Achyrocline satureioides (Lam) DC extracts acting as enzyme modulators: digestion, inflammation, and hemostasis / Extratos de Achyrocline satureioides (Lam) DC atuando como moduladores enzimáticos: digestão, inflamação e hemostasia

Achyrocline satureioides is popularly known for its richness in phenolic compounds and medicinal properties (anti-inflammatory, analgesic, and hepatoprotective). The present study aimed at broadening the knowledge about the pharmacological potential exerted by the aqueous and ethanolic extracts of A. satureioides. These extracts were characterized by HPLC and tested for their modulatory action on phospholipases A2 and proteases of snake venoms. In addition, they were tested on the activities of digestive enzymes. Snake venoms were used as tools since they have enzymes with high functional and structural homology to human enzymes. The results demonstrate that the extracts of A. satureioides act as enzymatic inhibitors or potentiators, interfering in processes related to the hemostasis, such as coagulation and thrombus dissolution. In addition, the anti-genotoxic activity and inhibitions exerted on digestive enzymes suggests their potential use in the prevention and/or treatment of several pathologies. New studies could provide information on how the compounds present in the extracts and the different enzymes interact.

A Achyrocline satureioides é popularmente conhecida por sua riqueza em compostos fenólicos e por suas propriedades medicinais (anti-inflamatória, analgésica e hepatoprotetora). No presente estudo, com o objetivo de ampliar o conhecimento sobre o potencial farmacológico exercido por esses extratos, os extratos aquoso e etanólico de A. satureioides foram caracterizados por HPLC e testados quanto à sua ação modulatória sobre as fosfolipases A2 e proteases de peçonhas de serpentes. Além disso, também foram testados em atividades de enzimas digestivas. As peçonhas de serpentes foram usadas como ferramentas por apresentarem enzimas com alta homologia funcional e estrutural às humanas. Os resultados demonstram que os extratos de A. satureioides atuam como inibidores ou potencializadores enzimáticos, interferindo em processos relacionados à hemostasia, como coagulação e dissolução do trombo. Além do mais, destacam seu potencial antigenotóxico e as inibições exercidas sobre as enzimas digestivas direcionando seu potencial de uso na prevenção e/ou tratamento de diversas patologias. Novos estudos poderão fornecer informações sobre os mecanismos de interação entre os compostos presentes nos extratos e as diferentes enzimas.

Thrombolysis, clotting, and genotoxic activities modulated by essential oils extracted from Lippia alba

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action

Metabolites from Induratia spp. modulating key enzymes in human hemostasis.

Induratia spp. fungi have been poorly evaluated for their non-volatile secondary metabolites. In the present work, we evaluated the effects of non-volatile secondary metabolites released into the culture medium by Induratia spp. upon toxic alterations induced by Bothrops spp. venoms. B. atrox venom phospholipase was inhibited by Induratia spp. around 12 and 16%. The extracts of the two strains inhibited 12-25% of the hemolysis induced by B.moojeni venom. Thrombolysis was inhibited by 30-60% by the compounds present in both extracts. The coagulation induced by B. moojeni venom was prolonged by 26-41 s by the action of the extracts of I. coffeana. The fungal extracts did not exert any cytotoxic effect, nor did they induce any alteration in the other evaluated substrates show the potential use of non-volatile metabolites produced by the fungi evaluated as enzyme modulators, especially for proteases with a fundamental role in human hemostasis.

Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells.

The venoms of wasps are a complex mixture of biologically active compounds, such as low molecular mass compounds, peptides, and proteins. The aim of the study was to evaluate the action of wasp venoms, Polybia occidentalis and Polybia fastidiosa, on the DNA of human leukocytes and on the cell cycle and genetic material of the plant model Lactuca sativa L. (lettuce). The cultured leukocytes were treated with the venoms and then evaluated by the comet assay. On another assay, seeds were exposed to a venom solution; the emitted roots were collected and the occurrence of cell cycle alterations (CCAs) and DNA fragmentation were evaluated by agarose gel electrophoresis and TUNEL assay. The results demonstrated that the venom of both wasps induces several CCAs and reduces the mitotic index (MI) on treated cells. They induced damage on human leukocytes DNA. High frequencies of fragments were observed in cells exposed to P. occidentalis venom, while those exposed to P. fastidiosa showed a high frequency of non-oriented chromosome. Both venoms induced the occurrence of various condensed nuclei (CN). This alteration is an excellent cytological mark to cell death (CD). Additionally, CD was evidenced by positive signals in TUNEL assay, by DNA fragmentation in agarose gel electrophoresis with vegetal cells, and by DNA fragmentation of the human leukocytes evaluated. Furthermore, human leukocytes exposed to the venom of P. fastidiosa had high rate of damage. The data demonstrate that both vegetal and human cells are adequate to evaluate the genotoxicity induced by venoms.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

Enzymatic Modulators from Induratia spp.

