to determine the effect of an ethyl acetate extract of Mikania micrantha stems (EAMMS) in hypercholesterolemia-induced rats. Rats were divided into a normal group (NC) and hypercholesterolemia induced groups: hypercholesterolemia control group (PC), simvastatin group (SV) (10 mg/kg) and EAMMS extract groups at different dosages of 50, 100 and 200 mg/kg, respectively. Blood serum and tissues were collected for haematological, biochemical, histopathological, and enzyme analysis. Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, malondialdehyde (MDA) level, as well as enzymes of HMG-CoA reductase (HMGCR) and acetyl-CoA acetyltransferase 2 (ACAT2), were measured. Feeding rats with high cholesterol diet for eight weeks resulted in a significantly (p < 0.05) increased of TC, TG, LDL-C, AST, ALT and MDA levels. Meanwhile, the administration of EAMMS extract (50, 100 and 200 mg/kg) and simvastatin (10 mg/kg) significantly reduced (p < 0.05) the levels of TC, TG, LDL-C and MDA compared to rats in the PC group. Furthermore, all EAMMS and SV-treated groups showed a higher HDL-C level compared to both NC and PC groups. No significant difference was found in the level of ALT, AST, urea and creatinine between the different dosages in EAMMS extracts. Treatment with EAMMS also exhibited the highest inhibition activity of enzyme HMGCR and ACAT2 as compared to the control group. From the histopathological examination, liver tissues in the PC group showed severe steatosis than those fed with EAMMS and normal diet. Treatment with EAMMS extract ameliorated and reduced the pathological changes in the liver. No morphological changes showed in the kidney structure of both control and treated groups. In conclusion, these findings demonstrated that EAMMS extract has anti-hypercholesterolemia properties and could be used as an alternative treatment for this disorder.

Comparison of allelopathic effects of two typical invasive plants: Mikania micrantha and Ipomoea cairica in Hainan island.

Mikania micrantha and Ipomoea cairica are two invasive plants widely distribute and seriously damage in Hainan island. In this study, the leaves extracts of two weeds were collected and determined for their allelopathic potentials on Chrysanthemum coronarium. The phytotoxicity bioassay showed that when the extract concentration was 50 and 100 mg/ml, the inhibited effects of M. micrantha on growth of C. coronarium were greater than by I. cairica. However, when the extract concertation at 400 mg/ml, the opposite inhibited effects were observed. We speculated this phenomenon was caused by different allelopathic compounds. Therefore, using gas chromatography-mass spectrometry, 19 and 23 compounds were identified respectively, benzoic acid and cinnamic acid were the main components in the two leaves extracts, which were selected to carry out the further bioassays. Subsequent bioassay results showed the effects of two allelochemicals on morphological index and chlorophyll content and POD activity were all negative to C. coronarium, whereas the content of MDA and activity of SOD, CAT represented adverse changes. Moreover, the inhibitions by cinnamic acid were generally greater than those by benzoic acid. Thus, the phenolic acids played the most crucial roles in the allelopathic effccts of M. micrantha and I. cairica leaves extracts.

New cadinane sesquiterpenoids from Mikania micrantha.

Chemical investigation of the aerial parts of Mikania micrantha led to the isolation of eight sesquiterpenoids and ten diterpenoids, including five cadinane sesquiterpenoids (1-5), three bisabolene sesquiterpenoids (6 - 8), nine ent-kaurane diterpenoids (9-17), and an abietane diterpenoid (18). Among them, 1 - 3 are new and feature a rare lactone or furan ring derived from C-6 isopropyl group side chain. Compound 18 was isolated from genus Mikania for the first time, and was also the first example of abietane-type diterpenoids from this plant. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HRESIMS, and ECD). All compounds were examined for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, and compound 18 exhibited pronounced inhibition on NO production (IC50 = 11.04 µM), being comparable to the positive control, quercetin (IC50 = 11.15 µM).

GC-MS-FID characterization and antibacterial activity of the Mikania cordifolia essential oil and limonene against MDR strains.

