The Essential Oil from Conyza bonariensis (L.) Cronquist (Asteraceae) Exerts an In Vitro Antimelanoma Effect by Inducing Apoptosis and Modulating the MAPKs, NF-κB, and PKB/AKT Signaling Pathways.

The characterization and cytotoxicity of the essential oil from Conyza bonariensis (L.) aerial parts (CBEO) were previously conducted. The major compound was (Z)-2-lachnophyllum ester (EZ), and CBEO exhibited significant ROS-dependent cytotoxicity in the melanoma cell line SK-MEL-28. Herein, we employed the Molegro Virtual Docker v.6.0.1 software to investigate the interactions between the EZ and Mitogen-Activated Protein Kinases (MAPKs), the Nuclear Factor kappa B (NF-κB), and the Protein Kinase B (PKB/AKT). Additionally, in vitro assays were performed in SK-MEL-28 cells to assess the effect of CBEO on the cell cycle, apoptosis, and these signaling pathways by flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using MAPKs inhibitors. CBEO induced a significant increase in the sub-G1 peak, as well as biochemical and morphological changes characteristic of apoptosis. The in-silico results indicated that EZ interacts with Extracellular Signal-Regulated Kinase 1 (ERK1), c-Jun N-terminal Kinase 1 (JNK1), p38α MAPK, NF-κB, and PKB/AKT. Moreover, CBEO modulated the ERK1/2, JNK, p38 MAPK, NF-κB, and PKB/AKT activities in SK-MEL-28 cells. Furthermore, CBEO's cytotoxicity against SK-MEL-28 cells was significantly altered in the presence of MAPKs inhibitors. These findings support the in vitro antimelanoma effect of CBEO through apoptosis induction, and the modulation of ERK, JNK, p38 MAPK, NF-κB, and PKB/AKT activities.

Chemical Composition, In Vitro Antitumor Effect, and Toxicity in Zebrafish of the Essential Oil from Conyza bonariensis (L.) Cronquist (Asteraceae).

The essential oil from Conyza bonariensis (Asteraceae) aerial parts (CBEO) was extracted by hydrodistillation in a Clevenger-type apparatus and was characterized by gas chromatography-mass spectrometry. The antitumor potential was evaluated against human tumor cell lines (melanoma, cervical, colorectal, and leukemias), as well as non-tumor keratinocyte lines using the MTT assay. The effect of CBEO on the production of Reactive Oxygen Species (ROS) was evaluated by DCFH-DA assay, and a protection assay using the antioxidant N-acetyl-L-cysteine (NAC) was also performed. Moreover, the CBEO toxicity in the zebrafish model was assessed. The majority of the CBEO compound was (Z)-2-lachnophyllum ester (57.24%). The CBEO exhibited selectivity towards SK-MEL-28 melanoma cells (half maximal inhibitory concentration, IC50 = 18.65 ± 1.16 µg/mL), and induced a significant increase in ROS production. In addition, the CBEO's cytotoxicity against SK-MEL-28 cells was reduced after pretreatment with NAC. Furthermore, after 96 h of exposure, 1.5 µg/mL CBEO induced death of all zebrafish embryos. Non-lethal effects were observed after exposure to 0.50-1.25 µg/mL CBEO. Additionally, significant alterations in the activity of enzymes associated with oxidative stress in zebrafish larvae were observed. These results provide evidence that CBEO has a significant in vitro antimelanoma effect by increasing ROS production and moderate embryotoxicity in zebrafish.

Toxicogenetic profile of the monoterpene alpha-terpineol on normal and tumor eukaryotic cells.

Alpha-terpineol is a monoterpene alcohol found in essential oils from medicinal plants with some well-known pharmacological activities and widely used in cosmetics. However, the toxicological effects and additional pharmacological activities need to be clarified. Thus, the study evaluated the toxic, cytotoxic, genotoxic, hemolytic, and oxidative potential of alpha-terpineol in non-clinical bioassays. Different concentrations of alpha-terpineol were used in bioassays, including MTT (50, 100, 200, and 400 µg/mL), Artemia salina (6.25-400 µg/mL), Allium cepa (10, 50, and 100 µg/mL), comet assay (100, 200, and 500 µg/mL), cytokinesis-block micronucleus (100, 250, and 500 µg/mL), confocal microscopy for apoptosis quantification (100 and 500 µg/mL), hemolysis and Saccharomyces cerevisiae central disk test (10, 35, and 75 µg/mL). For the MTT test, alpha-terpineol was more cytotoxic on melanoma murine B16-F10 cells rather than macrophages. For A. salina test, alpha-terpineol showed LC50 of 68.29 and 76.36 µg/mL for 24 h and 48 h of exposure time, respectively. Meanwhile, alpha-terpineol was also cytotoxic to meristematic cells, which revealed inhibition of cellular division and mutagenic action by formation of bridges and delayed anaphases. The compound increased damage index and frequency of damage corroborated by the presence of micronuclei, bridges and nuclear buds at 500 µg/mL, but it caused neither hemolysis, oxidative damage on the S. cerevisiae nor cell death in normal fibroblasts. The findings indicate alpha-terpineol has cytotoxic potential by cytogenetic and molecular mechanisms associated with apoptosis and probable target effects against melanoma cells.