In the present work, ethyl acetate extracts, consisting of non-volatile compounds, from the culture of endophytic fungi isolated from coffee plants, Induratia coffeana and Induratia yucatanensis, were prospected in enzyme modulation tests that act in human hemostasis. Dry extracts of the fungi were diluted in dimethyl sulfoxide p.a. 99.9% (DMSO), and then tested. Bothrops atrox venom was used as an enzyme source and tool to induce the activities. Prior to the evaluation of the activities, incubations of the extracts with the venom were performed in the proportions 1: 0.01, 1: 0.25, 1: 0.5, and 1: 1 (venom: extract; mass: mass). The extracts of all fungi promoted a significant increase in the clotting time induced by the venom, which was even longer when the extracts were previously incubated with the citrated plasma. The activity of phospholipases A2 did not significantly change when evaluated in the presence of fungal extracts. However, the evaluated extracts inhibited proteases by 73% and 30% in the thrombolytic and caseinolytic tests, respectively. In addition, the extracts did not induce cytotoxicity on human erythrocytes when evaluated in the absence of the venom. Thus, it is possible to suggest the presence of specific interactions between molecules present in extracts of Induratia spp. and venom proteases, highlighting non-volatile metabolites as promising sources of compounds of medical and scientific interest.

Mitochondriotropic action and DNA protection: Interactions between phenolic acids and enzymes.

The protective action of caffeic (CA) and syringic (SA) acids on the genotoxicity exercised by snake venoms was investigated in this study. Molecular interactions between phenolic acids and the enzyme succinate dehydrogenase were also explored. In the electrophoresis assay, SA did not inhibit the genotoxicity induced by the venom. However, CA partially inhibited DNA degradation. In the comet assay, SA and CA exerted an inhibitory effect on the venom-induced fragmentation. Succinate dehydrogenase presented, in computational analyzes, favorable energies to the molecular bond to both the malonic acid and the phenolic compounds evaluated. In the enzymatic activity assays, SA inhibited succinate dehydrogenase and interfered in the interaction of malonic acid. Meanwhile, CA potentiated the inhibition exerted by the malonic acid. The results suggest transient interactions between toxins present in venoms and phenolic acids, mainly by hydrogen interactions, which corroborate with the data from previous works.

Exploring the structural and functional aspects of the phospholipase A2 from Naja spp.

Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A2 (PLA2) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLA2s from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA2 Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA2 called ß-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K+ and Ca++ channels or even interfere with the Na+/K+/ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA2 can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLA2s result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLA2s can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies.

Molecular interactions between p-coumaric acid and snake venom toxins.

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.

Spent pot liner from aluminum industry: genotoxic and mutagenic action on human leukocytes.

Spent pot liner (SPL) is a toxic solid waste generated in the aluminum mining and processing industry. SPL is considered as an environmental pollution agent when is dumped on environment. Thus, it is important to access its toxicological risk for the exposed organisms. The comet assay and micronucleus test are efficient tests to detect genotoxic/mutagenic compounds by DNA damage observation. Therefore, in the present study, the genotoxic potential of SPL was evaluated through the micronucleus and comet assay on human leukocytes. After ethics committee approval (COEP-UFLA n°. CAAE 11355312.8.0000.5060), blood aliquots collected from healthy volunteers were exposed to increasing concentrations of SPL (from 0.1 to 80 g L-1). All SPL treatments, including the lowest concentration applied (0.1 g L-1), significantly increased the micronucleus frequency. The frequency of DNA damage was determined by visual scores (from 0 to 4) and the results were expressed on percentage of damage and arbitrary units (AU). CaCl2 (0.01 M) was applied as negative control (NC) and doxorubicin (10 µg mL-1) as positive control (PC). It was observed a dose-dependency between SPL treatments: as SPL concentration for cell incubation increases, the frequency of damage on DNA also increases. Cells incubated on the NC presented nucleoids class 0 to 2, while those exposed to SPL presents nucleoids class 0 to 4. SPL-incubated cells increasing significantly the frequency of nucleoids class 4. For the PC, the UA of damage was 267.74, which is lower than the one observed for the treatments with high doses of SPL (40-287.40 g L-1 and 80-315.30 g L-1). Thus, it was demonstrated that the SPL is a genotoxic agent that induces DNA damage on exposed organisms.

Prospection of Enzyme Modulators in Aqueous and Ethanolic Extracts of Lippia sidoides Leaves: Genotoxicity, Digestion, Inflammation, and Hemostasis.

