Aurelianolides from Aureliana fasciculata var. fasciculata Trigger Apoptosis With Caspase Activation in Human Leukemia Cells.

BACKGROUND/AIM: Aurelianolide A and B were identified and isolated from Aureliana fasciculata var. fasciculata leaves. Withanolides are naturally occurring C-28 steroidal lactone triterpenoids with cytotoxic and anticancer properties, among other relevant pharmacological activities. Herein we have described, for the first time, the cytotoxic effects of aurelianolides on human cancer cells. MATERIALS AND METHODS: Aurelianolide A and B were tested on human leukemia cell lines: THP-1, MOLT-4, Jurkat, K562 and K562-Lucena 1. RESULTS: For aurelianolide A, MOLT-4 had the lower IC50 (1.17 µM) and for aurelianolide B, Jurkat was the most susceptible cell line (IC50 2.25 µM). On the other hand, the multidrug resistant (MDR) cell line K562-Lucena 1 showed higher IC50 for both aurelianolides. Using 293T, a non-tumor embryonic kidney cell line, we observed an excellent selectivity index for both aurelianolides, from 2.24 (aurelianolide B in K562-Lucena 1) to 45.5 (aurelianolide A in MOLT-4). Aurelianolide A and B activated caspase 3/7 with consequent induction of apoptosis on Jurkat and K562-Lucena 1 cell lines. We have not observed induction of necrosis. CONCLUSION: Aurelianolides A and B have important cytotoxic effects on human leukemia cell lines by the activation of the caspase pathway.

Piper tectoniifolium Kunth: A New Natural Source of the Bioactive Neolignan (-)-Grandisin.

The Piper species are a recognized botanical source of a broad structural diversity of lignans and its derivatives. For the first time, Piper tectoniifolium Kunth is presented as a promising natural source of the bioactive (-)-grandisin. Phytochemical analyses of extracts from its leaves, branches and inflorescences showed the presence of the target compound in large amounts, with leaf extracts found to contain up to 52.78% in its composition. A new HPLC-DAD-UV method was developed and validated to be selective for the identification of (-)-grandisin being sensitive, linear, precise, exact, robust and with a recovery above 90%. The absolute configuration of the molecule was determined by X-ray diffraction. Despite the identification of several enantiomers in plant extracts, the major isolated substance was characterized to be the (-)-grandisin enantiomer. In vascular reactivity tests, it was shown that the grandisin purified from botanical extracts presented an endothelium-dependent vasorelaxant effect with an IC50 of 9.8 ± 1.22 µM and around 80% relaxation at 30 µM. These results suggest that P. tectoniifolium has the potential to serve as a renewable source of grandisin on a large scale and the potential to serve as template for development of new drugs for vascular diseases with emphasis on disorders related to endothelial disfunction.

Potential use of extract of the plant Schwartiza brasiliensis (choisy) bedell ex gir.-Cañas against the toxic effects of the venom of Bothrops jararaca or B. jararacussu.

Envenomation by snakes is a worldwide health public issue, and antivenoms are less efficient in neutralizing local toxic effects. Thus, more efficient therapies to treat patients deserve attention, and plants have been extensively tested. So, the aim of this work was to evaluate the effect of the aqueous fraction of the plant Schwartzia brasiliensis to inhibit some toxic activities of Bothrops jararaca or B. jararacussu venom. S. brasiliensis inhibited coagulant, hemolytic, proteolytic, hemorrhagic, edematogenic, and lethal activities of both venoms, regardless if plant was mixed together with venoms or injected after them as well as the route of administration (intravenous, oral or subcutaneous) of the plant. The S. brasiliensis extract showed no toxicity to mice or red blood cells. Thus, S. brasiliensis may be useful as an alternative treatment for snakebite envenomation and aid antivenom therapy to neutralize relevant toxic activities in patients bitten by Bothrops species.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Cytotoxicity and selectiveness of Brazilian Piper species towards oral carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the ten most common types of cancer worldwide. Plants of the genusPiper are used in traditional medicine to treat cancer, and they have a vast diversity of phytochemicals with cytotoxic potential. Purpose and Study Design: In this work, we analyzed the cytotoxic and selective potential of extracts and semipurified fractions of Piper mollicomum (PM), Piper truncatum (PT), Piper cernuum (PC), Piper arboreum (PA), and Piper cabralanum (PCa) using three different OSCC cell lines (SCC4, SCC9 and SCC25), and we measured their in vivo toxicities and conducted chemical analyses of their active fractions. RESULTS: The dichloromethane fractions of the crude methanolic extracts of the leaves of PM(-L-D), PC(-L-D) and PCa(-L-D) exhibited notable IC50 values of 94.2, 47.2 and 47.5 µg/mL, respectively, and all three of these extracts were more active than carboplatin (172.3 µg/mL). The most selective fraction was PC-L-D, which exhibited SI > 4.5; less than 5% hemolysis; and no significant alterations in in vivo acute toxicology. The major constituents in active fractions were lignans (PC-L-D and PCa-L-D) and chromenes (PM-L-D). CONCLUSION: PC-L-D demonstrated great potential for further development as an anticancer drug and could be the key to developing more effective and less toxic therapies against oral cancer.