The present study evaluated the effect of the essential oil of Mikania cordifolia (EOMc) and its major constituent limonene alone or associated with antibacterial drugs against Multidrug Resistant Bacteria (MDR). To evaluate the antibacterial activity, the minimum inhibitory concentrations (MIC) of the oil and limonene against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were determined. The antibiotic-modulating activity was assessed using subinhibitory concentrations (MIC/8) of these substances in combination with conventional antibacterial drugs. Although no relevant antibacterial activity of the natural products was detected, both substances modulated the action of antibiotics against resistant bacteria. The EOMc demonstrated the best modulating effect against P. aeruginosa, presenting synergistic effects when associated with gentamicin and norfloxacin. In addition, the oil reduced the MIC of norfloxacin against E. coli as well as reduced the MIC of gentamicin against S. aureus. On the other hand, the best effect of limonene was obtained against S. aureus. Thus, it is concluded that the essential oil Mikania cordifolia and the isolated compound limonene do not have clinically significant antibacterial effect, but modulate the action of antibiotics against MDR bacteria.

In Vitro Anticoagulant Activity of Mikania laevigata: Deepening the Study of the Possible Interaction Between Guaco and Anticoagulants.

Mikania laevigata, popularly known in Brazil as guaco, is widely used for respiratory disorders. As this plant is rich in coumarins, there is evidence of indications that it may cause bleeding and therefore should not be used concomitantly with anticoagulants. The basis of this information is very theoretical, with no clinical evidence of such contraindication. Thus, the aim of this study was to evaluate the in vitro effect of M. laevigata extract on blood coagulation through prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests, fibrinogen plasma concentration, and the new thrombin generation test, which investigate, with high sensibility, hemostatic changes (CAAE 60904316.6.0000.5149), besides evaluating its qualitative micromolecular composition, providing scientific evidence to support the management of patients taking warfarin. Ethanolic extracts of guaco leaves were incubated with a plasma pool of healthy individuals at concentrations of 1.67, 2.26, and 2.86 mg/mL. The presence of flavonoids, tannins, coumarins, and triterpenes was demonstrated by selective reagents in thin layer chromatography. Benzoylgrandifloric acid, cinnamoylgrandifloric acid, o-coumaric acid, coumarin, and quercetin-3-ß-glucoside were identified by coinjection in ultraperformance liquid chromatography. The extract at all concentrations prolonged TP and aPPT and reduced the potential for endogenous thrombin potential by the thrombin generation test. The control plasma had endogenous thrombin potential = 1465 nM/min, and after the addition of M. laevigata extract (2.26 mg/mL), this value was reduced to 1087 nM/min, indicating a lower generation of thrombin. Related to fibrinogen plasma concentration, concentrations of 2.26 and 2.86 mg/mL were effective in reducing plasma fibrinogen levels. These results allow us to conclude that the guaco extract demonstrated an anticoagulant effect in vitro, possibly interfering with intrinsic, extrinsic, and common coagulation pathways. A discussion on the contribution of the identified substances to the activity is also present.

Evaluation of genotoxicity and coumarin production in conventional and organic cultivation systems of Mikania glomerata Spreng.

Mikania glomerata Sprengel, popularly known as "guaco," is used in Brazilian folk medicine for several inflammatory and allergic conditions. Besides, the popular use "guaco" is indicated by the Brazilian Ministry of Health as a safe and effective herbal medicine. The biological activity of M. glomerata extracts is due to the presence of the coumarins, a large family of phenolic substances found in plants and is made of fused benzene and α-pyrone rings. Considering that there are few data on the biological effects of the extracts of M. glomerata, mainly in genetic level, this work aims to evaluate, in vitro, the genotoxicity and coumarin production in M. glomerata in conventional and organic growing. The data showed that the organic culture system showed double the concentration of coumarin being significantly more productive than the conventional system. Besides, the results of comet assay suggest that extracts of M. glomerata cultivated in a conventional system was genotoxic, increased DNA damage levels while the organic extracts seem to have antigenotoxic effect possibly due to the concentration of coumarins. Additional biochemical investigations are necessary to elucidate the mechanisms of action of M. glomerata extracts, which were found to have a role in protection against DNA damage.