Cytotoxic and mutagenic effects of the food additive tartrazine on eukaryotic cells.

BACKGROUND: Among the food additives used in the food industry, food dyes are considered the most toxic. For instance, tartrazine (TRZ) is a food colorant commercially available with conflicting data regarding its cytotoxic, genotoxic, and mutagenic effects. Therefore, this study aimed to evaluate the cytotoxic and mutagenic potential of TRZ using different eukaryotic cells (in vitro). METHODS: This study employed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), brine shrimp lethality, Allium cepa and Saccharomyces cerevisiae tests. Different concentrations of TRZ and different exposure times were used in this study. RESULTS: The results demonstrate that TRZ induced a concentration-dependent toxic effect on the test systems. It also exerted cytotoxicity in fibroblasts and human gastric cells. In addition, TRZ showed mutagenic effects on the A. cepa test system. However, its toxicogenic effects may not relate to the oxidizing activity, which was confirmed by the S. cerevisiae test model. CONCLUSION: Taken together, TRZ exerted toxicogenic effects on the test systems. Therefore, it may be harmful to health, especially its prolonged use may trigger carcinogenesis.

Apoptotic and antioxidant effects in HCT-116 colorectal carcinoma cells by a spiro-acridine compound, AMTAC-06.

BACKGROUND: Acridine compounds have been described as promising anticancer agents. Previous studies showed that (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a spiro-acridine compound, has antitumor activity on Ehrlich tumor and low toxicity. Herein, we investigated its antitumor effect against human cells in vitro. METHODS: MTT assay was used to assess cytotoxicity of AMTAC-06 (3.125-200 µM) against tumor and non-tumor cells, and the half-maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated. The effects on the cell cycle (propidium iodide-PI-staining), apoptosis (Annexin V-FITC/PI double staining by flow cytometry), and production of reactive oxygen species, ROS (DCFH assay) were also evaluated. Statistical analysis was achieved using ANOVA followed by Tukey's post-test. RESULTS: AMTAC-06 showed higher cytotoxicity against colorectal carcinoma HCT-116 cells (IC50: 12.62 µM). The SI showed that AMTAC-06 was more selective for HCT-116 cells (HaCaT SI: 1.41; PBMC SI: 0.62) than doxorubicin (HaCaT SI: 0.10; PBMC SI: 0.01). AMTAC-06 (15 and 30 µM) induced an increase in the sub-G1 peak (p < 0.000001) and cell cycle arrest in S phase (p = 0.003547). Moreover, treatment with this compound (15 and 30 µM) resulted in increased early (p < 0.000001) and late apoptotic cells (p < 0.000001). In addition, there was a reduction on ROS production (p < 0.000001). CONCLUSIONS: AMTAC-06 presents anticancer activity against HCT-116 cells by regulating the cell cycle, inducing apoptosis and an antioxidant action.

Methods of macrophages activation and their modulation for the prospection of new antileishmania drugs: a review

Leishmaniasis are a group of parasitic zoonoses provoked by protozoa from Leishmania genus and belonging to the group of neglected tropical diseases. The search and development for new drugs is necessary not only to investigate the activity against only the parasite, but also to investigate the possible synergistic effect of new drugs with the immune response of the host. In the present review, macrophages are pointed out as potential targets of the investigation of new antileishmanial drugs, and some methodologies in order to assess their activation as response to Leishmania-infected cells are presented. Macrophages are an important role in the cellular immune response, since they are cells from mononuclear phagocytic system, the first line of defense of the host, against parasites from Leishmania genus. Phagocytic capacity, lysosomal activity, increase of nitric oxide and intracellular calcium levels are parameters regarding assessment of macrophages activation which allow them to be more hostile in order to solve the infection and lead the patient to cure. In this context, we bring 19 substances already investigated and that activate macrophages, what makes them promising in the antileishmanial treatment. Therefore, assessment of macrophages activation, are important tools for discovery of immunomodulatory compounds which have potential to act in synergism with host immune response. Such compounds might be promising as monotherapy in the treatment of leishmaniasis, as well as being used as adjuvants in vaccines and/or in combination with conventional drugs.