Leishmanicidal Activity of Withanolides from Aureliana Fasciculata var. Fasciculata.

Leishmaniasis is the generic denomination to the neglected diseases caused by more than 20 species of protozoa belonging to the genus Leishmania. The toxic and parenteral-delivered pentavalent antimonials remain to be the first-line treatment. However, all the current used drugs have restrictions. The species Aureliana fasciculata (Vell.) Sendtner var. fasciculata is a native Brazilian species parsimoniously studied on a chemical point of view. In this study, the antileishmanial activity of A. fasciculata was evaluated. Among the evaluated samples of the leaves, the dichloromethane partition (AFfDi) showed the more pronounced activity, with IC50 1.85 µg/ml against promastigotes of L. amazonensis. From AFfDi, two active withanolides were isolated, the Aurelianolides A and B, with IC50 7.61 µM and 7.94 µM, respectively. The withanolides also proved to be active against the clinically important form, the intracellular amastigote, with IC50 2.25 µM and 6.43 µM for Aurelianolides A and B, respectively. Furthermore, withanolides showed results for in silico parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) similar to miltefosine, the reference drug, and were predicted as good oral drugs, with the advantage of not being hepatotoxic. These results suggest that these compounds can be useful as scaffolds for planning drug design.

Separation of Volatile Metabolites from the Leaf-Derived Essential Oil of Piper mollicomum Kunth (Piperaceae) by High-Speed Countercurrent Chromatography.

The technique of high-speed countercurrent chromatography was applied to the isolation of compounds in essential oil derived from the leaves of Piper mollicomum species. Plant leaves (200.0 g) were submitted to hydrodistillation in a modified Clevenger apparatus. The resulting crude leaf essential oil was analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine the profile of the components. The purified fractions were composed of monoterpenes and sesquiterpenes such as camphor (85.0 mg at 98.5% purity), (E)-nerolidol (100.0 mg at 92.8% purity), and camphene (150.0 mg at 82.0% purity). A minor component of the essential oil, bornyl acetate (16.2 mg at 91.2% purity) was also isolated in the one-step separation protocol in 2 h. The countercurrent chromatography technique proved to be a fast and efficient method for the separation of volatile metabolites that conserved the solvent while delivering various fractions of high purity.

Isolation of a larvicidal compound from Piper solmsianum C.DC. (Piperaceae).

The Aedes aegypti mosquito is one of the major vectors of arboviruses. These diseases have re-emerged and the insecticides used nowadays are toxic to mammals and environment and have only been effective in the short-term. In this context, natural products are an alternative. The genus Piper has many active compounds against arthropods, including neolignans. The present study evaluated the larvicidal potential of the n-hexanic extract of Piper solmsianum and eupomatenoid-6, identified by GC-MS and NMR techniques, from this extract against Ae. aegypti. The crude extract (100 µg/mL) killed 80% and 98.3% of larvae in the first and third day, respectively. Eupomatenoid-6 exhibited LD50 of 19.33 µM and LD90 of 28.68 µM and was then assayed in human fibroblast cells (MRC5), showing an IC50 of 39.30 µM with estimated LD50 of 42.26 mmol/kg. Our results indicate eupomatenoid-6 as a potent insecticide with relatively low toxicity for mammals.

Non-Clinical Studies for Evaluation of 8-C-Rhamnosyl Apigenin Purified from Peperomia obtusifolia against Acute Edema.