Influence of environmental factors on the volatile composition of two Brazilian medicinal plants: Mikania laevigata and Mikania glomerata.

INTRODUCTION: Mikania laevigata Sch.Bip. ex Baker and Mikania glomerata Spreng. are medicinal plants popularly known as guaco, used for inflammatory diseases of the respiratory system, included in pharmaceutical formularies and often used without distinction. However, several studies show that the chemical composition varies between these species, as well as in plants are subjected to different environmental stresses. Few studies have been carried out with the volatile compounds of guaco, even less about the changes in volatile composition due to abiotic variation. OBJECTIVE: The aim of this work was to evaluate how volatile compounds vary according to the seasons and at different times of the day and if these compounds are influenced by the variations in the growth conditions such as: temperature, luminosity and water. METHODS: The headspace volatiles of the leaves were sampled by solid phase micro extraction and analyzed by gas chromatography-mass spectrometry. Untargeted metabolomic analysis of the resulting chromatograms and chemometrics was applied. The chemical profile of the volatiles of M. laevigata and M. glomerata were different; being clearly separated in the exploratory grouping analyzes (PCA), followed by analysis of variance of the marker compounds of both species. RESULTS: Only M. laevigata contained coumarin, considered to be the chemical marker of both species and to be responsible for the therapeutic activities. There was no significant difference between the morning and afternoon collections of either species. Coumarin, α-pinene and bicyclogermacrene were more intense in the volatiles of M. laevigata throughout the year and responsible for grouping the samples of this species. For M. glomerata, hexanal and 2-hexenal were responsible for grouping the samples and were more intense in all months. The growth conditions tested affected the intensity of specific compounds in the chromatograms. Some compounds were less intense with the increase of the temperature and in the plants subjected to full sunlight. However, certain volatile compounds-such as pinenes-were more intense in plants suffering drought. CONCLUSION: The variation in composition between species of guaco was greater than those observed in the seasonal and cultivation studies, indicating that these species cannot be used indistinctly.

Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi.

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Seasonal changes and solvent effects on fractionated functional food component yields from Mikania laevigata leaves.

Statistical mixture design extraction and fractionation of Mikania plant samples were carried out to quantitatively study seasonal and solvent composition effects as well as their interactions on secondary metabolites. The mixture design consisted of ethanol, acetone, dichloromethane and chloroform solvents and their binary, ternary and quaternary mixtures. Yields were measured for the crude extract and its neutral, organic, basic, polar and fiber fractions obtained with each solvent composition from samples harvested during 2010. Two-way ANOVA found statistically significant seasonal and solvent effects for the crude extract and all the fractions except for the polar fraction solvent. The best solvent extractor depends the harvest season. High crude, organic and polar fraction yields in the summer are correlated with the coumarin UV-vis absorbance at 274 nm. Crude yields of mixtures containing ethanol are correlated with the coumarin absorbances in summer, winter and spring whereas mixtures without ethanol show no significant correlation.

Pharmacological study of anti-inflammatory activity of aqueous extracts of Mikania glomerata (Spreng.) and Mikania laevigata (Sch. Bip. ex Baker).