Assessment of In Vitro Anti-melanoma Potential of Ephedranthus pisocarpus R.E.Fr.

BACKGROUND/AIM: Despite being a rare disease, melanoma is considered the most dangerous skin cancer due to its highly invasive and aggressive nature, and still requires for more effective treatments. The aim of this study was to evaluate the in vitro anti-melanoma potential of Ephedranthus pisocarpus R.E.Fr. (Annonaceae), a popular Brazilian plant with medicinal properties. MATERIALS AND METHODS: Initially, the ethanolic extract (EtOH) was obtained from E. pisocarpus leaves and later partitioned using increasing polarity solvents. The anti-melanoma potential of E. pisocarpus was assessed by spectrophotometry and its cytotoxicity determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and confocal microscopy. RESULTS: We demonstrated that the EtOH extract and fractions from E. pisocarpus had a moderate photoprotective action (FPS 3.0-5.0) against UVA radiation. Interestingly, the dichloromethane fraction presented higher anti-melanoma activity against B16-F10 (IC50=46.8 µg/ml) and SK-MEL-28 cells (IC50=40.1 µg/ml) and lesser toxicity on normal cells. Additionally, our study reported that spathulenol, one of the major constituents from E. pisocarpus, acts through an apoptosis-dependent mechanism in SK-MEL-28 cells. CONCLUSION: The present study demonstrated, for the first time, the in vitro anti-melanoma potential of E. pisocarpus against melanoma cells.

Anticancer Effect of a Spiro-acridine Compound Involves Immunomodulatory and Anti-angiogenic Actions.

BACKGROUND/AIM: Studies with acridine compounds have reported anticancer effects. Herein, we evaluated the toxicity and antitumor effect of the (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a promising anticancer spiro-acridine compound. MATERIALS AND METHODS: The toxicity of AMTAC-06 was evaluated on zebrafish and mice. Antitumor activity was assessed in Ehrlich ascites carcinoma model. Effects on angiogenesis, cytokine levels and cell cycle were also investigated. RESULTS: AMTAC-06 did not induce toxicity on zebrafish and mice (LD50 approximately 5000 mg/kg, intraperitoneally). No genotoxicity was observed on micronucleus assay. AMTAC-06 significantly reduced the total viable Ehrlich tumor cells and increased sub-G1 peak, suggesting apoptosis was triggered. Moreover, the compound significantly decreased the density of peritumoral microvessels, indicating an anti-angiogenic action, possibly dependent on the cytokine modulation (TNF-α, IL-1ß and IFN-γ). No significant toxicological effects were recorded for AMTAC-06 on tumor transplanted animals. CONCLUSION: AMTAC-06 has low toxicity and a significant antitumor activity.

Gallic and Ellagic Acids Are Promising Adjuvants to Conventional Amphotericin B for the Treatment of Cutaneous Leishmaniasis.

In this study, we demonstrated the potential associative effect of combining conventional amphotericin B (Amph B) with gallic acid (GA) and with ellagic acid (EA) in topical formulations for the treatment of cutaneous leishmaniasis in BALB/c mice. Preliminary stability tests of the formulations and in vitro drug release studies with Amph B, GA, Amph B plus GA, EA, and Amph B plus EA were carried out, as well as assessment of the in vivo treatment of BALB/c mice infected with Leishmania major After 40 days of infection, the animals were divided into 6 groups and treated twice a day for 21 days with a gel containing Amph B, GA, Amph B plus GA, EA, or Amph B plus EA, and the negative-control group was treated with the vehicle. In the animals that received treatment, there was reduction of the lesion size and reduction of the parasitic load. Histopathological analysis of the treatments with GA, EA, and combinations with Amph B showed circumscribed lesions with the presence of fibroblasts, granulation tissue, and collagen deposition, as well as the presence of activated macrophages. The formulations containing GA and EA activated macrophages in all evaluated parameters, resulting in the activation of cells of the innate immune response, which can generate healing and protection. GA and EA produced an associative effect with Amph B, which makes them promising for use with conventional Amph B in the treatment of cutaneous leishmaniasis.