Compound 8-C-rhamnosyl apigenin (8CR) induced a moderate reduction in the enzymatic activity of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus and cytosolic phospholipase A2 (cPLA2), but the compound also significantly inhibited the enzymatic activity of the enzyme cyclooxygenase. In vitro assays showed that the compound induced a slight change in the secondary structure of sPLA2 from Crotalus durissus terrificus snake venom. In vivo assays were divided into two steps. In the first step, the 8CR compound was administered by intraperitoneal injections 30 min prior to administration of sPLA2. In this condition, 8CR inhibited edema and myonecrosis induced by the sPLA2 activity of Crotalus durissus terrificus in a dose-dependent manner by decreasing interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and lipid peroxidation. This has been demonstrated by monitoring the levels of malondialdehyde (MDA) in rat paws after the course of edema induced by sPLA2. These results, for the first time, show that sPLA2 of Crotalus durissus terrificus venom induces massive muscle damage, as well as significant edema by mobilization of cyclooxygenase enzymes. Additionally, its pharmacological activity involves increased lipid peroxidation as well as TNF-α and IL-1ß production. Previous administration by the peritoneal route has shown that dose-dependent 8CR significantly decreases the enzymatic activity of cyclooxygenase enzymes. This resulted in a decrease of the amount of bioactive lipids involved in inflammation; it also promoted a significant cellular protection against lipid peroxidation. In vivo experiments performed with 8CR at a concentration adjusted to 200 µg (8 mg/kg) of intraperitoneal injection 15 min after sPLA2 injection significantly reduced sPLA2 edema and the myotoxic effect induced by sPLA2 through the decrease in the enzymatic activity of cPLA2, cyclooxygenase, and a massive reduction of lipid peroxidation. These results clearly show that 8CR is a potent anti-inflammatory that inhibits cyclooxygenase-2 (COX-2), and it may modulate the enzymatic activity of sPLA2 and cPLA2. In addition, it was shown that Crotalus durissus terrificus sPLA2 increases cell oxidative stress during edema and myonecrosis, and the antioxidant properties of the polyphenolic compound may be significant in mitigating the pharmacological effect induced by sPLA2 and other snake venom toxins.

HSCCC separation and enantiomeric distribution of key volatile constituents of Piper claussenianum (Miq.) C. DC. (Piperaceae).

High Speed Countercurrent Chromatography (HSCCC) technique was used for the preparative isolation of the major leishmanicidal compounds from the essential oils of Piper claussenianum species in Brazil. The essential oils from inflorescences of P. claussenianum were analyzed by GC-FID and GC-MS. The enantiomeric ratio of the major constituents of the P. claussenianum essential oils were determined using a Rt-DEXsm chiral capillary column by GC-FID analysis. It was found an enantiomeric excess of (+)-(E)-nerolidol in the leaves, and (+)-linalool and (+)-(E)-nerolidol in the inflorescences essential oil. The major volatile terpenes alcohols were isolated in preparative scale from inflorescences: linalool (320.0 mg) and nerolidol (95.0 mg) in high purity level. The HSCCC, a support-free liquid-liquid partition chromatographic technique, proved to be an effective and useful method for fast isolation and purification of hydrophobic and similarly structured bioactive components from essential oils of Piper species.

Echinodorus grandiflorus: Ethnobotanical, phytochemical and pharmacological overview of a medicinal plant used in Brazil.

Echinodorus grandiflorus (Cham. & Schltdl.) Micheli is a native Brazilian species used in traditional practices for the treatment of several conditions such as inflammatory diseases, arthritis and hypertension. Through a systematic review of the accumulated knowledge about the species E. grandiflorus, the botanical, phytochemistry, ethnobotanical and pharmacological properties of this medicinal plant demonstrates its potential to naturally provide anti-inflammatory and anti-oxidant with a special emphasis on anti-hypertensive and cardioprotective effects. The body of literature reports that the chemical composition of crude E. grandiflorus extracts are notably composed of diterpenoids and flavonoids metabolites. Pharmacological studies have shown that oral treatments using the hydroalcoholic extracts of leaves from this plant has a significant anti-inflammatory, anti-hypertensive, diuretic and cardioprotective effects in rats with no toxicity. The holistic activities of complex extracts are corroborated by the individuals mechanisms of action, as well as, synergistic benefits attributed to the isolated chemical major constituents in this species. In light of the serious health concerns ascribed, it is important to investigate medicinal plant species with histories of traditional use for circulatory problems to meet the growing demands by scientifically validating their use and safety.

Larvicidal activity of Ottonia anisum metabolites against Aedes aegypti: A potential natural alternative source for mosquito vector control in Brazil.