ETHNOPHARMACOLOGICAL RELEVANCE: Mikania glomerata Spreng. (MG) and Mikania laevigata Sch. Bip. ex Baker (ML), popularly known as guaco, are medicinal plants similar in morphology, chemical composition and medicinal uses. Both species are often used and sold without distinction; however, it is believed that their chemical composition is different. AIM: Thus, the aim of this study is to investigate if the aqueous extract of MG and ML present similar anti-inflammatory activity to the point of being used interchangeably. MATERIAL AND METHODS: Different doses of both extracts and coumarin were given to rats in different experimental models to assess the anti-inflammatory activity between these two species. For this, the animals were submitted to paw edema, pleurisy and degranulation of peritoneal mast cell and the extracts were also characterized by Ultra High Efficiency Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). RESULTS: The chromatographic method showed that ML presents ten times more coumarin than MG. Oral administration of MG, ML and coumarin inhibited paw edema induced by carrageenan (400 mg/kg, 55% inhibition; 400 mg/kg, 57% inhibition; 75 mg/kg, 38% inhibition; p < 0.05, respectively). MG, ML and coumarin treatment also inhibited the edema induced by compound 48/80 (400 mg/kg, 56% inhibition; 400 mg/kg, 69% inhibition; 75 mg/kg, 40% inhibition; p < 0.05, respectively). MG, ML and coumarin did not prevent mast cell degranulation and the consequent histamine release in Wistar rat peritoneal mast cells induced by compound 48/80. MG did not inhibit cell infiltration in pleurisy nor the highest dose tested, while ML decreased the leukocyte migration (200 and 400 mg/kg, 23% and 30% inhibition; p < 0.001, respectively) and, to a lesser extent, coumarin also reduced cell infiltration (10, 50 and 75 mg/kg; 15%, 16% and 17% inhibition; p < 0.001, respectively). CONCLUSION: The variation of the results of the anti-inflammatory activity found in M. glomerata and M. laevigata demonstrates that these two species should not be used interchangeably. Coumarin, as already proven, has anti-inflammatory action however, we have suggested that it probably is not the only component responsible for this therapeutic effect in the extracts.

Kaurenoic acid and its sodium salt derivative: antibacterial activity against Porphyromonas gingivalis and their mechanism of action.

AIM: To evaluate the antibacterial activity of 12 kaurane-type diterpenes against a panel of bacteria that cause endodontic infection. METHODS & MATERIALS: We conducted tests against bacteria in the planktonic or in the sessile mode, cytotoxic assays for the most promising compounds against human normal lung fibroblast cells, and Porphyromonas gingivalis (ATCC 33277) proteomic analysis. RESULTS & CONCLUSION: Kaurenoic acid and its salt exhibited satisfactory antibacterial action against the evaluated bacteria. Proteomic analysis suggested that these compounds might interfere in bacterial metabolism and virulence factor expression. Kaurane-type diterpenes are an important class of natural products and should be considered in the search for new irrigating solutions to treat endodontic infections.

Comparison of the Morphology, Anatomy, and Chemical Profile of Mikania glomerata and Mikania laevigata.

Mikania glomerata and Mikania laevigata are medicinal plants popularly used in Brazil for colds and asthma. Although several studies report differences in their composition, they are frequently used indistinctly, which may be due to misidentification of the species or the plant part used. Herein, the chemical, anatomical, and morphological profiles of both species were evaluated, seeking to differentiate them. Due to the pattern of secondary metabolites found in M. glomerata and M. laevigata, the chemical profile of the leaves was the characteristic that best differentiated the two species. Coumarin was present in all the leaf samples of M. laevigata but absent or in low concentration in leaf samples of M. glomerata. The stems presented coumarin in both species, albeit in different concentrations. The quantification of chlorogenic and dicaffeoylquinic acids was crucial for the separation of the two species, as higher concentrations are found in M. glomerata. The anatomic structure of the leaves, stem, and petioles of both species was conserved, even when exposed to different environments, but presented only subtle differences between species. The morphology of the leaves presented different characteristics for each species but altered when exposed to shade. Therefore, the chemical analysis together with the morphological characteristics of plants grown in full sunlight may be used for the correct identification of these species.

Decontamination of Mikania glomerata Leaves by Gamma Irradiation: Coumarin Determination by HPLC-DAD, Microbiological Control and Genotoxicological Studies.