Retinol palmitate and ascorbic acid: Role in oncological prevention and therapy.

Cancer development has been directly related to oxidative stress. During chemotherapy, some cancer patients use dietary antioxidants to avoid nutritional deficiencies due to cancer treatment. Among the antioxidants consumed, there are vitamins, including retinyl palmitate (PR) and ascorbic acid (AA), which have the capacity to reduce free radicals formation, protect cellular structures and maintain the cellular homeostasis. This systematic review evaluated the antioxidant and antitumor mechanisms of retinol palmitate (a derivative of vitamin A) and/or ascorbic acid (vitamin C) in cancer-related studies. Ninety-seven (97) indexed articles in the databases PubMed and Science Direct, published between 2013 and 2017, including 23 clinical studies (5 for every single compound while 13 in interaction) and 74 non-clinical studies (37 for retinol palmitate, 36 for ascorbic acid and 1 in interaction) were considered. Antioxidant and antitumor effects, with controversies over dosage and route of administration, were observed for the test compounds in their isolated form or associated in clinical studies. Prevention of cancer risks against oxidative damage was seen in lower doses of retinol palmitate and/or vitamin C. However, at high doses, they can generate reactive oxygen species, cytotoxicity and apoptosis in test systems. Non-clinical studies using cell lines have allowed understanding the mechanisms related to antioxidants and antitumor effects of the isolated compounds, however, studies on vitamin interactions, acting as antioxidants and/or antitumor are still rare and controversial. More studies, mainly related to modulation of antineoplastic drugs are needed for understanding the risks and benefits of their use during treatment in order to achieve effectiveness in cancer therapy and patient's quality of life.

Gallic and ellagic acids: two natural immunomodulator compounds solve infection of macrophages by Leishmania major.

Leishmaniasis is a complex of parasitic protozoan diseases caused by more than 20 different species of parasites from Leishmania genus. Conventional treatments are high costly, and promote a sort of side effects. Besides, protozoan resistance to treatments has been reported. Natural products have been investigated as a source of new therapeutic alternatives, not only acting directly against the parasite but also being able to synergistically act on the host immune system in order to control parasitemia. Gallic acid (GA) and ellagic acid (EA) are plant-derived phenolic compounds which are able to induce antiinflammatory, gastroprotective, and anticarcinogenic activities. Therefore, the antileishmania, cytotoxic, and immunomodulatory activities of GA and EA were evaluated in this study. Both GA and EA were able to inhibit the growth of Leishmania major promastigotes (effective concentration (EC50) values 16.4 and 9.8 µg/mL, respectively). The cytotoxicity against BALB/c murine macrophages for GA and EA was also assessed (CC50 values 126.6 and 23.8 µg/mL, respectively). Interestingly, GA and EA also significantly reduced the infection and infectivity of macrophages infected by L. major (EC50 values 5.0 and 0.9 µg/mL, respectively), with selectivity index higher than 20. Furthermore, both GA and EA induced high immunomodulatory activity evidenced by the increase of phagocytic capability, lysosomal volume, nitrite release, and intracellular calcium [Ca2+i] in macrophages. Further investigations are reinforced in order to evaluate the therapeutic effects of GA and EA in in vivo experimental infection model of leishmaniasis.

Essential oil composition and antinociceptive activity of Thymus capitatus.

CONTEXT: The essential oil (EO) from Thymus capitatus Hoff. et Link. (Lamiaceae) has been traditionally used for its medicinal properties, such as anti-inflammatory, analgesic, antioxidant and antimicrobial properties. OBJECTIVE: Characterize the constituents from T. capitatus EO and further evaluate the antinociceptive activity by in vivo and in vitro procedures. MATERIALS AND METHODS: Gas chromatography-mass spectrometry was used to identify and quantify the constituents of the T. capitatus EO. The antinociceptive activity was evaluated in vivo by the glutamate-induced nociception model in male Swiss mice (25 g), at doses of 3, 6 and 12 mg/kg, 1 h before evaluation of the licking time response (0-15 min). The mechanism of T. capitatus EO (1-500 µg/mL) on the isolated nerve excitability of Wistar rat (300 g) was assessed by the single sucrose technique. RESULTS AND DISCUSSION: The EO of T. capitatus presented 33 components, mainly monoterpenes and sesquiterpenes, carvacrol (ca. 80%) was its major constituent. T. capitatus EO induced antinociception in orally treated mice (3, 6, and 12 mg/kg) reducing the licking time from control (100.3 ± 11.9 s) to 84.8 ± 12.2, 62.7.6 ± 9.9, and 41.5 ± 12.7 s, respectively (n = 8; p < 0.05). Additionally, we have demonstrated that T. capitatus EO (500 µg/mL) decreased the compound action potential amplitude (VCAP) of about 80.0 ± 4.3% from control recordings (n = 4; p < 0.05). Such activity was presumably mediated through a voltage-gated Na+ channels. CONCLUSIONS: The present study demonstrated the antinociceptive activity of Thymus capitatus essential oil, which acts via peripheral nervous excitability blockade.