The aqueous and ethanolic extracts of Lippia sidoides Cham. were chemically characterized and tested for their action on enzymes involved in processes such as inflammation, blood coagulation, and digestion. Both extracts potentiated the activity of phospholipases A2 present in the venom of Bothrops atrox in 12 % and completely inhibited the hemolysis induced by B. jararacussu and B. moojeni venoms in the proportions between 1 : 0.5 and 1 : 5 (venom/extracts (w/w)). They inhibited the thrombolysis induced by B. moojeni (10 to 25 %), potentiated the thrombolysis induced by the Lachesis muta muta venom (30 to 80 %), prolonged the coagulation time induced by B. moojeni and L. muta muta venoms, and presented antigenotoxic action. Both extracts reduced the activity of α-glycosidases, the aqueous extract inhibited lipases, and the ethanolic extract inhibited α-amylases. The results demonstrate the modulatory action of the extracts on proteases, phospholipases, and digestive enzymes. In addition, the rich phenolic composition of these extracts highlights their potential for nutraceutical use.

Snake Venom Disintegrins: An Overview of their Interaction with Integrins.

Disintegrins are non-enzymatic proteins that interfere on cell-cell interactions and signal transduction, contributing to the toxicity of snake venoms and play an essential role in envenomations. Most of their pharmacological and toxic effects are the result of the interaction of these molecules with cell surface ligands, which has been widely described and studied. These proteins may act on platelets, leading to hemorrhage, and may also induce apoptosis and cytotoxicity, which highlights a high pharmacological potential for the development of thrombolytic and antitumor agents. Additionally, these molecules interfere with the functions of integrins by altering various cellular processes such as migration, adhesion and proliferation. This review gathers information on functional characteristics of disintegrins isolated from snake venoms, emphasizing a comprehensive view of the possibility of direct use of these molecules in the development of new drugs, or even indirectly as structural models.

The protective effect exerted by ascorbic acid on DNA fragmentation of human leukocytes induced by Lachesis muta muta venom.

The objective of this study was to evaluate the genotoxic and mutagenic effects of the toxins present in Lachesis muta muta's venom on human peripheral blood leukocytes and the protective potential of ascorbic acid on DNA fragmentation. The venom of L. muta muta was incubated in different concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, and 120 µg/mL) with human blood to evaluate DNA fragmentation using the comet, agarose gel electrophoresis, and micronucleus assays. In these concentrations evaluated, the venom of L. muta muta induced genotoxicity (comet assay and agarose gel electrophoresis) and mutagenicity (micronucleus test), but they were not cytotoxic, as they did not change the rate of cell proliferation after cytokinesis blockade with cytochalasin B. The ascorbic acid significantly inhibited the genotoxicity induced by L. muta muta venom in the proportions evaluated (1:0.1 and 1:0.5, venom/ascorbic acid - w/w). Thus, future studies are needed to elucidate the protective mechanisms of ascorbic acid on the genotoxic effects induced by toxins present in snake venoms.

Protective effect of ß-D-glucan and glutamine on the genomic instability induced by Cytarabine/Ara-C in BALB/c mice.

Prophylactic antibiotics and growth promoters have been substituted, mainly for livestock, by immunomodulators and intestinal health promoters - such as ß-D-glucans and glutamine. The aim of this study was to verify the beneficial effects of ß-D-glucans and glutamine against Cytarabine/Ara-C, evaluating the DNA damage in leukocytes, the leukogram, and the mitotic index of intestinal crypts cells. Balb/C mice received treatment with ß-D-glucan (80 mg/Kg), glutamine (150 mg/Kg), or both, for 21 days. On the last two days of this period, Ara-C was administered (1.8 mg/animal) by intraperitoneal injection every 12 h. The animals submitted to the treatment with Ara-C presented the highest genotoxic index, a significant leukopenia, and a decrease in the mitotic index of the intestinal crypts cells. Treatment with ß-D-glucan protected the leukocytes against DNA fragmentation induced by Ara-C. Glutamine alone promoted maintenance of the mitotic index and, in association with ß-Dglucan, reduced leukopenia. Thus, the use of ß-D-glucan and glutamine proved to be beneficial to intestinal tropism. This can happen once the damage to the genetic material, prevented by the treatments with ß-D-glucan and glutamine, can result in genotoxicity. Not only this, but it might be capable of turning into a mutagenesis, with consequential physiopathological alterations.

Anticancer Properties of Essential Oils: An Overview.

BACKGROUND: Essential oils are complex mixtures of low molecular weight compounds extracted from plants. Their main constituents are terpenes and phenylpropanoids, which are responsible for their biological and pharmaceutical properties, such as insecticidal, parasiticidal, antimicrobial, antioxidant, anti-inflammatory, analgesic, antinociceptive, anticarcinogenic, and antitumor properties. Cancer is a complex genetic disease considered as a serious public health problem worldwide, accounting for more than 8 million deaths annually. OBJECTIVE: The activities of prevention and treatment of different types of cancer and the medicinal potential of essential oils are addressed in this review. CONCLUSION: Several studies have demonstrated anti-carcinogenic and antitumor activity for many essential oils obtained from various plant species. They may be used as a substitution to or in addition to conventional anti-cancer therapy. Although many studies report possible mechanisms of action for essential oils compounds, more studies are necessary in order to apply them safely and appropriately in cancer therapy.