Gamma irradiation as a decontaminating physical agent could be an important tool in the production chain of herbal medicines by improving the microbiological quality of raw materials and the safety of final products. This study was undertaken to investigate the genotoxic potential and eventual chemical modifications of a batch of Mikania glomerata raw material decontaminated by different doses of gamma irradiation (2.0, 3.5, and 5.0 kGy), using a cesium-137 source. DNA damage was assessed in vitro by agarose gel electrophoresis in regard to double-chain breaks of plasmid pUC 9.1 DNA and in vivo by micronucleus test in bone marrow cells of Wistar rats. Cytotoxicity in bone marrows was assessed by scoring polychromatic and normochromatic erythrocytes ratio. An HPLC-DAD method was adapted and validated for the enhancement of coumarin selectivity from the other matrix constituents. The microbial load was satisfactorily reduced, leading to sterilization at the highest dose. Genotoxic and cytotoxic effects were not increased in the in vitro and in vivo models. The concentration of coumarin and the chromatographic profiles of the hydroalcoholic plant extracts (ethanol 70% v/v) were not modified after such process. Therefore, this work suggests that gamma irradiation of M. glomerata raw material is suitable and safe for microbiological control purposes at the employed doses.

Assessment of sesquiterpene lactones isolated from Mikania plants species for their potential efficacy against Trypanosoma cruzi and Leishmania sp.

Four sesquiterpene lactones, mikanolide, deoxymikanolide, dihydromikanolide and scandenolide, were isolated by a bioassay-guided fractionation of Mikania variifolia and Mikania micrantha dichloromethane extracts. Mikanolide and deoxymikanolide were the major compounds in both extracts (2.2% and 0.4% for Mikania variifolia and 21.0% and 6.4% for Mikania micrantha respectively, calculated on extract dry weight). Mikanolide, deoxymikanolide and dihydromikanolide were active against Trypanosoma cruzi epimastigotes (50% inhibitory concentrations of 0.7, 0.08 and 2.5 µg/mL, for each compound respectively). These sesquiterpene lactones were also active against the bloodstream trypomastigotes (50% inhibitory concentrations for each compound were 2.1, 1.5 and 0.3 µg/mL, respectively) and against amastigotes (50% inhibitory concentrations for each compound were 4.5, 6.3 and 8.5 µg/mL, respectively). By contrast, scandenolide was not active on Trypanosoma cruzi. Besides, mikanolide and deoxymikanolide were also active on Leishmania braziliensis promastigotes (50% inhibitory concentrations of 5.1 and 11.5 µg/mL, respectively). The four sesquiterpene lactones were tested for their cytotoxicity on THP 1 cells. Deoxymikanolide presented the highest selectivity index for trypomastigotes (SI = 54) and amastigotes (SI = 12.5). In an in vivo model of Trypanosoma cruzi infection, deoxymikanolide was able to decrease the parasitemia and the weight loss associated to the acute phase of the parasite infection. More importantly, while 100% of control mice died by day 22 after receiving a lethal T. cruzi infection, 70% of deoxymikanolide-treated mice survived. We also observed that this compound increased TNF-α and IL-12 production by macrophages, which could contribute to control T. cruzi infection.

Phenolics from Mikania micrantha and Their Antioxidant Activity.

A phytochemical study on the aerial parts of Mikania micrantha led to the isolation of two new phenolic compounds, benzyl 5-O-ß-d-glucopyranosyl-2,5-dihydroxybenzoate (1) and (7S,8R)-threo-dihydroxydehydrodiconiferyl alcohol 9-acetate (2), together with twelve known compounds, benzyl 2-O-ß-d-glucopyranosyl-2,6-dihydroxybenzoate (3), 4-allyl-2,6-dimethoxyphenol glucoside (4), (+)-isolariciresinol (5), icariol A2 (6), 9,10-dihydroxythymol (7), 8,9,10-trihydroxythymol (8), caffeic acid (9), p-coumaric acid (10), ethyl protocatechuate (11), procatechuic aldehyde (12), 4-hydroxybenzoic acid (13), and hydroquinone (14). Their structures were elucidated on the basis of extensive spectroscopic analysis. Except 8 and 9, all the other compounds were isolated from this plant species for the first time. The antioxidant activity of those isolated compounds were evaluated using three different assays. Compounds 1, 2, 3, 9, 10, 13, and 14 demonstrated significant 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) free radical cation scavenging activity ranging from SC50 0.31 to 4.86 µM, which were more potent than l-ascorbic acid (SC50 = 10.48 µM). Compounds 5, 9, 11, and 12 exhibited more potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (SC50 = 16.24-21.67 µM) than l-ascorbic acid (39.48 µM). Moreover, the ferric reducing antioxidant power (FRAP) of compounds 2, 5, 9, and 11 were discovered to be also comparable to or even more potent than l-ascorbic acid.