Rosewood oil induces sedation and inhibits compound action potential in rodents.

AIM OF THE STUDY: Aniba rosaeodora is an aromatic plant which has been used in Brazil folk medicine due to its sedative effect. Therefore, the purpose of the present study was to evaluate the sedative effect of linalool-rich rosewood oil in mice. In addition we sought to investigate the linalool-rich oil effects on the isolated nerve using the single sucrose-gap technique. MATERIALS AND METHODS: Sedative effect was determined by measuring the potentiation of the pentobarbital-induced sleeping time. The compound action potential amplitude was evaluated as a way to detect changes in excitability of the isolated nerve. RESULTS: The results showed that administration of rosewood oil at the doses of 200 and 300 mg/kg significantly decreased latency and increased the duration of sleeping time. On the other hand, the dose of 100 mg/kg potentiated significantly the pentobarbital action decreasing pentobarbital latency time and increasing pentobarbital sleeping time. In addition, the effect of linalool-rich rosewood oil on the isolated nerve of the rat was also investigated through the single sucrose-gap technique. The amplitude of the action potential decreased almost 100% when it was incubated for 30 min at 100 microg/ml. CONCLUSIONS: From this study, it is suggested a sedative effect of linalool-rich rosewood oil that could, at least in part, be explained by the reduction in action potential amplitude that provokes a decrease in neuronal excitability.

Antinociceptive activity of (-)-carvone: evidence of association with decreased peripheral nerve excitability.

(-)-Carvone is a monoterpene ketone that is the main active component of Mentha plant species like Mentha spicata. This study aimed to investigate the antinociceptive activity of (-)-carvone using different experimental models of pain and to investigate whether such effects might be involved in the nervous excitability elicited by others monoterpenes. In the acetic acid-induced writhing test, we observed that (-)-carvone-treated mice exhibited a significant decrease in the number of writhes when 100 and 200 mg/kg was administered. It was also demonstrated that (-)-carvone inhibited the licking response of the injected paw when 100 and 200 mg/kg was administered (i.p.) to mice in the first and second phases of the formalin test. Since naloxone (5 mg/kg, s.c.), an opioid antagonist, showed no influence on the antinociceptive action of (-)-carvone (100 mg/kg), this suggested nonparticipation of the opioid system in the modulation of pain induced by (-)-carvone. Such results were unlikely to be provoked by motor abnormality, since (-)-carvone-treated mice did not exhibit any performance alteration on the Rota-rod apparatus. Because the antinociceptive effects could be associated with neuronal excitability inhibition, we performed the single sucrose gap technique and observed that (-)-carvone (10 mM) was able to reduce the excitability of the isolated sciatic nerve through a diminution of the compound action potential amplitude by about 50% from control recordings. We conclude that (-)-carvone has antinociceptive activity associated with decreased peripheral nerve excitability.

Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents.

Citronellol is one monoterpene alcohol, which is present in the essential oils of various aromatic plant species. This study evaluated the neuroprotective activity of citronellol on pentylenetetrazol- and picrotoxin-induced convulsions and maximal electroshock-induced seizures in mice. Administration of citronellol significantly reduced the number of animals of convulsion induced by pentylenetetrazol and eliminated the extensor reflex of maximal electroshock-induced seizures test in about 80% of the experimental animals. In addition, administration of citronellol showed protection in the pentylenetetrazol and picrotoxin tests by increasing the latency of clonic seizures. We also investigated the effect of citronellol in the rat isolated nerve using the single sucrose-gap technique. We showed that the amplitude of the compound action potential decreased more than 90% when the monoterpene was incubated for 30 min at 6.4 mM and we did not verify any effect on the repolarization of the compound action potential. Taken together, our results demonstrated an anticonvulsant activity of the citronellol that could be, at least in part, explained by the diminution of the action potential amplitude.