BACKGROUND & OBJECTIVES: Aedes aegypti mosquito is the principal vector of the viruses responsible for urban yellow fever, dengue, dengue haemorrhagic fever, as well as Zika and chikungunya in Brazil. The present study was aimed to investigate the insecticidal potential of the extract and fractions of Ottonia anisum, along with special metabolites isolated from it, as natural alternatives against larvae (L3) of Ae. aegypti, vector of potentially deadly tropical infections in Brazil. METHODS: The plant species O. anisum was collected in March 2015, at Xerιm area, in Rio de Janeiro City, Brazil. Crude extracts and the isolated pure compounds were screened for toxicity against Ae. aegypti larvae (L3). Bioassays were performed on 20 larvae (L3) of Ae. aegypti in triplicate. The samples were dissolved in a mixture of acetone and DMSO at final concentrations of 1-200 µg/ml. The toxicity of the solutions was evaluated towards the growth and development of Ae. aegypti larvae till emergence of adults. RESULTS: The crude hexane extract showed 100% larval mortality 24 h after treatment at a concentration of 200 µg/ml. The bioassays using 1-butyl-3,4-methylenedioxybenzene revealed 100% mortality among L3 larvae, 24 h afterthe treatment at a concentration of 30 µg/ml, the LC recorded was 1.6 µg/ml. At concentration of 10 µg/ml, the L3 larval mortality recorded was 92%. INTERPRETATION & CONCLUSION: The metabolite 1-butyl-3,4-methylenedioxybenzene showed potent toxicity against Ae. aegypti larvae. This arylbutanoid agent could be used as a natural alternative adjuvant pesticide, in new compositions that would be environmentally safer.

Laboratory evaluation of Clusia fluminensis extracts and their isolated compounds against Dysdercus peruvianus and Oncopeltus fasciatus

ABSTRACT The effects of the hexanic extracts of the fruits and flowers of Clusia fluminensis Planch. & Triana, Clusiaceae, as well as their main constituents, the triterpene lanosterol and the benzophenone clusianone, were evaluated on hemipterans Dysdercus peruvianus and Oncopeltus fasciatus. The topical treatments of insects with the hexanic extracts significantly affected the survival of O. fasciatus, but not that of D. peruvianus. Concomitantly, extracts delayed the development of both hemipterans. Moreover, isolated lanosterol significantly reduced both the survival and development of O. fasciatus and D. peruvianus, while clusianone only reduce the survival of D. peruvianus and marginally inhibited the development of both insects. The results show the specific activity of lanosterol and clusianone against the two evaluated insect species and indicate the potential of compounds derived from C. fluminensis for the development of specific biopesticides for the control of agricultural pests. Subsequent work will examine the mode of action of lanosterol and clusianone isolates from C. fluminensis.

Local Anesthetic Activity from Extracts, Fractions and Pure Compounds from the Roots of Ottonia anisum Spreng. (Piperaceae).

Piperaceae species can be found worldwide in tropical and subtropical areas and many of them have been used for centuries in traditional folk medicine and in culinary. In Brazil, species of Piperaceae are commonly used in some communities as local anesthetic and analgesic. Countrified communities have known some species of the genus Ottonia as "anestesia" and it is a common habit of chewing leaves and roots of Ottonia species to relief toothache. The purpose of this study is to report our findings on new molecules entities obtained from the roots of Ottonia anisum Spreng, in which local anesthetic activity (sensory blockage) is demonstrated for the first time in vivo guinea pig model. Phytochemical investigation led to the isolation of three amides (pipercallosidine, piperine and valeramide) and in an enriched mixture of seven amides (valeramide, 4,5-dihydropiperlonguminine, N-isobutil-6-piperonil-2-hexenamide, piperovatine, dihydropipercallosidine, pipercallosidine and pipercallpsine). Our findings demonstrated the anesthetic potential for the methanolic extract from roots, its n-hexane partition and amides from O. anisum and it is in agreement with ethnobotanical survey.

Bioactivities of the ethanol extract from Ageratum fastigiatum branches: antioxidant, antinociceptive and anti-inflammatory.

The present study was designed to investigate the antioxidant, antinociceptive and anti-inflammatory activities of the ethanol extract from Ageratum fastigiatum branches. Phytochemical screening and total phenol and flavonoid contents were determined. The antioxidant activity was assessed by 2,2-diphenyl-1-pycrilhydrazin (DPPH) and iron reducing power methods. The antinociceptive effect was evaluated using the acetic acid-induced writhing, formalin, hot plate and tail immersion assays; while the carrageenan-induced paw edema and pleurisy tests were performed to examine the anti-inflammatory activity against acute inflammation. The extract revealed the presence of flavonoids, tannins, coumarins, terpenes, sterols and saponins. Expressive levels of total phenols and flavonoids and a promising antioxidant effect were quantified. At the doses of 50, 100 and 200 mg/kg, the extract inhibited the writhing, reduced both phases of paw licking time and increased the reaction time on the hot plate. In the tail immersion test, the extract (50, 100 and 200 mg/kg) caused a significant inhibition of pain. In these doses, the paw edema, exudate volume and leucocyte mobilization were significantly reduced. These results suggest that A. fastigiatum can be an active source of substances with antioxidant, antinociceptive and anti-inflammatory activities, adding scientific support to the appropriate use in the Brazilian folk medicine.