Mikania glomerata Sprengel extract and its major compound ent-kaurenoic acid display activity against bacteria present in endodontic infections.

The search for new, effective and safe antimicrobial compounds from plant sources has continued to play an important role in the maintenance of human health since ancient times. Such compounds can be used to help to eradicate microorganisms from the root canal system, preventing/healing periapical diseases. Mikania glomerata (Spreng.), commonly known as "guaco," is a native climbing plant from Brazil that displays a wide range of pharmacological properties. Many of its activities have been attributed to its phytochemical composition, which is mainly composed of diterpenes, such as ent-kaurenoic acid (KA). The present study evaluated the potential activity of an ent-kaurenoic-rich (KA) extract from Mikania glomerata (i.e. Mikania glomerata extract/MGE) and its major compound KA against bacteria that can cause endodontic infections. Time-kill assays were conducted and the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), anti-biofilm activity, and synergistic antimicrobial activity of MGE and KA were determined. The MGE exhibited MIC and MBC values, which ranged from 6.25 to 100 µg/mL and 12.5 to 200 µg/mL respectively. The MIC and MBC results obtained for the KA, ranged from 3.12 to 100 µg/mL and 3.12 to 200 µg/mL respectively. Time-kill and anti-biofilm activity assays conducted for KA at concentrations between 3.12 and 12.5 µg/mL exhibited bactericidal activity between 6 and 72 h of incubation and 50% inhibition of biofilm formation for Porphyromonas gingivalis (clinical isolate), Propionibacterium acnes (ATCC 6919), Prevotella nigrescens (ATCC 33563), P. melaninogenica (ATCC 25845), Aggregatibacter actinomycetemcomitans (ATCC 43717). For synergistic antimicrobial activity, KA combined with chlorhexidine dichlorohydrate (CHD) had an additive effect with increased efficacy against P. gingivalis (clinical isolate) compared to CHD alone. It was concluded that M. glomerata extract and its major compound ent-kaurenoic acid (KA) showed in vitro antibacterial activity, the latter being a potential biofilm inhibitory agent. They may play important roles in the search for novel sources of agents that can act against bacteria present in endodontic infections.

Magnetic nanoparticles and high-speed countercurrent chromatography coupled in-line and using the same solvent system for separation of quercetin-3-O-rutinoside, luteoloside and astragalin from a Mikania micrantha extract.

A new in-line method of magnetic nanoparticles (MNPs) coupled with high-speed countercurrent chromatography (HSCCC) using a same solvent system during the whole separation process was established to achieve the rapid separation of flavonoids from Mikania micrantha. The adsorption and desorption capacities of five different MNPs for flavonoid standards and Mikania micrantha crude extract were compared and the most suitable magnetic nanoparticle Fe3O4@SiO2@DIH@EMIMLpro was selected as the in-line MNP column. An in-line separation system was established by combining this MNP column with HSCCC through a six-way valve. The comparison between two solvent systems n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v) and ethyl acetate-methanol-water (25:1:25, v/v) showed that the latter solvent system was more suitable for simultaneously in-line separating three flavonoids quercetin-3-O-rutinoside, luteoloside and astragalin from Mikania micrantha. The purities of these three compounds with the ethyl acetate-methanol-water solvent system were 95.13%, 98.54% and 98.19% respectively. Results showed the established in-line separation system of MNP-HSCCC was efficient, recyclable and served to isolate potential flavonoids with similar polarities from natural complex mixtures. The in-line combination of magnetic nanoparticles with high-speed countercurrent chromatography eluting with the same solvent system during the whole separation process was established for the first time.