Composition of the essential oils from leaves of Piper lepturum Kunth (C.DC.) var. lepturum and Piper lepturum var. angustifolium (Miq.) Yunck. from Brasil / Composición de los aceites esenciales de hojas de Piper lepturum Kunth (C.DC.) var. lepturum y Pip

The essential oils of Brazilian Piper lepturum var. lepturum and Piper lepturum var. angustifolium (Piperaceae) were obtained by hydrodistillation and analyzed by flame-detector gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC/MS). According to GC and GC/MS analysis, the essential oils are mostly composed by sesquiterpenes hydrocarbons. beta-Guaiene (29.96 percent) was the principal component in the essential oil of P. lepturum var. lepturum and beta-Bisabolene (17.72 percent) was the principal components in the essential oil of P. lepturum var. angustifolium.

Los aceites esenciales de las especies brasileñas Piper lepturum var. lepturum y Piper lepturum var. angustifolium fueron obtenidos por hidrodestilación y analizados utilizando cromatografía gas líquido con detector de ionización de llama (CG) y cromatografía gas líquido acoplada a un detector de masas (CG/EM). De acuerdo con los análisis de CG y CG/EM, los aceites esenciales muestran como componente principal beta-Guaieno (29,96 por ciento) en el aceite esencial de P. lepturum var. lepturum y beta-Bisaboleno (17,71 por ciento) en el aceite esencial de P. lepturum var. angustifolium.

Antiviral and Immunomodulatory Effects of Norantea brasiliensis Choisy on Dengue Virus-2.

BACKGROUND/AIMS: Severe dengue fever is a result of exacerbated immune responses and no specific treatments are available. We evaluated the antiviral and immunomodulatory effects of Norantea brasiliensis Choisy. METHODS: Human adherent monocytes infected in vitro with dengue virus (DENV)-2 were incubated with the crude ethanol extract from leaves (NB1) or 3 derived fractions: dichloromethane (NB3), ethyl acetate (NB5), and butanolic (NB6) partitions. The antiviral and immunomodulatory activities were determined by intracellular detection of DENV antigen within monocytes and by secreted NS1 viral protein and cytokines. RESULTS: The crude extract alone exhibited both antiviral activities (intracellular and secreted antigens) and all fractions derived from this extract modulated NS1 production. Regarding the immunomodulatory effect, among the secreted factors, TNF-α was inhibited by NB3 and NB6; IL-6 was inhibited by NB1, NB3, and NB6; IL-10 by NB1 and NB3; and IFN-α by NB6. The crude extract (NB1) presented the best antiviral effect, whereas the dichloromethane fraction (NB3) presented an immunomodulatory effect in the inflammatory and anti-inflammatory cytokines. CONCLUSION: During in vitro DENV infection, N. brasiliensis Choisy exerts both antiviral and immunomodulatory effects that are likely associated, considering that less viral load may lead to less immunostimulation.

Interferometry as a tool for evaluating effects of antimicrobial doses on Mycobacterium bovis growth.

Interferometry was used together with the conventional microplate resazurin assay to evaluate the antimycobacterial properties of essential oil (EO) from fruits of Pterodon emarginatus and also of rifampicin against Mycobacterium bovis. The aim of this work is not only to investigate the potential antimycobacterial activity of this EO, but also to test the interferometric method in comparison with the conventional one. The Minimum Inhibitory Concentration (MIC) values of EO (625 µg/mL) and rifampicin (4 ng/mL) were firstly identified with the microplate method. These values were used as parameters in Drug Susceptibility Tests (DST) with interferometry. The interferometry confirmed the MIC value of EO identified with microplate and revealed a bacteriostatic behavior for this concentration. At 2500 µg/mL interferometry revealed bactericidal activity of the EO. Mycobacterial growth was detected with interferometry at 4 ng/mL of rifampicin and even at higher concentrations. One important difference is that the interferometric method preserves the sample, so that after weeks of quantitative observation, the sample can be used to evaluate the bactericidal activity of the tested drug.